UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Db-cAMP applied to the Red Nucleus in rat improves the regenerative response of the rubrospinal tract… Lane, René 2012

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2012_fall_lane_rene.pdf [ 16.35MB ]
Metadata
JSON: 24-1.0073272.json
JSON-LD: 24-1.0073272-ld.json
RDF/XML (Pretty): 24-1.0073272-rdf.xml
RDF/JSON: 24-1.0073272-rdf.json
Turtle: 24-1.0073272-turtle.txt
N-Triples: 24-1.0073272-rdf-ntriples.txt
Original Record: 24-1.0073272-source.json
Full Text
24-1.0073272-fulltext.txt
Citation
24-1.0073272.ris

Full Text

      DB-CAMP APPLIED TO THE RED NUCLEUS IN RAT IMPROVES THE REGENERATIVE RESPONSE OF THE RUBROSPINAL TRACT AFTER CERVICAL SPINAL CORD INJURY  by Ren? Lane    A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in The Faculty of Graduate Studies (Neuroscience)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) October 2012 ? Ren? Lane, 2012   ii  Abstract  A high cervical injury to the rubrospinal tract (RST) generally results in cellular and nuclear atrophy of the rubral neurons, abortive axonal regeneration and eventual retraction from the forming injurious scar and enlarging cavity.  The neuronal cell body response to axonal injury plays an important role in the failure of central nervous system (CNS) neurons to regenerate.  Adult mammalian CNS neurons fail to re-express a variety of genes and signalling factors, such as cAMP, after axotomy that are seen at increased levels in regenerating peripheral neurons or in developing CNS neurons.    By mimicking the sustained increase in cAMP levels, prior to or during an upper cervical (C3/4) crush injury of the dorsolateral funiculus in adult male Sprague-Dawley rats using the membrane permeant analogue dibutyryl cAMP (db-cAMP) near the vicinity of rubrospinal neurons, will enhance the regenerative cell body response in order to promote axonal sprouting or regeneration to and across the site of a spinal cord injury. Two experimental groups, acute and pre-treatment, were used.  Both groups received either 25mM db-cAMP (treatment) or vehicle (control) solutions delivered via 14 day mini osmotic-pumps.  The pretreated group had db-cAMP infused beginning one week prior to injury for two weeks while the acutely treated group received concurrent treatment with injury.  The rubrospinal tract was anterogradedly traced with biotinylated dextran amine (BDA) for quantification purposes. Camera lucida like reconstructions (CLLRs) were created for enhanced visualization and density calculation of the RST. Changes in behaviour were assessed using the vertical exploration test.   Application of db-cAMP to the RN in both the acute and pretreated groups increased the number of labelled rubrospinal fibres in the gray matter and proximal to the site of injury in comparison to the controls.  Db-cAMP treatment did not reduce lesion sizes nor were there any visibly traced fibres caudal to the site of injury in any of the treatment groups.  Unexpectedly, pre-treatment of db-cAMP conferred no advantages over acute treatment.  Behavioural analysis of spontaneous forelimb usage as indicated by the vertical exploration test revealed no significant differences between any of the db-cAMP and control groups.   iii  Preface  All animal experiments and procedures were conducted in accordance to the guidelines set out by the Canadian Council for Animal Care (CCAC) as well as conforming to the rules provided by the University of British Columbia Animal Care Ethics Committee, certificate number: A03-0112.  Surgeries conducted by Dr. Jie Liu.      iv  Table of contents  Abstract ........................................................................................................................................... ii Preface............................................................................................................................................ iii Table of contents ............................................................................................................................ iv List of tables ................................................................................................................................. viii List of figures ................................................................................................................................. ix Abbreviations, nomenclature and symbols .................................................................................... xi Acknowledgements ...................................................................................................................... xiv Dedication ..................................................................................................................................... xv 1 INTRODUCTION .................................................................................................................. 1 1.1 Opening statement ............................................................................................................ 1 1.2 Spinal Cord Injury ? brief introduction, research imperative and statistics ..................... 1 1.3 Injury and regeneration in the CNS versus the PNS ? a brief historical perspective and introduction ................................................................................................................................. 3 1.4 cAMP ? history ................................................................................................................ 4 1.5 Molecular basis of cAMP production and signalling - general overview ........................ 5 1.5.1 Notable components of the cAMP cascade .............................................................. 6 1.6 cAMP ? involvement in axonal regeneration and neuronal survival - literature review 12 1.6.1 cAMP involvement in PNS regeneration ................................................................ 12 1.6.2 cAMP involvement in CNS regeneration ............................................................... 12 1.6.3 cAMP involvement in conditioning lesions ............................................................ 13 1.6.4 cAMP involvement in neuronal survival ................................................................ 14 1.7 Barriers to regeneration .................................................................................................. 16 1.8 cAMP involvement with inhibitors to regeneration ....................................................... 16 1.8.1 Axonal inhibitory molecules and receptors ............................................................ 17 1.9 SCI therapeutic strategies ............................................................................................... 20 1.10 The molecular role and potential benefits of increased levels of cAMP in axonal/neurite growth and regeneration.................................................................................... 21 1.10.1 Increased cAMP levels attenuate activated p75NTRs axonal inhibitory effects....... 22 1.10.2 Increased cAMP levels activate CREB to overcome the inhibitory effects of Nogo?20 ............................................................................................................................... 22 1.10.3 Increases in cAMP increase polyamine levels ........................................................ 23 v  1.10.4 Inflammation induced regeneration in the optic nerve with inflammatory proteins (i.e. Ocm) depends on cAMP ................................................................................................ 24 1.10.5 Ocm requires elevated cAMP levels when used as a regeneration enhancer in other nervous systems .................................................................................................................... 24 1.10.6 Activated Epacs by cAMP allows neurite extension on inhibitory substrates........ 25 1.10.7 Increased cAMP may also increase neurite outgrowth via unknown cAMP pathways ............................................................................................................................... 25 1.11 Rationale and experimental design ............................................................................. 29 1.11.1 Experimental design overview ................................................................................ 29 1.11.2 Red Nucleus (RN) and Rubrospinal tract (RST) ? rationale and anatomy ............. 30 1.11.3 Function of the RN and choice of behaviour test ................................................... 31 1.11.4 Importance of the ?cell body response?; the RN response to injury and as a treatment target ..................................................................................................................... 32 1.11.5 Overall experimental rationale and design ............................................................. 33 1.12 Aims and hypotheses .................................................................................................. 35 2 MATERIALS AND METHODS .......................................................................................... 37 2.1 Animals .......................................................................................................................... 37 2.1.1 Animal model guidelines ........................................................................................ 37 2.1.2 Animal model.......................................................................................................... 37 2.1.3 Animal care ............................................................................................................. 37 2.1.4 Surgery .................................................................................................................... 37 2.1.5 Tracing .................................................................................................................... 39 2.1.6 Perfusion and tissue harvesting ............................................................................... 39 2.2 Drug and tissue preparation and/or processing; immunohistochemistry ....................... 39 2.2.1 Experimental drug solutions ................................................................................... 39 2.2.2 Mini-osmotic pump preparation ............................................................................. 40 2.2.3 Immunohistochemistry ........................................................................................... 41 2.2.4 Tissue cutting and mounting ................................................................................... 41 2.3 Behaviour ....................................................................................................................... 42 2.3.1 Behaviour testing ? vertical exploration test .......................................................... 42 2.4 Quantification ................................................................................................................. 43 2.4.1 Quantification of axons ........................................................................................... 43 2.4.2 Lesion areas and perimeters .................................................................................... 48 2.5 Statistics ......................................................................................................................... 48 2.6 Experimental design overview and timeline .................................................................. 49 vi  2.6.1 Acute treatment experimental overview and timeline ............................................ 49 2.6.2 Pre-treatment experimental overview and timeline ................................................ 49 3 RESULTS ? ACUTE TREATMENT WITH DB-CAMP .................................................... 51 3.1 Introduction .................................................................................................................... 51 3.2 Db-cAMP treatment did not affect lesion cavity sizes ................................................... 51 3.3 Db-cAMP treated animals displayed overall greater numbers of BDA labelled axons/sprouts than vehicle treated animals ............................................................................... 52 3.4 RST axonal/sprout/fibre density was significantly higher in db-cAMP treated versus control animals .......................................................................................................................... 60 3.5 Db-cAMP treated animals had a significant increase in RST projections into the gray matter 61 3.6 RST axon/sprout density in the gray matter is greater for the db-cAMP treated group in comparison the control?s. .......................................................................................................... 68 3.7 Cross sectional views show greater numbers of traced RST axons/sprouts travelling to the gray matter for db-cAMP treated groups. ........................................................................... 69 3.8 Db-cAMP treatment provided no functional benefit versus the vehicle treated groups in the forelimb usage test .............................................................................................................. 71 4 RESULTS ? PRE-TREATMENT WITH DB-CAMP ONE WEEK PRIOR TO INJURY.. 74 4.1 Introduction .................................................................................................................... 74 4.2 Pretreatment of db-cAMP did not reduce lesion cavity sizes ........................................ 74 4.3 Db-cAMP treated animals have greater numbers of traced RST axon/sprouts than vehicle treated animals .............................................................................................................. 76 4.4 RST axonal/sprout density is significantly higher in db-cAMP treated versus control animals ...................................................................................................................................... 83 4.5 Pretreated db-cAMP groups show more RST axon/sprouts/fibres in the gray matter ... 85 4.6 RST axonal sprout density is higher for db-cAMP treated animals in comparison the controls. ..................................................................................................................................... 92 4.7 Cross sectional views show more RST projections to the gray matter for db-cAMP groups. ....................................................................................................................................... 94 4.8 Pre-treatment of db-cAMP provided no functional benefits in the forelimb usage test 96 5 DISCUSSION ....................................................................................................................... 99 5.1 Treatment of db-cAMP results in more RST axons/sprouts in comparison to the vehicle treated group ............................................................................................................................. 99 5.2 Increased numbers of visibly BDA labelled RST axons/sprouts in acute or pre- treated groups unlikely due to differences in tracing efficacy .............................................................. 99 5.3 Db-cAMP may increase axonal sprouting by eliciting a cell body response which can contribute to overcoming inhibitors of regeneration .............................................................. 102 vii  5.4 Is db-cAMP an effective method of mimicking increased cAMP levels? Are other analogues or methods more or less efficacious? ..................................................................... 105 5.4.1 The decomposition product butyrate can introduce potential experimental confounds ............................................................................................................................ 105 5.4.2 Mb-cAMP is the biologically active decomposition product of db-cAMP .......... 106 5.4.3 Db-cAMP and its decomposition products may not act similarly to native cAMP 107 5.4.4 C8 substituted cAMP analogues may be more efficacious than N6 cAMP analogues in some circumstances ........................................................................................................ 107 5.4.5 Circumstances where N6 cAMP analogues are more advantageous and efficacious than C8 cAMP analogues.................................................................................................... 108 5.4.6 Caution needs to be exercised with compounds that increase levels of native cAMP as well 109 5.5 Pre-treatment of db-cAMP provides no additional advantages to acute treatment ...... 111 5.6 Isolating behavioural changes involving only the RST poses difficulties in a dorsolateral funiculus crush injury.......................................................................................... 112 5.7 Significance of db-cAMP as a treatment for spinal cord injury ................................... 117 5.7.1 Enhancement of RST sprouting as a potential therapeutic goal ........................... 117 5.7.2 Cell body treatment of db-cAMP to the RN via canula is currently not a viable treatment paradigm ............................................................................................................. 119 5.8 Future experimental directions ..................................................................................... 120 5.8.1 Future use of elevating cAMP will require enhanced specificity either in part or in whole 120 5.8.2 Combinatorial cAMP treatments may be more efficacious in evoking a regenerative response .......................................................................................................... 122 5.8.3 Treatment of additional neuronal tracts and/or a much more sensitive behavioural technique is needed in order to determine functional benefit in DLF injuries ................... 123 5.8.4 The molecular inhibitory complexes in the RST need to be further characterized 125 5.9 Conclusions .................................................................................................................. 126 References: .................................................................................................................................. 127 Appendix ..................................................................................................................................... 169    viii  List of tables  Table I: Summary of notable neuronal studies involving axon regeneration or survival using altered levels of cAMP................................................................................................................ 169       ix  List of figures  Figure 1.1 Molecular diagram and 3D ball and stick model of cAMP (cyclic adenosine 3?,5?-monophosphate) .............................................................................................................................. 5 Figure 1.2  Prototypical initiation of the cAMP second messenger system and cAMP production....................................................................................................................................................... 11 Figure 1.3 Molecular representation of the conversion of ATP to cAMP .................................... 11 Figure 1.4  Schematic diagram illustrating the involvement of cAMP in CNS axonal injury and regeneration with identified inhibitory molecules ........................................................................ 27 Figure 1.5 Summary flowchart of cAMP?s molecular involvement in axon growth or regeneration................................................................................................................................... 29 Figure 2.1 Schematic depicting the experimental procedure. ....................................................... 38 Figure 2.2 Molecular diagram of adenosine-3?,5?-cyclic monophosphate N6, O2-dibutyryl-sodium salt (db-cAMP) ................................................................................................................. 40 Figure 2.3  Schematic illustrating the axonal quantification procedure. ...................................... 46 Figure 2.4  Timelines of acute and pre-treatment experiments .................................................... 50 Figure 3.1 Db-cAMP treatment does not affect lesion injury size ............................................... 52 Figure 3.2 The db-cAMP treated group shows greater density of the BDA traced RST and axons/fibres in comparison to the vehicle treated group .............................................................. 55 Figure 3.3  (Inset of Figure 3.2) the db-cAMP treated group exhibits more BDA labelled RST axons/sprouts/fibres than the vehicle treated group ...................................................................... 57 Figure 3.4 Camera lucida like reconstruction (CLLR) reveals a greater number of labelled RST axons/sprouts/fibres distally to proximal to the lesion edge for the db-cAMP treated group ...... 58 Figure 3.5 (Inset of Figure 3.4) CLLRs show greater numbers of axons/sprouts/fibres of the RST versus vehicle treated groups rostral and proximal to the lesion .................................................. 59 Figure 3.6 RST axon/sprout density increases significantly from the lesion epicentre rostrally for the db-cAMP treated group ........................................................................................................... 61 Figure 3.7 RST axon/sprout density is greater for the db-cAMP treated group in the gray matter....................................................................................................................................................... 63 Figure 3.8 (Inset of Figure 3.7) the db-cAMP treated group shows more RST axons/sprouts in the gray matter .............................................................................................................................. 65 Figure 3.9 CLLRs reveal greater visible RST axon/sprout density in the gray matter for the db-cAMP treated group ...................................................................................................................... 66 Figure 3.10 (Inset of Figure 3.9) CLLRs reveal greater RST axon/sprout density in gray matter for the db-cAMP treated group ..................................................................................................... 68 Figure 3.11 RST axon/sprout density in the gray matter is greater for the db-cAMP treated group....................................................................................................................................................... 69 Figure 3.12 Spinal cord cross sections display greater numbers of RST axon/sprouts to the gray matter for the db-cAMP treated group .......................................................................................... 70 Figure 3.13  (Inset of Figure 3.12) the db-cAMP treated group shows more traced RST axon/sprouts projecting to the gray matter .................................................................................... 71 Figure 3.14  db-cAMP provides no functional benefit as indicated by the vertical exploration test....................................................................................................................................................... 73 Figure 4.1 Pre-treatment of db-cAMP did not reduce lesion sizes ............................................... 76 Figure 4.2  Pretreated db-cAMP groups show moderately more labelled RST axon/sprout/fibres over controls.................................................................................................................................. 78 x  Figure 4.3 (Inset of Figure 4.2) Pretreated db-cAMP group shows more traced RST axons/sprouts rostrally .................................................................................................................. 80 Figure 4.4 CLLRs show more RST axons/sprouts/fibres for the pretreated db-cAMP group ..... 81 Figure 4.5  (Inset of Figure 4.4) CLLRs show a greater number of traced RST axons/sprouts rostrally and proximal to the lesion edge for the pretreated db-cAMP group .............................. 83 Figure 4.6  RST axon/sprout density increases significantly from the lesion epicentre for the pretreated db-cAMP group ........................................................................................................... 84 Figure 4.7 No significant RST axon/sprout density differences between acute and pretreated vehicle (control) groups ................................................................................................................ 85 Figure 4.8 RST axon/sprout density in the gray matter is greater for the pretreated db-cAMP  group ............................................................................................................................................. 87 Figure 4.9 (Inset of Figure 4.8) the pretreated db-cAMP group shows more RST axons/sprouts rostrally in gray matter .................................................................................................................. 89 Figure 4.10  CLLRs show more RST axon/sprout/fibres in the gray matter for the pretreated db-cAMP group .................................................................................................................................. 90 Figure 4.11  CLLRs reveal greater rostral axon/sprout density for the pretreated db-cAMP group....................................................................................................................................................... 92 Figure 4.12  RST axon/sprout density is greater for the pretreated db-cAMP group ................... 93 Figure 4.13 RST axon/sprout into the gray matter shows no significant differences between acute and pretreated vehicle (control) groups ........................................................................................ 94 Figure 4.14 Spinal cord cross sections show greater numbers of RST axon/sprouts to the gray matter for the pretreated db-cAMP group ..................................................................................... 95 Figure 4.15 (Inset of Figure 4.14) The pretreated db-cAMP group displays more RST axon/sprouts projecting to the gray matter .................................................................................... 96 Figure 4.16  Pre-treatment of db-cAMP provides no functional improvement as indicated by the vertical exploration test ................................................................................................................. 98    xi  Abbreviations, nomenclature and symbols  Abbreviation/Nomenclature Description 8Br-cAMP 8-bromo-cAMP  8-CPT-cAMP 8-(4-chlorophenylthio)-cAMP  AC Adenylyl or Adenlate Cyclase Acute Tx Acute treatment AKAP A Kinase Anchoring Protein Arg Arginase ATF1 Activating Transcription Factor ATP Adenosine Triphosphate BDA Biotinylated Dextran Amine BDNF Brain Derived Neurotrophic Factor C Catalytic Subunit C# Cervical (# = Cervical spinal column vertebral number; e.g. C3) c-Abl Protein Kinase CaM  Calmodulin cAMP Cyclic Adenosine 3?,5?-Monophosphate cAMP-GEF cAMP-Guanine Nucleotide Exchange Factors CBP CREB Binding Protein CCAC Canadian Council on Animal Care cGMP Cylic Guanosine Monophosphate CHO Chinese Hamster Ovary Cell CL  Conditioning Lesion CLLR Camera Lucida Like Reconstruction CNS Central Nervous System CNTF Ciliary Neurotrophic Factor CRD Cysteine Rich Domain CRE  cAMP Response Element CREB cAMP Response Binding Protein (CREB)  CSPG Chondroitin Sulfate Proteogylcan CST Corticospinal Tract D/D Dimerization Domain db-cAMP Dibutyryl Cyclic Adenosine 3?,5?-Monophosphate (Adenosine-3?,5?-cyclic Monophosphate N6, O2-dibutyryl-Sodium Salt ) DLF Dorsolateral Funiculus DNA Deoxyribonucleic Acid DRG Dorsal Root Ganglia E# Embryonic day # Epac Exchange Protein Directly Activated by cAMP EphB3 Ephrin B3 FGF Fibroblast Growth Factor xii  Abbreviation/Nomenclature Description Forskolin 7 beta-Acetoxy-8,13-epoxy-1alpha,6 beta,9 alpha-trihydroxy-labd-14-en-11-one GAP-43 Growth Associated Protein - 43 GDNF Glial Derived Neurotrophic Factor GEF Guanine Nucleotide Exchange Factor GFAP Glial Fibrillary Acidic Protein Gi? G-inhibitory GPCR G Protein Coupled Receptor GPI Glycosylphosphatidylinositol GTP Guanosine Triphosphate G?s G-?-stimulatory  HEK-293 Human Embryonic Kidney 293 Cell HGF Hepatocyte Growth Factor IBMX 3-Isobutyl-1-methylxanthine KH7 2-(1H-benzimidazol-2-ylthio)-2-[(5-bromo-2-hydroxyphenyl)methylene]hydrazide, propanoic acid L# Lumbar (# = lumbar spinal column vertebral number) L-MAG Large Myelin Associated Glycoprotein LN-1 Laminin-1 MAG Myelin Associated Glycoprotein MAP-2 Microtubule Associated Protein - 2 MEPP Miniature Endplate Potential Mesopram (R)-5-(4-Methoxy-3-propoxyphenyl)-5-methyl-2-oxazolidinone MLC Myosin Light Chain N.S. Nervous System (PNS or CNS) NEP1-40 Nogo Extracellular Peptide 1-40 NGF Nerve Growth Factor NgR Nogo Receptor NT Neurotrophin NT-3 Neurotrophin-3 NT-4 Neurotrophin-4 OD Ornithine Decarboxylase (used in Figure 1.4 only) ODC Ornithine Decarboxylase OMgp Oligodendrocyte Myelin Glycoprotein Ocm Oncomodulin P# Postnatal day # PBS Phosphate Buffered Solution P-CREB Phospo CREB PDE Phosphodiesterase PI-PLC Phosphatidylinositol Specific Phospholipase PKA Protein Kinase A PKI Protein Kinase Inhibitory xiii  Abbreviation/Nomenclature Description PLGA Polylactic-co-Glycolic Acid PolII RNA Polymerase II PNS Peripheral Nervous System PP Phosphatase Pre Tx Pre-treatment R Regulatory Subunit Rap Ras-Proximate RGC Retinal Ganglion Cell Rho Ras Homology Protein RhoA-GDP RhoA-Guanosine Triphosphate RhoA-GDP-GDI RhoA-GDP-Guanosine Dissociation Inhibitor rMAG recombinant MAG RN  Red Nucleus RNA Ribonucleic Acid ROCK Rho-Kinase Rolipram (R,S)-4-[3-(Cyclopentyloxy)-4-methoxy-phenyl]-2-pyrrolidinone RST Rubrospinal Tract SCI Spinal Cord Injury SEM  Standard Error of the Mean Sema3A/4D Semaphorin3A or Semaphorin4D Ser Serine T# Thoracic (# = thoracic spinal column vertebral number; e.g. T8) TAF TBP Associated Factors TBP TATA Binding Protein TFII Transcription Factor II Thr Threonine TNFR Tumour Necrosis Factor Receptor TNF? Tumour Necrosis Factor ? T?1 Tubulin - T?1  Wave-1 Wiskott-Aldrich Syndrome Protein Y27632 (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide     xiv  Acknowledgements  I would like to thank all the members of the Tetzlaff lab who lent me their ears and so generously provided me with their precious time, ideas, thoughts, opinions and support.  I will always remember our captivating discussions that lasted well into some nights. I would especially like to thank Dr. Wolfram Tetzlaff for giving me this tremendous opportunity.   I would like to extend a special gratitude to Dr. Jie Liu for lending his expertise and providing such unstinting tutelage.    I wish to thank my supervisory committee, Dr. Venessa Auld and Dr. Timothy O?Connor, for their unwavering patience, expert advice, thoughtful discussions and courteous feedback.    This work was made possible by the generous financial support of the Christopher Reeve Foundation and the Canadian Institutes of Health Research.     xv  Dedication  To the composers of the Baroque era whose music inspired, soothed and motivated me throughout my studies.1  1 INTRODUCTION 1.1 Opening statement  The overarching intent and purpose of these experiments is to add to the body of knowledge attempting to find regenerative treatments for spinal cord injury. While the primary focus of this thesis will investigate the outcome of augmenting the cyclic nucleotide messenger system in the red nucleus as a potential regenerative concept treatment in response to a cervical injury, the scope of the thesis, however, will also include research from bio-molecular, biochemical and genetics disciplines in addition to the neuroscience experiments presented here in order to further the understanding of the potential molecular mechanisms of these experiments.  1.2 Spinal Cord Injury ? brief introduction, research imperative and statistics  Spinal cord injury (SCI) is an often permanent and severely debilitating condition.  Its root causes can arise from vehicular crashes (24%), industrial accidents (28%), athletic activities (16%), falls (9%), birth defects (3%), natural disasters (1%), victims of violence (4%) and various diseases (6%) (Christopher and Dana Reeve Foundation, 2012a, 2012b).  Depending where the trauma has occurred, those with loss of function to the trunk and legs are referred to as paraplegic, while those with paralysis from the neck down are categorized as being quadriplegic or tetraplegic (Farry & Baxter, 2010; The Rick Hansen Institute, 2011). This often devastating condition comes with severe emotional, mental, medical and financial obligations to the individual, their families and society as a whole.  Individuals with SCI not only may have to cope with the accompanying physical impairment, but may also be confronted with difficulties with caring for themselves, having to cope with the change or loss of employment and pastimes, and may experience societal prejudice (Christopher and Dana Reeve Foundation, 2012b; Somers, 2001). Many of those suffering with SCI may also experience: depression, pain, sexual health issues, cardiovascular, autonomic dysreflexia, respiratory, gastrointestinal, integumentary and genitourinary problems (Christopher and Dana Reeve Foundation, 2012b; Christopher Reeve Foundation, 2009; Farry & Baxter, 2010; The Rick Hansen Institute, 2011).  Fortunately, the past few decades have seen a dramatic increase in both the understanding of the mechanisms involved and the number of potential therapies targeted towards people with SCI (Baptiste, Fehlings, John, & Andrew, 2007; Blesch & Tuszynski, 2009; Hawryluk, Rowland, Kwon, & Fehlings, 2008; 2  Kwon, Tetzlaff, Grauer, Beiner, & Vaccaro, 2004). Indeed, even though SCI for some can be as severe as to be life threatening, many can go on to live healthy, complete and productive lives (Christopher and Dana Reeve Foundation, 2012a, 2012b; Somers, 2001).  In addition to those secondary disorders, the financial burden to the individual, their families and society can be enormous.  SCI affects over 85,556 Canadians (Farry & Baxter, 2010) and approximately 1,275,000 persons in the United States (Christopher and Dana Reeve Foundation, 2012b).  Each year there are about 4,259 new cases in Canada (Farry & Baxter, 2010) and 12,000 in the U.S. (Christopher Reeve Foundation, 2009; The Rick Hansen Institute, 2011).  The average ages of the injured in Canada comprise of two bimodal peaks: those between the ages of 20 and 30, and those above the age of 70 (Farry & Baxter, 2010).  In the United States, the average age of someone living with a SCI is 48 (Christopher and Dana Reeve Foundation, 2012b).  The financial cost of a SCI to an individual ranges from $228,566 (U.S.) for an incomplete motor functional injury to $775,567 (U.S.) for a high tetraplegic (C1-C4) injury.  Each subsequent year the cost of care ranges from $16,018 to $138,923 (U.S.) depending on the level of injury.  A twenty five year old person with a high tetraplegic C1-C4 SCI is expected to pay $3,059,184 in the U.S. over his or her lifetime (Christopher and Dana Reeve Foundation, 2012a, 2012b; NSCISC, 2008; Rick Hansen Foundation, 2009).   The cost to the injured individual in Canada ranges between $1.6 million (paraplegic) to $3.0 million (tetraplegic).  In sum, the total economic impact of SCI is reported to be in excess of $3.6 billion in Canada and $40.5 billion in the U.S. (Christopher and Dana Reeve Foundation, 2012b; Rick Hansen Foundation, 2009; The Rick Hansen Institute, 2011).      SCI is without question a monumental hurdle for the individual afflicted; whether it is physical, psychosocial, social or financial. It also represents a challenge for the health care system, its infrastructure and researchers searching for viable treatments. Despite the tremendous advances in SCI research, efficacious treatments or even cures have yet to be fully put into practice.  Clearly, the importance of finding viable treatments for SCI remains paramount.    3  1.3 Injury and regeneration in the CNS versus the PNS ? a brief historical perspective and introduction  It was already clear according to one of the earliest known Egyptian scrolls, a medical and surgical papyrus written in approximately 1,600 B.C. containing medical knowledge from texts as old as 2,500 ? 3,000 B.C., that repair of the mammalian spinal cord when injured was extremely limited either intrinsically or with intervention (Wilkins, 1964, 2011).  