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Peripheral plasticity after spinal cord injury and ramifications for cardiovascular function Ramer, Leanne Margaret 2012

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PERIPHERAL PLASTICITY AFTER SPINAL CORD INJURY AND RAMIFICATIONS FOR CARDIOVASCULAR FUNCTION  by Leanne Margaret Ramer  MSc, The University of British Columbia, 2003.  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Zoology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April, 2012  © Leanne Margaret Ramer, 2012  Abstract Cardiovascular problems create life-long challenges for people living with spinal cord injury (SCI). When SCI occurs above the sixth thoracic segment (T6), it isolates spinal circuitry governing the critical splanchnic vascular bed, and creates the conditions for autonomic dysreflexia (AD), episodic hypertension instigated by sensory stimulation below the level of SCI. Most experiments investigating mechanisms of AD describe plasticity in the injured spinal cord. In this dissertation, I examined injury-induced changes at two peripheral loci critical to AD, the dorsal root ganglion (DRG) and mesenteric arteries. I used adult Wistar rats and performed complete transection SCI at T3 or T10: while both injuries produce hind limb paralysis, only the former is accompanied by AD. In the DRG, I found that T3 SCI triggered somatic hypertrophy in a specific subset of nociceptors, those expressing the capsaicin receptor (TRPV1). SCI-induced hypertrophy occurred in DRGs caudal to SCI and was most pronounced in lumbosacral ganglia. Intriguingly, SCI-induced hypertrophy was much more pronounced after T3 than T10 SCI. Importantly, when I used capsaicin to selectively eliminate TRPV1-positive projections to the lumbosacral spinal cord, the severity of AD was dramatically reduced. Next I examined glial, immune and vascular constituents of the lumbar DRG following SCI. I found that T3, but not T10 SCI activated satellite cells and macrophages in the DRG, and provoked mast cell accumulation in the adjacent spinal nerve. SCI at both levels promoted angiogenesis in the DRG and ingrowth of sympathetic ganglionic axons.  ii  In the superior mesenteric artery (SMA), I used in vitro myography to examine the role of cyclooxygenase (COX) enzymes in phenylephrine (PE) hyper-responsiveness after T3 SCI. I found that PE hypersensitivity was reversed by specific inhibitors of COX-2 and that COX-2 was upregulated in the SMA after T3 SCI. In an additional set of experiments, I found that recurrent episodes of AD, induced intentionally during recovery from SCI, exacerbated PE hyper-responsiveness in the SMA. These findings identify SCI-induced changes in the periphery that may contribute to AD by augmenting sensory input to the spinal cord or sympatheticallymediated vasoconstriction. These SCI-provoked effects may represent new therapeutic targets to treat AD.  iii  Preface All experiments in this dissertation were approved by the Animal Care Committee of the University of British Columbia, under certificates numbered A09-0334 and A10-0129. Detailed animal care procedures for rats with spinal cord injury (presented in Chapters 2-5) have been published: Ramsey JB*, Ramer LM*, Inskip JA, Alan N, Ramer MS, Krassioukov AV. (2010) Care of rats with complete high-thoracic spinal cord injury. Journal of Neurotrauma 27(9):1709-22. *Authors contributed equally.  In sections throughout, the thesis draws on material from a published review: Inskip JA*, Ramer LM*, Ramer MS, Krassioukov AV. (2009) Autonomic assessment of animals with spinal cord injury: tools, techniques and translation. Spinal Cord 47(1):235. *Authors contributed equally.  Chapters 2-5 present work on collaborative projects.  Chapter 2 is in preparation for publication, with the following author list: Ramer LM, van Stolk AP, Inskip JA, Ramer MS, Krassioukov AV. I co-designed the experiments (with AVK, APvS and MSR). Working with JAI, with assistance from APvS, I performed surgeries and animal care: these were split approximately equally between me and JAI. MSR performed intrathecal capsaicin injections. Tissue harvesting, preparation, immunohistochemistry, imaging and analysis  iv  were split approximately equally between me and APvS, with assistance and guidance from MSR and AVK. I wrote the chapter, with assistance from AVK and MSR.  Chapter 3 is in preparation for publication, with the following author list: Ramer LM, Crawford MA, Inskip JA, van Stolk AP, Steeves JD, Ramer MS, Krassioukov AV. I co-designed the experiments (with AVK, JAI, JDS and MSR). Working with JAI, I performed surgeries and animal care: these were split approximately equally between me and JAI. Tissue harvesting, preparation, immunohistochemistry, imaging and analysis were split approximately equally between me, MAC and APvS, with assistance and guidance from MSR and AVK. I wrote the chapter, with assistance from AVK, JDS and MSR.  Chapter 4 is in preparation for publication, with the following author list: Ramer LM, Alan N, Inskip JA, Golbidi S, Ramer MS, Laher I, Krassioukov AV. I co-designed the experiments (with NA, AVK, JAI, and IL). Working with JAI, I performed surgeries, animal care, and in vivo cardiovascular assessment: these were split approximately equally between me and JAI. SG performed in vitro myography. NA performed most of the data analysis, in consultation with me and JAI. I wrote the chapter, with assistance from NA, JAI and AVK; the published version was edited by all authors.  A version of Chapter 5 has been published. Alan N*, Ramer LM*, Inskip JA, Golbidi S, Ramer MS, Laher I, Krassioukov AV. (2010) Recurrent autonomic dysreflexia  v  exacerbates vascular dysfunction after spinal cord injury. The Spine Journal 10(12):1108-17. *NA and LMR contributed equally to this manuscript. I co-designed the experiments (with NA, AVK, JAI, and IL). Working with JAI, I performed surgeries, animal care, and in vivo cardiovascular assessment: these were split approximately equally between me and JAI. SG performed in vitro myography. NA performed most of the data analysis, in consultation with me and JAI. I wrote the manuscript, with assistance from NA, JAI, and AVK; the published version was edited by all authors.  vi  Table of Contents Abstract ............................................................................................................................. ii	
   Preface .............................................................................................................................. iv	
   Table of Contents ............................................................................................................ vii	
   List of Figures ................................................................................................................ xiv	
   List of Abbreviations .................................................................................................... xvii	
   Dedication ....................................................................................................................... xxi	
   Chapter 1: Introduction .......................................................................................................... 1	
   1.1	
   Overview ....................................................................................................................... 1	
   1.2	
   Neural cardiovascular control: relevant and interesting aspects ................................... 3	
   1.2.1	
   Supraspinal cardiovascular integration ................................................................................. 4	
   1.2.2	
   Autonomic control of the heart ............................................................................................. 7	
   1.2.3	
   Autonomic control of the vasculature ................................................................................ 11	
    1.3	
   Impaired cardiovascular regulation following SCI ..................................................... 22	
   1.4	
   Autonomic dysreflexia ................................................................................................ 26	
   1.4.1	
   Clinical significance of autonomic dysreflexia .................................................................. 27	
   1.4.2	
   Animal models of autonomic dysreflexia ........................................................................... 29	
    1.5	
   The dorsal root ganglion.............................................................................................. 33	
   1.5.1	
   First-order sensory neurons ................................................................................................ 34	
   1.5.2	
   Glial environment of the dorsal root ganglion.................................................................... 35	
   1.5.3	
   Vascular environment of the dorsal root ganglion ............................................................. 37	
    1.6	
   Spinal segments of the sensory-sympathetic reflex .................................................... 38	
   1.6.1	
   Sensory circuits in the dorsal horn ..................................................................................... 39	
   1.6.2	
   Spinal sympathetic transmission ........................................................................................ 39	
    vii  1.7	
   Sensory and sympathetic plasticity after spinal cord injury: the search for the cause of autonomic dysreflexia ........................................................................................................... 41	
   1.7.1	
   Spinal cord injury-induced plasticity in the spinal cord ..................................................... 42	
   1.7.2	
   Spinal cord injury-induced changes in the blood vessels ................................................... 46	
    1.8	
   Experimental overview and hypotheses ...................................................................... 47	
   1.8.1	
   Differential effects of high- and low-thoracic spinal cord injury ....................................... 47	
   1.8.2	
   Injury-induced changes in the dorsal root ganglion ........................................................... 47	
   1.8.3	
   Injury-induced changes in the mesenteric arteries ............................................................. 49	
    Chapter 2: Plasticity of capsaicin-sensitive sensory neurons mediating autonomic dysreflexia following spinal cord injury................................................................................ 51	
   2.1	
   Synopsis....................................................................................................................... 51	
   2.2	
   Introduction ................................................................................................................. 51	
   2.3	
   Materials and methods................................................................................................. 54	
   2.3.1	
   Spinal cord injury surgery .................................................................................................. 54	
   2.3.2	
   Post-operative animal care.................................................................................................. 56	
   2.3.3	
   Survival times ..................................................................................................................... 56	
   2.3.4	
   Intrathecal capsaicin injection ............................................................................................ 57	
   2.3.5	
   Cardiovascular assessment ................................................................................................. 57	
   2.3.6	
   Tissue processing and immunohistochemistry ................................................................... 58	
   2.3.7	
   Cardiovascular data analysis .............................................................................................. 60	
   2.3.8	
   Image analysis .................................................................................................................... 60	
   2.3.9	
   Statistics .............................................................................................................................. 62	
    2.4	
   Results ......................................................................................................................... 63	
   2.4.1	
   Complete high-thoracic spinal cord injury provoked hypertrophy in sensory neurons that express the capsaicin receptor. ....................................................................................................... 63	
   2.4.2	
   Spinal cord injury -induced hypertrophy was most pronounced in lumbosacral sensory ganglia ............................................................................................................................................ 70	
    viii  2.4.3	
   Capsaicin-sensitive afferents hypertrophied and upregulated the capsaicin receptor after spinal cord injury. .......................................................................................................................... 73	
   2.4.4	
   Injury-induced hypertrophy was modest after low-thoracic spinal cord injury ................. 75	
   2.4.5	
   Dramatic somatic hypertrophy in capsaicin-sensitive afferents was not reflected in plasticity of their central projections.............................................................................................. 77	
   2.4.6	
   Intrathecal capsaicin attenuated colo-rectal distension-induced autonomic dysreflexia .... 81	
    2.5	
   Discussion ................................................................................................................... 85	
   2.5.1	
   Spinal cord injury-induced hypertrophy was restricted to a subset of capsaicin-sensitive neurons ........................................................................................................................................... 85	
   2.5.2	
   Spinal cord injury-induced hypertrophy was particularly dramatic in caudal ganglia, far distal to injury ................................................................................................................................ 86	
   2.5.3	
   Injury-induced hypertrophy was not accompanied by pronounced intraspinal sprouting.. 88	
   2.5.4	
   Injury-induced hypertrophy was more pronounced after high-thoracic than low-thoracic spinal cord injury ........................................................................................................................... 88	
   2.5.5	
   Capsaicin-sensitive afferents contributed substantially to induction, but not development, of colo-rectal distension-induced autonomic dysreflexia .............................................................. 91	
   2.5.6	
   Conclusion .......................................................................................................................... 92	
    Chapter 3: Cellular responses in lumbar dorsal root ganglia after high- and low-thoracic spinal cord injury .................................................................................................................... 94	
   3.1	
   Synopsis....................................................................................................................... 94	
   3.2	
   Introduction ................................................................................................................. 94	
   3.3	
   Materials and methods................................................................................................. 96	
   3.3.1	
   Spinal cord injury surgery and post-operative care ............................................................ 96	
   3.3.2	
   Tissue processing and immunohistochemistry ................................................................... 97	
   3.3.3	
   Toludine blue staining ........................................................................................................ 98	
   3.3.4	
   Image analysis .................................................................................................................... 98	
   3.3.5	
   Statistics .............................................................................................................................. 99	
    3.4	
   Results ....................................................................................................................... 100	
   ix  3.4.1	
   High- but not low-thoracic spinal cord injury activated satellite cells in lumbosacral dorsal root ganglia .................................................................................................................................. 100	
   3.4.2	
   High- but not low-thoracic spinal cord injury stimulated immune cells in and around the lumbar dorsal root ganglion ......................................................................................................... 106	
   3.4.3	
   Capillary density in the dorsal root ganglion increased after high- and low-thoracic spinal cord injury .................................................................................................................................... 112	
   3.4.4	
   Sympathetic axons grew into lumbar dorsal root ganglia caudal to high- and low-thoraic spinal cord injury ......................................................................................................................... 112	
    3.5	
   Discussion ................................................................................................................. 119	
   3.5.1	
   Activation of satellite cells caudal to high-thoracic spinal cord injury ............................ 119	
   3.5.2	
   Activation of immune cells caudal to high-thoracic spinal cord injury ........................... 121	
   3.5.3	
   Angiogenesis in dorsal root ganglia caudal to high- and low-thoracic spinal cord injury 122	
   3.5.4	
   Sympathetic sprouting caudal to high- and low-thoracic spinal cord injury .................... 123	
   3.5.5	
   Conclusion ........................................................................................................................ 124	
    Chapter 4: Cyclooxygenase-2 contributes to vascular hypersensitivity following spinal cord injury ............................................................................................................................. 125	
   4.1	
   Synopsis..................................................................................................................... 125	
   4.2	
   Introduction ............................................................................................................... 125	
   4.3	
   Materials and methods............................................................................................... 128	
   4.3.1	
   Spinal cord injury surgery and post-operative care .......................................................... 128	
   4.3.2	
   In vitro myography ........................................................................................................... 128	
   4.3.3	
   Myography data analysis .................................................................................................. 130	
   4.3.4	
   Tissue processing and immunohistochemistry ................................................................. 130	
   4.3.5	
   Image analysis .................................................................................................................. 132	
   4.3.6	
   Statistics ............................................................................................................................ 132	
    4.4	
   Results ....................................................................................................................... 132	
    x  4.4.1	
   Phenylephrine-induced vasoconstriction was heightened in arteries from animals with spinal cord injury ......................................................................................................................... 132	
   4.4.2	
   Acetylcholine-induced vasodilation was functional but altered in arteries from animals with spinal cord injury ................................................................................................................. 133	
   4.4.3	
   Spinal cord injury-induced phenylephrine hypersensitivity was reversed by inhibitors of cyclooxygenase-2 ......................................................................................................................... 136	
   4.4.4	
   Cyclooxygenase-2 expression increased in mesenteric arteries, but not aorta, caudal to spinal cord injury ......................................................................................................................... 137	
    4.5	
   Discussion ................................................................................................................. 143	
   4.5.1	
   Mesenteric arteries from rats with spinal cord injury were hypersensitive to phenylephrine 143	
   4.5.2	
   Mesenteric arteries from rats with spinal cord injury were hyposensitive to acetylcholine 145	
   4.5.3	
   Cyclooxygenase-2-derived prostanoids contribute to phenylephrine hypersensitivity following spinal cord injury ......................................................................................................... 146	
   4.5.4	
   Conclusion ........................................................................................................................ 148	
    Chapter 5: Recurrent autonomic dysreflexia exacerbates vascular dysfunction following spinal cord injury .................................................................................................................. 150	
   5.1	
   Synopsis..................................................................................................................... 150	
   5.2	
   Introduction ............................................................................................................... 151	
   5.3	
   Materials and methods............................................................................................... 153	
   5.3.1	
   Spinal cord injury surgery and post-operative care .......................................................... 153	
   5.3.2	
   Repetitive colo-rectal distension ...................................................................................... 153	
   5.3.3	
   Cardiovascular assessment ............................................................................................... 153	
   5.3.4	
   In vitro myography ........................................................................................................... 154	
   5.3.5	
   Cardiovascular data analysis ............................................................................................ 154	
   5.3.6	
   Myography data analysis .................................................................................................. 155	
   5.3.7	
   Statistics ............................................................................................................................ 155	
   xi  5.4	
   Results ....................................................................................................................... 155	
   5.4.1	
   Repetitive colo-rectal distension exacerbated spinal cord injury-induced vascular dysfunction ................................................................................................................................... 155	
   5.4.2	
   The effect of repetitive colo-rectal distension on phenylephrine response was not a product of endothelial dysfunction .............................................................................................. 158	
   5.4.3	
   Repetitive colo-rectal distension over two weeks did not exacerbate cardiovascular dysfunction following spinal cord injury ..................................................................................... 160	
    5.5	
   Discussion ................................................................................................................. 164	
   5.5.1	
   Rationale for repetitive colo-rectal distension .................................................................. 164	
   5.5.2	
   Recurrent autonomic dysreflexia exacerbated phenylephrine hyper-responsiveness ...... 166	
   5.5.3	
   Recurrent autonomic dysreflexia did not alter endothelial-dependent vasodilation ........ 167	
   5.5.4	
   Reconciling the vascular effects of recurrent autonomic dysreflexia with the cardiovascular outcome of spinal cord injury .............................................................................. 169	
   5.5.5	
   Conclusion ........................................................................................................................ 170	
    Chapter 6: Discussion .......................................................................................................... 173	
   6.1	
   Summary and conclusions ......................................................................................... 173	
   6.1.1	
   Spinal cord injury-induced changes in the dorsal root ganglion ...................................... 173	
   6.1.2	
   Spinal cord injury-induced changes in the mesenteric artery ........................................... 174	
   6.1.3	
   Significance of the findings .............................................................................................. 175	
   6.1.4	
   Spinal cord injury-induced changes in the dorsal root ganglion ...................................... 175	
   6.1.5	
   Spinal cord injury-induced changes in the mesenteric artery ........................................... 176	
    6.2	
   Strengths .................................................................................................................... 177	
   6.3	
   Limitations................................................................................................................. 178	
   6.3.1	
   Breadth versus depth ........................................................................................................ 178	
   6.3.2	
   Proliferation versus hypertrophy of glia in the dorsal root ganglion ................................ 179	
   6.3.3	
   Relying on immunohistochemistry for protein expression studies .................................. 179	
    6.4	
   Clinical ramifications ................................................................................................ 181	
    xii  6.4.1	
   Effects of recurrent autonomic dysreflexia ...................................................................... 181	
   6.4.2	
   COX-2 activity in arteries caudal to spinal cord injury .................................................... 182	
   6.4.3	
   Cellular changes in the dorsal root ganglion after spinal cord injury ............................... 182	
   6.4.4	
   Capsaicin-sensitive neurons caudal to spinal cord injury ................................................. 183	
    6.5	
   Future directions ........................................................................................................ 184	
   6.5.1	
   Cellular changes in the dorsal root ganglion after spinal cord injury ............................... 184	
   6.5.2	
   COX-2 activity in arteries caudal to spinal cord injury .................................................... 185	
   6.5.3	
   Effects of recurrent autonomic dysreflexia ...................................................................... 187	
    6.6	
   A final word on peripheral plasticity following spinal cord injury ........................... 188	
    References...................................................................................................................... 189	
    xiii  List of Figures  Figure 1.1 The spinal reflex that mediates autonomic dysreflexia .................................. 32	
   Figure 2.1 High-thoracic (T3) spinal cord injury provoked hypertrophy of neurons in the dorsal root ganglion .......................................................................................................... 64	
   Figure 2.2 High-thoracic (T3) spinal cord injury induced selective hypertrophy of sensory neurons expressing the capsaicin receptor (TRPV1) and the artemin receptor (GFRα3) ............................................................................................................................ 67	
   Figure 2.3 Spinal cord injury had no effect on medium-to-large sized DRG neurons expressing heavy neurofilament (NF200) ........................................................................ 69	
   Figure 2.4 Capsaicin-sensitive dorsal root ganglion neurons increased in diameter caudal to, but not rostral to high-thoracic spinal cord injury ....................................................... 72	
   Figure 2.5 Capsaicin-sensitive neurons in the dorsal root ganglion exhibited increased TRPV1 signal intensity following high-thoracic spinal cord injury................................. 74	
   Figure 2.6 Low-thoracic (T10) spinal cord injury elicited only modest changes in size of the most caudal capsaicin-sensitive dorsal root ganglion neurons ................................... 76	
   Figure 2.7 Hypertrophy of capsaicin-sensitive afferents caudal to high-thoracic spinal cord injury was not accompanied by pronounced plasticity of their spinal projections ... 80	
   Figure 2.8 Intrathecal capsaicin attenuated colo-rectal distention (CRD) -induced autonomic dysreflexia in animals with T3 spinal cord injury .......................................... 84	
   Figure 3.1 Satellite cell expression of glial fibrillary acidic protein (GFAP) increased in lumbar dorsal root ganglia after high thoracic (T3), but not low-thoraic (T10) spinal cord injury ............................................................................................................................... 103	
    xiv  Figure 3.2 Satellite cell expression of neuroglycan 2 proteoglycan (NG2) increased in lumbar dorsal root ganglia after T3, but not T10 spinal cord injury .............................. 105	
   Figure 3.3 High-thoracic, but not low-thoracic spinal cord injury stimulated macrophages in the lumbar dorsal root ganglion............................................................ 109	
   Figure 3.4 Mast cells accumulated in the spinal nerve, immediately distal to dorsal root ganglia, after high- but not low-thoracic spinal cord injury ........................................... 111	
   Figure 3.5 Both high- and low-thoracic spinal cord injury induced angiogenesis in lumbar dorsal root ganglia .............................................................................................. 116	
   Figure 3.6 Both high- and low-thoracic spinal cord injury provoked sympathetic sprouting in lumbar dorsal root ganglia .......................................................................... 118	
   Figure 4.1 Arteries from animals with high-thoracic (T3) spinal cord injury were hypersensitive to phenylephrine ..................................................................................... 134	
   Figure 4.2 Endothelial-dependent vasodilation was functional, but altered, in arteries from animals with high-thoracic spinal cord injury ....................................................... 135	
   Figure 4.3 Inhibition of cyclooxygenase-2 reversed phenylephrine hypersensitivity in arteries from animals with SCI ....................................................................................... 140	
   Figure 4.4 Cyclooxygenase-2 expression was increased in superior mesenteric arteries from animals with high-thoracic spinal cord injury ....................................................... 142	
   Figure 5.1 Repetitive colo-rectal distension during recovery from high-thoracic (T3) spinal cord injury potentiated phenylephrine-induced vasoconstriction ........................ 157	
   Figure 5.2 Endothelial-dependent vasodilation was not affected by repetitive colo-rectal distension over two weeks following T3 spinal cord injury ........................................... 159	
    xv  Figure 5.3 Repetitive colo-rectal distension did not alter baseline cardiovascular parameters examined at one month following T3 spinal cord injury ............................. 161	
   Figure 5.4 Autonomic dysreflexia evoked by colo-rectal distension was less pronounced in animals that experienced repeated bouts of distension during recovery from T3 spinal cord injury....................................................................................................................... 163	
    xvi  List of Abbreviations ACh AD AMCA ANOVA ATP bpm BS CAA CCD CGRP CNS COX CRD DAP DGC Dmnx DRG EC50 EDHF GDNF GFAP GFRα3 GS HR IB4 Iba-1 IL-1β IMG IML LUT MAP MAP-2 NA NE NET NF200 NG2 NGF NO NPY OH P2X P2Y PB  Acetylcholine Autonomic dysreflexia Aminomethylcoumarin Analysis of variance Adenosine tri-phosphate Beats per minute Baroreflex sensitivity Central autonomic area Charge-coupled device Calcitonin gene-related peptide Central nervous system Cyclooxygenase Colo-rectal distension Diastolic arterial pressure Dorsal grey commissure Dorsal motor nucleus of the vagus Dorsal root ganglion Half-maximal effective concentration Endothelium-derived hyperpolarizing factor Glial cell line-derived neurotrophic factor Glial fibrillary acidic protein Glial cell line-derived neurotrophic factor receptor alpha 3 Glutamine synthetase Heart rate Lectin from Bandeiraea simplicifolia Ionized calcium binding adaptor molecule 1 Interleukin 1 beta Inferior mesenteric ganglion Intermediolateral cell column Lower urinary tract Mean arterial pressure Microtubule-associated protein 2 Nucleus ambiguus Norepinephrine Norepinephrine transporter Heavy neurofilament Neuroglycan 2 Nerve growth factor Nitric oxide Neuropeptide tyrosine Orthostatic hypotension Purinergic receptor subtype X Purinergic receptor subtype Y Phosphate buffer xvii  PBS PE PF PGE2 PGI2 PNS PSS RECA-1 RIP RSNA RVLM RVMM SA SAD SAP SCI SMA SP SPN SSEA4 TH TNF-α TrkA TrkC TRP TRPV1 TRPV2 TxA2 VIP  Phosphate-buffered saline Phenylephrine Paraformaldehyde Prostaglandin E2 Prostaglandin I2 Peripheral nervous system Physiological saline solution Rat entothelial cell antigen 1 Antibody clone name: anti-oligodendrocytes Renal sympathetic nerve activity Rostroventrolateral medulla Rostroventromedial medulla Sino-atrial Sino-aortic denervation Systolic arterial pressure Spinal cord injury Superior mesenteric artery Substance P Spinal preganglionic neuron Stage-specific embryonic antigen 4 Tyrosine hydroxylase Tumor necrosis factor alpha Tropomyosin receptor kinase A Tropomyosin receptor kinase C Transient receptor potential Transient receptor potential vanilloid 1 Transient receptor potential vanilloid 2 Thromboxane A2 Vasoactive intestinal polypeptide  xviii  Acknowledgements Ora na azu nwa. (Nigerian Igbo: It takes a village to raise a child.) Love me when I least deserve it, because that's when I really need it. (Swedish Proverb)  I was fortunate to receive funding from the following agencies to support my doctoral research: the Michael Smith Foundation of Health Research, the National Research Council of Canada, the Heart and Stroke Foundation of BC and the Yukon, ICORD, the Faculty of Graduate Studies and the Vancouver Coastal Health Research Institute.  In addition, I gratefully acknowledge the time, effort, support and kindness of so many people who have helped me along the way. I can only mention a handful. They are:  •  My supervisor, Dr. Andrei Krassioukov, for teaching me about efficiency and effectiveness, and about finding time for the finer things in life.  •  My supervisor, Dr. John Steeves, for his tireless support of our family over the last decade at ICORD.  •  My supervisory committee, Drs. Bill Milsom, Bill Sheel and Colin Brauner, for their guidance and reassurance throughout this process, and for feedback that vastly improved the quality of this dissertation.  •  My other mentors at UBC, including Drs. Ismail Laher and Joanne Nakonechny, for generously sharing their knowledge and expertise.  •  Peter van Stolk, Nima Alan and Byron Ramsey, for invaluable assistance and intellectual input – and for their patience as I learn to be a supervisor.  xix  •  Cheryl Niamath, for help on countless occasions – every student at ICORD is in her debt.  •  Dr. Wolfram Tetzlaff, for his astute scientific thought, which enriches the training environment at ICORD dramatically.  •  My newest and talented colleague, Dr. Chris West, for reassuring me that future directions of my work lie in good hands.  •  Jessica Inskip, for excellent companionship, for gracefully ignoring my floundering and for pretending to let me lead: who better to share a brain with?  •  My friends and family, but especially Amy Newell, Barbara Beale, Sandra Malfair and Neil Barclay, for their good judgment, honesty and encouragement.  •  My parents – biological and otherwise – for indefatigable support, particularly my father, whose under-stated wisdom I appreciate more with every conversation.  •  My daughter, for her fierce independence and her persistence, and for teaching me how to cope with adversity.  •  My husband, for his patience, his mentorship, and his unrelenting enthusiasm for discovery, which I find, inspiring, exciting and exhausting.  xx  Dedication  To my family and friends, who celebrate my accomplishments, overlook my failures and put up with me in between: Tempest Bebende!  xxi  Chapter 1: Introduction  1.1  Overview Spinal cord injury (SCI) impairs all types of communication between the brain  and body below the lesion. Although motor, sensory, and autonomic nerves are affected, the outcome of SCI is still commonly described in terms of motor function (i.e., as paraplegia or tetraplegia). The pervasive mental association between SCI and paralysis is reflected in our media and reinforced by researchers: among publications on experimental SCI, studies examining only motor outcomes outnumber studies considering autonomic or sensory function by about five-to-one (Inskip et al., 2009). Motor function has likely garnered such attention simply because it is the easiest type of function to measure in animal models and to explain to the public and our peers. However, an increasing body of data suggests that our motor-centric view of SCI is rather negligent. In a landmark paper published in 2004 (Anderson, 2004), a survey of individuals living with SCI revealed that recovery of autonomic functions far outranked recovery of walking movement as priorities for improving quality of life. Autonomic disorders, including those associated with pulmonary, renal, and lower urinary tract (LUT) function, have historically represented the primary causes of mortality following SCI (Whiteneck et al., 1992). Recently, cardiovascular complications have emerged as the leading cause of death in individuals with chronic SCI (Garshick et al., 2005). While this is true for many segments of the population, people with SCI develop cardiovascular disease at younger ages and at greater rates than the able-bodied population (Wahman et al., 2010; Myers et al., 2007; Nash and Mendez, 2007). Even those individuals with SCI  1  that classify themselves as active and healthy have unique and pronounced cardiovascular risks (Edwards et al., 2008). This dissertation focuses on one aspect of cardiovascular dysfunction after SCI, a condition called autonomic dysreflexia (AD), characterized by episodic hypertension induced by sensory stimulation below the level of injury. When SCI occurs above T6, it isolates the spinal circuits that govern the splanchnic vascular bed from supraspinal regulatory centres, creating the conditions for AD. However, AD typically develops over time after SCI: its post-injury evolution suggests that, while the loss of supraspinal control is prerequisite, AD is a product of injury-induce changes that take time to develop. At this point, the bulk of the literature on the mechanisms of AD investigates changes in spinal circuits. The only putative peripheral mechanism identified to date is vascular hyper-responsiveness to sympathetic neurotransmitters; how this develops is also unknown. My dissertation examines injury-induced changes at two peripheral loci that are critical to AD, namely the sensory neurons that reside in dorsal root ganglia (DRGs) and the mesenteric arteries. In the DRG, I found that a specific subset of sensory neurons in distal DRGs exhibits hypertrophy after high-thoracic SCI (Chapter 2) and that the same subset is critical in triggering AD (Chapter 2). I also found that the cellular environment of DRGs far distal to the site of SCI is dramatically altered following high-thoracic SCI (Chapter 3). In mesenteric arteries, I found that COX-2 is involved in vascular hyperresponsiveness to phenylephrine (a sympathetic mimetic) (Chapter 4). Finally, I found that recurrent AD induced intentionally over two weeks after SCI exacerbated vascular hyper-responsiveness (Chapter 5).  2  In Chapter 1, I begin with an overview of neural cardiovascular control, moving from the brainstem to the periphery: while I acknowledge the existence of grey matter rostral to the brainstem in most people, I omit it for purposes of this review. I also review the features of the splanchnic circulation that render it so crucial to blood pressure regulation and discuss some general features of the vasculature, such as the vascular endothelium. Armed with this background, I next describe cardiovascular dysfunction following SCI, in a system that is isolated from supraspinal centres and running amok. I proceed to describe anatomy and function of the spinal reflex that governs AD, with the bulk of attention devoted to the DRG and sensory neurons. Finally, I review the literature on injury-induced changes associated with AD to this point, before outlining the hypotheses that prompted the experiments outlined in subsequent data chapters.  1.2  Neural cardiovascular control: relevant and interesting aspects  At one stage early in my life, I was highly motivated to become a great flamenco guitar player. I went to Spain to learn how to play and my teacher said after a year or so that I should go down to the village after dinner and play; if they liked my playing, the girls would dance. Sadly, no-one danced… so, I decided to have a go at Science. (Geoffrey Burnstock, who discovered ATP was a neurotransmitter) The autonomic nervous system coordinates activity of smooth muscle in the heart and the peripheral circulation to optimize regional blood supply while maintaining systemic arterial pressure within a relatively narrow range. Superimposed on local mechanisms of vascular regulation, tonic neurogenic and reflex autonomic aspects of central neural activity rapidly adjust regional blood flow to suit a multitude of physiological conditions (Thomas, 2011; Inskip et al., 2009; Guyenet, 2006). In this section, I introduce the aspects of supraspinal and spinal outflow, input, and integration 3  that are relevant to cardiovascular function after SCI. In an attempt to avoid a tired discussion of autonomic cardiovascular control mediated by antagonistic actions of acetylcholine (ACh) and norepinephrine (NE), I include interesting aspects of cotransmission, featuring the physiological dance partners of the classical neurotransmitters. As Gail Thomas noted in her recent review (Thomas, 2011) autonomic control of the cardiovascular system represents an exception to the dogmatic notion that the parasympathetic and sympathetic nervous systems exert opposite but equal influence over the organ systems. Parasympathetic cardiovascular control is limited to the heart and a very few blood vessels, while sympathetic control extends to the heart, the contractile blood vessels, the adrenal glands and the kidneys, with profound influence on cardiovascular homeostasis.  1.2.1  Supraspinal cardiovascular integration The activity of autonomic nerves that govern cardiovascular function is regulated  by neural networks in the medulla oblongata. Anatomical experiments, including classical transneuronal tracing experiments using pseudo-rabies viruses to map neural circuits, revealed that most SPNs receive input from the same supraspinal areas: the rostral ventrolateral medulla (RVLM), the rostral ventromedial medulla (RVMM), the medullary raphe nuclei and the hypothalamus (Jansen et al., 1995; Sved et al., 2001). Of these, the most important site of cardiovascular integration is the RVLM (Ross et al., 1984), which provides a largely monosynaptic adrenergic projection to SPNs. Sufficiency and necessity of the RVLM in tonic and reflexive blood pressure regulation has been illustrated using pharmacological/electrical stimulation and chemical ablation,  4  respectively (Dampney et al., 2003; Dampney et al., 2002; Guyenet, 2012; Dampney et al., 2000). The collective conclusion of these experiments is that RVLM efferents provide tonic output to generate resting sympathetic tone, required to maintain resting blood pressure, and are activated in response to hypotension. In order to respond appropriately to preserve cardiovascular homeostasis, the RVLM integrates afferent input from several sites. Inputs from limbic, cortical, and midbrain structures allow the autonomic nervous system to alter circulation in response to emotional reactions, such as fright or stress (Blessing, 2012). Hypothalamic afferents projecting to the RVLM adjust the circulation appropriately for behaviour and the environment (Blessing, 2012), with the caveat that thermoregulatory function of the cutaneous circulation is predominately governed by the RVMM and the raphe nuclei (Morrison, 2011). Two populations of afferents, chemoreceptors and baroreceptors, provide input to the RVLM (via the nucleus of the solitary tract; NTS) to participate in short-term blood pressure regulation. Both types of receptors are located in the carotid bodies and the aortic arch and extend axons to the NTS in the glossopharyngeal and vagus nerves. Chemoreceptors are activated by hypoxia and hypercapnia, and their activation reflexively evokes an increase in ventilation and sympathetically-mediated vasoconstriction outside the heart and brain (Dampney et al., 2002; Kara et al., 2003). The arterial baroreceptors are stretch-sensitive afferents in the vessel wall. When activated by distension (i.e., by an increase in arterial blood pressure), the baroreceptors convey the information to neurons in the NTS, and from there to neurons in the RVLM to reduce sympathetic output to the heart and vessels. Projections from the NTS also travel to the neighbouring parasympathetic neurons in the caudal VLM, to increase  5  parasympathetic output to the heart (Kara et al., 2003). This sympatho-inhibitory reflex is known as the arterial baroreflex.  