As scribed in the papyrus case Thirty-Three illustrated the futile prognosis of spinal cord injury:  ... If thou examinest a man having a crushed vertebra in his neck (and) thou findest that one vertebra has fallen into the next one, while he is voiceless and cannot speak; his falling head downward has caused that one vertebra crush into the next one; (and) shouldst thou find that he is unconscious of his two arms and his two legs because of it... ...Thou shouldst say concerning him: "One having a crushed vertebra in his neck; he is unconscious of his two arms (and) his two legs, (and) he is speechless. An ailment not to be treated."... (Breasted, 1991; Wilkins, 2011)  In the early 1900?s Santiago Ram?n y Cajal, a pioneer in the field of Neuroscience, documented the regenerative differences between the mammalian central and peripheral nervous systems.  Of injury to the peripheral nervous system he wrote:   It is well known that, when one cuts a nervous cord in a young animal, the peripheral extremity thus severed from its central portion degenerates and dies immediately, resorbing its remnants; months later the intermediate scar and the peripheral nervous stump show numerous newly-formed fibres; in the end, totally or partially, the sensibility and motility of the paralyzed member are re-established (May, 1928; Ram?n y Cajal, 1991, p. 3).  It was evident that injuries to the peripheral nervous system (PNS) could result in functional repair. Unfortunately, the encouraging reparative capabilities of the PNS were not as apparent in the central nervous system (CNS). Following the observations of Dustin (1910) who had performed grafting and sectioning experiments in canine spinal cords, Ram?n y Cajal observed and noted the fate of the axons proximal to the site of injury:  ...his [Dustin?s] analysis of the late phenomena of spinal regeneration and degeneration. ...he notes that, no matter what the lapse of time, the nerve sprouts do not cross the scar, and cannot, therefore, restore the paths that have been destroyed,...(May, 1928; Ram?n y Cajal, 1991, p. 579)  4  While this once dismal outlook was an accepted belief for the outcome of most injuries in the CNS, especially of the spinal cord; the view was brought into question when it was demonstrated that when the injured CNS is presented with a supportive environment such as the PNS, the CNS displays surprising growth characteristics (David & Aguayo, 1981; Leoz & Arcaute, 1913; May, 1928; Pollard, McLeod, & Gye, 1973; Ram?n y Cajal, 1991; Richardson, McGuinness, & Aguayo, 1980; Tello, 1911, 1923).  For example, David & Aguayo (1981) used peripheral nerve ?bridges?, autologous sciatic nerves, to join the medulla oblongata and the upper thoracic spinal cord in Sprague-Dawley rats.  They demonstrated that growth of some CNS axons exceeded 30mm in the PNS graft.  Encouragingly, all these studies indicate that CNS injury may not necessarily be faced with a poor regenerative outcome, but one with renewed axonal growth and repair given the right environmental conditions and treatments.     1.4 cAMP ? history  Cyclic adenosine monophosphate (cAMP -cyclic adenosine 3?,5?-monophosphate; Figure 1.1) was discovered as the original, and now ubiquitous and prototypical, second messenger by Drs. Earl Sutherland and Ted Rall while investigating liver and heart metabolism (Rall & Sutherland, 1958; Sutherland & Rall, 1958). Ever since the landmark work that earned Sutherland a Nobel prize, the role of cAMP as a second messenger has been extensively studied over the last 50 years (Sutherland, 1971).   Some of the regulatory and cellular processes linked to cAMP include: cell growth, synaptic release of neurotransmitters, gene transcription and regulation, ion channel conductivity, phosphorylation, apoptosis, survival, cellular growth, memory, metabolism, olfaction, cellular maintenance, immune function and differentiation (for reviews see: Chin et al., 2002; Daniel, Walker, & Habener, 1998; Frey, Huang, & Kandel, 1993; Greengard, 2001; Skalhegg & Tasken, 2000; Sklar, Anholt, & Snyder, 1986; Svenningsson et al., 2004; Wong et al., 2000).  All these processes and outcomes, stimulatory or inhibitory, also depend on the cell type, genetic expression and cellular environment (Chin, et al., 2002).   5   Figure 1.1 Molecular diagram and 3D ball and stick model of cAMP (cyclic adenosine 3?,5?-monophosphate)  (Created from images in the public domain; Public domain: Protein Data Bank; Federal Government under the terms of Title 17, Chapter 1, Section 105 of the US Code).     1.5 Molecular basis of cAMP production and signalling - general overview  The prototypical cAMP signal pathway begins with an effector molecule (hormone, neurotransmitter, drug, lipid mediator or chemokine etc.) that binds to the extracellular portion of a G-protein coupled receptor (GPCR).  This 7 transmembrane GPCR or transducer is usually associated with a G-?-stimulatory (G?s) and ?? subunit that causes the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) which is performed by G?s (Serezani, Ballinger, Aronoff, & Peters-Golden, 2008). At this point the G?s detaches from the ?? subunit and stimulates the adenylyl or adenylate cyclase (AC) subunit or effector (Figure 1.2).  AC then catalyzes the reaction by converting adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cAMP) and pyrophosphate (PPi) (Figure 1.3) (Cooper, 2003).  The accumulating cAMP proceeds to stimulate effector targets in the cell; effectors such as protein kinase A (PKA).  PKA phosphorylates numerous targets usually at serine (Ser) and threonine (Thr) amino acid residues on target proteins (Taylor et al., 2008).  Targets can be cytosolic or nuclear.  Nuclear targets usually include the cAMP response binding protein (CREB) which can result in gene transcription (Sands & Palmer, 2008). The entire process results in a cascading 6  action. Cessation of the cascade is performed by phosphodiesterases (PDEs) that degrade cAMP (Kleppisch, 2009).   1.5.1 Notable components of the cAMP cascade  1.5.1.1 Adenylate Cyclase (AC)  AC is the enzyme responsible for catalyzing the conversion of ATP to cAMP. Currently ten isoforms have been identified; nine of which are membrane bound and one is deemed to be soluble.  ACs are generally grouped into one of four categories based on their regulatory properties. Group I AC?s include ACI, III, IIX and are stimulated by Gs? and Ca2+, group II ACs comprise of ACII, IV, VII and is stimulated by G??, group III consists of ACV,VI and is also G?? stimulated while ACIX is the sole member of group IV (Sadana & Dessauer, 2009).  The general structure of ACs is similar to that of the ABC cassette transporter proteins; two six transmembrane segments connected together with a large catalytic cytoplasmic loop.  The two catalytic cytoplasmic loops commonly contain around 230-250 amino acid residues with 40% homology across current known ACs (Krupinski et al., 1989; Sunahara, Dessauer, & Gilman, 1996; Sunahara & Taussig, 2002; Tesmer, Sunahara, Gilman, & Sprang, 1997). There are many ways that ACs are activated, inhibited and regulated; only a few will be considered here.  Briefly, Gs?-GTP stimulates all isoforms of AC.  G-inhibitory (Gi?), or Gi?1,2,3, Gz?, and Go? selectively inhibit specific AC isoforms (Sadana & Dessauer, 2009; Taussig, Iniguez-Lluhi, & Gilman, 1993).  For example, G?z selectively inhibits ACI and ACV (Kozasa & Gilman, 1995), while Gi?1,2,3 preferentially inhibits ACV and ACVI (Taussig, Tang, Hepler, & Gilman, 1994). ACI is inhibited by Go? and Gi?1,2,3 while ACII remains unaffected (Taussig, et al., 1994).  Ca2+ / calmodulin (CaM), depending on the pathway and the mode of increase, also has selective effects on certain ACs.   ACI and ACVIII are stimulated by Ca2+- CaM while ACIII is inhibited (Xia & Storm, 1997).  Generally, ACs are usually tissue specific and therefore particular isoforms are localized to certain tissue types; however, there are exceptions. All AC isoforms including the soluble AC (SAC) are found in the brain (Alasbahi & Melzig, 2012; Defer, Best-Belpomme, & Hanoune, 2000; Sunahara & Taussig, 2002; Tresguerres, Levin, & Buck, 2011; Wu et al., 2006).  Moreover, adding to the complexity to  the tissue distribution of specific isoforms of ACs, AC isoform tissue specificity changes over the life cycle of an organism as 7  Visel & Alvarez-Bolado et al (2006) demonstrated in embryonic (e14.5), postnatal (P7) and P57 C57BL/6 mice. Additionally, some ACs are also regulated by feedback from PKAs or PKCs. For example, ACV and ACVI are inhibited by feedback from PKA (Iwami et al., 1995).  1.5.1.2 Protein Kinase A (PKA)  PKA, one of the first proteins discovered to interact with cAMP (Walsh, Perkins, & Krebs, 1968),  consists of two catalytic domains and two regulatory domains (Cheng, Ji, Tsalkova, & Mei, 2008; Skalhegg & Tasken, 2000; Taylor et al., 1999).  PKA, when inactive, is known as a holoenzyme. In the inactive state, PKA is a tetrameric protein that consists of two regulatory (R) subunits and two catalytic (C) subunits.  The subunits consist of various combinations of the isoforms of regulatory subunits RI?, RI?, RII? and RII? and similarly, a combination of different C subunit isoforms. The C subunits exist as either C?, C?, C? or various other splice forms of C?,?.  When R and C subunits combine to form the holoenyme, they are generally characterized as either PKAI or PKAII holoenzymes (Skalhegg & Tasken, 2000; Tasken et al., 1997). The various isoforms of PKA are also tissue specific; PKAII (RII??) is somewhat ubiquitous, PKAI (RI?) is generally located primarily in the CNS brain and spinal cord, whereas, for example, PKAII (RII?) is found in the heart and PKAII (RII?) is detected in liver and adipose tissue (Skalhegg & Tasken, 2000).  These various isoforms create another level of regulation and specificity as they elute or activate at different NaCl levels, pH, temperature, cAMP levels, and concentration of their own subunits (Skalhegg & Tasken, 2000).  Taken together, the basic principle behind the operation of PKA is that cAMP binds the R subunit in a cooperative manner (i.e. as more cAMP binding sites are filled [there are two cAMP binding sites on each R subunit]  the additional cAMP binding sites confer greater binding accessibility), that when occupied with cAMP, the C subunit dissociates and either phosphorylates its target enzymes in the cytosol or enters the nucleus to phosphorylate various nuclear targets (enzymes and transcription factors etc.) (Tasken, et al., 1997).  In the nucleus, protein kinase inhibitory (PKI), serves as a C unit regulator. PKI binds the C subunit in the nucleus and provides the C subunit with an ?exit? signal to leave the nucleus and cause the C subunit to re-associate with the R subunits and reform the holoenzyme (Skalhegg & Tasken, 2000; Spaulding, 1993).  Further specificity for cAMP signalling and proximity substrate targeting by PKA is facilitated by A kinase anchoring proteins (AKAPs).  8   1.5.1.3 A-Kinase Anchoring Proteins (AKAPs)  AKAPs were originally discovered as contaminants in the process of trying to purify PKA (Lohmann, DeCamilli, Einig, & Walter, 1984; Sarkar, Erlichman, & Rubin, 1984). These proteins come from a family of 50 related proteins all with the unique ability to bind PKA (Dodge-Kafka, Bauman, & Kapiloff, 2008).  These scaffolding proteins play in important role in cAMP signalling in that they bind specific PKA?s in order to proximally locate them to specific cellular substrates or organelles. For example, Fraser & Tavalin et al. (1998) demonstrated that AKAP18 targeted the PKA to a proximal L-type calcium channel in the membrane. Furthermore, they also coordinate the coupling of additional participants of the cAMP signalling complex; such as, PDEs, phosphatases (PPs), ACs etc. (Coghlan et al., 1995; Dodge-Kafka et al., 2005; Dodge et al., 2001; Klauck et al., 1996). The R subunits of PKA bind AKAP?s through an amphipathic helix motif or docking and dimerization domain (D/D) of the AKAP (Newlon et al., 2001).  Taken together, specific AKAP?s occupy certain tissues and specific subcellular locations and have particular properties or functions.  For example, Westphal & Soderling et al. (2000) discovered that the Wiskott-Aldrich syndrome protein (WAVE-1) was an AKAP that bound PKA and the tyrosine kinase c-Abl to sites specific to actin reorganization. As previously noted, AKAP?s also coordinate the activity of PDE?s, the phosphodiesterases responsible for hydrolyzing cAMP.  1.5.1.4 Phosphodiesterases (PDEs)  PDEs, like ACs, AKAPs and PKAs, have tissue specific isoforms.  The spatiotemporal dynamics of PDEs help control the cAMP cascade and also facilitate in the creation of cAMP microdomains (for review see: Kleppisch, 2009).  Essentially, PDEs not only reduce the levels of cAMP thereby quelling the cascade, but PDEs also create compartmentalized microdomains of cAMP which focuses the signal of the original effector molecule to a specific substrate or target (Baillie, 2009; Tresguerres, et al., 2011). Thus far, PDEs of the mammalian superfamily are encoded by some 21 genes and grouped into 11 families (PDE1-PDE11) based on residue sequence homology, substrate specificity, pharmacological properties and regulatory characteristics.  PDEs 4,7,8 hydrolyze cAMP only and PDEs 5,6,9 hydrolyze cyclic guanosine 9  mono phosphate (cGMP) specifically, while PDEs 1,2,3,10,11 hydrolyze both cAMP and cGMP (Beavo, Conti, & Heaslip, 1994; Conti & Beavo, 2007; Kleppisch, 2009; Tasken & Aandahl, 2004).  In general, one mechanism by which PDEs exert their actions is by being activated by the very same PKAs that target specific substrates. For instance, Sette & Conti (1996) demonstrated that when PKA is activated by cAMP, PDE4D3, a variant of PDE4, is subsequently phosphorylated by PKA as well thereby either terminating or desensitizing the stimulatory cascade.  Thus, PDEs serve an important role, not only in attenuating the effects of PKA, but other receptors or proteins that elevated cAMP activates, such as, the exchange-protein-directly-activated-by-cAMP (Epac). 1.5.1.5 Exchange Protein Directly Activated by cAMP (Epacs)  Epacs were originally coined by de Rooij & Zwartkruis et al. (1998) and are also known as cAMP-guanine nucleotide exchange factors (cAMP-GEFs) (Kawasaki et al., 1998).  Epacs generally comprise of domains that are homologous to guanine-nucleotide-exchange factors (GEFs) and have a cAMP binding site.  Since Epacs have direct cAMP binding sites, their actions can be independent from PKA.  However, there are instances that Epac and PKA can perform synergistically or act antagonistically (Christensen et al., 2003).  Essentially, Epacs exert their effects when they are bound to by cAMP.  When cAMP bound, Epacs activate the Ras-like GTPase superfamily of enzymes such as Rap1 and Rap2 [Ras-proximate 1 & 2] (Cheng, et al., 2008; de Rooij, et al., 1998).  Some of the functions of Epac include cell adhesion (Bos et al., 2003; Rangarajan et al., 2003), exocytosis (Ozaki et al., 2000; Roscioni, Elzinga, & Schmidt, 2008; Seino & Shibasaki, 2005), and neurite outgrowth (Christensen, et al., 2003; Monaghan, Mackenzie, Plevin, & Lutz, 2008).  EpacI and EpacII, like that of ACs, PDEs and AKAPs, are also spatiotemporal and tissue specific (Kawasaki, et al., 1998).  Epacs have additional nuclear functions. For example, Epacs have been shown to suppress genes when activated.  Fuld & Borland et al. (2005) demonstrated that when Epac1 was selectively activated it suppressed several genes including the glutamate receptor GluR1, the TNF? receptor TNFR1 and the p53 phosphoprotein. Conversely Shi et al. (2006) established that the cAMP-response element (CRE) binding protein (CREB) can be activated through the Epac section of the cAMP pathway.   10  1.5.1.6 cAMP Response Element Binding protein (CREB)  CREB is in essence a protein that imparts cAMP?s signal transduction process in order to produce genetic responsiveness.  Structurally CREB contains a basic region of amino acid residues used for deoxyribonucleic acid (DNA) binding and a zipper region of a heptad of leucine residues that aid in either hetero or homodimerization of similar family members.  Family members that include the cAMP response element modulator (CREM) and the activating transcription factor (ATF1). (for reviews please see: Mayr & Montminy, 2001; Mayr, Canettieri, & Montminy, 2001; Shaywitz & Greenberg, 1999).  In general, CREB is activated when cAMP binds to the PKA holoenzyme which dissociates and releases the C subunit that enters the nucleus and phosphorylates CREB at, for example, serine 133 (for CREB 347) or serine 119 (for CREB 327) (Gonzalez & Montminy, 1989).  Phospho-CREB (P-CREB) goes on to complex with co-activator CREB-binding-protein (CBP) which in turn associates with RNA polymerase II (Pol II) (Kwok et al., 1994; Mayr, et al., 2001). CREB has additional domains that can bind other factors and contribute towards transcriptional activation. Factors such as transcription factor-II-D (TFIID), TFIIB or TATA-binding protein (TBP)-associated factors (TAFs) that bind downstream to TATA boxes (Shaywitz & Greenberg, 1999). Transcription occurs when the complex binds to the CRE promoter or close variants thereof (Montminy, Sevarino, Wagner, Mandel, & Goodman, 1986).  Cessation of gene transcription generally occurs by the actions of serine/threonine phosphatases (PP) that dephosphorylate P-CREB (for review see: Daniel, et al., 1998).        11   Figure 1.2  Prototypical initiation of the cAMP second messenger system and cAMP production Initiation of the cAMP second messenger system begins with an effecter molecule binding to the G protein coupled receptor (GPCR). The exchange of GDP for GTP results in the dissociation of the G?s and G?? subunits.  G?s subsequently associates with AC resulting in the conversion of ATP to cAMP (Created from images in the public domain; Protein Data Bank; Public domain: Federal Government under the terms of Title 17, Chapter 1, Section 105 of the US Code).         Figure 1.3 Molecular representation of the conversion of ATP to cAMP Conversion from ATP to the cyclic form results in the products cAMP and pyrophosphate.  12  1.6 cAMP ? involvement in axonal regeneration and neuronal survival - literature review  1.6.1 cAMP involvement in PNS regeneration  Initial forays about the involvement of cAMP in axonal regeneration were performed in the PNS.  Dorsal root ganglia from 8.5 day old chick embryos cultured on collagen coated coverslips combined with N6,O2-dibutyryl cyclic adenosine mono-phosphate (db-cAMP) or cAMP, extended their axons by greater than two fold versus the controls (Roisen, Murphy, Pichichero, & Braden, 1972).   Moreover, db-cAMP had an even greater effect on neurite extensions than that of cAMP alone.  Db-cAMP applied to a sciatic crush injury in Holtzman rats in vivo increased axonal nerve regeneration and provided an expedited return of sensorimotor function in comparison to the control animals (Pichichero, Beer, & Clody, 1973).  Kilmer and Carlsen (1984) using Rana pipiens with a sciatic nerve injury as a model and forskolin (a labdane diterpene from Coleus forskohlii) to increase cAMP levels, demonstrated a 40% increase in nerve regeneration. Kilmer and Carlsen (1987) replicated their previous work in the mammalian system using a variety of methods to increase cAMP levels in order to augment axonal regeneration with equal success.      1.6.2 cAMP involvement in CNS regeneration  Incipient neuronal studies incorporating the role of cAMP in axonal regeneration with CNS inhibitors were conducted in vitro.  DRG (dorsal root ganglia) neurons that are previously exposed to the neurotrophins BDNF (brain derived neurotrophic factor) or GDNF (glial cell line-derived neurotrophic factor) have been shown to be able to extend their neurites when plated on a substrate expressing MAG (myelin associated glycoprotein), a CNS inhibitor. This neurotrophin priming increases endogenous levels of cAMP by two fold.  Interestingly, DRGs exposed to only db-cAMP also overcame the effects of MAG based inhibition (Cai, Shen, De Bellard, Tang, & Filbin, 1999).  P4 and younger DRGs show enhanced neurite outgrowth when grown on CHO (Chinese Hampster Ovary) cells expressing MAG. In contrast, P5 and older DRGs are strongly inhibited by the CNS inhibitor.  Intriguingly, it appears that in P5 and older DRGs, the endogenous levels of cAMP decline dramatically in comparison with P4 and younger DRGs (Cai et al., 2001).  It is therefore possible that elevated cAMP levels in the cell body contribute towards the neuron?s ability to overcome inhibitory proteins or molecules. 13   The use of phosphodiesterase inhibitors, such as the drug rolipram, to increase cAMP levels have been shown to be somewhat efficacious in CNS axonal regeneration.  C3/4 hemisected rat spinal cords treated with rolipram displayed improved axonal regeneration (i.e. >100 fold increase in the numbers of serotonergic fibres growing into a transplant of embryonic spinal tissue (E14)) and functional recovery as indicated by the vertical exploration test in comparison to the control groups (Nikulina, Tidwell, Dai, Bregman, & Filbin, 2004).    Downstream effectors of the cAMP second messenger system have been shown to contribute to CNS axonal regeneration.  cAMP response element binding proteins (CREBs) are activated when elevated levels of cAMP initiate the second messenger cascade.  For example, constitutively active CREB via an adenovirus expressing VP-CREB injected into L4 DRGs 4 days prior to injury has been shown to promote regeneration of dorsal column axons in T6/7 lesioned rats (Gao et al., 2004).    1.6.3 cAMP involvement in conditioning lesions  Richardson & Issa?s (1984) landmark study revealed the significance of a peripheral ?conditioning? lesion in axonal regeneration in the CNS. L4 and L5 ganglia regenerated in abundance only when the ipsilateral sciatic nerve was cut.  Preconditioning lesions in PNS sciatic nerves  performed 1-2 weeks prior to a T6-8 dorsal column lesion displayed axonal growth into the spinal cord to and beyond the lesion (Neumann & Woolf, 1999). Prior peripheral lesions performed before test lesions have been shown to not only increase cAMP levels by three fold in DRGs, but also prevent inhibition by CHO cells expressing MAG.  Furthermore, the addition of db-cAMP to DRGs without a conditioning lesion was sufficient to also overcome MAG induced inhibition in vitro.  Pre injection of db-cAMP into DRGs, in vivo, 1 week preceding the lesion of the dorsal column at T6-7 revealed an overall increase in axonal growth into the spinal cord in comparison to the control group (Qiu et al., 2002). In a similar experiment, injection of db-cAMP into lumber DRGs performed 48hrs before a T6-7 dorsal column lesion, also resulted in axonal growth to and around the site of injury (Neumann, Bradke, Tessier-Lavigne, & Basbaum, 2002).  CNS injuries in the optic nerve also benefit from increased levels of cAMP.  Pre or post injection of db-cAMP into the rat eye before or after an optic nerve crush 14  promoted the regeneration of retinal ganglion cells (RGCs) beyond the site of injury (Monsul et al., 2004).  Additionally, while it has been shown that preconditioning injuries (i.e. PNS sciatic nerve crush in adult fisher rats) performed one week prior to the test lesion (bilateral transaction of the dorsal columns at the C3 level) significantly increased cAMP levels in the soma; recent evidence has been brought forward to show that the same ?preconditioned injury? can be performed as a ?postconditioned? injury as much as one day after the test injury culminating with the same regenerative outcome.  It should be noted, however, that ?postconditioned? lesions made more than one day after the test injury do not result in a significant regenerative response.  Furthermore, in that case, all of the ?conditioned? lesions, whether performed a week prior or on the day of the test injury, resulted in elevated sustained levels of cAMP in the cell body for approximately one week (Blesch et al., 2012).  1.6.4 cAMP involvement in neuronal survival  In addition to cAMP?s ability to overcome CNS inhibitors and augment axonal regeneration, cAMP has been implicated in increasing neuronal survival as well in CNS injury.  Various cAMP analogs have been shown to be capable of replacing NGF?s survival inducing capabilities in cultured rat DRGs and superior cervical ganglia (Rydel & Greene, 1988). Rolipram, a selective phosphodiesterase inhibitor that increases cAMP levels, has been shown to protect CA1 neurons from ischemic damage (Kato, Araki, Itoyama, & Kogure, 1995).  It is believed that the elevation of cAMP reduces the levels TNF-?, a proinflammatory cytokine (Endres et al., 1991; Greten, Eigler, Sinha, Moeller, & Endres, 1995; Molnar-Kimber, Yonno, Heaslip, & Weichman, 1993; Molnar-Kimber, Yonno, Heaslip, & Weichman, 1992; Semmler, Wachtel, & Endres, 1993).  Indeed, rolipram reduces TNF-? levels and the excitotoxic neuronal damage caused by quinolinic acid in the rat striatum (Block, Schmidt, Nolden-Koch, & Schwarz, 2001). While higher levels of TNF-? may be implicated with neuronal damage, it should be noted, however, that a clear role for TNF-? in either neuronal death or the enhancement of neuronal survival has yet to be fully elucidated (Cheng, Christakos, & Mattson, 1994; Kwon, Tetzlaff, et al., 2004).  It has also been demonstrated that NGF dependent PC 12 cells are not only rescued from apoptotic cell death by the administration of db-cAMP or other cAMP analogues, but the survival of the cells depend on the increased levels of cAMP after being NGF/db-cAMP deprived (Michel, Vyas, & Agid, 1995).  Moreover, embryonic spinal motor neurons survived longer in culture 15  with an increase in the levels of cAMP only in comparison to culture mediums containing either individual or combinations of the peptide trophic factors such as BNDF (brain derived neurotrophic factor), CNTF (ciliary neurotrophic factor), FGF (fibroblast growth factor), GDNF (glial-derived neurotrophic factor) or HGF (hepatocyte growth factor) (Hanson, Shen, Wiemelt, McMorris, & Barres, 1998).    For a summary of experiments employing altered cAMP levels to increase either axonal growth, regeneration, or survival in-vitro and/or in-vivo, see Table I in the Appendices.   16  1.7 Barriers to regeneration  Ram?n y Cajal had exhaustively documented many ineffectual outcomes of CNS injury in comparison to PNS injuries (May, 1928). In his incipient investigations, Ram?n y Cajal (1928) had already indicated that the myelin or white matter of the CNS could restrict CNS regeneration. It was years later that investigators began to examine the components of myelin in order to understand the molecular nature of axonal inhibition. Over the years, some of these myelin inhibitors discovered included Nogo (Nogo-A), Nogo-66 (Caroni & Schwab, 1988a, 1988b; Fournier, GrandPre, & Strittmatter, 2001; GrandPre, Li, & Strittmatter, 2002; GrandPre, Nakamura, Vartanian, & Strittmatter, 2000), Myelin Associated Glycoprotein (MAG) (DeBellard, Tang, Mukhopadhyay, Shen, & Filbin, 1996; Li et al., 1996; McKerracher et al., 1994; Mukhopadhyay, Doherty, Walsh, Crocker, & Filbin, 1994), Oligodendrocyte myelin glycoprotein (OMgp) (Kottis et al., 2002; Wang et al., 2002), Semaphorin Sema4D (Moreau-Fauvarque et al., 2003), and Ephrin B3 (EphB3) (Benson et al., 2005).  However, not all inhibitors of axonal regeneration in SCI are myelin associated. SCI embodies an enormous intertwined cascading series of events that involve physical, biochemical, genetic, immunological, vascular and cellular changes that ultimately leads to a mostly abortive regenerative condition. Physical and chemical barriers also include the so called ?glial scar?. Comprised mainly of reactive astrocytes, microglia/macrophages and proteoglycans, this, when stained for GFAP (a general, but not exclusive, astrocytic marker), intermeshed stellate appearing barrier creates a formidable physical and chemical obstruction between the fluid filled cavity and the confines of the spinal cord (for reviews see: Fawcett & Asher, 1999; Silver & Miller, 2004; Yiu & He, 2006).    1.8 cAMP involvement with inhibitors to regeneration  cAMP can participate in a prominent role, either directly or indirectly, in the convergence of the intracellular downstream pathways of the some of the inhibitors to regeneration.  Intrinsic levels of cAMP are usually altered either directly via G protein, adenylyl cyclase activated pathways or indirectly by the outcomes of other activated pathways which ?crosstalk? with cAMP pathways.  Much study has been undertaken to elucidate the receptors to the inhibitors of regeneration, and importantly what downstream biochemical pathways they invoke and how they mediate their 17  ability to prevent, or in some cases stimulate, regeneration. Various molecules and downstream events believed to be inhibitory to axonal regeneration are discussed below. How changes in cAMP levels alter the functions of these inhibitory molecules and their downstream events is discussed in Section 1.10. 1.8.1 Axonal inhibitory molecules and receptors  1.8.1.1 Myelin based inhibitor Nogo-A  One of two protein fragments found mainly in CNS, not PNS myelin, were discovered to be non-permissive for neurite growth (Caroni & Schwab, 1988b).  The 250kD protein was identified as Nogo-A (GrandPre, et al., 2000), a member of the reticulon family, and Chen et al. (2000) reported that recombinant Nogo-A was the target of the antibody IN-1. Injected Anti Nogo-A IN-1 antibody was shown to increase axonal in-growth in damaged optic nerves (Caroni & Schwab, 1988a).  The extracellular portion comprising of 66 residues (Nogo-66) when brought into contact with DRG (dorsal root ganglion) cells, produced strong growth cone collapse (GrandPre, et al., 2000).      1.8.1.2 Myelin associated glycoprotein (MAG)  Adult and developing DRG cells and cerebellar neurons cultured on Chinese hamster ovary (CHO) cells transfected with L-MAG cDNA displayed differing effects. Adult cells were strongly inhibited while MAG was permissive, in contrast, for developing DRGs. Antibodies against MAG removed the inhibitory effects of MAG for the adult cells (Mukhopadhyay, et al., 1994). Neurites from NG108-15 cells (cells with motor neuron properties) failed to extend on the polylysine coated plates containing recombinant MAG, however neurite growth was restored to within 63% of control levels when MAG was immunodepleted (McKerracher, et al., 1994). Li et al?s. (1996) study supported the findings that MAG was inhibitory by demonstrating that the growth cones of postnatal day 1 (P1) hippocampal neurons collapsed when exposed to polystyrene beads covered with rMAG (recombinant MAG).  rMAG that was denatured abolished this collapse.     18  1.8.1.3 Oligodendrocyte myelin glycoprotein (OMgp)  It was originally postulated that when sensory DRG?s, retinal cells, and sympathetic neurons cocultured with glia, that both myelin and oligodendrocytes were inhibitory (Schwab & Caroni, 1988).  It was, however, unclear which components in either the myelin or the oligodendrocytes were responsible for the restrictive neurite behaviour.  Wang et al. (2002) used phosphatidylinositol specific phospholipase C (PI-PLC) to isolate OMgp in order to demonstrate that it caused growth cone collapse and neurite inhibition in neurite outgrowth assays.  Furthermore, OMgp, human or mouse derived, causes neurite inhibition on OMgp polylysine coated wells (Kottis, et al., 2002).  1.8.1.4 Nogo-66 receptor (NgR)   Identified as the Nogo-66 receptor (NgR), this receptor when bound to by the extracellular or luminal domain of Nogo causes the inhibition of neurite outgrowth and growth cone collapse of DRGs (Fournier, GrandPre, Gould, Wang, & Strittmatter, 2002; Fournier, et al., 2001; GrandPre, et al., 2000).  NgRs (specifically NgR1) are unique in that structurally unrelated proteins or molecules such as MAG, OMgP and Nogo bind to and activate the NgR complex (Wang, Kim, Sivasankaran, Segal, & He, 2002; Wang, Koprivica, et al., 2002).  The 66 amino acid domain located in the extracellular segment of Nogo-A, binds to NgR and is responsible for neurite and growth cone collapse of chick DRGs and mouse cerebellar neuron cultures (Fournier, et al., 2001). NgRs have a leucine rich domain which is indicative of a structural framework that lends itself towards protein-protein interactions (Kobe & Kajava, 2001).   Additionally, NgRs are also GPI anchored without cytoplasmic domains, which raises questions about which receptor complexes may be formed (Fournier, et al., 2001).  Wong et al. (2002) established that the p75 neurotrophin receptor (p75NTR) co-immunoprecipitates with NgR in HEK-293 cell membrane extracts.  1.8.1.5 p75NTR  Structurally, p75NTR can be characterized as a 75KDa glycoprotein that belongs to the tumor necrosis superfamily (Gong, Cao, Yu, & Jiang, 2008).  Extracellularly it has a cysteine rich domain (CRD) which is IgG like, and intracellularly, it contains a death domain (Blochl & 19  Blochl, 2007; Liepinsh, Ilag, Otting, & Ibanez, 1997).  It is known to bind to all proneurotrophins (proNTs) and neurotrophins (NTs) such as BDNF (brain derived neurotrophic factor), NT-3, NT-4 (Dechant, Rodriguez-Tebar, & Barde, 1994; Reichardt, 2006; Rodriguez-Tebar, Dechant, & Barde, 1990, 1991; Rodriguez-Tebar, Dechant, Gotz, & Barde, 1992).  The downstream signalling events of p75NTR have been shown to produce a multitude of neuronal outcomes, such as apoptosis, reduced growth cone motility, and survival among others (Haase, Pettmann, Raoul, & Henderson, 2008; Lu, Pang, & Woo, 2005; Miller & Kaplan, 2001).  Repulsive growth cone turning of Xenopus axons occurs when MAG binds NgR that is in close apposition to p75NTR (Wong, et al., 2002).  It was also established that the binding by MAG, OMgp or Nogo-A (Nogo-66) to NgR causes neurite inhibition through the downstream signalling events of p75NTR (Wang, Kim, et al., 2002).  1.8.1.6 TROY and LINGO-1  It was demonstrated that NgR can still wield its restrictive axonal growth effects in cells not showing detectable levels of p75NTR.  The discovered molecule TROY (TAJ), a TNF receptor family member, formed a tripart complex similar to the p75NTR complex with NgR and LINGO-1 that also produced axonal inhibitory effects by myelin inhibitors (Park, Yiu, Kaneko, Wang, Chang, & He, 2005; Shao et al., 2005).   1.8.1.7 The Rho pathway with the p75NTR complex     During SCI Nogo-A (Nogo-66), MAG and OMgp can bind to NgR1 which associates closely to p75NTR (for a complete molecular overview see Figure 1.4).  Close apposition of the two receptors with LINGO-1 allows p75NTR to exert its downstream effects.  Activated p75NTR recruits the RhoA-GDP (RhoA-guanosine diphosphate) and RhoA-GDP-GDI (Rho-GDP-dissociation inhibitor) complex (Sasaki & Takai, 1998; Yamashita, Higuchi, & Tohyama, 2002; Yamashita & Tohyama, 2003; Yamashita, Tucker, & Barde, 1999). In this situation RhoA-GDP is considered to be the inactive form of Rho while RhoA-GTP is the active form; RhoA-GDP-GDI not only keeps RhoA in the cytosol until p75NTR displaces RhoA from Rho-GDI, but also prevents guanine nucleotide exchange factors (GEFs) from displacing GDP for GTP (Yamashita & Tohyama, 2003). Once p75NTR is associated with the RhoA-GDP-GDI complex, Rho-GDP is 20  released into the cytosol where it is activated by GEFs.  In addition, it should be noted that another model purports that activated p75NTR stabilizes RhoA-GDP-GDI until cleaved by ?-secretase extracellulary (Hooper, Karran, & Turner, 1997) and ?-secretase (Kopan & Ilagan, 2004) intracellulary (Domeniconi et al., 2005; Kanning et al., 2003). Subsequently, RhoA-GTP then activates membrane bound Rho-kinases (ROCKs).  Activated Rho and now phosphorylated Rho-kinase phosphorylates myosin light chain (MLC) and inhibits the Rac/cdc-42 pathways leading to actomyosin contractility and eventual neurite retraction (Amano, Fukata, & Kaibuchi, 2000; Amano et al., 1996; Hirose et al., 1998). Rho-kinase also phosphorylates LIM-kinase, a serine/threonine kinase with a zinc-finger structure (Arber et al., 1998; Kadrmas & Beckerle, 2004), which in turn, phosphorylates cofilin, a protein that depolymerizes actin (Arber, et al., 1998; Moon & Drubin, 1995), thereby inactivating it. While the exact mechanism of Rho/ROCK activated growth cone collapse has yet to be fully elucidated, phosphorylation of cofilin appears necessary (Aizawa et al., 2001).  There are also other inhibitory proteins, molecules and receptors that converge on the Rho pathway.  For instance, CSPGs can exert their inhibitory effects through the Rho/ROCK pathway (Borisoff et al., 2003; Monnier, Sierra, Schwab, Henke-Fahle, & Mueller, 2003).  Schweigreiter et al. (2004) made apparent that versican V2 utilitizes another pathway that converges with the Rho pathway in order to inhibit neurites of DRGs in vitro. Other molecules and receptors implicated in the Rho pathway include plexin-B1 (Semaphorin4D, an axon guidance molecule, is the ligand) (Aurandt, Vikis, Gutkind, Ahn, & Guan, 2002) and Ephrin-A5. Ephrin-A5 has been shown to cause growth cone collapse in retinal ganglion cell cultures via Rho-kinase (Wahl, Barth, Ciossek, Aktories, & Mueller, 2000).    1.9 SCI therapeutic strategies   Therapeutic approaches directed towards functional repair of SCI are undoubtedly daunting tasks. Almost a century of research, with the greatest proportion of knowledge gathered in recent decades, has uncovered a significant amount of information with regards to factors that influence, for example, axonal inhibition and regeneration, cellular death, or the general pathophysiology of SCI.  Even with the current knowledge of the components involved in the degenerative process of SCI, there still are many proteins, molecules or systems that have yet to be either revealed or of the currently identified ones - their functions fully elucidated.  SCI is an injury that changes with time. As time progresses, the requirements and challenges in treating 21  SCI need to be altered as well.  Indeed, it appears that no single strategy may be completely sufficient in providing true functional recovery in SCI.    There are many strategies and areas for treating SCI. Neuroprotective approaches attempt to attenuate the continuing secondary damage while preventing further neuronal and axonal loss after SCI (for review see: Kwon, Fisher, Dvorak, & Tetzlaff, 2005).  Neuroregenerative methods try to prime and maintain axonal regeneration, sprouting and plasticity by either overcoming inhibitors to regeneration or quelling the effects of the inhibitory factors, or by stimulating intrinsic ?regenerative programs? or initiating/maintaining the expression of growth promoting genes (for reviews see: Blesch & Tuszynski, 2009; Kwon, et al., 2005; Rowland, Hawryluk, Kwon, & Fehlings, 2008; Ruff, McKerracher, & Selzer, 2008).  Cellular neuro-reconstructive and bridging therapies endeavour to replace damaged and missing oligodendrocytes, while bridging treatments pursue ways to traverse the fluid filled cystic cavity that develops in SCI.  Functional restoration regimes attempt reinstate the lost or severely reduced propagative conduction signals that the damaged axons now have.  Finally, targeting and axonal guidance therapies strive to guide the newly formed axons to the appropriate targets (for overviews see: Rowland, et al., 2008; Schwab et al., 2006). The focus of this thesis is on a proof of principle treatment where the resultant data may someday contribute towards devising a potential regenerative therapy for SCI.   1.10 The molecular role and potential benefits of increased levels of cAMP in axonal/neurite growth and regeneration  Increased levels of cAMP in neurons with damaged axons have been shown to be one of several contributing factors toward augmented axon/neurite growth and regeneration (see Table I in the Appendices for details).  Over the last few decades some of the molecular participants and pathways involved with cAMP and/or its pathways in axon growth and regeneration have been detailed.  Listed below are some of molecules and the mechanisms by which elevated cAMP levels augment axon regeneration. For a detailed graphically summarized molecular map of this section see Figure 1.4., and for a molecular flowchart listing the molecules that either increase, inhibit, mimic or reduce cAMP levels along with the molecules affected by changes in cAMP levels which contribute towards axon regeneration see Figure 1.5.      22  1.10.1 Increased cAMP levels attenuate activated p75NTRs axonal inhibitory effects   Of the known myelin based inhibitors, Nogo-A (Nogo-66), MAG and OMgp, all three interact through the p75NTR complex (Wang, Kim, et al., 2002; Wong, et al., 2002; Yamashita, et al., 2002).  P75NTR and effector Rho with its related pathway components are notable intervention targets for overcoming axonal inhibition.  It has been shown that increased levels of cAMP activate PKA which phosphorylates RhoA preventing it from binding to Rho kinase (Dong, Leung, Manser, & Lim, 1998).  Downstream molecules of ROCK such as MLC and LIM kinases will then also be inhibited helping prevent growth cone retraction.  By increasing the levels of cAMP with db-cAMP, it has been demonstrated that PKA phosphorylates the RhoA-GDP-GDI complex thereby redirecting it away from the cellular membrane and back into the cytosol (Lang et al., 1996).  As p75NTR is located in the cellular membrane the efficacy by which RhoA is displaced from the GDI complex and subsequent activation by GEFs is markedly reduced thereby inhibiting downstream events of Rho. Raising cAMP levels will additionally compensate for the cAMP reduction produced by the axonal inhibitor initiated p75NTR activation of Gi which inhibits ACs that produce cAMP (Cai, et al., 1999).    1.10.2 Increased cAMP levels activate CREB to overcome the inhibitory effects of Nogo?20  A Nogo-A specific region, Nogo?20 (Nogo-A specific region; NiG-?20 aa544-725; 181aa), has been shown to cause in vitro growth cone collapse and neurite inhibition of rat DRGs (P6) independently of NgR (Oertle et al., 2003).  Furthermore, it has been demonstrated that Nogo?20 activated the GTPase RhoA in rat (P7) cerebellar granule cells and inhibited neurite outgrowth in vitro (Nieder?st, Oertle, Fritsche, McKinney, & Bandtlow, 2002).  Recently it has been shown that Nogo?20 is endocytosed at the growth cone via a clathrin independent mechanism in rat hippocampal neurons (E19). Once internalized Nogo?20 activates the small GTPase RhoA and is retrogradedly transported to the cell body (in this case DRG cell bodies).  Nogo?20 also decreased the amount of phosphorylated CREB (pCREB) in the cell bodies of DRGs.  The application of 1mM db-cAMP to cerebellar granule neurons (CGNs) elevated pCREB levels and nearly fully restored Nogo?20 neurite outgrowth inhibition (Joset, Dodd, Halegoua, & Schwab, 2010). 23  1.10.3 Increases in cAMP increase polyamine levels   Increases in the levels of polyamines have been implicated in both neuronal growth and neurons that have undergone axonal injury (Seiler, Sarhan, & Roth-Schechter, 1984; Tetzlaff & Kreutzberg, 1985).  While the exact functions of polyamines remain unclear (Morgan, 1998; Pegg & Casero, 2011); polyamines are vital for cell growth (Chattopadhyay, Park, & Tabor, 2008; Pegg, 1986; Pegg & Casero, 2011).  Cells either lacking in polyamines or are polyamine depleted show losses in microtubules and actin filaments, as well as major chromosomal aberrations (Knuutila & Pohjanpelto, 1983; Pohjanpelto & Knuutila, 1982, 1984; Pohjanpelto, Virtanen, & Holtta, 1981).  It has been shown that axotomized rat facial neurons increase ornithine decarboxylase (ODC), a rate limiting enzyme in the initial stage of polyamine synthesis, which in turns increases polyamine levels as part of the regenerative response (Tetzlaff & Kreutzberg, 1985).  Exogenous applications of polyamines accelerate the regeneration of crush injured rat peripheral sympathetic neurons (Dornay, Gilad, Shiler, & Gilad, 1986), increase survival of axonally injured sympathetic neurons (Gilad & Gilad, 1988), promote regeneration of injured hippocampal neurons in vitro (Chu, Saito, & Abe, 1995) and precipitate motor function recovery after facial nerve injury in rats (Gilad, Tetzlaff, Rabey, & Gilad, 1996).  Interestingly, ODC activity is increased by increased levels of cAMP (Byus & Russell, 1975) in a PKA dependent manner (Byus, Costa, Sipes, Brodie, & Russell, 1976; Byus & Russell, 1976).  Arginase-1 (Arg I), the enzyme that catalyzes the reaction of the amino acid arginine to ornithine and subsequently the polyamines; Arg-I mRNA and protein are upregulated 22 fold in axotomized superior cervical ganglia (SCG) (Boeshore et al., 2004).  The addition of db-cAMP to cerebellar neurons grown on MAG expressing CHO cells increased Arg-I levels two fold.  This resulted in an increase in polyamine synthesis as well as the ability of the cerebellar neurons being able to extend neurites over an inhibitory substrate (Cai et al., 2002).  Furthermore, activation of the transcription factor CREB is required for the upregulation of Arg I.  Constitutively active CREB in DRG neurons promotes regeneration of lesioned dorsal column axons (Gao, et al., 2004).   24  1.10.4 Inflammation induced regeneration in the optic nerve with inflammatory proteins (i.e. Ocm) depends on cAMP  Macrophages, when activated, are known to promote the regeneration of CNS neurons. Of the many factors released by macrophages, some are detrimental and others are beneficial to the survival and regenerative abilities of neurons (Gensel et al., 2009).  One of the macrophage expressed and secreted factors shown to be growth promoting in retinal ganglion cells is Oncomodulin (Ocm).  Ocm is an 11.7-kDA Ca2+-binding protein secreted by macrophages (Yin et al., 2009; Yin et al., 2006).   Ocm levels are seen to sharply rise when inflammation is induced by either an eye lens injury or when an inflammatory induced chemical such as Zymosan is used (Yin, et al., 2009).  Optic nerve crush injuries in adult mice, in vivo, require Ocm to be present, regardless of the inflammatory mechanism used with the injury, in order for significant axon regeneration to occur. Using the same paradigm, but blocking the action of Ocm abolishes axon regeneration in the optic nerve (Yin, et al., 2009).  Of importance, however, is that augmented optic nerve regeneration in mice requires not only Ocm, but mannose and elevated levels of cAMP (Benowitz & Yin, 2010; Kurimoto et al., 2010; Yin, et al., 2009; Yin, et al., 2006).  cAMP operates by prolonging the binding of Ocm to receptors in the inner retina of mice in vivo (Kurimoto, et al., 2010).   Adult rats that received an optic nerve crush injury with intraocular injections of polylactic-co-glycolic acid (PLGA) microspheres containing Ocm and Sp-8-Br-cAMP produced a 5-7 fold increase in regeneration versus the control group that received empty PLGA microspheres. Sp-8-Br-cAMP by itself produced a 2 fold increase in regeneration whereas Ocm was without effect when used alone to treat the optic nerve crush injury thus highlighting the requirement for increased cAMP levels (Yin, et al., 2006).     1.10.5 Ocm requires elevated cAMP levels when used as a regeneration enhancer in other nervous systems   The regenerative and functional role of Ocm in augmenting axonal growth after injury was initially investigated in damaged optic systems (for details see section 1.10.4).  Recent research examined whether or not Ocm could enhance axonal regeneration after axon injury in other nervous systems.  Using adult female Sprague-Dawley rat DRGs as models for nervous system injury, Harel and colleagues (2012) applied Ocm (200ng/?l) and/or db-cAMP (50mM) to the DRGs (cervical, lumbar or thoracic) either at the time of axon injury, or preconditionally one week prior to injury or harvest.  Both the in vitro and in vivo outcomes resulted in similar 25  conclusions.  When plated on poly-L-lysine with laminin (5?g/ml) and aggrecan (0.7mg/ml) containing an inhibitory proteoglycan rim, only cells treated with both Ocm and db-cAMP grew across the axonally restrictive rim.  Similarly, in the in vivo experiments, DRGs treated with both Ocm and db-cAMP had significantly more axonal growth cross the DREZ (dorsal root entry zone) than with either treatment alone. Moreover, preconditioning with Ocm and db-cAMP, both in vivo and in vitro, resulted in a greater degree of regeneration versus unconditioned treatment.  What is important to note is that Ocm relies on elevated levels of cAMP in order to exert its regenerative influences.  1.10.6 Activated Epacs by cAMP allows neurite extension on inhibitory substrates  Epacs serve as alternative effector targets for cAMP and are known to act through the Rap-1 pathway (Bos, 2006).  It has further been established that not only are Epacs developmentally regulated in the rat nervous system, but developing axons are attracted towards gradients of Epac agonists.  Epac1 expression is elevated at neonatal and embryonic ages, but declines precipitously in adulthood.  Moreover, embryonic DRG neurons cultured on adult rat spinal cord, extended lengthy neurites while those of adult DRGs were inhibited.  Activation of Epac with 8-CPT-2?-O-Me-cAMP allowed adult DRGs to extend neurites on the inhibitory substrate in a non PKA dependent manner (Murray & Shewan, 2008).   1.10.7 Increased cAMP may also increase neurite outgrowth via unknown cAMP pathways  Finally, not all cAMP signalling events occur through the known effector targets such as PKA or Epacs; some have yet to be elucidated.  It has been demonstrated, for example, that elevation of cAMP promotes ?6?1 integrin dependant neurite outgrowth of retinal neurons independently of the Epac and Rap-1 pathway as well as the PKA pathway (Ivins, Parry, & Long, 2004).      26   27  Figure 1.4  Schematic diagram illustrating the involvement of cAMP in CNS axonal injury and regeneration with identified inhibitory molecules This diagram represents a hypothetical compilation of how the cAMP second messenger system may be molecularly involved in both axonal regeneration and inhibition with known inhibitory molecules and components of their downstream pathways.  See text for a complete overview and specific details. Diagrammatic components and their actions: BDNF elevates cAMP levels (Cai, et al., 1999), TrkB receptors recruited to the plasma membrane by increases in cAMP (Meyer-Franke et al., 1998), elevated cAMP binds to Epacs which activate CREB (Shi, et al., 2006), activated CREB is sufficient to overcome inhibition by MAG (Gao, et al., 2004), elevated cAMP increases Arg-1 expression which increase polyamine levels (Cai, et al., 2002), elevated cAMP activates PKA which increase Ornithine Decarboxylase levels that increase the synthesis of polyamines (Byus & Russell, 1975; Byus & Russell, 1976), MAG inhibits axonal regeneration (McKerracher, et al., 1994; Mukhopadhyay, et al., 1994), OMgp inhibits axonal regeneration (Kottis, et al., 2002; Wang, Koprivica, et al., 2002), Nogo-66 causes growth cone collapse (GrandPre, et al., 2002; GrandPre, et al., 2000), Bound NgR causes inhibition of neurite outgrowth and growth cone collapse (Fournier, Gould, Liu, & Strittmatter, 2002; Fournier, et al., 2001; GrandPre, et al., 2002), p75NTR is associated with NgR and exerts its inhibitory axonal regenerative actions when NgR binds either MAG, Nogo-66, OMgp (Wang, Kim, et al., 2002), LINGO-1 forms a tripart complex with p75NTR and NgR, and is required for axonal inhibition by myelin inhibitors (Mi et al., 2004),  TROY (TAJ) forms a tripart complex with NgR/LINGO-1 and causes axonal regenerative inhibition when NgR is bound by myelin inhibitors (Park, Yiu, Kaneko, Wang, Chang, & He, 2005; Shao, et al., 2005), p75NTR activates Gi to reduce cAMP levels (Cai, et al., 1999),  RhoA-GDP-GDI is stabilized until cleaved by ?-secretase extracellulary (Hooper, et al., 1997) and ?-secretase intracellularly (Domeniconi, et al., 2005; Kanning, et al., 2003; Kopan & Ilagan, 2004), activated p75NTR recruits RhoA-GDP-GDI to release RhoA-GTP (Sasaki & Takai, 1998; Yamashita, et al., 2002; Yamashita & Tohyama, 2003; Yamashita, et al., 1999), RhoA-GTP activates ROCK ((Amano, et al., 2000; Amano, et al., 1996; Hirose, et al., 1998), ROCK phosphorylates LIM-kinase (Arber, et al., 1998; Kadrmas & Beckerle, 2004), LIM-kinase inactivates Cofilin as part of a mechanism involved in actin depolymerization (Aizawa, et al., 2001; Arber, et al., 1998; Moon & Drubin, 1995), elevated cAMP activates PKA that prevents RhoA-GDP-GDI from associating with p75NTR thereby preventing ROCK from exerting its downstream effects (Dong, et al., 1998; Lang, et al., 1996), activated Epacs allow neurite extension on inhibitory substrates (Murray & Shewan, 2008).  Db-cAMP has been shown to: augment CNS regeneration (Monsul, et al., 2004; Neumann, et al., 2002; Pearse et al., 2004), overcome MAG inhibition (Cai, et al., 1999; Neumann, et al., 2002), mimic conditioning lesions in order to enhance regeneration (Neumann, et al., 2002; Qiu, et al., 2002), increase Arg-1 thereby increasing polyamine levels in order to overcome inhibitors of regeneration (Cai, et al., 2002).    28   29  Figure 1.5 Summary flowchart of cAMP?s molecular involvement in axon growth or regeneration This diagrammatic flowchart is split into two sections. The two boxes on the left hand side of the image indicate the inducers (intrinsic or extrinsic) and the inhibitors or reducers (intrinsic or extrinsic) of cAMP.  The three boxes located on the right hand side of the diagram show the molecules that are either dependent on, increase, or are increased by cAMP, or act synergistically with or are reduced by elevated cAMP levels in order to augment axon regeneration. References for the diagram: 1 Posternak et al., (1962); 2 Buxton & Brunton (1983), Klein et al., (1989); 3 Huai et al., (2003), Miro et al., (2002), Nikulina et al., (2004); 4 Qui et al., (2002); 5 Tanaka et al., (2007); 6 Cai et al., (1999); 7 Gordon & Gordon (2010); 8 Ahmed et al., (2009); 9 Kleppisch et al., (2009); 10 Hansen et al., (2001), Christensen et al., (2003); Aglah et al., (2008); 11 Cai et al., (1999); 12 Joset et al., (2010); 13 Shelly et al., (2011); 14 Lane et al., (2004), Lane et al., (2005); 15 Lane et al., (2004), Lane et al., (2005); 16 Skalhegg et al., (1998), Cai et al., (1999); 17 Murray et al., (2008); 18 Murray et al., (2009); 19 Cao et al., (2006); 20 Wu et al., (2006); 21 Cai et al., (2002); 22 Yin et al., (2006), Kurimoto et al., (2010); 23 Yin et al., (2006); 24 Byus et al., (1975); Byus et al., (1976); 25 Ahmed et al., (2009); 26 Gao et al., (2004); 27 Ahmed et al.,(2009); 28 Murray et al., (2009); 29 Kurimoto et al., (2010); 30 Hellstrom et al., (2011), Ahmed et al.,(2009); 31 Kurimoto et al., (2010); 32 Dong et al., (1998), Lang et al., (1996); 33 Joset et al., (2010); 34 Park et al., (2009).  1.11 Rationale and experimental design  1.11.1 Experimental design overview  One of the goals of my experiments is to add to the body of knowledge in contributing towards the development of a regenerative treatment for spinal cord injury.   Using Sprague-Dawley rats as injury and treatment models, the red nucleus was treated prior to or accompanied rubrospinal tract damage. As previously noted, reduction of cAMP levels in the cell bodies of injured axons are believed to contribute to the inability of axons to successfully regenerate. Hence, as a treatment regime, an analogue of cAMP, dibutyryl cyclic adenosine mono-phosphate (db-cAMP), was delivered to the red nucleus in order to mimic cAMP elevation. Two treatment groups were used. An acutely treated group received db-cAMP at the same time as the injury while a pretreated group was given db-cAMP one week prior to the injury.  The overall assumption of these experiments was that sustained elevation of cAMP via analogue should stimulate or maintain the regenerative response of the red nucleus.     30  1.11.2 Red Nucleus (RN) and Rubrospinal tract (RST) ? rationale and anatomy  For these experiments I chose the rat RN and RST as a model for spinal cord injury (SCI), axonal damage and target for a regenerative treatment.  Both the RN and RST have clearly defined anatomical locations that allow them to be both suitable and useful targets for manipulations  in experimental design (i.e. drug therapy, injury etc.) as they can be readily identified with a tracer such as BDA (biotinylated dextran amine), and therefore be visualized and quantified for regenerative success or failure.  As a model for SCI or axonal damage, it is known that the RST displays both regenerative potential, for example, by being able to grow into a peripheral nerve graft (Richardson, Issa, & Aguayo, 1984) and general axonal abortive characteristics, such as, by not being able to cross the site of injury (Barron, Banerjee, Dentinger, Scheibly, & Mankes, 1989).    The RN is well defined and has a distinct anatomical arrangement in the motor system of mammals.  It is comprised of an anterior parvocellular and posterior magnocellular region (Canedo, 1997; Keifer & Houk, 1994) although the distinction is not as clear in rats (Yamaguchi & Goto, 2006).  The parvocellular region receives its main input from the cerebral cortex (Humphrey, Gold, & Reed, 1984; Humphrey & Rietz, 1976; Keifer & Houk, 1994) while the predominant input to the magnocellular region in rats is from the contralateral nucleus interpositus of the cerebellum (Angaut, Batini, Billard, & Daniel, 1986; Daniel, Angaut, Batini, & Billard, 1988; Keifer & Houk, 1994).  The cells of origin of the RST in rat are found primarily in the caudal section (magnocellular) of the RN.  Furthermore, the RN cells that give projections to the cervical spinal levels are located in the medial and dorsal sections of the RN (Murray & Gurule, 1979).  From the RN, the majority of the RST decussates in the ventral mesencephalic tegmentum (tegmental decussation) (Brown, 1974) although there is also a minor uncrossed ipsilateral projection (Holstege, 1987; Kennedy, 1987).  The contralateral axons of the RST continue to course through the dorsolateral funiculus of the spinal cord while in close apposition to the lateral corticospinal tract and terminate in Rexed?s lamina IV,V, VI, dorsal portions of VII, ventral portions of IV and IX in rats (Antal et al., 1992; Brown, 1974; Kuchler, Fouad, Weinmann, Schwab, & Raineteau, 2002).    31  1.11.3 Function of the RN and choice of behaviour test  The complete functions of the RN have yet to be fully realized and functional investigations remain ongoing (Onodera & Hicks, 2009, 2010).  Additionally, the development of the RN differs between mammals; for example, the size of the parvocellular portion of the RN of mammals increases as bipedalism becomes more pronounced as less emphasis is directed towards the forelimbs? use for locomotion (Canedo, 1997; Onodera & Hicks, 2009, 2010).  In primates the RN is involved in reaching to grasp movements (van Kan & McCurdy, 2002b) as well has hand preshaping during reaching to grab movements (van Kan & McCurdy, 2001, 2002a).  The RN in cats is hypothesized not to be involved in complex motor movements but instead acts as a link between supraspinal and spinal centres (Arshavsky, Orlovsky, & Perret, 1988; Rho, Lavoie, & Drew, 1999).   Moreover, it is felt that the ventral spinocerebellar tract adds to the activity of  the lumbar projecting rubrospinal neurons in the cat  (Orsal, Perret, & Cabelguen, 1988).  In rats, the complete role the RN has also yet to be completely determined and is still being investigated (Hermer-Vazquez et al., 2004; Whishaw & Gorny, 1996). Unilateral RN lesions in the rat result in decreased forelimb lifting and food pellet arpeggio grasping capabilities (Whishaw, Gorny, & Sarna, 1998), and influences ongoing overground locomotion (Muir & Whishaw, 2000).  Cervical dorsolateral funiculotomies that ablates the RST have also been shown to impact general reach to grasp tasks (Stackhouse, Murray, & Shumsky, 2008).  Behavioural studies also suggest that the RN in the rat operates to influence general control of groups of muscles in the forearm and paw, and less so in the precision aspect of those movements (Kuchler, et al., 2002).    The vertical exploration or ?cylinder? test (Choi-Lundberg et al., 1998; Liu et al., 1999; Schallert, Fleming, Leasure, Tillerson, & Bland, 2000; Schallert & Lindner, 1990) was used for determining the behavioural outcome of the db-cAMP versus vehicle treated C3/4 dorsal lateral funiculus crushed rats.  One of the primary reasons the vertical exploration test was chosen was because this metric and derivations thereof have been used as sensitive indicators of chronic forelimb use asymmetries (Humm, Kozlowski, James, Gotts, & Schallert, 1998; Jin, Fischer, Tessler, & Houle, 2002; Jones & Schallert, 1994; Liu, et al., 1999; Lundblad et al., 2002; MacLellan, Shuaib, & Colbourne, 2002; Murray et al., 2002; Tillerson et al., 2002; Tillerson et al., 2001; Vergara-Aragon, Gonzalez, & Whishaw, 2003; Voorhies & Jones, 2002) where 32  asymmetry of contact with the plexiglass cylinder by the forelimb is considered to be an index of motor system integrity (Gharbawie, Whishaw, & Whishaw, 2004). This particular test was also chosen for several other reasons: it does not require training of the rats thereby introducing potential confounds; it does not require food deprivation procedures (Schallert, et al., 2000); it does not require non afflicted limb binding or forced use of impaired limbs; only the animals? natural exploratory behaviours are observed and therefore their actions may be deemed more autonomous.  As well, this particular behavioural test can be conducted under extremely low light conditions. Potentially this may reduce the animal?s stress levels with the aim of further encouraging their natural exploring behaviour.  1.11.4 Importance of the ?cell body response?; the RN response to injury and as a treatment target  The significance of the ?cell body response? to axonal injury was made more apparent by the seminal reviews of Cragg (1970) and Grafstein (1975).  Cragg (1970) had documented the physiological changes in the neuronal cell body after axotomy including increased RNA and protein synthesis, neurofilament changes, apoptosis and survival; even in cells that did not regenerate, while Grafstein (1975) reviewed the possible contributions of the neuronal cell body response to axonal regeneration.    The response of rubrospinal neurons to a high cervical level (C2/3) lateral funiculotomy in the rat spinal cord is that of cytoplasmic, nuclear and nucleolar atrophy (Barron, et al., 1989).   Despite the resultant cellular atrophy of the rat rubral neurons, the initial response is regenerative, that is, the mRNA levels of the regeneration associated genes (RAGs) such as Growth Associated Protein 43 (GAP-43) and T?1 tubulin are increased while in contrast, initial synthesis of neurofilament proteins decrease. By week two there is a decrease in actin and tubulin mRNA levels with a concomitant increase in cellular atrophy (Tetzlaff, Alexander, Miller, & Bisby, 1991).  To underscore the importance of treating the cell body, it was demonstrated that infusion of the neurotrophic factors BDNF or NT4/5 near the vicinity of the RN in rats that received a C3/4 left transection of the dorsolateral funiculus not only prevented neural atrophy but increased the number of regenerating axons into a peripheral nerve graft (Kobayashi et al., 1997).  Of importance, however, is that the presentation of a permissive environment is not sufficient for a regenerative response if there is minimal to no cell body 33  response. Thoracic rubrospinal axotomies in the rat displayed only marginal increases in GAP-43 and T?1 signals in the RN, and did not regenerate into peripheral nerve grafts (Fernandes, Fan, Tsui, Cassar, & Tetzlaff, 1999).  However, to further emphasize the significance of treating the cell body in order to augment axonal regeneration, our group, using a chronically injured model where rats received a transection of the dorsolateral funiculus at the C4 level, demonstrated that infusion of BDNF to the cell bodies of the RN one year later, rescued them from atrophy, increased the levels of GAP-43 and T?1 and promoted axon regeneration into a peripheral nerve graft (Kwon et al., 2002).  In contrast, using a similar chronically injured animal model, BDNF of various concentrations applied to the site of injury two months after injury failed to reverse RN neuronal atrophy, neither promoted a regenerative response into a peripheral nerve graft nor increased levels of GAP-43 and T?1 (Kwon et al., 2004).    1.11.5 Overall experimental rationale and design  For my experimental design I assiduously determined the anatomical target area for treatment, selection of drug and duration of delivery, and timing of treatment in relation to injury.  In the case of the RN we previously demonstrated regenerative success when treatment was delivered at the cell body level (Kobayashi, et al., 1997; Kwon, et al., 2002) and not at the site of injury (Kwon, Liu, et al., 2004).  Based on our prior results treatment in my experiments would be directed to the RN and not to the lesion in the spinal cord.   For the treatment model, I chose to elevate cAMP levels with the membrane permeant analogue dibutyryl-cAMP (db-cAMP) for several reasons.  Firstly, we previously reported that BDNF elevated the regeneration associated genes GAP-43 and T?1 at the level of the RN cell body which permitted axonal regeneration into a peripheral nerve graft.  It has also been shown that application of BDNF to neurons increases endogenous levels of cAMP (Cai, et al., 1999) and activates cAMP downstream effectors such as CREB (Finkbeiner et al., 1997).  It is therefore plausible that a potential regenerative treatment at the level of the rubrospinal cell bodies could be used by mimicking increased cAMP levels using the cAMP analogue db-cAMP.  Secondly, it has been made known that elevation of cAMP has been correlated with regenerative success in axonal injury (see section 1.10 for details).  Finally, we have shown that application of db-cAMP 34  to the RN increases mRNA levels of the regeneration associated genes GAP-43 and T?1 in comparison to their uninjured contralateral controls (Lane, et al., 2005; Lane, et al., 2004).    In order to treat the RN, drug infusion or delivery of db-cAMP can be discrete or continuous.  Others have reported regenerative success with single or double bolus applications of db-cAMP (Blesch, et al., 2012; Harel, et al., 2012; Monsul, et al., 2004; Neumann, et al., 2002; Pearse, et al., 2004; Qiu, et al., 2002). I instead opted for a constant delivery of the drug over a specified period for the following reasons. During embryonic neuronal development in rats endogenous cAMP levels remain high only to decline dramatically during P1-P5 and generally remain low throughout the adult period which also coincides with the inability of axons to overcome inhibitors of myelin such as MAG (Cai, et al., 2001).  Furthermore, within one day of a spinal cord injury, it has been shown that endogenous levels of cAMP significantly decline below baseline levels for at least two weeks in injured neurons (Pearse, et al., 2004).  Given that during neuronal development cAMP remains elevated and unimpeded by myelin inhibitors and that a spinal cord injury in adult rat results in diminished cAMP levels with little to no regeneration, it is conceivable that unimpeded increased cAMP levels could be effective in maintaining or augmenting a regenerative response.   Two treatment groups were prepared: acute and pre-treatment. The acutely treated group received 14 days of continuous delivery of db-cAMP at and after the rubrospinal tract (RST) injury.  The pretreated group were given db-cAMP for one week prior to and one week post injury.  The assumption here was that pre-treating the RN with db-cAMP either enhanced or invoked a regenerative response similar those in seen in conditioning lesions after injury. While the mechanism of a conditioning injury is unclear, elevations in cAMP are believed to involved (Blesch, et al., 2012; Carlsen, 1982b; Neumann, et al., 2002; Qiu, et al., 2002).  Indeed, it has been shown that pre-treatment of db-cAMP prior to dorsal column injury increased the regenerative response of the injured axons (Neumann, et al., 2002; Qiu, et al., 2002).  Furthermore, conditioning lesions show sustained elevated levels of cAMP (2 fold) within a day in the cell bodies of injured axons (i.e. DRGs) for at least a week (Blesch, et al., 2012).  35  1.12 Aims and hypotheses  Aim 1:  To visualize, describe and quantify the axonal changes of the RST after treating the RN with a membrane permeant cAMP analogue (db-cAMP) for two weeks via mini osmotic pump after a C3/4 dorsolateral funiculus crush injury in-vivo using the rat as a model of SCI.  As elevations in cAMP have been reported to improve axonal regeneration in various PNS and CNS neurons after axotomy, I therefore hypothesize the following after elevating db-cAMP levels in the RN following a RST crush injury: - Infusion of db-cAMP near the RN will: o increase axonal sprouting and/or regeneration and/or prevent dieback of the RST o show axonal regeneration/sprouting in and beyond the site of injury o reduce the lesion size  Aim 2:  To evaluate the efficacy of pre-treating the RN of a rat with db-cAMP one week before and continuing for one week after a C3/4 RST dorsolateral crush injury in vivo.  Pre-treatment of db-cAMP in CNS injuries has been shown to increase axonal regeneration, I therefore hypothesize that: - Infusion of db-cAMP near the RN one week prior to RST injury will: o increase axonal sprouting and/or regeneration and/or prevent dieback of the RST o show axonal regeneration/sprouting in and beyond the site of injury o axonal sprouting/regeneration and/or dieback will be similar to or greater than the results obtained by the acute injury group o reduce the lesion size  Aim 3:  To assess whether or not acute or pre-treatment of db-cAMP near the RN promotes sufficient axonal regeneration or sprouting of the RST in order to improve the behavioural outcome of the injured forearm of the rat post injury using the vertical exploration test to discern any behavioural improvements or degradations.  I hypothesize that: - Elevated db-cAMP in the RN will: o Improve injured forearm cylinder touching behaviour in comparison to a control treatment  36  Aim 4: To improve the method by which axons/sprouts/fibres are quantified. There are many techniques to quantify axons each having their strengths and weaknesses.  The objective of this approach is to produce a reliable, reproducible and comparable method that does not rely on proprietary comparisons that are limited to single test samples, but instead are able to generate quantifiable percentages of axon/sprout/fibre densities based on specific criteria.   37  2 MATERIALS AND METHODS  2.1 Animals  2.1.1 Animal model guidelines  All animal experiments and procedures were conducted in accordance to the guidelines set out by the Canadian Council for Animal Care (CCAC) as well as conforming to the rules provided by the University of British Columbia Animal Care Ethics Committee. Experimental endpoints were approved by our animal care committee.  2.1.2  Animal model  Adult male Sprague-Dawley rats (200-250g) were chosen for these experiments and provided by the Animal Care Centre (UBC, 6199 South Campus Road, Vancouver, BC, V6T 1W5).   2.1.3 Animal care  The Sprague-Dawley rats were housed in cages lined with standard cedar chip bedding and were fed a standard rodent diet with two readily accessible water containers per cage.  Animals consumed water and standard rodent feed ad libitum.  The rats were housed in a 12hr dark/light cycle.  Daily inspections of the animal cages and animals were performed to ensure that the health and living environment of the animals met or exceeded the guidelines set out by the CCAC. Cage linings were replaced at least every second day.   2.1.4 Surgery  The adult male Sprague-Dawley rats (200-250g) were anaesthetized with an intraperitoneal injuection of Ketalean (72 mg/kg; Ketamine hydrochloride by Bimeda-MTC DIN 00612316, Cambridge, Ontario, Canada) mixed with Rompun (9 mg/kg; Xylazine Hydrochloride, Bayer Inc., DIN 02169592, Etobicoke, Ontario, Canada).  The rats were placed in a stereotaxic frame and laminectomized at the 3-4 cervical level. Custom manufactured forceps (#5) were used to unilaterally crush the dorsolateral funiculus containing the rubrospinal tract on the left side for   38  20s. Mini osmotic pumps (Alzet Model 2002, 14day-0.5 ?L/hr) containing either db-cAMP or vehicle were placed under the back skin, while the pump cannula was placed stereotactically.  