1.2.1.1  The arterial baroreflex The arterial baroreflex is an extremely effective negative feedback system: it  buffers beat-to-beat fluctuations in blood pressure and rapidly adjusts blood pressure to preserve vital organ perfusion in the face of physiological demand (such as moving from supine to upright). The effectiveness of this reflex is based on the relationship between baroreceptor activity and arterial pressure, which is sigmoidal, with a threshold pressure for baroreceptor activation of ~50mmHg and a maximal response of ~180mmHg. A similar, but inverse relationship exists between arterial blood pressure and sympathetic nerve activity (Thomas, 2011). The important result is that small increases in arterial pressure elicit large reductions in sympathetic nerve activity. The relationship between arterial pressure and activity in the neural components of the baroreflex is described as baroreflex sensitivity (BS). The definition of BS has been refined repeatedly, with baroreceptor activity measured by direct recordings from afferents such as the aortic nerve defined as baroreceptor sensitivity to distinguish it from central gain, which reflects changes in responsiveness of any portion of the brainstem circuitry (Chen et al., 1995). The most convenient definition of BS is the relationship between arterial pressure and heart rate, which allows BS to be easily measured (Smyth et al., 1969). This working definition was used to establish the link between diminished BS and hypertension (Bristow et al., 1969). There is a significant confound to this definition, since hypertension produces arterial stiffening, which in turn  6  renders the baroreceptors less sensitive; Karemaker raised this issue in a provocative commentary (Karemaker, 2002). The jury is still out on whether BS represents cause or effect in most types of cardiovascular pathology, but the link between essential hypertension and BS may render its acronym particularly apt. Another important property of the baroreflex is its set point, the baseline blood pressure it aims to achieve. This set point can be acutely or chronically altered. During exercise, the set point is adaptively altered to permit blood pressure and heart rate to be elevated to meet performance (Fadel and Raven, 2011). Chronic re-set has, like sensitivity, been linked to cardiovascular pathology, particularly to the development of hypertension. More recently, however, the role of the baroreflex in long-term blood pressure regulation has been challenged, in large part due to data demonstrating that elimination of the baroreceptors through sino-aortic denervation (SAD) had little effect on basal levels of sympathetic nerve activity and arterial pressure (Osborn et al., 2005). A recent view that has emerged is that there is a neural set-point governing long-term blood pressure control, through renal sympathetic nerve activity (RSNA), but that it is independent of baroreceptor input to the brainstem (Osborn et al., 2005); however, debate surrounding this issue continues to thrive (Barrett and Malpas, 2005).  1.2.2 1.2.2.1  Autonomic control of the heart Parasympathetic control of the heart Parasympathetic efferents governing the heart originate in the brainstem, their  cell bodies arising from two nuclei: the dorsal motor nucleus of the vagus (DmnX), adjacent to the fourth ventricle, and the nucleus ambiguus (NA), dorsolateral to the  7  inferior olive. The relative contribution of these nuclei to cardiac control has been hotly debated (Cheng et al., 2004): for our purposes here, it suffices to say that both nuclei play significant roles. The axons of cells bodies in the DmnX and NA travel to local cardiac ganglia in the vagus nerve (cranial nerve X), which exits the CNS at the level of the medulla. The vagus is therefore the only element of autonomic cardiovascular control that is spared in all levels of SCI. Parasympathetic ganglionic axons release acetylcholine, which acts on muscarinic cholinergic receptors on cardiac effector cells of the nodes and Purkinje fibers to decrease heart rate and conduction velocity (Carlsten et al., 1957; Berntson et al., 1992; Jones, 2001) (i.e. chronotropy and dromotropy, respectively). Historically, the role of parasympathetic activity in dictating cardiac contractility was thought to be minimal, except in hibernating animals (O'Shea and Evans, 1985), but recent data have altered this view: we now know that ventricles in at least some mammals are innervated by parasympathetic cholinergic axons (Ulphani et al., 2010), and that vagal activity has significant inotropic effects (Lewis et al., 2001). Multiple interesting additions enrich this classical understanding of parasympathetic cardiac control, including the roles of parasympathetic co-transmitters. For example, somatostatin-14 is a neuropeptide that is co-stored and released with acetylcholine in parasympathetic ganglionic neurons, and exerts bi-directional inotropic effects on ventricular myocytes, determined by the receptor subtype expressed in the myocyte (Murray et al., 2001; Bell et al., 2008). Vasoactive intestinal peptide (VIP), another parasympathetic co-transmitter, actually has potent positive chronotropic effects, and mediates the paradoxical parasympathetic tachycardia observed in some species  8  under muscarinic blockade (Markos and Snow, 2006). In addition, VIP is 50-100 times more potent than acetycholine as a vasodilator (Henning and Sawmiller, 2001): in terms of cardiac function, VIP directly dilates coronary arteries to increase blood flow to the heart (Feliciano and Henning, 1998). Although the roles of co-transmitters fine-tune parasympathetic cardiac regulation, the net effects of parasympathetic activity on cardiac function are the same as those of acetylcholine: reduced heart rate and reduced stroke volume.  1.2.2.2  Sympathetic control of the heart Sympathetic activity has an opposite, stimulatory effect on the heart. In contrast  to the parasympathetic preganglionic neurons, with cell bodies residing in brainstem nuclei, sympathetic preganglionic neurons governing the heart originate in the intermediolateral cell column (IML) of the upper-thoracic spinal cord (T1-T5). These cell bodies and their axons are damaged directly when SCI occurs between T1 and T5. When SCI occurs in the cervical region (i.e., rostral to T1), sympathetic preganglionic neurons (SPNs) are isolated from supraspinal control centres in the ventral medulla. In order to facilitate the discussion of sympathetic cardiovascular regulation, Guyenet (2006) classified the cardiovascular sympathetic efferents into three functional groups, as those that are thermosensitive, glucosensitive or barosensitive. The first two groups, innervating cutaneous vessels and the adrenal medulla, respectively, represent small groups of efferents with specific homeostatic roles. The majority of cardiovascular sympathetic efferents are “baro”-sensitive; Guyenet used this term to describe sympathetic efferents that exhibit ongoing activity at rest, which is rapidly modulated by  9  a number of autonomic reflexes, including the arterial baroreflex. These sympathetic efferents generate sympathetic tone and mediate rapid fluctuations in blood pressure (Janig and Habler, 2003), both of which are central to cardiovascular dysfunction after SCI. I will therefore confine the remainder of this discussion to this population of sympathetic efferents (i.e., those that are regulated reflexively). As a non-trivial aside, these efferents also innervate the kidneys to participate in long-term neural regulation of blood pressure (DiBona and Kopp, 1997). Spinal SPNs extend axons in the ventral root, which branch into the white ramus communicans to adjacent ganglia of the paravertebral chain. Sympathetic ganglionic neurons exit the ganglia in the grey ramus, and travel in mixed peripheral nerves to their target tissue in the heart: these axons release norepinephrine (NE), which acts on βadrenergic receptors in cardiac effectors of all heart tissues with positive chronotropic, dromotropic, inotropic and lusitropic effects. The molecular mechanisms underlying each effect are thoroughly reviewed by Bers and Despa (2009). To summarize a complex story, β-adrenergic receptor activation hastens depolarization in the sinoatrial (SA) node, increases conduction in the atrio-ventricular (AV) node, and activates (via PKA) multiple calcium (Ca2+) cycling proteins in cardiac myocytes to increase Ca2+ membrane current and Ca2+ release and reuptake in the sarcoplasmic reticulum. Akin to the cardiac parasympathetic nerves, sympathetic NE release and activity in the heart is modulated by co-transmitters. Adenosine triphosphate (ATP) serves as a signaling molecule in sympathetic nerves throughout the CNS and PNS, although its relative contribution varies with species and pathology. The discovery of ATP as a sympathetic neurotransmitter marked a dramatic shift in neurophysiology, as it over-  10  turned the dogma that nerves used only one transmitter (Burnstock, 2004). In the heart, ATP is thought to act on ionotropic (P2X) and metabotropic (P2Y) receptors, both expressed in cardiac myocytes (Vassort, 2001), to exert its positive inotropic effects. While ATP essentially augments NE-mediated inotropy, another co-transmitter, neuropeptide Y (NPY) has a more complex cardiac function: in many mammals, NPY has been implicated in the antagonism between parasympathetic and sympathetic cardiac control (Protas and Robinson, 2008). Cholinergic and adrenergic nerves not only exert opposite effects on target cardiac tissue; they also reciprocally inhibit release of the opposing neurotransmitter. The best-documented example of this effect is inhibition of vagal bradycardia via strong stimulation of the cardiac sympathetic nerves (Potter, 1985). Intense sympathetic activity induces release of NPY from sympathetic nerve varicosities, which acts at pre-synaptic Y2 receptors on cholinergic nerves in the sinoatrial node to inhibit ACh release and attenuate vagal bradycardia (Herring et al., 2008).  1.2.3  Autonomic control of the vasculature In contrast to the heart, which is under the dual and opposing control of the  parasympathetic and sympathetic nervous systems, the contractile elements of the peripheral vasculature – arteries, arterioles, and veins – are typically governed by the sympathetic nervous system alone. A few anomalous vessels – such as those of the sexual organs, the brain, orofacial skin and muscles and the pulmonary system (Giuliano et al., 1995; Ishii et al., 2010; Watanabe et al., 2008; Sudo et al., 2009; Taktakishvili et al., 2010; Kummer, 2011) – are actively vasodilated via parasympathetic activity. While this regional parasympathetic vasodilation is physiologically important, it has little direct  11  contribution to systemic blood pressure regulation. Other crucial influences on vascular tone include the peptidergic sensory nerves (described for the splanchnic vasculature) and the vascular endothelium.  1.2.3.1  Sympathetic control of the vasculature Spinal sympathetic neurons governing vascular tone have cell bodies in the IML  of the thoracolumbar spinal cord, from T1-L2. Abnormalities of blood pressure control after SCI depend on the level of injury, which dictates the proportion of sympathetic effectors that are damaged and/or isolated from their supraspinal governing centres. Sympathetic post-ganglionic neurons project (in the grey ramus and mixed peripheral nerves) to the adventitial-medial border of arteries, arterioles and veins. In most vessels sympathetic innervation does not penetrate the media, and is restricted to the superficial layer of smooth muscle cells, such that NE only reaches deeper smooth muscle via diffusion. (The exceptions to this rule appear to be dictated by physiological necessity; for example, sympathetic axons penetrate deep into the media in the carotid arteries of the giraffe; Nilsson et al., 1988). Prevailing dogma remains that sympathetic ganglionic axons innervating smooth muscle do not form specialized neuromuscular junctions that are comparable to those of motor axons innervating skeletal muscle. However, ultrastructural studies of the vasculature indicate that sympathetic axons supplying vascular smooth muscle do form neuromuscular junctions of sorts, with presynaptic vesicle aggregation, and in some cases, junctional clefts (Luff, 1996). The density and structural arrangement of sympathetic innervation contributes to the heterogeneity among vascular beds (as well as species, gender and age, and even  12  different segments of the same vessel), in terms of the vascular response to sympathetic activity (Burnstock, 2008). The vascular distribution of adrenergic receptors also dictates the vascular response. NE released from sympathetic axons acts on α1-adrenoreceptors in vascular smooth muscle to increase intracellular Ca2+ and elicit contraction. Amidst a metaphorical dog’s breakfast of often conflicting in vitro, in vivo, and knock-out data, most authors agree that α1A-adrenoreceptors expressed on the vascular smooth muscle are the most crucial determinants of vascular tone (Guimaraes and Moura, 2001). Secondary regulatory mechanisms include α2-adrenoreceptors expressed on sympathetic nerves (i.e., pre-junctional receptors), which inhibit NE release, and β-adrenoreceptors in the vascular smooth muscle, which respond primarily to circulating catecholamines, such that their effects are negligible at rest but pronounced during exercise (Rohrer et al., 1998). Adrenoreceptors are also expressed in the vascular endothelium, where their activation elicits release of nitric oxide (NO) to attenuate sympathetically-mediated vasoconstriction (Cocks and Angus, 1983; Guimaraes and Moura, 2001). While receptor heterogeneity has greatly complicated the task of making generalizations about the roles of adrenoreceptor sub-types in blood pressure control, it confers remarkable capability to fine-tune vascular function. A final aspect of vascular heterogeneity that bears mention is the variable contribution of co-transmitters to sympathetic vasoconstriction. As in the heart, the majority of sympathetic nerves in blood vessels co-release NPY and ATP with NE. NPY acts on Y1 receptors on vascular smooth muscle to potentiate the contractile effects of NE and ATP (Racchi et al., 1997). Evidence in rat mesenteric arteries suggests that NPY  13  exerts its effects largely by inhibiting vasodilation: in vitro, NPY abolished the vasodilating effects of the adenylyl cyclase activator forskolin (Prieto et al., 2000). In a few specific vascular beds, including the cerebral and the coronary circulation, NPY can induce direct vasoconstriction (Edvinsson, 1985; Macho et al., 1989). ATP has a dual action on most vessels: it acts on P2X receptors in the vascular smooth muscle to evoke depolarization and vasoconstriction, and it acts on P2Y receptors in the vascular endothelium to elicit vasodilator release (Thomas, 2011; Gao et al., 1999). The differential kinetics of the P2X receptors (ATP-gated ion channels) and the α1adrenoreceptors (G-protein-coupled receptors) are likely critical to the complementary action of these transmitters on vascular (and cardiac) smooth muscle (Mackeprang et al., 1992). ATP induces depolarization of the smooth muscle within milliseconds, while intracellular Ca2+ stores are mobilized over seconds after NE binding to initiate and sustain contractile machinery. While these mechanisms render ATP and NE synergistic, the relative proportions of ATP and NE released as functional co-transmitters at the vascular neuroeffector junction vary with species and vessel. For example, the contribution of ATP to neurally-evoked contractions in rat mesenteric arteries increases as vessel diameter decreases (Gitterman and Evans, 2001). The relative proportion of ATP as a functional sympathetic neurotransmitter appears to increase as blood pressure (vascular tone) increases. In both small mesenteric arteries (Rummery et al., 2007) and the whole perfused mesentery (Pakdeechote et al., 2007) isolated from rats, ATP acts as an important sympathetic neurotransmitter under raised pressure/tone conditions, where the perfusion pressure is closer to that found in vivo. The vessel diameter and pressuredependence of the contribution of ATP in vasoconstriction has important ramifications  14  for interpreting experimental data, particularly those derived from isometric in vitro myography: more on this in Chapters 4 and 5.  1.2.3.1.1  The splanchnic circulation  The peripheral vasculature is not homogenous in terms of its contribution to blood pressure regulation. The vessels supplying the splanchnic organs – the stomach, liver, intestines, pancreas and spleen – make a disproportionate contribution to blood pressure control. In humans at rest, the splanchnic circulation receives about 25% of the total cardiac output and contains about one-third of the total blood volume, making it the largest reservoir of blood in the circulatory system (Rowell, 2012). In addition, the splanchnic vasculature is densely innervated and sensitive to changes in sympathetic activity. Classical human studies revealed that these vessels contract powerfully when the sympathetic nervous system is reflexively activated (e.g., through lower body negative pressure, hemorrhage or exercise (Qamar and Read, 1987; Lundgren, 1983) to increase peripheral resistance and mobilize their blood reserve. In humans, Greenway (1983) demonstrated that up to 27% of total blood volume can be mobilized from the splanchnic venous bed by sympathetic stimulation. In anesthetized cats, Greenway and Lister (Greenway and Lister, 1974) found that the splanchnic vascular bed mobilized or pooled up to 65% of the blood volume removed or infused, respectively. More recent studies have used noninvasive Doppler ultrasound to study blood flow in the superior mesenteric artery (SMA), which supplies blood to the whole small intestine (Chaudhuri et al., 1991). These experiments confirmed the importance of the splanchnic contribution to cardiovascular homeostasis by documenting significant reductions in SMA flow during  15  routine physiological sympatho-activation (e.g., during mental arithmetic, isometric exercise and head-up tilt). The properties of the mesenteric veins – the capacitance units of this reservoir – are central to the splanchnic contribution to systemic blood pressure. In humans, the veins of the splanchnic bed are more densely innervated than the corresponding arteries (Birch et al., 2008), which runs counter to the general pattern of sympathetic innervation throughout the circulatory system (Todd, 1980). In rats, there are fewer sympathetic nerves at the adventitial-media border of veins than arteries (Furness and Marshall, 1974; Nilsson et al., 1986; Park et al., 2007); however, as noted by Park and colleagues (2007), this does not necessarily translate into reduced innervation density, since the rat mesenteric veins have a thinner medial layer. In contrast, human mesenteric veins have a thick muscular media (Birch et al., 2008). It is a generally-accepted notion that the mesenteric veins play a more significant role in governing capacitance in humans than in experimental animals, due to the gravitational difficulties of upright stance (Hainsworth, 1986). While it is clear that both sides of the mesenteric circulation contribute substantially to blood pressure regulation in most mammals, the great majority of experimental studies of cardiovascular disease and dysregulation investigate the properties of the rat mesenteric arteries; at least one recent study suggests that pathological changes (in an animal model of hypertension) occur in mesenteric arteries, but not in veins (Park et al., 2010). Differential control of arteries and veins (i.e., of flow and volume) is a general property of the vascular system, but the effects of this control are more pronounced in the splanchnic circulation due to the large blood volume at its disposal. In general, venous  16  capacitance vessels are more sensitive to sympathetic activity (i.e., they respond at lower frequencies of stimulation) than arterial resistance vessels, which require higher frequency stimulation to elicit the same degree of constriction (Kreulen, 1986; Hottenstein and Kreulen, 1987). Separate control of vascular resistance and capacitance is a product of both sympathetic and sensory innervation of the splanchnic vasculature. (The latter, the substantial sensory innervation of the vasculature, is another general feature that I have conveniently ignored to this point, but will arrive at shortly.) The most glaring mechanism for differential control of splanchnic arteries and veins is their innervation by separate sympathetic ganglionic neurons. This was demonstrated for the inferior mesenteric artery and vein by retrograde labeling of sympathetic neurons in the inferior mesenteric ganglion (IMG) (Browning et al., 1999): in this ganglion, there is some viscerotopy, with arterial neurons in the centre of the IMG and venous neurons around the edges. Viscerotopical organization is also evident in the pelvic ganglia containing sympathetic neurons that innervate the LUT and the genitals (Keast and de Groat, 1989; Tabatai et al., 1986), and creates intra-ganglionic functional subgroups of sympathetic neurons to provide another level of sympathetic control (Janig and McLachlan, 1992). Different sympathetic neurotransmitters contribute differentially in mesenteric arteries and veins. Electrophysiological recordings of neuroeffector transmission in mesenteric arteries and veins (Suzuki, 1981; Kreulen, 1986; Hottenstein and Kreulen, 1987), and more recently amperometry combined with video imaging (Park et al., 2010), have provided insight into neurotransmitter-based control of capacitance and resistance in this crucial vascular bed. Under low-frequency conditions, NE signaling via α1-  17  adrenoreceptors mediates venous vasoconstriction, while ATP acting at P2X receptors is the predominate agent of contraction in the mesenteric arteries (Hottenstein and Kreulen, 1987). With repetitive (or high-frequency) sympathetic stimulation, NE acting at α1adrenoreceptors contributes increasingly to arterial vasoconstriction, while ATP (acting at P2Y receptors) and NPY (acting at Y1 receptors) contribute increasingly to venous contraction (Kreulen, 2003). Some of these differential responses can be attributed to the distribution of neurotransmitter receptors. For example, P2X receptors are expressed in mesenteric arteries while P2Y receptors are expressed in mesenteric veins (Galligan et al., 2001). Along the same vein (if you will forgive the pun), recent findings using antagonists that are specific for the adrenoreceptor subtypes indicate that α2adrenoreceptors play an important, venous-specific role in potentiating NE signaling at α1-adrenoreceptors (Sporkova et al., 2010). Neurotransmitter dynamics at the vascular neuroeffector junction has also emerged as an important contributor to mesenteric vasoconstriction, under both physiological and (particularly) pathological conditions (Kreulen, 2003). Most data explore the role of the NE transporter (NET), a specific transporter in sympathetic nerves that removes approximately 95% of NE that is secreted into the vascular junction (Axelrod and Kopin, 1969; Axelrod, 1971). For several decades, various lines of evidence have raised the possibility that altered NET expression and/or function contribute to hypertension (Esler et al., 1980; de Champlain et al., 1966; Goldstein et al., 1983); this suggestion has also crept into SCI research as an explanation for episodic hypertension. In terms of essential and age-related hypertension, however, this hypothesis has not been borne out by experimental or clinical data. The most consistent findings demonstrate increases in sympathetic nerve firing, suggesting that NE  18  release contributes more substantially to pathological sympathetic activation than impaired NE uptake (Eisenhofer, 2001). The sensory innervation of the vasculature is the easiest portion for most students (or at least for this student) to overlook. These nerves are sometimes referred to as “sensory-motor” nerves, a contradictory and confusing adjective that should be abandoned. The vasculature, including the mesenteric vasculature, is innervated by peptidergic sensory neurons originating in the dorsal root ganglion (DRG). These nerves are capsaicin-sensitive (i.e., they express the capsaicin receptor, transient receptor potential vanilloid-1 or TRPV1) and release substance P, calcitonin gene-related peptide (CGRP) and the ubiquitous ATP to elicit vasodilation. Of these, CGRP is the predominant – or at least the best-characterized – vasodilator (Bell and McDermott, 1996). A subset of sensory neurons in the DRG produce CGRP and project peripherally to blood vessels and centrally to the spinal cord, with central processes terminating on both second order sensory neurons in the superficial dorsal horn and sympathetic circuitry in the IML (Marti et al., 1987). Most blood vessels are surrounded by a network of CGRP-positive nerves at the adventitial-media border, with some medial penetration. The mesenteric arteries, particularly the SMA, are densely innervated by CGRP-positive fibres; this innervation tapers off toward the precapillary arterioles and is scant in mesenteric veins (Holzer and Lippe, 1992). In rat and human mesenteric arteries, CGRP acts on a G-protein-coupled receptor (called, in a ridiculous feat of nomenclature, the calcitonin receptor-like receptor) in smooth muscle to evoke potent vasodilation (Kawasaki et al., 1988; Brain et al., 1985; DiPette et al., 1989). It also acts on receptors in the vascular endothelium to elicit nitric  19  oxide release. Multiple lines of evidence implicate CGRP-mediated vasodilation (or a lack thereof) in cardiovascular pathology. Initial observations established that the density of CGRP-positive nerves innervating the mesenteric arteries decreases in hypertension and aging (Kawasaki et al., 1990; Li and Duckles, 1993). Extensions of this work demonstrated that vasodilatory function of CGRP-positive sensory nerves is impaired in the mesenteric arteries of rats with experimentally-induced diabetes and concomitant hypertension (Ralevic et al., 1993). However, more recent data suggest that CGRPmediated vasodilation in the mesentery tends to be adaptive in the face of cardiovascular pathology. For example, sensory-evoked vasodilation is augmented in response to chronic infusion of purinoreceptor agonists (Relevic et al., 1996) and CGRP receptors are upregulated in salt-induced hypertension (Li and Wang, 2003). Potentially germane to SCI, Phillips and colleagues (Phillips et al., 2000) demonstrated that CGRP-mediated vasodilation increased proportionally with increased pre-constriction, while endothelialdependent vasodilation was inhibited. This suggests that under (pathological) conditions of intensive vasoconstriction, endothelial-dependent vasodilation may be less important than vasodilation mediated by sensory neurons. In the CGRP-positive sensory innervation of the mesenteric vasculature, then, we have a system which differentially supplies the arteries and veins and is altered in many different types of cardiovascular pathology. Unfortunately, amongst the excitement surrounding plasticity of the central (spinal) terminals of CGRP-positive sensory axons, vasodilation by mesenteric afferents has not been investigated after SCI.  20  1.2.3.2  The vascular endothelium In addition to vasodilation evoked by sensory nerves, a major determinant of  vascular tone is vasodilation in response to locally-derived cues, particularly those produced by the vascular endothelium. This single layer of endothelial cells, lining the inner surface of all blood vessels, is no longer viewed as an inert structure but a dynamic organ, crucial to normal cardiovascular function. In response to shear stress and ACh, the arterial endothelium produces nitric oxide (NO), which induces vasodilation (Furchgott and Zawadzki, 1980). Other vasoactive molecules produced by the endothelium include the prostanoids, produced from arachidonic acid by the enzyme cyclooxygenase (COX). COX-derived vasoactive prostanoids exert their effects by acting on specific G-protein coupled receptors, expressed predominately on vascular smooth muscle, but also on the vascular endothelium and blood cells (Norel, 2007). The best-characterized prostanoids in vascular function are prostaglandin I2 (PGI2), which elicits vasodilation, and thromboxane A2 (TxA2), which induces vasoconstriction. Thromboxane A2 (TxA2) is a potent vasoconstrictor, which acts on the TP receptor expressed on vascular smooth muscle cells to mobilize intracellular calcium and elicit contraction. In addition to PGI2 and TxA2, there is increasing evidence documenting a critical cardiovascular role for prostaglandin E2 (PGE2), which acts on four EP receptors (EP1-EP4) to exert diverse effects. In keeping with the theme of vascular heterogeneity, the specific contribution of various prostanoids to vascular dilation and constriction varies with arterial bed, species, and physiological conditions. In addition to its role in vascular tone, the endothelium modulates vascular permeability, platelet activation and aggregation, inflammation and immune modulation, vascular smooth muscle cell proliferation and angiogenesis  21  (Herrmann and Lerman, 2001). Endothelial dysfunction is a marker of vascular disease and plays an important role in the initiation and progression of disease (Widlansky et al., 2003).  1.3  Impaired cardiovascular regulation following SCI  This is how ghastly it is. It starts with a dreadful inevitability – with visual disturbance, flashing lights, and then rapidly everything goes black and dark green. Voices grow dim; you grow hot and claustrophobic; and if no one can tip your wheelchair back in time and raise your legs in front of you, you will vomit… it’s scary, debilitating and stops you doing pretty much everything you need to do. (Melanie Reid, journalist, describing orthostatic hypotension after her SCI) With these aspects of neural cardiovascular control in mind – the supraspinal regulation of blood pressure, the autonomic control of the heart and blood vessels and the importance of the splanchnic circulation – we now turn to the cardiovascular consequences of SCI. The primary mechanism of cardiovascular dysfunction after SCI is damage of the descending projections from the RVLM to the SPNs. If damage to these pathways is functionally complete, SCI isolates SPNs below the level of injury from their supraspinal control centres, with two immediate effects. SPNs below the injury lose tonic input from the RVLM and sympathetic tone to the associated end organs drops. In addition, SPNs below SCI are no longer regulated by arterial baro- or chemoreflexes. Since the vagus nerve exits the CNS at the level of the brainstem, the afferent limb of these reflexes remain operational after SCI, and the parasympathetic control of the heart remains baro- and chemosensitive. It is a common mis-step to describe the cardiovascular end-organs as being “denervated” following SCI; rather, they are isolated from the supraspinal centres that render their function homeostatic.  22  The importance of the splanchnic circulation in blood pressure regulation creates a level-specific effect of SCI on cardiovascular function. Injuries at or above the sixth thoracic level (T6) eliminate descending control over the SPNs that govern (among other organs) the splanchnic vascular bed. As a result, the ramifications of altered neural cardiovascular control are most profound for individuals with injury above T6 (which I often describe as ‘high SCI’). Even within this group, the duration and severity of the effects of SCI on resting blood pressure and reflex blood pressure control are proportional to the level of injury and the severity of damage to the RVLM spinal projections (Sidorov et al., 2008; Claydon and Krassioukov, 2006; Mathias, 2006; Krassioukov and Claydon, 2006). In order to simplify this discussion, I will describe the cardiovascular ramifications of SCI that is “autonomically complete”; that is, an injury that produces complete and permanent functional disconnect between the RVLM and SPNs (Claydon and Krassioukov, 2006). The loss of tonic sympathetic activity to sympathetic preganglionic neurons results in severe hypotension and bradycardia in acute SCI, creating a syndrome described as neurogenic shock (Krassioukov and Claydon, 2006). This term is reserved for the cardiovascular consequences of SCI, and is not interchangeable with the term spinal shock, which describes the marked reduction or absence of sensory-motor reflexes below the injury (Atkinson and Atkinson, 1996; Ditunno et al., 2004). In clinical use, the end of neurogenic shock is usually marked as the time when intervention (pharmacological or volume-boosting) is no longer required to maintain minimally acceptable blood pressure (Hadley et al., 2002). While there is some recovery post-SCI (possibly due to adaptive changes, such as increases in blood volume), individuals with  23  cervical SCI typically have permanent hypotension and bradycardia. As a frame of reference, an individual with chronic cervical SCI might have a blood pressure of 90/50 mmHg and a heart rate of 55 beats per minute (bpm) at supine rest (Claydon and Krassioukov, 2006). Compared to humans, neurogenic shock is generally described as being less pronounced in experimental animals with SCI (Krassioukov and Claydon, 2006); however, this is unlikely to be due to differences in neural cardiovascular control. The most likely explanation is simply the difference in injury level; T3 essentially represents the rostral limit for severe SCI in experimental animals that survive beyond the acute stages of SCI. There are obviously other relevant differences between experimental and clinical scenarios; in the laboratory, experimental SCI is induced without concomitant trauma to other regions (including the brain) and rodents are mobile and active on the same day of their injury, limiting the effects of deconditioning. While low resting blood pressure and heart rate complicate matters for individuals with high SCI, the more disruptive problems stem from impairments in reflex blood pressure regulation. Individuals with high SCI develop orthostatic hypotension (OH), a syndrome that develops in other autonomic disorders and is defined as a reduction in blood pressure (≥20mmHg systolic) with assumption of upright posture (Freeman et al., 2011). The primary neural mechanism of OH is the loss of reflex vasoconstriction with baroreceptor unloading (Mathias, 1995). In SCI, hypotension in upright posture is exacerbated by paralysis in the legs which permits venous pooling, cardiovascular deconditioning due to bed rest and (possibly) hyponatremia (Frisbie and Steele, 1997; Faghri et al., 2001; Claydon et al., 2006b; Vaziri, 2003). The debilitating symptoms of OH are associated with cerebral hypoperfusion and include fatigue, light-  24  headedness, dizziness, blurred vision, nausea and syncope (Frisbie and Steele, 1997). Tachycardia often occurs, induced by parasympathetic inactivation via the baroreflex; however, the increased heart rate is not typically sufficient to compensate for the dramatic reduction in stroke volume. We have demonstrated that rats with high SCI develop OH; in response to passive upright (90º) tilt, rats with T3 SCI exhibit a pronounced drop in blood pressure (Inskip et al., 2009). Analogous to OH in humans, OH was more severe at one week after T3 SCI than at one month, and OH was not observed in animals with T10 SCI (Inskip, Ramer and Krassioukov, unpublished observations). Related to OH (at least in terms of neural mechanism) is the impaired reflex response to exercise in individuals with high SCI (Jacobs and Nash, 2004; Claydon et al., 2006a). Individuals with SCI above T1 (and even above T3-T4) exhibit diminished cardiac acceleration: in one study, maximal heart rates observed across a cohort of subjects with SCI at or above T6 was approximately 130 bpm (Jacobs and Nash, 2004). Individuals with cervical SCI also develop counter-productive exercise-induced hypotension, which interferes with rehabilitation and exercise and is often accompanied by symptoms of presyncope (Dela et al., 2003; Claydon et al., 2006a). Both OH and exercise-induced hypotension appear to improve somewhat over time after SCI. However, determining the time course of these conditions cannot rely on symptoms alone: people with high SCI are remarkably tolerant to hypotension (Illman et al., 2000; Claydon et al., 2006a), possibly due to adaptive changes in auto-regulation of cerebral blood flow (Johnson, 1976; Gonzalez et al., 1991). Despite marked physiological tolerance, resting hypotension, exacerbated by bouts of exertion-induced and positional  25  hypotension profoundly alter quality of life: even subclinical OH is associated with dramatic impairments in cognition and outlook (Perlmuter et al., 2011). These conditions create the troughs of the pathologically wide blood pressure range experienced by people with high SCI; the peaks are of this physiological roller coaster occur due to another condition, called autonomic dysreflexia (AD).  1.4  Autonomic dysreflexia In AD, visceral or somatic sensory input below the level of SCI stimulates  sympathetic outflow to the vasculature to increase vascular resistance (and thus blood pressure) (Maiorov et al., 1997; Krassioukov and Claydon, 2006). In many ways, AD represents an exact opposite of its hypotensive counterpart, OH. While OH tends to be most severe in the acute stages of SCI, AD typically develops over time after injury (Teasell et al., 2000; Mathias and Frankel, 1988). The hypertension in AD results from high sympathetic tone, due to unopposed activity in sympathetic preganglionic neurons that have lost their regulating connections from the RVLM. The blood pressure increase during AD activates the arterial baroreceptors, and reflexive bradycardia is a hallmark of AD. It bears mention, however, that the actual chromotropic effect is dictated by injury level: when injury occurs between T1 and T6, the heart receives substantial and simultaneous input from sympathetic and parasympathetic nerves during AD, and bradycardia, tachycardia or cardiac arrhythmia can result (Guttmann et al., 1965).  26  1.4.1  Clinical significance of autonomic dysreflexia The most distressing symptom of AD, a headache that is often described as  excruciating, is a direct result of baroreceptor activation, as vessels above the injury level vasodilate to oppose the rise in blood pressure. Symptoms of AD – anxiety, flushing, sweating and nausea – can be intensely unpleasant, and severe episodes produce exhaustion that lasts for days, an aspect of poorly-controlled AD that is rarely discussed but is extremely debilitating. Like OH, the severity of AD cannot be reliably determined by symptoms alone, since remarkable hypertension occurs in the absence of noteworthy symptoms (Guttmann et al., 1965; Ekland et al., 2008; Kirshblum et al., 2002; Linsenmeyer et al., 1996). One study demonstrated that individuals with high SCI experience systolic blood pressures in excess of 170 mmHg during routine bowel care, without symptoms (Kirshblum et al., 2002). The most common precipitating events of clinical AD are visceral stimuli. Rectal and colonic stimulation are potent inducers of AD, but bladder-related events (such as kinked or clogged catheters) account for a large majority of AD episodes that require clinical intervention (Shergill et al., 2004). However, any sensory stimulus, visceral or somatic, can evoke AD. Noxious stimuli, such as the sensory activation associated with an ingrown toenail, a urinary tract infection, a broken bone or contractions in childbirth, can induce AD (Teasell et al., 2000; Edvardsson and Persson, 2010; Burns and Clark, 2004); in these instances, AD may serve as a useful indicator of something amiss in the insensate portion of the body. However, the severity of hypertension does not necessarily reflect the potency of the stimulus. For example, pronounced hypertension can be  27  induced by sexual stimulation (Anderson et al., 2007; Forsythe and Horsewell, 2006), breast-feeding (Dakhil-Jerew et al., 2008) or even a tight belt or shoelace (Teasell et al., 2000) in an individual with high SCI. It is the unpredictability of AD, the variable relationship between stimulus and hypertension, which renders it acutely dangerous. Even among those with severe cervical SCI, the clinical presentation of AD is variable, and ranges from uncomfortable symptoms to life-threatening crises (Teasell et al., 2000). While most episodes of AD can be controlled by the individual with SCI or by a caregiver, by eliminating the inciting stimulus (i.e., via bladder emptying, bowel evacuation, or other measures), some episodes of AD require urgent pharmacological intervention due to their malignant presentation (Elliott and Krassioukov, 2006). Unpredictable and uncontrollable AD has emerged as a major safety concern in Paralympic competition, where athletes intentionally induce AD as a form of physiological doping, to overcome their impaired cardiovascular response to exercise and improve their performance (Mills and Krassioukov, 2011; Bhambhani et al., 2010). Apart from the acute danger associated with episodes of AD, the chronic effects of AD on the cardiovascular system represent an insidious threat. The repetitive and significant blood pressure elevations due to AD are distinct from sustained hypertension in able-bodied individuals. Individuals with high SCI experience dramatic fluctuations in blood pressure. For example, daily systolic blood pressure might range from 80 mmHg at rest to ≥170 mmHg during a bowel routine (Kirshblum et al., 2002). The upper end of this range is conservative: systolic blood pressure during sexual activity can exceed 300mmHg (McBride et al., 2003). A major concern with this pathological scenario is possible shear injury to the blood vessel endothelium that could predispose these  28  individuals to cardiovascular complications. Such lability in blood pressure is unique to SCI, and its long-term effects over the lifetime of individuals with SCI are unknown. In 2002, Steins and co-authors proposed that instability in blood pressure could contribute to vascular injury and consequently result in a greater risk for arterial disease in individuals with SCI: this premise is tested experimentally in Chapter 5.  1.4.2  Animal models of autonomic dysreflexia In animal models, AD is most commonly induced by colo-rectal distension  (CRD), a relatively easy and non-invasive technique that reliably induces AD in rats and mice (Mayorov et al., 2001; Maiorov et al., 1997; Krassioukov and Weaver, 1995a; Jacob et al., 2003). I have found that CRD induces AD, indicated by increases of 25-60 mmHg in systolic blood pressure and accompanying (although more variable) bradycardia, in conscious rats with T3 SCI (Chapters 2,5). The same stimulus does not induce significant hypertension in rats with T10 SCI (Inskip, Ramer and Krassioukov, unpublished observations). In rats with cervical or high-thoracic SCI, AD has also been documented in response to bladder distension, noxious and innocuous somatic stimulation of the tail and hind limbs, gastric distension, and vagino-cervical distension (Krassioukov and Weaver, 1995; Krassioukov et al., 2002; Sansone et al., 2007; Leal et al., 2008; Inskip, Ramer and Krassioukov, unpublished observations). A unique model of AD provoked episodic hypertension via intramuscular injection of hypertonic saline (Lujan et al., 2010b); importantly, these experiments used cholera toxin B subunit conjugated to saporin to eliminate mesenteric- projecting sympathetic neurons, demonstrating their importance in AD.  29  With a picture of clinical and experimental AD in mind, I now turn to a description of the critical components of the spinal reflex arc that mediates AD. For each anatomical compartment of the reflex – dorsal root ganglion (DRG), spinal cord, sympathetic ganglion and the sympathetic neurovascular junction – I will review the normal anatomy and function, and describe plasticity that occurs in this reflex after SCI. The functional connectivity of this reflex arc has been verified by viral (pseudorabies) tracer injections into the DRG, which produces progressive infection of sympathetic ganglionic neurons, SPNs and neurons in the dorsal horn (Hofstetter et al., 2005).  30  31  Figure 1.1 The spinal reflex that mediates autonomic dysreflexia The spinal reflex that mediates autonomic dysreflexia (AD) is comprised of sensory neurons, spinal circuits, sympathetic ganglionic neurons and blood vessels. Autonomic dysreflexia is most commonly triggered by sensory stimulation of the urinary bladder or the colon/rectum. In these instances, sensory information enters the lumbar spinal cord (in the rat) via sensory neurons in the lumbar dorsal root ganglia (DRGs) (pink). Propriospinal circuits (represented schematically in blue) transmit sensory information (indirectly, through associated interneurons) to sympathetic preganglionic neurons (SPNs) in the thoraco-lumbar spinal cord. Critical regulatory SPNs (red) govern the mesenteric arterial bed, a capacitance unit capable of mobilizing stored blood upon physiological demand. Spinal cord injuries above T6 isolate SPNs that supply the mesenteric arterial bed from supraspinal control. As a result, these SCI above T6 is generally associated with AD. Logically, the severity of AD (and other cardiovascular problems) increases as the injury level ascends, and an increasing proportion of SPNs in the thoracic spinal cord lose contact with their regulatory centres. Modified from Inskip & Ramer et al., 2009.  32  1.5  The dorsal root ganglion  Primary sensory neurons transduce environmental stimuli and transmit the resulting information to the central nervous system (CNS). Their cell bodies reside in cranial nerve ganglia and dorsal root ganglia (DRGs); since I am primarily interested in sensory neurons below the level of SCI, I limit this discussion to the latter group. Mammalian primary sensory neurons are pseudounipolar, with a single axon emanating from their cell body and bifurcating within the DRG: one branch travels peripherally in the peripheral nerve, and one branch travels centrally in the dorsal root. The DRG can, therefore, be divided into a cell layer (sensory neuron somata) and a fibre layer (axons). Dorsal roots increase in length along the rostral-caudal axis and DRGs are caudally shifted relative to the spinal cord, so DRGs are named for the spinal segment to which they are connected. Most DRGs contain both somatic (exteroceptive) and visceral (interoceptive) afferents. In terms of the sensory stimuli for AD, we are most concerned with the notable receptive fields below the level of injury. Sensory neurons innervating the skeletal muscles of the rat hind limb reside in the L4 and L5 DRGs (Baron et al., 1988). Sensory neurons innervating the rat colon and rectum are distributed among DRGs spanning T12 to S3 (Baron and Janig, 1991) with some reports indicating that they are most abundant in the S1 DRG (Keast and de Groat, 1992) and others demonstrating that thoracolumbar DRGs contribute most significantly (Christianson et al., 2009). The rat L6 and S1 DRGs contain afferents for many pelvic viscera, including the urinary bladder, penis, clitoris and urethra (Vera and Nadelhaft, 1992; Keast and de Groat, 1992; Yoshimura et al., 2003; Cruz et al., 2004).  