Using skull landmarks, bregma and lambda were levelled horizontally; the tip of the cannula was directed 6mm below the level of the dura by initially using a dental drill to create an opening in the bone 6mm posterior to bregma and 1.7mm lateral to the midline (Figure 2.1).  Dental cement was used to secure the cannula in place, while two miniature screws near the cannula were used to secure the dental cement to the bone.  The animals were placed on heating pads post-surgery and monitored.   Figure 2.1 Schematic depicting the experimental procedure.  Both acute and pre-treatment groups received 14day-0.5 ?L/hr mini osmotic-pumps containing either db-cAMP or vehicle solutions with the canula placed in the vicinity of the RN.  The injury model and procedure consisted of a crush injury to the dorsolateral funiculus containing the RST using modified forceps.  The acutely treated groups received the mini osmotic-pump and injury simultaneously while the pretreated groups received the mini osmotic-pump 1 week prior to injury.    39  2.1.5 Tracing  At day 28 (acute treatment) and day 35 (pre-treatment), the animals were re-anaesthetized (using the same surgical procedures as section 2.1.4) and had 0.7?L BDA (biotin dextran amine, 25%, Molecular Probe) anterogradedly stereotactically  injected in the vicinity of the Red Nucleus (7mm below the dura, 6mm posterior to bregma, 1.7mm lateral to midline) using an electronic pump (World Precision Instrument, Inc., Model UMP2) with the micro-syringe controller (World Precision Instrument, Inc., Model UMC4).    2.1.6 Perfusion and tissue harvesting  At day 35 (acute treatment) and day 42 (pre-treatment) the animals were deeply anaesthetized with chloral hydrate (BDH Chemicals, Toronto, Ontario, Canada).  The animal was transcardially perfused with 150ml of PBS (1X), followed by 300ml paraformaldehyde (4% in PBS) perfusion fixation solution.  The brainstems and spinal cords were removed and placed in 4% paraformaldehyde to post fix overnight.  The tissue was cryoprotected with daily changes of sucrose solutions (12%, 18%, and 24% respectively in PBS (1X) at pH 7.4).  The brains and spinal cords were cut into sections and mounted with Tissue Tek? (Sakura Fineteck U.S.A. Inc., Torrance, CA) mounting medium.  The cortices were cut and mounted with the rostral side apposing the mounting medium, while the spinal cords were cut approximately 12 ? 2mm in length with the lesion being at the centre of the section. The spinal cord sections were mounted horizontally.  Additionally, small cervical sections of spinal cord (for cross sectional cutting) 2-3mm in length at level C1/2, were placed vertically and perpendicularly adjacent to the rostral end of the mounted horizontal spinal cord. Once all the tissue specimens were mounted, they were frozen and stored at -80oC.    2.2 Drug and tissue preparation and/or processing; immunohistochemistry  2.2.1 Experimental drug solutions  The experimental solution (db-cAMP) consisted of 25 mM Adenosine-3?,5?-cyclic Monophosphate N6, O2-dibutyryl-Sodium Salt (Figure 2.2; Calbiochem, Lot?s B51511, B58714) and was prepared in 20mM PBS and 20?l penicillin/streptomycin. The vehicle solution 40  comprised of 20 mM PBS and 20?l penicillin/streptomycin. The 25mM db-cAMP and vehicle solutions were filtered using 0.2?m syringe filters (Nalgene).        Figure 2.2 Molecular diagram of adenosine-3?,5?-cyclic monophosphate N6, O2-dibutyryl-sodium salt (db-cAMP) (Created from images in the public domain; Protein Data Bank; Public domain: Federal Government under the terms of Title 17, Chapter 1, Section 105 of the US Code).     2.2.2 Mini-osmotic pump preparation  All mini-osmotic pump preparatory work was performed under sterile conditions. Alzet (DURECT Corporation, P.O. Box 530, Cupertino, CA 95015-0530) Model 2002 mini-osmotic pumps (200 ?L volume, 0.5?L/hr flow rate, 14 day) were used for the experiments.  All Alzet mini-osmotic pump flow modulators (Model 2002 [#0002486]) were connected to 3.5mm medical vinyl tubing (SCI intravenous, BB317-85, Size V/3, Scientific Commodities Inc.) and subsequently connected to 8mm canulae (Plastics One Inc, 813280PSPCXC/3280P/SPC).  The mini-osmotic pumps (Model 2002) were filled completely with either db-cAMP or vehicle 41  solutions using a BD 1ml syringe (Becton, Dickinson & Company) with a tuberculin slip tip. The filled mini-osmotic pumps were then incubated overnight at 37o C (Forma Scientifica Incubator).  2.2.3  Immunohistochemistry  For the RST, Biotinylated dextran amine was visualized with Cy3-conjuagated Streptavidin (Jackson ImmunoResearch; 1:400).  Essentially, the slides were placed in dark moisture chambers at room temperature for 2hrs followed by 3 ? 5 min washes of 0.01M PBS (pH 7.4).   The lesions were visualized using anti-GFAP antibodies.  Generally, the previously frozen and cut sections (see section 2.2.4) were thawed until they reached room temperature and rehydrated in 0.01M PBS (@ 7.4pH) for 15-30min. In order to increase the signal to noise ratio, the sections were incubated in 10% NGS (Normal Goat Serum [goat anti mouse]) in 0.01PBS + 0.1M TX100 for 30mins.  The primary antibody, monoclonal anti-glial fibrillary acidic protein (GFAP; Sigma G3893, St. Louis, Missouri), at a dilution 1:400 was pipetted on the sections and incubated in dark, moist chambers for 24hrs. The slides were then washed 3 times (5 min) in 0.01M  PBS.  The secondary antibodies conjugated to Alex350 (blue) [Molecular Probes Inc., Eugene, OR], were applied to the sections for 2hrs (1:200 dilution).      2.2.4 Tissue cutting and mounting  Horizontally mounted spinal cords with cervical cross sections were cut consecutively dorsally to ventrally in 20?m thick sections on a Microm HM505E cryostat. The internal temperature of the Cryostat that produced the best and/or most consistent results was determined to be -28o to -30oC. Preparatory work included freezing Tissue Tek? (Sakura Fineteck U.S.A. Inc., Torrance, CA) on a base and adjusting the stage so that the base with the mounting medium, when cut, remained horizontal to the surface of the mount. The frozen specimen was placed on the levelled, frozen mounting medium using small amounts of Tissue Tek? to tack the corners of the paper mount to the base. Additional mounting medium was added to create a rectangular border (approximately 2mm of additional width radially) around the specimen. The purpose for this additional step is to protect the edges of the specimen when the blade makes initial contact. Additionally it pre-emptively helps to ensure that the cut section moves in an unrestricted 42  manner beneath the stage glass.  Adjustments to the stage can therefore be made before the tissue specimen is cut or potentially damaged.  Cutting was also conducted at approximately 40-60o angles to the horizontal. The sections were mounted on Superfrost Plus slides (Fisher Scientific, U.S.A.) in groups of three for a total of nine sections per slide.  The mounting of the midbrain sections on the base utilized the same mounting procedures as that of the spinal cords.  Midbrain sections were cut in 20?m sections starting from the beginning of the Red Nucleus (caudal to rostral) and mounted every 5th paired section.  2.3 Behaviour  2.3.1 Behaviour testing ? vertical exploration test  The vertical exploration test was used for behavioural testing which generally adhered to the procedures described by Schallert & Lindner (1990),  Choi-Lundberg et al., (1998), Liu et al., (1999) and Schallert, Fleming et al. (2000).  The behaviour test used a transparent acrylic cylinder (305mm diameter x 305mm height) that was placed in markers on a testing bench.  Two mirrors were placed behind the cylinder and adjusted in order to provide clear rear views of the animal's paws when the animal no longer faced the camera directly.  Each test began with the cylinder being washed and deodorized prior to behaviour testing of each animal.  Each rat was placed in the cylinder with the room then darkened. The tests were conducted in a near zero lux (dark) environment and digitally filmed with an infrared digital video camera (Model: Sony DCD-403, Sony of Canada, Ltd. Toronto, ON).  The exploratory behaviour of the rats increased when the vertical exploration test was conducted in near complete darkness (personal observation).  Each paw touch, left, right or both, was counted using a hand held manual counter for at least  a minimum of 20 scores. A score is listed as either an independent paw touch in the case of left or right paws, or a simultaneous touch of both paws. All digitally recorded video was replayed in slow motion and/or frame by frame basis for data acquisition where all paw touches were recorded.  Both observer and recorder of data were blinded to the treatment groups the animals were in.   43  2.4 Quantification    2.4.1 Quantification of axons  2.4.1.1 Image capture and photomerging  20?m thick horizontal cryostat cut sections of the spinal cord were photographed using Q-Imaging cameras and Northern Eclipse Version 6 (Empix Imaging, Inc., Mississauga, Ontario) image capturing software on a Zeiss Axioplan 2 microscope (Objective 10x).  Multiple pictures (image properties: 1,360 x 1,032 pixels at 300dpi) were taken that comprised of contiguous, slightly overlaid images of the injury site to approximately 7mm from the crush epicenter rostrally, and to 2mm caudally from the epicenter, and perpendicularly past the midline of the spinal cord. These images were photomerged using Adobe Photoshop CS4 Extended  (Adobe Systems Incorporated, San Jose, California) using the advanced blending options (Figure 2.3).  Any spinal cord sections that appeared off axis to the horizontal were rotated to the horizontal.   2.4.1.2 Digital extraction of the labelled axons  The blended, photomerged images of the spinal cord were opened in Adobe Photoshop CS4 Extended and converted to 8 bit grayscale. Using the selection tools, carefully selected areas around and including the axons were selected and layer copied.  The purpose for selectively choosing specific sections is to ensure that the amounts of artefacts are kept to a minimum and obvious artefacts are not inadvertently included in the quantification process. This process is repeated until all the visible axons/sprouts were layer copied.  The layers were then thresheld to provide the maximal axonal visibility with the least amount of background interference. Any missing axons visible in the source image and not in the new image were manually traced (2px width) and added.    Using the automatic selection tool the lesion centre was outlined. With the selection in place, the foreground colour was chosen, and the border automatically filled in with the border selection applet. The overlaid border was then layer copied. In order to create the lesion epicentre for quantification purposes, the line tool was utilized to draw a perpendicular line to the horizontal section thereby bisecting the lesion.  Employing a custom script, the layers were then inverted, 44  the backgrounds were made transparent and the image was flattened while the source was removed.  Figure 2.3 illustrates the visual appearance of the axons displayed as a binary image.   The images were saved into two groups: one for creating the images for quantification and the other to create stacked images akin to camera lucida.  The images used for quantification included the lesion epicentre marker and not the lesion border outline.  Conversely, the lesion border outline, and not the lesion epicentre marker, was included in the stacked images group.  2.4.1.3 Converting the visible axons into numerical data  The quantification images were opened in Photoshop CS4 Extended and the edge filter was applied to all the axons in the image.  The rational for this approach is twofold: firstly, it de-emphasizes overly large diameter axons by reducing the amount of visible data for quantification, and secondly, large diameter axons are essentially normalized to no more than two pixels in width.  Axons that already appear two pixels in width remain unaffected by this filter.  Using a script, the images were processed as noted above and saved.   The saved images were opened in ImageJ Version 1.41i (National Institutes of Health, Public Domain Software).  The binary conversion process was used to convert the image into a single bit. This step is important as all the visible pixels representing the axons must have the same bit colour value while the background has none.  A sweep of the cursor through the image confirmed the bit value (pixel colour value) of the displayed axons to be identical to each visually represented axon while the background registered a zero value. The program?s set scale was adjusted to the known resolution distance in the image. The appropriate distance units, in this case, ?m, were used.  Using the dimensions of 1,200?m as the height, and 3,000?m as the length, the selection rectangle was placed on the image. The boundaries of the rectangle serve as the border by which the data is recorded.  The selection rectangle is then moved so that the leading edge is aligned with the lesion epicentre (previously marked).  The plot profile setting is then used which converts the number of pixels in each column to a proportional value based on the colour depth of the pixels bound by the frame (Figure 2.3).  For example, if the selection rectangular frame height is 1,200?m and in a vertical column 50% of the image was occupied by pixels with a colour value of 255, the resulting total would be 127.5.  Given that the height of the 45  frame, pixel colour value and pixel count are known, one can calculate the percentage of axons in a defined space.  Before all the pixels are converted into density percentages, a control image is used to confirm the output of the plot profiler.  An image of known proportions, in this case 1,200?m by 4,000?m was created with six vertical lines and two horizontal lines of both known distance and fixed pixel colour values.  Heights of the calibration lines were 25, 33, 50, 66, 75 and 100 percent of the fixed vertical height, while the horizontal lines were 500?m and 1,000?m in length, with a height of 5% of the fixed vertical height.  Again the plot profiler was used to count the pixels in the image and output the results. Once converted into distance, the values of the generated output should match the known distances of the lines in the control image.  With the validity of the output of the control image being confirmed, the raw pixel data was transferred to an Excel 2007 SP1 (Microsoft Office 2007, Microsoft Canada Co., Mississauga, ON) spreadsheet.       46    Figure 2.3  Schematic illustrating the axonal quantification procedure. Multiple images (horizontal sections) of the area containing the dorsolateral funiculus including the injury are assembled to form a longitudinal figure as shown by the overlay on the spinal cord.  These consecutive images are then processed to display only the BDA traced axons and the lesion area. The lesion area is then quantified for both area and perimeter length.  A bounding box of 1,200?m high and 3,000?m in length is placed from the lesion epicentre rostrally over the visualized BDA labelled axons.  The captured image is then converted into numerical data representing axonal density over distance.  47  2.4.1.4 Creating normalizing values for axonal density  Due to the variability in the BDA tracing efficacy of the rubrospinal tract, normalizing values needed to be created that would be used to calculate the axonal density.  Consecutive, partially overlaid images of the cross section of the spinal cord were opened in Photoshop CS4 Extended .  The pictures were photomerged using the advanced blending option and saved.  The images were opened in Photoshop CS4 Extended and the counting tool selected. The area containing the dorso-lateral funiculus was magnified to maximally fill the visible working area of the monitor.  Blinded to the experimental treatments, three people manually counted the number of visible BDA labelled rubrospinal tracts and recorded their counts into an Excel spreadsheet. Tracts that could not be easily discerned as separate tracts were counted as one tract.  Each group of the three independent counts per animal were averaged to account for discrepancies.  The normalized values were generated by employing the animal with the highest rubrospinal tract count value and dividing the others animals? lower values into it.  Both the pre-treatment and acute groups used their own normalizing maxima as the groups had differing timelines, surgery dates and experimental durations.   2.4.1.5 Processing the raw data into percentages of axonal density  The raw data produced by ImageJ (1.41i) from the procedure in section 2.4.1.3 was transferred into Excel 2007 SP1.  Each animal section had its own vertical columns in the spreadsheet; axonal density versus distance for a total of six sections per animal for RST axonal density calculations, and three sections per animal for gray matter sprouting axonal density calculations.  A second spreadsheet page was opened to generate the summarized and normalized density values.  This was done by summating the raw data for each distance point, dividing by the pixel colour value and multiplying by the rectangular selection height.  The resulting value is then normalized by multiplying by the generated normalized cross sectional values from section 2.4.1.4.  The final total is then averaged to produce a fixed density value proportional to the height of the selection rectangle. In order to generate the data for the graph, averages of the axonal density were calculated for each 100?m.  Additionally, density percentages (i.e. axonal density of a given perpendicular distance over length) were produced.  This was done by 48  dividing the axonal density by the height of the selection rectangle and multiplying by 100 to generate the percentage.   2.4.1.6 Creating the axonal density versus distance graphs  SPSS 16 (SPSS Inc., Chicago, Illinois) was used to import the data from section 2.4.1.5 to calculate the treatment group averages with the corresponding standard error of the means.  Graphs were created using Sigmaplot 11 (Systat Software Inc., San Jose, CA) and Excel 2007 (Microsoft).    2.4.2 Lesion areas and perimeters  In order to calculate the lesion area and perimeter sums, the section with the largest lesion area was selected as the initiating point.  Four addition sections were added to complete the group for the calculation.  The additional sections were from ?200?m and ?400?m from the lesion cavity epicentre.  ImageJ (1.41i) with the area calculator plugin was used to perform the measurements. The set scale setting was set to mm while the area and perimeter output options were set in the area calculator.  A test image of known proportions was used to verify the validity of the area calculator.  Summations were chosen as well as averages because the lesion or cavity is approximately spherical in shape and axial distances away from the centre result in cross sectional areas being smaller. Since the cavity shape is unknown, summations could be more helpful for comparison purposes. The perimeter summation was also an added measurement used in order to compensate for circumstances where the cavity may have collapsed on the slide.  In these cases, for example in a collapsed lesion area, the area would decrease but the perimeter edge should remain consistent.   2.5 Statistics  All statistical calculations were performed using SPSS 16.  Independent samples T-test was also used to test the equality of means when comparing the lesion sizes between the treated and untreated groups.  Repeated measures ANOVA was used to determine whether the effect of distance on density is different for the two treatment groups (db-cAMP versus vehicle treated) as well as whether the effect of the treatment (db-cAMP) is independent of distance.  If Mauchly?s 49  test of sphericity could not be performed, the Huynh-Feldt epsilon correction method was used instead.  In all statistical calculations the level of significance was set at p?0.05.   2.6 Experimental design overview and timeline 2.6.1 Acute treatment experimental overview and timeline  In the acute injury model, adult male Sprague-Dawley rats received a crush of the dorsolateral funiculus at the C3/4 level thereby damaging the RST.  At the same time, a 14day-0.5 ?L/hr mini osmotic-pump containing either vehicle or db-cAMP solutions was surgically implanted in the animal. The mini osmotic-pumps directed their contents via a canula that was placed in the vicinity of the red nucleus.  The vertical exploration test was employed for behavioural testing.  All animals were tested prior to injury to establish a baseline score, and were subsequently tested each week after surgery for a total of 5 weeks after injury.  One week prior to tissue harvesting, BDA was injected near the RN to trace the RST (Figure 2.4).   2.6.2 Pre-treatment experimental overview and timeline  For the pre-treatment group, Sprague-Dawley rats initially received only a 14day-0.5 ?L/hr mini osmotic-pump containing either vehicle or db-cAMP solutions surgically implanted with a canula directing the solution in proximity of the RN.   One week after the mini osmotic-pump implantation, the animals received the same crush injury of the dorsolateral funiculus as the acute treatment group.  Behavioural testing, using the vertical exploration test, was conducted prior to both mini osmotic-pump implantation and injury in order to establish a baseline score. This was done after the mini osmotic-pump implantation, and for 5 weeks post injury.  BDA tracer was injected in the vicinity of the RN in order to visualize the RST one week prior to tissue harvesting (Figure 2.4).   50   Figure 2.4  Timelines of acute and pre-treatment experiments Acutely treated groups received concurrent dorsolateral funiculus injuries and mini osmotic-pump implantations while pretreated groups acquired db-cAMP or vehicle containing mini pumps 1 week prior to injury. Behaviour tests were performed weekly with a baseline line test prior to injury and/or RN treatment.    51  3 RESULTS ? ACUTE TREATMENT WITH DB-CAMP 3.1 Introduction  Injury induced reduction (Pearse, et al., 2004) or endogenously low (Cai, et al., 2001) levels of cAMP can contribute to the failure of axons to appreciably regenerate in the mammalian spinal cord after injury.  Conversely, raising the levels of cAMP via analogue can augment axon regeneration after injury in the PNS (Gershenbaum & Roisen, 1980; Kilmer & Carlsen, 1984; Pichichero, et al., 1973; Roisen, et al., 1972) and the CNS (spinal cord) (Cai, et al., 2002; Neumann, et al., 2002; Qiu, et al., 2002). While increased cAMP levels have been shown to improve the regenerative response in the spinal cord after injury, treatment in these cases consisted of single or double bolus applications of db-cAMP external to the CNS.  In this acutely treated experiment, db-cAMP will be continuously infused near the vicinity of the rubrospinal neuronal cell bodies in order to maintain increased levels of the cyclic nucleotide. I hypothesized that the duration limited sustained increase of db-cAMP would augment the regenerative response of the injured rubrospinal system.    3.2 Db-cAMP treatment did not affect lesion cavity sizes In the acute treatment paradigm, db-cAMP was being used potential regenerative treatment for RST axons following a crush injury.  To ascertain whether or not the application of db-cAMP to the RN had affected the damaged RST axons by creating either an enhanced regenerative response or prevented retraction thereby subsequently altering the enlarging lesion cavity and scar, the cavity was measured. Using anti-GFAP to demarcate the lesion border, the area sum, area average, perimeter sum and perimeter average were measured.  The horizontal section containing the largest lesion size including sections that were ?200?m and ?400?m away were used to calculate both the area sum and perimeter sum of the injury site.  The perimeter sum was an additional measurement used to ensure that there was no discrepancy in the area sum measurement due to irregularities (i.e. collapse) in the lesion area.  There were no significant differences between the db-cAMP (area sum 3.85?0.42mm2; perimeter sum 17.49?0.98mm; average area 0.77?0.07mm2 and perimeter 3.50?0.12mm) and vehicle (area sum 3.41?0.71mm2; perimeter sum 15.99?1.57mm; average area 0.68?0.07mm2 and perimeter 3.22?0.61mm) treated groups (Figure 3.1).  The average lesion cavity size did, however, increase in comparison to a 52  similar experimental group where the overall experimental period was two weeks shorter in duration (unpublished data).   Figure 3.1 Db-cAMP treatment does not affect lesion injury size Db-cAMP and Vehicle treated groups show no differences in lesion size.  Vehicle and db-cAMP groups indicate no significant differences in either area sum (A), average area (B), perimeter sum (C) or average perimeter distance (D).     3.3 Db-cAMP treated animals displayed overall greater numbers of BDA labelled axons/sprouts than vehicle treated animals  Db-cAMP was applied to the RN at the same time the dorsolateral funiculus containing the RST received a crush injury at the C3/4 level. I hypothesized that the use of db-cAMP to mimic 53  increased cAMP levels in the RN during a RST axonal injury would initiate or augment an enhanced regenerative response.  In order to visualize and quantify any morphological changes to the injured RST, 0.7 ?L BDA (biotinylated dextran amine) was injected in close proximity to the RN one week prior to tissue harvesting.  The visible BDA labelled RST in Figure 3.2 (II, IV, VI), and Figure 3.3 (inset C,D,G,H,J), exemplified by arrows (Figure 3.2 I,II) denoting the RST, revealed a denser RST with greater numbers of axon/sprouts/fibres (typified by arrowheads [Figure 3.2 I,II; Figure 3.3 A,C,D]) for the db-cAMP treated group versus the control group.    As seen in inset D,J (Figure 3.3), there are prominent numbers of axons/sprouts/fibres in close proximity to the lesion edge in the db-cAMP treated group.  In contrast, the control group has reduced numbers of RST axons/sprouts/fibres coming from the RST as seen in inset B,I (Figure 3.3).    The individual photomicrographs displaying the BDA labelled RST in this experiment only show up to 20?m of depth of the spinal cord.  Furthermore, contrast between the labelled RST and the background can be reduced in some images thus making quantitative and qualitative assessment difficult.  By digitally extracting the areas of interest, in this case the visibly labelled RST and the lesion edge, and compositing multiple images into a single image thereby increasing the depth, a more complete view of the injured RST can be obtained.  Digital ?camera lucida? like reconstructions (CLLRs) of the same horizontal sections were created using five 20?m vertically contiguous sections.  These newly constructed images provide more detail and better illustrate any differences that the labelled RST and fibres may have between the db-cAMP and the control group.    Apparent density of the RST in db-cAMP treated animals is greater, both rostrally (Figure 3.4 II; arrows), and proximal to the lesion (Figure 3.4 II, arrow by lesion) in comparison to the vehicle treated group (Figure 3.4 I; arrows for RST and RST proximal to the lesion edge).   Axons/fibres proximal to the lesion edge were also greater in number for the db-cAMP treated group (Figure 3.4 II; arrowheads indicate examples of axons/sprouts, Figure 3.5 II; D,F) in contrast to the control group (Figure 3.4 I; arrowheads,  Figure 3.5, B,E).  Rostrally, density of the labelled RST and the axons/sprouts are noticeably greater for the db-cAMP treated group (Figure 3.4 II;  Figure 3.5, C) versus the control group (Figure 3.4 II;  Figure 3.5, A).   54    55     Figure 3.2 The db-cAMP treated group shows greater density of the BDA traced RST and axons/fibres in comparison to the vehicle treated group Images above represent horizontal views of the dorsolateral funiculus containing the BDA labelled RST and lesion cavity for the acutely treated groups. The RST axons/sprouts/fibres are displayed in white in the above images and run horizontally rostrally (left) to caudally (right) while branching axons/sprouts/fibres generally course perpendicularly from the RST. Arrows indicate examples of labelled tracts of the RST while arrowheads exemplify branching axons/sprouts/fibres as shown in I,II.  Roman numerals in the above images represent horizontal sections of different animals.  Animals treated with db-cAMP or vehicle are shown in II,IV,VI and I,III,V respectively.  Db-cAMP treated groups (II,IV,VI) exhibit more axon/sprouts than vehicle treated groups (I,III,V). Asterisks denote the lesion cavity epicentre as shown in I,II. Images I,III,V (vehicle) and II,IV,VI (db-cAMP) represent micrographs from six different animals.  Inset boxes = 1mm2. Scale bar = 500?m.    56    57    Figure 3.3  (Inset of Figure 3.2) the db-cAMP treated group exhibits more BDA labelled RST axons/sprouts/fibres than the vehicle treated group Images A,B,E,F,I are magnified inset images from Figure 3.2 (I,III,V) and represent vehicle treated groups.  Db-cAMP treated animals are shown in inset images C,D,G,H,J from Figure 3.2 (II,IV,VI).  Db-cAMP treated animals show more axons/sprouts/fibres (as exemplified by arrowheads in C,D [db-cAMP] versus A,B [vehicle]) from the RST both rostrally (C,G) and proximal to the lesion edge (D,H,J) than vehicle treated animals rostrally (A,E) and proximal to the lesion edge (B,F,I).  Overall, db-cAMP treated groups show more traced RST axons/sprouts/fibres proximal to the lesion edge; Image (D) versus (B), Image (J) versus (I) [db-cAMP versus Vehicle respectively]. Each image = 1mm2.  Scale bar = 200?m.    58      Figure 3.4 Camera lucida like reconstruction (CLLR) reveals a greater number of labelled RST axons/sprouts/fibres distally to proximal to the lesion edge for the db-cAMP treated group Images II and IV are camera lucida like reconstructions (CLLR) of traced RST axons/sprouts/fibres of the acutely treated db-cAMP group whereas images I and III represent vehicle treated groups. Db-cAMP treated groups show increased RST axonal density (II: arrows) over vehicle treated groups (I: arrows).  The density of axons/sprouts/fibres (II:arrowheads) is increased caudally to proximal to the lesion edge in db-cAMP treated groups II, IV in comparison to vehicle groups (I:arrowheads), III.  Images I,III (vehicle) and II,IV (db-cAMP) are compiled from four different animals.  Inset boxes = 1mm2. Scale bar = 500?m.  59      Figure 3.5 (Inset of Figure 3.4) CLLRs show greater numbers of axons/sprouts/fibres of the RST versus vehicle treated groups rostral and proximal to the lesion Db-cAMP treated groups exhibit increased axon/sprout/fibre density proximal to lesion (D,F) in contrast to vehicle groups (B,E).  Similarly, RST axon/sprout density rostral from the lesion is more pronounced for db-cAMP treated groups (C) versus vehicle treated groups (A). Scale bar = 200?m. 60  3.4 RST axonal/sprout/fibre density was significantly higher in db-cAMP treated versus control animals  One of the aims of this experiment was to create a quantification method to measure the density of the traced RST.  This method of quantification reduces visualized fibre thickness bias and normalizes the RST count between animals.  As described in section 2.4, the percentage of normalized RST axons/sprouts within a 1,200?m (height/vertical) x 3,000?m (distance/horizontal) bounding box were quantified.   Figure 3.6 indicates that as early as 200?m from the lesion epicentre the average axonal/sprouting density of db-cAMP animals (0.1?0.08%) begins to increase significantly more than the controls (0.03?0.0 %) and plateaus at approximately double the density at 3,000 ?m (8.4?1.0% db-cAMP versus 4.2?1.0% vehicle; p  ? 0.0004).    61     Figure 3.6 RST axon/sprout density increases significantly from the lesion epicentre rostrally for the db-cAMP treated group The db-cAMP treated group shows increased RST and RST axon/sprout density from the lesion centre rostrally versus the vehicle control group; p ? 0.0004.  Data is presented as percentage of average totally axon density per 100?m.  n=5 (db-cAMP), n=6 (vehicle). Error bars = SEM.  3.5 Db-cAMP treated animals had a significant increase in RST projections into the gray matter  The RST normally projects to the gray matter.  I postulated that application of the db-cAMP to the RN should produce an enhanced regenerative response; I therefore examined the gray matter for any differences in the projections from the RSTs between db-cAMP and vehicle treated groups. Inspection of the gray matter (RST omitted in the images) of the vehicle treated groups (Figure 3.7; I,III) revealed a significantly reduced presence of labelled RST axons/sprouts over the db-cAMP treated groups. Moreover, the RST axons/sprouts to the gray matter in the db-cAMP treated group were visibly denser from the lesion rostrally.  This was further evidenced by 62  the CLLRs of the same images. The CLLRs of the db-cAMP groups show greater visible axon/sprout density  rostrally (Figure 3.9 II, IV; Figure 3.10 C,G) to proximal to the lesion edge (Figure 3.9 II, IV; Figure 3.10 D,H) versus the vehicle treated group rostrally (Figure 3.9 I, III; Figure 3.10 A,E) to proximal to the lesion edge  (Figure 3.9 I, III; Figure 3.10 B,F).  Furthermore, greater RST fibre density in the db-cAMP group can be seen proximal to the lesion edge (Figure 3.10 D,H) whereas the vehicle treated group show limited appearance of the traced RST axons/sprouts proximal to the lesion edge (Figure 3.10 B,F).  In no cases were any traced RST fibres visible caudal to the lesion.   63      Figure 3.7 RST axon/sprout density is greater for the db-cAMP treated group in the gray matter  Significantly more traced RST axons/sprouts can be seen coursing in the gray matter (RST has been omitted in the above images) rostrally to proximal to the lesion edge for the db-cAMP treated group (II,IV) in comparison to the vehicle treated group (I,III) which shows a dearth of labelled axons/sprouts. Images I,III (vehicle) and II, IV (db-cAMP) are micrographs from four different animals. Scale bar = 500?m.    64   65  Figure 3.8 (Inset of Figure 3.7) the db-cAMP treated group shows more RST axons/sprouts in the gray matter Both rostral (C,G) and proximal to the lesion edge (D,H) images show more traced RST axons/sprouts for the db-cAMP treated group  in contrast to the vehicle treated group where there is a noticeable reduction of axons/sprouts rostrally (A,E) and proximal to the lesion edge (B,F).  Scale bar = 200?m.     66      Figure 3.9 CLLRs reveal greater visible RST axon/sprout density in the gray matter for the db-cAMP treated group Visible RST axon/sprout density in the gray matter is more apparent rostrally to proximal to the lesion edge for the db-cAMP treated group (II, IV) in comparison to the vehicle treated group (I,III).  In contrast, there is a noticeable decrease in the axon/sprout density rostrally to proximal to the lesion edge of the vehicle treated group (I,III).  Images I,III (vehicle) and II,IV (db-cAMP) are compiled from four different animals. Scale bar = 500?m. 67    68  Figure 3.10 (Inset of Figure 3.9) CLLRs reveal greater RST axon/sprout density in gray matter for the db-cAMP treated group The db-cAMP treated group shows significant RST axon/sprout density both rostral (C,G) and proximal (D,H) to the lesion.  The vehicle treated group displays modest axon/sprout density rostrally (A,E) from the lesion, but little density proximal to the lesion (B,F).  Scale bar = 200?m.  3.6 RST axon/sprout density in the gray matter is greater for the db-cAMP treated group in comparison the control?s.  Traced axon/sprouts in the gray matter were quantified using the same method as section 3.4. Shown quantitatively, there is a significant difference in the densities of traced RST axons/sprouts in the gray matter between the two groups (Figure 3.11, p ? 0.0005). The quantified BDA traced axon/sprout density in the gray matter of the vehicle treated group increases to approximately 1% 1,500?m from the lesion epicentre and remains less than 2% density until 3,000?m (Figure 3.11).  In contrast, the axon/sprout density of the db-cAMP group increases steadily from the lesion epicentre to approximately 8% 3,000 ?m from the lesion centre (Figure 3.11).      69   Figure 3.11 RST axon/sprout density in the gray matter is greater for the db-cAMP treated group The RST axon/sprout density of the db-cAMP treated group is significantly higher in the gray matter from the lesion centre caudally in comparison to the vehicle treated group. p  ? 0.0005. Data is presented as percentage of average totally axonal density per 100?m. n=5 (db-cAMP), n=6 (vehicle). Error bars = SEM. 3.7 Cross sectional views show greater numbers of traced RST axons/sprouts travelling to the gray matter for db-cAMP treated groups.  In addition to horizontal views, cross sectional images of the spinal cord displaying the labelled RST at approximately level C2 were photographed.  Db-cAMP animals show greater numbers of axon/sprouts projecting into the gray matter; mostly to Rexed laminae IV,V,VI (Figure 3.12;Inset Figure 3.13; B,D,F) in comparison to the control group (Figure 3.12; Inset Figure 3.13; A,C,E).   70      Figure 3.12 Spinal cord cross sections display greater numbers of RST axon/sprouts to the gray matter for the db-cAMP treated group The db-cAMP treated group shows greater numbers of traced axons/sprouts coursing towards and into the gray matter (I,II,III cross sections on the right).  The control group shows markedly less visible RST axon/sprouts towards and into the gray matter (I,II,III cross sections on the left).  Images are spinal cord cross sections at approximately level C2 of six different animals. Scale bar = 500?m.     71   Figure 3.13  (Inset of Figure 3.12) the db-cAMP treated group shows more traced RST axon/sprouts projecting to the gray matter Prominently visible axon/sprouts can be seen projecting from the RST coursing to the gray matter in the db-cAMP treated group (B,D,F).  In contrast, the visible BDA labelled RST of the vehicle group shows only modest numbers of visible axons/sprouts to the gray matter (A,C,E). Scale bar = 200?m.  3.8 Db-cAMP treatment provided no functional benefit versus the vehicle treated groups in the forelimb usage test   A lesion in the dorsolateral funiculus containing the rubrospinal tract will produce a distal forelimb behavioural deficit (Kuchler, et al., 2002; Whishaw, et al., 1998). The average number of touches recorded per animal each test was approximately 44 (across all groups); 20 touches is generally considered sufficient for quantification. Baseline scores (Figure 3.14) were generally 72  uniform between both the vehicle (n=6) and db-cAMP (n=6) treated groups. The commonly reported score, average left plus both touches, for the baseline was 79.1 ? 0.02% for the vehicle treated group and 79.6 ? 0.03% for the db-cAMP treated group. As shown in Figure 3.14, the acutely treated db-cAMP treated animals scored lower in contrast to the control group in weeks 1 through 3 (35.3 ? 0.05, 27.5 ? 0.06 and 33.41 ? 0.07% versus 48.9 ? 0.11, 46.8 ? 0.12 and 49.2 ? 0.09% respectively). However, while the db-cAMP group appeared to perform worse during the first three weeks of treatment in comparison to the control group, the difference did not reach significance.  During weeks 4 and 5 the db-cAMP groups scored on par in performance with the vehicle treated groups (45.5 ? 0.12 and 42.36 ? 0.07% versus 52.0 ? 0.06 and 42.34 ? 0.05% respectively; Figure 3.14) however there were no statistical differences between the two groups. In summary, as indicated by the vertical exploration test results, the db-cAMP treatment neither provided any functional benefit nor did it produce any further deficits either.     73     Figure 3.14  db-cAMP provides no functional benefit as indicated by the vertical exploration test The db-cAMP treated group appeared to perform worse during weeks 1 through 3 but this did not reach statistical significance. Conversely, Weeks 4 and 5 indicated an increase in behavioural performance for the acutely treated db-cAMP group relative to the vehicle treated group although this too did not reach statistical significance. All behavioural test scores used the commonly reported score of percentage of left + both cylinder touches.  n=5 (db-cAMP), n=6 (vehicle).    74  4 RESULTS ? PRE-TREATMENT WITH DB-CAMP ONE WEEK PRIOR TO INJURY  4.1 Introduction  Pre-conditioning lesions prior to axonal injury can create an enhanced regenerative response thereby augmenting the axons? reparative response (McQuarrie & Grafstein, 1973).  Moreover, this procedure has been successfully used in advancing regeneration in the CNS (Blesch, et al., 2012; Neumann & Woolf, 1999; Reich, Burmeister, Schmidt, & Grafstein, 1990; Richardson & Issa, 1984).  Interestingly, it has been shown that cAMP levels increase after a preconditioning axon lesion and that prior application of a cAMP analogue, such as db-cAMP, without a preceding injury can mimic the effects of a preconditioning lesion (Neumann, et al., 2002; Qiu, et al., 2002).  On this basis, I postulated that application of db-cAMP near the red nucleus prior to, during and after injury to the dorsolateral funiculus would produce an enhanced regenerative response. 4.2 Pretreatment of db-cAMP did not reduce lesion cavity sizes  In the pre-treatment paradigm, db-cAMP was infused near the rubral cell bodies one week prior to injury to provide increased levels of the cyclic nucleotide before injury. To determine whether or not pre-treatment of db-cAMP to the RN had affected the damaged RST axons by creating either an enhanced regenerative response or prevented retraction and thereby subsequently altering the enlarging lesion cavity and scar, the size of the cavity was measured. Anti-GFAP was used to delineate the lesion border and measured were: the area sum, area average, perimeter sum and perimeter average.  Horizontal sections ?200?m and ?400?m including the largest diameter/circumference centre lesion (i.e. initial measuring point) were used to calculate both the area sum and perimeter sum of the injury site. Although it appears that the lesion areas and perimeters of db-cAMP treated groups appear smaller than the control groups; there were no significant differences between the db-cAMP (area sum 2.86?0.73mm2; perimeter sum 14.02?1.95mm; average area 0.57?0.07mm2 and perimeter 2.80?0.19mm) and vehicle (area sum 3.34?0.60mm2; perimeter sum 15.81?1.53mm; average area sum 0.70?0.06mm2 and 3.16?0.14mm) treated groups (Figure 4.1).  Furthermore, it should also be noted that there were 75  no significant differences in lesion areas or perimeter distances across experimental groups (i.e. pre-treatment versus acute) either (see Figure 3.1 for comparison).  76    Figure 4.1 Pre-treatment of db-cAMP did not reduce lesion sizes  There are no significant differences in the area or perimeter between db-cAMP or vehicle treated groups.  Vehicle and db-cAMP groups indicate no significant differences in either area sum (A), average area (B), perimeter sum (C) or average perimeter distance (D).      4.3 Db-cAMP treated animals have greater numbers of traced RST axon/sprouts than vehicle treated animals  0.7 ?L BDA (biotinylated dextran amine) was injected in close proximity to the red nucleus in order to visualize and quantify the injured RST.  Slightly more labelled RST axons/sprouts can be seen coursing towards the gray matter rostrally for pretreated db-cAMP groups (Figure 4.2 II, 77  IV; Figure 4.3 C,G) versus vehicle groups (Figure 4.2 I,III; Figure 4.3 A,E).  Proximal to the lesion site, the RST is marginally more prominent in the pretreated db-cAMP group (Figure 4.2 II,IV; Figure 4.3 D,H) in comparison to the control group (Figure 4.2 I,III; Figure 4.3 B,F).   Rostrally, the CLLRs indicate a greater presence of RST axons/sprouts for the pretreated db-cAMP group (Figure 4.4 II,IV; Figure 4.5 C,G) in comparison to the control group (Figure 4.4 I,III; Figure 4.5 A,E), however, the differences become less distinct when compared proximal to the lesion border.  The pretreated db-cAMP group (Figure 4.4 II,IV; Figure 4.5 D,H) shows slightly  more traced RST fibres proximal to the lesion edge in comparison to the vehicle group (Figure 4.4 I,III; Figure 4.5 B,F)   78     Figure 4.2  Pretreated db-cAMP groups show moderately more labelled RST axon/sprout/fibres over controls The above horizontal views of pretreated db-cAMP groups (II,IV) reveal slightly more RST axon/sprouts/fibres (rostrally [left]) than pretreated vehicle groups (I,III).  Proximal to the lesion edge, marginally more RST axons/sprouts/fibres can be seen for db-cAMP pretreated groups (II,IV) versus vehicle pretreated groups. Images presented represent four different animals. Scale bar = 500?m.    79    80  Figure 4.3 (Inset of Figure 4.2) Pretreated db-cAMP group shows more traced RST axons/sprouts rostrally Rostrally, the numbers of RST axons/sprouts are more prominent in the pretreated db-cAMP group (C,G) in contrast to the vehicle group (A, E).  Proximal to the lesion edge, the traced RST is marginally more apparent for the pretreated db-cAMP group (D,H) versus the vehicle group (B,F).  Each image = 1mm2.  Scale bar = 200?m.   81     Figure 4.4 CLLRs show more RST axons/sprouts/fibres for the pretreated db-cAMP group RST axons/sprouts/fibres of the pretreated db-cAMP group (II,IV) are more apparent rostrally than the control group (I,III).  Proximal to the lesion, the traced RST is somewhat more distinct for the pretreated db-cAMP group (II,IV) in contrast to the control group (I,III).  Inset boxes = 1mm2.  CLLRs shown are comprised from four different animals. Scale bar = 500?m.    82    83  Figure 4.5  (Inset of Figure 4.4) CLLRs show a greater number of traced RST axons/sprouts rostrally and proximal to the lesion edge for the pretreated db-cAMP group The pretreated db-cAMP group displays greater numbers of RST axons/sprouts (C,G) rostrally in comparison to the pretreated vehicle group (A,E).  Proximal to the lesion edge, the traced RST is more prominent for the pretreated db-cAMP group (D,H) versus the control group (B,F). Scale bar = 200?m.    4.4 RST axonal/sprout density is significantly higher in db-cAMP treated versus control animals  Figure 4.6 indicates that the axonal density between the db-cAMP and vehicle pretreated groups differs little until 1,500?m from the lesion epicentre. The density of the db-cAMP treated group reaches an overall average of approximately 7.5% between 2,000-3,000?m from the lesion, while the control group?s average density reached a ceiling of approximately 4.8% in the same distance (p ? 0.007).   The overall density between 2,000-3,000?m, however, for the pretreated db-cAMP group is lower, approximately 7.5% (Figure 4.6), in contrast to the acutely treated db-cAMP group whose RST axonal/sprouting density averaged 9.5% (Figure 3.6).  Interestingly, RST axonal density levels between acute and pretreated vehicle groups were extremely similar (Figure 4.7).    84   Figure 4.6  RST axon/sprout density increases significantly from the lesion epicentre for the pretreated db-cAMP group The pretreated db-cAMP group shows increased RST axon/sprout density from the lesion centre rostrally in contrast to the control group; p ? 0.007. Data is presented as percentage of average total axonal density per 100?m.  n=4 (db-cAMP), n=5 (vehicle). Error bars = SEM.  85   Figure 4.7 No significant RST axon/sprout density differences between acute and pretreated vehicle (control) groups  Comparison between the acute and pre-treatment paradigms of vehicle treated groups indicate that there are no significant differences between RST axon/sprout densities.  Data is presented as percentage of average totally axonal density per 100?m.  n=6 (acute vehicle), n=5 (pretreated vehicle). Error bars = SEM.  4.5 Pretreated db-cAMP groups show more RST axon/sprouts/fibres in the gray matter  The number of visible RST axons/sprouts/fibres in the gray matter seen in pretreated db-cAMP treated animals (Figure 4.8 II,IV; Figure 4.9 C,G) was greater in comparison to the control group (Figure 4.8 I,III; Figure 4.9 A,E).  Proximal to the lesion edge, neither the pretreated db-cAMP group (Figure 4.8 II,IV; Figure 4.9 D,H) nor the control group (Figure 4.8 I,III; Figure 4.9 B,F) displayed any substantial numbers of labelled axons/sprouts.   The CLLRs reveal more traced RST axons/fibres in the gray matter for the pretreated db-cAMP group (Figure 4.10 II,IV) versus the control group (Figure 4.10 I,III).  RST axons/sprouts/fibres can be seen rostrally to proximal to the lesion border for the pretreated db-cAMP group (Figure 4.10 II,IV) whereas the pretreated vehicle group shows greatly reduced numbers of RST 86  axons/sprouts/fibres in the same range (Figure 4.10 I,III).  This is further exemplified in Figure 4.11 (D,H) where the pretreated db-CAMP groups shows more axons/sprouts proximal to the lesion while the control group shows few (Figure 4.11; B) to none (Figure 4.11; F).  Rostrally, traced RST axon/sprout density in the gray matter of the pretreated db-cAMP group (Figure 4.11; C,G) is much greater than that of the control group (Figure 4.11; A,E).    87     Figure 4.8 RST axon/sprout density in the gray matter is greater for the pretreated db-cAMP  group The pretreated db-cAMP group shows more labelled RST axons/sprouts/fibres rostrally to proximal to the lesion edge in the gray matter (II,IV) versus the pretreated vehicle group (I,III).  RST has been omitted in the above images.  Few to no labelled axons/sprouts are visible proximal to the lesion edge in the gray matter for all groups (I-IV).  Above images represent four different animals.  Scale bar = 500?m. 88    89  Figure 4.9 (Inset of Figure 4.8) the pretreated db-cAMP group shows more RST axons/sprouts rostrally in gray matter Rostral images (C,G) reveal greater numbers of labelled RST axons/sprouts in the gray matter in contrast to the control group (A,E).  The pretreated db-cAMP group (D,H) and the control group (B,F) display little to no visibly labelled RST axons/sprouts proximal to the lesion edge.  Scale bar = 200?m.     90     Figure 4.10  CLLRs show more RST axon/sprout/fibres in the gray matter for the pretreated db-cAMP group Proximal to the lesion edge and rostrally the pretreated db-cAMP shows significantly more numbers of traced RST axons/sprouts/fibres in the gray matter (II,IV) in contrast to the control group (I,III).  Visible axonal density decreases rostrally to proximal to the lesion edge for all groups (I-IV), but much more so for the pretreated vehicle groups (I,III).  Above CLLRs represent compilations of four different animals. Scale bar = 500?m. 91    92  Figure 4.11  CLLRs reveal greater rostral axon/sprout density for the pretreated db-cAMP group The pretreated db-cAMP group shows noticeably higher numbers of traced RST axons/sprouts/fibres rostrally in the gray matter (C,G) versus the control group (A,E).  Proximal to the lesion edge, the pretreated db-cAMP group reveals a small amount of visible axons/sprouts (D,H) in contrast to the pretreated vehicle group which shows few (B) to none (F).  Scale bar = 200?m. 4.6 RST axonal sprout density is higher for db-cAMP treated animals in comparison the controls.  Figure 4.12 shows RST axon/sprout density in the gray matter of the db-cAMP group to be greater than that of the vehicle treated groups. While the difference in densities between the two groups is significant (p ? 0.05), the disparity between the two is not as large as seen, for example, between the acute groups.  At 1,500?m (Figure 4.12) the RST axonal/sprouting density is < 1% for the control group whereas the db-cAMP group?s density is approximately 2.6%. Between 2,000-3,000?m this increases to an average of 3.4% for the pretreated db-cAMP group while the vehicle pretreated group remains < 2% (Figure 4.12).  This is in stark contrast to the acutely treated db-cAMP treated group whose RST axonal/sprouting density exceeds 8% between 2,500 and 3,000?m (Figure 3.11).  Intriguingly, axonal/sprout densities in the gray matter between pre and acutely treated vehicle groups is remarkably similar (Figure 4.13).    93     Figure 4.12  RST axon/sprout density is greater for the pretreated db-cAMP group The pretreated db-cAMP group displays an overall higher density of axons/sprouts in the gray matter from the lesion centre rostrally in comparison to the control group.  p ? 0.05. Data is presented as percentage of average totally axonal density per 100?m.  n=4 (db-cAMP), n=5 (vehicle). Error bars = SEM.   94   Figure 4.13 RST axon/sprout into the gray matter shows no significant differences between acute and pretreated vehicle (control) groups Compared directly, both acute and pretreated vehicle groups show very similar levels of RST axon/sprout density into the gray matter; differences between the two groups are not significant. Data are presented as percentage of average totally axonal density per 100?m. n=6 (acute vehicle), n=5 (pretreated vehicle). Error bars = SEM. 4.7 Cross sectional views show more RST projections to the gray matter for db-cAMP groups.  At approximately level C2, cross sectional images of the spinal cord show traced RST projections into the gray matter. The pretreated db-cAMP group (Figure 4.14; Figure 4.15 B,D,F) displayed greater numbers of axons/sprouts coursing towards and into the dorsal horn of the spinal cord versus the control group (Figure 4.14; Figure 4.15 A,C,E).    95        Figure 4.14 Spinal cord cross sections show greater numbers of RST axon/sprouts to the gray matter for the pretreated db-cAMP group In images I,II,III, the pretreated db-cAMP group (cross sections on the right hand side) shows a greater number of RST axons/sprouts coursing towards the gray matter than the control group (cross sections on the left hand side).  Images are of spinal cord cross sections from six animals taken at approximately level C2.  Scale bar = 500?m. 96   Figure 4.15 (Inset of Figure 4.14) The pretreated db-cAMP group displays more RST axon/sprouts projecting to the gray matter Greater numbers of traced axon/sprouts can be seen projecting from the RST coursing to the gray matter in the pretreated db-cAMP  group (B,D,F).  In comparison, the RST of the vehicle group shows only modest numbers of projections to the gray matter (A,C,E). Scale bar = 200?m.  4.8 Pre-treatment of db-cAMP provided no functional benefits in the forelimb usage test  The baseline scores (Figure 4.16) for the pretreated groups, as with the acute groups, remained similar between the pretreated db-cAMP group (n=4) and the control group (n=6).  The baseline scores, using the commonly reported score average left plus both touches, were 81.6 ? 0.02% and 76.1 ? 0.03% for the vehicle and db-cAMP pretreated group respectively.  One week after the mini osmotic-pump implantation (no injury), the animals were re-evaluated. Db-cAMP and vehicle pretreated groups scored 64.3 ? 0.11% and 83.6 ? 0.04% respectively.  The db-cAMP 97  treated group had a significant degradation in performance (p = 0.02) in comparison to the control group.  Behavioural test scores during weeks 1 through 5 for the vehicle group initially declined, then stabilized scoring 39.2?0.13, 51.0?0.13, 42.6?0.11, 52.7?0.09 and 46.3?0.10% respectively.  The db-cAMP treated group scored similarly to the controls in weeks 1 to 3 with 28.0?0.13, 50.16?0.13 and 45.3?0.09% (left+both touches) respectively, however, weeks 4 and 5 indicated a drop in performance versus the control group with 31.1?0.09 and 24.2?0.12% (left + both touches) respectively.  Nevertheless, it should be noted that with the exception of the scores for the behaviour measured during the ?Pump only? segment, there were no statistical differences in reaching/touching behaviour between the groups at any other time points.   98      Figure 4.16  Pre-treatment of db-cAMP provides no functional improvement as indicated by the vertical exploration test Initial baseline scores were similar between the pretreated db-cAMP and vehicle groups, however the db-cAMP group performed worse (64.3 ? 0.11%) in the ?Pump Only? (no injury) week versus the controls (83.6 ? 0.04%; p=0.02). Week 1 represented the reaching test scores one week post injury of the dorsolateral funiculus.  Despite the db-cAMP group scoring lower during weeks 1,2,4 and 5, there were no significant differences between the scores. n=4 (db-cAMP), n=6 (vehicle). Error bars = SEM.    99  5 DISCUSSION  5.1 Treatment of db-cAMP results in more RST axons/sprouts in comparison to the vehicle treated group  Rats treated with db-cAMP prior to or while receiving a crush injury to the dorsolateral funiculus displayed greater numbers of traced RST axons/sprouts in comparison to the control group. The density of the RST was visually more pronounced rostral to proximal to the lesion edge than that of the control groups.  The numbers of RST axons/sprouts/fibres travelling towards and in the gray matter were also more abundant than the vehicle treated group rostral to and proximal to the lesion edge.  Moreover, these axons/sprouts were significantly more noticeable near the lesion cavity.  Unfortunately in neither db-cAMP nor vehicle treated groups, in either the acute or pre-treatment paradigms, were any traced RST axons, sprouts or fibres found beyond the rostral lesion edge. There was also no evidence of any traced RST axons/sprouts caudal to the lesion cavity either.  In addition, despite the appearance of greater numbers of RST axons/sprouts proximal to the lesion edge for db-cAMP groups, this had little influence on the overall lesion sizes.  Lesion size differences between the treated and control groups were remained similar. Therefore, treatment of db-cAMP did not reduce the enlarging injury cavity.  Overall, however, the findings of this study are very encouraging as either increased axonal sprouting or prevention of axonal/ retraction are helpful additions to the momentous task of spinal cord reparation.   5.2 Increased numbers of visibly BDA labelled RST axons/sprouts in acute or pre- treated groups unlikely due to differences in tracing efficacy  It is important to note, however, that consideration be given to whether or not the differences in visible RST axon/sprouting/fibre densities were due in part to overall BDA tracing differences between the two groups. Concerns with interpreting the result that either acute or pre-treatment with db-cAMP increases RST axon/sprout density versus the vehicle treated groups are due to: a) whether or not new axons/sprouts/fibres have formed in response to treatment, b) existing RST axons, sprouts or fibres having reduced retraction or are spared, or c) BDA labelling being more effective in db-cAMP treated animals.  In order to confirm that either an increased axonal 100  sprouting response has occurred or that axons/sprouts have reduced retraction/increased sparing, the validity of premise c has to be brought into question.       It is conceivable, but somewhat less likely, that the BDA labelling of the RST is more efficacious in db-cAMP treated animals versus the vehicle treated animals. Firstly, all data were normalized to significantly reduce tracing bias. Secondly, the general efficacy of using BDA tracer applied to the RN is approximately 2-5% (Raineteau, Fouad, Bareyre, & Schwab, 2002) based on a total of 3,000 RST fibres (Liu, et al., 1999).  I found no statistical differences in the total number of cross sectioned labelled RST fibres (all diameters) between any of the four groups tested (229?26 [vehicle] labelled fibres versus 209?26 [db-cAMP] labelled fibres; p>.80).  Thirdly, the tracer mechanism by which a dextran amine such as BDA labels the axons over longer distances is by axonal transport (Dolleman-Van der Weel, Wouterlood, & Witter, 1994; Ferguson, Xian, Barati, & Rush, 2001; Fritzsch, 1993; Glover, Petursdottir, & Jansen, 1986; Kobbert et al., 2000; Nance & Burns, 1990; Reiner et al., 2000; Schmued, Kyriakidis, & Heimer, 1990; Veenman, Reiner, & Honig, 1992; Wouterlood & Jorritsma-Byham, 1993).  If there were any drug treatments that affect the functions of axonal transport, the amount of labelling by an actively transported tracer such as BDA could be affected as well. For example it was demonstrated that when an axonally transported tracer such as HRP (horse radish peroxidase) is used with a transport inhibiting drug such as colchicine, both transport and therefore tracing were significantly reduced (Krah & Meller, 1999; Meller, 1992).  It has been shown that db-cAMP inhibits fast axonal transport in frog DRG cells through an indirect mechanism (Edstr?m, 1977).  Moreover, the application of db-cAMP to enhance axonal regeneration in crushed optic nerves resulted in the decrease of fast axonal transport in a dose dependent manner, however, enhanced initial sprout formation remained unaffected (McQuarrie & Grafstein, 1983).  Therefore, if db-cAMP reduced the rate of axonal transport or even inhibited it, then it is conceivable that BDA delivery and distribution would be disturbed as well. Fourthly, conditioning lesions have been shown to increase axonal outgrowth rates during regeneration (Bisby & Pollock, 1983; McQuarrie, Grafstein, & Gershon, 1977; Tetzlaff & Bisby, 1989) whereas the application of db-cAMP to increase axonal regeneration has been shown to increase outgrowth rates either modestly (Kilmer & Carlsen, 1987) or not all; even in cases where db-cAMP increased and maintained T?1 and ?III tubulin levels for 28 days in contrast to the conditioning lesion?s group 101  which had already returned to baseline levels (Han, Shukla, Subramanian, & Hoffman, 2004).  Proteins that are synthesized in the cell body are delivered to the axon by either slow or fast axonal transport (Bisby, 1978; Black & Lasek, 1980; Ochs, 1972; Ochs, Sabri, & Johnson, 1969). Hence if BDA, in this case, was transported by slow transport, the use of db-cAMP should not increase the rate of slow axonal transport in comparison to the vehicle treated group by much, thereby, minimizing any labelling advantages it may produce.  Fifthly, if transport is indeed affected by db-cAMP, it raises questions about the state or configuration of the components in the axon. It has been suggested that the use of db-cAMP to augment axonal regeneration in the CNS also increases the calibre of the axons (Pearse, et al., 2004).  Indeed, an anecdotal examination of Figure 3.13 and Figure 4.15 generally show visibly larger diameters of the traced RST in the db-cAMP groups in comparison to the controls. Naturally, there could be instances where visual bloom or flare could occur which are caused by photographing anomalies of the fluorescent labelling. As well any closely apposed tracts could also appear as one larger tract. In either case ultrastructural examinations would be required to confirm the true diameters of the labelled fibres.  Nevertheless, it has been brought forward that axonal calibre is based either on neurofilament (NF) number (Friede, 1972; Friede & Samorajski, 1970; Hoffman, Griffin, & Price, 1984; Hoffman, Thompson, Griffin, & Price, 1985; Lasek, Oblinger, & Drake, 1983) or related to the degree of NF phosphorylation in the axon (de Waegh, Lee, & Brady, 1992).  It has been reported that axonal injury causes a reduction in the production of NFs.  For example, a rat facial nerve crush results in the decreased synthesis of NFs (Tetzlaff, Bisby, & Kreutzberg, 1988) and a rat rubrospinal tract transection at the cervical level produced lower levels of NF mRNA (Tetzlaff, et al., 1991). It has also been postulated that the reduction of NFs might be beneficial for a regenerating axon as the decrease of NFs in the axon may allow for the increase in the transport rate of tubulins needed for axonal outgrowth due to decreased NF interactions with tubulin (Tetzlaff, Leonard, Krekoski, Parhad, & Bisby, 1996).  Thus, if there were more NFs in the RST due to db-cAMP treatment, this too should reduce or interfere with axonal transport thereby potentially impeding BDA labelling efficacy in db-cAMP treated animals. On the other hand, increased NF phosphorylation may have augmented the axonal calibre of the RST.  Goldstein et al. (1987) revealed that an escalation in the levels of NF phosphorylation occurs after nerve transection, while Pestronk et al. (1990) reported a correlative increase in axonal sprouting with NF phosphorylation which may be indicative of increased 102  plasticity.  If so, this should appear as more RST fibres being labelled for the db-cAMP treated animals as this should be indicative of increased axonal sprouting and not simply due to improvements in tracing efficacy.   It is clear that axonal transport in db-cAMP treated animals is not inhibited as successful BDA labelling of the RST has occurred. However, it appears more likely that the visible labelling is characteristic of an increased sprouting response and less likely due to increases in tracing efficacy for the db-cAMP.  5.3 Db-cAMP may increase axonal sprouting by eliciting a cell body response which can contribute to overcoming inhibitors of regeneration  Surprisingly, the initial response of the rubrospinal neurons to a high cervical rubrospinal tract funiculotomy is regenerative (Tetzlaff, et al., 1991).  mRNA expression of regeneration associated genes (RAGs) such as T?1 tubulin and GAP-43 whose expression has been closely associated with regenerating axons (Hoffman, 1989; Jacobson, Virag, & Skene, 1986; Kalil & Skene, 1986; Miller, Naus, Durand, Bloom, & Milner, 1987; Miller, Tetzlaff, Bisby, Fawcett, & Milner, 1989; Skene, 1989; Skene & Willard, 1981a, 1981b) is seen to increase from the onset following RST injury only to be proceeded later by rubral neuronal atrophy (Tetzlaff, et al., 1991).  By using a very similar method of drug delivery, application of db-cAMP to the rat RN via mini-osmotic pump after a C3/4 crush of the RST prevented cellular atrophy of the cells of the red nucleus (Lane, et al., 2005; Lane, et al., 2004).  Moreover, in situ hybridization of T?1 and GAP-43 mRNA levels in the RN were increased in comparison to the controls as well (Lane, et al., 2005; Lane, et al., 2004).    Under injury only conditions it is conceivable that the elevated RAGs (T?1 and GAP-43) may be part of a regenerative response that is ultimately curtailed by many inhibitory factors and molecules in the environment. Molecules such as MAG, OMgp and Nogo-66 that bind to the Nogo receptor (NgR1) which colocalize with either p75NTR or TROY (TAJ), and Lingo-1 may be contributors to this abortive response (for details about the inhibitory molecules, receptors and pathways see section 1.8.1).  It has been previously reported that p75 expression initially increased in rat rubral neurons sustaining a high cervical injury only to decline to lower levels at 103  later stages; however, the abortive response of the RST remained ongoing despite the reduction in the levels of p75 (Kobayashi, et al., 1997; Tetzlaff et al., 1994).  It is possible that another molecule that participates in a similar tripart complex like that of p75NTR may be another contributor that inhibits regeneration.  Indeed, TROY(TAJ) has been found to not only bind to NgR1 (Park, Yiu, Kaneko, Wang, Chang, & He, 2005; Shao, et al., 2005), but also activate RhoA (a contributor to the Rho/ROCK inhibitory pathway) similarly to p75NTR when exposed to myelin based inhibitors (Shao, et al., 2005).  It has also been demonstrated that the activation of the TROY(TAJ) complex causes neurite inhibition even in cells lacking or have reduced expression of p75 (Park, Yiu, Kaneko, Wang, Chang, & He, 2005).  Furthermore, expression of TROY(TAJ) has been found in many areas, for example, in the mouse CNS, including the spinal cord (Mi, 2008).  Treatment with db-cAMP in these experiments may be beneficial in overcoming the downstream effects of the activated TROY(TAJ) or p75NTR complexes.  Increases in cAMP activate PKA and the use of db-cAMP has been found to adequately activate PKA as well (Neelon & Birch, 1973). Hence, activated PKA then phosphorylates the RhoA-GDP-GDI complex; a complex with downstream targets, such as ROCK, that are responsible for neurite inhibition. This then essentially curtails the inhibitory effect of the Rho-ROCK pathway (Dong, et al., 1998; Lang, et al., 1996).   Initiation of gene transcription by CREB has also been shown to be an effective method of augmenting axonal growth in the inhibitory CNS environment.  Application of db-cAMP to the RN may have induced the transcription factor CREB to maintain the axonal sprouting response seen in the acute and pretreated groups.  Increases in CREB expression, for instance, helped promote regeneration of DRG neurons of lesioned dorsal column axons (Gao, et al., 2004).  Moreover, specific genes require activated CREB before expression.  Arg-1, for example, requires phosphorylated CREB before being transcribed (Gao, et al., 2004).  Arg-1 is the enzyme that catalyzes the reaction of arginine to ornithine, where ornithine is the precursor to the polyamines (Morgan, 1998).  The complete functions of polyamines are not yet fully understood (Pegg & Casero, 2011), however, increases in polyamine synthesis or application of polyamines have been demonstrated to be beneficial for axonal regeneration (Chu, et al., 1995; Dornay, et al., 1986; Edbladh, Edstrom, & Persson, 1990; Gilad, et al., 1996; Kauppila, 1992; Tetzlaff & Kreutzberg, 1985; Wong & Mattox, 1991).  It has been made apparent that Arg-1 acts 104  downstream from cAMP to increase polyamine levels. Moreover, the use of db-cAMP has been shown to increase the levels of both Arg-1 and subsequently polyamines in DRGs in a manner sufficient to overcome myelin inhibitors and increase neurite outgrowth (Cai, et al., 2002).  Additionally, ornithine decarboxylase (ODC), the rate limiting enzyme in polyamine synthesis which catalyzes the decarboxylation of ornithine to putrescine, the initial polyamine, has a very short half-life, is extremely labile and is present in low amounts in the cytosol (Ghoda, van Daalen Wetters, Macrae, Ascherman, & Coffino, 1989; Lu, Stanley, & Pegg, 1991).  It has been shown that in transected facial motor axons, ODC activity increases by 3 fold hours after injury (Tetzlaff & Kreutzberg, 1985) and the increases in ODC are mediated by changes in cAMP levels (Byus & Russell, 1975).  Furthermore, these changes involve the cAMP pathway downstream target PKA (Byus, et al., 1976; Byus & Russell, 1976).  It is conceivable that the use of db-cAMP with its decomposition products in the acute and pre-treatment paradigms play a role in elevating the short lived enzyme, ODC, and correspondingly increase polyamine synthesis to help promote regeneration.     It has become ostensible, as previously noted, that elevations in cAMP are correlated with growing or regenerating axons.  Growing embryonic axons have natively high levels of endogenous cAMP which diminish to background levels in adulthood. This increase in cAMP allows the neurites to overcome inhibitors of myelin while the same adult neuronal cellular population becomes hampered (Cai, et al., 2001).  In contrast to the CNS, the adult peripheral nervous system displays strong recovery from injury. Indeed, successful regenerating previously injured PNS motor neurons show significant elevations of cAMP in the cell body and proximal to the injury during recovery. What is more, additional increases in native cAMP has been shown to enhance the regenerative response (Kilmer & Carlsen, 1984).  It has become apparent that increasing cAMP levels in axotomized neurons may have beneficial regenerating effects; however, the method by which to increase cAMP levels or mimic the elevations of cAMP needs to be carefully examined for design and experimental limitations.  Early use of db-cAMP in axonal regeneration studies were mired in controversy.  Application of db-cAMP proved favourable in crushed or hemisected nerves in rats (Gershenbaum & Roisen, 1980; Pichichero, et al., 1973), in contrast, however, the use of db-cAMP in similar experiments failed to elicit any 105  measureable benefits (Black & Lasek, 1979; McQuarrie, et al., 1977).  Naturally and of concern is, are there limitations or caveats when using db-cAMP as an analogue of native cAMP?    5.4 Is db-cAMP an effective method of mimicking increased cAMP levels? Are other analogues or methods more or less efficacious?  There are numerous ways to approximate increased cAMP levels in a given population of cells. One of the methods used include the use of cAMP analogues such as db-cAMP that Posternak et al (1962) had originally synthesized.  