33  1.5.1  First-order sensory neurons The DRG contains a remarkably heterogeneous population of sensory neurons.  From the time of Ramon y Cajal, neuroscientists have attempted to sort the primary sensory neurons; they have been classified according to size, cytological criteria, conduction velocity and neurochemical phenotype (Willis and Coggeshall, 2004). In my mind, these neurons elude useful categorization: neurochemical classification has produced a Ven diagram of dizzying complexity under normal conditions, which becomes impenetrable when neurons in the DRG alter their neurochemical profile in response to injury, inflammation and disease (Sandkuhler, 2009). Nonetheless, the field has clung to a few basic distinctions, based on the type of information conveyed by different populations of sensory neurons. Neurons that detect noxious stimuli are predominately small and unmyelinated. Small nociceptive neurons can be divided into two major groups. Peptidergic nociceptive neurons express neuropeptides such as CGRP and substance P. These neurons also express the tyrosine kinase neurotrophin receptor TrkA, and are therefore sensitive to nerve growth factor (NGF). Non-peptidergic nociceptive neurons lack TrkA, CGRP, and SP but bind the plant lectin IB4 (from Bandeiraea simplicifolia), and express the glial cell line-derived neurotrophic factor (GDNF) receptor components. A growing body of evidence suggests that these populations are functionally distinct (Braz et al., 2005; Fang et al., 2006; Choi et al., 2007). Neurons that detect innocuous mechanical and proprioceptive stimuli are mainly large cells with myelinated axons. These do not express peptidergic neurochemicals, nor do they bind IB4; they are typically identified by their expression of  34  the heavy neurofilament NF200. TrkC, the receptor for neurotrophin-3, is expressed almost exclusively by large sensory neurons. The astute reader will notice that this description of sensory neurons in the DRG is plagued by “predominately”, “mainly” and “almost”. Fortunately, our tools have improved over the last decade, and we now have antibodies raised against functional ion channels in sensory neurons, which provide (more) reliable information about the function of a sensory neuron in a fixed section of tissue. These tools extend a longstanding effort to categorize sensory neurons in terms of function (Carr and Nagy, 1993). A prime example is the transient receptor potential (TRP) family of ion channels. Many of the TRP channels were first identified in nociceptors as the mediators of the somatosensory effects of natural products such as capsaicin, mustard oil and menthol. TRP channels have now been implicated in physiology and disease in almost every organ system, and have emerged as important therapeutic targets (Moran et al., 2011).  1.5.2  Glial environment of the dorsal root ganglion Sensory neurons in the DRG have a unique glial environment, comprised mainly  of satellite cells. These surround sensory somata, such that each soma is enclosed by a sheath of several glial cells that are “intimately applied” (in the words of Cajal) to the neuronal plasma membrane (Pannese, 1981; Hanani, 2005). Satellite cells are readily identified immunohistochemically by their expression of glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS), which catalyses the conversion of glutamate to glutamine (Woodham et al., 1989; Miller et al., 2002). Sensory axons in the fibre layer associate with Schwann cells (myelinating and non-myelinating), but these are largely  35  excluded from the cell layer of the DRG. The relationship between sensory neurons and satellite cells does seem both persistent and intimate, since even in cultures of cells from the DRG, satellite cells surround sensory neurons: recent experiments revealed that nearly all neurons in early culture were surrounded by satellite cells, reminiscent of the in vivo arrangement (Belzer et al., 2011). Although satellite cells represent the dominant glial population in all peripheral ganglia – sensory, sympathetic and parasympathetic – research on their functional properties is largely restricted to studies of the sensory ganglia (Hanani, 2010). Satellite cells create a microenvironment around sensory neurons and isolate neurons from one another within the DRG. Crucially, the distance between satellite cells and neurons is small, approximately 20 nm (Pannese, 2010): when glia and neurons release small amounts of substance into this microenvironment, the effective concentration is (presumably) significant (Hanani, 2010). This close association has prompted significant investigation into the role of satellite cells in processing of afferent information, with lucrative results. Satellite cells produce inflammatory mediators and neurotrophins and express receptors for these (Hanani, 2005). For example, they express TrkA and can bind and internalize NGF, which may allow them to act as a reservoir for NGF in the DRG (Pannese and Procacci, 2002). Similarly, they express P2Y and P2X adrenoreceptors (Weick et al., 2003; Ceruti et al., 2008; Kushnir et al., 2011) and can release ATP to act on their associated neurons (Suadicani et al., 2010). Recent findings have characterized a pivoltal role for purinergic signaling in the communication between sensory neurons and satellite cells. Zhang and colleagues (Zhang et al., 2007) used Ca2+ imaging to demonstrate that sensory neuron somata release ATP to communicate with  36  and activate satellite cells, a peripheral counterpart to the astrocyte response to synaptic input in the CNS (Wang et al., 2006). Like glia in the CNS, satellite cells in the DRG are “activated” (to produce signaling and other proteins in increased amounts or de novo) in response to nerve injury and to local or systemic inflammation (Takeda et al., 2009). The emerging roles of satellite glia as regulators of sensory neuron function are discussed in Chapter 3 (Discussion).  1.5.3  Vascular environment of the dorsal root ganglion Most vessels supplying the DRG arise from the radicular arteries at each spinal  segment. Elegant anatomical studies in humans and monkeys demonstrated that the cell layer of the DRG has a dense capillary bed relative to most grey matter in the CNS (Bergmann, 1941). Bergmann surmised that this was due to high metabolic demands of the tightly-packed sensory neurons of the DRG. Studies using cytochrome oxidase activity as an indicator of oxidative metabolism, which is closely-coupled to neuronal activity (Wong-Riley, 1989) appear to have borne out this supposition (Liu et al., 1990; Karmy et al., 1991). The metabolic demands of sensory neurons appear to be met in part by autoregulation of blood flow in the DRG (i.e., by local mechanisms that couple blood flow to metabolic demands) (McManis et al., 1997). Blood flow measurements by autoradiography demonstrated that in rats at rest, blood flow in the L5 DRG was approximately twice that in the sciatic nerve. More significantly, blood flow to the L5 DRG (and the superior cervical ganglion) did not vary with blood pressure; flow remained constant over a wide range of systolic blood pressures (50-140 mmHg). In  37  contrast, blood flow in the sciatic nerve varied with systemic blood pressure, in keeping with previous work demonstrating that peripheral nerves have little capacity for autoregulation (McManis and Low, 1988). Recent data (confirmed in part by my work) describe peptidergic, cholinergic and adrenergic innervation of the intraganglionic vasculature in the DRG (Kobayashi et al., 2010; Kobayashi et al., 2009). Blood flow to the DRG has unpredictable effects on afferent activity; after injury of their peripheral process, primary afferents exhibit spontaneous activity when blood flow is reduced (Habler et al., 2000). This becomes a real head-scratcher, of particular relevance for autonomic dysreflexia, when we consider viral tracing data that demonstrate synaptic contacts between primary afferents and the sympathetic ganglionic neurons regulating sympathetic outflow to corresponding DRGs (Hofstetter et al., 2005).  1.6  Spinal segments of the sensory-sympathetic reflex The sympathetic preganglionic neurons (SPNs) innervate sympathetic ganglionic  neurons (and chromaffin cells in the adrenal medulla) and are the source of sympathetic outflow exiting the CNS. The SPNs and their afferent and interneuronal inputs constitute the spinal components of the sensory-sympathetic reflex arc. All available evidence – degeneration studies and infection with viral tracers – argues against direct connections between primary afferents and SPNs (Petras and Cummings, 1972). Therefore, the sensory circuits in the dorsal represent the crucial link between afferent input and sympathetic output.  38  1.6.1  Sensory circuits in the dorsal horn The majority of primary afferents project to the dorsal horn, where they synapse  on second-order sensory neurons. The exceptions are afferents transducing light touch and proprioception, with centripetal axons that project to dorsal column nuclei: in thoracic and lumbar DRGs, these constitute fewer than 15% of sensory neurons (Giuffrida and Rustioni, 1992). The spinal grey matter is divided into anatomical and functional divisions or laminae (Rexed, 1952); the dorsal horn occupies laminae I-VI, with laminae I being most superficial. The neuropil of the dorsal horn is distinctively designed for local integration. Second-order sensory neurons are surrounded (and drastically outnumbered) by interneurons (Chung et al., 1984). Sensory neurons also have extensive local axonal arbours: although they are often referred to as “projection” neurons, with axons extending long distances to the thalamus, they do not merely pass a signal along to the brain. Dendritic remodeling in these circuits occurs as a consequence of both SCI and peripheral nerve injury: this dendritic plasticity has important implications for sensory signaling and has recently been identified as a substrate of neuropathic pain (Tan and Waxman, 2011; Tan et al., 2011).  1.6.2  Spinal sympathetic transmission SPNs are cholinergic and reside in the thoracic and upper lumbar spinal cord. The  largest collection of SPNs is the intermediolateral cell column (IML), in the lateral horn at the border between grey and white matter. SPNs are also found in the central autonomic area (CAA), which is above the central canal in lamina X, in the intercalated nucleus, which is by definition between the IML and the CAA, and in the dorsolateral  39  funiculus, within the white matter. SPNs in the IML are clumped in “nests” at each spinal segment; they are connected rostrocaudally by their long dendrites (Llewellyn-Smith, 2009). In rats, there is general rostrocaudal topography to the IML, such that SPNs in T1 innervate neurons in the most rostral sympathetic ganglia. However, the spinal distributions of SPNs supplying different targets frequently overlap, so each sympathetic ganglion (and the adrenal gland) receives a multi-segmental SPN input, with one segment contributing the predominant innervation (Strack et al., 1988). For most targets, the main source of sympathetic innervation is the IML; the inferior mesenteric ganglion, with neurons innervating the distal ileum and colon, is an exception, as approximately 75% of its sympathetic innervation is derived from SPNs in the CAA (Strack et al., 1988). The SPNs of the IML receive monsynaptic input from the cardiovascular regulatory centres in the brainstem (i.e., from the RVLM and the caudal raphe nuclei) (Zagon and Smith, 1993; Bacon et al., 1990; Deuchars et al., 1995; Deuchars et al., 1997). They also receive significant input from excitatory and inhibitory spinal interneurons, demonstrated by the persistence of these inputs caudal to complete SCI (Llewellyn-Smith et al., 1997; Llewellyn-Smith et al., 2005). The interneurons that are retrogradely labeled by tracer injections into the sympathetic ganglia (Cabot et al., 1994), adrenal medulla (Clarke et al., 1998) or kidney (Tang et al., 2004), are often referred to as “sympathetic interneurons” or “sympathetically-related interneurons”: this terminology indicates that they are synaptically coupled to SPNs, although they may also modulate sensory or somatic efferent transmission. The tracing reveals that the majority of these sympathetic interneurons synapse locally on SPNs, without traversing the spinal  40  cord longitudinally (Tang et al., 2004), reinforcing the segmental organization of the sympathetic nervous system (Laskey and Polosa, 1988). Interneurons with activity that is positively or negatively correlated with sympathetic nerve activity are described as sympathetically-correlated interneurons (Chau et al., 2000; Miller et al., 2001; Tang et al., 2003). Interestingly, SPNs at different rostrocaudal levels appear to differ in proportions of supraspinal versus interneuronal inputs. When the spinal cord was completely transected at T4, SPNs in T8 exhibited profound denervation 14 days after injury (Llewellyn-Smith and Weaver, 2001), while SPNs in T12-L2, which govern the pelvic viscera, retained the bulk of their innervation (Llewellyn-Smith et al., 2005; Llewellyn-Smith et al., 2006).  1.7  Sensory and sympathetic plasticity after spinal cord injury: the search for the  cause of autonomic dysreflexia O how they cling and wrangle, some who claim For preacher and monk the honored name! For, quarreling, each to his view they cling. Such folk see only one side of a thing. (The Blind Men and the Elephant) The mechanisms underlying AD continue to be debated and have provided fodder for many manuscripts and theses (including this one) over the past decade. The central feature of AD that has captured the attention of neuroscientists and vascular biologists is that it develops and progresses over time after SCI. This is true in both rats and humans, although AD onset typically occurs earlier in rats (Krassioukov and Weaver, 1995a; Teasell et al., 2000). In our model (rats with complete T3 SCI), AD is more severe three  41  months after injury than at one month after injury (Inskip, Ramer and Krassioukov, unpublished observations). The implication is that AD is not solely a consequence of the loss of descending control over spinal sensory-sympathetic circuits: while this is the prerequisite for AD, the condition likely develops due to injury-induced changes that develop over time (and might be halted or reversed through timely post-injury intervention). The majority of research investigating potential mechanisms for AD has focused on injury-induced changes (or plasticity) in relevant spinal neural circuits, namely, sensory projections to the dorsal horn, sympathetic preganglionic neurons or sympathetically-correlated interneurons, below the level of injury. Smaller collections of studies have investigated the effects of SCI on the cardiovascular end organs and peripheral ganglia; in terms of the former, I limit this discussion to injury-induced changes in the resistance vessels, although the reader should also be aware that SCI triggers important changes in the heart. For example, recent findings describe increased sympathetic innervation density in the left ventricle after high-thoracic SCI in rats (Lujan et al., 2010a). DiCarlo and colleagues speculate that this injury-induced plasticity may contribute to dangerous arrhythmia (Lujan and DiCarlo, 2007; Lujan et al., 2009).  1.7.1 1.7.1.1  Spinal cord injury-induced plasticity in the spinal cord Primary afferent sprouting in the dorsal horn Sprouting of the central branches of primary afferents within the dorsal horn of  the spinal cord has garnered the lion’s share of attention in the search for a neural substrate for AD. The premise of this line of enquiry is that if primary afferents expand  42  their territory in the dorsal horn, they can increase (or establish de novo) their inputs to SPNs (or associated excitatory interneurons). The idea originated in the study of neuropathic pain (Liu and Chambers, 1958; Woolf et al., 1992). The original and highlycited study reported that under painful conditions induced by peripheral nerve injury, large sensory neurons sprouted centrally to occupy the superficial laminae of the dorsal horn, normally the territory of nociceptors (Liu and Chambers, 1958). The notion that proprioceptors and mechanoreceptors could pathologically subserve nociception readily gained steam as a mechanism for neuropathic pain. A few years later, however, it was revealed that peripheral nerve injury altered the tracer uptake of nociceptors; after injury, a subset of nociceptors take up and transport cholera toxin B subunit, a specific marker for non-nociceptive sensory neurons in uninjured conditions (Tong et al., 1999; Bao et al., 2002; Shehab et al., 2003). Although it was spurious tracing rather than sprouting that had been observed and reported (by nearly every prestigious laboratory studying pain in the world), the link between sensory hyperexcitability and sensory sprouting in the dorsal horn stuck, and soon found a home in SCI research. Krassioukov and Weaver et al. were first to identify sensory sprouting (of nociceptors, this time) in the dorsal horn after SCI and to draw a causal link between sensory sprouting and the development of AD. Concurrent with the development of AD in rats with high-thoracic SCI (Krassioukov and Weaver, 1995a; Maiorov et al., 1997), peptidergic afferents (i.e., those immunoreactive for CGRP) increase their central terminal arbors in the dorsal horn of the thoracolumbar spinal cord (Krenz and Weaver, 1998; Weaver et al., 2001). Electron microscopy demonstrated that SCI-induced afferent plasticity represented the reciprocal of the proposed (but flawed) scenario described  43  above: after SCI, nociceptors sprouted to occupy deeper spinal laminae, into the normal territory of non-nociceptive transmission (Wong et al., 2000). An intrathecally-delivered neutralizing antibody directed against endogenous nerve growth factor (NGF) both prevented this sensory sprouting and reduced the severity of AD in rats two weeks after SCI (Krenz et al., 1999). These findings were extended by Rabchevsky and colleagues in 2006; using genetic techniques to manipulate peptidergic sprouting at different levels of the spinal cord, they demonstrated that sprouting in the lumbosacral dorsal horn was tied to the severity of AD (Cameron et al., 2006). More recently, retrograde tracing from the distal colon indicated that at least some of these sprouting afferents are colonic (and thus stimulated by the CRD used to evoke AD in each of these studies; Hou et al., 2009). The effects of peptidergic sprouting in distal spinal segments (L6-S1) appear to be potentiated by concomitant plasticity in the propriospinal circuitry that links afferent input to L6-S1 with SPNs in the thoracolumbar cord (Hou et al., 2008; Rabchevsky, 2006). In addition to contributing to AD, spinal sensory plasticity has been linked to other conditions of sensory hyperexcitability after SCI, including bladder dysfunction (Zinck and Downie, 2008; Zinck et al., 2007) and pain (Christensen and Hulsebosch, 1997; Macias et al., 2006).  1.7.1.2  Injury-induced plasticity in spinal sympathetic transmission Moving around the horn (so to speak), there are also important changes in the  interneurons that are functionally linked to sympathetic outflow from the CNS. Elegant electrophysiological studies demonstrated that the functional properties of sympathetically-correlated interneurons were altered over time after SCI (Weaver et al.,  44  2002; Krassioukov et al., 2002; Schramm, 2006). Specifically, the activity of these interneurons was increased by stimulation of more of the body surface and decreased by stimulation of less of the body surface in rats with chronic SCI than in rats with acute SCI. These changes accompanied the development of enhanced pressor responses to colon distension, and suggest that interneurons may play in role in exaggerated spinal reflexes, by providing increased excitatory input to SPNs through temporal summation (Chau et al., 1997). On the efferent side of this reflex, histological findings in both rats (Krassioukov and Weaver, 1995b; Krassioukov and Weaver, 1996) and humans (Krassioukov et al., 1999) with SCI demonstrated that thoracic sympathetic preganglionic neurons (SPNs) caudal to SCI undergo transient atrophy after injury. Over time, SPNs in both species regain their normal morphology and re-establish their dendritic arbour. The transient atrophy in SPNs correlates with the development of AD, which can be observed acutely although this is clinically rare (Krassioukov, 2004), is typically absent for a sub-acute period, and develops in the weeks or months post-SCI. Transient atrophy of SPNs may contribute to the slow onset of AD; however, this explanation is less satisfying for AD evoked by stimuli of the pelvic viscera or hind limbs. These afferent stimuli enter the spinal cord at the lumbosacral levels (L1-S1) and the SPNs at these levels (L1-L2) do not atrophy or exhibit denervation following SCI (Llewellyn-Smith et al., 2005). This does not preclude the possibility that afferent input entering distally might interact with thoracic SPNs via propriospinal connections, but in my mind, transient atrophy does not fit as well as progressive plasticity for a potential mechanism for AD.  45  1.7.2  Spinal cord injury-induced changes in the blood vessels On the other side of the neurovascular equation, considerably less is known about  SCI-induced changes that might contribute to AD. Most available data characterize changes in arterial structure and function in arteries below the level of SCI. In the structural category, one relatively rapid response to paralysis after SCI is the remodeling of arteries, as the cardiovascular system adapts to revised metabolic demands. In people, remodeling of peripheral arteries in paralyzed limbs occurs quickly; for example, reductions in diameter and flow in the femoral artery occur within 6 weeks of paralysis due to SCI (de Groot et al., 2006). Recent, preliminary findings suggest that the chronic stages of SCI are accompanied by systemic arterial stiffening, indicated by an increase in aortic pulse wave velocity (Miyatani et al., 2009). This may prove to be clinically important, since stiffening of the arterial walls is a significant independent risk factor for the development of cardiovascular disease in the able-bodied population (Blacher and Safar, 2005; Safar et al., 2003). In terms of vascular function, both endothelial and smooth muscle function have been investigated in arteries caudal to SCI. One might reasonably expect (as I did) that the lability of blood pressure in high SCI might damage the endothelium and consequently impair endothelial-dependent vasodilation. In preeclampsia, another condition characterized by episodic hypertension in relatively young individuals, endothelial damage can ensue and persist for months-to-years after pregnancy (Chambers et al., 2001; Agatisa et al., 2004). However, all available evidence, including studies of flow-mediated dilation in people with SCI and in vitro studies on arteries from rats with SCI herein (Chapter 5) indicate that endothelial-mediated vasodilation is preserved after  46  SCI (de Groot et al., 2004; Thijssen et al., 2008; Kooijman et al., 2008). The predominant abnormality in resistance vessels below SCI is NE/PE hyper-responsiveness, which has been documented in both people and animals, and proposed to contribute to AD. The literature describing the extent and mechanisms of PE hyper-responsiveness in vessels caudal to SCI is reviewed in Chapter 4 (Discussion).  1.8 1.8.1  Experimental overview and hypotheses Differential effects of high- and low-thoracic spinal cord injury I investigated changes at two segments of the spinal reflex that couples sensory  input to hypertension caudal to SCI. My experimental model was the adult Wistar rat, in which I performed a complete transection of the spinal cord at the third (T3) or tenth (T10) thoracic level. Given that we study AD, injury level-dependent effects of SCI have always been a theme in our laboratory. Over the last several years, the importance of injury level has been reinforced through our findings. T3 and T10 SCI are similar in motor outcome (both produce spastic paraplegia); in addition, both injuries produce similar dysfunction of the LUT. Despite this, we have found that some consequences of injury occur as a result of T3 but not T10 SCI (Inskip et al., 2010; Chapters 2-3).  1.8.2  Injury-induced changes in the dorsal root ganglion In the DRG, I first examined injury-induced hypertrophy in sensory neurons  following SCI (Chapter 2). The neurotrophic hypothesis, one of the principal tenets of neuroscience, states that neurons depend on cues produced in limiting amounts in target tissues for survival, growth and (as we now know) normal function. Despite the dramatic  47  changes in targets of sensory neurons caudal to SCI – atrophy of skeletal muscle, hypertrophy of smooth muscle, such as the detrusor and likely many other uncharacterized changes – remarkably little is known about the phenotypic fate of sensory neurons. I took a systematic immunohistochemical approach to studying one of the defining phenotypic features of sensory neurons – soma size – in different populations of sensory neurons after T3 SCI. After characterizing hypertrophy in a very specific subset of sensory neurons, I examined the effect of rostro-caudal level and injury level; I also examined the density of central projections of hypertrophied neurons and the role of those neurons in AD. I hypothesized that SCI would induce nociceptor hypertrophy and/or terminal sprouting and that these effects would be more pronounced after high-thoracic injury. After identifying a specific subset of sensory neurons that responded to SCI, I hypothesized that these would be involved in triggering and/or development of AD. My next piece of work in the DRG characterized changes in the cellular environment of sensory neurons (Chapter 3). I examined lumbar (L4 and L5) DRGs from animals that survived for one month after T3 or T10 SCI. I chose the L4/L5 DRGs because they are a relatively simple system: they contain sensory neurons that are almost exclusively somatic, supplying the skin and muscles of the hind limbs. They have also been used as a control in studies characterizing changes in other, visercal DRGs after low-thoracic SCI (Zvarova et al., 2005). From a practical standpoint, they are very large and have a continuous cell layer compared to L6/S1 DRGs, which facilitates densitometric quantification of relatively large structures (such as blood vessels). I used histochemical and immunohistochemical techniques to examine satellite cells, immune  48  cells and blood vessels in the DRG after T3 and T10 SCI. I also examined aberrant ingrowth of sympathetic ganglionic axons into the DRG. I hypothesized that SCI would activate glial and immune cells in the DRG, promote angiogenesis and trigger sympathetic sprouting, and that these effects would be more pronounced after T3 SCI than after T10 SCI.  1.8.3  Injury-induced changes in the mesenteric arteries In the arteries, I did not (perhaps unfortunately) examine the effects of high-  versus low-thoracic SCI. When we designed these experiments, we consulted Dr. James Brock, one of only a few investigators in the world studying arterial function in experimental animals with SCI. Dr. Brock predicted that, while he had not done the experiment, mesenteric arteries would not exhibit PE hypersensitivity caudal to lowthoracic SCI. To date, his hypothesis remains to be tested. My first study on mesenteric arteries examined the role of cyclooxygenase (COX) enzymes in PE hyper-responsiveness (Chapter 4). I used in vitro myography, performed by my collaborators Saeid Golbidi and Ismail Laher, to examine vasoconstriction and vasodilation, and the role of COX enzymes in arterial behaviour following SCI. I also used immunohistochemistry to examine COX-2 expression in mesenteric arteries. I hypothesized that COX-2 activity mediated PE hyperresponsiveness in mesenteric arteries after SCI. My second study on mesenteric arteries examined the effects of repeated episodes of AD, induced intentionally over a period of two weeks following SCI. I hypothesized that shear stress on the vasculature due to recurrent AD would  49  damage the vascular endothelium, exacerbating both PE hyper-responsiveness and AD. I used in vitro myography (Laher lab) and cardiovascular physiology (Krassioukov Lab) to examine the effects of recurrent AD after T3 SCI.  50  Chapter 2: Plasticity of capsaicin-sensitive sensory neurons mediating autonomic dysreflexia following spinal cord injury  2.1  Synopsis Spinal cord injury (SCI) triggers profound changes in visceral and somatic targets  of sensory neurons below the level of injury. Despite this, little is known about the influence of injury to the spinal cord on sensory ganglia. One of the defining characteristics of sensory neurons is the size of their cell body: for example, nociceptors are smaller in size than mechanoreceptors or proprioceptors. In these experiments, I tested the hypothesis that SCI would trigger hypertrophy in nociceptors below the injury. I found that a specific subpopulation of nociceptors, those that express the capsaicin receptor (transient receptor potential vanilloid-1 or TRPV1), underwent significant hypertrophy. Injury-induced hypertrophy was most pronounced in caudal ganglia and following higher SCI. By causing selective degeneration of spinally-projecting TRPV1 axons, I also showed that these neurons are crucial to the induction of autonomic dysreflexia (AD), hypertension elicited (in this instance) by colo-rectal distension (CRD).  2.2  Introduction The original formulation of the neurotrophic hypothesis by Viktor Hamburger  and Rita Levi Montalcini (1949) asserted that the survival of neurons depends on cues produced in limiting amounts in target tissues. The subsequent discovery of nerve growth factor (NGF) as a survival factor for sympathetic and sensory neurons (Cohen et al., 1954) not only validated this hypothesis, but also extended the role of neurotrophic  51  factors beyond developmental neuronal survival: target-derived NGF and numerous other neurotrophic molecules maintain the phenotype of neurons during adulthood. This has been arguably best demonstrated in experiments on primary afferent neurons of the dorsal root ganglion (DRG), a heterogeneous population of neurons responsible for transmission of information from the periphery (somatic) and internal milieu (visceral) to the spinal cord and brainstem. Early classification of DRG neuronal subsets was based on cytoplasmic staining with standard histological techniques and electrophysiological recording followed by dye-filling of neurons (Willis and Coggeshall, 2004). These methods revealed “small dark” neurons which have slowly-conducting axons (corresponding to thermoceptors and nociceptors, approximately 70% of all neurons), and “large light” cells with rapidlyconducting fibres (30% of all DRG neurons, representing skin and muscle mechanoreceptors). In terms of their neurochemistry, DRG neurons are remarkably diverse, and much effort has gone into establishing particular neurochemical phenotypes as functional proxies (Willis and Coggeshall, 2004). Among the small dark cells, for example, there are peptidergic and non-peptidergic subtypes, which also happen to be sensitive to different neurotrophic factors (NGF, and glial cell line-derived neurotrophic factor, GDNF, respectively) (Braz et al., 2005). Ascribing functional relevance to this neurochemical distinction has proven difficult, in part because both populations express transducers of thermal and noxious stimuli: one example is the capsaicin-sensitive cation channel TRPV1, a remarkably versatile receptor that is also activated by protons and noxious heat (Caterina et al., 1997; Tominaga et al., 1998).  52  DRG neurons undergo marked changes in neuronal phenotype following their disconnection from their target tissues by axotomy. Importantly, and in-line with the neurotrophic hypothesis, these changes are mimicked by depletion of the relevant factors in the absence of axotomy, and reversed by supplying axotomized neurons with trophic support from exogenous sources (Rich et al., 1984; Yip et al., 1984; Johnson and Yip, 1985; Wong and Oblinger, 1991; Matheson et al., 1997; Bennett et al., 1998). Uninjured DRG neurons undergo hypertrophy following in vivo delivery of NGF or GDNF (Ramer et al., 2001; Ramer et al., 2003). Likewise, transgenic over-expression of NGF (Goodness et al., 1997) and the GDNF family member, artemin (Elitt et al., 2006) cause sensory neuronal hypertrophy. Spinal cord injury (SCI), while not disconnecting DRG neurons from their peripheral targets, nevertheless has profound effects on multiple tissues which might be expected to influence neuronal phenotype. Among the most dramatic of these are skeletal muscle atrophy, and in the lower urinary tract (LUT), detrusor hypertrophy. In the latter case, increased NGF production by the bladder has been correlated with hypertrophy of innervating DRG neurons (Yoshimura, 1999). For complete lesions above T5/6, SCI is almost always accompanied by cardiovascular disturbances including orthostatic hypotension (OH; a sudden fall in blood pressure upon assuming an upright position) and autonomic dysreflexia (AD; potentially life-threatening elevations in blood pressure triggered by sensory stimulation below the injury) (Krassioukov and Claydon, 2006). There is increasing evidence that blood vessels, a peripheral target of sensory as well as sympathetic axons, also undergo SCI-induced alterations which may evoke phenotypic  53  changes in their innervating neurons (Alan et al., 2010; Rummery et al., 2010; Tripovic et al., 2011; McLachlan and Brock, 2006; Al Dera et al., 2011). Here I took a systematic immunohistochemical approach to studying one of the defining phenotypic features of sensory neurons – soma size – in different populations of sensory neurons after T3 SCI. I characterized hypertrophy in a specific subset of nociceptors, those that are sensitive to capsaicin and artemin. I examined the effect of rostro-caudal level and injury level; I also examined the density of central projections of hypertrophied neurons and the role of those neurons in AD. I hypothesized that SCI would induce nociceptor hypertrophy and/or terminal sprouting and that these effects would be more pronounced after high-thoracic injury. After identifying a specific subset of sensory neurons that responded to SCI, I hypothesized that these would be involved in triggering and/or development of AD.  2.3 2.3.1  Materials and methods Spinal cord injury surgery Complete transection of the spinal cord at the third (T3) and tenth (T10) thoracic  segments was performed in adult male Wistar rats (250-350g, Charles River Laboratories, Inc., Wilmington, Canada). T3 SCI promotes the development of cardiovascular dysfunction, including AD, while T10 SCI does not. Sham surgeries were performed at T3 and were identical up to and including durotomy, without transection of the cord.  54  Rats were housed in a secure conventional rodent facility, on a 12-h reversed light-dark cycle. The surgical procedures and post-operative animal care have been described in detail (Ramsey et al., 2010), but the essential steps are included here. Animals received prophylactic enrofloxacin (Baytril; 10 mg/kg, s.c., Associated Veterinary Purchasing; AVP, Langley, Canada) for three days prior to SCI surgery. On the day of surgery, anesthesia was induced with ketamine hydrochloride (Vetalar®; 70 mg/kg, i.p., University of McGill Animal Resources Centre, Montreal, Canada) and medetomidine hydrochloride (Domitor®; 0.5 mg/kg, i.p., AVP). Enrofloxacin (10 mg/kg, s.c.), buprenorphine (Temgesic®; 0.02 mg/kg, s.c., U. of McGill) and ketoprofen (Anafen®, 5 mg/kg, s.c., AVP) were administered pre-operatively. After the skin at the surgical site was shaved, scrubbed, and treated with iodine, the animal was placed in the prone position. The spinal cord was exposed via a midline incision in the skin and superficial muscles, and blunt dissection of the muscles overlying the C8-T3 (T3 transection) or T8-T11 (T10 transection) vertebrae. At the T2-T3 intervertebral space or following a small laminectomy at T9, connective tissues were removed, the dura was opened and the spinal cord was completely transected with surgical scissors. Complete transection was verified by the clear separation and retraction of the cut ends of the cord, visualized under the surgical microscope. After hemostasis was achieved, the muscle and skin were closed with continuous, 4-0 Vicryl sutures, and interrupted 4-0 Prolene sutures, respectively.  55  2.3.2  Post-operative animal care Animals received warmed Lactated Ringer’s solution (5 ml, s.c.) and recovered in  a temperature-controlled environment (Animal Intensive Care Unit, HotSpot for Birds, Los Angeles, CA). Anesthesia was reversed with atipamezole hydrochloride (Antisedan; 1 mg/kg, s.c., Novartis, Mississauga, Canada). Enrofloxacin (10 mg/kg, s.c.), buprenorphine (0.02 mg/kg, s.c.), and ketoprofen (5 mg/kg, s.c.) were administered once per day for three days following SCI. Home cages for animals with complete T3 SCI were fitted with rubber matting (under woodchips, to facilitate movement), low-reaching water bottles and food scattered on the cage bottom to encourage foraging (Ramsey et al., 2010). Animals were supported with an enriched diet, including meal replacement shake (Ensure; Abbott, Saint-Laurent, Canada), nutritive transport gel (Charles River), fruit, cereals, commercially available rat treats and kibble (LabDiet, Rodent Diet 5001). The bladder was manually expressed 3-4 times daily until spontaneous bladder function returned (about 10 days post-injury). Animal health was formally monitored daily for the first two weeks after SCI and every two days thereafter, using objective criteria to assess body weight, activity level, social behaviour, healing at the surgery site and clinical signs of morbidity.  2.3.3  Survival times Animals survived for one or three months after SCI. In our initial experiments  examining SCI-induced hypertrophy in the DRG (data in Fig. 2.1-2.3,2.5), T3 sham- and spinal cord-injured animals survived for three months. In all subsequent experiments  56  (data in Fig. 2.4, 2.6-2.8), T3 and T10 sham and spinal cord-injured animals survived for one month following surgery.  2.3.4  Intrathecal capsaicin injection At 48 hours (early) or 28 days (late) after SCI, animals were anesthetized with  inhalant isoflurane (AErrane®, AVP; 5% induction, 2-3% maintenance). The skin overlying the lumbar enlargement of the spinal cord was shaved, scrubbed and treated with iodine. The lumbar enlargement was exposed via midline incision in the skin and superficial muscles, blunt dissection of deeper muscles, and a midline laminectomy. Polyurethane tubing (PU-30; Instech, Plymouth Meeting, USA) was introduced subdurally and 50µg of capsaicin (Sigma-Aldrich Inc., St. Louis, USA) in 10µl 50% dimethyl sulfoxide (DMSO; Sigma) was injected intrathecally. Control animals received 50% DMSO only, at 28 days after SCI. The muscle and skin were closed with sutures (40 Vicryl and 4-0 Prolene, respectively).  2.3.5  Cardiovascular assessment Thirty days after SCI, a PU-30 cannula was implanted into the left carotid artery  in all animals for continuous beat-to-beat blood pressure recording. Arterial cannulation was performed under isoflurane anesthesia and the cannula was tunneled subcutaneously to exit the skin dorsally, through small incision at the base of the skull. The cannula was filled with a lock solution of 1:10 heparin (Hepalean®, AVP) and 5% dextrose in Lactated Ringer’s.  57  Two hours after carotid cannulation, the cannula was connected to a fluid-filled pressure transducer (SP844, MEMScAP, Norway). Animals were conscious and the cannula was long enough to permit them to move freely in a cage during cardiovascular assessment. Beat-to-beat arterial pressure was monitored using PowerLab and Chart™ 5 for Windows (ADInstruments, Colorado Springs, USA). When blood pressure was stable (typically 5-10 minutes after connecting the cannula to the transducer), baseline blood pressure was recorded over five minutes. The severity of CRD-induced AD was examined using a protocol that is wellestablished in our laboratory (Krassioukov and Weaver, 1995a). A pediatric silicone balloon-tipped catheter (Fr10; Coloplast, Denmark) was inserted into the rectum and secured to the tail. After stabilization of arterial pressure, the colon was distended via inflation of 2 ml of air, over 10 seconds. Distension was maintained for one minute; upon deflation of the balloon, arterial pressure was allowed to recover over 10 minutes. Blood pressure was recorded during two episodes of AD in each animal, with a minimum of 10 minutes of recovery intervening.  2.3.6  Tissue processing and immunohistochemistry Animals were euthanized with an overdose of chloral hydrate (1 g/kg, i.p.) and  perfused through the heart with room temperature phosphate-buffered saline (PBS) followed by cold 4% paraformaldehyde (PF). Spinal segments and DRGs were removed, post-fixed in 4% PF for 12 hours and cryoprotected in 20% sucrose in 0.1M phosphate buffer for ≥24 hours. Tissue was embedded in Tissue Tek (Fisher Scientific, Ottawa,  58  Canada), frozen over liquid nitrogen, sectioned on a cryostat at 16 µm (DRG) or 20 µm (cord), thaw-mounted on to glass slides and stored at -80ºC. For immunohistochemistry, slides were incubated in 10% normal donkey serum in PBS plus Triton X-100 (0.1%) for 20 min. I used five antibodies to delineate different subsets of nociceptors: these included antibodies raised against TRPV1 (Neuromics, Edina, MN; 1:2,000), the ionotropic ATP purinoceptor P2X3 (Millipore, Billerica, MA; 1:1,000), Substance P (Neuromics, 1:1,000), the glycoprotein isolectin B4 (IB4; Neuromics; 1:2,000), the glial cell line-derived neurotrophic factor family receptor α 3 (GFRα3; Neuromics; 1:500). Subsets of non-nociceptive sensory neurons were identified by expression of the vanilloid transient receptor potential vanilloid-2 (TRPV2; Abcam, Cambridge, UK; 1:200), parvalbumin (Millipore, Etobicoke, Canada; 1:1,000), stagespecific embryonic antigen-4 (SSEA-4; Stem Cell Technologies, Vancouver, Canada; 1:100), and heavy neurofilament (NF200; Millipore, 1:500). Pan-neuronal markers – microtubule-associated protein-2 (MAP-2; Abcam, 1:5:000) or β-III-tubulin (Neuromics; 1:500) – were used to label all neuronal profiles in the DRG. All primary antibodies were applied in PBS plus Triton X overnight. After three 15-minute washes in PBS, secondary antibodies raised in donkey and conjugated to Cy3 (Jackson ImmunoResearch, West Grove, USA), Alexa 488 (Invitrogen, Eugene, USA), or AMCA (7-amino-4-methylcoumarin-3-acetic acid; Jackson ImmunoResearch) were applied at 1:200-1:400, in PBS-Triton X for 2 hours. Epifluorescent images of DRGs were captured with an Axioplan 2 microscope (Zeiss, Jena, Germany) with a digital camera (Q Imaging, Burnaby, Canada) and Northern Eclipse software (Empix Imaging Inc., Mississauga, Canada). Confocal images of TRPV1-positive axons in the spinal cord  59  were captured on a spinning disk confocal microscope (an inverted Zeiss AxioObserver Z.1 equipped with an AxioCam CCD camera). All images for each antigen used for quantitative analysis were captured at identical imaging settings.  2.3.7  Cardiovascular data analysis Systolic and diastolic blood pressures (SAP and DAP) were obtained from  maxima (max) and minima (min) respectively of beat-to-beat blood pressure recordings. Mean arterial pressure (MAP) was calculated as 1/3max+2/3min and heart rate (HR) was calculated from inter-beat interval. Prior to data analysis, raw beat-to-beat blood pressure data were examined in the Chart view; false max/min readings created by muscle spasm (large, high-frequency deflections such as those appearing in Figure 8) were manually eliminated for each animal. Data were analyzed using SigmaPlot (SPSS Inc., Ashburn, USA). In SigmaPlot, raw pressure and HR values were averaged over one second, such that measurements represent a one second average, not a single beat. For each animal, baseline cardiovascular parameters represent the average of at least three minutes of recording time. The CRD-evoked changes in SAP and HR represent the average of two consecutive distensions for each animal.  2.3.8  Image analysis All images were analyzed using SigmaScan Pro 5.0 (SPSS Inc.). For analysis, L4  and L5 DRGs were pooled for each animal (denoted as L4/L5 throughout) and L6 and S1 DRGs were pooled for each animal (L6/S1 throughout). The size-frequency distributions of sensory neurons in the DRG were determined using recursive translation (Rose and  60  Rohrlich, 1988), which converts neuronal profiles in section to the cellular population from which they were drawn (as described previously; Ramer et al., 2001). In five randomly-selected sections from each DRG, all neuronal profiles were traced manually to create an artificial overlay. The average intensity and feret diameter of each object (profile) identified by the overlay was then measured automatically. For proportional frequency measurements, the threshold intensity for expression was set manually for each image. Size-frequency and intensity-frequency distributions were generated to examine SCI-induced shifts in afferent size and TRPV1 signal intensity, respectively. In the spinal cord, measurements of the distribution and intensity of TRPV1positive axons in the dorsal horn were taken from tiled confocal projected z-stacks of the L4/L5 and L6/S1 dorsal horn in cross section. Terminal density was measured as a function of depth at two sites in the dorsal horn (mid and medial) as described previously (Ramer et al., 2001; Ramer et al., 2004; MacDermid et al., 2004; Scott et al., 2005). Images were passed through a Laplacian omnidirectional edge-detection filter, which optimizes the signal-to-noise ratio and corrects for variations in background staining. Terminal profiles in filtered images were selected with a threshold overlay. In order to give all immunopositive pixels equal weight regardless of brightness, the threshold overlay was treated as a new image. Density measurements for each animal represent average density at each depth across five sections. Density of TRPV1-positive axons in the spinal parasympathetic nucleus was measured from confocal images of the L6/S1 dorsal grey commissure (DGC). These images were filtered through horizontal and vertical Sobel edge-detection filters, with the results added through image math prior to thresholding. Density was measured in the  61  spinal parasympathetic nucleus by selecting an area of grey matter that was centered on the midline, immediately ventral to the dorsal corticospinal tract and rostral to the central canal. Density measurements for each animal represent average density across 3-5 sections.  