For these experiments db-cAMP was chosen to mimic the increased levels of cAMP for several reasons: it is membrane permeant which increases its ability to gain entry into cells (Henion, Sutherland, & Posternak, 1967), it is resistant to breakdown by several phosphodiesterases thereby maintaining an elevated concentration in the cell (Blecher, 1971; Henion, et al., 1967; Menahan, Hepp, & Wieland, 1969; Moore, Iorio, & McManus, 1968), it is and has often been used to mimic increases in cAMP levels, and it is a crystallized solid and therefore known initial predetermined experimental concentrations in solution can be prepared.  Of concern, however, is how well does db-cAMP approximate the actions of native cAMP and are there any potential deficits in using this compound.  5.4.1 The decomposition product butyrate can introduce potential experimental confounds  There are several potential issues with using db-cAMP to mimic elevations of cAMP.  To begin with, when db-cAMP is introduced into a buffered solution of PBS 1X it decomposes into db-cAMP, N6-monobutyryl cAMP (mb-cAMP), cAMP and butyrate.  The decomposition rate for these products is approximately 4% for 60 mins of incubation (Swislocki, 1970).  The decomposition concern is further exacerbated when db-cAMP enters the cell and is further metabolized to N6-mb-cAMP, N6-mb-AMP, O2?-mb-cAMP, O2?-mb-AMP cAMP, AMP, ADP, ATP and butyrate (Hsle, Kawashima, O'Neill, & Schroder, 1975; O'Neill, Schroder, & Hsle, 1975). Any experimental by-products could introduce potential confounds to the research in question. With an experimental concentration of 25 mM of db-cAMP in PBS, 1 mM of butyrate would be generated in as little as one hour. Butyrate has been shown to influence or interfere with the effects of second messenger systems.  For example, it has been reported that 1mM of sodium butyrate can stimulate PKC (protein kinase C) by 3 fold and induce differentiation in K562 human erythroleukemia cells (Rivero & Adunyah, 1998).  Sodium butyrate, known to be 106  an inhibitor of histone deacetylase,  has also been shown to dramatically enhance IL-6  differentiation of transformed human B cell SKW 6.4 cells where in comparison, sodium butyrate had little effect on IL-4 differentiated SKW 6.4 cells suggesting there may be mechanistic or synergistic link between IL-6 and sodium butyrate (Kawamoto, Gohda, Iji, Fujiwara, & Yamamoto, 1998).   Moreover, sodium butyrate can operate independently of cAMP to strongly inhibit DNA synthesis and cell proliferation (Feng, Ge, Akyhani, & Liau, 1996).  It can increase chromatin-associated kinase activity thereby altering the accessibility of DNA nucleases which affects gene expression (Kitzis, Tichonicky, Defer, & Kruh, 1980).  Yusta et al. (1988) demonstrated that while the use of db-cAMP can partially mimic the actions of elevated cAMP, some of the effects such as histone acetylation, are clearly mediated by the newly formed by-product butyrate.  Finally, butyrate can produce other deleterious effects such as apoptosis.  Salminen et al., (1998) demonstrated that sodium butyrate at concentrations of 1-10 mM  activated caspase-3, a marker of apoptotic cell death (Gillardon, Bottiger, Schmitz, Zimmermann, & Hossmann, 1997; Medina et al., 1997; Yakovlev et al., 1997), and caused nuclear fragmentation in cerebellar granule neurons from 7 day old Wistar rats and mouse Neuro-2a neuroblastoma cells. It has also been shown that 3mM sodium butyrate caused internucleosomal DNA fragmentation followed by a substantial decrease in cell viability within 48hrs of treatment (Soldatenkov, Prasad, Voloshin, & Dritschilo, 1998).  5.4.2 Mb-cAMP is the biologically active decomposition product of db-cAMP  Another concern with db-cAMP is that its native form does not normally activate PKA.  It requires enzymes such as N6-butyryl aminohydrolase and O2?-butyrylesterase to catalyze the conversion to mb-cAMP and cAMP (Blecher & Hunt, 1972).  Mb-cAMP, specifically N6-mb-cAMP, is generally considered to be the most biologically active form of the derivatives of db-cAMP (Blecher & Hunt, 1972; Henion, et al., 1967; Hsle, et al., 1975; Kaukel & Hilz, 1972; Kaukel, Mundhenk, & Hilz, 1972; Neelon & Birch, 1973; O'Neill, et al., 1975; Posternak, et al., 1962; Posternak & Weimann, 1974). It is approximately 3 fold less effective than cAMP in stimulating protein kinase activity while db-cAMP is more than 3,000 times less effective (Neelon & Birch, 1973). In addition, mb-cAMP is much more resistant to phosphodiesterases than the other degradation products O2?-mb-cAMP and cAMP which are removed relatively quickly from the cell (O'Neill, et al., 1975).  107   5.4.3 Db-cAMP and its decomposition products may not act similarly to native cAMP  A further complication with the use of db-cAMP may be linked to its catabolic products.  As previously noted, intracellularly, db-cAMP can be split into several by-products, some with differing affinities to cAMP binding sites. Since the cyclic derivatives are not structurally identical to native cAMP, they may not function analogously to it either. Indeed, it has been reported that the use of db-cAMP as a cyclic adenosine monophosphate analogue has resulted in not just a similar or even muted response compared with native cAMP, but with an outcome that had a substantial antagonistic effect (Hilz & Tarnowski, 1970; Kaukel, Fuhrmann, & Hilz, 1972; Solomon, Brush, & Kitabchi, 1970).  For example, 1mM db-cAMP decreased glycogen content in HeLa S3 cells while increases in native cAMP increased glycogen content (Kaukel, Fuhrmann, et al., 1972).  Moreover, when the db-cAMP was compared with other methods of increasing cAMP, such as with the use of PDE inhibitors, vastly differing outcomes were reported between the two methods attempting to mimic increased or augmented cAMP levels.  For example, when db-cAMP (4mM) was added to rat diaphragms in order to increase the frequency of miniature endplate potentials (MEPP), there was no change in frequency; however, when theophylline, a PDE inhibitor (5.6mM) was introduced to the preparation instead in order to increase native levels of cAMP, there was a sizeable increase in the MEPP frequency and amplitude (Miyamoto & Breckenridge, 1974).      5.4.4 C8 substituted cAMP analogues may be more efficacious than N6 cAMP analogues in some circumstances  The comparison of db-cAMP with other cAMP analogues or other methods of increasing cAMP has also yielded dissimilar results. In some cases the use of db-cAMP against other cAMP analogues has produced comparable outcomes, and in other cases it has been without effect.  For instance, db-cAMP failed to activate PKA and phosphorylate microtubule-associated protein 2 (MAP2) while cAMP and cAMP analogues such as 1-NO-cAMP, 6- Cl-cAMP and 8-Br-cAMP did (Richter-Landsberg & Jastorff, 1985).   Identical concentrations of cAMP analogues 8-benzylthio-cAMP or 8-(4-aminobuylamino)-cAMP produced a profound change in neuronal electrical activity while db-cAMP of the same concentration generated no change at all (Treistman & Levitan, 1976).  Additionally, in a similar study that preceded Treistman et als? 108  (1976) work, using the same model, Gainer & Barker (1975) originally suggested that cAMP was an unlikely mechanism in their model of neuronal electrical activity because their use of db-cAMP was without effect.  Furthermore, Rydel & Greene (1988) noted that C-8 cAMP analogues such as 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP) and 8-bromo-cAMP (8Br-cAMP) were much more efficacious than N6 substituted analogues like db-cAMP and N6-mb-cAMP in promoting neurite outgrowth and long term survival in rat superior cervical ganglion cells (P1-3) and DRGs (E15).   5.4.5 Circumstances where N6 cAMP analogues are more advantageous and efficacious than C8 cAMP analogues  The negative or ineffective outcomes of the studies noted above utilizing db-cAMP might imply that this compound may not be a suitable analogue for cAMP.  Indeed, certain conditions or precautions need to be adhered to in order try to provide the best possible outcomes, but an equal amount of care needs to be observed when using other cAMP analogues or alternative methods of increasing or mimicking increased cAMP levels.  C8 cAMP analogues may been shown to be more efficacious than N6 cAMP analogues in certain circumstances as previously noted,  however, like the N6 cAMP analogues, they too are not a direct replacement for native cAMP (Butt et al., 1995; Schwede et al., 2000).  For instance, C8 analogues such as 8-Cl-cAMP and 8-NH2-cAMP have been shown to cause pro-apoptotic effects instead of the reversible cell cycle arrest that the increases that native cAMP produces (Vintermyr et al., 1995).  Furthermore, 8-CPT-2?-O-Me-cAMP increased neurite length with increasing concentrations up to 1mM but significantly reduced neurite length when concentrations reached 10mM.  Conversely,  forskolin (AC activator) which elevates endogenous levels of cAMP, at increasing concentrations starting at 0.1?M and reaching as high as 10?M produced no such deleterious effects (Xu et al., 2012).  It is becoming increasingly evident that the use of cAMP analogues of either moiety be it of the N6 or C8 variety to augment cAMP levels that the focus needs to be on molecular target specificity.  It has been demonstrated, for example, that in general N6 cAMP analogues preferentially target site A2 while C8 and C2 cAMP analogues generally prefer site A1 of PKA isozymes (Beebe, Holloway, Rannels, & Corbin, 1984; Corbin et al., 1982; Schwede, et al., 2000).  Specific bulky N6 cAMP analogues have also been shown to produce higher PKA type II 109  potency while conversely certain C8 cAMP analogues have increased PKA type I affinity (?greid et al., 1985). Knowing the molecular specificity beforehand will be helpful in designing experiments that employ and require increased levels of cAMP.    5.4.6 Caution needs to be exercised with compounds that increase levels of native cAMP as well   It is apparent that due to the various specific molecular affinities that different cAMP analogues have for definitive targets that alternative methods for increasing native cAMP have also been employed.  Approaches include using adenylyl cyclase (AC) activators such as forskolin (7 beta-Acetoxy-8,13-epoxy-1alpha,6 beta,9 alpha-trihydroxy-labd-14-en-11-one) or phosphodiesterases inhibitors like the generalized PDE inhibitor IBMX (3-Isobutyl-1-methylxanthine) or the PDE4 specific inhibitors rolipram ((R,S)-4-[3-(Cyclopentyloxy)-4-methoxy-phenyl]-2-pyrrolidinone) and mesopram ((R)-5-(4-Methoxy-3-propoxyphenyl)-5-methyl-2-oxazolidinone).  The appeal of using these compounds is that native cAMP is increased without sacrificing target specificity.  Unfortunately, the use of these drugs to augment cellular cAMP levels is also not without its limitations. Forskolin, for example, inhibits glucose transport independently of the actions of cAMP (Joost, Habberfield, Simpson, Laurenza, & Seamon, 1988; Joost & Steinfelder, 1987; Kim, Sergeant, & Shukla, 1986; Mills, Moreno, & Fain, 1984).  In addition, both forskolin and IBMX directly inhibited glucose transport while db-cAMP or cAMP had no effect in rat plasma membrane vesicles (Kashiwagi, Huecksteadt, & Foley, 1983).  Herness et al. (1997) also demonstrated that forskolin inhibits potassium channels independently of cAMP activation. Furthermore, forskolin potency varies widely for different adenylyl cyclases (Sutkowski, Tang, Broome, Robbins, & Seamon, 1994).  For instance, ACVI had a 18 fold increase in activity in contrast to ACII which only produced a 2 fold increase when activated by forskolin (Pieroni et al., 1995).  Interestingly, ACIX is generally insensitive to activation by forskolin (Antoni et al., 1998; Paterson, Smith, Harmar, & Antoni, 1995; Premont et al., 1996; Yan, Huang, Andrews, & Tang, 1998). As ACIX is found prominently in mouse brain and midbrain (Visel, et al., 2006) and throughout the rat brain (Premont, et al., 1996), use of forskolin to activate this AC might not be efficacious.  Additionally, forskolin does not activate sACs (solubleAC) (Forte, Bylund, & Zahler, 1983).  This is significant because sAC is required for Netrin-1 signalling and axonal growth. The use of KH7 (2-(1H-benzimidazol-2-ylthio)-2-[(5-bromo-2-hydroxyphenyl) 110  methylene] hydrazide, propanoic acid) to block the actions of sAC abrogated growth cone and filopodia elaboration in plated rat E15-16 dorsal spinal cord explants (Wu, et al., 2006).   On the other hand, phosphodiesterase (PDE) inhibitors are not without their own disadvantages.  IBMX, a non-specific PDE inhibitor, has been reported to be ineffective against PDEs 8 (Hayashi et al., 1998) and 9 (Soderling, Bayuga, & Beavo, 1998).   Expression of PDEs 8 and 9 have been shown to be expressed in human brain (Hayashi, et al., 1998; Soderling, et al., 1998).  Moreover, PDE8A (an isoform of PDE8) has been shown to be strongly present in the Sprague-Dawley rat RN and therefore use of IBMX to inhibit these PDEs could be ineffectual (Kruse, M?ller, & Kruuse, 2011).  Additionally, use of a PDE specific inhibitor such as rolipram could also be limiting in trying to increase overall cAMP levels.  Rolipram is a PDE4 inhibitor (Huai, et al., 2003) and cells with higher expression levels of other cAMP specific PDE inhibitors could attenuate increases in cAMP levels.  PDE7, for example, is a cAMP specific PDE (Miro, Perez-Torres, Palacios, Puigdomenech, & Mengod, 2001) that is insensitive to rolipram (Martinez et al., 2000).  PDE7 is expressed in many areas of the rat including the brain, red nucleus (RN) and spinal cord (Miro, et al., 2001; Reyes-Irisarri, Perez-Torres, & Mengod, 2005) and expression of PDE7 has been shown to colocalize with that of PDE4 (Miro, et al., 2001).  Again, use of only rolipram to increase cAMP levels in cells also expressing PDE7 may not be completely effective.  Additionally, in some experiments PDE4 inhibitors have been continuously applied (such as with a pump) in order to maintain elevated levels of cAMP.  Unfortunately, in some cases the endogenous levels of cAMP quickly returned to background levels despite continuous application of the inhibitor.  For example, mesopram (a PDE4 inhibitor infused at 10?l/hr at a concentration of 2.6mg/kg/day) was used to augment cAMP levels in rat (Fisher) DRG cells (C3).  While the pump delivered its contents for a week, cAMP levels dropped to background levels after 2days. A single conditioning lesion to the same cells kept cAMP levels elevated for 7 days (Blesch, et al., 2012).            What has become noticeable about the use of any method to increase cAMP levels is that no particular method is without impediments. Clearly the cellular environment contains specific ACs, PDEs, AKAPs and cAMP specific targets in order to create cAMP microdomains to help focus the outcome of cAMP elevation (for reviews see: Baillie, 2009; Cooper, 2003; Jarnaess & Tasken, 2007; Tasken & Aandahl, 2004; Tresguerres, et al., 2011; Willoughby & Cooper, 2007).  111  Future research employing methods to increase cAMP levels should carefully address which approach or approaches of increasing cAMP levels are the most suitable to use.      5.5 Pre-treatment of db-cAMP provides no additional advantages to acute treatment  Conditioning lesions (CL), that is, prior injuries performed before the test injury, have demonstrated remarkable regenerative enhancements in both the PNS (Arntz, Kanje, & Lundborg, 1989; Bisby & Pollock, 1983; Carlsen, 1983; McQuarrie, Grafstein, Dreyfus, & Gershon, 1978; McQuarrie, et al., 1977) and the CNS (McQuarrie & Grafstein, 1981; Neumann & Woolf, 1999; Richardson & Issa, 1984).  In fact, there are instances where pre conditioning injuries are required for regenerative success in the CNS after axonal injury (Richardson & Issa, 1984).  While the exact molecular mechanism of a CL is not fully understood, elevations in cAMP are presumed to play a pivotal role in the enhancement of the regenerative response (Carlsen, 1982b; Neumann, et al., 2002; Qiu, et al., 2002).  Moreover, db-cAMP has been used to echo elevations in cAMP in order to mimic the effects of a conditioning lesion. The use of db-cAMP to mimic a CL did result in regenerative success of injured central sensory axons despite the inhibitory environment of the damaged CNS (Neumann, et al., 2002; Qiu, et al., 2002).      The results of pre-treating (see section 4) the RN with db-cAMP one week prior to injury did culminate in a visibly denser RST with greater numbers of RST axon/sprout/fibres projecting into the gray matter in comparison to the vehicle pretreated group.  Of interest, however, is that pre-treatment did not eventuate in a similar or greater outcome to the acute treated groups, but one with a lower overall visible RST density and axonal sprouting projections into the gray matter.  Inspection of the RST axonal density data between the pre and acute treated groups not only show a decline in overall densities for the pretreated group, but also seem to indicate a response indicative of a lower available concentration of db-cAMP in the pre-treated group.  The mini-osmotic pumps exhausted their contents 14 days after implantation. The pretreated group had ended its db-cAMP infusion one week after injury with three weeks of declining db-cAMP levels thereon after while the acutely treated groups received two full weeks of db-cAMP infusion after injury and two weeks of db-cAMP decline.  A cursory examination of the results between the pre and acute treated groups suggest that the outcome differences could possibly be due to differences in the concentration of db-cAMP and its by-products remaining in the RN.  It 112  has been shown that increases in cAMP or cAMP-like levels (up to a certain concentration) increase neurite outgrowth or regeneration in a dose dependent manner (Gao, Nikulina, Mellado, & Filbin, 2003; Nikulina, et al., 2004; Xu, et al., 2012; Yin, et al., 2006), or overcome myelin inhibition only when a cAMP concentration threshold is exceeded (Gao, et al., 2003).  The acutely groups received available db-cAMP for a longer period in comparison to the pretreated groups after injury.  Thus, it appears that pre-treatment with db-cAMP garnered no additional advantages compared with the acutely treated group.  It is conceivable that pre-treatment with db-cAMP may elicit a similar response to that of a CL; however, the data is more indicative of a decreased concentration of available db-cAMP and its by-products.         5.6 Isolating behavioural changes involving only the RST poses difficulties in a dorsolateral funiculus crush injury  There are several impediments in measuring behavioural differences involving injury to the dorsolateral funiculus containing the RST when the RN is the target of treatment.  Firstly, the function of the RN and its resultant behavioural outcome needs to be understood in order to measure deficits after RST axonal trauma. Secondly, since there are anatomical differences in the RN between species; the behavioural function of the RN for the species being measured needs to be known.  Thirdly, a crush injury of the dorsolateral funiculus may not only damage the RST, but other projections that influence motor behaviour as well.  Lastly, selecting a behavioural metric that is sensitive enough to discern alterations in behavioural outcome involving changes of the RST.    The RN and RST are phylogenetically older than the pyramidal neurons and accompanying tracts, such as the CST, and have undergone significant evolutionary changes with regards to functions and connections over time (Burman, Darian-Smith, & Darian-Smith, 2000; Keifer & Houk, 1994; Lavoie & Drew, 2002; Massion, 1967; Massion, 1988; Onodera & Hicks, 1999; Onodera & Hicks, 2009, 2010; Onodera, Nitatori, & Hicks, 2004; ten Donkelaar, 1988).  The ?classical? or older motor area of the RN is the magnocellular region that made its appearance in vertebrates with limbs or limb-like structures (e.g. fins). As the development of a more complex cerebellum along with the overarching expansion of the parvocellular RN due to the arising need for more sophisticated hand and forelimb control did the diminution of the magnocellular RN occur (ten Donkelaar, 1988).  That is not to say that the magnocellular RN is not apparent or in 113  use in mammals for motor functions, but that skillful body movements and muscles requiring sensitive control have been supplanted by the pyramidal tract as the primary form of motor control.  As the magnocellular RN innervates mostly large motor units it is functionally limited in a wider range of  motor control capabilities (Onodera & Hicks, 1999).  The magnocellular region of the RN is proportionately larger in mammals involved with quadrupedal locomotion and declines in size with the increasing development of bipedalism (Massion, 1988; Nathan & Smith, 1982; ten Donkelaar, 1988).   In humans, for example, the functions of the magnocellular RN are considered to be largely rudimentary as its role has largely been superseded by the phylogenetically newer pyramidal system; however, partially erect anthropoid primates still retain a well-developed magnocellular RN for terrestrial quadrupedal locomotion. Moreover, they require that the feet of their lower limbs be able to pre shape in order to grasp so that they can ambulate above ground in their arboreal environment (Onodera & Hicks, 2010).   It should be noted that while the parvocellular RN is distinct from the magnocellular RN in humans, the distinction is not as clear in some proportionately ?lower? mammals such as the rat (Yamaguchi & Goto, 2006).    As noted, in humans the RN consists mainly of a larger parvocellular and almost imperceptible magnocellular RN (Massion, 1988; Nathan & Smith, 1982; Onodera & Hicks, 2009, 2010; Yamaguchi & Goto, 2006).  The parvocellular RN is connected to the ipsilateral principal olivary nucleus via the central tegmental tract (Ausim Azizi, 2007) and is part of the Guillain-Mollaret Triangle that includes the dentate nucleus (Lavezzi, Corna, Matturri, & Santoro, 2009).  While the ?pure? motor functions of the human RN and specifically the magnocellular portion have been almost completely subsumed by the pyramidal system, it is believed that the RN nucleus in association with the principal olive, functions to participate in sensorimotor functions involved in novel or unexpected stimuli detection along with other higher cognitive functions in association with working memory (Habas, Guillevin, & Abanou, 2010).    In primates, the magnocellular RN has been reported to be involved in distal limb movement (Houk, Gibson, Harvey, Kennedy, & van Kan, 1988), specifically the metacarpi-phalangeal extension in order to pre-shape the hand at the appropriate phase in a reach to grasp extension (van Kan & McCurdy, 2001, 2002b).  Specifically, the magnocellular RN functions during the 114  production of a coordinated whole-limb movement in order to group the digit extensions while much less so in isolated grouped digit movements (van Kan & McCurdy, 2002a).    In an almost exclusively quadrupedal mammal such as the cat, the magnocellular RN is well developed and suited for quadrupedalism whereas the subnuclei of the dorsomedialis and ventrolateralis of  parvocellular RN are poorly developed (Onodera, 1984).  As such the feline RN discharges in the swing phase (i.e. transport and placement) of the limbs during unobstructed locomotion and significantly more so when obstacles are presented.  Furthermore, adjustments of the motor neurons during the stance phase also resulted in RN activity (Lavoie & Drew, 2002).  Recent work by Zelenin et al. (2010) reported that the rubrospinal system in the cat participates in postural correction through the transmission of supraspinal commands. Furthermore, the majority of the somatosensory feedback to the RN in order to control body posture comes from the target (intra) limb. This intra-limb postural coordination capability schematic by the RN loosely parallels the corticospinal system (Zelenin, et al., 2010).    The rat, on the other hand, is a mammal that is both quadrupedal and possesses the capacity to grasp or reach to grasp (Whishaw & Gorny, 1996).   Certain similarities and differences can be seen when comparing the structure and projections to and from the RN in the rat with other quadrupedal animals not capable of similar digit flexion and grasping such as the cat.  For instance, both share similarities in topographic olivary projections from the mesodiencephalic nuclei commonly seen in quadrupeds but do not share neuroanatomical homologies in the nucleus of the posterior commissure (Onodera & Hicks, 2009).  Furthermore, it appears that the numbers of rubrospinal fibres that distribute collaterals to different levels of the spinal cord is greater in the rat when compared to the cat (Huisman, Kuypers, & Verburgh, 1982).  Chemical ablation studies involving the rat RN have yielded more information in the quest to determine the motor function of the RN.  Destruction of the rat RN attenuated the arpeggio movement in a reach to grasp food pellet exercise. It is believed that the RN acts as a framework in which the motor cortex can provide more fractionated responses. The ablated RN eliminates any pausing during the reach to grab movement phase (Whishaw & Gorny, 1996).  Like the cat, RN lesions also impair over ground locomotion in the rat. The RN in the rat is thought to provide correctional adjustments during locomotion. The chemical ablation of the left RN in the rat 115  resulted in an asymmetric gait during locomotion throughout the duration of the study (Muir & Whishaw, 2000). The authors also caution that similar behavioural deficits can be seen in unilateral pyramidal tract transections in the rat as compared to unilateral RN ablations, and that the observed behavioural changes could also be due to a generalized compensatory strategy in response to a unilateral CNS injury (Muir & Whishaw, 1999a, 1999b, 2000).     In rats, the role and behavioural contributions of the RN and RST are still under investigation and there are still challenges in deciphering RN contributions to motor behaviour.  In creating a lesion of the dorsolateral funiculus at the C4 level in rat and using the food pellet reaching test to measure behaviour, it was reported that damage to the dorsolateral funiculus containing the RST results in severe pellet reaching performance compared with uninjured controls.  Moreover, the rats were also unable to execute a grasp and close the digits around the pellets (Schrimsher & Reier, 1993).  A more recent study agreed with the finding that lesions of the dorsolateral funiculus result in an impaired grasping ability in the rat (Stackhouse, et al., 2008).  Also, it has been reported that in rats, unlike cats and monkeys, the dorsolateral funiculus does not contain, for example, the pathways of the corticospinal tract (CST) (Kennedy, 1990; Kennedy & Humphrey, 1987; Vahlsing & Feringa, 1980).    In contrast, however, it has been shown that ablation of the RN in rat has neither an effect on the success of the food pellet reaching test, nor does it affect the ability to grasp (Whishaw & Gorny, 1996; Whishaw, et al., 1998; Whishaw, Tomie, & Ladowsky, 1990).  It has also been shown that lateral lesions of the spinal cord (C5) in the rat result in recovery of grip strength suggesting the medial pathways may be important for grasping (Anderson, Gunawan, & Steward, 2007).  The concern with performing an injury of the dorsolateral funiculus is that damage may not only affect the RST, but also to a minor corticospinal projection and long-tract ascending sensory pathways which have been shown to be present in the DLF (Br?samle & Schwab, 1997; Casale, Light, & Rustioni, 1988; Goodman & Jarrard, 1966; Joosten, Schuitman, Vermelis, & Dederen, 1992; Kanagal & Muir, 2008, 2009; Liang, Moret, Wiesendanger, & M., 1991; Muir, Webb, Kanagal, & Taylor, 2007; Schreyer & Jones, 1982; Schrimsher & Reier, 1993; Weidner, Ner, Salimi, & Tuszynski, 2001).  Severing the dorsal roots at the level of C5-T2 in rats resulted in the loss of food pellet grasping ability (Saling, Sitarova, Vejsada, & Hnik, 1992).  Moreover,  in 116  rat RN ablation studies, it has been purported that part of the function of the RN may be to provide extensor tone in the limb thereby providing a framework which the paw can move in an arpeggio movement in order to grasp, for instance, a food pellet (Whishaw & Gorny, 1996; Whishaw, et al., 1998).    It is evident that distinguishing behavioural changes due to sprouting or regeneration of the injured RST will be onerous in an injury of the dorsolateral funiculus.  As previously noted, RN ablations alone may not affect success in pellet reaching tasks, however, lesions to the dorsolateral funiculus do (Muir, et al., 2007).  Moreover, the use of additional behavioural tests can produce results that are hard to analyze. For example, the results of behavioural metrics such as the rotating bar task may be difficult to interpret as lesions of the dorsolateral funiculus eventually conclude in similar to pre injury performance in the behavioural task (Kennedy & Humphrey, 1987).   In a further example, T8 RST lesioned rats showed a dramatic improvement in the rope test compared to dorsally hemisected rats that did not improve or CST lesioned rats which remained constant in their performance (Hendriks et al., 2006).   In my experiments I chose the vertical exploration test to quantify any behavioural improvements or deficits in relation to either regenerative or degenerative changes in the RST.  The vertical exploration test has been shown to be a sensitive metric in chronic limb use asymmetries (Liu, et al., 1999) and is considered to be an index of motor system integrity when used as a measure of asymmetry in forelimb cylinder wall contact (Gharbawie, et al., 2004).  It has been widely used as instrument to measure forelimb function (Biernaskie & Corbett, 2001; Bland, Pillai, Aronowski, Grotta, & Schallert, 2001; Bland et al., 2000; Choi-Lundberg, et al., 1998; DeBow, Davies, Clarke, & Colbourne, 2003; Jin, et al., 2002; Jones & Schallert, 1994; Karhunen, Virtanen, Schallert, Sivenius, & Jolkkonen, 2003; Liu, et al., 1999; MacLellan, et al., 2002; Murray, et al., 2002; Nikulina, et al., 2004; Schallert, Kozlowski, Humm, & Cocke, 1997; Shumsky et al., 2003; Soblosky, Song, & Dinh, 2001; Tillerson, et al., 2002; Tillerson, et al., 2001; Vergara-Aragon, et al., 2003; Voorhies & Jones, 2002) and has also been used to measure forelimb behavioural changes involving injury to the dorsolateral funiculus containing the RST (Bretzner, Liu, Currie, Roskams, & Tetzlaff, 2008; Bretzner et al., 2010; Cao et al., 2008; Liu, et al., 1999; Wu et al., 2009).  The vertical exploration test exploits the rat?s natural propensity to 117  explore featureless environments therefore it confers several advantages over other behavioural paradigms.  For instance, other behavioural regimes may require aversive motivational tactics such as food or water deprivation which may introduce significant confounds. Rats that are totally deprived of water or are completely fasted do not produce conspicuous behavioural characteristics (Schallert, Hernandez, & Barth, 1989; Schallert, Pendergrass, & Farrar, 1982), or induce experiential plasticity through repeated testing thereby altering the outcome (Jones, Hawrylak, Klintsova, & Greenough, 1998; Will & Kelche, 1992), or allow the animal to create compensatory strategies thereby attaining successful outcomes at the expense of the intended behavioural measurement of the afflicted system in question (Jones, Chu, Grande, & Gregory, 1999; Jones & Schallert, 1994; McKenna & Whishaw, 1999; Schallert & Jones, 1993; Whishaw, Pellis, Gorny, & Pellis, 1991; Whishaw, Woodward, Miklyaeva, & Pellis, 1997).  A further advantage of using the vertical exploration test is that its inter-rater reliability score (r>0.95) is high (Schallert, et al., 2000).  Despite the vertical exploration test?s wide spread use, it has met with some criticism. Stackhouse et al. (2008) reported both a recovery and a lack of forelimb deficit within two weeks after a dorsolateral funiculus aspiration (C3/4) in the rat as indicated by the results of the vertical exploration test.  The behavioural results as indicated by the scores of the vertical exploration test in my experiments did not reveal any recovery back to baseline levels in any of the four groups tested.       It is clear that distinguishing behavioural improvements involving RST recovery in models involving dorsolateral funiculus injuries will be challenging. Future studies evaluating this paradigm may benefit from additional novel assessment procedures to and beyond the currently used battery of behavioural measurement techniques.  5.7 Significance of db-cAMP as a treatment for spinal cord injury  5.7.1 Enhancement of RST sprouting as a potential therapeutic goal  As a potential regenerative treatment, the results of this study are very encouraging. To begin with, the application of db-cAMP to the RN produced a remarkable increase in the overall density of RST axon/fibre sprouting to the gray matter in comparison to the control groups.  While the caudal injured end of the RST neither regenerated nor were any tracts visible caudal to 118  the lesion cavity; prominent visibly labelled fibres appeared proximal to the rostral side of the lesion while projecting to the gray matter.  This is significant because firstly, regeneration, and to some degree, sprouting, are rather limited in the adult CNS spinal cord (Kuang & Kalil, 1990; Prendergast & Misantone, 1980), and secondly, an increase in sprouting by the RST may be indicative of functional reorganization or of that of a compensatory sprouting response which perhaps could result in partial behavioural recovery (Liu, et al., 1999; Murray, et al., 2002; Raineteau, et al., 2002).   The regenerative capacity of the rubrospinal system is one of the lowest amongst different CNS neuronal populations (Blesch & Tuszynski, 2009; Schiwy, Brazda, & Muller, 2009). Even though the overall regenerative capability of the RST is poor after axonal damage in the adult mammal, its initial response to injury (cervical) is the upregulation of regeneration associated genes, such as GAP-43 and T?1 (Tetzlaff, et al., 1991; Tetzlaff, et al., 1994), and has been shown to sprout albeit briefly (Prendergast & Misantone, 1980; Tetzlaff, et al., 1991).  Either increases in, maintenance of or increases/changes in sprouting or plasticity of the RST are perhaps important objectives for potential therapeutic treatments for spinal cord injury. Encouragingly, it has been reported that increases in sprouting in the RST have been correlated with improved behavioural functional recovery (Cao, et al., 2008; Liu, et al., 1999; Murray, et al., 2002).  Moreover, the sprouting response of the damaged mammalian RST can be further augmented by specific treatment interventions. For example, application of IN-1 (Raineteau, et al., 2002), NEP1-40 (Cao, et al., 2008) or BDNF (Murray, et al., 2002) to the RST has been shown to increase regeneration or sprouting, while the application of BDNF to the RN furthered regeneration of the RST into peripheral nerve grafts (Kobayashi, et al., 1997) even when applied one year later (Kwon, et al., 2002).  Additionally, it has been demonstrated that in some cases sprouting of RST is not arbitrary, but purposefully directed towards specific targets (Raineteau, et al., 2002).  Caution, however, still needs to be observed when applying certain treatment regimens to the RN and/or RST in order to enhance the regenerative response as this may result in a maladaptive effect such as a decreased sensory threshold (Bretzner, et al., 2008).  Fortunately, certain strategies have shown that it possible to increase the sprouting response of the RST without apparent deleterious effects.  For example, it has been reported that application 119  of BDNF to the injured RST increased the sprouting response while decreasing hypersensitivity (Shumsky, et al., 2003).    ?True? regeneration or axonal regeneration after injury that follows its previous trajectory and re-establishes its original connection remains a formidable task.  Sprouting, while also a complex issue that is also not completely understood, currently reveals propitious regenerative potential. Indeed, others have demonstrated that sprouting from other CNS populations, such as the CST, have resulted in improved behavioural recovery (Bareyre et al., 2004; Fouad, Pedersen, Schwab, & Br?samle, 2001; Grill, Murai, Blesch, Gage, & Tuszynski, 1997; Thallmair et al., 1998). Given that it has been reported that enhanced sprouting from the RST, CST and other systems have resulted improved behavioural recovery; augmented sustained sprouting may be a viable potential treatment goal.  Optimistically, it has been made known that as little as either 8% (Kakulas, 2004) or 10-15% (Dietz, 2006) of the spared axons in either humans or rodents respectively, for example, is all that is required for functional improvement. Increased plasticity by sprouting to or from these remaining pathways could serve to advance functional recovery. Therefore increased sprouting of the RST by the application of db-cAMP as a treatment represents a positive step in the regimen of trying to restore motor function after spinal cord injury.   5.7.2  Cell body treatment of db-cAMP to the RN via canula is currently not a viable treatment paradigm  Delivery of db-cAMP via mini osmotic-pump with a canula directed towards the RN currently represents a proof of principle treatment.  The sustained use of stationary canulae inserted through the brain causes severe irreparable damage, parenchymal destruction and inflammation (Kwon et al., 2007).  Although it is imperative that increased tissue damage be limited as much as possible when implementing a therapy or drug treatment, creating a safe focussed delivery treatment will need to be of consideration when designing future treatment strategies.  The canula technique conferred the advantage of yielding a known concentration of a drug solution within reasonable proximity of the target area, in this case, the RN.  Unfortunately, the use of alternative procedures such as systemic delivery methods of increasing cAMP levels via analogues of cAMP,  or PDEs or AC activators have several disadvantages.  Therapeutic or 120  efficacious dosage will be difficult to determine due to the reduction of target specificity.  Concomitantly, this raises concerns about the drug/compound reaching the area of interest with sufficient potency in order to produce the desired effect. Additionally, systemic application of compounds that invoke second messenger systems may produce adverse effects in other cellular systems (i.e. non-neuronal cells etc.) in addition to the intended target systems.  Finally, the biological fate of a systemically administered compound invoking ubiquitous second messenger systems may be difficult to monitor.  Presented with these challenges, upcoming therapies will need to have increased specificity and safety.  5.8 Future experimental directions  5.8.1 Future use of elevating cAMP will require enhanced specificity either in part or in whole  As previously noted, the regenerative response has coincided with elevations in cAMP in the injured axon.  Unfortunately, treatments that increase cAMP levels also create an experimental conundrum with advantages and disadvantages.  On the one hand, raised cAMP levels in the injured axon have previously been shown to positively affect a multitude of areas in the cell; many of which have contributed to the enhancement of the sprouting or regenerative response (see section 1.10 for details).  However, it is probably due in part to this blanket approach that the net regenerative or sprouting effect of treated injured axons occurs.  Thus a major advantage of manipulating cAMP levels or using cAMP analogues as a cAMP substitute in axonal injury is that it affects many systems.  OD (Byus, et al., 1976; Byus & Russell, 1975), CREB (Gao, et al., 2004), RhoA (Dong, et al., 1998; Lang, et al., 1996), Arg-1 (Cai, et al., 2002), and Gi (Cai, et al., 1999), for example, are some of the eventual targets of increased cAMP (native or artificially with the use of cAMP analogues) demonstrated to either augment a regenerative response and/or overcome inhibitors to regeneration.  On the other hand, the paradoxical nature of augmenting cAMP levels belies specificity.  As many molecules are either controlled or affected by cAMP directly or indirectly, some of these molecular objectives may not contribute to an enhanced regenerative outcome; quite possibly cause an antagonistic response.  Herein lay the challenges. Upcoming investigations will need to: determine what targets of cAMP are responsible for 121  supporting the regenerative response, which targets of cAMP negatively impact the response, genes and gene products involved, and where in the cell this occurs.  While it is true that cAMP is a ubiquitous second messenger, its native actions are tightly controlled temporally and spatially within in a cell.  Furthermore, different effectors may produce similar cellular increases in levels of cAMP in the same cells but with completely dissimilar outcomes (Brunton, Hayes, & Mayer, 1981; Buxton & Brunton, 1983; Hayes, Brunton, & Mayer, 1980).  Cells achieve this specificity by creating spatial and temporal intracellular cAMP microdomains with particular AKAPs (Michel & Scott, 2002), PKAs (Skalhegg & Tasken, 2000; Tasken, et al., 1997), EPACs (Kawasaki, et al., 1998), ACs (Sunahara, et al., 1996; Sunahara & Taussig, 2002; Tresguerres, et al., 2011),  and PDEs (Kleppisch, 2009) etc..  With this knowledge at least two avenues of subsequent investigations into the involvement of cAMP in generating and/or maintaining a regenerative response can be performed.  An initial area of research could investigate which molecular targets are affected by elevated cAMP levels and how and where in the cells those increased concentration changes occur.  Different cellular populations have different isoforms and combinations of PDEs, EPACs, AKAPs, PKAs and ACs.  The molecular prevalence of the defined combinations and locations of these molecules in a regenerating neuron will be helpful in devising a therapeutic strategy.  For instance, it has been shown, that in a peripheral nerve injury ACs are transported to regenerating nerve tips and proximal to the injury site suggesting that cAMP not only participates in the overall regenerative response but does so by forming micro regional concentrations of activity (Carlsen, 1982a, 1982b; Carlsen & Anderson, 1982).  Knowing where the cAMP microdomains appear may provide a more precise understanding of the mechanisms involved.    A subsequent part of the investigation could address the efficacy of a pseudo specific cAMP response. Rather than focusing a regime directly on all the specific cAMP molecular participants in a damaged axon; targeting a generalized subset of molecules involved in the cAMP cascade may be sufficient in eliciting or maintaining a regenerative response with minimal deleterious effects.  An example of a target for intervention may be tissue specific PDEs.  Specific and 122  generalized PDE inhibitors have existed for decades (Nicholson, Challiss, & Shahid, 1991) and have been used to treat different pathological conditions with varying degrees of success.  Rolipram, a PDE4 inhibitor, has successfully been used as a treatment modality in injured CNS systems in-vivo in animals and also in-vitro in cell cultures under experimental conditions (Block, et al., 2001; Block, Tondar, Schmidt, & Schwarz, 1997; Hannila & Filbin, 2008; Kato, et al., 1995; Koopmans et al., 2009; Nikulina, et al., 2004).  Rolipram, however, when therapeutically administered systemically invokes extreme nausea and vomiting (Dyke & Montana, 1999; Robichaud, Savoie, Stamatiou, Tattersall, & Chan, 2001) as a side effect thereby requiring dosage adjustments below efficacious levels.  Fortunately, novel PDE4 inhibitors with reduced side effects (Aoki et al., 2001; Dal Piaz & Giovannoni, 2000; Dastidar et al., 2009), are constantly being developed as an alternative to rolipram. These new variants have yet to be examined in spinal cord injury models in animals. It should be noted as well that it is possible to use a PDE inhibitor as a treatment modality with reduced secondary effects. For instance, sildenafil citrate, a potent selective cGMP PDE5 inhibitor (Boolell et al., 1996) has successfully been used for on demand erectile dysfunction with a relatively good safety profile (Samarzija et al., 2009).  While it is true that specific PDE inhibitors, such as the PDE5 inhibitors in this example, also affect other cell populations or systems (Konstantinos & Petros, 2009), the effects may not be detrimental, but can be beneficial for other systems as well (Konstantinos & Petros, 2009; Vargas-Origel et al., 2009; Vlachopoulos, Terentes-Printzios, Ioakeimidis, Rokkas, & Stefanadis, 2009).    In short, a more complete understanding of both the molecular players and their responses of induced increased cAMP levels will aid in devising a more targeted therapy to augment or maintain a regenerative response in axonal injury.  5.8.2 Combinatorial cAMP treatments may be more efficacious in evoking a regenerative response  As noted above, achievement of maintaining a regenerative response in the CNS after axonal injury using altered cAMP levels may require targeting several of the second messenger members. Cells contain variants of PDEs and PKAs etc. and altering a single isoform or using a cAMP analogue that preferentially targets certain sites may not be sufficient to elicit or maintain 123  the desired regenerative response.  Using more than one approach to change cAMP levels may be more efficacious.  Indeed, several studies have shown benefits of using PDE inhibitors with either cAMP stimulating drugs or cell specific cAMP analogues in comparison with either single method alone.  For instance, IBMX and forskolin increased survival of spinal motor neurons in culture more than either method of increasing cAMP alone (Hanson, et al., 1998).  Also, neither forskolin nor IBMX alone promoted neurite outgrowth of late embryonic rat retinal neurons plated on laminin-1 (LN-1) while the combination of both did (Ivins, et al., 2004). Moreover, it has been shown that the use of structurally dissimilar cAMP analogues in certain circumstances act synergistically to create a much more potent effect than the use of either structural group alone (Beebe, et al., 1984).  Future experimentation with different cAMP analogues and/or cAMP inducers or maintainers in an animal spinal cord injury model may produce an enhanced regenerative response over single methods of increasing cAMP.    5.8.3 Treatment of additional neuronal tracts and/or a much more sensitive behavioural technique is needed in order to determine functional benefit in DLF injuries  Targeting the RN as a treatment choice for high cervical RST axonal injury has the advantage of being anatomically distinct which allows for visible differentiation from other tracts. Hence morphological changes can be observed over time. One of the difficulties in choosing to treat this tract, however, is determining which behavioural metric is sensitive enough to measure any improvements or degradations in functional outcome following treatment.  As there is controversy over which behavioural measurement technique accurately reports functional differences after DLF injury involving the RST (Muir, et al., 2007; Muir & Whishaw, 2000; Schrimsher & Reier, 1993; Stackhouse, et al., 2008; Whishaw & Gorny, 1996; Whishaw, et al., 1998; Whishaw, et al., 1990), upcoming experiments examining functional distinctive differences of treatments to injuries of the DLF will need to refine or add more behavioural tests, and/or treat the other tracts as well. Unfortunately, damage to the DLF in the rat not only affects the RST, but a portion of the CST and ascending systems as well (Br?samle & Schwab, 1997; Casale, et al., 1988; Goodman & Jarrard, 1966; Joosten, et al., 1992; Kanagal & Muir, 2008, 2009; Liang, et al., 1991; Muir, et al., 2007; Schreyer & Jones, 1982; Schrimsher & Reier, 1993; Weidner, et al., 2001). In this experiment, I found no behavioural functional improvement as indicated by the results of the vertical exploration test despite the significant increase in the 124  density of labelled RST fibres of the treated group.  Interestingly, when Cao and colleagues (2008) administered a Nogo-66 receptor antagonist peptide via intrathecal catheter to a C4 lateral funiculotomy, they too revealed enhanced RST density in the treated group as well as a significant behavioural improvement as indicated by the vertical exploration test.  It should be noted that they placed the catheter directly above the injury thereby exposing several different tracts to the Nogo-66 receptor antagonist peptide.  It is conceivable that the recorded behavioural improvement is due to the treatment of several tracts and not just the RST.  Recently, our lab performed a series of experiments comparing the local effects of db-cAMP (0.5 ?g/?L/hr) infused near the cell bodies of RST neurons or with rolipram (0.4 ?mol/kg/hr) delivered systemically both with or without transplanted OEC cells to rats that received injuries to the cervical DLF.  Both the db-cAMP and rolipram treated animals regardless of whether they received the transplanted cells had attenuated RST retraction compared with the controls; however, the systemically treated rolipram group in combination with the OEC transplanted cells performed better in the vertical exploration test despite that no RST axons were found caudal to the site of injury (Bretzner, et al., 2010).  It is plausible that the systemic elevation of cAMP has an effect on other descending or ascending tracts, or an enhanced interactive effect between the elevated cAMP levels with transplanted cells and the RST and/or with other systems.     Studies that use systemic treatments to show either axon regeneration or behavioural outcome improvements with regards to a specific axonal tract in the DLF may need to treat and investigate the other tracts independently and/or in combination. Some previous RN ablation only studies have not shown obvious gross motor deficits (Papaioannou, 1971; Whishaw, et al., 1990) while more recent studies have (Muir & Whishaw, 2000; Whishaw & Gorny, 1996).  Moreover, no firm consensus has been established about the functional contribution of the RST in lesions involving either the RST (i.e. injury to the DLF) or both the RST and CST (i.e. injury to the dorsal columns as well) in injuries in rats (Hendriks, et al., 2006; Kanagal & Muir, 2009; Krajacic, Weishaupt, Girgis, Tetzlaff, & Fouad, 2010).  It is clear that a more comprehensive understanding of the functions of the RN along with the RST needs to be determined and will benefit future studies.   125  5.8.4 The molecular inhibitory complexes in the RST need to be further characterized  Studies have been conducted investigating the relation and function of, for example, the p75NTR and NgR complex in axonal inhibition (Wang, Kim, et al., 2002; Wong, et al., 2002).  Moreover, it appears that myelin inhibitory molecules act through NgR that are in close apposition to p75NTR during axonal damage (Wang, Kim, et al., 2002). Yet in our laboratory examinations we found that despite the initial increase of p75 levels in the RST after high cervical injury, the levels declined to low levels over time (Kobayashi, et al., 1997; Tetzlaff, et al., 1994).  What is more, axon regeneration remains inhibited even with the low p75 levels. This suggests that not only are there other inhibitory mechanisms in effect, but ones that may or may not include involvement of NgR.  In cases where p75 levels are low or absent with inhibitory molecules still exerting their effects, it has been shown that NgR also forms another inhibitory tripart complex with another molecule, TROY/TAJ, thereby continuing its restrictive effects (Park, Yiu, Kaneko, Wang, Chang, He, et al., 2005; Shao, et al., 2005).     Based on this information further investigations into the role of NgR can be undertaken.  Firstly, is NgR expressed on the RST in sufficient concentration to participate in regenerative inhibition after injury?  Evidence has been brought forward to suggest it is.  RST fibres crossed the site of injury in NgR -/- mice while none did in NgR +/- ones (Kim, Liu, Park, & Strittmatter, 2004), while peptide receptor antagonists against NgR on the RST in rat resulted in an increased sprouting response with functional improvement (Cao, et al., 2008).  Secondly, does NgR form a different yet similar tripart complex like the one with p75NTR and are the downstream effects sensitive to increases in cAMP concentration?  Answers to these questions may continue to provide treatment regimes that are helpful to improving the regenerative response after spinal cord injury.   126  5.9 Conclusions   Elevations of cyclic adenosine monophosphate (cAMP) have been correlated with axonal regenerative success in the peripheral nervous system (PNS) after injury, the developing mammalian central nervous system (CNS) and preconditioned lesions in the CNS. These experiments investigated the hypotheses that sustained elevation of cAMP using the analogue dibutyryl cyclic adenosine monophosphate (db-cAMP) delivered to the red nucleus (RN) after an injury to the dorsolateral funiculus would facilitate a regenerative response in the rubrospinal system.  Results indicated that db-cAMP improved the regenerative response as there were more labelled fibres proximal to the lesion edge and in the gray matter versus the control groups.  Unexpectedly, pre-treatment with db-cAMP, however, did not produce equal or better results in comparison with the acutely treated group and in neither case were any fibres found caudal to the injury site nor did any treatments decrease the lesion cavity size.  Regardless, the significant increase of rubrospinal tract fibres in the gray matter may be indicative of a sprouting response contributing towards functional reorganization. If so, this is an encouraging step in the quest for improving axonal regeneration. Unfortunately, the vertical exploration test failed to yield any discernible behavioural improvements despite the greater numbers of rubrospinal tract fibres in the db-cAMP treated groups.  Encouragingly, the lack of behavioural improvement may have had more to do with the untreated injured lateral corticospinal and ascending sensory tracts rather than the treated rubrospinal tract.   In sum, altering neuronal cAMP levels may be a helpful addition towards devising a potential regenerative therapy for spinal cord injury.        127  References:  Aglah, C., Gordon, T., & Posse de Chaves, E. I. (2008). cAMP promotes neurite outgrowth and extension through protein kinase A but independently of Erk activation in cultured rat motoneurons. Neuropharmacology, 55(1), 8-17. doi: 10.1016/j.neuropharm.2008.04.005 Ahmed, Z., Berry, M., & Logan, A. (2009). ROCK inhibition promotes adult retinal ganglion cell neurite outgrowth only in the presence of growth promoting factors. Mol Cell Neurosci, 42(2), 128-133. doi: 10.1016/j.mcn.2009.06.005 Ahmed, Z., Jacques, S. J., Berry, M., & Logan, A. (2009). Epidermal growth factor receptor inhibitors promote CNS axon growth through off-target effects on glia. Neurobiol Dis, 36(1), 142-150. doi: S0969-9961(09)00176-4 [pii] 10.1016/j.nbd.2009.07.016 [doi] Aizawa, H., Wakatsuki, S., Ishii, A., Moriyama, K., Sasaki, Y., Ohashi, K., Sekine-Aizawa, Y., Sehara-Fujisawa, A., Mizuno, K., Goshima, Y., & Yahara, I. (2001). Phosphorylation of cofilin by LIM-kinase is necessary for semaphorin 3A-induced growth cone collapse. Nat Neurosci, 4(4), 367-373. doi: 10.1038/86011 86011 [pii] Alasbahi, R. H., & Melzig, M. F. (2012). Forskolin and derivatives as tools for studying the role of cAMP. Pharmazie, 67(1), 5-13.  Amano, M., Fukata, Y., & Kaibuchi, K. (2000). Regulation and Functions of Rho-Associated Kinase. Experimental Cell Research, 261(1), 44-51. doi: 10.1006/excr.2000.5046 Amano, M., Ito, M., Kimura, K., Fukata, Y., Chihara, K., Nakano, T., Matsuura, Y., & Kaibuchi, K. (1996). Phosphorylation and Activation of Myosin by Rho-associated Kinase (Rho-kinase). J. Biol. Chem., 271(34), 20246-20249. doi: 10.1074/jbc.271.34.20246 Anderson, K. D., Gunawan, A., & Steward, O. (2007). Spinal pathways involved in the control of forelimb motor function in rats. Exp Neurol, 206(2), 318-331. doi: S0014-4886(07)00205-1 [pii] 10.1016/j.expneurol.2007.05.024 [doi] Angaut, P., Batini, C., Billard, J. M., & Daniel, H. (1986). The cerebellorubral projection in the rat: Retrograde anatomical study. Neuroscience Letters, 68(1), 63-68. doi: 10.1016/0304-3940(86)90230-2 Antal, M., Sholomenko, G. N., Moschovakis, A. K., Storm-Mathisen, J., Heizmann, C. W., & Hunziker, W. (1992). The termination pattern and postsynaptic targets of rubrospinal fibers in the rat spinal cord: a light and electron microscopic study. J Comp Neurol, 325(1), 22-37. doi: 10.1002/cne.903250103 Antoni, F. A., Palkovits, M., Simpson, J., Smith, S. M., Leitch, A. L., Rosie, R., Fink, G., & Paterson, J. M. (1998). Ca2+/calcineurin-inhibited adenylyl cyclase, highly abundant in forebrain regions, is important for learning and memory. J Neurosci, 18(23), 9650-9661.  128  Aoki, M., Fukunaga, M., Sugimoto, T., Hirano, Y., Kobayashi, M., Honda, K., & Yamada, T. (2001). Studies on mechanisms of low emetogenicity of YM976, a novel phosphodiesterase type 4 inhibitor. J Pharmacol Exp Ther, 298(3), 1142-1149.  Arber, S., Barbayannis, F. A., Hanser, H., Schneider, C., Stanyon, C. A., Bernard, O., & Caroni, P. (1998). Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature, 393(6687), 805-809. doi: 10.1038/31729 Arntz, C., Kanje, M., & Lundborg, G. (1989). Regeneration of the rat sciatic nerve after different conditioning lesions: effects of the conditioning interval. Microsurgery, 10(2), 118-121.  Arshavsky, Y. I., Orlovsky, G. N., & Perret, C. (1988). Activity of rubrospinal neurons during locomotion and scratching in the cat. Behav Brain Res, 28(1-2), 193-199. doi: 0166-4328(88)90096-4 [pii] Aurandt, J., Vikis, H. G., Gutkind, J. S., Ahn, N., & Guan, K. L. (2002). The semaphorin receptor plexin-B1 signals through a direct interaction with the Rho-specific nucleotide exchange factor, LARG. Proc Natl Acad Sci U S A, 99(19), 12085-12090. doi: 142433199 [pii]10.1073/pnas.142433199 [doi] Ausim Azizi, S. (2007). . . . And the olive said to the cerebellum: organization and functional significance of the olivo-cerebellar system. Neuroscientist, 13(6), 616-625. doi: 10.1177/1073858407299286 Baillie, G. S. (2009). Compartmentalized signalling: spatial regulation of cAMP by the action of compartmentalized phosphodiesterases. FEBS J, 276(7), 1790-1799. doi: EJB6926 [pii] 10.1111/j.1742-4658.2009.06926.x [doi] Baptiste, D. C., Fehlings, M. G., John, T. W., & Andrew, I. R. M. (2007). Update on the treatment of spinal cord injury Progress in Brain Research (Vol. Volume 161, pp. 217-233): Elsevier. Bareyre, F. M., Kerschensteiner, M., Raineteau, O., Mettenleiter, T. C., Weinmann, O., & Schwab, M. E. (2004). The injured spinal cord spontaneously forms a new intraspinal circuit in adult rats. Nat Neurosci, 7(3), 269-277. doi: 10.1038/nn1195 Barron, K. D., Banerjee, M., Dentinger, M. P., Scheibly, M. E., & Mankes, R. (1989). Cytological and cytochemical (RNA) studies on rubral neurons after unilateral rubrospinal tractotomy: the impact of GM1 ganglioside administration. J Neurosci Res, 22(3), 331-337. doi: 10.1002/jnr.490220313 Beavo, J. A., Conti, M., & Heaslip, R. J. (1994). Multiple cyclic nucleotide phosphodiesterases. Mol Pharmacol, 46(3), 399-405.  Beebe, S., Holloway, R., Rannels, S., & Corbin, J. (1984). Two classes of cAMP analogs which are selective for the two different cAMP-binding sites of type II protein kinase demonstrate synergism when added together to intact adipocytes. J. Biol. Chem., 259(6), 3539-3547.  129  Benowitz, L. I., & Yin, Y. (2010). Optic nerve regeneration. Arch Ophthalmol, 128(8), 1059-1064. doi: 128/8/1059 [pii] 10.1001/archophthalmol.2010.152 [doi] Benson, M. D., Romero, M. I., Lush, M. E., Lu, Q. R., Henkemeyer, M., & Parada, L. F. (2005). Ephrin-B3 is a myelin-based inhibitor of neurite outgrowth. Proc Natl Acad Sci U S A, 102(30), 10694-10699. doi: 0504021102 [pii] 10.1073/pnas.0504021102 [doi] Biernaskie, J., & Corbett, D. (2001). Enriched rehabilitative training promotes improved forelimb motor function and enhanced dendritic growth after focal ischemic injury. J Neurosci, 21(14), 5272-5280. doi: 21/14/5272 [pii] Bisby, M. A. (1978). Fast axonal transport of labeled protein in sensory axons during regeneration. Exp Neurol, 61(2), 281-300. doi: 0014-4886(78)90247-9 [pii] Bisby, M. A., & Pollock, B. (1983). Increased regeneration rate in peripheral nerve axons following double lesions: enhancement of the conditioning lesion phenomenon. J Neurobiol, 14(6), 467-472. doi: 10.1002/neu.480140607 Black, M. M., & Lasek, R. J. (1979). Slowing of the rate of axonal regeneration during growth and maturation. Exp Neurol, 63(1), 108-119.  Black, M. M., & Lasek, R. J. (1980). Slow components of axonal transport: two cytoskeletal networks. J Cell Biol, 86(2), 616-623.  Bland, S. T., Pillai, R. N., Aronowski, J., Grotta, J. C., & Schallert, T. (2001). Early overuse and disuse of the affected forelimb after moderately severe intraluminal suture occlusion of the middle cerebral artery in rats. Behav Brain Res, 126(1-2), 33-41. doi: S0166432801002431 [pii] Bland, S. T., Schallert, T., Strong, R., Aronowski, J., Grotta, J. C., & Feeney, D. M. (2000). Early exclusive use of the affected forelimb after moderate transient focal ischemia in rats : functional and anatomic outcome. Stroke, 31(5), 1144-1152.  Blecher, M. (1971). Biological effects and catabolic metabolism of 3', 5'-cyclic nucleotides and derivatives in rat adipose tissue and liver. Metabolism, 20(1), 63-77.  Blecher, M., & Hunt, N. H. (1972). Enzymatic Deacylation of Mono- and Dibutyryl Derivatives of Cyclic Adenosine 3', 5'-Monophosphate by Extracts of Rat Tissues. J. Biol. Chem., 247(23), 7479-7484.  Blesch, A., Lu, P., Tsukada, S., Alto, L. T., Roet, K., Coppola, G., Geschwind, D., & Tuszynski, M. H. (2012). Conditioning lesions before or after spinal cord injury recruit broad genetic mechanisms that sustain axonal regeneration: Superiority to camp-mediated effects. Experimental Neurology, 235(1), 162-173. doi: 10.1016/j.expneurol.2011.12.037 Blesch, A., & Tuszynski, M. H. (2009). Spinal cord injury: plasticity, regeneration and the challenge of translational drug development. Trends in Neurosciences, 32(1), 41-47. doi: 10.1016/j.tins.2008.09.008 130  Blochl, A., & Blochl, R. (2007). A cell-biological model of p75NTR signaling. J Neurochem, 102(2), 289-305. doi: JNC4496 [pii] 10.1111/j.1471-4159.2007.04496.x [doi] Block, F., Schmidt, W., Nolden-Koch, M., & Schwarz, M. (2001). Rolipram reduces excitotoxic neuronal damage. Neuroreport, 12(7), 1507-1511.  Block, F., Tondar, A., Schmidt, W., & Schwarz, M. (1997). Delayed treatment with rolipram protects against neuronal damage following global ischemia in rats. Neuroreport, 8(17), 3829-3832.  Boeshore, K. L., Schreiber, R. C., Vaccariello, S. A., Sachs, H. H., Salazar, R., Lee, J., Ratan, R. R., Leahy, P., & Zigmond, R. E. (2004). Novel changes in gene expression following axotomy of a sympathetic ganglion: a microarray analysis. J Neurobiol, 59(2), 216-235. doi: 10.1002/neu.10308 Boolell, M., Allen, M. J., Ballard, S. A., Gepi-Attee, S., Muirhead, G. J., Naylor, A. M., Osterloh, I. H., & Gingell, C. (1996). Sildenafil: an orally active type 5 cyclic GMP-specific phosphodiesterase inhibitor for the treatment of penile erectile dysfunction. Int J Impot Res, 8(2), 47-52.  Borisoff, J. F., Chan, C. C., Hiebert, G. W., Oschipok, L., Robertson, G. S., Zamboni, R., Steeves, J. D., & Tetzlaff, W. (2003). Suppression of Rho-kinase activity promotes axonal growth on inhibitory CNS substrates. Mol Cell Neurosci, 22(3), 405-416.  Bos, J. L. (2006). Epac proteins: multi-purpose cAMP targets. Trends Biochem Sci, 31(12), 680-686. doi: S0968-0004(06)00292-1 [pii] 10.1016/j.tibs.2006.10.002 [doi] Bos, J. L., de Bruyn, K., Enserink, J., Kuiperij, B., Rangarajan, S., Rehmann, H., Riedl, J., de Rooij, J., van Mansfeld, F., & Zwartkruis, F. (2003). The role of Rap1 in integrin-mediated cell adhesion. Biochem Soc Trans, 31(Pt 1), 83-86. doi: 10.1042/  Breasted, J. H. (1991). The Edwin Smith Surgical Papyrus (Vol. 2). Chicago: Oriental Institue Publications 4.  Chicago: The University of Chicago Press, 1930. Bretzner, F., Liu, J., Currie, E., Roskams, J. A., & Tetzlaff, W. (2008). Undesired effects of a combinatorial treatment for spinal cord injury; transplantation of olfactory ensheathing cells and BDNF infusion to the red nucleus. European Journal of Neuroscience, 28(9), 1795-1807. doi: 10.1111/j.1460-9568.2008.06462.x Bretzner, F., Plemel, J. R., Liu, J., Richter, M., Roskams, A. J., & Tetzlaff, W. (2010). Combination of olfactory ensheathing cells with local versus systemic cAMP treatment after a cervical rubrospinal tract injury. J Neurosci Res, 88(13), 2833-2846. doi: 10.1002/jnr.22440 Br?samle, C., & Schwab, M. E. (1997). Cells of origin, course, and termination patterns of the ventral, uncrossed component of the mature rat corticospinal tract. The Journal of Comparative Neurology, 386(2), 293-303. doi: 10.1002/(SICI)1096-9861(19970922)386 131  Brown, L. T. (1974). Rubrospinal projections in the rat. J Comp Neurol, 154(2), 169-187. doi: 10.1002/cne.901540205 Brunton, L. L., Hayes, J. S., & Mayer, S. E. (1981). Functional compartmentation of cyclic AMP and protein kinase in heart. Adv Cyclic Nucleotide Res, 14, 391-397.  Burman, K., Darian-Smith, C., & Darian-Smith, I. (2000). Geometry of rubrospinal, rubroolivary, and local circuit neurons in the macaque red nucleus. J Comp Neurol, 423(2), 197-219.  Butt, E., Beltman, J., Becker, D. E., Jensen, G. S., Rybalkin, S. D., Jastorff, B., & Beavo, J. A. (1995). Characterization of cyclic nucleotide phosphodiesterases with cyclic AMP analogs: topology of the catalytic sites and comparison with other cyclic AMP-binding proteins. Mol Pharmacol, 47(2), 340-347.  Buxton, I. L., & Brunton, L. L. (1983). Compartments of cyclic AMP and protein kinase in mammalian cardiomyocytes. J Biol Chem, 258(17), 10233-10239.  Byus, C. V., Costa, M., Sipes, I. G., Brodie, B. B., & Russell, D. H. (1976). Activation of 3':5'-cyclic AMP-dependent protein kinase and induction of ornithine decarboxylase as early events in induction of mixed-function oxygenases. Proc Natl Acad Sci U S A, 73(4), 1241-1245.  Byus, C. V., & Russell, D. H. (1975). Ornithine decarboxylase activity: control by cyclic nucleotides. Science, 187(4177), 650-652.  Byus, C. V., & Russell, D. H. (1976). Possible regulation of ornithine decarboxylase activity in the adrenal medulla of the rat by a cAMP-dependent mechanism. Biochemical Pharmacology, 25(14), 1595-1600.  Cai, D., Deng, K., Mellado, W., Lee, J., Ratan, R. R., & Filbin, M. T. (2002). Arginase I and polyamines act downstream from cyclic AMP in overcoming inhibition of axonal growth MAG and myelin in vitro. Neuron, 35(4), 711-719.  Cai, D., Qiu, J., Cao, Z., McAtee, M., Bregman, B. S., & Filbin, M. T. (2001). Neuronal cyclic AMP controls the developmental loss in ability of axons to regenerate. J Neurosci, 21(13), 4731-4739.  Cai, D., Shen, Y., De Bellard, M., Tang, S., & Filbin, M. T. (1999). Prior exposure to neurotrophins blocks inhibition of axonal regeneration by MAG and myelin via a cAMP-dependent mechanism. Neuron, 22(1), 89-101.  Canedo, A. (1997). Primary motor cortex influences on the descending and ascending systems. Prog Neurobiol, 51(3), 287-335. doi: S0301-0082(96)00058-5 [pii] Cao, Y., Shumsky, J. S., Sabol, M. A., Kushner, R. A., Strittmatter, S., Hamers, F. P. T., Lee, D. H. S., Rabacchi, S. A., & Murray, M. (2008). Nogo-66 Receptor Antagonist Peptide (NEP1-40) Administration Promotes Functional Recovery and Axonal Growth After 132  Lateral Funiculus Injury in the Adult Rat. Neurorehabil Neural Repair, 22(3), 262-278. doi: 10.1177/1545968307308550 Cao, Z., Gao, Y., Bryson, J. B., Hou, J., Chaudhry, N., Siddiq, M., Martinez, J., Spencer, T., Carmel, J., Hart, R. B., & Filbin, M. T. (2006). The cytokine interleukin-6 is sufficient but not necessary to mimic the peripheral conditioning lesion effect on axonal growth. J Neurosci, 26(20), 5565-5573. doi: 26/20/5565 [pii] 10.1523/JNEUROSCI.0815-06.2006 [doi] Carlsen, R. C. (1982a). Axonal transport of adenylate cyclase activity in normal and axotomized frog sciatic nerve. Brain Res, 232(2), 413-424.  Carlsen, R. C. (1982b). Comparison of adenylate cyclase activity in segments of rat sciatic nerve with a condition/test or test lesion. Exp Neurol, 77(2), 254-265.  Carlsen, R. C. (1983). Delayed induction of the cell body response and enhancement of regeneration following a condition/test lesion of frog peripheral nerve at 15 degrees C. Brain Res, 279(1-2), 9-18.  Carlsen, R. C., & Anderson, L. J. (1982). Subcellular distribution of axonally transported adenylate cyclase: effect of nerve constriction and comparison with acetylcholinesterase. J Neurochem, 39(5), 1467-1477.  Caroni, P., & Schwab, M. E. (1988a). Antibody against myelin-associated inhibitor of neurite growth neutralizes nonpermissive substrate properties of CNS white matter. Neuron, 1(1), 85-96.  Caroni, P., & Schwab, M. E. (1988b). Two membrane protein fractions from rat central myelin with inhibitory properties for neurite growth and fibroblast spreading. J Cell Biol, 106(4), 1281-1288.  Casale, E. J., Light, A. R., & Rustioni, A. (1988). Direct projection of the corticospinal tract to the superficial laminae of the spinal cord in the rat. J Comp Neurol, 278(2), 275-286. doi: 10.1002/cne.902780210 Chattopadhyay, M. K., Park, M. H., & Tabor, H. (2008). Hypusine modification for growth is the major function of spermidine in Saccharomyces cerevisiae polyamine auxotrophs grown in limiting spermidine. Proc Natl Acad Sci U S A, 105(18), 6554-6559. doi: 10.1073/pnas.0710970105 Chen, M. S., Huber, A. B., van der Haar, M. E., Frank, M., Schnell, L., Spillmann, A. A., Christ, F., & Schwab, M. E. (2000). Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature, 403(6768), 434-439. doi: 10.1038/35000219 Cheng, B., Christakos, S., & Mattson, M. P. (1994). Tumor necrosis factors protect neurons against metabolic-excitotoxic insults and promote maintenance of calcium homeostasis. Neuron, 12(1), 139-153.  133  Cheng, X., Ji, Z., Tsalkova, T., & Mei, F. (2008). Epac and PKA: a tale of two intracellular cAMP receptors. Acta Biochim Biophys Sin (Shanghai), 40(7), 651-662.  Chin, K. V., Yang, W. L., Ravatn, R., Kita, T., Reitman, E., Vettori, D., Cvijic, M. E., Shin, M., & Iacono, L. (2002). Reinventing the wheel of cyclic AMP: novel mechanisms of cAMP signaling. Ann N Y Acad Sci, 968, 49-64.  Choi-Lundberg, D. L., Lin, Q., Schallert, T., Crippens, D., Davidson, B. L., Chang, Y. N., Chiang, Y. L., Qian, J., Bardwaj, L., & Bohn, M. C. (1998). Behavioral and cellular protection of rat dopaminergic neurons by an adenoviral vector encoding glial cell line-derived neurotrophic factor. Exp Neurol, 154(2), 261-275. doi: S0014-4886(98)96887-X [pii] 10.1006/exnr.1998.6887 [doi] Christensen, A. E., Selheim, F., de Rooij, J., Dremier, S., Schwede, F., Dao, K. K., Martinez, A., Maenhaut, C., Bos, J. L., Genieser, H. G., & Doskeland, S. O. (2003). cAMP analog mapping of Epac1 and cAMP kinase. Discriminating analogs demonstrate that Epac and cAMP kinase act synergistically to promote PC-12 cell neurite extension. J Biol Chem, 278(37), 35394-35402. doi: 10.1074/jbc.M302179200 Christopher and Dana Reeve Foundation. (2012a). Costs of Living with Spinal Cord Injury  Retrieved Mar 1, 2012, 2012, from http://www.christopherreeve.org/site/c.mtKZKgMWKwG/b.5184189/k.5587/Paralysis_Facts__Figures.htm Christopher and Dana Reeve Foundation. (2012b). One Degree of Separation: Paralysis and Spinal Cord Injury in the United States (pp. 28). Short Hills, NJ. Christopher Reeve Foundation. (2009). Paralysis and Its Impact - Paralysis Resource Center  Retrieved February 5, 2009, from http://www.christopherreeve.org/site/c.mtKZKgMWKwG/b.4453145/k.8A22/What_Causes_Paralysis_and_its_Impact.htm Chu, P. J., Saito, H., & Abe, K. (1995). Polyamines promote regeneration of injured axons of cultured rat hippocampal neurons. Brain Res, 673(2), 233-241. doi: 0006-8993(94)01419-I [pii] Coghlan, V. M., Perrino, B. A., Howard, M., Langeberg, L. K., Hicks, J. B., Gallatin, W. M., & Scott, J. D. (1995). Association of Protein Kinase A and Protein Phosphatase 2B with a Common Anchoring Protein. Science, 267(5194), 108-111.  