2.3.9  Statistics For cardiovascular data, baseline parameters and stimulus-evoked changes were  compared among groups using a one-way analysis of variance (ANOVA). The HolmSidak Test was used for pair-wise comparisons when significant differences were detected. For size distribution data from the DRG, Kolmogorov−Smirnov (K-S) goodness-of-fit tests were used to determine whether neuronal cumulative size-frequency distributions differed between groups (sham- versus spinal cord-injured). Proportions of neurons labeled with each antigen were compared using Student’s t-test. Density of TRPV1-positive terminals in the dorsal horn of sham- and spinal cord-injured animals was compared using a one-way ANOVA on ranks, followed by Dunn’s test to detect pair-wise differences. Density of TRPV1-positive terminals in the DGC of sham-injured and SCI rats was compared between groups using Student’s t-test. In assessing the effects of early and late intrathecal capsaicin on TRPV1 axons in the DGC, a one-way ANOVA was used. For all physiological and anatomical analyses, group averages represent 5-7 animals per group, results are expressed as mean±standard error of the mean (SEM), image and data analyses were performed in a blinded fashion (using coded image and data files) and P values less than .05 were considered significant.  62  2.4 2.4.1  Results Complete high-thoracic spinal cord injury provoked hypertrophy in sensory  neurons that express the capsaicin receptor. Neuronal phenotype, of which size is a defining characteristic, is governed in large part by trophic infleunces of target tissues. These undergo profound changes following SCI, including atrophy of skeletal muscle and bladder hypertrophy. Therefore, I examined changes in the size distribution of all sensory neurons in lumbosacral DRGs three months after T3 complete SCI. Neuronal profiles in the L4/L5 DRG were labelled with neuron-specific β-III-tubulin (Fig. 2.1A) and size-frequency analysis was performed following recursive translation (Fig. 2.1B). There was a subtle but statistically significant right-shift (i.e., hypertrophy) in the size distribution of all sensory neurons in DRGs of animals with T3 SCI.  63  Figure 2.1 High-thoracic (T3) spinal cord injury provoked hypertrophy of neurons in the dorsal root ganglion (A) βIII-tubulin immunohistochemistry illustrating neuronal profiles in the L5 DRG. (B) Size-frequency distribution of pooled L4/L5 DRG neurons, reconstructed from profile distributions using recursive translation. There was a small but significant rightward shift in size-frequency distribution in animals with T3 SCI (P<0.05, K-S goodness-of-fit test). Scale bar = 70 µm.  64  I next sought to determine which population(s) of sensory neurons responded to T3 SCI. I began by examining subsets of small-diameter sensory neurons in the L4/L5 DRG (Figure 2.2). Peptidergic (NGF-sensitive) nociceptors were identified by expression of Substance P (SP) (Fig. 2.2A), while non-peptidergic nociceptors were identified by binding of the glycoprotein isolectin B4 (IB4, from Bandeiraea simplicifolia) and the ionotropic ATP purinoceptor P2X3 (Fig. 2.2B,C). At three months after T3 SCI, neither of these minimally-overlapping populations of nociceptors exhibited hypertrophy after T3 SCI, suggesting that sensory hypertrophy caudal to T3 SCI was selective to another subset of sensory neurons. In rats, TRPV1-expression occurs in subsets of both peptidergic and nonpeptidergic nociceptors (Tominaga et al., 1998). TRPV1 expression also defines a subpopulation of neurons which are neither P2X3-expressing/IB4-binding nor neuropeptide-expressing (Michael and Priestley, 1999). When I examined the size distribution of TRPV1-positive neurons in the L4/L5 DRG, I found a pronounced hypertrophy in DRGs from animals with T3 SCI (Fig. 2.2D). TRPV1-positive cells increased in size after SCI, but their proportional frequency did not change (Fig. 2.2D, inset). Approximately 50% of TRPV1-positive sensory neurons also express the arteminspecific glial cell line-derived neurotrophic factor family receptor α 3 (GFRα3) (Baloh et al., 1998; Bennett et al., 2006). GFRα3-positive sensory neurons (of which >80% express TRPV1 (Bennett et al., 2006), also exhibited hypertrophy, in the absence of change in proportional frequency, in DRGs distal to T3 SCI (Fig. 2.2E).  65  66  Figure 2.2. High-thoracic (T3) spinal cord injury induced selective hypertrophy of sensory neurons expressing the capsaicin receptor (TRPV1) and the artemin receptor (GFRα3) (A) Substance P (SP) –positive DRG neurons and their size-frequency distributions. (B) IB4-binding DRG neurons. (C) P2X3-positive DRG neurons. (D) TRPV1-positive DRG neurons. (E) GFRα3-expressing DRG neurons, known to express TRPV1. The overall proportions of immunopositive neurons did not change for any subpopulation. Scale bar = 50 µm. Asterisks indicate P<0.05, K-S goodness-of-fit test.  67  I performed size-frequency analysis on medium-to-large sensory neurons in the same (L4/L5) DRG by examining neurons expressing heavy neurofilament (NF200) (Fig. 2.3). At three months after T3 SCI, there was no evidence of injury-induced hypertrophy in NF200-positive cells, nor did the proportion of NF200-expressing cells change (Fig. 2.3A). In agreement with previous findings (e.g., Yamamoto et al., 2008), I found that the large majority of TRPV1-positive neurons were NF200-negative; only occasional neurons co-expressed TRVP1 and NF200 (Fig. 2.3B, arrow). The extent of TRPV1 and NF200 co-expression did not increase after T3 SCI (Fig. 2.3B). To confirm that SCI-induced hypertrophy was specific to small-diameter DRGs, I also performed size-frequency analysis on three sub-populations of medium-to-large sensory neurons in the L4/L5 DRG (data not shown). Expression of the vanilloid transient receptor potential vanilloid-2 (TRPV2) was used to identify larger sensory neurons that are heat-sensitive but TRPV1-negative (Caterina et al., 1999; Tamura et al., 2005). Proprioceptors and cutaneous mechanoreceptors were labelled with parvalbumin (Celio, 1990) and the stage-specific embryonic antigen-4 (Dodd et al., 1984), respectively. In contrast to SCI-induced hypertrophy in TRPV1-expressing nociceptors, there were no detectable changes in the size distributions of these subpopulations of medium- to-large DRG neurons following T3 SCI.  68  Figure 2.3. Spinal cord injury had no effect on medium-to-large sized DRG neurons expressing heavy neurofilament (NF200) (A) NF200-positive neurons did not undergo SCI-induced hypertrophy, nor did the proportion of neurons expressing NF200 change. (B) Hypertrophy of TRPV1-expressing DRG neurons was not accompanied by increased co-localization of TRPV1 and NF200. Arrow: DRG neuron immunopositive for both TRPV1 and NF200. Scale bar = 70 µm.  69  2.4.2  Spinal cord injury -induced hypertrophy was most pronounced in  lumbosacral sensory ganglia Since my initial observations were made in DRGs far distal to T3 SCI (Fig. 2.12.3, L4/L5 DRG), I examined the extent of SCI-induced hypertrophy in TRPV1-positive neurons at different rostro-caudal levels (Fig. 2.4). I examined tissue harvested one month after T3 durotomy (sham injury) or SCI (Figures 2.4-2.6). Of the levels examined, SCI-induced hypertrophy was restricted to ganglia below the injury: TRPV1-positive afferents did not exhibit hypertrophy in the T1 DRG, despite their proximity to the injury site (Fig. 2.4A). Equidistant but caudal to SCI (in the T5 DRG), the size distribution of TRPV1-positive sensory neurons was right-shifted relative to sham-injured controls (Fig. 2.4B). However, the effect of SCI was most dramatic in lumbosacral DRGs, remote from the site of injury. TRPV1-positive cells exhibited pronounced hypertrophy in both L4/L5 and L6/S1 DRGs (Fig. 2.4C,D). The rightward shift in size distribution was most dramatic in L6/S1 DRGs, containing afferents innervating the urinary bladder and the distal colon (Nadelhaft and Booth, 1984).  70  71  Figure 2.4. Capsaicin-sensitive dorsal root ganglion neurons increased in diameter caudal to, but not rostral to high-thoracic spinal cord injury (A)-(D). Size-frequency distributions of TRPV1-positive neurons from a rostral DRG (T1) and caudal (T5, L4/L5, L6/S1) DRGs. The increase in size is the most pronounced in the most caudal ganglia. Asterisks indicate P<0.05, K-S goodness-of-fit test.  72  2.4.3  Capsaicin-sensitive afferents hypertrophied and upregulated the capsaicin  receptor after spinal cord injury. While there was no change in the proportion of sensory neurons expressing TRPV1 after T3 SCI, this does not negate an intracellular upregulation of TRPV1. To investigate this possibility, I examined the intensity of TRPV1 expression in lumbosacral DRGs. Sections of L4/L5 and L6/S1 DRG from sham-injured controls and animals with T3 SCI were processed for TRPV1 immunohistochemistry and examined at the microscope by a blinded investigator, with all imaging parameters (exposure time, gain and offset) set at constant levels across groups. With identical immunohistochemical processing and imaging, there was an obvious increase in intensity of TRPV1 expression in DRGs from animals with SCI (Fig. 2.5). This was confirmed through blind quantification: when images were processed to generate size-intensity distributions of TRPV1-positive neuronal profiles, the intensity distribution in DRGs from animals with T3 SCI was right-shifted relative to sham-injured controls (Fig. 2.5A,B). Akin to SCIinduced hypertrophy, the shift in TRPV1 signal intensity was most pronounced in the L6/S1 DRGs.  73  Figure 2.5. Capsaicin-sensitive neurons in the dorsal root ganglion exhibited increased TRPV1 signal intensity following high-thoracic spinal cord injury. (A) Size-intensity scatter plots of TRPV1-positive DRG neurons showing a marked upward scatter with T3 SCI. The increase in TRPV1 intensity was particularly pronounced in L6/S1 DRGs. There was a significant increase in signal intensity after T3 SCI for both L4/L5 DRGs and L6/S1 DRGs (K-S goodness-of-fit tests on cumulative intensity-frequency distributions). Scale bar = 50 µm.  74  2.4.4  Injury-induced hypertrophy was modest after low-thoracic spinal cord  injury The large majority of work in animal models of SCI examines injury-induced changes after low-thoracic SCI (Ramsey et al., 2010). For example, data describing SCIinduced changes in DRGs with bladder-projecting afferents (L6/S1), in the absence of changes in DRGs with somatic afferents (L4/L5), were derived from rats with lowthoracic SCI (Zvarova et al., 2005). I therefore examined the size distribution of TRPV1positive afferents in lumbosacral DRGs from animals with T10 complete SCI (Fig. 2.6). One month after T10 complete SCI, TRPV1-positive neurons did not exhibit hypertrophy in L4/L5 DRGs; size distribution of TRPV1-expressing neurons was similar between animals with T10 SCI and animals with durotomy only performed at the same level (sham; Fig. 2.6A). In the L6/S1 DRGs, there was a small but significant right-ward shift in the size distribution of TRPV1-positive neurons after T10 SCI (6B). Interestingly, hypertrophic changes induced by low-thoracic SCI were much less dramatic than those triggered by T3 SCI (compare Figs. 2.6B and 2.4D). This was surprising, given that both injuries induce hind limb paralysis and lower urinary tract (LUT) dysfunction.  75  Figure 2.6. Low-thoracic (T10) spinal cord injury elicited only modest changes in size of the most caudal capsaicin-sensitive dorsal root ganglion neurons (A) No difference in size distributions of TRPV1-expressing DRG neurons in L4/L5 DRGs. (B) There was a small but significant rightward shift in the size-frequency distribution of L6/S1 DRG neurons (P<0.05, K-S goodness-of-fit test), but this was much less dramatic than that which occurred after T3 SCI (see Fig. 2.6).  76  2.4.5  Dramatic somatic hypertrophy in capsaicin-sensitive afferents was not  reflected in plasticity of their central projections Multiple studies have demonstrated that severe SCI triggers intraspinal sprouting of nociceptors (Krenz and Weaver, 1998; Weaver et al., 2001) and that sprouting of CGRP-expressing afferents in the dorsal horn is correlated with severity of AD (Krenz et al., 1999; Cameron et al., 2006). SCI also prompts a subset of DRG neurons, those expressing the pituitary adenylate cyclase activation peptide (PACAP) to expand their territory in the lumbosacral dorsal horn in segments containing visceral circuitry (L1, L2, L6 and S1) (Zvarova et al., 2005). Since PACAP and CGRP partially co-localize with TRPV1 in DRG neurons (Moller et al., 1993), I measured the density of TRPV1expressing terminals in the L4/L5 and L6/S1 dorsal horn, the central projections of afferents exhibiting the most pronounced hypertrophy after T3 SCI. Working from tiled mosaics of confocal z-stack projections (Fig. 2.7A), I detected a slight but significant increase in density of TRPV1-positive terminals in the superficial laminae at two locations in the L4/L5 dorsal horn. Density of TRVP1-positive projections was increased superficially, in lamina I of the lateral dorsal horn and lamina II-III of the medial dorsal horn. There was no evidence of TRPV1-positive afferents sprouting into deeper laminae after SCI. There was also no difference in density of TRPV1-expressing afferents in the L6/S1 dorsal horn between sham-injured animals and animals with T3 SCI (Fig 2.7B). These results indicate that the CGRP- and PACAP –positive axons which were previously shown to sprout after SCI are not those which contain TRPV1. I also examined density of TRPV1-positive projections to the dorsal grey commissure (DGC) in the L6/S1 spinal cord, a region that receives input from visceral  77  afferents, including those in the distal colon (Hou et al., 2009; Morgan et al., 1981). Densitometric analysis demonstrated that there was no effect of SCI on projections to the DGC (Fig. 2.7B). Since I was working from cross-sections of spinal cord, I did not attempt quantitative measurements of TRPV1-positive projections to the lateral parasympathetic preganglionic nucleus (which is rostro-caudally periodic in the lumbosacral spinal cord; Morgan et al., 1981). There were no qualitatively-apparent changes in the density of TRPV1-positive projections to parasympathetic preganglionic neurons after T3 SCI.  78  79  Figure 2.7. Hypertrophy of capsaicin-sensitive afferents caudal to high-thoracic spinal cord injury was not accompanied by pronounced plasticity of their spinal projections (A) In the L4/L5 dorsal horn, there was a small but significant increase in TRPV1positive axon density in the medial (med.) and lateral (lat.) parts of the most superficial laminae in animals with T3 SCI (boxed regions are shown enlarged). There was no evidence of TRPV1-positive axon extension into deeper laminae. (B) In the L6/S1 cord, there were no differences between sham-injured or T3 SCI animals in the dorsal horn, or in the dorsal grey commissure (DGC, boxed image enlarged to the right), the terminal field of the visceral medial collateral pathway. Asterisks: P<0.05, Student’s t-test. Scale bars = 200 µm.  80  2.4.6  Intrathecal capsaicin attenuated colo-rectal distension-induced autonomic  dysreflexia The dramatic effects of T3 SCI on TRPV1-expressing afferents in L6/S1 DRGs prompted me to examine their contribution to the development of AD (Fig. 2.8). I administered 50 µg of capsaicin in a single intrathecal injection at the L4 spinal cord, 28 days after T3 SCI (“late Cap.”) or 48 hours after T3 SCI (“early Cap.”). The “early” group was included to test the possibility of a contribution of TRPV1 to the emergence of AD: since TRPV1-positive sensory neurons exhibit spontaneous activity de novo following SCI (Bedi et al., 2010), I hypothesized that early capsaicin treatment might have particularly pronounced effects on development of AD. Vehicle injections (“Veh.”) were performed 28 days after SCI. Consistent with previous findings, capsaicin injection produced a permanent degeneration of spinally-projecting TRPV1-positive axons (Fig. 2.8A; Yaksh et al., 1979). Efficacy of capsaicin was confirmed via densitometric measurements within the DGC (Fig. 2.8A), which demonstrated a rapid and sustained depletion of TRPV1positive projections. At 30 days after T3 SCI, carotid cannulae were implanted for beatto-beat blood pressure measurements (Fig. 2.8B). Capsaicin injection had no effects on blood pressure or heart rate at rest (Fig. 2.8C). Animals that received capsaicin after T3 SCI exhibited much less severe AD in response to colo-rectal distension (CRD) (Fig. 2.8C). Refuting my hypothesis, there was no effect of differential timing of capsaicin injection following SCI: early and late capsaicin treatment produced equivalent reductions in CRD-induced hypertension and bradycardia. In both groups, the CRD-  81  evoked change in SAP was reduced by approximately 50% relative to vehicle-treated animals, and CRD-induced bradycardia was dramatically attenuated.  82  83  Figure 2.8. Intrathecal capsaicin attenuated colo-rectal distention (CRD) -induced autonomic dysreflexia in animals with T3 spinal cord injury (A) A single intrathecal bolus of capsaicin (10 µl of 5mg/ml in 50% DMSO) resulted in permanent degeneration of spinally-projecting TRPV1-positive axons. Quantification shows TRPV1 axon density in the dorsal grey commissure (DGC). Asterisk indicates significant difference between vehicle-treated T3 SCI animals and both capsaicin-treated groups (P<0.05, one-way ANOVA). Scale bar = 200 µm. (B) Beat-to-beat changes in blood pressure in response to colo-rectal distension (horizontal bars) from animals treated with intrathecal vehicle (Veh.), early capsaicin (48 hours following T3 SCI, early Cap.), and late capsaicin (48 hours prior to physiological recording, 28 days post-T3 SCI, late Cap.). (C) Quantitative cardiovascular responses to CRD in vehicle and capsaicintreated rats, 30 days post-SCI. Capsaicin treatment, whether administered 48h or 28d after SCI, mitigated CRD-induced increases in systemic arterial pressure (SAP) and decreases in heart rate (HR). Resting SAP and HR were unaffected by intrathecal capsaicin. Asterisks: P<0.05, one-way ANOVA .  84  2.5  Discussion These experiments demonstrate injury-induced hypertrophy in a specific subset  of TRPV1-positive sensory neurons caudal to SCI. The response was most pronounced in lumbosacral DRGs and after high-thoracic SCI. Finally and notably, eliminating the central projections of TRPV1-expressing axons after T3 SCI via intrathecal capsaicin injection had pronounced mitigating effects on the severity of CRD-induced AD. Here I discuss the potential mechanisms of this SCI-provoked hypertrophy, its relationship to level of injury and the relevance for sensory-autonomic dysfunction following SCI.  2.5.1  Spinal cord injury-induced hypertrophy was restricted to a subset of  capsaicin-sensitive neurons The selectivity of sensory neuron hypertrophy following SCI provides important clues about the underlying trophic mechanism. While the results demonstrate T3 SCIinduced hypertrophy of TRPV1-positive DRG neurons, it is clear that this is not universally true: that is, there must be a specific subset of TRPV1 neurons which responds to SCI by increasing in size. This assertion is based on the fact that a rightward shift in size-frequency distribution is detectable in the analysis of all (βIII-tubulinlabeled) neurons (Fig. 2.1), but not for IB4/P2X3 or SP subpopulations (Fig. 2.2), each of which partially co-localizes with TRPV1 (Tominaga et al., 1998). Thus, the TRPV1expressing neurons of interest express neither standard peptidergic nor non-peptidergic nociceptor markers (Michael and Priestley, 1999); these would have been included in the analysis of βIII tubulin-positive profiles (i.e., all DRG neurons), but omitted from those  85  involving SP, P2X3 and IB4-positive cells. Thus, hypertrophy is largely or entirely restricted to a unique population of TRPV1-expressing neurons. Analysis of neurons expressing GFRα3 (~85% of which co-express TRPV1; (Bennett et al., 2006) provides a further clue to the identity of this specific subset of nociceptors. The hypertrophic neurons are most likely sensitive to artemin, a GDNF family neurotrophic factor. Previous experiments have shown that peripheral overexpression of artemin in mouse keratinocytes not only leads to hypertrophy of DRG neurons, but also to upregulation of TRPV1 mRNA and increased capsaicin sensitivity (Elitt et al., 2006). Artemin is therefore a likely candidate for inducing hypertrophy following SCI. While there are no data describing expression of endogenous artemin after SCI, the pattern of injury-induced hypertrophy is suggestive.  2.5.2  Spinal cord injury-induced hypertrophy was particularly dramatic in  caudal ganglia, far distal to injury The ganglia in which size changes occurred also give cause for speculation on the molecular underpinnings of this response to injury. The inflammatory response to SCI is prolonged and well-characterized (Alexander and Popovich, 2009). The character of the inflammatory milieu evolves over time and is accompanied by production of cytokines and trophic factors which could act on the ganglia attached to the cord to induce hypertrophy. However, injury-induced hypertrophy was absent or modest in T1 and T5 DRGs, respectively, which argues against a central role for a factor produced at the site of SCI.  86  Pronounced hypertrophy occurred far distal to the site of T3 SCI, in lumbosacral DRGs. Interestingly, TRPV1-positive somata were enlarged in DRGs supplying predominately somatic (L4/L5) and predominately visceral (L6/S1) peripheral targets. This scenario represents a departure from the phenotypic changes that are restricted to visceral afferents after low-thoracic injury (Kruse et al., 1995; Qiao and Vizzard, 2002). TRPV1-expressing sensory neurons innervate both skeletal muscle and smooth muscle in a variety of targets, including the colon and the bladder (Skryma et al., 2011; Malin et al., 2011; Everaerts et al., 2008). The structure and function of these target tissues are dramatically altered following SCI. Paralyzed skeletal muscle undergoes rapid and profound atrophy (Qin et al., 2010). In contrast, the smooth (detrusor) muscle of the bladder becomes hypertrophic over time following supraconal SCI, due to the combined effects of detrusor hyperactivity and detrusor-sphincter dyssynergia (Yoshimura, 1999). Less is known about remodeling of smooth muscle in the lower gastrointestinal (GI) tract after SCI. However, given the pronounced changes in lower GI function after SCI, including increased transit time that manifests clinically as constipation (Brading and Ramalingam, 2006), it seems reasonable to speculate that intrinsic smooth muscle of the distal colon also undergoes inactivity-induced remodeling following injury. In sum, the diverse peripheral targets of TRPV1-positive lumbosacral DRG afferents exhibit either atrophy (or inactivity-induced changes) or hypertrophy after SCI. It would be very surprising indeed if such a variety of peripheral changes did not result in alterations in neurotrophic factor expression.  87  2.5.3  Injury-induced hypertrophy was not accompanied by pronounced  intraspinal sprouting The pronounced somatic hypertrophy in TRPV1-expressing afferents was not accompanied by an equally dramatic expansion of their central terminals in the spinal dorsal horn. Previous findings report that there is no change in spinal distribution of TRPV1-positive projections after low-thoracic SCI (Cruz et al., 2008), and I observed a relatively minor increase in density that was restricted to the superficial laminae of the L4/L5 dorsal horn after T3 SCI. While somatic hypertrophy and axonal sprouting do not necessarily occur together, most trophic factors are capable of eliciting both. For example, in bladder afferents caudal to low-thoracic SCI, bladder- and/or spinal cordderived NGF is thought to mediate somatic hypertrophy (Yoshimura et al., 2006; Seki et al., 2002; Yoshimura, 1999), and intraspinal NGF also contributes to SCI-induced sprouting of peptidergic nociceptors in the dorsal horn (Krenz et al., 1999; Cameron et al., 2006). The absence of robust sprouting of TRPV1 axons in the spinal cord suggests that the stimulus is peripheral, possibly in the DRG itself. An intraganglionic source of artemin, for example, might be satellite cells in the DRG: artemin is expressed in Schwann cells, which are phenotypically similar to satellite cells, and is upregulated in Schwann cells after peripheral nerve injury (Baloh et al., 1998).  2.5.4  Injury-induced hypertrophy was more pronounced after high-thoracic than  low-thoracic spinal cord injury The differential effects of complete SCI at T3 and T10 are interesting, given the similarities in many aspects of functional outcome. For example, T3 and T10 SCI induce  88  hind limb paralysis and bladder dysfunction that is grossly similar: the bladder is initially areflexic and requires manual emptying until reflexive micturition is restored. The issue of LUT function is certainly relevant, since TRPV1-positive sensory neurons are critically involved in physiological bladder function (Araki, 2011) and appear to contribute to bladder dysfunction after SCI (Cruz et al., 2008).TRPV1-expressing afferents also mediate a number of pathological phenomena in the LUT, including bladder pain and overactive bladder (Eid, 2011; Kissin and Szallasi, 2011); recent findings suggest that these afferents also participate in pelvic organ cross-sensitization (Asfaw et al., 2011). However, SCI-induced changes were not restricted to bladder afferents after T3 SCI, since hypertrophy was also apparent in L4/L5 DRGs. One explanation might be that signals from the hypertrophic bladder contribute to injuryinduced hypertrophy, but the predominant trigger is present in high-, but not low-thoracic SCI. One notable difference in high- versus low-thoracic SCI that has been identified lies in the immune response to injury. In mice, immune suppression induced by SCI is level-dependent, such that mice with T3 SCI exhibit impaired antibody synthesis and elevated splenic norepinephrine, neither of which develop in mice with T9 injury (Lucin et al., 2007). If level-dependent immune suppression also occurs in rats with SCI, the inflammatory response in the DRG may also vary with level of injury, influenced by systemic activity of the immune system. The limited data that are available describe immune cell infiltration in DRGs caudal to T8 SCI (McKay and McLachlan, 2004): in this study, intra-ganglionic immune cell density was highest in DRGs closer to the lesion site (i.e., greater in T12 DRGs than in L6 DRGs). This pattern does not seem to fit with  89  my results, but may be different after T3 SCI; alternatively, other factors modifying the environment of the DRG (such as satellite cells activation) may not vary in step with the local immune response. The most dramatic difference in the functional outcomes of T3 and T10 SCI is the development of AD after the former, but not the latter injury. In AD, sensory stimulation evokes sympathetic contractions of vascular smooth muscle. It is not insignificant that artemin is developmentally expressed in vascular smooth muscle where it acts as a guidance cue for sympathetic (and probably also sensory) axons (Honma et al., 2002). In essential hypertension, chronic constriction of the blood vessels induces maladaptive remodeling of the vasculature (Rizzoni et al., 2009; Rizzoni et al., 2007; Rehman and Schiffrin, 2010). Remodeling resulting in an increase in media-lumen ratio can occur via different mechanisms, including rearrangement of the same wall material around a narrowed lumen (eutrophic remodeling) or vascular smooth muscle cell growth (hypertrophic remodeling) (Intengan and Schiffrin, 2001). While less is known about the effects of intermittent or episodic hypertension on vascular structure, one recent study indicates that carotid intima-media thickness is increased in individuals with SCI (MatosSouza et al., 2009). The possibility of artemin upregulation in vascular smooth muscle has yet to be explored, but may be sufficient to explain the more pronounced hypertrophy of TRPV1/GFRα3 neurons following T3 SCI than injury at T10. Injury-induced changes in the vasculature need not be systemic: the heterogeneity of arterial changes among vascular beds has been described in other pathological conditions, such as a high-fat diet (Bhattacharya et al., 2008). A differential SCI-induced effect in different vascular beds is  90  another consideration, and may relate to differential hypertrophy in sensory neurons at different levels of the neuraxis.  2.5.5  Capsaicin-sensitive afferents contributed substantially to induction, but not  development, of colo-rectal distension-induced autonomic dysreflexia Selective elimination of TRPV1-expressing afferents in the spinal dorsal horn dramatically reduced the severity of AD. Peripheral projections of TRPV1-positive afferents in the DRGs innervating the rectum and distal colon are found in smooth muscle and the mucosa and are mechanosensitive and/or chemosensitive (Berthoud et al., 2001; Ward et al., 2003; Lynn and Blackshaw, 1999). The capsaicin-sensitive, mechanosensitive subset are known to respond to CRD (Brierley et al., 2005). Intrathecal capsaicin administered injected at L4 eliminated the central projections of TRPV1positive spinal colonic afferents, which constitute approximately 50% of the lumbosacral colonic DRG neurons (Brierley et al., 2005). This is reflected in my data, demonstrating that CRD following intrathecal capsaicin injection still activates a subset of mechanosensitive afferents to elicit AD, although AD is dramatically reduced in severity (Fig. 2.8). I hypothesized that early elimination of TRPV1-expressing afferents would have even more pronounced effects on AD (than late capsaicin, administered at 28 days postSCI). This premise was based in part on recent findings demonstrating spontaneous activity arising in the soma develops in DRGs after SCI (Bedi et al., 2010). There are several striking similarities between the patterns of de novo spontaneous activity and hypertrophy that emerge following SCI. Both phenomena develop caudal, but not rostral  91  to SCI, and are most pronounced in distal DRGs, remote from the site of injury. Both occur in nociceptors, and most intriguingly, a high percentage of afferents that exhibited spontaneous activity after T10 SCI were capsaicin-sensitive (Bedi et al., 2010). Cumulatively, these findings suggest that SCI has specific effects on TRPV1-expressing primary afferents. In bladder afferents, SCI-induced somatic hypertrophy is accompanied by increased excitability, including reduced thresholds for activation (Yoshimura, 1999). If hypertrophy is an anatomical surrogate for spontaneous activity after SCI, injuryinduced ongoing activity in TRPV1-positive neurons might be more even more pronounced and/or prevalent after T3 SCI. However, early and late capsaicin treatment had equivalent effects on AD (Fig. 2.8B). This suggests that TRPV1-positive afferents are involved in triggering AD, but not in its development over time following SCI. Given the evidence for spontaneous activity in capsaicin-sensitive afferents, this is a finding with important clinical implications, since it suggests that spontaneous activity in primary afferents does not sensitize central sensory circuits to promote the development of AD. It will be important to examine the extent of spontaneous activity after high thoracic SCI to verify this assertion.  2.5.6  Conclusion Previous work has identified numerous mechanisms that might contribute to  induction and progression of AD, and the list of putative mechanisms includes injuryinduced changes in the vasculature and multiple components of the spinal sensorysympathetic circuitry caudal to SCI (Krassioukov et al., 1999; Krenz and Weaver, 1998;  92  Krenz et al., 1999; Brock et al., 2006; McLachlan and Brock, 2006). In terms of sensory plasticity, prior findings demonstrate that severity of AD is closely correlated to the extent of intraspinal nociceptor sprouting (Cameron et al., 2006). However, this is the first study to demonstrate AD mediated by a specific subset of afferents that exhibit pronounced somatic, but only slight central, injury-induced plasticity. Given the array of pronounced changes in peripheral targets of sensory neurons after SCI, it is not surprising that they respond to injury. Plasticity occurring outside the CNS may represent a new and more accessible target for limiting sensory-autonomic dysfunction following SCI.  93  Chapter 3: Cellular responses in lumbar dorsal root ganglia after highand low-thoracic spinal cord injury  3.1  Synopsis The dorsal root ganglia (DRGs) at the L4/L5 level contain somatic sensory  neurons innervating the skin and muscles of the hind limbs. In these experiments, I examined L4 and L5 DRGs from animals that survived for one month after T3 or T10 SCI. I was interested in injury-induced changes in the local environment of the sensory neuron. I hypothesized that SCI would activate glial and immune cells in the DRG, promote angiogenesis and trigger ingrowth of sympathetic ganglionic axons, which are normally excluded from the immediate environs of sensory somata. I found that SCI stimulated angiogenesis in the DRG and stimulated sympathetic ganglionic axons to grow into the DRG and among sensory neurons. Intriguingly, other effects of SCI varied between T3 and T10 injury. T3 SCI provoked activation of satellite cells, the primary glial constituent of the DRG, while T10 SCI did not. Similarly, T3 SCI provoked macrophage activation and mast cell accumulation, while T10 did not. These findings demonstrate that injury to the thoracic spinal cord has pronounced effects on distal ganglia and that high-thoracic and low-thoracic SCI are heterogeneous in their effects on sensory neurons.  3.2  Introduction Driven by the holy grails of achieving regeneration and overcoming paralysis in  the mammalian central nervous system (CNS), spinal cord injury (SCI) research has  94  evolved in a remarkably CNS-focused manner. Attention has been devoted to almost every component of the injured spinal cord, including neuronal, axonal, glial, inflammatory, vascular and extracellular matrix elements. In contrast, remarkably little is known about SCI-induced effects on peripheral ganglia. For example, apart from transient atrophy of cholinergic terminals in pelvic ganglia (Takahara et al., 2007), there are no data describing injury-induces changes in sympathetic ganglia. This seems remarkable, given the dramatic remodeling that has been observed after lesions of the white rami (or the proximal lumbar trunk), which denervate sympathetic ganglionic neurons (McLachlan and Brock, 2006). There are some data describing changes in DRGs caudal to SCI, the bulk of which describe changes in afferents projecting to the hypertrophic bladder. Sensory neurons innervating the bladder of animals with SCI exhibit hypertrophy and increased excitability in vitro (Kruse et al., 1995; Yoshimura and de Groat, 1997). They also upregulate a number of neuropeptides, including galanin and vasoactive intestinal peptide (Zvarova et al., 2004; Zvarova et al., 2005; Vizzard, 2006). Interestingly, these studies tend to report SCI-induced changes in L6-S1 (visceral) DRGs, but not in L4-L5 (somatic) DRGs (Qiao and Vizzard, 2002). There are only a few reports of SCI-induced changes in other DRGs (i.e., those not connected to the bladder). These report increased expression of mRNA for NGF in thoracic DRGs (Brown et al., 2007) and increased accumulation of T-cells and macrophages in thoracolumbar DRGs (McKay and McLachlan, 2004). Both of these studies report changes in ganglia caudal to SCI, with the most pronounced responses occurring in DRGs proximal to the site of injury.  95  Recent data demonstrate spontaneous activity in a subpopulation of sensory neurons caudal to SCI (Bedi et al., 2010). In this instance, the incidence of spontaneous activity was most pronounced in lumbar L4/L5 DRGs, far distal to the site of injury. I have also characterized neuronal changes in L4/L5 DRGs, namely, hypertrophy that was specific to TRPV1-expressing neurons and that was more pronounced after T3 than T10 SCI (Chapter 2). Injury to the peripheral process of DRGs induces a host of well-characterized changes, including glial activation, immune cell recruitment and ingrowth of sympathetic ganglionic axons (Klusakova and Dubovy, 2009). Although it is now well-recognized that activation of satellite glia and immune cells crucially regulate sensory neuron function (Scholz and Woolf, 2007; Gosselin et al., 2010), there are no data describing how these cellular elements of the DRG respond to SCI. In these experiments, I examined satellite cells, immune cells, blood vessels and sympathetic ganglionic axons in the L4/L5 DRG after T3 and T10 SCI. I hypothesized that SCI would activate glial and immune cells in the DRG, promote angiogenesis and trigger sympathetic sprouting, and that these effects would be more pronounced after T3 SCI than after T10 SCI.  3.3 3.3.1  Materials and methods Spinal cord injury surgery and post-operative care The procedures for complete transection of the T3 and T10 spinal cord, sham-  injury and post-operative animal care were identical to those described previously (Ramsey et al., 2010) (Chapter 2).  96  3.3.2  Tissue processing and immunohistochemistry Animals were euthanized and perfused with fixative as described in Chapter 2.  DRGs were removed, post-fixed in 4% PF for 12 hours and cryoprotected in 20% sucrose in 0.1M phosphate buffer for ≥24 hours. Tissue was embedded in Tissue Tek (Fisher Scientific, Ottawa, Canada), frozen over liquid nitrogen, sectioned on a cryostat at 16 µm, thaw-mounted on to glass slides and stored at -80ºC. For immunohistochemistry, slides were incubated in 10% normal donkey serum in PBS plus Triton X-100 (0.1%) for 20 min. Satellite glial cells in the DRG were labeled using primary antibodies raised against neuroglycan 2 (NG2; Millipore; 1:1000), glial fibrillary acidic protein (GFAP; Dako; 1:1000) and the RIP antibody (Millipore; 1:500). Macrophages in the DRG were identified by their expression of ionized calcium binding adaptor molecule 1 (Iba-1; Milipore, 1:500). Sympathetic ganglion axons were labeled with an antibody raised against tyrosine hydroxylase (TH; Millipore, 1:1000) and capillaries were labeled with an antibody raised against the rat endothelial cell antigen-1 (RECA-1; Millipore; 1:500). Primary antibodies were applied in PBS plus Triton X overnight. After three 15minute washes in PBS, secondary antibodies raised in donkey and conjugated to Cy3 (Jackson ImmunoResearch, West Grove, USA) or Alexa 488 (Invitrogen, Eugene, USA) were applied at 1:200 in PBS-Triton X for 2 hours. Epifluorescent images of TH and RECA-1 were captured with an Axioplan 2 microscope (Zeiss, Jena, Germany) with a digital camera (Q Imaging, Burnaby, Canada) and Northern Eclipse software (Empix Imaging Inc., Mississauga, Canada). Confocal images of NG2, GFAP, RIP, Iba-1 were captured on a spinning disk confocal microscope (an inverted Zeiss AxioObserver Z.1  97  equipped with an AxioCam CCD camera). All images for each antigen used for quantitative analysis were captured at identical imaging settings.  3.3.3  Toludine blue staining Toludine stock solution was prepared by dissolving 1g Toludine blue (Sigma-  Aldrich Inc., St. Louis, USA) in 100ml 70% ethanol. Slides were stained in fresh Toludine blue working solution (5ml stock solution plus 45ml 1% NaCl, pH~2.3) for two minutes and washed in dH2O (three times, 15 min. each). Slides were dehydrated through three changes of ethanol (10 dips each), cleared in xylene and and mounted in Permount (Fisher). Mast cells were counted at the microscope. Although mast cells were not encountered in the DRG, they were present in the spinal nerve at the distal pole of the ganglia where the grey ramus joins the mixed nerve. Mast cells were therefore counted in the first mm of DRG-attached spinal nerve. Brightfield images for display purposes were captured with an upright microscope (Leica DM5000 B; Leica, Concord, Ontario) with a digital camera and integrated imaging software (both from Leica).  3.3.4  Image analysis All fluorescent images were analyzed using SigmaScan Pro 5.0 (SPSS Inc.). For  analysis, L4 and L5 DRGs were pooled for each animal (denoted as L4/L5 throughout). Three-to-five sections were randomly selected from each DRG for analysis: that is, sections were selected under the ultraviolet filter, without examining expression of the antigen of interest. For toluidine blue staining, the first five sections with an adequate length of spinal nerve were used for mast cell counting.  98  In order to measure glial density in the cell layer of the DRG, images (projected 2 micron-thick optical z-stacks of the L4/L5 DRG) of GFAP, NG2, and Iba-1 were processed with horizontal and vertical Sobel edge-detection filters, with the results added through image math prior to thresholding. The area occupied by thresholded cells was divided by the area of the DRG cell layer in each image. Capillary density in the cell layer of the DRG was examined from images of RECA-1, via manual tracing of RECA1-positive capillaries. In order to measure density of sympathetic axons, a grid was placed over the DRG such that grid lines were orthogonal to the long axis of the DRG and the lines were spaced 100 µm apart. The number of intersections between grid lines and sympathetic axons was counted and normalized to the short axis distance at that point of the DRG.  3.3.5  Statistics Proportional area (or mast cell number) in sham- and spinal cord-injured animals  was compared using a one-way analysis of variance (ANOVA), followed by Dunn’s test to detect pair-wise differences. For all analyses, group averages represent 5-7 animals per group, results are expressed as mean±standard error of the mean (SEM), image and data analyses were performed in a blinded fashion (using coded image and data files) and P values less than .05 were considered significant.  99  3.4 3.4.1  Results High- but not low-thoracic spinal cord injury activated satellite cells in  lumbosacral dorsal root ganglia Satellite cells enclose sensory somata to create a microenvironment around each neuron and critically regulate sensory neuron activity (Jasmin et al., 2010). Satellite cells respond vigorously to injury or inflammation of the peripheral process of DRG neurons by upregulation of a number of proteins, including GFAP (Stephenson and Byers, 1995; Woodham et al., 1989). Satellite cells also express NG2 (Rezajooi et al., 2004). SCI induces upregulation of NG2 (and GFAP) in astrocytes neighbouring the injury site (Busch et al., 2010), but their expression in distal DRGs has not been characterized after CNS injury. I used GFAP and NG2 immunohistochemistry to examine satellite cell activation in L4/L5 DRGs caudal to T3 and T10 SCI (Fig. 3.1,3.2, respectively). The pattern of GFAP-expression in L4/L5 DRGs from sham-injured controls (Fig. 3.1A) was consistent with previous findings (Zhou et al., 1999). Only a few satellite cells in each section of the DRG expressed GFAP in their processes, encircling sensory neurons (Fig. 3.1A). Occasional GFAP-positive outlines of satellite cells bodies were visible in each section (Fig. 3.1A). There was a dramatic increase in GFAP expression in L4/L5 DRGs harvested one month after T3 SCI (Fig. 3.1B,D). GFAP-positive processes were abundant in the cell layer of the DRG and encircled many sensory neurons. In contrast, GFAP expression subsequent to T10 SCI was not elevated above control levels (Fig. 3.1C). Densitometric measurements of GFAP expression in the cell layer of the DRG revealed that GFAP expression in animals with T10 SCI was comparable to that in sham-  100  injured controls (Fig. 3.1D). GFAP expression among sensory neurons was higher after T3 SCI than after T10 SCI. NG2 expression was detected on satellite cells surrounding a small subset of sensory neurons in sham-injured controls (Fig. 3.2A). The only other structure that was NG2-positive in the parenchyma of the DRG was the node of Ranvier (Fig. 3.2A, inset); NG2 expression was restricted to the nodal gap, as reported previously (Martin et al., 2001). Analogous to GFAP expression, NG2 immunoreactivity was dramatically increased in L4/L5 DRGs from animals with T3 SCI (Fig. 3.1B,D). One month after T3 SCI, an increased proportion of DRG neurons were encircled by NG2-positive satellite cells. The SCI-induced increase in NG2 reactivity did not occur after T10 SCI (Fig. 3.1C,D). The density of NG2 expression in the L4/L5 DRG caudal to T10 SCI was indistinguishable from that in sham-injured controls. Therefore, the effects of SCI on glial activation in the DRG are level-dependent: despite the fact that I was examining DRGs located far distal to the site of thoracic SCI, high-thoracic SCI provokes activation of satellite cells, while low-thoracic SCI does not.  101  102  Figure 3.1 Satellite cell expression of glial fibrillary acidic protein (GFAP) increased in lumbar dorsal root ganglia after high thoracic (T3), but not low-thoraic (T10) spinal cord injury (A) Few satellite cells were GFAP-positive in sham-injured L4/L5 DRGs. (B) GFAP expression in the L4/L5 DRGs increased dramatically after T3 SCI; GFAP-positive processes encircled many sensory neurons. (C) GFAP expression was comparable to that in sham-injured controls following T10 SCI. (D) Quantification of GFAP density among sensory neurons somata revealed a statistically significant increase (asterisk; one-way ANOVA) in L4/L5 DRGs following T3, but not T10 SCI. GFAP expression after T3 SCI was also significantly increased compared to T10 SCI (no symbol). P=0.06 reflects the comparison between T10 SCI and sham-injured controls. Scale bar = 50 µm.  103  104  Figure 3.2 Satellite cell expression of neuroglycan 2 proteoglycan (NG2) increased in lumbar dorsal root ganglia after T3, but not T10 spinal cord injury (A) Reminiscent of the pattern of GFAP expression, few satellite cells were NG2positive in L4/L5 DRGs from sham-injured controls. (B) NG2 expression in L4/L5 DRGs increased substantially after T3 SCI and many sensory neurons were surrounded by NG2-positive processes. (C) T10 had no effect on NG2 expression in L4/L5 DRGs. (D) Quantification of NG2 density in the cell layer of the DRG demonstrated a statistically significant increase (asterisk; one-way ANOVA) in L4/L5 DRGs following T3, but not T10 SCI. Scale bar = 50 µm.  105  3.4.2  High- but not low-thoracic spinal cord injury stimulated immune cells in  and around the lumbar dorsal root ganglion Iba-1 is a macrophage/microglia-specific calcium binding protein (Imai et al., 1996) and microglia upregulate Iba-1 when activated by nerve injury (Ito et al., 1998) and by direct application of cytokines (Imai and Kohsaka, 2002). I used Iba-1 immunohistochemistry to examine the response of macrophages in the lumbar DRG after high- and low-thoracic SCI (Fig. 3.3). Iba-1-expressing cells were abundant in the cell layer of L4/L5 DRGs from sham-injured controls (Fig. 3.3A), indicating that a considerable population of macrophages patrols the DRG under physiological conditions. The density of Iba-1 expression among sensory neurons increased after T3 SCI (Fig. 3.3B,D); the increase may be a product of upregulation, proliferation or (most likely) both processes occurring in parallel. In step with glial products GFAP and NG2, Iba-1 expression did not increase after T10 SCI (Fig. 3.3C). As observed in sham-injured controls, Iba-1-positive cells were distributed throughout the cell layer of DRGs caudal to T10 SCI, and the density of Iba-1 expression was similar between sham and T10 SCI groups (Fig. 3.3D). Mast cells are best-known for their roles in allergic/hypersensitivity responses and are rapid instigators of inflammation. Correspondingly, they are normally prominent at or near points of entry, in the skin, in the mucosa of the airways and the gastrointestinal tract (Galli et al., 2005). However, they are also resident in most vascularized tissues, including nerves, albeit at low density under physiological conditions (Mizisin and Weerasuriya, 2011). I examined mast cell density in L4/L5 DRGs one month after T3 and T10 SCI. Mast cells appeared red-purple (metachromatic,  106  appearing as small, black cells, Fig. 3.4) after toludine blue staining. In sham-injured controls, mast cells were scarce in the spinal nerve immediately distal to the DRG (Fig. 3.4A). Many more mast cells were encountered at the same location in animals with T3 SCI (Fig. 3.4B): while mast cells did not invade the DRG parenchyma, they were present at the distal pole of the DRG in all tissue sections from animals with T3 SCI. Conversely, mast cell accrual was not evident in DRGs from animals with T10 SCI (Fig. 3.4C). Mast cell numbers were counted at the microscope using coded slides but the inter-group differences were obvious: quantification revealed that T3, but not T10 SCI provoked a dramatic increase in mast cell density in the spinal nerve adjacent to L4/L5 DRGs (Fig. 3.4D).  107  108  Figure 3.3 High-thoracic, but not low-thoracic spinal cord injury stimulated macrophages in the lumbar dorsal root ganglion (A) Iba1-labeled macrophages in a sham-injured L5 DRG. (B) The density of Iba1immunoreactive cells and their processes increased in L4/L5 DRGs following T3 SCI (an L5 DRG is shown). (C) Iba1 density was unchanged following T10 SCI. (D) Quantification of Iba1 density revealed a statistically significant increase (asterisk) in L4/5 DRGs following T3, but not T10 SCI. Scale bar = 50 µm.  109  110  Figure 3.4 Mast cells accumulated in the spinal nerve, immediately distal to dorsal root ganglia, after high- but not low-thoracic spinal cord injury (A) Few if any mast cells (small black cells) were present in the spinal nerve just distal to the L4/L5 DRG in sham-operated animals. (B) There was a dramatic increase in mast cell numbers following T3 SCI. (C) Mast cells in the spinal nerve were rare following T10 SCI. (D) Quantification of mast cell density (# mast cells per mm of DRG-adjacent) in the spinal nerve revealed a significant increase (asterisk, one-way ANOVA) in L4/5 DRGs, but only following T3 SCI. Scale bar = 70 µm.  111  3.4.3  Capillary density in the dorsal root ganglion increased after high- and low-  thoracic spinal cord injury The DRG is richly-supplied with blood vessels; as described in Chapter 1, capillary density may vary with inflammation. I examined the density of blood vessels in DRGs caudal to T3 and T10 SCI using the pan-endothelial cell-specific antibody RECA1 (Duijvestijn et al., 1992). RECA-1-labeling identified the capillary network within the cell layer of the DRG. In sham-injured controls, RECA-1-positive vessels were encountered throughout the cell layer of the DRG, between sensory neurons (Fig. 3.5A). The density of RECA-1-positive capillaries amongst sensory somata of the L4/L5 DRGs was increased after both T3 (shown; Fig. 3.5B) and T10 SCI. RECA-1-expressing profiles were manually traced and density expressed as proportional area within the cell layer of the DRG (Fig. 3.5C). This quantification demonstrated a clear increase in density of RECA-1-positive capillaries within the DRG after SCI, indicating that SCI at both levels provoked intra-ganglionic angiogenesis.  3.4.4  Sympathetic axons grew into lumbar dorsal root ganglia caudal to high- and  low-thoraic spinal cord injury After SCI, sympathetic ganglionic neurons (SGNs) caudal to injury undergo important functional changes, reflected in enhanced contractile vascular responses to nerve activity that are pre-junctionally mediated (McLachlan and Brock, 2006). They are also likely to undergo significant local remodelling within sympathetic ganglia, as they do following decentralization (McLachlan and Brock, 2006; Zaidi and Matthews, 1999). Given the glial, immune cell and vascular effects of thoracic SCI in lumbosacral DRGs, I  112  used TH immunohistochemistry to investigate the density of sympathetic axons in the cell layer of lumbosacral DRGs caudal to T3 and T10 SCI. In DRGs from sham-injured animals, sympathetic axons penetrating the cell layer were associated with blood vessels or, more rarely, TH-positive DRG neurons (Fig. 3.6A). While they coursed through the fibre layer of the DRG, they did not extend out of the fibre layer in large numbers to surround sensory neurons. Despite the presence of blood vessels throughout the cell layer, some supplied by sympathetic axons even in sham-injured controls (not shown), I did not observe sympathetic axons “jumping off” blood vessels to grow among sensory neurons in the absence of SCI. In contrast, DRGs from some animals with T3 (Fig. 3.6B) and T10 SCI were teeming with sympathetic axons. Sympathetic axons exited the fibre layer to grow among sensory neurons, sometimes in considerable numbers. In some sections, small THpositive axon profiles were encountered throughout the cell layer of the DRG. Notably, sympathetic axons encircled sensory neurons to form pericellular baskets. These baskets or “nests” were encountered in cup and spool morphologies (Fig. 3.6B, arrows), characteristic of Cajal’s early description following peripheral nerve injury. In some instances, sympathetic axons were juxtaposed to neuronal membranes (Fig. 3.6C); in others, the sympathetic axons encircling sensory neurons were clearly growing among satellite cells, and not in direct contact with the neuron (Fig. 3.6C, bottom). Sympathetic axons were also present on capillaries surrounding sensory neurons (Fig. 3.6B). Projections of TH-expressing axons often emanated from capillaries into the cell layer and extended processes among sensory neurons (not shown). I quantified density of sympathetic axons in the DRG by counting grid crossings of axons penetrating the DRG,  113  taking the grey ramus as zero (Fig. 3.6C). While the extent of sympathetic sprouting was variable among animals, quantification confirmed that both T3 and T10 SCI provoked significant sympathetic ingrowth to the L4/L5 DRG.  114  115  Figure 3.5 Both high- and low-thoracic spinal cord injury induced angiogenesis in lumbar dorsal root ganglia (A) RECA1 labeling of endothelial cells reveals the capillary network among neurons in the cell body layer of the L5 DRG from a sham-injured control. (B) Capillary network density was increased in L5 DRGs following both T3 SCI (shown) and T10 SCI (not shown). (C) The density of RECA1-positive capillaries was significantly increased (asterisks) in L4/5 DRGs following both T3 and T10 SCI; differences were detected via one-way ANOVA. Scale bar = 70 µm.  116  117  Figure 3.6 Both high- and low-thoracic spinal cord injury provoked sympathetic sprouting in lumbar dorsal root ganglia (A) In DRGs from sham-operated animals, sympathetic axons were only present around blood vessels; TH-positive DRG neurons were rare. (B) SCI results in invasion of the DRG by sympathetic axons. In some cases the sympathetic sprouts grew among satellite cells (bottom arrow in B); in others sympathetic axons were juxtaposed to neuronal membranes (arrow, right, in B); still in other cases, sympathetic axons were present on capillary networks surrounding neurons (left arrow in B). (C) High-magnification images of sympathetic axons (red) apposing DRG somata (top) or among satellite cells (bottom). (D) Densitometric analysis of sympathetic axons in the DRG reveals significant ingrowth (asterisks) in L4/L5 DRGs following both T3 and T10 SCI. Scale bars: 70 µm in A, 50 µm in C.  118  3.5  Discussion These data reveal multiple effects of high- and low-thoracic SCI on lumbar  DRGs. Despite being far-removed from the site of injury, L4/L5 DRGs clearly respond to SCI. SCI stimulated angiogenesis in the lumbar DRG and prompted TH-expressing sympathetic ganglionic axons to grow into the DRG and among sensory neurons. Intriguingly, other effects of SCI varied between T3 and T10 injury. T3 SCI provoked satellite cell activation, indicated by increased density of GFAP and NG2 expression in the cell layer of the DRG, while T10 SCI had no effect on GFAP or NG2 in the DRG. Similarly, Iba-1 expression in the DRG increased after injury at T3 but not T10, and mast cells accumulated in the spinal nerve immediately distal to the DRG after T3 but not T10 SCI. Here I discuss the potential ramifications of these injury-induced changes.  3.5.1  Activation of satellite cells caudal to high-thoracic spinal cord injury Activation of satellite cells in the DRG, indicated by an increase of both GFAP  and NG2 expression, succeeded T3 but not T10 SCI. The pattern of glial activation after T3 SCI was consistent with previous reports of GFAP and NG2 expression after peripheral nerve injury (Otoshi et al., 2010): GFAP/NG2-positive satellite cells were scarce in DRGs from sham-injured controls and relatively abundant in the cell layer following T3 SCI. More sensory neurons were encircled by GFAP/NG2-positive glia after T3 SCI; since we know from ultrastructural analyses that every sensory neuron is surrounded by a satellite cell (Hanani, 2005), this pattern represents an upregulation of GFAP/NG2. Importantly, I did not perform a direct comparison between peripheral nerve  119  injury and T3 SCI, so I cannot comment reliably on the relative levels of glial activation under both circumstances. In this study, we examined only the extent of glial activation after SCI, but the potential functional ramifications merit some discussion.The emerging roles of satellite (and spinal) glia in induction and maintenance of neuropathic pain has prompted one group to coin the term “gliopathic” pain (Ohara et al., 2009). Some of the most persuasive evidence for the role of satellite cells in pain is derived from models of trigeminal nerve (oral or facial) pain (Takeda et al., 2009). Satellite cells in the trigeminal ganglia respond to peripheral injury or inflammation by upregulating glial fibrillary acidic protein (GFAP) and proinflammatory cytokines such as interleukin-1β (IL-1β; Stephenson and Byers, 1995; Li et al., 2005). In this model, intra-ganglionic glial production of IL-1β has been implicated in nociceptor hyper-excitability and spontaneous activity (Takeda et al., 2007; Takeda et al., 2008). Satellite cells in DRGs also upregulate inflammatory cytokines under pathological conditions. For example, when activated by local application of autologous nucleus pulposus, an animal model of lumbar disc herniation, satellite cells in lumbar DRGs expressed tumour necrosis factor alpha (TNF-α), which may contribute to concomitant pain (Otoshi et al., 2010). While it remains to be seen whether satellite cells produce cytokines in the DRG following T3 SCI, it represents a means by which satellite cells might profoundly modify afferent input to the injured spinal cord.  120  3.5.2  Activation of immune cells caudal to high-thoracic spinal cord injury I found that immune cell activation in the DRG also varied with level of SCI:  activation of macrophages, monitored via expression of Iba-1, increased following T3 but not T10 SCI. Microglia are estimated to represent 10-20% of the total cell population in the adult CNS (Graeber, 2010) but may constitute a larger proportion of cells in the DRG; Iba-1-expressing macrophages were present throughout the cell layer, at considerable density in DRGs from uninjured controls. The pattern of Iba-1 expression in my sham-injured controls appears to be largely consistent with expression of major histocompatibility complex (MHC) II following sham SCI (McKay and McLachlan, 2004). In that study, low-thoracic SCI provoked an increase in expression of MHC II that was most pronounced in proximal DRGs (i.e., MHCII expression increased in the T12 DRG, but not the L6 DRG, following T8 SCI). This pattern differs from my observations in the L4/L5 DRG, which exhibited pronounced macrophage activation after T3 but not T10 SCI. Hu & McLachlan also observed increased MHC II expression in L4/L5 DRGs, so it is likely to be the difference in injury level that is relevant. As described in Chapter 2, immunological effects of SCI are level-dependent in mice; for example, T3 SCI increased levels of splenic norepinephrine (NE), an effect that did not accompany T10 SCI (Lucin et al., 2007). Another important difference between high- and low-thoracic SCI is the development of autonomic dysreflexia (AD) in the former, but not the latter scenario. Lymphoid organs are innervated by sympathetic ganglionic axons, which enter via the vasculature and release NE which acts on lymphocytes and antigen-presenting cells to influence leukocyte activation, cytokine  121  production and immune cell trafficking (Rice et al., 2001; Sanders and Straub, 2002). While there has been no clear link between AD and immune function to date, there is certainly the potential for sympathetic discharge caudal to high-thoracic SCI to modulate immune function. Like satellite cells, both macrophages and mast cells influence sensory neuron function (Moalem and Tracey, 2006). Macrophage-derived cytokines have welldocumented, sensitizing effects on sensory neurons (Zimmermann, 2001). The bestcharacterized cytokines acting on sensory neurons after neurotrauma are tumour necrosis factor α (TNF-α) and interleukin-1β (IL-1β) (Miller et al., 2009). Both are rapidly and dramatically upregulated in the DRG after peripheral nerve injury (Uceyler et al., 2007); while their expression in DRGs caudal to SCI has not been investigated, gliosis and macrophage activation make it likely that both will be present at elevated levels. Mast cells are normally associated with nociceptor sensitization in the periphery (Ren and Dubner, 2010). It is difficult to speculate on the functional relevance of mast cells in the spinal nerve, but their presence suggests that they are responding to a DRG-derived cue that is present after T3, but not T10 SCI.  3.5.3  Angiogenesis in dorsal root ganglia caudal to high- and low-thoracic spinal  cord injury As described in Chapter 1, the cell layer of the DRG is supplied by a dense capillary bed. The density of RECA-1-positive capillaries increased after both T3 and T10 SCI. To my knowledge, this is the first demonstration of injury-induced angiogenesis in the DRG. However, based on our knowledge of inflammation-induced  122  angiogenesis in cancer (Ono, 2008), this could be considered a predictable event. Recent data from an animal model of disk herniation suggest that angiogenesis occurs in the L5 DRG in response to nucleus pulposus application (Miyoshi et al., 2011), although it is the upregulation of cues for angiogenesis rather than vascular sprouting that is demonstrated. In addition to its density, the vasculature of the DRG is notable for its permeability. Capillaries in the DRG are relatively leaky; that is, they are fenestrated and lack tight endothelial cell junctions (Jacobs et al., 1976). In contrast to the environment of neurons in the CNS which is generally regarded as immune-privileged, the DRG parenchyma is not protected by a blood-brain barrier. This was conclusively demonstrated experimentally via intravenous injection of autologous antibodies (immunoglobulins; IgG) against horse radish peroxidase, which diffused into the parenchyma of the DRG, but not the brain or spinal cord (Azzi et al., 1990). Provided that capillaries in the DRG retain their leakiness after SCI, the consequence of angiogenesis could be increased access of blood-borne immune cells to the DRG.  3.5.4  Sympathetic sprouting caudal to high- and low-thoracic spinal cord injury Both T3 and T10 SCI provoked ingrowth of noradrenergic sympathetic  ganglionic axons to the cell layer of the DRG. Sprouting of sympathetic axons in the DRG is a notorious consequence of peripheral nerve injury (Chung et al., 1993; McLachlan et al., 1993; Ramer et al., 1999). Sympathetic sprouting, including the formation of terminal arborizations or baskets among sensory neurons in the dorsal root ganglion (DRG), results from ligation or transection of peripheral nerves, and has been associated with neuropathic pain resulting from these and other injuries (Ramer et al.,  123  1998; Ramer and Bisby, 1999; Deng et al., 2000). The causal link between sympathetic sprouting and heightened/spontaneous activity in sensory neurons remains debatable: however, recent findings have kept the debate alive. In a tyrosine hydroxylase reporter mouse, neurons with sympathetic baskets accounted for a large proportion of sponataneous activity in sensory neurons after damage to their peripheral axons (Xie et al., 2011). In addition, in vitro stimulation of sympathetic axons in DRGs isolated after peripheral nerve injury enhanced spontaneous activity in sensory neurons (Xie et al., 2010). These findings raise the possibility that, despite being relatively rare, sympathetic baskets in DRGs caudal to SCI may have local, excitatory effects on sensory neurons.  3.5.5  Conclusion Although they were named for glue, the glial (and other non-neuronal) elements  of the nervous system have long been recognized as more than an inert cement in which neurons are embedded (Allen and Barres, 2009). In the sensory nervous system, the past two decades have witnessed a surge in exploration of roles for non-neuronal cells in sensory neuron excitability (Scholz and Woolf, 2007; Gosselin et al., 2010). Here I report pronounced reactions of non-neuronal components of the DRG that are specific to highthroacic SCI. I also document SCI-induced angiogenesis and ingrowth of sympathetic axons in distal DRGs. The ganglionic effects of SCI merit further investigation: SCIinduced plasticity in the peripheral nervous system has the potential contribute to sensory and autonomic dysfunction after SCI.  124  Chapter 4: Cyclooxygenase-2 contributes to vascular hypersensitivity following spinal cord injury  4.1  Synopsis One of the best-characterized effects of spinal cord injury (SCI) on the blood  vessels is hypersensitivity to sympathetic neurotransmitters and their mimetics. Blood vessels below the level of SCI in both humans and experimental animals exhibit enhanced vasoconstriction in response to norepinephrine and phenylephrine (PE). The mechanism(s) underlying the enhanced response to PE caudal to SCI are not well understood. In these experiments, I used in vitro myography to test the hypothesis that cyclooxygenase (COX) activity contributes to PE hypersensitivity following SCI. I found that superior mesenteric arteries from rats with SCI were hypersensitive to PE and hyposensitive to ACh. Using COX antagonists, I found that COX-2 activity contributes to PE hypersensitivity. I also found that COX-2 was upregulated in superior mesenteric arteries from rats with SCI. These findings implicate COX-2 activity in heightened splanchnic vasoconstriction after SCI and suggest that COX-2 might be a novel therapeutic target for treatment of autonomic dysreflexia (AD).  4.2  Introduction Blood pressure regulation at the level of the resistance vessels is achieved through  a balance of neural activity, hormonal control, and intrinsic properties of the vascular smooth muscle and endothelium. Spinal cord injury (SCI) perturbs this balance. Severe SCI isolates sympathetic preganglionic neurons (SPNs) in the thoracolumbar spinal cord  125  from their regulatory centres in the brainstem (Llewellyn-Smith et al., 2006). The cardiovascular ramifications are most pronounced when SCI occurs at or above T6, isolating SPNs that govern the splanchnic circulation. This scenario creates the conditions for autonomic dysreflexia (AD), episodic hypertension elicited by sensory input that enters the central nervous system (CNS) caudal to SCI. AD affects a large proportion of individuals with high SCI; in addition to the acute danger of hypertensive crises (Vaidyanathan et al., 2011; Eltorai et al., 1992; Calder et al., 2009), the chronic effects of AD may damage or alter the cardiovascular end organs (Steins et al., 1995) (Chapter 5). Although clinical awareness of the dangers of AD is gradually increasing (McGillivray et al., 2009; Jackson and Acland, 2011), the debate surrounding its underlying mechanisms continues to flourish (Weaver, 2002). We know that SCI triggers plasticity in several divisions of the central and peripheral nervous system that regulate sympathetic tone (Chapters 2-3) (Krassioukov and Weaver, 1996; Krassioukov and Weaver, 1995b; Krenz and Weaver, 1998; Krenz et al., 1999; McLachlan and Brock, 2006). While injury-induced changes in the nervous system are obviously relevant, characterizing effects of SCI on the other half of the cardiovascular equation – in the target organs – is of equal importance. Although less is known about effects of SCI on cardiovascular end-organs, abnormalities in cardiac and vascular structure and function have been reported in individuals with SCI (Nash et al., 1991; Hopman et al., 2002; Arnold et al., 1995). Recent experiments in animal models reveal marked vascular dysfunction within the critical splanchnic vascular bed. One pioneering group has been leading work in this  126  area, and has reported that mesenteric arteries from rats with chronic high-thoracic SCI are hypersensitive to the α1-adrenoreceptor agonist phenylephrine (PE) (Brock et al., 2006). In this study, PE hypersensitivity was attributed in part to impaired action of the neuronal norepinephrine transporter (which also takes up PE). While this is an intriguing finding, other mechanisms may contribute to arterial PE hypersensitivity caudal to SCI. A prime candidate is dysfunction of the vascular endothelium, the single layer of endothelial cells lining the vascular system (Khazaei et al., 2008b). In addition to forming a semi-permeable “blood-tissue” barrier (Irie and Tavassoli, 1991), vascular endothelial cells sense changes in hemodynamic forces and respond by releasing vasoactive substances. The classical and predominant endothelium-derived vasodilator is nitric oxide (NO), which is released in response to shear stress and chemical stimuli, including acetylcholine (ACh) (Furchgott and Zawadzki, 1980; Pohl et al., 1986). Most available evidence argues against endothelial dysfunction in people with SCI (Thijssen et al., 2008), but for obvious reasons these data are derived from the peripheral arteries. Nitric oxide production is governed by NO synthase and NO synthesis is impaired in a host of pathological conditions, including diabetes, atherosclerosis, hypertension and cigarette smoking (Kietadisorn et al., 2011). In addition to NO, other endotheliumderived factors play a critical role in dictating vascular tone under both physiological and pathological conditions (Davel et al., 2011). Another family of molecules notoriously linked with cardiovascular disease is the cyclooxygenases. These (COX) enzymes catalyze the rate-limiting step in the synthesis of prostanoids, a family of important biological mediators which includes the prostaglandins, prostacyclin, and thromboxane. In the blood vessels, prostanoids  127  influence vascular tone by promoting local vasodilation or vasoconstriction. They also regulate a variety of other, critical processes, including platelet aggregation, local inflammatory response, and leukocyte-endothelial cell adhesion (Iniguez et al., 2008). Here I used in vitro myography to test the hypothesis that COX activity mediated PE hyper-responsiveness in mesenteric arteries after SCI. I examined PE-mediated vasoconstriction and ACh-induced vasodilation in superior mesenteric arteries from animals with T3 complete SCI. I also examined the effects of COX-inhibitors on PEinduced vasoconstriction. I found that COX-2 inhibitors normalized PE-induced vasoconstriction in arteries from rats with SCI. Vascular immunohistochemistry indicated that COX-2 expression was increased in arteries from rats with SCI relative to age-matched controls, implicating COX-2 and its prostanoid products in PE hypersensitivity following SCI.  4.3 4.3.1  Materials and methods Spinal cord injury surgery and post-operative care The procedures for complete transection of the T3 spinal cord, sham-injury and  post-operative animal care were identical to those described previously (Chapters 2-3) (Ramsey et al., 2010). In these experiments, all rats survived for one month following T3 SCI.  4.3.2  In vitro myography I used in vitro myography to examine vasoconstriction in response to PE and  vasodilation in response to acetylcholine (ACh) in superior mesenteric arteries.  128  Following cardiovascular assessment, the superior mesenteric artery (SMA) was isolated under deep anesthesia. The arteries were immersed in ice-cold physiological salt solution (PSS), cleared of fat and connective tissue, cut into 2 mm rings and mounted isometrically onto a four-channel wire myograph (JP Trading, Aarhus, Denmark). Each ring was immersed in PSS gassed with 95% O2 and 5% CO2. Four arterial rings were prepared for each animal, such that vascular responses for each animal represented an average of four replicates, studied in parallel. Arterial rings were equilibrated for one hour, with the PSS replaced at 15-20 minute intervals. During this time, the resting tension was gradually increased to an optimal value determined from normalization curves for each vessel segment. Tissues were maintained at this level of pre-load for 20-30 minutes. Following equilibration, arterial rings were constricted with potassium chloride (KCl; 80 mM) to ensure viability of the samples and to facilitate normalization of developed force. Subsequent to PSS wash and restoration of basal tension, PE was added in a cumulative manner (1nM to 10µM) to generate concentration-response curves. In order to examine the PE response in the presence of inhibitors of COX enzymes, indomethacin, a non-selective COX inhibitor, SC-560, a COX-1 specific inhibitor, or NS-398, a COX-2 specific inhibitor was added to the bath. All inhibitors were from Sigma-Aldrich (Oakville, Canada) and applied at 100µM. After one hour of equilibration with a COX inhibitor, the PE concentration-response curve was repeated. Assessment of endothelium-dependent vasodilation was performed in arteries from separate groups of animals (i.e., not in those treated with COX inhibitors). Arterial  129  segments were treated with 1µM PE to establish a stable contraction and exposed to cumulative additions of ACh (1nM to 10µM).  4.3.3  Myography data analysis For each arterial ring, raw forces were normalized to force induced by KCl, and  maximum force of contraction (Emax) and half-maximal effective concentration of PE (EC50) were determined before and after addition of the COX inhibitor. Vasodilator responses in each segment were expressed as percent relaxation of PE-induced contraction. GraphPad Prism (GraphPad Software Inc., La Jolla, CA) was used for curvefitting and concentration-response analysis. PE and ACh concentration-response curves for each animal (with response values representing the average of 4 arterial rings) were fitted to a variable slope log[agonist] versus response curve. Maximum force of contraction (Emax) and half-maximal effective concentration of PE and ACh (EC50) were measured for each animal and averaged for each group.  4.3.4  Tissue processing and immunohistochemistry Segments of mesenteric artery immediately adjacent to those used for in vitro  myography as well as segments of descending thoracic aorta were rinsed in PSS, postfixed in 4% paraformaldehyde (PF) for 12 hours and cryoprotected in 20% sucrose in 0.1M phosphate buffer (PB) for 24 hours. Vas deferens was used as positive control tissue; it was harvested from uninjured rats which were perfused through the heart with PBS and 4% PF, post-fixed in 4% PF and cryo-protected in 20% sucrose in 0.1M PB. Arteries and vas deferens were embedded in the same blocks (so that every section  130  contained both arteries from the same animal, plus a section of vas deferens) in Tissue Tek (Fisher Scientific), frozen over liquid nitrogen, sectioned on a cryostat at 6 µm, thaw-mounted on to glass slides and stored at -80ºC. Prior to immunolabeling, slides were coded to preserve blindness. Slides were incubated in 0.1%H2O2 in phosphate-buffered saline (PBS) for 10 min. to quench endogenous peroxidase activity. After washes in PBS (3 washes, 15 min. each), slides were incubated in 10% normal donkey serum in PBS plus Triton X-100 (0.1%) for 20 min. Primary antibody directed against COX-2 (Cayman Chemical, Ann Arbor, US) was applied at 1:200 in PBS plus Triton X overnight. Biotinylated donkey anti-rabbbit secondary antibody (Invitrogen) was applied at 1:500 in PBS-Triton X for 2 hours, with PBS washes before and after. Slides were incubated with Avidin: Biotinylated enzyme Complex (Vectastain Elite ABC Kit; Vector Laboratories, Burlington, Canada) for 30 min., washed in PBS, and incubated with fresh 3,3'-diaminobenzidine (DAB) substrate solution for two minutes. DAB incubation was performed in small batches (≤5 slides) to ensure that development time remained consistent; the development time was determined using the vas deferens. The reaction was stopped with dH2O; slides were dehydrated through three changes of ethanol, cleared in xylene and mounted in Permount (Fisher). Brightfield images were captured with an upright microscope (Leica DM5000 B; Leica, Concord, Ontario) with a digital camera and integrated imaging software (both from Leica). Blindness was preserved throughout imaging and analysis and all images were captured at identical settings.  131  4.3.5  Image analysis COX-2 expression in images of arterial sections (≥6 per animal) was scored by a  blind investigator, using a 4-point scale (1-weakest; 4-strongest). This method has been used previously to provide a semi-quantitative estimate of vascular COX-2 expression (Li et al., 2008). The scale was established after imaging, by selecting images as representative of each level of expression (1-4).  4.3.6  Statistics Student’s t-test was used to compare Emax and EC50 between groups (i.e., between  arteries from sham- and spinal cord-injured rats). A two-way repeated measures analysis of variance (ANOVA) was used to compare the response to COX inhibitors between arteries from sham- and spinal cord-injured rats, and Bonferroni’s test was used for pairwise comparisons. The Wilcoxon rank-sum test was used to compare COX-2 expression in arteries from both groups. In all cases, P values less than 0.05 were considered significant and results are expressed as mean ± standard error of the mean (SEM).  4.4 4.4.1  Results Phenylephrine-induced vasoconstriction was heightened in arteries from  animals with spinal cord injury Previous work demonstrated PE hypersensitivity in second-order mesenteric arteries seven weeks after T4 SCI (Brock et al., 2006). Since I was working with the SMA rather than its daughter resistance branches, I first examined PE-induced vasoconstriction to determine whether it was also potentiated in the SMA after SCI. Raw  132  force recorded in response to PE was normalized internally (i.e., for each arterial ring) and expressed as percentage of contraction induced by 80mM KCl (Fig. 4.1A). The EC50 for PE was smaller in the SMA from animals with SCI (Fig. 4.1B). There was no difference in Emax for PE between arteries from sham-injured animals and animals with SCI (Fig. 4.1C). All of these findings in the SMA are consistent with previous findings in second-order mesenteric arteries (Brock et al., 2006), indicating that high-thoracic SCI has similar effects on the SMA and smaller resistance arteries of the splanchnic bed.  4.4.2  Acetylcholine-induced vasodilation was functional but altered in arteries  from animals with spinal cord injury Endothelium-derived vasodilators oppose the activity of α-adrenoceptor agonists to limit vasoconstriction (Jin et al., 2011). Endothelial-dependent vasodilation is impaired in a number of pathological scenarios, but has not been examined in animal models of SCI. Arteries isolated from animals with T3 SCI and T3 durotomy (sham) were treated with 1µM PE to establish a stable contraction, and then exposed to cumulative addition of ACh. ACh-induced vasodilation was normalized for each arterial segment and expressed as percentage relaxation of PE-induced contraction (Fig. 4.2A). The EC50 for ACh was higher in the SMA from animals with SCI (Fig. 4.2B). There was no difference in the Emax for ACh (Fig. 4.2C). Thus, the SMA was hyposensitive to ACh after T3 SCI, but endothelial-dependent vasodilation was not markedly impaired as it is in animal models of diabetes (Sachidanandam et al., 2009; Khazaei et al., 2008a) and hypertension (Silva et al., 2011).  133  Figure 4.1 Arteries from animals with high-thoracic (T3) spinal cord injury were hypersensitive to phenylephrine (A) Concentration-response curves for PE in mesenteric arteries from animals with T3 durotomy (sham) and T3 SCI. (B) At one month after injury, the EC50 for PE was lower in arteries from animals with SCI. (C) There was no effect of SCI on maximum response to PE (normalized to KCl-induced contraction). Values represent mean±SEM; asterisk indicates P<0.05, Student’s t-test.  134  Figure 4.2 Endothelial-dependent vasodilation was functional, but altered, in arteries from animals with high-thoracic spinal cord injury (A) Concentration-response curves for ACh in mesenteric arteries from animals with T3 durotomy (sham) and T3 SCI. (B) At one month after injury, the EC50 for ACh was higher in arteries from animals with SCI. Values represent mean±SEM; asterisk indicates p<0.05, Student’s t-test.  135  4.4.3  Spinal cord injury-induced phenylephrine hypersensitivity was reversed by  inhibitors of cyclooxygenase-2 In addition to endothelium-derived NO, vasoactive prostanoids produced in the endothelium and the vascular smooth muscle critically regulate local vascular tone under both physiological and pathological conditions (Smith, 1986; Sellers and Stallone, 2008). I examined the contribution of vasoactive prostanoids to SCI-induced PE hypersensitivity by inhibiting COX enzyme activity in the SMA. As an initial step, concentrationresponse curves for PE were performed before and after addition of indomethacin, a nonselective COX inhibitor (Fig. 4.3A). Consistent with previous findings in rat mesenteric arteries (Mendizabal et al., 2011), addition of indomethacin diminished the relative force of PE-induced vasoconstriction. Importantly, incubation with indomethacin normalized PE sensitivity in the SMA from animals with SCI. Prior to addition of indomethacin, the EC50 for PE was lower in arteries from animals with SCI compared to arteries from sham-injured controls (Fig. 4.3A). After indomethacin incubation (+ indo), the PE EC50 was similar between arteries from sham-injured controls and those from animals with SCI. In order to determine whether the effects of indomethacin were due to COX-1 or COX-2, we used the specific inhibitors SC560 (COX-1) (Fig. 4.3B) and NS398 (COX-2) (Fig. 4.3C). Consistent with the data from my previous groups of animals, the EC50 for PE (prior to COX inhibitor treatment) was reduced in animals with T3 SCI. Incubation with SC560 reduced the relative force of PE-induced vasoconstriction in both arteries from both groups of animals (Fig. 4.3B). The effects of the COX-1 inhibitor on the PE EC50 proved rather difficult to interpret; the effects on arteries from sham-injured  136  controls varied considerably. Nonetheless, SC560 incubation did not abolish the difference in PE EC50 between arteries from sham-injured controls and arteries from animals with SCI. NS398, the COX-2 specific inhibitor, also reduced the force of PEinduced contraction in both groups (Fig. 4.3C). However, NS398 normalized the EC50 in arteries from animals with SCI; after NS398, PE EC50 was no longer different between arteries from animals with SCI and sham-injury (Fig. 4.3C).  4.4.4  Cyclooxygenase-2 expression increased in mesenteric arteries, but not aorta,  caudal to spinal cord injury COX-2 is considered the inducible COX isoform and vascular COX-2 upregulation is a hallmark of cardiovascular disease (Guo et al., 2005; Cipollone et al., 2001). COX-2 upregulation has been reported in the injured CNS, in the endothelium of blood vessels at the site of SCI (Adachi et al., 2005) but its expression has not been characterized in the peripheral vasculature following SCI. I used DAB immunohistochemistry to examine COX-2 expression in the SMA and descending aorta from animals with T3 SCI (Fig. 4.4). COX-2 expression was apparent by deposition reaction product in the epithelium of the distal rat vas deferens (Fig. 4.4A), where COX2 is constitutively expressed at high levels (McKanna et al., 1998). Reaction product was absent when the primary antibody was omitted (Fig. 4.4B). In the SMA, COX-2 expression was scant in arteries from sham-injured controls (Fig. 4.4C, C’). Under identical immunohistochemical and imaging conditions, COX-2 expression was appreciably more abundant in the SMA from animals with SCI (Fig. 4.4D). COX-2 expression was dispersed throughout the media and was apparent in the vascular  137  endothelium (Fig. 4.4D’). COX-2 expression in images of sections of aorta and the SMA was scored by a blind investigator using a 4-point scale, as has been used previously (Li et al., 2008). COX-2 expression in the descending aorta was equivalent between animals with sham injury and animals with T3 SCI (Fig. 4.4E). In the SMA, COX-2 expression was increased in animals with SCI (Fig. 4.4E).  138  139  Figure 4.3 Inhibition of cyclooxygenase-2 reversed phenylephrine hypersensitivity in arteries from animals with SCI (A) Concentration-response curves and EC50 values for PE in mesenteric arteries from animals with T3 durotomy (sham) and T3 SCI, before and after incubation with indomethacin. Prior to addition of indomethacin, the EC50 for PE was lower in arteries from animals with SCI. After indomethacin incubation (+ indo), the EC50 for PE was similar between arteries from sham-injured controls and those from animals with SCI. (B,C) Concentration-response curves and EC50 values for PE in mesenteric arteries from animals with T3 durotomy (sham) and T3 SCI, before and after incubation with SC-560 (B), a specific COX-1 inhibitor or NS-398 (C), a specific COX-2 inhibitor. Prior to addition of NS-398, the EC50 for PE was lower in arteries from animals with SCI. After incubation with NS-398 (+ NS-398), the EC50 for PE was similar between arteries from sham-injured controls and those from animals with SCI. Values represent mean±SEM; asterisk indicates P<0.05, two-way repeated measures ANOVA followed by Bonferroni’s test to detect pair-wise differences.  140  141  Figure 4.4 Cyclooxygenase-2 expression was increased in superior mesenteric arteries from animals with high-thoracic spinal cord injury (A) The specificity of COX-2 expression observed with DAB immunohistochemistry was confirmed in the epithelium of the distal rat vas deferens; (B) reaction product was absent when the primary antibody was omitted. (C) In the SMA from sham-injured animals, COX-2 expression was scant. (D) Under identical immunohistochemical and imaging conditions, COX-2 expression was appreciably more abundant in the SMA from animals with SCI. (D’) COX-2 expression was dispersed throughout the media and was apparent in the vascular endothelium. (E) COX-2 expression in images of sections of aorta and the SMA was scored by a blind investigator using a 4-point scale. In the SMA, but not the descending aorta, COX-2 expression was increased in animals with SCI. Values represent mean±SEM; asterisk indicates P<0.05, Wilcoxon rank-sum test.  142  4.5  Discussion I examined PE-induced vasoconstriction and ACh-induced vasodilation in  mesenteric arteries from animals with T3 complete SCI. Arteries from animals with SCI were hypersensitive to PE and hyposensitive to ACh, but endothelial-mediated vasodilation was functional. COX-2 inhibition normalized PE hypersensitivity in arteries from spinal cord-injured rats; in addition, arterial immunohistochemistry indicated that COX-2 expression was increased in arteries from rats with SCI. These findings implicate COX-2 in the heightened response to sympathetic mimetics in arteries caudal to high SCI.  4.5.1  Mesenteric arteries from rats with spinal cord injury were hypersensitive to  phenylephrine The SMA from rats with T3 SCI exhibited PE hypersensitivity relative to arteries from-sham injured controls (Fig. 4.1). This finding is consistent with previous findings in second-order mesenteric arteries from rats with high-thoracic SCI (Brock et al., 2006). Sympathetic hypoactivity after SCI results in a reduction in circulating norepinephrine (Schmid et al., 1998; Claydon and Krassioukov, 2006). It therefore might be interpreted as adaptive that the vascular response to norepinephrine (NE) and its mimetics is potentiated caudal to SCI. However, NE/PE hyper-responsiveness (i.e., vasoconstriction elicited by the same amount of neurotransmitter and/or mimetic is more powerful and/or persistent) is a feature of many animal models of cardiovascular disease (Lais and Brody, 1975; Lais and Brody, 1978; Feletou et al., 1994); in a pathological light, exaggerated  143  vasoconstriction in response to adrenoreceptor activation is an obvious candidate for under-pinning AD. The first evidence for NE hyper-responsiveness came from clinical research demonstrating that tetraplegics exhibit enhanced pressor responses to intravenous NE (Arnold et al., 1995; Mathias et al., 1976) and PE (Krum et al., 1992). Vascular hyperresponsiveness was subsequently confirmed in animal models of SCI: in both tail (Yeoh et al., 2004) and mesenteric (Brock et al., 2006) arteries from rats with SCI, contractions evoked via stimulation of the perivascular nerves were more forceful than those in arteries from uninjured controls. Recent data demonstrate similar hyper-reactivity in tail veins caudal to SCI (Tripovic et al., 2011). While all of the data agree on some variation of NE/PE hyper-responsiveness after SCI, the search for a specific mechanism underlying this phenomenon has proven more difficult, and may vary among vascular beds. In tail arteries from rats with SCI, the vascular smooth muscle is generally hyper-reactive. Nerve-evoked contractions are dramatically enhanced, with force of contractions increased up to 25-fold relative to arteries from uninjured animals (Yeoh et al., 2004). This was accompanied by evidence for an increased role of ATP, enhanced NE release, and a near doubling of contraction amplitude in response to extracellular K+. In mesenteric arteries from rats with SCI, adrenergic but not purinergic signaling was enhanced, and there was no effect of SCI on the response to extracellular K+ (Brock et al., 2006). An important distinction between these data sets is pre- versus post-junctional effects of NE/PE: in mesenteric arteries, PE hyper-responsiveness was abolished by the NE uptake blocker desmethylimipramine, indicating that the increased neurally-evoked response after SCI was due to a reduction  144  in neuronal uptake of NE (Brock et al., 2006). Recent data enrich the story further, indicating a role for purinergic transmission in enhanced nerve-evoked contraction in saphenous arteries caudal to SCI (Rummery et al., 2010). An interesting new development highlights a role for vascular L-type Ca2+ channels in enhanced neurovascular transmission caudal to SCI (Tripovic et al., 2011). My data indicate that in the splanchnic vasculature, COX-2 activity mediates PE hyper-responsiveness after highthoracic SCI.  4.5.2  Mesenteric arteries from rats with spinal cord injury were hyposensitive to  acetylcholine I found that ACh-induced vasodilation was functional, but altered in mesenteric arteries from rats with T3 SCI. After maximal concentration with PE, the ACh EC50 in arteries from spinal cord-injured animals was increased relative to that in arteries from sham-injured controls (Fig. 4.2). This is suggestive, but clearly inconsistent with the overt endothelial dysfunction observed in animal models of diabetes (Sachidanandam et al., 2009; Khazaei et al., 2008a) and hypertension (Silva et al., 2011): in these instances, the maximum vasodilation induced by ACh is dramatically reduced in arteries from experimental animals relative to healthy controls. This renders the intermediate arterial behaviour I observed following SCI difficult to interpret; however, some discussion of recent findings surrounding roles of endothelial-derived relaxing factors in the mesenteric arteries is still warranted. The endothelium regulates vascular tone through the release of endotheliumderived vasodilators. Removal or dysfunction of the endothelium augments  145  vasoconstriction elicited by a number of stimuli, including α-adrenoreceptor agonists (Martin et al., 1986; Jin et al., 2011). Recent data highlight the relevance of endotheliumderived relaxing cues in the mesenteric arteries under conditions of sustained adrenoreceptor-mediated vasoconstriction. In an ex vivo preparation measuring perfusion pressure across the whole mesenteric vascular bed, vasoconstriction induced by methoxamine (an α-1-adrenoreceptor agonist) decreased by 20-40% over time (Jin et al., 2008). This time-dependent decrease was not observed following high K+; further experiments demonstrated that the effect was endothelium-dependent, and was not altered by L-NAME or indomethacin. Using K+-channel and gap junction blockers, this group has provided evidence that the endothelial-mediated opposition to sustained vasoconstriction in the mesenteric arteries is due to endothelium-derived hyperpolarizing factor (EDHF) (Jin et al., 2008; Jin et al., 2011). Earlier experiments, using in vitro myography to examine mesenteric arteries from rats (Shimokawa et al., 1996) and eNOS knockout mice (Scotland et al., 2001) demonstrated that the contribution of EDHF increases as vessel diameter decreases. Given my finding that ACh-induced vasodilation is mildly impaired at the level of the SMA, it would be interesting to conduct these (whole vascular bed) experiments in animals with high-thoracic SCI.  4.5.3  Cyclooxygenase-2-derived prostanoids contribute to phenylephrine  hypersensitivity following spinal cord injury Both of the main isoforms of COX play crucial roles in blood pressure regulation. COX-1 is constitutively expressed in most tissues, including vascular endothelium and smooth muscle. Vascular COX-2 expression is relatively low under  146  physiological conditions (Fig. 4.4C), but it is induced in response to a wide range of stimuli, including pro-inflammatory cytokines, bacterial products, hypoxia, shear stress and mechanical stretch (Iniguez et al., 2008). The classical demarcation identifying COX-1 as a constitutive, homeostatic enzyme and COX-2 as an inducible, pathological enzyme likely represents an oversimplification. Recent findings suggest that in the vasculature (and in most systems), both isoforms contribute to both homeostasis and pathology (Norel, 2007; Vanhoutte, 2009). Nonetheless, my findings demonstrate a role for COX-2 in PE hypersensitivity following SCI. A growing body of evidence implicates changes in vascular COX-2 in cardiovascular dysfunction. For example, COX-2 upregulation and activity in the vasculature is thought to contribute to atherosclerosis (Cipollone et al., 2001). Intriguingly, COX-2 upregulation in peripheral vasculature is associated with vascular PE hypersensitivity in several animal models of cardiovascular disease (Guo et al., 2005; Adeagbo et al., 2005; Virdis et al., 2009). There are other examples of a demonstrated link between vascular COX-2 and PE hypersensitivity in the mesenteric arteries. In a mouse model of type 2 diabetes (db/db mice), NS-398 attenuated PE hypersensitivity of mesenteric arteries in vitro (Guo et al., 2005). In rats with salt-induced hypertension, NS398 reversed PE hypersensitivity of mesenteric arteries in vitro; in addition, systemic administration of NS-398 inhibitor alleviated hypertension in vivo (Adeagbo et al., 2005). My data demonstrate that COX-2 activity is critically involved in PE hypersensitivity in the SMA after T3 SCI (Fig. 4.3) and that COX-2 is upregulated in the SMA after T3 SCI (Fig. 4.4). COX-2 upregulation and/or activation may represent a general feature of vascular sympathetic hyper-responsiveness under pathological cardiovascular conditions.  147  COX-derived vasoactive prostanoids exert their effects by acting on specific Gprotein-coupled receptors, expressed predominately on vascular smooth muscle, but also on the vascular endothelium and on blood cells (Norel, 2007). This begs the question: which COX-2-derived prostanoid(s) is/are involved in potentiated vasoconstriction after SCI? The best-characterized prostanoids in vascular function are prostaglandin I2 (PGI2), which elicits vasodilation, and thromboxane A2 (TxA2), which induces vasoconstriction. Thromboxane A2 (TxA2) is a potent vasoconstrictor, which acts on the TP receptor expressed on vascular smooth muscle cells to mobilize intracellular calcium and elicit contraction. In addition to PGI2 and TxA2, there is increasing evidence documenting a critical cardiovascular role for prostaglandin E2 (PGE2), which acts on four EP receptors (EP1-EP4) to exert diverse effects. In rat mesenteric arteries, PGE2 acts on EP3 receptors in the vascular smooth muscle to stimulate vasoconstriction (Kobayashi et al., 2011). Vascular expression and/or activity of both TxA2 and PGE2 increase in a wide range of pathological conditions, including hypertension and diabetes, and both have been implicated in vascular inflammation (Feletou et al., 2010a; Feletou et al., 2010b; Legler et al., 2010). Therefore, it appears that we have two new candidate molecules (and more, if we include their vascular receptors) to investigate as mediators of enhanced sympathetically-mediated vasoconstriction following SCI.  4.5.4  Conclusion My findings demonstrate that COX-2 activity contributes to vascular hyper-  responsiveness caudal to SCI. More work is required to determine which COX-2-derived prostanoids are involved and the extent to which these might be manipulated to improve  148  the cardiovascular outcome of SCI. However, these data indicate that compounds already in clinical use, such as celecoxib (a COX-2 inhibitor), may be of benefit in treating AD. Current management of AD is based largely on prevention and conservative management of acute episodes (raising the head and removing the precipitating stimulus) (Krassioukov et al., 2009). If non-pharmacological measures do not relieve hypertension, acute pharmacological intervention is initiated, using short-acting hypotensives such as nifedipine, nitrates and captopril. Unfortunately, due to blood pressure instability in individuals with SCI, hypotensives can trigger a cycle of low and high blood pressure episodes, and a lengthy pharmacological struggle to stabilize blood pressure. Specific antagonists targeting COX-2, its downstream effectors or their receptors may represent an attractive alternative for controlling AD in chronic SCI.  149  Chapter 5: Recurrent autonomic dysreflexia exacerbates vascular dysfunction following spinal cord injury  5.1  Synopsis An often unappreciated consequence of spinal cord injury (SCI) is remarkable  blood pressure lability. Impaired neural control of the blood pressure creates dramatic hypoand hypertension in response to physiological stimuli. There is very limited information on how these fluctuations in blood pressure affect the structure and function of the blood vessels over time after injury. In these experiments, I tested the hypothesis that shear stress due to hypertension during recurrent episodes of autonomic dysreflexia (AD) would damage the vascular endothelium and therefore exacerbate PE hyper-responsiveness and ultimately, the severity of AD. In my experimental animals, I induced AD on a daily basis via colorectal distension (CRD) over two weeks after T3 SCI. I found that this manipulation exacerbated PE hyper-responsiveness in mesenteric arteries examined one month after SCI. The effects of recurrent AD were not due to impaired endothelial function, since AChevoked vasodilation was similar in arteries from SCI-CRD animals and SCI-only controls. Interestingly, arterial hyper-responsiveness following recurrent AD was not accompanied by an increase in baseline blood pressure or severity of CRD-induced AD. These findings demonstrate that increased frequency of AD over even a brief period alters the functional properties of the blood vessels after SCI.  150  5.2  Introduction Spinal cord injury (SCI) disrupts descending autonomic pathways and consequently  impairs cardiovascular homeostasis (Krassioukov et al., 1999; Furlan et al., 2003). Deficits in cardiovascular control are particularly marked in individuals with cervical or high thoracic SCI (Krassioukov and Claydon, 2006; Curt et al., 1997; Krum et al., 1991; Munakata et al., 1997). A large proportion of individuals with SCI above the sixth thoracic level (T6) experience autonomic dysreflexia (AD), episodic hypertension elicited by sensory stimulation below the level of injury (Lindan et al., 1980; Helkowski et al., 2003). Many individuals with SCI also experience episodes of low blood pressure due to orthostatic hypotension (OH). While OH is commonly associated with the acute phase of injury (Sidorov et al., 2008; Illman et al., 2000), it persists in up to 70% of individuals with chronic SCI (Claydon and Krassioukov, 2006). Episodes of AD and OH frequently occur numerous times per day, such that blood pressure fluctuates dramatically: for example, systolic blood pressure can fall to 60mmHg during OH (Claydon and Krassioukov, 2006; Mathias, 1995) and exceed 300mmHg during AD (Elliott and Krassioukov, 2006; McBride et al., 2003). The effects of such pronounced changes in blood pressure on the vasculature and cardiovascular function over the lifetime of individuals with SCI are unknown. However, several lines of evidence suggest that chronic blood pressure lability is likely to be detrimental. For example, animal studies reveal that experimental manipulations of arterial hemodynamics to increase shear stress produce deterioration of the vascular endothelium (Fry, 1968). In addition, preeclampsia, an intermittent hypertensive state that occurs during pregnancy, is associated with impaired flow-mediated dilation (FMD; an indicator of vascular endothelial function (Khan et al.,  151  2005; Chambers et al., 2001; Agatisa et al., 2004). Impaired endothelial function in preeclampsia, hypertension which occurs for a short period in relatively young females, provides persuasive evidence that even short periods of episodic hypertension induce vascular dysfunction. More than a decade ago, Steins and colleagues proposed that instability in blood pressure could contribute to vascular injury, and consequently result in greater risk for arterial disease in individuals with SCI (Steins et al., 1995). As described and discussed in Chapter 4, the principal vascular abnormality that has been described after SCI is hyper-responsiveness to PE, which may contribute to the development of AD (Brock et al., 2006; McLachlan and Brock, 2006). Here I sought to determine whether the opposite is also true; that is, whether recurrent AD might exacerbate vascular dysfunction following SCI. I examined the effects of recurrent AD on vascular function following high-thoracic (T3) SCI. In one group of animals, I induced AD on a daily basis via colo-rectal distension (CRD) over two weeks after complete transection of the T3 spinal cord. At one month postinjury, I examined severity of CRD-induced AD in animals exposed to CRD during recovery from SCI and in SCI-only controls. I then characterized vasoactive properties of mesenteric arteries from both groups of animals using in vitro myography.  152  5.3  Materials and methods  5.3.1 Spinal cord injury surgery and post-operative care The procedures for complete transection of the T3 spinal cord and post-operative animal care were identical to those described previously (Chapters 2-4) (Ramsey et al., 2010). In these experiments, all rats survived for one month following T3 SCI.  5.3.2 Repetitive colo-rectal distension For 14 days after SCI, one group of animals received daily bouts of colo-rectal distension (CRD; 30 minutes) (SCI-CRD). In these animals, the colon was distended once per day using a pediatric Foley catheter: the catheter was inserted into the rectum, secured to the tail with tape and inflated with 2ml of air over 10 seconds. The inflated balloon was maintained in the colon of the conscious, freely-moving animal for 30 minutes. The control group was comprised of sex- and age-matched spinal cord-injured animals that did not undergo repetitive CRD.  5.3.3 Cardiovascular assessment Thirty days after SCI, a cannula was implanted into the carotid artery in all animals for beat-to-beat blood pressure recording. As described in Chapter 2, the cannulation was performed under isoflurane anesthesia. Cannulae were tunneled subcutaneously to exit at the base of the skull and filled with a lock solution of 1:10 heparin in 5% dextrose. As outlined in Chapter 2, baseline cardiovascular parameters and severity of AD were examined two hours after carotid artery cannulation. Beat-to-beat arterial pressure was monitored using PowerLab and Chart™ 5 for Windows (ADInstruments). Animals were  153  conscious and allowed to move freely in a cage throughout the recording session. Baseline blood pressure was recorded over five minutes; blood pressure during two episodes of CRD-evoked AD (separated by 10 minutes of recovery) was recorded for each animal.  5.3.4 In vitro myography I used in vitro myography to examine vasoconstriction in response to PE and vasodilation in response to acetylcholine (ACh), an endothelial-dependent dilator, in superior mesenteric arteries. Following cardiovascular assessment, superior mesenteric arteries (SMAs) were isolated from deeply-anesthetized SCI-CRD animals and SCI-only controls. Concentration-response curves for PE and ACh were derived as described in Chapter 4.  5.3.5 Cardiovascular data analysis Analysis of beat-to-beat blood pressure recordings was performed as described in Chapter 2. In brief, raw beat-to-beat pressure data were averaged over one second, systolic and diastolic blood pressure (SAP and DAP) were obtained from maxima and minima (respectively) and mean arterial pressure (MAP) and heart rate (HR) were calculated. Baseline cardiovascular parameters were averaged over at least three minutes of recording time and CRD-evoked changes in SAP and HR represent the average of two consecutive distensions for each animal.  154  5.3.6 Myography data analysis Analysis of arterial force changes in response to PE and ACh was performed as described in Chapter 4. For comparing PE-induced vasoconstriction between groups, raw force of contraction induced by PE was normalized to contractile force induced by KCl in each arterial segment. Maximum force of contraction (Emax) and half-maximal effective concentration of PE (EC50) were determined for arteries from each group. Vasodilator responses in each artery were expressed as percent relaxation of PE-induced contraction and Emax and EC50 of ACh were determined for arteries from each group.  5.3.7 Statistics Baseline cardiovascular data and CRD-evoked changes in SAP and HR were compared between groups using Student’s t-test. For myography data, GraphPad Prism was used for curve-fitting and concentration-response analysis. Student’s t-test or one-way analysis of variance (ANOVA) with multiple comparisons using the Bonferroni test were performed where appropriate. In all cases, P values less than 0.05 were considered significant and results are expressed as mean ± standard error of the mean (SEM).  5.4  Results  5.4.1 Repetitive colo-rectal distension exacerbated spinal cord injury-induced vascular dysfunction Using in vitro myography, I examined vasoactive responses in mesenteric arteries from animals exposed to repeated CRD (SCI-CRD) and from T3 SCI-only controls. When exposed to cumulative addition of PE, mesenteric arteries from SCI-CRD animals exhibited  155  enhanced vasoconstriction compared to arteries from the SCI-only group (Fig. 5.1A). Raw force recorded in response to PE was normalized internally (i.e., for each arterial segment) and expressed as percentage of contraction induced by 80mM KCl. Arteries from animals exposed to repetitive CRD exhibited larger maximal contraction in response to PE (Emax; Fig. 5.1A,B). The sensitivity to PE (EC50; Fig. 5.1C) did not differ between arteries from SCI-only controls and SCI-CRD animals. The slopes of the Hill equation fits were not different between groups.  156  Figure 5.1 Repetitive colo-rectal distension during recovery from high-thoracic (T3) spinal cord injury potentiated phenylephrine-induced vasoconstriction (A) Phenylephrine (PE) concentration-response curves for mesenteric arteries from SCIonly controls (SCI) and those from animals that underwent repetitive colo-rectal distension (SCI-CRD) revealed that arteries from SCI-CRD animals exhibited larger responses to PE. Maximum vasoconstriction (in response to 10-5 M PE; Emax, B) was greater in arteries from SCI-CRD animals. Repetitive CRD had no effect on half maximal effective concentration of PE (EC50, C). For each arterial segment, force of PE-induced vasoconstriction was normalized to force induced by 80 mM KCl. Values represent mean±SEM. Asterisk indicates P<0.05, one-way ANOVA (A), Student’s t-test (B).  157  5.4.2 The effect of repetitive colo-rectal distension on phenylephrine response was not a product of endothelial dysfunction Since blood pressure oscillations can damage the vascular endothelium (Khan et al., 2005; Fry, 1968), I hypothesized that PE hyper-responsiveness in arteries from SCI-CRD rats was due to endothelial dysfunction. Arteries from SCI-only controls and SCI-CRD animals were treated with 1µM PE to establish a stable contraction, and then exposed to cumulative addition of ACh (Fig. Fig. 5.2A). Vasodilator responses were normalized for each arterial segment, and expressed as percentage relaxation of PE-induced contraction. The concentration-response curves for ACh were similar for arteries from SCI-only controls and SCI-CRD animals, and the slopes of the Hill equation fits did not differ between groups. There was also no difference in maximal vasodilation induced by ACh (at 10-5 M; Fig. 5.2B) or EC50 of ACh (Fig. 5.2C). Thus, endothelial-dependent vasodilation was similar between arteries from SCI-only controls and SCI-CRD animals, and was not affected by repetitive CRD performed over two weeks following T3 SCI.  158  Figure 5.2 Endothelial-dependent vasodilation was not affected by repetitive colorectal distension over two weeks following T3 spinal cord injury (A) Acetylcholine (ACh) concentration-response curves were similar for mesenteric arteries from SCI-only controls and those from animals that underwent repetitive colorectal distension (SCI-CRD). There was no difference in maximum vasodilation induced by ACh (at 10-5 M ACh; Emax, B) or half maximal effective concentration of ACh (EC50, C) between arteries from SCI and SCI-CRD animals. For each arterial segment, ACh-induced vasodilation was normalized to force at maximum vasoconstriction (with 10-5 M PE). Values represent mean±SEM.  159  5.4.3 Repetitive colo-rectal distension over two weeks did not exacerbate cardiovascular dysfunction following spinal cord injury Given that repetitive CRD increased PE hyper-responsiveness in arteries from animals with SCI, I examined cardiovascular function in SCI-only controls and in animals that received repetitive CRD (SCI-CRD). SCI-CRD animals received 30 minutes of CRD per day during the first two weeks after SCI. One month after T3 SCI, carotid cannulae were implanted for beat-to-beat blood pressure recording. Baseline blood pressure and heart rate did not differ between SCI-only controls and SCI-CRD animals: SAP, DAP, MAP and heart rate (HR) at rest were similar between SCI-CRD animals and SCI-only controls (Figure 5.3). I also examined the severity of AD induced by CRD in SCI-only controls and in animals that received daily bouts of CRD over two weeks following SCI. At one month after SCI, both groups of animals exhibited AD, as indicated by the presence of hypertension and bradycardia in response to one-minute of CRD (Fig. 5.4D). However, CRD-induced hypertension was less pronounced in animals that experienced recurrent CRD during recovery from SCI (Fig. 5.4A-C). The reduced pressor response to CRD in SCI-CRD animals was reflected in SAP, DAP and MAP (Fig. 5.4D). The CRD-evoked change in HR was not different between SCI-CRD and SCI animals (Fig. Fig. 5.4D).  160  Figure 5.3 Repetitive colo-rectal distension did not alter baseline cardiovascular parameters examined at one month following T3 spinal cord injury Animals that underwent repetitive colorectal distension (SCI-CRD) and SCI-only controls (SCI) received a carotid cannula one month after SCI. Arterial blood pressure was collected in conscious animals at rest, over 3-5 minutes; baseline systolic, diastolic, and mean arterial pressure (SAP, DAP, and MAP, respectively) and heart rate (HR) represent the average over this period. HR was calculated from duration of interbeat intervals. Arterial pressure and HR were not significantly different between SCI and SCI-CRD animals. Values represent mean±SEM.  161  162  Figure 5.4 Autonomic dysreflexia evoked by colo-rectal distension was less pronounced in animals that experienced repeated bouts of distension during recovery from T3 spinal cord injury Autonomic dysreflexia (AD) was induced via one minute of colo-rectal distension (CRD) at one month after SCI. Beat-to-beat intra-arterial blood pressure was recorded before, during, and after CRD in SCI-only controls (SCI; A,B) and in animals that underwent repetitive CRD for two weeks following SCI (SCI-CRD; A,C). (D) CRD-induced changes in systolic, diastolic, and mean arterial pressure (SAP, DAP, and MAP) and heart rate (HR) represent the average of two distensions per animal, separated by a minimum of 10 minutes. Although SCI-CRD animals exhibited AD at one month following SCI, the CRD-induced hypertension was less pronounced, compared to animals that had not been exposed to this stimulus during recovery from SCI. Bradycardia was similar between SCI-only and SCICRD animals. Values represent mean±SEM. Asterisk indicates P<0.05.  163  5.5  Discussion In these experiments, I investigated the effects of recurrent CRD-induced AD on  vascular function in animals with SCI. In one group of animals, AD was induced daily via CRD during two weeks after T3 transection: this manipulation exacerbated PE hyperresponsiveness in mesenteric arteries examined one month after SCI. The effects of recurrent AD were not due to impaired endothelial function, since ACh-evoked vasodilation was similar in arteries from SCI-CRD animals and SCI-only controls. Interestingly, arterial hyper-responsiveness following recurrent AD was not accompanied by an increase in baseline blood pressure or severity of CRD-induced AD. Here I discuss the rationale for and relevance of repetitive CRD, the importance of vascular dysfunction following SCI, and the apparently paradoxical relationship between vascular effects of recurrent AD and cardiovascular outcome of SCI, the latter of which may be due to vasoactive neurotransmitters released from colorectal afferents.  5.5.1  Rationale for repetitive colo-rectal distension Repetitive induction of AD in rats with high SCI mimics the spontaneous and  frequent episodes of AD experienced by individuals with similar injuries. Individuals with high SCI experience AD on a daily basis, triggered by a variety of stimuli, including bladder filling, catheterization, presence of infection, bowel routine, and sexual intercourse; in addition, AD can be self-induced (boosting). While bladder distension is the most common precipitant of clinical AD (Lindan et al., 1980; Shergill et al., 2004), CRD represents a more feasible and better-characterized (Krassioukov et al., 2002;  164  Mayorov et al., 2001) experimental manipulation for testing the effects of recurrent AD on vascular function. CRD is a potent and noninvasive stimulus for AD in rats with severe highthoracic SCI (Maiorov et al., 1997; Krassioukov and Weaver, 1995a; Hou et al., 2008; Laird et al., 2006). In uninjured rats, CRD can induce a transient increase in blood pressure: this pressor response is typically due to movement and associated with tachycardia (Krassioukov and Weaver, 1995a). In rats with SCI, CRD induces AD, indicated by pronounced and persistent hypertension and accompanying bradycardia which resolve over time when distension is alleviated. This maneuver mimics some of the most common causes of AD clinically, such as constipation and fecal impaction (Teasell et al., 2000). Recurrent CRD during the acute and sub-acute period following SCI is a clinically-relevant scenario: individuals with acute SCI often experience constipation, due to immobility, bed-rest and opiates (Krogh et al., 2000). Although the ENS does have some intrinsic functional capacity, this is significantly impaired when it loses central coordination following SCI: as a result, increased colonic transit time also contributes to constipation after SCI (Camilleri and Bharucha, 1996; Stone et al., 1990; Glickman and Kamm, 1996). Clinically, AD is typically associated with chronic SCI (although it can occur in the acute stage of injury, particularly following severe high-cervical SCI (Krassioukov et al., 2003). In rats with severe high-thoracic SCI, CRD-induced AD is pronounced at 24 hours after injury (Krassioukov and Weaver, 1995a; Osborn et al., 1990). The magnitude of the CRD-evoked pressor response is less marked (but still significantly elevated above resting levels) at one week following SCI (Mayorov et al., 2001; Krassioukov and  165  Weaver, 1995a). After one week, CRD-induced AD begins to increase in severity again; when assessed at two weeks following SCI (and in my experience, for at least three months thereafter) AD initiated by CRD is pronounced, and manifests as both hypertension and bradycardia that resolve gradually over minutes when distension is alleviated (Krassioukov and Weaver, 1995a; Hou et al., 2008; Krassioukov et al., 2002; Leman et al., 2000; Mayorov et al., 2001; Laird et al., 2009). Thus, although I did not monitor blood pressure during daily CRD in this study, I am confident that AD was induced during each session.  5.5.2  Recurrent autonomic dysreflexia exacerbated phenylephrine hyper-  responsiveness In my experiments, daily bouts of CRD-induced AD during the first two weeks post-SCI augmented the vascular response to PE, as indicated by a larger Emax in arteries from SCI-CRD animals than in arteries from SCI-only controls. Since vascular function was assessed 30 days post-injury, or 16 days after daily CRD was terminated, recurrent AD appears to have persistent effects on vascular function. At present, the mechanism by which repetitive CRD alters vascular function remains unknown. Intriguingly, the effect of repetitive CRD on the vasculature is distinct from the effect of T3 SCI (Chapter 4). Compared to arteries from sham-injured controls, the SMA from rats with SCI exhibited a reduction in the EC50 of PE: this finding is consistent with previous results in second-order mesenteric arteries (Brock et al., 2006). However, arteries from rats that experienced repetitive CRD exhibited an increase in PE Emax, with no change in EC50. It is tempting to interpret these differential effects as reflecting  166  differential mechanisms of vascular response. For example, arteries from animals with SCI-CRD may undergo hypertrophic remodeling as a result of increased exposure to high pressure conditions, as described in Chapter 1. However, when I compared the responses to 80mM KCl, arteries from animals with SCI exhibited a more pronounced contraction (in raw force) than arteries from SCI-CRD animals. Admittedly, this is difficult to interpret, since the size of the SMA varies considerably along its length: comparing raw force of contraction among arterial segments implies that the segments were removed at identical locations in every experiment, reproducibility that is challenging to achieve in an ex vivo system. The extent of SCI and/or CRD-induced remodeling of the mesenteric arteries is currently under investigation in our laboratory, working from fixed tissue and using appropriate normalization (e.g., using media-to-lumen ratio).  5.5.3  Recurrent autonomic dysreflexia did not alter endothelial-dependent  vasodilation While endothelial damage is a plausible contributor to PE hyper-responsiveness following repetitive CRD, in this study, endothelium-mediated vasodilation was functional and comparable between arteries from SCI-CRD animals and SCI-only controls. In interpreting this finding, it is critical to consider the duration of recurrent AD in this experiment. Animals were exposed to recurrent AD - and the vascular endothelium exposed to increased shear stress - for 30 minutes per day over two weeks following SCI. I also acknowledge that spontaneous episodes of AD occurred in both groups of animals due to daily manual bladder expression (unpublished observations); I would expect these episodes to be comparable in frequency and severity for both groups.  167  Nonetheless, this (two-week) period of repetitive CRD is a relatively short amount of time, particularly when considering the potential for recurrent AD due to colo-rectal stimulation over the lifetime of an individual with SCI. The time required for bowel care varies dramatically, but at a conservative estimate, an individual with high SCI might spend 60 minutes on bowel care every other day (House and Stiens, 1997). AD is commonly reported to occur during bowel care (Stone et al., 1990) and is likely underreported, as “silent” or asymptomatic AD is also triggered by routine bowel management (Kirshblum et al., 2002). Therefore, while dysfunction of the vascular endothelium did not underlie PE hyper-responsiveness following two weeks of recurrent AD in rats with T3 SCI, endothelial damage might result from recurrent AD sustained over longer periods in chronic SCI. In the absence of endothelial dysfunction, an alternate and intriguing explanation for vascular hyper-responsiveness in animals with recurrent AD is that heightened response to PE represents a persistent compensatory effect, to preserve vascular tone in the face of ongoing release of vasoactive substances. In rats, the CRD maneuver increases intracolonic pressure by 25-35 mmHg (Krassioukov and Weaver, 1995a) and activates colo-rectal mechanoreceptors. Many of these afferents contain calcitonin generelated peptide (CGRP) and substance P (SP), both potent vasodilators, and have collateral branches supplying blood vessels (Blackshaw et al., 2007). Afferent activation triggers both central propagation of action potentials and release of CGRP and SP via an axon reflex. If afferents activated by repetitive CRD have branches to mesenteric arteries, the arteries may augment their PE response to preserve cardiovascular homeostasis. This effect could also be mediated by cross-excitation of colorectal afferents and mesenteric  168  afferents. Cross-excitation of primary afferents is a well-characterized phenomenon (Shinder et al., 1998; Amir and Devor, 1996) in which stimulation of a sub-population of sensory neurons excites adjacent neurons that reside in the same dorsal root ganglion (Devor and Wall, 1990). While the notion that sensory activity has important implications for cardiovascular function is hardly new (Okajima and Harada, 2006), little is known about the relationship between sensory stimulation and cardiovascular control following SCI.  5.5.4  Reconciling the vascular effects of recurrent autonomic dysreflexia with the  cardiovascular outcome of spinal cord injury In these experiments, arterial PE hyper-responsiveness in animals exposed to recurrent CRD-induced AD over two weeks following SCI did not increase the severity of AD. In fact, when CRD-evoked AD was examined at one month after injury, hypertension was reduced by approximately 50% in SCI-CRD rats relative to SCI-only controls. Thus, although CRD-induced AD was still present in animals exposed to repetitive CRD, its severity was attenuated relative to animals that were not previously exposed to the stimulus. Although there was no significant difference in heart rate response between groups, pronounced bradycardia was evident in SCI-CRD animals. An increase in baroreflex sensitivity could underlie reduced severity of CRD-induced AD, but this premise requires further investigation. Another possible explanation for this result is that some form of “habituation”, defined here as reduced response of a reflex with repeated activation, occurred as a result of repeated CRD. The site of habituation remains obscure. However, adaptation of  169  stretch receptors in the colon and rectum, or activity-induced plasticity in sensory or sympathetic spinal circuits may be involved. If cross-excitation between colorectal mechanonociceptors and mesenteric afferents does occur within the dorsal root ganglion, this would explain the blunted response to CRD. Once cross-excitation is established through repetitive CRD, CRD provokes both centrally-directed action potentials (signaling distension) and release of CGRP/SP at the mesenteric artery (inducing vasodilation). If this is the case, I would expect a novel stimulus to evoke more pronounced AD in SCI-CRD animals. The possibility of habituation within reflexes triggering AD and/or cross-excitation between different populations of afferents remains speculative at this point. However, it does reconcile well with our clinical knowledge of AD, in that individuals with chronic SCI often experience severe AD in response to a novel stimulus, such as sexual stimulation (Ekland et al., 2008).  5.5.5  Conclusion It is important to recognize that AD represents a non-physiological, frequent,  and abrupt increase in blood pressure, and thus is potentially damaging, regardless of amplitude. In these experiments, a relatively short period of recurrent AD (two weeks) produced persistent PE hyper-responsiveness. Since sympathetic innervation provides the main source of vascular tone to resistance vessels, PE hyper-reactivity may set the stage for severe AD when the system is challenged with novel stimuli. For example, recurrent AD triggered by colo-rectal stimulation might result in more pronounced AD evoked by a pressure sore. In addition, PE hyper-responsiveness may represent only one of multiple  170  forms of vascular adaptation to labile blood pressure following SCI. It is widely-accepted that chronic constriction of the blood vessels, particularly at the level of small vessels, can result in locally damaging increases in blood flow velocity and turbulence that result in shear stress and maladaptive remodeling of the vasculature (Davies, 2009; Schiffrin, 2004). I do not yet know how recurrent AD affects the structure of the vasculature, or how such structural adaptations might contribute to cardiovascular complications, which are the leading cause of morbidity and mortality in individuals with high SCI (Strauss et al., 2006; Garshick et al., 2005). While many clinicians, care-givers, and individuals with SCI appreciate the common triggers and symptoms of AD, asymptomatic AD complicates diagnosis and treatment. We now know that pronounced hypertension may occur during an episode of AD in the absence of any symptoms (Ekland et al., 2008; Kirshblum et al., 2002; Linsenmeyer et al., 1996). While we do not know how frequently “silent” AD occurs, the available data suggest that it is common among individuals with chronic SCI (Kirshblum et al., 2002). In the absence of symptoms, episodes of hypertension are prolonged, and the long-term cardiovascular consequences may be severe. Vigilant self or assisted bowel, bladder, and skin care to prevent AD are of paramount importance. In addition, research directed at reducing the incidence of AD - even when patients do not complain of symptoms - during routine activities or medical procedures should be considered a high priority. My findings suggest that the ramifications of recurrent AD extend beyond the immediate danger and discomfort associated with acute episodes of hypertension. The long-term effects of AD merit further investigation; however, the potential for  171  damaging effects of chronic and recurrent AD should be a consideration for individuals living with SCI.  172  Chapter 6: Discussion 6.1  Summary and conclusions In this dissertation, I examined SCI-induced changes in two portions of the  spinal reflex that mediates autonomic dysreflexia (AD). My work focused in injuryinduced effects in the dorsal root ganglion (DRG), home of the sensory neurons that initiate AD in response to a wide range of stimuli, and mesenteric arteries, a sample of the effectors that vasocontrict caudal to SCI to produce hypertension.  6.1.1  Spinal cord injury-induced changes in the dorsal root ganglion In Chapter 2, I tested the hypothesis that SCI would promote nociceptor  hypertrophy and/or terminal sprouting and that these effects would be more pronounced after high-thoracic SCI than after low-thoracic SCI. After a comprehensive survey of sub-populations of sensory neurons, I found that only those expressing the capsaicin receptor TRPV1 exhibited somatic hypertrophy caudal to SCI. The effects of SCI were most pronounced in ganglia far distal to injury, and in support of my hypothesis, were more pronounced after T3 than T10 SCI. I conclude that high-thoracic SCI had selective effects on TRPV1-expressing sensory neurons in distal sensory ganglia. Prompted by the selectivity of SCI-induced effects on TRPV1-expressing nociceptors, I used capsaicin to selectively eliminate TRPV1-positive projections to the lumbosacaral dorsal horn. I hypothesized that these would be involved in triggering and/or development of AD. To test this hypothesis, I administered capsaicin at two different time points in different groups of animals: I reasoned that eliminating TRPV1positive axons early (48 hours after SCI) might have more dramatic effects on AD than  173  eliminating TRPV1-positive projections later (28 days) following injury.I found that both treatments dramatically but equivalently attenuated the severity of AD. I conclude that TRPV1-positive afferents play a critical role in triggering, but not in development of AD. Given that non-neuronal elements of the DRG play a critical role in dictating function of sensory neurons, I next examined the effects of SCI on the other components of the DRG (Chapter 3). I hypothesized that SCI would activate glial and immune cells in the DRG, promote angiogenesis and trigger sympathetic sprouting, and that these effects would be more pronounced after T3 SCI than after T10 SCI. I found that T3 but not T10 SCI provoked glial activation and activated macrophages in lumbar DRGs. I also found that T3 (but not T10) SCI prompted mast cell accumulation in the vicinity of the lumbar (L4/L5) DRG. Both injuries induced angiogenesis and sympathetic axon ingrowth in the cell layer of the lumbar DRG. I conclude that high-thoracic SCI has more pronounced effects on non-neuronal elements of distal DRGs than lowthoracic SCI.  6.1.2  Spinal cord injury-induced changes in the mesenteric artery In Chapter 4, I used in vitro myography (performed in the laboratory of Dr.  Ismail Laher) to test the hypothesis that cyclooxygenase (COX) enzymes mediated phenylephrine (PE) hyper-responsiveness in mesenteric arteries after SCI. I found that COX-2 inhibitors reversed PE hypersensitivity in the SMA from animals with T3 SCI. I also found that COX-2 was uprgegulated in the SMA from animals with T3 SCI. I  174  conclude that COX-2 activity contributed to vascular PE hyper-responsiveness caudal to T3 SCI. My final set of experiments (Chapter 5) examined the effects of recurrent episodes of AD. I hypothesized that shear stress on the vasculature due to recurrent AD would damage the vascular endothelium, exacerbating both PE hyperresponsiveness and AD. I intentionally induced AD over two weeks during recovery from SCI, and examined both severity of AD and arterial function one month after SCI. Animals that were exposed to recurrent AD exhibited attenuated AD in response to the same stimulus. Endothelial-mediated vasodilation was unchanged in arteries from animals that experienced recurrent AD; however, recurrent AD exacerbated PE hyperresponsiveness (by another, unknown mechanism). I conclude that repetitive AD over a relatively short (two week) period can alter the properties of the vasculature.  6.1.3  Significance of the findings  6.1.4  Spinal cord injury-induced changes in the dorsal root ganglion These data demonstrate that SCI has dramatic, injury-level-dependent effects on  the sensory nervous system. For the first time, they demonstrate that the effects of highand low-thoraic SCI on the sensory nervous system are dramatically different. Given that effects of high- and low-thoracic SCI on the DRG differ so dramatically, this begs the question of what other effects might vary between the two injuries. This is of particular concern when we consider that the overwhelming majority of SCI research is conducted in animal models of low-thoracic injury (Ramsey et al., 2010).  175  As a secondary implication, the data reveal multiple effects of high-thoracic SCI on DRGs that might contribute to both pain and AD following SCI. This also suggests that we, as a field, need to zoom out, away from the injured spinal cord, to consider injury-induced effects in peripheral ganglia and the targets that they supply.  6.1.5  Spinal cord injury-induced changes in the mesenteric artery The findings of this dissertation implicate COX-2 activity as a key mediator of  PE hyper-responsveness in the vasculature caudal to SCI. This represents unique mechanistic insight into PE hyper-responsiveness: despite the fact that this was identified as a consequence of SCI more than 35 years ago (Mathias et al., 1976), the roles of vascular COX and/or prostanoids have not been considered. COX-2 upregulation caudal to SCI raises the intriguing possibility that it might represent a therapeutic target for preventing the development of AD following injury. If further (in vivo) experiments support this hypothesis, it will be the first example of a clinically-relevant treatment to prevent the development (rather than treat an acute episode) of AD. The data herin also demonstrate that recurrent episodes of AD can alter the vasculature caudal to SCI. This is the first demonstration of AD-induced changes in the vasculature, and represents a launch pad for an extremely important area of investigation, examining how a lifetime of pathological blood pressure lability alters the cardiovascular end organs.  176  6.2  Strengths The overall strength of the dissertation is its breadth. The work herein describes  a host of changes in sensory ganglia caudal to SCI, identifies a population of sensory neurons that selectively respond to injury and contribute substantially to AD, discovers a vascular mechanism for vascular hyper-responsiveness and reveals consequences of repetitive episodes of AD during recovery from SCI. It represents a starting point for multiple projects investigating peripheral mechanisms of AD. Another strength of this dissertation is the multi-disciplinary nature of the work. In order to study cardiovascular dysfunction after SCI, a working knowledge of cardiovascular physiology, neuroscience, and (in these experiments) vascular pharmacology is required. This has been a considerable challenge. I appreciate the opportunity to combine cardiovascular physiology with neuroscience, and the opportunity to see the chasm between fields: everyone cannot be an expert in everything, and this imposes unfortunate limits on the work that gets done. Designing experiments with a cardiovascular physiologist, a sensory neuroscientist and a vascular pharmacologist is a bit like herding cats, but the advantages far outweigh the challenges. Finally, this dissertation represents a considerable body of work in a notoriously challenging animal model. When I began this work, I wondered why so few laboratories worked with animal models of high-thoracic SCI and (on a related note) examined autonomic outcomes. I have been approached many times at conferences since we published our paper on care of rats with high-thoracic SCI, with thanks, comments and questions on how we do what we do (and also, why we do what we do). In the wee hours of the morning, while administering medications, soft food and enemas, I have often  177  wondered myself. Overcoming challenges associated with animal care were fundamental to the success of these experiments. I hope that our efforts in this regard also represent a meaningful contribution to the SCI literature.  6.3  Limitations Incorporating a long list of limitations into the dissertation is something akin to  painting a target on your back during hunting season. However, I will address a few areas that have potential to raise the reader’s ire.  6.3.1  Breadth versus depth The dissertation is strong in breadth but lacking in depth: in some ways, the lack  of deep probing into any single molecule or mechanism for AD is unsatisfying. This is due in part to technical limitations, but also due to the nature of the experiments: certainly the experiments characterizing the DRG represent entirely new ground in SCI research and are therefore intrinsically exploratory. While I have never compared the effects of peripheral nerve injury (PNI) and SCI directly, I would say from reading the literature that the effects of SCI are more subtle than the effects of PNI. This does not render the effects of SCI less relevant, but it does make them more difficult to observe and to measure: if I had only examined animals with low-thoracic SCI, I might have missed them (or dismissed them) altogether.  178  6.3.2  Proliferation versus hypertrophy of glia in the dorsal root ganglion In Chapter 3, I describe glial and immune cell activation, but do not delineate  the mechanism: is the increase in expression due to proliferation or to the per cell upregulation of characteristic markers? Addressing this question correctly requires a huge amount of investment for a questionably relevant return. While people routinely attempt to count glia packed densely in tissue section under the light microscope, my own experience with this has been patently unsuccessful. One way to count cells reliably is to use confocal microscopy to reliably identify cells with nuclei or co-localized with a proliferation marker, such as Ki-67 (Vukojevic et al., 2009). (In fact, I attempted the latter experiment, but Ki-67 expression seemed extremely variable across animals, even within groups. Perhaps this is why there are no published studies that use it in the rodent DRG.) But the amount of work to count densely-packed glia in three-dimensions is considerable, and to what end? The biologically-relevant consequence of glial activation is the increased presence of glia and glial products. The most interesting question that remains is: what are the functional effects of (micro)glial activation in the DRG? I propose experiments to answer this question at the end of the dissertation.  6.3.3  Relying on immunohistochemistry for protein expression studies In Chapters 2-4, I use immunohistochemistry to characterize changes in  expression in ganglia and blood vessels caudal to SCI. It some cases, it would strengthen the findings if they were supported with immunoblotting. The most obvious example is the COX-2 expression in the vasculature, and I can assure the reader that this omission was not for lack of trying: although the Western blot confirming COX-2 in the vas  179  deferens was convincing, I could not detect COX-2 in arteries, even when arteries from multiple animals were pooled. It has been a frustrating and futile exercise. I have now identified alternate methods of measuring arterial COX-2 expression that I could (and likely will) try, including enzyme-linked immunosorbent assay measurements of arterial homogenate and plasma. Hopefully these will prove more successful. I am not convinced, however, that immunoblotting would add a great deal to my characterization of SCI-induced changes in the DRG. In Chapter 3, the observations are extremely clear at the immunohistochemical level: more importantly, viewing the tissue in section is essential to identify changes in the relevant (cellular) compartment of the DRG. Similarly, in Chapter 2, I found that sensory neurons upregulate TRPV1. This is demonstrated indirectly (or if you prefer, semi-quantitatively) via immunohistochemistry, but at least this method allows one to discern changes in subpopulations of sensory neuronal and glial somata. This issue is particularly germane for GFAP-expression, which is upregulated in Schwann cells, including those in the fibre layer of the DRG, in response to nerve injury (Kirsch et al., 1998)  180  6.4  Clinical ramifications In biomedical research, it is common to have a “so what?” section in the  dissertation: at the end of the day, do your findings have any relevance (immediate, future or potential) for the condition you are modeling in the laboratory? Although I’m not doing clinical research, I’ve had many opportunities to speak about my research with clinical audiences, including people with SCI. These discussions have been incredibly interesting and have influenced the direction of my work. For the sake of variety here, I’ll work from back-to-front and begin with Chapter 5.  6.4.1  Effects of recurrent autonomic dysreflexia Of the experiments in this dissertation, Chapter 5 has the most immediate  clinical ramifications. Taken superficially, the data convey a straightforward message: even though you may not die from an episode of AD, repeated episodes can change your blood vessels forever. Based on my interactions with individuals with SCI over the last six years, this is an extremely important message. I have heard fascinating stories about how people use their AD. Some people use it as an early warning system, to alert them of a full bladder or bowel. Alarmingly, some people use it as an indicator of how long they can delay emptying their bladder or bowel. I have heard from people who dread their bowel routine so much that they only empty their bowels when their AD becomes intolerable. I have spoken with people who use AD as a substitute for sexual pleasure. I have also spoken with people who use AD to overcome an impaired exercise response; while this is well-documented in the literature (Krassioukov, 2012), it has been surprising to learn about the ingenuity and intensity of stimuli that individuals with SCI are using in  181  their own home in order to improve exercise performance. (I will leave the details to the reader’s imagination, but electrodes and testicles are involved.) My study on recurrent AD in the laboratory was a small study, over a short period of time. Nonetheless, recurrent AD altered the mesenteric arteries, raising the issue of what could happen over the lifetime of an individual with SCI.  6.4.2  COX-2 activity in arteries caudal to spinal cord injury In Chapter 4, I found that COX-2 is upregulated in arteries caudal to SCI and  contributes to vascular hyper-responsiveness. These data also have obvious translational potential, the extent of which depends on the outcome of future work (described a few paragraphs onward). If COX-2 inhibitors attenuate the development of AD in animal models, they could be tested in people with SCI. The caveat to long-term use in people with SCI is the link between COX-2 inhibitors and adverse cardiovascular events (Solomon et al., 2005); it is conceivable that people with SCI might be at greater risk for some of these events, such as thromboses in paralyzed limbs. The safety of COX-2 inhibitors (and related compounds) would obviously need to be tested in people with SCI.  6.4.3  Cellular changes in the dorsal root ganglion after spinal cord injury The data in Chapter 3 are descriptive, and may or may not be clinically relevant.  These observations form the foundation for further experiments characterizing the effects of SCI on glial, vascular and ectopic (sympathetic) axonal components of the DRG.  182  These data represent a starting point for research into peripheral mechanisms of AD, rather than an end-point for clinical translation.  6.4.4  Capsaicin-sensitive neurons caudal to spinal cord injury In Chapter 2, I identify a specific population of sensory neurons that both  hypertrophy in response to SCI and contribute substantially to triggering CRD-induced AD. The clinical relevance of these findings is more remote, but still visible on the horizon: it depends in part on whether TRVP1-positive sensory neurons also trigger AD related to bladder distension/inflammation. The data from TRPV1-null mice are suggestive in this regard: genetic deletion of TRPV1 attenuated firing of bladder afferents in response to bladder filling in an ex vivo preparation (Daly et al., 2007). In combination, distension of the bladder and colon account for a large proportion of clinical episodes of AD (Shergill et al., 2004; Krassioukov, 2012). My data represent a proof-of-principle: I would not advise permanently eliminating a population of sensory neurons in people via intrathecal application of a neurotoxin. However, a competitive TRPV1 antagonist has been tested for safety in people (Chizh et al., 2007). This compound (SB-705498) might represent an attractive alternative to vasodilator agents for treatment of AD.  183  6.5  Future directions  “The dignity of movement of an iceberg is due to only one ninth of it being above water.” (Ernest Hemingway) It is a humbling exercise to look back over several years of experiments and wonder what on earth you have been doing. The work in this dissertation sets the stage for a number of important and exciting experiments, and I contemplated calling this section “cool stuff that I really wish I could do”. In biology, we are never at a loss for questions, only time: like most bodies of work, I think that this dissertation has generated more questions than answers. Here I outline which questions I think are most important, and what I would do to answer them.  6.5.1  Cellular changes in the dorsal root ganglion after spinal cord injury In Chapter 3, I describe a number of SCI-induced changes in the DRG – glial and  immune cell activation, angiogenesis and sympathetic sprouting – which profoundly alter the environment of sensory neurons. There are many aspects of this response that could be characterized, including the time course following injury, differential effects in different DRGs (e.g., those rostral and caudal to SCI) and the extent of proliferation versus hypertrophy, as I have already discussed. However, I would relegate this work to the back burner (or perhaps to the second aim of a grant, if I happen to be facing a deadline and lacking imagination), in favour of two more important questions. Do activated glia and immune cells increase excitability of sensory neurons? If so, how? These are questions that are extremely difficult to answer in vivo, and would be better addressed in a reduced (cell culture) system. It would be interesting to harvest glia from  184  animals with SCI (perhaps using laser capture microdissection), expand them in culture and co-culture them with sensory neurons from uninjured animals. In this system, one could record directly from sensory neurons and apply agonists and antagonists targeting glial-derived products. These experiments would be technically challenging, but possible, and would provide real insight into the causal relationship between glial activation and sensory neuron function in DRGs caudal to SCI.  6.5.2  COX-2 activity in arteries caudal to spinal cord injury As I have alluded to multiple times throughout the dissertation, Chapter 4 has  obvious next-step experiments that could dramatically enhance the impact and relevance of the findings. In these experiments, we found that COX-2 activity participates in vascular hyper-responsiveness to sympathetic mimetics caudal to SCI. This raises two obvious questions. The first is mechanistic: which COX-2-derived vasoactive prostanoid(s) mediate(s) its effects after SCI? The second has more clinical relevance: can systemic treatment with a COX-2 or prostanoid inhibitor alleviate AD? In order to address the first question, I would first use immunoblotting of superior and first-order mesenteric arteries to compare vascular expression of thromboxane A2 (TxA2) synthase and prostaglandin E2 (PGE2) synthase in animals with T3 SCI and shaminjured controls. (I am assuming, for the sake of experimental design, that I have overcome the technical problems associated with vascular immunoblotting and/or have had the foresight to hire a molecular biologist). As a reminder, PGE2 is often classified as a vasodilator, but recent data demonstrate that it provokes vasoconstriction in the rat mesenteric artery (Kobayashi et al., 2011). In parallel, I would examine expression of the  185  TxA2 receptor and the EP1 and EP3 PGE2 receptors; both of the latter have been implicated in PGE2-mediated vasoconstriction (Kobayashi et al., 2011; Rutkai et al., 2009). These experiments address the related question of whether SCI regulates expression of these receptors in the vasculature. If I were submitting a grant proposal, I would propose to use the ex vivo perfused mesenteric vascular bed to compare perfusion pressure across the bed (and PE response) in the presence of (first) a COX-2 inhibitor (e.g., celecoxib) and (second) commerciallyavailable antagonists that are selective for the TP (TxA2) receptor or EP1 and EP3 (PGE2). Assuming that I could, by hook, crook or collaboration, get reliable expression data, my hypotheses and priorities would be guided by the expression studies. However, if I were working happily in my own laboratory, I would skip the ex vivo preparation and move straight to administering COX-2 and/or prostanoids antagonists in vivo, to examine the effects on AD. There are several reasons for this. First and foremost, the in vivo model is ours, and I can do all of the work myself. In addition, the results of Chapter 5 demonstrate that PE hyper-responsiveness and severity of AD don’t necessarily vary together. However, PE response is only one variable in the circuit that links sensory input to vasoconstriction. Finally, systemic treatment with a COX-2/prostanoid inhibitor gives us the best chance of seeing something interesting, which could be wholly unrelated to my original hypothesis and observations to date. One the one hand, this is a messy experiment: COX-2 inhibition could have effects on many organ systems affected by SCI. On the other hand, aspirin was the most widely-used drug in the world well before we understood how it worked (Vane and Botting, 2003).  186  6.5.3  Effects of recurrent autonomic dysreflexia In Chapter 5, I found that intentionally inducing AD during recovery from SCI  augmented PE-induced vasoconstriction in the superior mesenteric artery (SMA). Paradoxically, it also reduced the severity of AD examined at one month after SCI. Given that sympathetically-evoked vasoconstriction in the splanchnic bed plays a role in AD (and all evidence, as well as the working knowledge of cardiovascular physiology, suggests that it does) this indicates that there has been some type of adaptation of the sensory branch of the reflex. A simple experiment to test this hypothesis would be to intentionally induce AD via CRD as before, and then compare the severity of CRDinduced AD with AD induced by another stimulus (e.g., bladder distension) at the end of the experiment. A horrendously complicated (though elegant) experiment to test this hypothesis would be to use the in vitro mouse colon preparation developed by Blackshaw and colleagues (Brierley et al., 2004) to determine whether the properties of colonic afferents are altered by repetitive episodes of CRD. Of the experiments I propose here, I am happiest to pass the torch on repetitive CRD to one of my fellow graduate students: performing repetitive CRD is messy, tedious and irritating, for both the experimenter and the subject.  187  6.6  A final word on peripheral plasticity following spinal cord injury In this dissertation, I have found that SCI has profound effects at at least two  peripheral sites that are relevant to AD. However, examining peripheral effects of SCI is clearly a daunting task. Most organ systems are bound to change dramatically in the wake of SCI, and have not been studied. My work suggests that at least some of these changes only succeed high-thoracic SCI, complicating the matter further. There is clear impetus to consider SCI as a whole-body condition, and to move beyond the lesion site as an area of interest.  188  References Adachi K, Yimin Y, Satake K, Matsuyama Y, Ishiguro N, Sawada M, Hirata Y, Kiuchi K (2005) Localization of cyclooxygenase-2 induced following traumatic spinal cord injury. Neurosci Res 51: 73-80. Adeagbo AS, Zhang X, Patel D, Joshua IG, Wang Y, Sun X, Igbo IN, Oriowo MA (2005) Cyclo-oxygenase-2, endothelium and aortic reactivity during deoxycorticosterone acetate salt-induced hypertension. J Hypertens 23: 1025-1036. Agatisa PK, Ness RB, Roberts JM, Costantino JP, Kuller LH, McLaughlin MK (2004) Impairment of endothelial function in women with a history of preeclampsia: an indicator of cardiovascular risk. Am J Physiol Heart Circ Physiol 286: H1389-H1393. Al Dera H, Habgood MD, Furness JB, Brock JA (2011) A prominent contribution of Ltype Ca2+ channels to cutaneous neurovascular transmission that is revealed after spinal cord injury augments vasoconstriction. Am J Physiol Heart Circ Physiol. Published online ahead of print. Alan N, Ramer LM, Inskip JA, Golbidi S, Ramer MS, Laher I, Krassioukov AV (2010) Recurrent autonomic dysreflexia exacerbates vascular dysfunction after spinal cord injury. Spine J 10: 1108-1117. Alexander JK, Popovich PG (2009) Neuroinflammation in spinal cord injury: therapeutic targets for neuroprotection and regeneration. Prog Brain Res 175: 125-137. Allen NJ, Barres BA (2009) Neuroscience: Glia - more than just brain glue. Nature 457: 675-677. Amir R, Devor M (1996) Chemically mediated cross-excitation in rat dorsal root ganglia. J Neurosci 16: 4733-4741. Anderson KD (2004) Targeting recovery: priorities of the spinal cord-injured population. J Neurotrauma 21: 1371-1383. Anderson KD, Borisoff JF, Johnson RD, Stiens SA, Elliott SL (2007) The impact of spinal cord injury on sexual function: concerns of the general population. Spinal Cord 45: 328-337. Araki I (2011) TRP channels in urinary bladder mechanosensation. Adv Exp Med Biol 704: 861-879. Arnold JM, Feng QP, Delaney GA, Teasell RW (1995) Autonomic dysreflexia in tetraplegic patients: evidence for alpha-adrenoceptor hyper-responsiveness. Clin Auton Res 5: 267-270. Asfaw TS, Hypolite J, Northington GM, Arya LA, Wein AJ, Malykhina AP (2011) Acute colonic inflammation triggers detrusor instability via activation of TRPV1 189  receptors in a rat model of pelvic organ cross-sensitization. Am J Physiol Regul Integr Comp Physiol 300: R1392-R1400. Atkinson PP, Atkinson JL (1996) Spinal shock. Mayo Clin Proc 71: 384-389. Axelrod J (1971) Noradrenaline: fate and control of its biosynthesis. Science 173: 598606. Axelrod J, Kopin IJ (1969) The uptake, storage, release and metabolism of noradrenaline in sympathetic nerves. Prog Brain Res 31: 21-32. Azzi G, Bernaudin JF, Bouchaud C, Bellon B, Fleury-Feith J (1990) Permeability of the normal rat brain, spinal cord and dorsal root ganglia microcirculations to immunoglobulins G. Biol Cell 68: 31-36. Bacon SJ, Zagon A, Smith AD (1990) Electron microscopic evidence of a monosynaptic pathway between cells in the caudal raphe nuclei and sympathetic preganglionic neurons in the rat spinal cord. Exp Brain Res 79: 589-602. Baloh RH, Tansey MG, Lampe PA, Fahrner TJ, Enomoto H, Simburger KS, Leitner ML, Araki T, Johnson EM, Jr., Milbrandt J (1998) Artemin, a novel member of the GDNF ligand family, supports peripheral and central neurons and signals through the GFRalpha3-RET receptor complex. Neuron 21: 1291-1302. Bao L, Wang HF, Cai HJ, Tong YG, Jin SX, Lu YJ, Grant G, Hokfelt T, Zhang X (2002) Peripheral axotomy induces only very limited sprouting of coarse myelinated afferents into inner lamina II of rat spinal cord. Eur J Neurosci 16: 175-185. Baron R, Janig W (1991) Afferent and sympathetic neurons projecting into lumbar visceral nerves of the male rat. J Comp Neurol 314: 429-436. Baron R, Janig W, Kollmann W (1988) Sympathetic and afferent somata projecting in hindlimb nerves and the anatomical organization of the lumbar sympathetic nervous system of the rat. J Comp Neurol 275: 460-468. Barrett CJ, Malpas SC (2005) Problems, possibilities, and pitfalls in studying the arterial baroreflexes' influence over long-term control of blood pressure. Am J Physiol Regul Integr Comp Physiol 288: R837-R845. Bedi SS, Yang Q, Crook RJ, Du J, Wu Z, Fishman HM, Grill RJ, Carlton SM, Walters ET (2010) Chronic spontaneous activity generated in the somata of primary nociceptors is associated with pain-related behavior after spinal cord injury. J Neurosci 30: 1487014882. Bell D, McDermott BJ (1996) Calcitonin gene-related peptide in the cardiovascular system: characterization of receptor populations and their (patho)physiological significance. Pharmacol Rev 48: 253-288.  190  Bell D, Zhao Y, McMaster B, McHenry EM, Wang X, Kelso EJ, McDermott BJ (2008) SRIF receptor subtype expression and involvement in positive and negative contractile effects of somatostatin-14 (SRIF-14) in ventricular cardiomyocytes. Cell Physiol Biochem 22: 653-664. Belzer V, Shraer N, Hanani M (2011) Phenotypic changes in satellite glial cells in cultured trigeminal ganglia. Neuron Glia Biol 1-7. Bennett DL, Boucher TJ, Michael GJ, Popat RJ, Malcangio M, Averill SA, Poulsen KT, Priestley JV, Shelton DL, McMahon SB (2006) Artemin has potent neurotrophic actions on injured C-fibres. J Peripher Nerv Syst 11: 330-345. Bennett DL, Michael GJ, Ramachandran N, Munson JB, Averill S, Yan Q, McMahon SB, Priestley JV (1998) A distinct subgroup of small DRG cells express GDNF receptor components and GDNF is protective for these neurons after nerve injury. J Neurosci 18: 3059-3072. Berntson GG, Quigley KS, Fabro VJ, Cacioppo JT (1992) Vagal stimulation and cardiac chronotropy in rats. J Auton Nerv Syst 41: 221-226. Bers DM, Despa S (2009) Sodium-potassium-ATPase: an integral player in the adrenergic fight-or-flight response. Trends Cardiovasc Med 19: 111-118. Berthoud HR, Lynn PA, Blackshaw LA (2001) Vagal and spinal mechanosensors in the rat stomach and colon have multiple receptive fields. Am J Physiol Regul Integr Comp Physiol 280: R1371-R1381. Bhambhani Y, Mactavish J, Warren S, Thompson WR, Webborn A, Bressan E, De Mello MT, Tweedy S, Malone L, Frojd K, Van D, V, Vanlandewijck Y (2010) Boosting in athletes with high-level spinal cord injury: knowledge, incidence and attitudes of athletes in paralympic sport. Disabil Rehabil 32: 2172-2190. Bhattacharya I, Mundy AL, Widmer CC, Kretz M, Barton M (2008) Regional heterogeneity of functional changes in conduit arteries after high-fat diet. Obesity (Silver Spring) 16: 743-748. Birch DJ, Turmaine M, Boulos PB, Burnstock G (2008) Sympathetic innervation of human mesenteric artery and vein. J Vasc Res 45: 323-332. Blacher J, Safar ME (2005) Large-artery stiffness, hypertension and cardiovascular risk in older patients. Nat Clin Pract Cardiovasc Med 2: 450-455. Blackshaw LA, Brookes SJ, Grundy D, Schemann M (2007) Sensory transmission in the gastrointestinal tract. Neurogastroenterol Motil 19: 1-19. Blessing WW (2012) Arterial pressure and blood flow to the tissues. In: The Lower Brainstem and Bodily Homeostasis pp 165-268. New York: Oxford University Press.  191  Brading AF, Ramalingam T (2006) Mechanisms controlling normal defecation and the potential effects of spinal cord injury. Prog Brain Res 152: 345-358. Brain SD, Williams TJ, Tippins JR, Morris HR, MacIntyre I (1985) Calcitonin generelated peptide is a potent vasodilator. Nature 313: 54-56. Braz JM, Nassar MA, Wood JN, Basbaum AI (2005) Parallel "pain" pathways arise from subpopulations of primary afferent nociceptor. Neuron 47: 787-793. Brierley SM, Carter R, Jones W, III, Xu L, Robinson DR, Hicks GA, Gebhart GF, Blackshaw LA (2005) Differential chemosensory function and receptor expression of splanchnic and pelvic colonic afferents in mice. J Physiol 567: 267-281. Brierley SM, Jones RC, III, Gebhart GF, Blackshaw LA (2004) Splanchnic and pelvic mechanosensory afferents signal different qualities of colonic stimuli in mice. Gastroenterology 127: 166-178. Bristow JD, Honour AJ, Pickering GW, Sleight P, Smyth HS (1969) Diminished baroreflex sensitivity in high blood pressure. Circulation 39: 48-54. Brock JA, Yeoh M, McLachlan EM (2006) Enhanced neurally evoked responses and inhibition of norepinephrine reuptake in rat mesenteric arteries after spinal transection. Am J Physiol Heart Circ Physiol 290: H398-H405. Brown A, Ricci MJ, Weaver LC (2007) NGF mRNA is expressed in the dorsal root ganglia after spinal cord injury in the rat. Exp Neurol 205: 283-286. Browning KN, Zheng Z, Kreulen DL, Travagli RA (1999) Two populations of sympathetic neurons project selectively to mesenteric artery or vein. Am J Physiol 276: H1263-H1272. Burns R, Clark VA (2004) Epidural anaesthesia for caesarean section in a patient with quadriplegia and autonomic hyperreflexia. Int J Obstet Anesth 13: 120-123. Burnstock G (2004) Cotransmission. Curr Opin Pharmacol 4: 47-52. Burnstock G (2008) Non-synaptic transmission at autonomic neuroeffector junctions. Neurochem Int 52: 14-25. Busch SA, Horn KP, Cuascut FX, Hawthorne AL, Bai L, Miller RH, Silver J (2010) Adult NG2-positive cells are permissive to neurite outgrowth and stabilize sensory axons during macrophage-induced axonal dieback after spinal cord injury. J Neurosci 30: 255265. Cabot JB, Alessi V, Carroll J, Ligorio M (1994) Spinal cord lamina V and lamina VII interneuronal projections to sympathetic preganglionic neurons. J Comp Neurol 347: 515-530.  192  Calder KB, Estores IM, Krassioukov A (2009) Autonomic dysreflexia and associated acute neurogenic pulmonary edema in a patient with spinal cord injury: a case report and review of the literature. Spinal Cord 47: 423-425. Cameron AA, Smith GM, Randall DC, Brown DR, Rabchevsky AG (2006) Genetic manipulation of intraspinal plasticity after spinal cord injury alters the severity of autonomic dysreflexia. J Neurosci 26: 2923-2932. Camilleri M, Bharucha AE (1996) Gastrointestinal dysfunction in neurologic disease. Semin Neurol 16: 203-216. Carlsten A, Folkow B, Hamburger CA (1957) Cardiovascular effects of direct vagal stimulation in man. Acta Physiol Scand 41: 68-76. Carr PA, Nagy JI (1993) Emerging relationships between cytochemical properties and sensory modality transmission in primary sensory neurons. Brain Res Bull 30: 209-219. Caterina MJ, Rosen TA, Tominaga M, Brake AJ, Julius D (1999) A capsaicin-receptor homologue with a high threshold for noxious heat. Nature 398: 436-441. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D (1997) The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389: 816824. Celio MR (1990) Calbindin D-28k and parvalbumin in the rat nervous system. Neuroscience 35: 375-475. Ceruti S, Fumagalli M, Villa G, Verderio C, Abbracchio MP (2008) Purinoceptormediated calcium signaling in primary neuron-glia trigeminal cultures. Cell Calcium 43: 576-590. Chambers JC, Fusi L, Malik IS, Haskard DO, De Swiet M, Kooner JS (2001) Association of maternal endothelial dysfunction with preeclampsia. JAMA 285: 16071612. Chau D, Johns DG, Schramm LP (2000) Ongoing and stimulus-evoked activity of sympathetically correlated neurons in the intermediate zone and dorsal horn of acutely spinalized rats. J Neurophysiol 83: 2699-2707. Chau D, Kim N, Schramm LP (1997) Sympathetically correlated activity of dorsal horn neurons in spinally transected rats. J Neurophysiol 77: 2966-2974. Chaudhuri KR, Thomaides T, Hernandez P, Alam M, Mathias CJ (1991) Noninvasive quantification of superior mesenteric artery blood flow during sympathoneural activation in normal subjects. Clin Auton Res 1: 37-42. Chen CY, DiCarlo SE, Scislo TJ (1995) Daily spontaneous running attenuated the central gain of the arterial baroreflex. Am J Physiol 268: H662-H669. 193  Cheng Z, Zhang H, Yu J, Wurster RD, Gozal D (2004) Attenuation of baroreflex sensitivity after domoic acid lesion of the nucleus ambiguus of rats. J Appl Physiol 96: 1137-1145. Chizh BA, O'Donnell MB, Napolitano A, Wang J, Brooke AC, Aylott MC, Bullman JN, Gray EJ, Lai RY, Williams PM, Appleby JM (2007) The effects of the TRPV1 antagonist SB-705498 on TRPV1 receptor-mediated activity and inflammatory hyperalgesia in humans. Pain 132: 132-141. Choi JS, Dib-Hajj SD, Waxman SG (2007) Differential slow inactivation and usedependent inhibition of Nav1.8 channels contribute to distinct firing properties in IB4+ and IB4- DRG neurons. J Neurophysiol 97: 1258-1265. Christensen MD, Hulsebosch CE (1997) Spinal cord injury and anti-NGF treatment results in changes in CGRP density and distribution in the dorsal horn in the rat. Exp Neurol 147: 463-475. Christianson JA, Bielefeldt K, Altier C, Cenac N, Davis BM, Gebhart GF, High KW, Kollarik M, Randich A, Undem B, Vergnolle N (2009) Development, plasticity and modulation of visceral afferents. Brain Res Rev 60: 171-186. Chung K, Kevetter GA, Willis WD, Coggeshall RE (1984) An estimate of the ratio of propriospinal to long tract neurons in the sacral spinal cord of the rat. Neurosci Lett 44: 173-177. Chung K, Kim HJ, Na HS, Park MJ, Chung JM (1993) Abnormalities of sympathetic innervation in the area of an injured peripheral nerve in a rat model of neuropathic pain. Neurosci Lett 162: 85-88. Cipollone F, Prontera C, Pini B, Marini M, Fazia M, De Cesare D, Iezzi A, Ucchino S, Boccoli G, Saba V, Chiarelli F, Cuccurullo F, Mezzetti A (2001) Overexpression of functionally coupled cyclooxygenase-2 and prostaglandin E synthase in symptomatic atherosclerotic plaques as a basis of prostaglandin E(2)-dependent plaque instability. Circulation 104: 921-927. Clarke HA, Dekaban GA, Weaver LC (1998) Identification of lamina V and VII interneurons presynaptic to adrenal sympathetic preganglionic neurons in rats using a recombinant herpes simplex virus type 1. Neuroscience 85: 863-872. Claydon VE, Hol AT, Eng JJ, Krassioukov AV (2006a) Cardiovascular responses and postexercise hypotension after arm cycling exercise in subjects with spinal cord injury. Arch Phys Med Rehabil 87: 1106-1114. Claydon VE, Krassioukov AV (2006) Orthostatic hypotension and autonomic pathways after spinal cord injury. J Neurotrauma 23: 1713-1725. Claydon VE, Steeves JD, Krassioukov A (2006b) Orthostatic hypotension following spinal cord injury: understanding clinical pathophysiology. Spinal Cord 44: 341-351. 194  Cocks TM, Angus JA (1983) Endothelium-dependent relaxation of coronary arteries by noradrenaline and serotonin. Nature 305: 627-630. Cohen S, Levi Montalcini R, Hamburger V (1954) A nerve growth stimulating factor isolated from sarcomas 37 and 180. Proc Natl Acad Sci USA 40: 1014-1018. Cruz CD, Charrua A, Vieira E, Valente J, Avelino A, Cruz F (2008) Intrathecal delivery of resiniferatoxin (RTX) reduces detrusor overactivity and spinal expression of TRPV1 in spinal cord injured animals. Exp Neurol 214: 301-308. Cruz Y, Zempoalteca R, Angelica LR, Pacheco P, Hudson R, Martinez-Gomez M (2004) Pattern of sensory innervation of the perineal skin in the female rat. Brain Res 1024: 97103. Curt A, Nitsche B, Rodic B, Schurch B, Dietz V (1997) Assessment of autonomic dysreflexia in patients with spinal cord injury. J Neurol Neurosurg Psychiatry 62: 473477. Dakhil-Jerew F, Brook S, Derry F (2008) Autonomic dysreflexia triggered by breastfeeding in a tetraplegic mother. J Rehabil Med 40: 780-782. Daly D, Rong W, Chess-Williams R, Chapple C, Grundy D (2007) Bladder afferent sensitivity in wild-type and TRPV1 knockout mice. J Physiol 583: 663-674. Dampney RA, Coleman MJ, Fontes MA, Hirooka Y, Horiuchi J, Li YW, Polson JW, Potts PD, Tagawa T (2002) Central mechanisms underlying short- and long-term regulation of the cardiovascular system. Clin Exp Pharmacol Physiol 29: 261-268. Dampney RA, Polson JW, Potts PD, Hirooka Y, Horiuchi J (2003) Functional organization of brain pathways subserving the baroreceptor reflex: studies in conscious animals using immediate early gene expression. Cell Mol Neurobiol 23: 597-616. Dampney RA, Tagawa T, Horiuchi J, Potts PD, Fontes M, Polson JW (2000) What drives the tonic activity of presympathetic neurons in the rostral ventrolateral medulla? Clin Exp Pharmacol Physiol 27: 1049-1053. Davel AP, Wenceslau CF, Akamine EH, Xavier FE, Couto GK, Oliveira HT, Rossoni LV (2011) Endothelial dysfunction in cardiovascular and endocrine-metabolic diseases: an update. Braz J Med Biol Res 44: 920-932. Davies PF (2009) Hemodynamic shear stress and the endothelium in cardiovascular pathophysiology. Nat Clin Pract Cardiovasc Med 6: 16-26. de Champlain J, Krakoff LR, Axelrod J (1966) A reduction in the accumulation of H3norepinephrine in experimental hypertension. Life Sci 5: 2283-2291. de Groot PC, Bleeker MW, Hopman MT (2006) Magnitude and time course of arterial vascular adaptations to inactivity in humans. Exerc Sport Sci Rev 34: 65-71. 195  de Groot PC, Poelkens F, Kooijman M, Hopman MT (2004) Preserved flow-mediated dilation in the inactive legs of spinal cord-injured individuals. Am J Physiol Heart Circ Physiol 287: H374-H380. Dela F, Mohr T, Jensen CM, Haahr HL, Secher NH, Biering-Sorensen F, Kjaer M (2003) Cardiovascular control during exercise: insights from spinal cord-injured humans. Circulation 107: 2127-2133. Deng YS, Zhong JH, Zhou XF (2000) BDNF is involved in sympathetic sprouting in the dorsal root ganglia following peripheral nerve injury in rats. Neurotox Res 1: 311-322. Deuchars SA, Morrison SF, Gilbey MP (1995) Medullary-evoked EPSPs in neonatal rat sympathetic preganglionic neurones in vitro. J Physiol 487 ( Pt 2): 453-463. Deuchars SA, Spyer KM, Gilbey MP (1997) Stimulation within the rostral ventrolateral medulla can evoke monosynaptic GABAergic IPSPs in sympathetic preganglionic neurons in vitro. J Neurophysiol 77: 229-235. Devor M, Wall PD (1990) Cross-excitation in dorsal root ganglia of nerve-injured and intact rats. J Neurophysiol 64: 1733-1746. DiBona GF, Kopp UC (1997) Neural control of renal function. Physiol Rev 77: 75-197. DiPette DJ, Schwarzenberger K, Kerr N, Holland OB (1989) Dose-dependent systemic and regional hemodynamic effects of calcitonin gene-related peptide. Am J Med Sci 297: 65-70. Ditunno JF, Little JW, Tessler A, Burns AS (2004) Spinal shock revisited: a four-phase model. Spinal Cord 42: 383-395. Dodd J, Solter D, Jessell TM (1984) Monoclonal antibodies against carbohydrate differentiation antigens identify subsets of primary sensory neurones. Nature 311: 469472. Duijvestijn AM, van Goor H, Klatter F, Majoor GD, van Bussel E, Breda Vriesman PJ (1992) Antibodies defining rat endothelial cells: RECA-1, a pan-endothelial cell-specific monoclonal antibody. Lab Invest 66: 459-466. Edvardsson B, Persson S (2010) Reversible cerebral vasoconstriction syndrome associated with autonomic dysreflexia. J Headache Pain 11: 277-280. Edvinsson L (1985) Characterization of the contractile effect of neuropeptide Y in feline cerebral arteries. Acta Physiol Scand 125: 33-41. Edwards LA, Bugaresti JM, Buchholz AC (2008) Visceral adipose tissue and the ratio of visceral to subcutaneous adipose tissue are greater in adults with than in those without spinal cord injury, despite matching waist circumferences. Am J Clin Nutr 87: 600-607.  196  Eid SR (2011) Therapeutic targeting of TRP channels--the TR(i)P to pain relief. Curr Top Med Chem 11: 2118-2130. Eisenhofer G (2001) The role of neuronal and extraneuronal plasma membrane transporters in the inactivation of peripheral catecholamines. Pharmacol Ther 91: 35-62. Ekland MB, Krassioukov AV, McBride KE, Elliott SL (2008) Incidence of autonomic dysreflexia and silent autonomic dysreflexia in men with spinal cord injury undergoing sperm retrieval: implications for clinical practice. J Spinal Cord Med 31: 33-39. Elitt CM, McIlwrath SL, Lawson JJ, Malin SA, Molliver DC, Cornuet PK, Koerber HR, Davis BM, Albers KM (2006) Artemin overexpression in skin enhances expression of TRPV1 and TRPA1 in cutaneous sensory neurons and leads to behavioral sensitivity to heat and cold. J Neurosci 26: 8578-8587. Elliott S, Krassioukov A (2006) Malignant autonomic dysreflexia in spinal cord injured men. Spinal Cord 44: 386-392. Eltorai I, Kim R, Vulpe M, Kasravi H, Ho W (1992) Fatal Cerebral-Hemorrhage Due to Autonomic Dysreflexia in A Tetraplegic Patient - Case-Report and Review. Paraplegia 30: 355-360. Esler M, Leonard P, Kelleher D, Jackman G, Bobik A, Skews H, Jennings G, Korner P (1980) Assessment of neuronal uptake of noradrenaline in humans: defective uptake in some patients with essential hypertension. Clin Exp Pharmacol Physiol 7: 535-539. Everaerts W, Gevaert T, Nilius B, De Ridder D (2008) On the origin of bladder sensing: Tr(i)ps in urology. Neurourol Urodyn 27: 264-273. Fadel PJ, Raven PB (2011) Human investigations into the arterial and cardiopulmonary baroreflexes during exercise. Exp Physiol. Faghri PD, Yount JP, Pesce WJ, Seetharama S, Votto JJ (2001) Circulatory hypokinesis and functional electric stimulation during standing in persons with spinal cord injury. Arch Phys Med Rehabil 82: 1587-1595. Fang X, Djouhri L, McMullan S, Berry C, Waxman SG, Okuse K, Lawson SN (2006) Intense isolectin-B4 binding in rat dorsal root ganglion neurons distinguishes C-fiber nociceptors with broad action potentials and high Nav1.9 expression. J Neurosci 26: 7281-7292. Feletou M, Huang Y, Vanhoutte PM (2010a) Vasoconstrictor prostanoids. Pflugers Arch 459: 941-950. Feletou M, Moreau N, Duhault J (1994) Vascular responsiveness in young, diabetic, and aging hyperinsulinemic rats. Life Sci 54: 1801-1813.  197  Feletou M, Vanhoutte PM, Verbeuren TJ (2010b) The thromboxane/endoperoxide receptor (TP): the common villain. J Cardiovasc Pharmacol 55: 317-332. Feliciano L, Henning RJ (1998) Vagal nerve stimulation releases vasoactive intestinal peptide which significantly increases coronary artery blood flow. Cardiovasc Res 40: 4555. Forsythe E, Horsewell JE (2006) Sexual rehabilitation of women with a spinal cord injury. Spinal Cord 44: 234-241. Freeman R, Wieling W, Axelrod FB, Benditt DG, Benarroch E, Biaggioni I, Cheshire WP, Chelimsky T, Cortelli P, Gibbons CH, Goldstein DS, Hainsworth R, Hilz MJ, Jacob G, Kaufmann H, Jordan J, Lipsitz LA, Levine BD, Low PA, Mathias C, Raj SR, Robertson D, Sandroni P, Schatz I, Schondorff R, Stewart JM, van Dijk JG (2011) Consensus statement on the definition of orthostatic hypotension, neurally mediated syncope and the postural tachycardia syndrome. Clin Auton Res 21: 69-72. Frisbie JH, Steele DJ (1997) Postural hypotension and abnormalities of salt and water metabolism in myelopathy patients. Spinal Cord 35: 303-307. Fry DL (1968) Acute Vascular Endothelial Changes Associated with Increased Blood Velocity Gradients. Circulation Research 22: 165-&. Furchgott RF, Zawadzki JV (1980) The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288: 373-376. Furlan JC, Fehlings MG, Shannon P, Norenberg MD, Krassioukov AV (2003) Descending vasomotor pathways in humans: correlation between axonal preservation and cardiovascular dysfunction after spinal cord injury. J Neurotrauma 20: 1351-1363. Furness JB, Marshall JM (1974) Correlation of the directly observed responses of mesenteric vessles of the rat to nerve stimulation and noradrenaline with the distribution of adrenergic nerves. J Physiol 239: 75-88. Galli SJ, Kalesnikoff J, Grimbaldeston MA, Piliponsky AM, Williams CM, Tsai M (2005) Mast cells as "tunable" effector and immunoregulatory cells: recent advances. Annu Rev Immunol 23: 749-786. Galligan JJ, Hess MC, Miller SB, Fink GD (2001) Differential localization of P2 receptor subtypes in mesenteric arteries and veins of normotensive and hypertensive rats. J Pharmacol Exp Ther 296: 478-485. Gao J, Zhao J, Rayner SE, Van Helden DF (1999) Evidence that the ATP-induced increase in vasomotion of guinea-pig mesenteric lymphatics involves an endotheliumdependent release of thromboxane A2. Br J Pharmacol 127: 1597-1602.  198  Garshick E, Kelley A, Cohen SA, Garrison A, Tun CG, Gagnon D, Brown R (2005) A prospective assessment of mortality in chronic spinal cord injury. Spinal Cord 43: 408416. Gitterman DP, Evans RJ (2001) Nerve evoked P2X receptor contractions of rat mesenteric arteries; dependence on vessel size and lack of role of L-type calcium channels and calcium induced calcium release. Br J Pharmacol 132: 1201-1208. Giuffrida R, Rustioni A (1992) Dorsal root ganglion neurons projecting to the dorsal column nuclei of rats. J Comp Neurol 316: 206-220. Giuliano F, Rampin O, Bernabe J, Rousseau JP (1995) Neural control of penile erection in the rat. J Auton Nerv Syst 55: 36-44. Glickman S, Kamm MA (1996) Bowel dysfunction in spinal-cord-injury patients. Lancet 347: 1651-1653. Goldstein DS, Horwitz D, Keiser HR, Polinsky RJ, Kopin IJ (1983) Plasma l[3H]norepinephrine, d-[14C]norepinephrine, and d,l-[3H]isoproterenol kinetics in essential hypertension. J Clin Invest 72: 1748-1758. Gonzalez F, Chang JY, Banovac K, Messina D, Martinez-Arizala A, Kelley RE (1991) Autoregulation of cerebral blood flow in patients with orthostatic hypotension after spinal cord injury. Paraplegia 29: 1-7. Goodness TP, Albers KM, Davis FE, Davis BM (1997) Overexpression of nerve growth factor in skin increases sensory neuron size and modulates Trk receptor expression. Eur J Neurosci 9: 1574-1585. Gosselin RD, Suter MR, Ji RR, Decosterd I (2010) Glial cells and chronic pain. Neuroscientist 16: 519-531. Graeber MB (2010) Changing face of microglia. Science 330: 783-788. Greenway CV (1983) Role of splanchnic venous system in overall cardiovascular homeostasis. Fed Proc 42: 1678-1684. Greenway CV, Lister GE (1974) Capacitance effects and blood reservoir function in the splanchnic vascular bed during non-hypotensive haemorrhage and blood volume expansion in anaesthetized cats. J Physiol 237: 279-294. Guimaraes S, Moura D (2001) Vascular adrenoceptors: an update. Pharmacol Rev 53: 319-356. Guo Z, Su W, Allen S, Pang H, Daugherty A, Smart E, Gong MC (2005) COX-2 upregulation and vascular smooth muscle contractile hyperreactivity in spontaneous diabetic db/db mice. Cardiovasc Res 67: 723-735.  199  Guttmann L, Frankel HL, Paeslack V (1965) Cardiac irregularities during labour in paraplegic women. Paraplegia 3: 144-151. Guyenet PG. (2012) Role of the ventral medulla oblongata in blood pressure regulation. In: Central Regulation of Autonomic Functions. (Loewy AD SK, ed), pp 145-167. New York: Oxford University Press. Guyenet PG (2006) The sympathetic control of blood pressure. Nat Rev Neurosci 7: 335346. Habler H, Eschenfelder S, Liu XG, Janig W (2000) Sympathetic-sensory coupling after L5 spinal nerve lesion in the rat and its relation to changes in dorsal root ganglion blood flow. Pain 87: 335-345. Hadley MN, Walters BC, Grabb PA, Oyesiku NM, Przybylski GJ, Resnick DK, Ryken TC, Mielke DH (2002) Guidelines for the management of acute cervical spine and spinal cord injuries. Clin Neurosurg 49: 407-498. Hainsworth R (1986) Vascular capacitance: its control and importance. Rev Physiol Biochem Pharmacol 105: 101-173. HAMBURGER V, LEVI-MONTALCINI R (1949) Proliferation, differentiation and degeneration in the spinal ganglia of the chick embryo under normal and experimental conditions. J Exp Zool 111: 457-501. Hanani M (2005) Satellite glial cells in sensory ganglia: from form to function. Brain Res Brain Res Rev 48: 457-476. Hanani M (2010) Satellite glial cells in sympathetic and parasympathetic ganglia: in search of function. Brain Res Rev 64: 304-327. Helkowski WM, Ditunno JF, Jr., Boninger M (2003) Autonomic dysreflexia: incidence in persons with neurologically complete and incomplete tetraplegia. J Spinal Cord Med 26: 244-247. Henning RJ, Sawmiller DR (2001) Vasoactive intestinal peptide: cardiovascular effects. Cardiovasc Res 49: 27-37. Herring N, Lokale MN, Danson EJ, Heaton DA, Paterson DJ (2008) Neuropeptide Y reduces acetylcholine release and vagal bradycardia via a Y2 receptor-mediated, protein kinase C-dependent pathway. J Mol Cell Cardiol 44: 477-485. Herrmann J, Lerman A (2001) The endothelium: dysfunction and beyond. J Nucl Cardiol 8: 197-206. Hofstetter CP, Card JP, Olson L (2005) A spinal cord pathway connecting primary afferents to the segmental sympathetic outflow system. Exp Neurol 194: 128-138.  200  Holzer P, Lippe IT (1992) Role of calcitonin gene-related peptide in gastrointestinal blood flow. Ann N Y Acad Sci 657: 228-239. Honma Y, Araki T, Gianino S, Bruce A, Heuckeroth R, Johnson E, Milbrandt J (2002) Artemin is a vascular-derived neurotropic factor for developing sympathetic neurons. Neuron 35: 267-282. Hopman MTE, Groothuis JT, Flendrie M, Gerrits KHL, Houtman S (2002) Increased vascular resistance in paralyzed legs after spinal cord injury is reversible by training. Journal of Applied Physiology 93: 1966-1972. Hottenstein OD, Kreulen DL (1987) Comparison of the frequency dependence of venous and arterial responses to sympathetic nerve stimulation in guinea-pigs. J Physiol 384: 153-167. Hou S, Duale H, Cameron AA, Abshire SM, Lyttle TS, Rabchevsky AG (2008) Plasticity of lumbosacral propriospinal neurons is associated with the development of autonomic dysreflexia after thoracic spinal cord transection. J Comp Neurol 509: 382399. Hou S, Duale H, Rabchevsky AG (2009) Intraspinal sprouting of unmyelinated pelvic afferents after complete spinal cord injury is correlated with autonomic dysreflexia induced by visceral pain. Neuroscience 159: 369-379. House JG, Stiens SA (1997) Pharmacologically initiated defecation for persons with spinal cord injury: effectiveness of three agents. Arch Phys Med Rehabil 78: 1062-1065. Illman A, Stiller K, Williams M (2000) The prevalence of orthostatic hypotension during physiotherapy treatment in patients with an acute spinal cord injury. Spinal Cord 38: 741747. Imai Y, Ibata I, Ito D, Ohsawa K, Kohsaka S (1996) A novel gene iba1 in the major histocompatibility complex class III region encoding an EF hand protein expressed in a monocytic lineage. Biochem Biophys Res Commun 224: 855-862. Imai Y, Kohsaka S (2002) Intracellular signaling in M-CSF-induced microglia activation: role of Iba1. Glia 40: 164-174. Iniguez MA, Cacheiro-Llaguno C, Cuesta N, Diaz-Munoz MD, Fresno M (2008) Prostanoid function and cardiovascular disease. Arch Physiol Biochem 114: 201-209. Inskip J, Plunet W, Ramer L, Ramsey JB, Yung A, Kozlowski P, Ramer M, Krassioukov A (2010) Cardiometabolic risk factors in experimental spinal cord injury. J Neurotrauma 27: 275-285. Inskip JA, Ramer LM, Ramer MS, Krassioukov AV (2009) Autonomic assessment of animals with spinal cord injury: tools, techniques and translation. Spinal Cord 47: 2-35.  201  Intengan HD, Schiffrin EL (2001) Vascular remodeling in hypertension: roles of apoptosis, inflammation, and fibrosis. Hypertension 38: 581-587. Irie S, Tavassoli M (1991) Transendothelial transport of macromolecules: the concept of tissue-blood barriers. Cell Biol Rev 25: 317-1. Ishii H, Niioka T, Izumi H (2010) Vagal visceral inputs to the nucleus of the solitary tract: involvement in a parasympathetic reflex vasodilator pathway in the rat masseter muscle. Brain Res 1312: 41-53. Ito D, Imai Y, Ohsawa K, Nakajima K, Fukuuchi Y, Kohsaka S (1998) Microgliaspecific localisation of a novel calcium binding protein, Iba1. Brain Res Mol Brain Res 57: 1-9. Jackson CR, Acland R (2011) Knowledge of autonomic dysreflexia in the emergency department. Emerg Med J 28: 866-869. Jacob JE, Gris P, Fehlings MG, Weaver LC, Brown A (2003) Autonomic dysreflexia after spinal cord transection or compression in 129Sv, C57BL, and Wallerian degeneration slow mutant mice. Exp Neurol 183: 136-146. Jacobs JM, Macfarlane RM, Cavanagh JB (1976) Vascular leakage in the dorsal root ganglia of the rat, studied with horseradish peroxidase. J Neurol Sci 29: 95-107. Jacobs PL, Nash MS (2004) Exercise recommendations for individuals with spinal cord injury. Sports Med 34: 727-751. Janig W, Habler HJ (2003) Neurophysiological analysis of target-related sympathetic pathways--from animal to human: similarities and differences. Acta Physiol Scand 177: 255-274. Janig W, McLachlan EM (1992) Characteristics of function-specific pathways in the sympathetic nervous system. Trends Neurosci 15: 475-481. Jansen AS, Nguyen XV, Karpitskiy V, Mettenleiter TC, Loewy AD (1995) Central command neurons of the sympathetic nervous system: basis of the fight-or-flight response. Science 270: 644-646. Jasmin L, Vit JP, Bhargava A, Ohara PT (2010) Can satellite glial cells be therapeutic targets for pain control? Neuron Glia Biol 6: 63-71. Jin X, Satoh-Otonashi Y, Zamami Y, Koyama T, Sun P, Kitamura Y, Kawasaki H (2008) Characterization of the inhibitory effect of vascular endothelium on agonist-induced vasoconstriction in rat mesenteric resistance arteries. J Pharmacol Sci 108: 95-103. Jin X, Satoh-Otonashi Y, Zamami Y, Takatori S, Hashikawa-Hobara N, Kitamura Y, Kawasaki H (2011) New molecular mechanisms for cardiovascular disease: contribution  202  of endothelium-derived hyperpolarizing factor in the regulation of vasoconstriction in peripheral resistance arteries. J Pharmacol Sci 116: 332-336. Johnson EM, Jr., Yip HK (1985) Central nervous system and peripheral nerve growth factor provide trophic support critical to mature sensory neuronal survival. Nature 314: 751-752. Johnson RH (1976) Orthostatic hypotension in neurological disease. Cardiology 61 suppl 1: 150-167. Jones JF (2001) Vagal control of the rat heart. Exp Physiol 86: 797-801. Kara T, Narkiewicz K, Somers VK (2003) Chemoreflexes--physiology and clinical implications. Acta Physiol Scand 177: 377-384. Karemaker JM (2002) Why do we measure baroreflex sensitivity the way we do? Clin Auton Res 12: 427-428. Karmy G, Carr PA, Yamamoto T, Chan SH, Nagy JI (1991) Cytochrome oxidase immunohistochemistry in rat brain and dorsal root ganglia: visualization of enzyme in neuronal perikarya and in parvalbumin-positive neurons. Neuroscience 40: 825-839. Kawasaki H, Saito A, Takasaki K (1990) Changes in calcitonin gene-related peptide (CGRP)-containing vasodilator nerve activity in hypertension. Brain Res 518: 303-307. Kawasaki H, Takasaki K, Saito A, Goto K (1988) Calcitonin gene-related peptide acts as a novel vasodilator neurotransmitter in mesenteric resistance vessels of the rat. Nature 335: 164-167. Keast JR, de Groat WC (1989) Immunohistochemical characterization of pelvic neurons which project to the bladder, colon, or penis in rats. J Comp Neurol 288: 387-400. Keast JR, de Groat WC (1992) Segmental distribution and peptide content of primary afferent neurons innervating the urogenital organs and colon of male rats. J Comp Neurol 319: 615-623. Khan F, Belch JJF, MacLeod M, Mires G (2005) Changes in endothelial function precede the clinical disease in women in whom preeclampsia develops. Hypertension 46: 1123-1128. Khazaei M, Moien-Afshari F, Kieffer TJ, Laher I (2008a) Effect of Exercise on Augmented Aortic Vasoconstriction in the db/db Mouse Model of Type-II Diabetes. Physiological Research 57: 847-856. Khazaei M, Moien-Afshari F, Laher I (2008b) Vascular endothelial function in health and diseases. Pathophysiology 15: 49-67.  203  Kietadisorn R, Juni RP, Moens AL (2011) Tackling endothelial dysfunction by modulating NOS-uncoupling: new insights in pathogenesis and therapeutic possibilities. Am J Physiol Endocrinol Metab. Kirsch M, Schneider T, Lee MY, Hofmann HD (1998) Lesion-induced changes in the expression of ciliary neurotrophic factor and its receptor in rat optic nerve. Glia 23: 239248. Kirshblum SC, House JG, O'connor KC (2002) Silent autonomic dysreflexia during a routine bowel program in persons with traumatic spinal cord injury: a preliminary study. Arch Phys Med Rehabil 83: 1774-1776. Kissin I, Szallasi A (2011) Therapeutic targeting of TRPV1 by resiniferatoxin, from preclinical studies to clinical trials. Curr Top Med Chem 11: 2159-2170. Klusakova I, Dubovy P (2009) Experimental models of peripheral neuropathic pain based on traumatic nerve injuries - an anatomical perspective. Ann Anat 191: 248-259. Kobayashi K, Murata T, Hori M, Ozaki H (2011) Prostaglandin E2-prostanoid EP3 signal induces vascular contraction via nPKC and ROCK activation in rat mesenteric artery. Eur J Pharmacol 660: 375-380. Kobayashi S, Mwaka ES, Baba H, Kokubo Y, Yayama T, Kubota M, Nakajima H, Meir A (2010) Microvascular system of the lumbar dorsal root ganglia in rats. Part II: neurogenic control of intraganglionic blood flow. J Neurosurg Spine 12: 203-209. Kobayashi S, Mwaka ES, Meir A, Uchida K, Takeno K, Miyazaki T, Kubota M, Nakajima H, Nomura E, Yoshizawa H, Baba H (2009) Vasomotion of intraradicular microvessels in rat. Spine (Phila Pa 1976 ) 34: 990-997. Kooijman M, Thijssen DH, de Groot PC, Bleeker MW, van Kuppevelt HJ, Green DJ, Rongen GA, Smits P, Hopman MT (2008) Flow-mediated dilatation in the superficial femoral artery is nitric oxide mediated in humans. J Physiol 586: 1137-1145. Krassioukov A (2004) Autonomic dysreflexia in acute spinal cord injury: incidence, mechanisms, and management. SCI Nurs 21: 215-216. Krassioukov A (2012) Autonomic dysreflexia: current evidence related to unstable arterial blood pressure control among athletes with spinal cord injury. Clin J Sport Med 22: 39-45. Krassioukov A, Claydon VE (2006) The clinical problems in cardiovascular control following spinal cord injury: an overview. Prog Brain Res 152: 223-229. Krassioukov A, Warburton DE, Teasell R, Eng JJ (2009) A systematic review of the management of autonomic dysreflexia after spinal cord injury. Arch Phys Med Rehabil 90: 682-695.  204  Krassioukov AV, Bunge RP, Pucket WR, Bygrave MA (1999) The changes in human spinal sympathetic preganglionic neurons after spinal cord injury. Spinal Cord 37: 6-13. Krassioukov AV, Furlan JC, Fehlings MG (2003) Autonomic dysreflexia in acute spinal cord injury: An under-recognized clinical entity. Journal of Neurotrauma 20: 707-716. Krassioukov AV, Johns DG, Schramm LP (2002) Sensitivity of sympathetically correlated spinal interneurons, renal sympathetic nerve activity, and arterial pressure to somatic and visceral stimuli after chronic spinal injury. J Neurotrauma 19: 1521-1529. Krassioukov AV, Weaver LC (1995a) Episodic hypertension due to autonomic dysreflexia in acute and chronic spinal cord-injured rats. Am J Physiol 268: H2077H2083. Krassioukov AV, Weaver LC (1995b) Reflex and morphological changes in spinal preganglionic neurons after cord injury in rats. Clin Exp Hypertens 17: 361-373. Krassioukov AV, Weaver LC (1996) Morphological changes in sympathetic preganglionic neurons after spinal cord injury in rats. Neuroscience 70: 211-225. Krenz NR, Meakin SO, Krassioukov AV, Weaver LC (1999) Neutralizing intraspinal nerve growth factor blocks autonomic dysreflexia caused by spinal cord injury. J Neurosci 19: 7405-7414. Krenz NR, Weaver LC (1998) Sprouting of primary afferent fibers after spinal cord transection in the rat. Neuroscience 85: 443-458. Kreulen DL (1986) Activation of mesenteric arteries and veins by preganglionic and postganglionic nerves. Am J Physiol 251: H1267-H1275. Kreulen DL (2003) Properties of the venous and arterial innervation in the mesentery. J Smooth Muscle Res 39: 269-279. Krogh K, Mosdal C, Laurberg S (2000) Gastrointestinal and segmental colonic transit times in patients with acute and chronic spinal cord lesions. Spinal Cord 38: 615-621. Krum H, Louis WJ, Brown DJ, Howes LG (1992) Pressor dose responses and baroreflex sensitivity in quadriplegic spinal cord injury patients. J Hypertens 10: 245-250. Krum H, Louis WJ, Brown DJ, Jackman GP, Howes LG (1991) Diurnal blood pressure variation in quadriplegic chronic spinal cord injury patients. Clin Sci (Lond) 80: 271-276. Kruse MN, Bray LA, de Groat WC (1995) Influence of spinal cord injury on the morphology of bladder afferent and efferent neurons. J Auton Nerv Syst 54: 215-224. Kummer W (2011) Pulmonary vascular innervation and its role in responses to hypoxia: size matters! Proc Am Thorac Soc 8: 471-476.  205  Kushnir R, Cherkas PS, Hanani M (2011) Peripheral inflammation upregulates P2X receptor expression in satellite glial cells of mouse trigeminal ganglia: a calcium imaging study. Neuropharmacology 61: 739-746. Laird AS, Carrive P, Waite PM (2006) Cardiovascular and temperature changes in spinal cord injured rats at rest and during autonomic dysreflexia. J Physiol 577: 539-548. Laird AS, Carrive P, Waite PM (2009) Effect of treadmill training on autonomic dysreflexia in spinal cord--injured rats. Neurorehabil Neural Repair 23: 910-920. Lais LT, Brody MJ (1975) Mechanism of vascular hyperresponsiveness in the spontaneously hypertensive rat. Circ Res 36: 216-222. Lais LT, Brody MJ (1978) Vasoconstrictor hyperresponsiveness: an early pathogenic mechanism in the spontaneously hypertensive rat. Eur J Pharmacol 47: 177-189. Laskey W, Polosa C (1988) Characteristics of the sympathetic preganglionic neuron and its synaptic input. Prog Neurobiol 31: 47-84. Legler DF, Bruckner M, Uetz-von Allmen E, Krause P (2010) Prostaglandin E2 at new glance: novel insights in functional diversity offer therapeutic chances. Int J Biochem Cell Biol 42: 198-201. Leman S, Bernet F, Sequeira H (2000) Autonomic dysreflexia increases plasma adrenaline level in the chronic spinal cord-injured rat. Neurosci Lett 286: 159-162. Lewis ME, Al Khalidi AH, Bonser RS, Clutton-Brock T, Morton D, Paterson D, Townend JN, Coote JH (2001) Vagus nerve stimulation decreases left ventricular contractility in vivo in the human and pig heart. J Physiol 534: 547-552. Li J, Wang DH (2003) High-salt-induced increase in blood pressure: role of capsaicinsensitive sensory nerves. J Hypertens 21: 577-582. Li M, Kuo L, Stallone JN (2008) Estrogen potentiates constrictor prostanoid function in female rat aorta by upregulation of cyclooxygenase-2 and thromboxane pathway expression. Am J Physiol Heart Circ Physiol 294: H2444-H2455. Li M, Shi J, Tang JR, Chen D, Ai B, Chen J, Wang LN, Cao FY, Li LL, Lin CY, Guan XM (2005) Effects of complete Freund's adjuvant on immunohistochemical distribution of IL-1beta and IL-1R I in neurons and glia cells of dorsal root ganglion. Acta Pharmacol Sin 26: 192-198. Li Y, Duckles SP (1993) Effect of age on vascular content of calcitonin gene-related peptide and mesenteric vasodilator nerve activity in the rat. Eur J Pharmacol 236: 373378. Lindan R, Joiner E, Freehafer AA, Hazel C (1980) Incidence and clinical features of autonomic dysreflexia in patients with spinal cord injury. Paraplegia 18: 285-292. 206  Linsenmeyer TA, Campagnolo DI, Chou IH (1996) Silent autonomic dysreflexia during voiding in men with spinal cord injuries. J Urol 155: 519-522. Liu CN, Chambers WW (1958) Intraspinal sprouting of dorsal root axons; development of new collaterals and preterminals following partial denervation of the spinal cord in the cat. AMA Arch Neurol Psychiatry 79: 46-61. Liu S, Wilcox DA, Sieber-Blum M, Wong-Riley M (1990) Developing neural crest cells in culture: correlation of cytochrome oxidase activity with SSEA-1 and dopamine-betahydroxylase immunoreactivity. Brain Res 535: 271-280. Llewellyn-Smith IJ (2009) Anatomy of synaptic circuits controlling the activity of sympathetic preganglionic neurons. J Chem Neuroanat 38: 231-239. Llewellyn-Smith IJ, Cassam AK, Krenz NR, Krassioukov AV, Weaver LC (1997) Glutamate- and GABA-immunoreactive synapses on sympathetic preganglionic neurons caudal to a spinal cord transection in rats. Neuroscience 80: 1225-1235. Llewellyn-Smith IJ, DiCarlo SE, Collins HL, Keast JR (2005) Enkephalinimmunoreactive interneurons extensively innervate sympathetic preganglionic neurons regulating the pelvic viscera. J Comp Neurol 488: 278-289. Llewellyn-Smith IJ, Weaver LC (2001) Changes in synaptic inputs to sympathetic preganglionic neurons after spinal cord injury. J Comp Neurol 435: 226-240. Llewellyn-Smith IJ, Weaver LC, Keast JR (2006) Effects of spinal cord injury on synaptic inputs to sympathetic preganglionic neurons. Prog Brain Res 152: 11-26. Lucin KM, Sanders VM, Jones TB, Malarkey WB, Popovich PG (2007) Impaired antibody synthesis after spinal cord injury is level dependent and is due to sympathetic nervous system dysregulation. Exp Neurol 207: 75-84. Luff SE (1996) Ultrastructure of sympathetic axons and their structural relationship with vascular smooth muscle. Anat Embryol (Berl) 193: 515-531. Lujan HL, Chen Y, DiCarlo SE (2009) Paraplegia increased cardiac NGF content, sympathetic tonus, and the susceptibility to ischemia-induced ventricular tachycardia in conscious rats. Am J Physiol Heart Circ Physiol 296: H1364-H1372. Lujan HL, DiCarlo SE (2007) T5 spinal cord transection increases susceptibility to reperfusion-induced ventricular tachycardia by enhancing sympathetic activity in conscious rats. Am J Physiol Heart Circ Physiol 293: H3333-H3339. Lujan HL, Palani G, DiCarlo SE (2010a) Structural neuroplasticity following T5 spinal cord transection: increased cardiac sympathetic innervation density and SPN arborization. Am J Physiol Regul Integr Comp Physiol 299: R985-R995.  207  Lujan HL, Palani G, Peduzzi JD, DiCarlo SE (2010b) Targeted ablation of mesenteric projecting sympathetic neurons reduces the hemodynamic response to pain in conscious, spinal cord-transected rats. Am J Physiol Regul Integr Comp Physiol 298: R1358-R1365. Lundgren O (1983) Role of splanchnic resistance vessels in overall cardiovascular homeostasis. Fed Proc 42: 1673-1677. Lynn PA, Blackshaw LA (1999) In vitro recordings of afferent fibres with receptive fields in the serosa, muscle and mucosa of rat colon. J Physiol 518 ( Pt 1): 271-282. MacDermid VE, McPhail LT, Tsang B, Rosenthal A, Davies A, Ramer MS (2004) A soluble Nogo receptor differentially affects plasticity of spinally projecting axons. Eur J Neurosci 20: 2567-2579. Macho P, Perez R, Huidobro-Toro JP, Domenech RJ (1989) Neuropeptide Y (NPY): a coronary vasoconstrictor and potentiator of catecholamine-induced coronary constriction. Eur J Pharmacol 167: 67-74. Macias MY, Syring MB, Pizzi MA, Crowe MJ, Alexanian AR, Kurpad SN (2006) Pain with no gain: allodynia following neural stem cell transplantation in spinal cord injury. Exp Neurol 201: 335-348. Mackeprang T, Andersson JE, Jensen SB, Rosenkvist HC (1992) [Sexological evaluation, referral and treatment in the county psychiatric care. A retrospective study of a sexological patient population referred to a department of general psychiatry]. Ugeskr Laeger 154: 719-724. Maiorov DN, Weaver LC, Krassioukov AV (1997) Relationship between sympathetic activity and arterial pressure in conscious spinal rats. Am J Physiol 272: H625-H631. Malin S, Molliver D, Christianson JA, Schwartz ES, Cornuet P, Albers KM, Davis BM (2011) TRPV1 and TRPA1 function and modulation are target tissue dependent. J Neurosci 31: 10516-10528. Markos F, Snow HM (2006) Vagal postganglionic origin of vasoactive intestinal polypeptide (VIP) mediating the vagal tachycardia. Eur J Appl Physiol 98: 419-422. Marti E, Gibson SJ, Polak JM, Facer P, Springall DR, Van Aswegen G, Aitchison M, Koltzenburg M (1987) Ontogeny of peptide- and amine-containing neurones in motor, sensory, and autonomic regions of rat and human spinal cord, dorsal root ganglia, and rat skin. J Comp Neurol 266: 332-359. Martin S, Levine AK, Chen ZJ, Ughrin Y, Levine JM (2001) Deposition of the NG2 proteoglycan at nodes of Ranvier in the peripheral nervous system. J Neurosci 21: 81198128.  208  Martin W, Furchgott RF, Villani GM, Jothianandan D (1986) Depression of contractile responses in rat aorta by spontaneously released endothelium-derived relaxing factor. J Pharmacol Exp Ther 237: 529-538. Matheson CR, Carnahan J, Urich JL, Bocangel D, Zhang TJ, Yan Q (1997) Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor for sensory neurons: comparison with the effects of the neurotrophins. J Neurobiol 32: 22-32. Mathias CJ (1995) Orthostatic hypotension: causes, mechanisms, and influencing factors. Neurology 45: S6-11. Mathias CJ (2006) Orthostatic hypotension and paroxysmal hypertension in humans with high spinal cord injury. Prog Brain Res 152: 231-243. Mathias CJ, Frankel HL (1988) Cardiovascular control in spinal man. Annu Rev Physiol 50: 577-592. Mathias CJ, Frankel HL, Christensen NJ, Spalding JM (1976) Enhanced pressor response to noradrenaline in patients with cervical spinal cord transection. Brain 99: 757-770. Matos-Souza JR, Pithon KR, Ozahata TM, Gemignani T, Cliquet A, Jr., Nadruz W, Jr. (2009) Carotid intima-media thickness is increased in patients with spinal cord injury independent of traditional cardiovascular risk factors. Atherosclerosis 202: 29-31. Mayorov DN, Adams MA, Krassioukov AV (2001) Telemetric blood pressure monitoring in conscious rats before and after compression injury of spinal cord. J Neurotrauma 18: 727-736. McBride F, Quah SP, Scott ME, Dinsmore WW (2003) Tripling of blood pressure by sexual stimulation in a man with spinal cord injury. J R Soc Med 96: 349-350. McGillivray CF, Hitzig SL, Craven BC, Tonack MI, Krassioukov AV (2009) Evaluating knowledge of autonomic dysreflexia among individuals with spinal cord injury and their families. J Spinal Cord Med 32: 54-62. McKanna JA, Zhang MZ, Wang JL, Cheng H, Harris RC (1998) Constitutive expression of cyclooxygenase-2 in rat vas deferens. Am J Physiol 275: R227-R233. McKay SM, McLachlan EM (2004) Inflammation of rat dorsal root ganglia below a midthoracic spinal transection. Neuroreport 15: 1783-1786. McLachlan EM, Brock JA (2006) Adaptations of peripheral vasoconstrictor pathways after spinal cord injury. Prog Brain Res 152: 289-297. McLachlan EM, Janig W, Devor M, Michaelis M (1993) Peripheral nerve injury triggers noradrenergic sprouting within dorsal root ganglia. Nature 363: 543-546.  209  McManis PG, Low PA (1988) Factors affecting the relative viability of centrifascicular and subperineurial axons in acute peripheral nerve ischemia. Exp Neurol 99: 84-95. McManis PG, Schmelzer JD, Zollman PJ, Low PA (1997) Blood flow and autoregulation in somatic and autonomic ganglia. Comparison with sciatic nerve. Brain 120 ( Pt 3): 445449. Mendizabal Y, Llorens S, Nava E (2011) Reactivity of the aorta and mesenteric resistance arteries from the obese spontaneously hypertensive rat: effects of glitazones. Am J Physiol Heart Circ Physiol 301: H1319-H1330. Michael GJ, Priestley JV (1999) Differential expression of the mRNA for the vanilloid receptor subtype 1 in cells of the adult rat dorsal root and nodose ganglia and its downregulation by axotomy. J Neurosci 19: 1844-1854. Miller CO, Johns DG, Schramm LP (2001) Spinal interneurons play a minor role in generating ongoing renal sympathetic nerve activity in spinally intact rats. Brain Res 918: 101-106. Miller KE, Richards BA, Kriebel RM (2002) Glutamine-, glutamine synthetase-, glutamate dehydrogenase- and pyruvate carboxylase-immunoreactivities in the rat dorsal root ganglion and peripheral nerve. Brain Res 945: 202-211. Miller RJ, Jung H, Bhangoo SK, White FA (2009) Cytokine and chemokine regulation of sensory neuron function. Handb Exp Pharmacol 417-449. Mills PB, Krassioukov A (2011) Autonomic function as a missing piece of the classification of Paralympic athletes with spinal cord injury. Spinal Cord 49: 768-776. Miyatani M, Masani K, Oh PI, Miyachi M, Popovic MR, Craven BC (2009) Pulse wave velocity for assessment of arterial stiffness among people with spinal cord injury: a pilot study. J Spinal Cord Med 32: 72-78. Miyoshi S, Sekiguchi M, Konno S, Kikuchi S, Kanaya F (2011) Increased expression of vascular endothelial growth factor protein in dorsal root ganglion exposed to nucleus pulposus on the nerve root in rats. Spine (Phila Pa 1976 ) 36: E1-E6. Mizisin AP, Weerasuriya A (2011) Homeostatic regulation of the endoneurial microenvironment during development, aging and in response to trauma, disease and toxic insult. Acta Neuropathol 121: 291-312. Moalem G, Tracey DJ (2006) Immune and inflammatory mechanisms in neuropathic pain. Brain Res Rev 51: 240-264. Moller K, Zhang YZ, Hakanson R, Luts A, Sjolund B, Uddman R, Sundler F (1993) Pituitary adenylate cyclase activating peptide is a sensory neuropeptide: immunocytochemical and immunochemical evidence. Neuroscience 57: 725-732.  210  Moran MM, McAlexander MA, Biro T, Szallasi A (2011) Transient receptor potential channels as therapeutic targets. Nat Rev Drug Discov 10: 601-620. Morgan C, Nadelhaft I, de Groat WC (1981) The distribution of visceral primary afferents from the pelvic nerve to Lissauer's tract and the spinal gray matter and its relationship to the sacral parasympathetic nucleus. J Comp Neurol 201: 415-440. Morrison SF (2011) 2010 Carl Ludwig Distinguished Lectureship of the APS Neural Control and Autonomic Regulation Section: Central neural pathways for thermoregulatory cold defense. J Appl Physiol 110: 1137-1149. Munakata M, Kameyama J, Kanazawa M, Nunokawa T, Moriai N, Yoshinaga K (1997) Circadian blood pressure rhythm in patients with higher and lower spinal cord injury: simultaneous evaluation of autonomic nervous activity and physical activity. J Hypertens 15: 1745-1749. Murray F, Bell D, Kelso EJ, Millar BC, McDermott BJ (2001) Positive and negative contractile effects of somatostatin-14 on rat ventricular cardiomyocytes. J Cardiovasc Pharmacol 37: 324-332. Myers J, Lee M, Kiratli J (2007) Cardiovascular disease in spinal cord injury: an overview of prevalence, risk, evaluation, and management. Am J Phys Med Rehabil 86: 142-152. Nadelhaft I, Booth AM (1984) The location and morphology of preganglionic neurons and the distribution of visceral afferents from the rat pelvic nerve: a horseradish peroxidase study. J Comp Neurol 226: 238-245. Nash MS, Bilsker S, Marcillo AE, Isaac SM, Botelho LA, Klose KJ, Green BA, Rountree MT, Shea JD (1991) Reversal of adaptive left ventricular atrophy following electrically-stimulated exercise training in human tetraplegics. Paraplegia 29: 590-599. Nash MS, Mendez AJ (2007) A guideline-driven assessment of need for cardiovascular disease risk intervention in persons with chronic paraplegia. Arch Phys Med Rehabil 88: 751-757. Nilsson H, Goldstein M, Nilsson O (1986) Adrenergic innervation and neurogenic response in large and small arteries and veins from the rat. Acta Physiol Scand 126: 121133. Nilsson O, Booj S, Dahlstrom A, Hargens AR, Millard RW, Pettersson KS (1988) Sympathetic innervation of the cardiovascular system in the giraffe. Blood Vessels 25: 299-307. Norel X (2007) Prostanoid receptors in the human vascular wall. ScientificWorldJournal 7: 1359-1374.  211  O'Shea JE, Evans BK (1985) Innervation of bat heart: cholinergic and adrenergic nerves innervate all chambers. Am J Physiol 249: H876-H882. Ohara PT, Vit JP, Bhargava A, Romero M, Sundberg C, Charles AC, Jasmin L (2009) Gliopathic pain: when satellite glial cells go bad. Neuroscientist 15: 450-463. Okajima K, Harada N (2006) Regulation of inflammatory responses by sensory neurons: molecular mechanism(s) and possible therapeutic applications. Curr Med Chem 13: 2241-2251. Ono M (2008) Molecular links between tumor angiogenesis and inflammation: inflammatory stimuli of macrophages and cancer cells as targets for therapeutic strategy. Cancer Sci 99: 1501-1506. Osborn JW, Jacob F, Guzman P (2005) A neural set point for the long-term control of arterial pressure: beyond the arterial baroreceptor reflex. Am J Physiol Regul Integr Comp Physiol 288: R846-R855. Osborn JW, Taylor RF, Schramm LP (1990) Chronic Cervical Spinal-Cord Injury and Autonomic Hyperreflexia in Rats. American Journal of Physiology 258: R169-R174. Otoshi K, Kikuchi S, Konno S, Sekiguchi M (2010) The reactions of glial cells and endoneurial macrophages in the dorsal root ganglion and their contribution to painrelated behavior after application of nucleus pulposus onto the nerve root in rats. Spine (Phila Pa 1976 ) 35: 264-271. Pakdeechote P, Rummery NM, Ralevic V, Dunn WR (2007) Raised tone reveals purinergic-mediated responses to sympathetic nerve stimulation in the rat perfused mesenteric vascular bed. Eur J Pharmacol 563: 180-186. Pannese E (1981) The satellite cells of the sensory ganglia. Adv Anat Embryol Cell Biol 65: 1-111. Pannese E (2010) The structure of the perineuronal sheath of satellite glial cells (SGCs) in sensory ganglia. Neuron Glia Biol 6: 3-10. Pannese E, Procacci P (2002) Ultrastructural localization of NGF receptors in satellite cells of the rat spinal ganglia. J Neurocytol 31: 755-763. Park J, Galligan JJ, Fink GD, Swain GM (2007) Differences in sympathetic neuroeffector transmission to rat mesenteric arteries and veins as probed by in vitro continuous amperometry and video imaging. J Physiol 584: 819-834. Park J, Galligan JJ, Fink GD, Swain GM (2010) Alterations in sympathetic neuroeffector transmission to mesenteric arteries but not veins in DOCA-salt hypertension. Auton Neurosci 152: 11-20.  212  Perlmuter LC, Sarda G, Casavant V, O'Hara K, Hindes M, Knott PT, Mosnaim AD (2011) A review of orthostatic blood pressure regulation and its association with mood and cognition. Clin Auton Res. Published online ahead of print. Petras JM, Cummings JF (1972) Autonomic neurons in the spinal cord of the Rhesus monkey: a correlation of the findings of cytoarchitectonics and sympathectomy with fiber degeneration following dorsal rhizotomy. J Comp Neurol 146: 189-218. Phillips JK, Hickey H, Hill CE (2000) Heterogeneity in mechanisms underlying vasodilatory responses in small arteries of the rat hepatic mesentery. Auton Neurosci 83: 159-170. Pohl U, Holtz J, Busse R, Bassenge E (1986) Crucial role of endothelium in the vasodilator response to increased flow in vivo. Hypertension 8: 37-44. Potter EK (1985) Prolonged non-adrenergic inhibition of cardiac vagal action following sympathetic stimulation: neuromodulation by neuropeptide Y? Neurosci Lett 54: 117121. Prieto D, Buus CL, Mulvany MJ, Nilsson H (2000) Neuropeptide Y regulates intracellular calcium through different signalling pathways linked to a Y(1)-receptor in rat mesenteric small arteries. Br J Pharmacol 129: 1689-1699. Protas L, Robinson RB (2008) Dissecting the NPY signaling cascade between cardiac sympathetic and parasympathetic nerves. J Mol Cell Cardiol 44: 470-472. Qamar MI, Read AE (1987) Effects of exercise on mesenteric blood flow in man. Gut 28: 583-587. Qiao L, Vizzard MA (2002) Up-regulation of tyrosine kinase (Trka, Trkb) receptor expression and phosphorylation in lumbosacral dorsal root ganglia after chronic spinal cord (T8-T10) injury. J Comp Neurol 449: 217-230. Qin W, Bauman WA, Cardozo C (2010) Bone and muscle loss after spinal cord injury: organ interactions. Ann N Y Acad Sci 1211: 66-84. Rabchevsky AG (2006) Segmental organization of spinal reflexes mediating autonomic dysreflexia after spinal cord injury. Prog Brain Res 152: 265-274. Racchi H, Schliem AJ, Donoso MV, Rahmer A, Zuniga A, Guzman S, Rudolf K, Huidobro-Toro JP (1997) Neuropeptide Y Y1 receptors are involved in the vasoconstriction caused by human sympathetic nerve stimulation. Eur J Pharmacol 329: 79-83. Ralevic V, Belai A, Burnstock G (1993) Impaired sensory-motor nerve function in the isolated mesenteric arterial bed of streptozotocin-diabetic and ganglioside-treated streptozotocin-diabetic rats. Br J Pharmacol 110: 1105-1111.  213  Ramer LM, Borisoff JF, Ramer MS (2004) Rho-kinase inhibition enhances axonal plasticity and attenuates cold hyperalgesia after dorsal rhizotomy. J Neurosci 24: 1079610805. Ramer MS, Bisby MA (1999) Adrenergic innervation of rat sensory ganglia following proximal or distal painful sciatic neuropathy: distinct mechanisms revealed by anti-NGF treatment. Eur J Neurosci 11: 837-846. Ramer MS, Bradbury EJ, McMahon SB (2001) Nerve growth factor induces P2X(3) expression in sensory neurons. J Neurochem 77: 864-875. Ramer MS, Bradbury EJ, Michael GJ, Lever IJ, McMahon SB (2003) Glial cell linederived neurotrophic factor increases calcitonin gene-related peptide immunoreactivity in sensory and motoneurons in vivo. Eur J Neurosci 18: 2713-2721. Ramer MS, Kawaja MD, Henderson JT, Roder JC, Bisby MA (1998) Glial overexpression of NGF enhances neuropathic pain and adrenergic sprouting into DRG following chronic sciatic constriction in mice. Neurosci Lett 251: 53-56. Ramer MS, Thompson SW, McMahon SB (1999) Causes and consequences of sympathetic basket formation in dorsal root ganglia. Pain Suppl 6: S111-S120. Ramsey JB, Ramer LM, Inskip JA, Alan N, Ramer MS, Krassioukov AV (2010) Care of rats with complete high-thoracic spinal cord injury. J Neurotrauma 27: 1709-1722. Rehman A, Schiffrin EL (2010) Vascular effects of antihypertensive drug therapy. Curr Hypertens Rep 12: 226-232. Relevic V, Rubino A, Burnstock G (1996) Augmented sensory-motor vasodilatation of the rat mesenteric arterial bed after chronic infusion of the P1-purinoceptor antagonist, DPSPX. Br J Pharmacol 118: 1675-1680. Ren K, Dubner R (2010) Interactions between the immune and nervous systems in pain. Nat Med 16: 1267-1276. Rexed B (1952) The cytoarchitectonic organization of the spinal cord in the cat. J Comp Neurol 96: 414-495. Rezajooi K, Pavlides M, Winterbottom J, Stallcup WB, Hamlyn PJ, Lieberman AR, Anderson PN (2004) NG2 proteoglycan expression in the peripheral nervous system: upregulation following injury and comparison with CNS lesions. Mol Cell Neurosci 25: 572-584. Rice PA, Boehm GW, Moynihan JA, Bellinger DL, Stevens SY (2001) Chemical sympathectomy increases the innate immune response and decreases the specific immune response in the spleen to infection with Listeria monocytogenes. J Neuroimmunol 114: 19-27.  214  Rich KM, Yip HK, Osborne PA, Schmidt RE, Johnson EM, Jr. (1984) Role of nerve growth factor in the adult dorsal root ganglia neuron and its response to injury. J Comp Neurol 230: 110-118. Rizzoni D, De Ciuceis C, Porteri E, Paiardi S, Boari GE, Mortini P, Cornali C, Cenzato M, Rodella LF, Borsani E, Rizzardi N, Platto C, Rezzani R, Rosei EA (2009) Altered structure of small cerebral arteries in patients with essential hypertension. J Hypertens 27: 838-845. Rizzoni D, Porteri E, Platto C, Rizzardi N, De Ciuceis C, Boari GE, Muiesan ML, Salvetti M, Zani F, Miclini M, Paiardi S, Castellano M, Rosei EA (2007) Morning rise of blood pressure and subcutaneous small resistance artery structure. J Hypertens 25: 16981703. Rohrer DK, Bernstein D, Chruscinski A, Desai KH, Schauble E, Kobilka BK (1998) The developmental and physiological consequences of disrupting genes encoding beta 1 and beta 2 adrenoceptors. Adv Pharmacol 42: 499-501. Rose RD, Rohrlich D (1988) Counting sectioned cells via mathematical reconstruction. J Comp Neurol 272: 365-386. Ross CA, Ruggiero DA, Park DH, Joh TH, Sved AF, Fernandez-Pardal J, Saavedra JM, Reis DJ (1984) Tonic vasomotor control by the rostral ventrolateral medulla: effect of electrical or chemical stimulation of the area containing C1 adrenaline neurons on arterial pressure, heart rate, and plasma catecholamines and vasopressin. J Neurosci 4: 474-494. Rowell LB (2012) The splanchnic circulation. In: The Peripheral Circulations pp 163192. Grune and Stratton Inc. Rummery NM, Brock JA, Pakdeechote P, Ralevic V, Dunn WR (2007) ATP is the predominant sympathetic neurotransmitter in rat mesenteric arteries at high pressure. J Physiol 582: 745-754. Rummery NM, Tripovic D, McLachlan EM, Brock JA (2010) Sympathetic vasoconstriction is potentiated in arteries caudal but not rostral to a spinal cord transection in rats. J Neurotrauma 27: 2077-2089. Rutkai I, Feher A, Erdei N, Henrion D, Papp Z, Edes I, Koller A, Kaley G, Bagi Z (2009) Activation of prostaglandin E2 EP1 receptor increases arteriolar tone and blood pressure in mice with type 2 diabetes. Cardiovasc Res 83: 148-154. Sachidanandam K, Hutchinson JR, Elgebaly MM, Mezzetti EM, Wang MH, Ergul A (2009) Differential effects of diet-induced dyslipidemia and hyperglycemia on mesenteric resistance artery structure and function in type 2 diabetes. J Pharmacol Exp Ther 328: 123-130.  215  Safar ME, Levy BI, Struijker-Boudier H (2003) Current perspectives on arterial stiffness and pulse pressure in hypertension and cardiovascular diseases. Circulation 107: 28642869. Sanders VM, Straub RH (2002) Norepinephrine, the beta-adrenergic receptor, and immunity. Brain Behav Immun 16: 290-332. Sandkuhler J (2009) Models and mechanisms of hyperalgesia and allodynia. Physiol Rev 89: 707-758. Schiffrin EL (2004) Remodeling of resistance arteries in essential hypertension and effects of antihypertensive treatment. Am J Hypertens 17: 1192-1200. Schmid A, Huonker M, Barturen JM, Stahl F, Schmidt-Trucksass A, Konig D, Grathwohl D, Lehmann M, Keul J (1998) Catecholamines, heart rate, and oxygen uptake during exercise in persons with spinal cord injury. J Appl Physiol 85: 635-641. Scholz J, Woolf CJ (2007) The neuropathic pain triad: neurons, immune cells and glia. Nat Neurosci 10: 1361-1368. Schramm LP (2006) Spinal sympathetic interneurons: their identification and roles after spinal cord injury. Prog Brain Res 152: 27-37. Scotland RS, Chauhan S, Vallance PJ, Ahluwalia A (2001) An endothelium-derived hyperpolarizing factor-like factor moderates myogenic constriction of mesenteric resistance arteries in the absence of endothelial nitric oxide synthase-derived nitric oxide. Hypertension 38: 833-839. Scott AL, Borisoff JF, Ramer MS (2005) Deafferentation and neurotrophin-mediated intraspinal sprouting: a central role for the p75 neurotrophin receptor. Eur J Neurosci 21: 81-92. Seki S, Sasaki K, Fraser MO, Igawa Y, Nishizawa O, Chancellor MB, de Groat WC, Yoshimura N (2002) Immunoneutralization of nerve growth factor in lumbosacral spinal cord reduces bladder hyperreflexia in spinal cord injured rats. J Urol 168: 2269-2274. Sellers MM, Stallone JN (2008) Sympathy for the devil: the role of thromboxane in the regulation of vascular tone and blood pressure. Am J Physiol Heart Circ Physiol 294: H1978-H1986. Shehab SA, Spike RC, Todd AJ (2003) Evidence against cholera toxin B subunit as a reliable tracer for sprouting of primary afferents following peripheral nerve injury. Brain Res 964: 218-227. Shergill IS, Arya M, Hamid R, Khastgir J, Patel HR, Shah PJ (2004) The importance of autonomic dysreflexia to the urologist. BJU Int 93: 923-926.  216  Shimokawa H, Yasutake H, Fujii K, Owada MK, Nakaike R, Fukumoto Y, Takayanagi T, Nagao T, Egashira K, Fujishima M, Takeshita A (1996) The importance of the hyperpolarizing mechanism increases as the vessel size decreases in endotheliumdependent relaxations in rat mesenteric circulation. J Cardiovasc Pharmacol 28: 703-711. Shinder V, Amir R, Devor M (1998) Cross-excitation in dorsal root ganglia does not depend on close cell-to-cell apposition. Neuroreport 9: 3997-4000. Sidorov EV, Townson AF, Dvorak MF, Kwon BK, Steeves J, Krassioukov A (2008) Orthostatic hypotension in the first month following acute spinal cord injury. Spinal Cord 46: 65-69. Silva DM, Gomes-Filho A, Olivon VC, Santos TM, Becker LK, Santos RA, Lemos VS (2011) Swimming training improves the vasodilator effect of angiotensin-(1-7) in the aorta of spontaneously hypertensive rat. J Appl Physiol 111: 1272-1277. Skryma R, Prevarskaya N, Gkika D, Shuba Y (2011) From urgency to frequency: facts and controversies of TRPs in the lower urinary tract. Nat Rev Urol 8: 617-630. Smith WL (1986) Prostaglandin biosynthesis and its compartmentation in vascular smooth muscle and endothelial cells. Annu Rev Physiol 48: 251-262. Smyth HS, Sleight P, Pickering GW (1969) Reflex regulation of arterial pressure during sleep in man. A quantitative method of assessing baroreflex sensitivity. Circ Res 24: 109121. Solomon SD, McMurray JJ, Pfeffer MA, Wittes J, Fowler R, Finn P, Anderson WF, Zauber A, Hawk E, Bertagnolli M (2005) Cardiovascular risk associated with celecoxib in a clinical trial for colorectal adenoma prevention. N Engl J Med 352: 1071-1080. Sporkova A, Perez-Rivera A, Galligan JJ (2010) Interaction between alpha(1)- and alpha(2)-adrenoreceptors contributes to enhanced constrictor effects of norepinephrine in mesenteric veins compared to arteries. Eur J Pharmacol 643: 239-246. Steins SA, Johnson MC, Lyman PJ (1995) Cardiac rehabilitation in patients with spinal cord injuries. Physical medicine and rehabilitation clinics of North America 6: 263-296. Stephenson JL, Byers MR (1995) GFAP immunoreactivity in trigeminal ganglion satellite cells after tooth injury in rats. Exp Neurol 131: 11-22. Stone JM, Nino-Murcia M, Wolfe VA, Perkash I (1990) Chronic gastrointestinal problems in spinal cord injury patients: a prospective analysis. Am J Gastroenterol 85: 1114-1119. Strack AM, Sawyer WB, Marubio LM, Loewy AD (1988) Spinal origin of sympathetic preganglionic neurons in the rat. Brain Res 455: 187-191.  217  Strauss DJ, Devivo MJ, Paculdo DR, Shavelle RM (2006) Trends in life expectancy after spinal cord injury. Archives of Physical Medicine and Rehabilitation 87: 1079-1085. Suadicani SO, Cherkas PS, Zuckerman J, Smith DN, Spray DC, Hanani M (2010) Bidirectional calcium signaling between satellite glial cells and neurons in cultured mouse trigeminal ganglia. Neuron Glia Biol 6: 43-51. Sudo E, Ishii H, Niioka T, Hirai T, Izumi H (2009) Parasympathetic vasodilator fibers in rat digastric muscle. Brain Res 1302: 125-131. Suzuki H (1981) Effects of endogenous and exogenous noradrenaline on the smooth muscle of guinea-pig mesenteric vein. J Physiol 321: 495-512. Sved AF, Cano G, Card JP (2001) Neuroanatomical specificity of the circuits controlling sympathetic outflow to different targets. Clin Exp Pharmacol Physiol 28: 115-119. Tabatai M, Booth AM, de Groat WC (1986) Morphological and electrophysiological properties of pelvic ganglion cells in the rat. Brain Res 382: 61-70. Takahara Y, Maeda M, Nakatani T, Kiyama H (2007) Transient suppression of the vesicular acetylcholine transporter in urinary bladder pathways following spinal cord injury. Brain Res 1137: 20-28. Takeda M, Takahashi M, Matsumoto S (2008) Contribution of activated interleukin receptors in trigeminal ganglion neurons to hyperalgesia via satellite glial interleukin1beta paracrine mechanism. Brain Behav Immun 22: 1016-1023. Takeda M, Takahashi M, Matsumoto S (2009) Contribution of the activation of satellite glia in sensory ganglia to pathological pain. Neurosci Biobehav Rev 33: 784-792. Takeda M, Tanimoto T, Kadoi J, Nasu M, Takahashi M, Kitagawa J, Matsumoto S (2007) Enhanced excitability of nociceptive trigeminal ganglion neurons by satellite glial cytokine following peripheral inflammation. Pain 129: 155-166. Taktakishvili OM, Lin LH, Vanderheyden AD, Nashelsky MB, Talman WT (2010) Nitroxidergic innervation of human cerebral arteries. Auton Neurosci 156: 152-153. Tamura S, Morikawa Y, Senba E (2005) TRPV2, a capsaicin receptor homologue, is expressed predominantly in the neurotrophin-3-dependent subpopulation of primary sensory neurons. Neuroscience 130: 223-228. Tan AM, Chang YW, Zhao P, Hains BC, Waxman SG (2011) Rac1-regulated dendritic spine remodeling contributes to neuropathic pain after peripheral nerve injury. Exp Neurol 232: 222-233. Tan AM, Waxman SG (2011) Spinal cord injury, dendritic spine remodeling, and spinal memory mechanisms. Exp Neurol. Published online ahead of print.  218  Tang X, Neckel ND, Schramm LP (2003) Locations and morphologies of sympathetically correlated neurons in the T(10) spinal segment of the rat. Brain Res 976: 185-193. Tang X, Neckel ND, Schramm LP (2004) Spinal interneurons infected by renal injection of pseudorabies virus in the rat. Brain Res 1004: 1-7. Teasell RW, Arnold JM, Krassioukov A, Delaney GA (2000) Cardiovascular consequences of loss of supraspinal control of the sympathetic nervous system after spinal cord injury. Arch Phys Med Rehabil 81: 506-516. Thijssen DH, Kooijman M, de Groot PC, Bleeker MW, Smits P, Green DJ, Hopman MT (2008) Endothelium-dependent and -independent vasodilation of the superficial femoral artery in spinal cord-injured subjects. J Appl Physiol 104: 1387-1393. Thomas GD (2011) Neural control of the circulation. Adv Physiol Educ 35: 28-32. Todd ME (1980) Development of adrenergic innervation in rat peripheral vessels: a fluorescence microscopic study. J Anat 131: 121-133. Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, Raumann BE, Basbaum AI, Julius D (1998) The cloned capsaicin receptor integrates multiple painproducing stimuli. Neuron 21: 531-543. Tong YG, Wang HF, Ju G, Grant G, Hokfelt T, Zhang X (1999) Increased uptake and transport of cholera toxin B-subunit in dorsal root ganglion neurons after peripheral axotomy: possible implications for sensory sprouting. J Comp Neurol 404: 143-158. Tripovic D, Al Abed A, Rummery NM, Johansen NJ, McLachlan EM, Brock JA (2011) Nerve-evoked constriction of rat tail veins is potentiated and venous diameter is reduced after chronic spinal cord transection. J Neurotrauma 28: 821-829. Uceyler N, Tscharke A, Sommer C (2007) Early cytokine expression in mouse sciatic nerve after chronic constriction nerve injury depends on calpain. Brain Behav Immun 21: 553-560. Ulphani JS, Cain JH, Inderyas F, Gordon D, Gikas PV, Shade G, Mayor D, Arora R, Kadish AH, Goldberger JJ (2010) Quantitative analysis of parasympathetic innervation of the porcine heart. Heart Rhythm 7: 1113-1119. Vaidyanathan S, Soni BM, Singh G, Hughes PL, Pulya K, Oo T (2011) Infarct of the right basal ganglia in a male spinal cord injury patient: adverse effect of autonomic dysreflexia. ScientificWorldJournal 11: 666-672. Vane JR, Botting RM (2003) The mechanism of action of aspirin. Thromb Res 110: 255258. Vanhoutte PM (2009) COX-1 and vascular disease. Clin Pharmacol Ther 86: 212-215. 219  Vassort G (2001) Adenosine 5'-triphosphate: a P2-purinergic agonist in the myocardium. Physiol Rev 81: 767-806. Vaziri ND (2003) Nitric oxide in microgravity-induced orthostatic intolerance: relevance to spinal cord injury. J Spinal Cord Med 26: 5-11. Vera PL, Nadelhaft I (1992) Afferent and sympathetic innervation of the dome and the base of the urinary bladder of the female rat. Brain Res Bull 29: 651-658. Virdis A, Colucci R, Versari D, Ghisu N, Fornai M, Antonioli L, Duranti E, Daghini E, Giannarelli C, Blandizzi C, Taddei S, Del Tacca M (2009) Atorvastatin prevents endothelial dysfunction in mesenteric arteries from spontaneously hypertensive rats: role of cyclooxygenase 2-derived contracting prostanoids. Hypertension 53: 1008-1016. Vizzard MA (2006) Neurochemical plasticity and the role of neurotrophic factors in bladder reflex pathways after spinal cord injury. Prog Brain Res 152: 97-115. Vukojevic K, Skobic H, Saraga-Babic M (2009) Proliferation and differentiation of glial and neuronal progenitors in the development of human spinal ganglia. Differentiation 78: 91-98. Wahman K, Nash MS, Lewis JE, Seiger A, Levi R (2010) Increased cardiovascular disease risk in Swedish persons with paraplegia: The Stockholm spinal cord injury study. J Rehabil Med 42: 489-492. Wang X, Lou N, Xu Q, Tian GF, Peng WG, Han X, Kang J, Takano T, Nedergaard M (2006) Astrocytic Ca2+ signaling evoked by sensory stimulation in vivo. Nat Neurosci 9: 816-823. Ward SM, Bayguinov J, Won KJ, Grundy D, Berthoud HR (2003) Distribution of the vanilloid receptor (VR1) in the gastrointestinal tract. J Comp Neurol 465: 121-135. Watanabe H, Ishii H, Niioka T, Yamamuro M, Izumi H (2008) Occurrence of parasympathetic vasodilator fibers in the lower lip of the guinea-pig. J Comp Physiol B 178: 297-305. Weaver LC (2002) What causes autonomic dysreflexia after spinal cord injury? Clin Auton Res 12: 424-426. Weaver LC, Marsh DR, Gris D, Meakin SO, Dekaban GA (2002) Central mechanisms for autonomic dysreflexia after spinal cord injury. Prog Brain Res 137: 83-95. Weaver LC, Verghese P, Bruce JC, Fehlings MG, Krenz NR, Marsh DR (2001) Autonomic dysreflexia and primary afferent sprouting after clip-compression injury of the rat spinal cord. J Neurotrauma 18: 1107-1119.  220  Weick M, Cherkas PS, Hartig W, Pannicke T, Uckermann O, Bringmann A, Tal M, Reichenbach A, Hanani M (2003) P2 receptors in satellite glial cells in trigeminal ganglia of mice. Neuroscience 120: 969-977. Whiteneck GG, Charlifue SW, Frankel HL, Fraser MH, Gardner BP, Gerhart KA, Krishnan KR, Menter RR, Nuseibeh I, Short DJ, . (1992) Mortality, morbidity, and psychosocial outcomes of persons spinal cord injured more than 20 years ago. Paraplegia 30: 617-630. Widlansky ME, Gokce N, Keaney JF, Jr., Vita JA (2003) The clinical implications of endothelial dysfunction. J Am Coll Cardiol 42: 1149-1160. Willis W.D., Coggeshall R.E. (2004) Sensory Mechanisms of the Spinal Cord: Primary Afferent Neurons and the Spinal Dorsal Horn. New York: Kluwer Academic/Plenum Publishers. Wong J, Oblinger MM (1991) NGF rescues substance P expression but not neurofilament or tubulin gene expression in axotomized sensory neurons. J Neurosci 11: 543-552. Wong ST, Atkinson BA, Weaver LC (2000) Confocal microscopic analysis reveals sprouting of primary afferent fibres in rat dorsal horn after spinal cord injury. Neurosci Lett 296: 65-68. Wong-Riley MT (1989) Cytochrome oxidase: an endogenous metabolic marker for neuronal activity. Trends Neurosci 12: 94-101. Woodham P, Anderson PN, Nadim W, Turmaine M (1989) Satellite cells surrounding axotomised rat dorsal root ganglion cells increase expression of a GFAP-like protein. Neurosci Lett 98: 8-12. Woolf CJ, Shortland P, Coggeshall RE (1992) Peripheral nerve injury triggers central sprouting of myelinated afferents. Nature 355: 75-78. Xie W, Strong JA, Mao J, Zhang JM (2011) Highly localized interactions between sensory neurons and sprouting sympathetic fibers observed in a transgenic tyrosine hydroxylase reporter mouse. Mol Pain 7: 53. Xie W, Strong JA, Zhang JM (2010) Increased excitability and spontaneous activity of rat sensory neurons following in vitro stimulation of sympathetic fiber sprouts in the isolated dorsal root ganglion. Pain 151: 447-459. Yaksh TL, Farb DH, Leeman SE, Jessell TM (1979) Intrathecal capsaicin depletes substance P in the rat spinal cord and produces prolonged thermal analgesia. Science 206: 481-483.  221  Yamamoto W, Sugiura A, Nakazato-Imasato E, Kita Y (2008) Characterization of primary sensory neurons mediating static and dynamic allodynia in rat chronic constriction injury model. J Pharm Pharmacol 60: 717-722. Yeoh M, McLachlan EM, Brock JA (2004) Tail arteries from chronically spinalized rats have potentiated responses to nerve stimulation in vitro. J Physiol 556: 545-555. Yip HK, Rich KM, Lampe PA, Johnson EM, Jr. (1984) The effects of nerve growth factor and its antiserum on the postnatal development and survival after injury of sensory neurons in rat dorsal root ganglia. J Neurosci 4: 2986-2992. Yoshimura N (1999) Bladder afferent pathway and spinal cord injury: possible mechanisms inducing hyperreflexia of the urinary bladder. Prog Neurobiol 57: 583-606. Yoshimura N, Bennett NE, Hayashi Y, Ogawa T, Nishizawa O, Chancellor MB, de Groat WC, Seki S (2006) Bladder overactivity and hyperexcitability of bladder afferent neurons after intrathecal delivery of nerve growth factor in rats. J Neurosci 26: 1084710855. Yoshimura N, de Groat WC (1997) Plasticity of Na+ channels in afferent neurones innervating rat urinary bladder following spinal cord injury. J Physiol 503 ( Pt 2): 269276. Yoshimura N, Seki S, Erickson KA, Erickson VL, Hancellor MB, de Groat WC (2003) Histological and electrical properties of rat dorsal root ganglion neurons innervating the lower urinary tract. J Neurosci 23: 4355-4361. Zagon A, Smith AD (1993) Monosynaptic projections from the rostral ventrolateral medulla oblongata to identified sympathetic preganglionic neurons. Neuroscience 54: 729-743. Zaidi ZF, Matthews MR (1999) Stimulant-induced exocytosis from neuronal somata, dendrites, and newly formed synaptic nerve terminals in chronically decentralized sympathetic ganglia of the rat. J Comp Neurol 415: 121-143. Zhang X, Chen Y, Wang C, Huang LY (2007) Neuronal somatic ATP release triggers neuron-satellite glial cell communication in dorsal root ganglia. Proc Natl Acad Sci U S A 104: 9864-9869. Zhou XF, Deng YS, Chie E, Xue Q, Zhong JH, McLachlan EM, Rush RA, Xian CJ (1999) Satellite-cell-derived nerve growth factor and neurotrophin-3 are involved in noradrenergic sprouting in the dorsal root ganglia following peripheral nerve injury in the rat. Eur J Neurosci 11: 1711-1722. Zimmermann M (2001) Pathobiology of neuropathic pain. Eur J Pharmacol 429: 23-37. Zinck ND, Downie JW (2008) IB4 afferent sprouting contributes to bladder dysfunction in spinal rats. Exp Neurol 213: 293-302. 222  Zinck ND, Rafuse VF, Downie JW (2007) Sprouting of CGRP primary afferents in lumbosacral spinal cord precedes emergence of bladder activity after spinal injury. Exp Neurol 204: 777-790. Zvarova K, Dunleavy JD, Vizzard MA (2005) Changes in pituitary adenylate cyclase activating polypeptide expression in urinary bladder pathways after spinal cord injury. Exp Neurol 192: 46-59. Zvarova K, Murray E, Vizzard MA (2004) Changes in galanin immunoreactivity in rat lumbosacral spinal cord and dorsal root ganglia after spinal cord injury. J Comp Neurol 475: 590-603.  223  

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