Conti, M., & Beavo, J. (2007). Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. Annu Rev Biochem, 76, 481-511. doi: 10.1146/annurev.biochem.76.060305.150444 Cooper, D. M. F. (2003). Regulation and organization of adenylyl cyclases and cAMP. Biochem. J., 375(3), 517-529. doi: 10.1042/bj20031061 134  Corbin, J. D., Rannels, S. R., Flockhart, D. A., Robinson-Steiner, A. M., Tigani, M. C., Doskeland, S. O., Suva, R. H., Suva, R., & Miller, J. P. (1982). Effect of cyclic nucleotide analogs on intrachain site I of protein kinase isozymes. Eur J Biochem, 125(2), 259-266.  Cragg, B. G. (1970). What is the signal for chromatolysis? Brain Research, 23(1), 1-21.  Dal Piaz, V., & Giovannoni, M. P. (2000). Phosphodiesterase 4 inhibitors, structurally unrelated to rolipram, as promising agents for the treatment of asthma and other pathologies. Eur J Med Chem, 35(5), 463-480. doi: S0223-5234(00)00179-3 [pii] Daniel, H., Angaut, P., Batini, C., & Billard, J. M. (1988). Topographic organization of the interpositorubral connections in the rat. A WGA-HRP study. Behav Brain Res, 28(1-2), 69-70. doi: 0166-4328(88)90078-2 [pii] Daniel, P. B., Walker, W. H., & Habener, J. F. (1998). Cyclic AMP signaling and gene regulation. Annu Rev Nutr, 18, 353-383. doi: 10.1146/annurev.nutr.18.1.353 Dastidar, S. G., Ray, A., Shirumalla, R., Rajagopal, D., Chaudhary, S., Nanda, K., Sharma, P., Seth, M. K., Balachandran, S., Gupta, N., & Palle, V. (2009). Pharmacology of a novel, orally active PDE4 inhibitor. Pharmacology, 83(5), 275-286. doi: 000209608 [pii] 10.1159/000209608 [doi] David, S., & Aguayo, A. J. (1981). Axonal elongation into peripheral nervous system "bridges" after central nervous system injury in adult rats. Science, 214(4523), 931-933.  de Rooij, J., Zwartkruis, F. J., Verheijen, M. H., Cool, R. H., Nijman, S. M., Wittinghofer, A., & Bos, J. L. (1998). Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature, 396(6710), 474-477. doi: 10.1038/24884 de Waegh, S. M., Lee, V. M., & Brady, S. T. (1992). Local modulation of neurofilament phosphorylation, axonal caliber, and slow axonal transport by myelinating Schwann cells. Cell, 68(3), 451-463. doi: 0092-8674(92)90183-D [pii] DeBellard, M. E., Tang, S., Mukhopadhyay, G., Shen, Y. J., & Filbin, M. T. (1996). Myelin-associated glycoprotein inhibits axonal regeneration from a variety of neurons via interaction with a sialoglycoprotein. Mol Cell Neurosci, 7(2), 89-101. doi: 10.1006/mcne.1996.0007 DeBow, S. B., Davies, M. L., Clarke, H. L., & Colbourne, F. (2003). Constraint-induced movement therapy and rehabilitation exercises lessen motor deficits and volume of brain injury after striatal hemorrhagic stroke in rats. Stroke, 34(4), 1021-1026. doi: 01.STR.0000063374.89732.9F [pii]10.1161/01.STR.0000063374.89732.9F [doi] Dechant, G., Rodriguez-Tebar, A., & Barde, Y. A. (1994). Neurotrophin receptors. Prog Neurobiol, 42(2), 347-352. doi: 0301-0082(94)90075-2 [pii] 135  Defer, N., Best-Belpomme, M., & Hanoune, J. (2000). Tissue specificity and physiological relevance of various isoforms of adenylyl cyclase. Am J Physiol Renal Physiol, 279(3), F400-416.  Dietz, V. (2006). Neuronal Plasticity After Spinal Cord Injury: Significance for Present and Future Treatments. J Spinal Cord Med, 29(5), 481-488.  Dodge-Kafka, K. L., Bauman, A., & Kapiloff, M. S. (2008). A-kinase anchoring proteins as the basis for cAMP signaling. Handb Exp Pharmacol(186), 3-14. doi: 10.1007/978-3-540-72843-6_1 Dodge-Kafka, K. L., Soughayer, J., Pare, G. C., Carlisle Michel, J. J., Langeberg, L. K., Kapiloff, M. S., & Scott, J. D. (2005). The protein kinase A anchoring protein mAKAP coordinates two integrated cAMP effector pathways. Nature, 437(7058), 574-578. doi: 10.1038/nature03966 Dodge, K. L., Khouangsathiene, S., Kapiloff, M. S., Mouton, R., Hill, E. V., Houslay, M. D., Langeberg, L. K., & Scott, J. D. (2001). mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module. EMBO J, 20(8), 1921-1930. doi: 10.1093/emboj/20.8.1921 Dolleman-Van der Weel, M. J., Wouterlood, F. G., & Witter, M. P. (1994). Multiple anterograde tracing, combining Phaseolus vulgaris leucoagglutinin with rhodamine- and biotin-conjugated dextran amine. J Neurosci Methods, 51(1), 9-21. doi: 0165-0270(94)90021-3 [pii] Domeniconi, M., Zampieri, N., Spencer, T., Hilaire, M., Mellado, W., Chao, M. V., & Filbin, M. T. (2005). MAG induces regulated intramembrane proteolysis of the p75 neurotrophin receptor to inhibit neurite outgrowth. Neuron, 46(6), 849-855. doi: 10.1016/j.neuron.2005.05.029 Dong, J.-M., Leung, T., Manser, E., & Lim, L. (1998). cAMP-induced Morphological Changes Are Counteracted by the Activated RhoA Small GTPase and the Rho Kinase ROKalpha. J. Biol. Chem., 273(35), 22554-22562. doi: 10.1074/jbc.273.35.22554 Dornay, M., Gilad, V. H., Shiler, I., & Gilad, G. M. (1986). Early polyamine treatment accelerates regeneration of rat sympathetic neurons. Exp Neurol, 92(3), 665-674.  Dustin, A. P. (1910). Le R?le de Tropismes et de l'Odog?n?se dans la R?g?n?ration du Syst?me Nerveux. Arch. de Biol, 25, 269-275.  Dyke, H. J., & Montana, J. G. (1999). The therapeutic potential of PDE4 inhibitors. Expert Opin Investig Drugs, 8(9), 1301-1325. doi: 10.1517/13543784.8.9.1301 Edbladh, M., Edstrom, A., & Persson, L. (1990). The role of ornithine decarboxylase and polyamines in regeneration of the frog sciatic nerve. Exp Neurol, 107(1), 63-68. doi: 0014-4886(90)90063-X [pii] 136  Edstr?m, A. (1977). Rapid axonal transport in vitro. Effects of derivatives of cyclic AMP and other agents acting on the cyclic AMP system. Journal of Neurobiology, 8(4), 371-380. doi: 10.1002/neu.480080408 Endres, S., Fulle, H. J., Sinha, B., Stoll, D., Dinarello, C. A., Gerzer, R., & Weber, P. C. (1991). Cyclic nucleotides differentially regulate the synthesis of tumour necrosis factor-alpha and interleukin-1 beta by human mononuclear cells. Immunology, 72(1), 56-60.  Farry, A., & Baxter, D. (2010). The Incidence and Prevalence of Spinal Cord Injury in Canada (pp. 57). Vancouver, BC. Fawcett, J. W., & Asher, R. A. (1999). The glial scar and central nervous system repair. Brain Res Bull, 49(6), 377-391.  Feng, P., Ge, L.-d., Akyhani, N., & Liau, G. (1996). Sodium butyrate is a potent modulator of smooth muscle cell proliferation and gene expression. Cell Proliferation, 29(5), 231-241.  Ferguson, I. A., Xian, C., Barati, E., & Rush, R. A. (2001). Comparison of wheat germ agglutinin-horseradish peroxidase and biotinylated dextran for anterograde tracing of corticospinal tract following spinal cord injury. J Neurosci Methods, 109(2), 81-89. doi: S0165027001003806 [pii] Fernandes, K. J. L., Fan, D.-P., Tsui, B. J., Cassar, S. L., & Tetzlaff, W. (1999). Influence of the axotomy to cell body distance in rat rubrospinal and spinal motoneurons: Differential regulation of GAP-43, tubulins, and neurofilament-M. The Journal of Comparative Neurology, 414(4), 495-510.  Finkbeiner, S., Tavazoie, S. F., Maloratsky, A., Jacobs, K. M., Harris, K. M., & Greenberg, M. E. (1997). CREB: a major mediator of neuronal neurotrophin responses. Neuron, 19(5), 1031-1047. doi: S0896-6273(00)80395-5 [pii] Forte, L. R., Bylund, D. B., & Zahler, W. L. (1983). Forskolin does not activate sperm adenylate cyclase. Molecular Pharmacology, 24(1), 42-47.  Fouad, K., Pedersen, V., Schwab, M. E., & Br?samle, C. (2001). Cervical sprouting of corticospinal fibers after thoracic spinal cord injury accompanies shifts in evoked motor responses. Current Biology, 11(22), 1766-1770. doi: 10.1016/S0960-9822(01)00535-8 Fournier, A., Gould, G., Liu, B., & Strittmatter, S. (2002). Truncated soluble Nogo receptor binds Nogo-66 and blocks inhibition of axon growth by myelin. J Neurosci, 22(20), 8876 - 8883.  Fournier, A. E., GrandPre, T., Gould, G., Wang, X., & Strittmatter, S. M. (2002). Nogo and the Nogo-66 receptor. Prog Brain Res, 137, 361-369.  Fournier, A. E., GrandPre, T., & Strittmatter, S. M. (2001). Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration. Nature, 409(6818), 341-346. doi: 10.1038/35053072 137  Fraser, I. D., Tavalin, S. J., Lester, L. B., Langeberg, L. K., Westphal, A. M., Dean, R. A., Marrion, N. V., & Scott, J. D. (1998). A novel lipid-anchored A-kinase Anchoring Protein facilitates cAMP-responsive membrane events. EMBO J, 17(8), 2261-2272. doi: 10.1093/emboj/17.8.2261 Frey, U., Huang, Y. Y., & Kandel, E. R. (1993). Effects of cAMP simulate a late stage of LTP in hippocampal CA1 neurons. Science, 260(5114), 1661-1664.  Friede, R. L. (1972). Control of myelin formation by axon caliber (with a model of the control mechanism). J Comp Neurol, 144(2), 233-252. doi: 10.1002/cne.901440207 Friede, R. L., & Samorajski, T. (1970). Axon caliber related to neurofilaments and microtubules in sciatic nerve fibers of rats and mice. Anat Rec, 167(4), 379-387. doi: 10.1002/ar.1091670402 Fritzsch, B. (1993). Fast axonal diffusion of 3000 molecular weight dextran amines. J Neurosci Methods, 50(1), 95-103. doi: 0165-0270(93)90060-5 [pii] Fuld, S., Borland, G., & Yarwood, S. J. (2005). Elevation of cyclic AMP in Jurkat T-cells provokes distinct transcriptional responses through the protein kinase A (PKA) and exchange protein activated by cyclic AMP (EPAC) pathways. Experimental Cell Research, 309(1), 161-173. doi: 10.1016/j.yexen.2005.05.016 Gainer, H., & Barker, J. L. (1975). Selective modulation and turnover of proteins in identified neurons of Aplysia. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 51(2), 221-227. doi: 10.1016/0305-0491(75)90212-6 Gao, Y., Deng, K., Hou, J., Bryson, J. B., Barco, A., Nikulina, E., Spencer, T., Mellado, W., Kandel, E. R., & Filbin, M. T. (2004). Activated CREB is sufficient to overcome inhibitors in myelin and promote spinal axon regeneration in vivo. Neuron, 44(4), 609-621. doi: 10.1016/j.neuron.2004.10.030 Gao, Y., Nikulina, E., Mellado, W., & Filbin, M. T. (2003). Neurotrophins elevate cAMP to reach a threshold required to overcome inhibition by MAG through extracellular signal-regulated kinase-dependent inhibition of phosphodiesterase. J Neurosci, 23(37), 11770-11777.  Gensel, J. C., Nakamura, S., Guan, Z., van Rooijen, N., Ankeny, D. P., & Popovich, P. G. (2009). Macrophages promote axon regeneration with concurrent neurotoxicity. J Neurosci, 29(12), 3956-3968. doi: 10.1523/jneurosci.3992-08.2009 Gershenbaum, M. R., & Roisen, F. J. (1980). The effects of dibutyryl cyclic adenosine monophosphate on the degeneration and regeneration of crush-lesioned rat sciatic nerves. Neuroscience, 5(9), 1565-1580.  Gharbawie, O. A., Whishaw, P. A., & Whishaw, I. Q. (2004). The topography of three-dimensional exploration: a new quantification of vertical and horizontal exploration, 138  postural support, and exploratory bouts in the cylinder test. Behav Brain Res, 151(1-2), 125-135. doi: 10.1016/j.bbr.2003.08.009 Ghoda, L., van Daalen Wetters, T., Macrae, M., Ascherman, D., & Coffino, P. (1989). Prevention of rapid intracellular degradation of ODC by a carboxyl-terminal truncation. Science, 243(4897), 1493-1495.  Gilad, G. M., & Gilad, V. H. (1988). Early polyamine treatment enhances survival of sympathetic neurons after postnatal axonal injury or immunosympathectomy. Brain Res, 466(2), 175-181.  Gilad, V. H., Tetzlaff, W. G., Rabey, J. M., & Gilad, G. M. (1996). Accelerated recovery following polyamines and aminoguanidine treatment after facial nerve injury in rats. Brain Res, 724(1), 141-144.  Gillardon, F., Bottiger, B., Schmitz, B., Zimmermann, M., & Hossmann, K. A. (1997). Activation of CPP-32 protease in hippocampal neurons following ischemia and epilepsy. Brain Res Mol Brain Res, 50(1-2), 16-22.  Glover, J. C., Petursdottir, G., & Jansen, J. K. (1986). Fluorescent dextran-amines used as axonal tracers in the nervous system of the chicken embryo. J Neurosci Methods, 18(3), 243-254.  Goldstein, M. E., Cooper, H. S., Bruce, J., Carden, M. J., Lee, V. M., & Schlaepfer, W. W. (1987). Phosphorylation of neurofilament proteins and chromatolysis following transection of rat sciatic nerve. J Neurosci, 7(5), 1586-1594.  Gong, Y., Cao, P., Yu, H.-j., & Jiang, T. (2008). Crystal structure of the neurotrophin-3 and p75NTR symmetrical complex. Nature, 454(7205), 789-793. doi: 10.1038/nature07089 Gonzalez, G. A., & Montminy, M. R. (1989). Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell, 59(4), 675-680. doi: 0092-8674(89)90013-5 [pii] Goodman, D. C., & Jarrard, L. E. (1966). Corticospinal pathways and their sites of termination in the albino rat. Paper presented at the American Association of Anatomists. Seventy-ninth Session. , University of California School of Medicine, San Francisco, California. Paper retrieved from  Gordon, T., Chan, K. M., Sulaiman, O. A., Udina, E., Amirjani, N., & Brushart, T. M. (2009). Accelerating axon growth to overcome limitations in functional recovery after peripheral nerve injury. Neurosurgery, 65(4 Suppl), A132-144. doi: 00006123-200910001-00022 [pii] 10.1227/01.NEU.0000335650.09473.D3 [doi]  Gordon, T., & Gordon, K. (2010). Nerve regeneration in the peripheral nervous system versus the central nervous system and the relevance to speech and hearing after nerve injuries. J Commun Disord, 43(4), 274-285. doi: S0021-9924(10)00037-7 [pii] 10.1016/j.jcomdis.2010.04.010 [doi] 139  Grafstein, B. (1975). The nerve cell body response to axotomy. Experimental Neurology, 48(3, Part 2), 32-51.  GrandPre, T., Li, S., & Strittmatter, S. M. (2002). Nogo-66 receptor antagonist peptide promotes axonal regeneration. Nature, 417(6888), 547-551. doi: 10.1038/417547a GrandPre, T., Nakamura, F., Vartanian, T., & Strittmatter, S. M. (2000). Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein. Nature, 403(6768), 439-444. doi: 10.1038/35000226 Greengard, P. (2001). The Neurobiology of Slow Synaptic Transmission. Science, 294(5544), 1024-1030. doi: 10.1126/science.294.5544.1024 Greten, T. F., Eigler, A., Sinha, B., Moeller, J., & Endres, S. (1995). The specific type IV phosphodiesterase inhibitor rolipram differentially regulates the proinflammatory mediators TNF-[alpha] and nitric oxide. International Journal of Immunopharmacology, 17(7), 605-610.  Grill, R., Murai, K., Blesch, A., Gage, F. H., & Tuszynski, M. H. (1997). Cellular delivery of neurotrophin-3 promotes corticospinal axonal growth and partial functional recovery after spinal cord injury. J Neurosci, 17(14), 5560-5572.  Gunning, P., Letourneau, P., Landreth, G., & Shooter, E. (1981). The action of nerve growth factor and dibutyryl adenosine cyclic 3':5'- monophosphate on rat pheochromocytoma reveals distinct stages in the mechanisms underlying neurite outgrowth. J. Neurosci., 1(10), 1085-1095.  Haase, G., Pettmann, B., Raoul, C., & Henderson, C. E. (2008). Signaling by death receptors in the nervous system. Curr Opin Neurobiol, 18(3), 284-291. doi: S0959-4388(08)00071-8 [pii] 10.1016/j.conb.2008.07.013 [doi] Habas, C., Guillevin, R., & Abanou, A. (2010). In vivo structural and functional imaging of the human rubral and inferior olivary nuclei: A mini-review. Cerebellum, 9(2), 167-173. doi: 10.1007/s12311-009-0145-1 Han, P. J., Shukla, S., Subramanian, P. S., & Hoffman, P. N. (2004). Cyclic AMP elevates tubulin expression without increasing intrinsic axon growth capacity. Exp Neurol, 189(2), 293-302. doi: 10.1016/j.expneurol.2004.03.010 Hannila, S. S., & Filbin, M. T. (2008). The role of cyclic AMP signaling in promoting axonal regeneration after spinal cord injury. Exp Neurol, 209(2), 321-332. doi: S0014-4886(07)00264-6 [pii] 10.1016/j.expneurol.2007.06.020 [doi] Hansen, M. R., Zha, X. M., Bok, J., & Green, S. H. (2001). Multiple distinct signal pathways, including an autocrine neurotrophic mechanism, contribute to the survival-promoting effect of depolarization on spiral ganglion neurons in vitro. J Neurosci, 21(7), 2256-2267.  140  Hanson, M. G., Jr., Shen, S., Wiemelt, A. P., McMorris, F. A., & Barres, B. A. (1998). Cyclic AMP elevation is sufficient to promote the survival of spinal motor neurons in vitro. J Neurosci, 18(18), 7361-7371.  Harel, R., Iannotti, C. A., Hoh, D., Clark, M., Silver, J., & Steinmetz, M. P. (2012). Oncomodulin affords limited regeneration to injured sensory axons in vitro and in vivo. Experimental Neurology, 233(2), 708-716. doi: 10.1016/j.expneurol.2011.04.017 Hawryluk, G. W., Rowland, J., Kwon, B. K., & Fehlings, M. G. (2008). Protection and repair of the injured spinal cord: a review of completed, ongoing, and planned clinical trials for acute spinal cord injury. Neurosurg Focus, 25(5), E14. doi: 10.3171/FOC.2008.25.11.E14 Hayashi, M., Matsushima, K., Ohashi, H., Tsunoda, H., Murase, S., Kawarada, Y., & Tanaka, T. (1998). Molecular cloning and characterization of human PDE8B, a novel thyroid-specific isozyme of 3',5'-cyclic nucleotide phosphodiesterase. Biochem Biophys Res Commun, 250(3), 751-756. doi: S0006-291X(98)99379-2 [pii] 10.1006/bbrc.1998.9379 [doi] Hayes, J. S., Brunton, L. L., & Mayer, S. E. (1980). Selective activation of particulate cAMP-dependent protein kinase by isoproterenol and prostaglandin E1. J Biol Chem, 255(11), 5113-5119.  Hellstrom, M., & Harvey, A. R. (2011). Retinal ganglion cell gene therapy and visual system repair. Curr Gene Ther, 11(2), 116-131. doi: CGT ABS- 65a [pii] Hendriks, W. T., Eggers, R., Ruitenberg, M. J., Blits, B., Hamers, F. P., Verhaagen, J., & Boer, G. J. (2006). Profound differences in spontaneous long-term functional recovery after defined spinal tract lesions in the rat. J Neurotrauma, 23(1), 18-35. doi: 10.1089/neu.2006.23.18 Henion, W. F., Sutherland, E. W., & Posternak, T. (1967). Effects of derivatives of adenosine 3',5'-phosphate on liver slices and intact animals. Biochim Biophys Acta, 148(1), 106-113. doi: 0304-4165(67)90284-X [pii] Hermer-Vazquez, L., Hermer-Vazquez, R., Moxon, K. A., Kuo, K. H., Viau, V., Zhan, Y., & Chapin, J. K. (2004). Distinct temporal activity patterns in the rat M1 and red nucleus during skilled versus unskilled limb movement. Behav Brain Res, 150(1-2), 93-107. doi: 10.1016/S0166-4328(03)00226-2 [doi] S0166432803002262 [pii] Herness, M. S., Sun, X. D., & Chen, Y. (1997). cAMP and forskolin inhibit potassium currents in rat taste receptor cells by different mechanisms. Am J Physiol, 272(6 Pt 1), C2005-2018.  Hilz, H., & Tarnowski, W. (1970). Opposite effects of cyclic AMP and its dibutyryl derivative on glycogen levels in HeLa cells. Biochem Biophys Res Commun, 40(4), 973-981. doi: 0006-291X(70)90999-X [pii] 141  Hirose, M., Ishizaki, T., Watanabe, N., Uehata, M., Kranenburg, O., Moolenaar, W. H., Matsumura, F., Maekawa, M., Bito, H., & Narumiya, S. (1998). Molecular Dissection of the Rho-associated Protein Kinase (p160ROCK)-regulated Neurite Remodeling in Neuroblastoma N1E-115 Cells. J. Cell Biol., 141(7), 1625-1636. doi: 10.1083/jcb.141.7.1625 Hoffman, P., Griffin, J., & Price, D. (1984). Control of axonal caliber by neurofilament transport. J. Cell Biol., 99(2), 705-714. doi: 10.1083/jcb.99.2.705 Hoffman, P. N. (1989). Expression of GAP-43, a rapidly transported growth-associated protein, and class II beta tubulin, a slowly transported cytoskeletal protein, are coordinated in regenerating neurons. J Neurosci, 9(3), 893-897.  Hoffman, P. N., Thompson, G. W., Griffin, J. W., & Price, D. L. (1985). Changes in neurofilament transport coincide temporally with alterations in the caliber of axons in regenerating motor fibers. J Cell Biol, 101(4), 1332-1340.  Holstege, G. (1987). Anatomical evidence for an ipsilateral rubrospinal pathway and for direct rubrospinal projections to motoneurons in the cat. Neurosci Lett, 74(3), 269-274.  Hooper, N. M., Karran, E. H., & Turner, A. J. (1997). Membrane protein secretases. Biochem J, 321 ( Pt 2), 265-279.  Houk, J. C., Gibson, A. R., Harvey, C. F., Kennedy, P. R., & van Kan, P. L. E. (1988). Activity of primate magnocellular red nucleus related to hand and finger movements. Behavioural Brain Research, 28(1-2), 201-206. doi: 10.1016/0166-4328(88)90097-6 Hsle, A. W., Kawashima, K., O'Neill, J. P., & Schroder, C. H. (1975). Possible role of adenosine cyclic 3':5'-monophosphate phosphodiesterase in the morphological transformation of Chinese hamster ovary cells mediated by N6,O2-dibutyryl adenosine cyclic 3':5"-monophosphate. J. Biol. Chem., 250(3), 984-989.  Huai, Q., Wang, H., Sun, Y., Kim, H. Y., Liu, Y., & Ke, H. (2003). Three-dimensional structures of PDE4D in complex with roliprams and implication on inhibitor selectivity. Structure, 11(7), 865-873.  Huisman, A. M., Kuypers, H. G. J. M., & Verburgh, C. A. (1982). Differences in collateralization of the descending spinal pathways from red nucleus and other brain stem cell groups in cat and monkey. In H. G. J. M. Kuypers & G. F. Martin (Eds.), Progress in Brain Research (Vol. Volume 57, pp. 185-217): Elsevier. Humm, J. L., Kozlowski, D. A., James, D. C., Gotts, J. E., & Schallert, T. (1998). Use-dependent exacerbation of brain damage occurs during an early post-lesion vulnerable period. Brain Res, 783(2), 286-292. doi: S0006-8993(97)01356-5 [pii] Humphrey, D. R., Gold, R., & Reed, D. J. (1984). Sizes, laminar and topographic origins of cortical projections to the major divisions of the red nucleus in the monkey. The Journal of Comparative Neurology, 225(1), 75-94. doi: 10.1002/cne.902250109 142  Humphrey, D. R., & Rietz, R. R. (1976). Cells of origin of corticorubral projections from the arm area of primate motor cortex and their synaptic actions in the red nucleus. Brain Research, 110(1), 162-169. doi: 10.1016/0006-8993(76)90217-1 Ivins, J. K., Parry, M. K., & Long, D. A. (2004). A novel cAMP-dependent pathway activates neuronal integrin function in retinal neurons. J Neurosci, 24(5), 1212-1216. doi: 10.1523/JNEUROSCI.4689-03.2004 Iwami, G., Kawabe, J.-i., Ebina, T., Cannon, P. J., Homcy, C. J., & Ishikawa, Y. (1995). Regulation of Adenylyl Cyclase by Protein Kinase A. J. Biol. Chem., 270(21), 12481-12484. doi: 10.1074/jbc.270.21.12481 Jacobson, R. D., Virag, I., & Skene, J. H. (1986). A protein associated with axon growth, GAP-43, is widely distributed and developmentally regulated in rat CNS. J Neurosci, 6(6), 1843-1855.  Jarnaess, E., & Tasken, K. (2007). Spatiotemporal control of cAMP signalling processes by anchored signalling complexes. Biochem Soc Trans, 35(Pt 5), 931-937. doi: BST0350931 [pii] 10.1042/BST0350931 [doi] Jin, Y., Fischer, I., Tessler, A., & Houle, J. D. (2002). Transplants of fibroblasts genetically modified to express BDNF promote axonal regeneration from supraspinal neurons following chronic spinal cord injury. Exp Neurol, 177(1), 265-275. doi: S001448860297980X [pii] Jones, T. A., Chu, C. J., Grande, L. A., & Gregory, A. D. (1999). Motor skills training enhances lesion-induced structural plasticity in the motor cortex of adult rats. J Neurosci, 19(22), 10153-10163.  Jones, T. A., Hawrylak, N., Klintsova, A. Y., & Greenough, W. T. (1998). Brain damage, behavior, rehabilitation, recovery, and brain plasticity. Mental Retardation and Developmental Disabilities Research Reviews, 4(3), 231-237.  Jones, T. A., & Schallert, T. (1994). Use-dependent growth of pyramidal neurons after neocortical damage. J Neurosci, 14(4), 2140-2152.  Joost, H., Habberfield, A., Simpson, I., Laurenza, A., & Seamon, K. (1988). Activation of adenylate cyclase and inhibition of glucose transport in rat adipocytes by forskolin analogues: structural determinants for distinct sites of action. Mol Pharmacol, 33(4), 449-453.  Joost, H., & Steinfelder, H. (1987). Forskolin inhibits insulin-stimulated glucose transport in rat adipose cells by a direct interaction with the glucose transporter. Mol Pharmacol, 31(3), 279-283.  Joosten, E. A. J., Schuitman, R. L., Vermelis, M. E. J., & Dederen, P. J. W. C. (1992). Postnatal development of the ipsilateral corticospinal component in rat spinal cord: A light and 143  electron microscopic anterograde HRP study. The Journal of Comparative Neurology, 326(1), 133-146. doi: 10.1002/cne.903260112 Joset, A., Dodd, D. A., Halegoua, S., & Schwab, M. E. (2010). Pincher-generated Nogo-A endosomes mediate growth cone collapse and retrograde signaling. J Cell Biol, 188(2), 271-285. doi: jcb.200906089 [pii] 10.1083/jcb.200906089 [doi] Kadrmas, J. L., & Beckerle, M. C. (2004). The LIM domain: from the cytoskeleton to the nucleus. Nat Rev Mol Cell Biol, 5(11), 920-931. doi: nrm1499 [pii] 10.1038/nrm1499 [doi] Kakulas, B. A. (2004). Neuropathology: the foundation for new treatments in spinal cord injury. Spinal Cord: The Official Journal Of The International Medical Society Of Paraplegia, 42(10), 549-563. doi: 10.1038/sj.sc.3101670 Kalil, K., & Skene, J. H. (1986). Elevated synthesis of an axonally transported protein correlates with axon outgrowth in normal and injured pyramidal tracts. J Neurosci, 6(9), 2563-2570.  Kanagal, S. G., & Muir, G. D. (2008). The differential effects of cervical and thoracic dorsal funiculus lesions in rats. Behav Brain Res, 187(2), 379-386. doi: 10.1016/j.bbr.2007.09.035 Kanagal, S. G., & Muir, G. D. (2009). Task-dependent compensation after pyramidal tract and dorsolateral spinal lesions in rats. Exp Neurol, 216(1), 193-206. doi: 10.1016/j.expneurol.2008.11.028 Kanning, K. C., Hudson, M., Amieux, P. S., Wiley, J. C., Bothwell, M., & Schecterson, L. C. (2003). Proteolytic processing of the p75 neurotrophin receptor and two homologs generates C-terminal fragments with signaling capability. J Neurosci, 23(13), 5425-5436. doi: 23/13/5425 [pii] Karhunen, H., Virtanen, T., Schallert, T., Sivenius, J., & Jolkkonen, J. (2003). Forelimb use after focal cerebral ischemia in rats treated with an alpha 2-adrenoceptor antagonist. Pharmacol Biochem Behav, 74(3), 663-669. doi: S0091305702010535 [pii] Kashiwagi, A., Huecksteadt, T., & Foley, J. (1983). The regulation of glucose transport by cAMP stimulators via three different mechanisms in rat and human adipocytes. J. Biol. Chem., 258(22), 13685-13692.  Kato, H., Araki, T., Itoyama, Y., & Kogure, K. (1995). Rolipram, a cyclic AMP-selective phosphodiesterase inhibitor, reduces neuronal damage following cerebral ischemia in the gerbil. European Journal of Pharmacology, 272(1), 107-110.  Kaukel, E., Fuhrmann, U., & Hilz, H. (1972). Divergent action of cAMP and dibutyryl cAMP on macromolecular synthesis in HeLa S3 cultures. Biochemical and Biophysical Research Communications, 48(6), 1516-1524. doi: 0006-291X(72)90886-8 [pii] 144  Kaukel, E., & Hilz, H. (1972). Permeation of dibutyryl cAMP into HeLa cells and its conversion to monobutyryl cAMP. Biochemical and Biophysical Research Communications, 46(2), 1011-1018.  Kaukel, E., Mundhenk, K., & Hilz, H. (1972). N6 -monobutyryladenosine 3':5'-mono phosphate as the biologically active derivative of dibutyryladenosine 3':5'-monophosphate in HeLa S3 cells. Eur J Biochem, 27(1), 197-200.  Kauppila, T. (1992). Polyamines enhance recovery after sciatic nerve trauma in the rat. Brain Res, 575(2), 299-303. doi: 0006-8993(92)90093-O [pii] Kawamoto, T., Gohda, E., Iji, H., Fujiwara, M., & Yamamoto, I. (1998). SKW 6.4 cell differentiation induced by interleukin 6 is stimulated by butyrate. Immunopharmacology, 40(2), 119-130.  Kawasaki, H., Springett, G. M., Mochizuki, N., Toki, S., Nakaya, M., Matsuda, M., Housman, D. E., & Graybiel, A. M. (1998). A family of cAMP-binding proteins that directly activate Rap1. Science, 282(5397), 2275-2279.  Keifer, J., & Houk, J. C. (1994). Motor function of the cerebellorubrospinal system. Physiol Rev, 74(3), 509-542.  Kennedy, P. R. (1987). Light labeling of red nucleus neurons following an injection of peroxidase-conjugated wheat germ agglutinin into the inferior olivary nucleus of the rat. Neuroscience Letters, 74(3), 262-268. doi: 10.1016/0304-3940(87)90307-7 Kennedy, P. R. (1990). Corticospinal, rubrospinal and rubro-olivary projections: a unifying hypothesis. Trends in Neurosciences, 13(12), 474-479. doi: 10.1016/0166-2236(90)90079-P Kennedy, P. R., & Humphrey, D. R. (1987). The compensatory role of the parvocellular division of the red nucleus in operantly conditioned rats. Neurosci Res, 5(1), 39-62. doi: 0168-0102(87)90022-8 [pii] Kilmer, S. L., & Carlsen, R. C. (1984). Forskolin activation of adenylate cyclase in vivo stimulates nerve regeneration. Nature, 307(5950), 455-457.  Kilmer, S. L., & Carlsen, R. C. (1987). Chronic infusion of agents that increase cyclic AMP concentration enhances the regeneration of mammalian peripheral nerves in vivo. Exp Neurol, 95(2), 357-367.  Kim, H., Sergeant, S., & Shukla, S. (1986). Glucose transport in human platelets and its inhibition by forskolin. J Pharmacol Exp Ther, 236(3), 585-589.  Kim, J., Liu, B., Park, J., & Strittmatter, S. (2004). Nogo-66 receptor prevents raphespinal and rubrospinal axon regeneration and limits functional recovery from spinal cord injury. Neuron, 44(3), 439 - 451. doi: 10.1016/j.neuron.2004.10.015 145  Kitzis, A., Tichonicky, L., Defer, N., & Kruh, J. (1980). Localization of phosphoproteins and of protein kinases in chromatin from butyrate treated HTC cells. Biochemical and Biophysical Research Communications, 97(2), 530-537. doi: 10.1016/0006-291X(80)90296-X Klauck, T. M., Faux, M. C., Labudda, K., Langeberg, L. K., Jaken, S., & Scott, J. D. (1996). Coordination of Three Signaling Enzymes by AKAP79, a Mammalian Scaffold Protein. Science, 271(5255), 1589-1592.  Klein, H. W., Kilmer, S., & Carlsen, R. C. (1989). Enhancement of peripheral nerve regeneration by pharmacological activation of the cyclic AMP second messenger system. Microsurgery, 10(2), 122-125.  Kleppisch, T. (2009). Phosphodiesterases in the central nervous system. Handb Exp Pharmacol(191), 71-92. doi: 10.1007/978-3-540-68964-5_5 Knuutila, S., & Pohjanpelto, P. (1983). Polyamine starvation causes parallel increase in nuclear and chromosomal aberrations in a polyamine-dependent strain of CHO. Exp Cell Res, 145(1), 222-226.  Kobayashi, N. R., Fan, D. P., Giehl, K. M., Bedard, A. M., Wiegand, S. J., & Tetzlaff, W. (1997). BDNF and NT-4/5 prevent atrophy of rat rubrospinal neurons after cervical axotomy, stimulate GAP-43 and Talpha1-tubulin mRNA expression, and promote axonal regeneration. J Neurosci, 17(24), 9583-9595.  Kobbert, C., Apps, R., Bechmann, I., Lanciego, J. L., Mey, J., & Thanos, S. (2000). Current concepts in neuroanatomical tracing. Prog Neurobiol, 62(4), 327-351. doi: S0301-0082(00)00019-8 [pii] Kobe, B., & Kajava, A. V. (2001). The leucine-rich repeat as a protein recognition motif. Current Opinion in Structural Biology, 11(6), 725-732.  Konstantinos, G., & Petros, P. (2009). Phosphodiesterase-5 inhibitors: future perspectives. Curr Pharm Des, 15(30), 3540-3551.  Koopmans, G. C., Deumens, R., Buss, A., Geoghegan, L., Myint, A. M., Honig, W. H., Kern, N., Joosten, E. A., Noth, J., & Brook, G. A. (2009). Acute rolipram/thalidomide treatment improves tissue sparing and locomotion after experimental spinal cord injury. Exp Neurol, 216(2), 490-498.  Kopan, R., & Ilagan, M. X. (2004). Gamma-secretase: proteasome of the membrane? Nat Rev Mol Cell Biol, 5(6), 499-504. doi: 10.1038/nrm1406 [doi] nrm1406 [pii] Kottis, V., Thibault, P., Mikol, D., Xiao, Z. C., Zhang, R., Dergham, P., & Braun, P. E. (2002). Oligodendrocyte-myelin glycoprotein (OMgp) is an inhibitor of neurite outgrowth. Journal Of Neurochemistry, 82(6), 1566-1569.  146  Kozasa, T., & Gilman, A. G. (1995). Purification of Recombinant G Proteins from Sf9 Cells by Hexahistidine Tagging of Associated Subunits. J. Biol. Chem., 270(4), 1734-1741. doi: 10.1074/jbc.270.4.1734 Krah, K., & Meller, K. (1999). Axonal and dendritic transport in Purkinje cells of cerebellar slice cultures studied by microinjection of horseradish peroxidase. Cell and Tissue Research, 295(1), 55-64.  Krajacic, A., Weishaupt, N., Girgis, J., Tetzlaff, W., & Fouad, K. (2010). Training-induced plasticity in rats with cervical spinal cord injury: effects and side effects. Behav Brain Res, 214(2), 323-331. doi: S0166-4328(10)00427-4 [pii] 10.1016/j.bbr.2010.05.053 [doi] Krupinski, J., Coussen, F. o., Bakalyar, H. A., Tang, W.-J., Feinstein, P. G., Orth, K., Slaughter, C., Reed, R. R., & Gilman, A. G. (1989). Adenylyl Cyclase Amino Acid Sequence: Possible Channel- or Transporter-Like Structure. Science, 244(4912), 1558-1564.  Kruse, L. S., M?ller, M., & Kruuse, C. (2011). Distribution of PDE8A in the nervous system of the Sprague-Dawley rat. Journal of Chemical Neuroanatomy, 42(3), 184-191. doi: 10.1016/j.jchemneu.2011.07.002 Kuang, R. Z., & Kalil, K. (1990). Specificity of corticospinal axon arbors sprouting into denervated contralateral spinal cord. The Journal of Comparative Neurology, 302(3), 461-472.  Kuchler, M., Fouad, K., Weinmann, O., Schwab, M. E., & Raineteau, O. (2002). Red nucleus projections to distinct motor neuron pools in the rat spinal cord. J Comp Neurol, 448(4), 349-359. doi: 10.1002/cne.10259 Kurimoto, T., Yin, Y., Omura, K., Gilbert, H. Y., Kim, D., Cen, L. P., Moko, L., Kugler, S., & Benowitz, L. I. (2010). Long-distance axon regeneration in the mature optic nerve: contributions of oncomodulin, cAMP, and pten gene deletion. J Neurosci, 30(46), 15654-15663. doi: 30/46/15654 [pii] 10.1523/JNEUROSCI.4340-10.2010 [doi] Kwok, R. P., Lundblad, J. R., Chrivia, J. C., Richards, J. P., Bachinger, H. P., Brennan, R. G., Roberts, S. G., Green, M. R., & Goodman, R. H. (1994). Nuclear protein CBP is a coactivator for the transcription factor CREB. Nature, 370(6486), 223-226. doi: 10.1038/370223a0 Kwon, B. K., Fisher, C. G., Dvorak, M. F., & Tetzlaff, W. (2005). Strategies to promote neural repair and regeneration after spinal cord injury. Spine, 30(17 Suppl), S3-13.  Kwon, B. K., Liu, J., Lam, C., Plunet, W., Oschipok, L. W., Hauswirth, W., Di Polo, A., Blesch, A., & Tetzlaff, W. (2007). Brain-derived neurotrophic factor gene transfer with adeno-associated viral and lentiviral vectors prevents rubrospinal neuronal atrophy and stimulates regeneration-associated gene expression after acute cervical spinal cord injury. Spine, 32(11), 1164-1173. doi: 00007632-200705150-00004 [pii] 10.1097/BRS.0b013e318053ec35 [doi]  147  Kwon, B. K., Liu, J., Messerer, C., Kobayashi, N. R., McGraw, J., Oschipok, L., & Tetzlaff, W. (2002). Survival and regeneration of rubrospinal neurons 1 year after spinal cord injury. Proc Natl Acad Sci U S A, 99(5), 3246-3251. doi: 10.1073/pnas.052308899 Kwon, B. K., Liu, J., Oschipok, L., Teh, J., Liu, Z. W., & Tetzlaff, W. (2004). Rubrospinal neurons fail to respond to brain-derived neurotrophic factor applied to the spinal cord injury site 2 months after cervical axotomy. Exp Neurol, 189(1), 45-57. doi: 10.1016/j.expneurol.2004.05.034 Kwon, B. K., Tetzlaff, W., Grauer, J. N., Beiner, J., & Vaccaro, A. R. (2004). Pathophysiology and pharmacologic treatment of acute spinal cord injury. Spine J, 4(4), 451-464. doi: 10.1016/j.spinee.2003.07.007 Lane, R., Liu, J., Plemel, J. R., Liu, W.-S., & Tetzlaff, W. (2005, December 14-18, 2005). db-cAMP Treatment of the Red Nucleus Promotes Regenerative Response of Rubrospinal Neurons but Fails to Improve Forelimb Usage. Paper presented at the Neurorehabilitation & Neural Repair 2006; 11th International Symposium on Neural Regeneration, Pacific Grove, CA. Lane, R., Lui, J., Plemel, J. R., Sutherland, D., Oschipok, L., Lam, C. K., & Tetzlaff, W. (2004, October 27, 2004, Wednesday, 4:00pm-5:00pm). Cell body treatment with cAMP attenuates atrophy, stimulates regeneration associated gene expression and promotes regenerative sprouting of rubrospinal axons after spinal cord injury. Paper presented at the Society For Neuroscience, San Diego. Lang, P., Gesbert, F., Delespine-Carmagnat, M., Stancou, R., Pouchelet, M., & Bertoglio, J. (1996). Protein kinase A phosphorylation of RhoA mediates the morphological and functional effects of cyclic AMP in cytotoxic lymphocytes. EMBO J, 15(3), 510-519.  Lasek, R. J., Oblinger, M. M., & Drake, P. F. (1983). Molecular biology of neuronal geometry: expression of neurofilament genes influences axonal diameter. Cold Spring Harb Symp Quant Biol, 48 Pt 2, 731-744.  Lavezzi, A. M., Corna, M., Matturri, L., & Santoro, F. (2009). Neuropathology of the Guillain-Mollaret Triangle (Dentato-Rubro-Olivary Network) in Sudden Unexplained Perinatal Death and SIDS. Open Neurol J, 3, 48-53. doi: 10.2174/1874205x00903010048 Lavoie, S., & Drew, T. (2002). Discharge characteristics of neurons in the red nucleus during voluntary gait modifications: a comparison with the motor cortex. J Neurophysiol, 88(4), 1791-1814.  Leoz, O., & Arcaute, L. R. (1913). Procesos regenerativos del nervio ?ptico y retina con ocasi?n de ingertos nerviosos. Trab. Lab. Invest. Biol. Univ. Madr., 11, 239-254.  Li, M., A., S., Li, C., Braun, P. E., McKerracher, L., Roder, J., Kater, S. B., & David, S. (1996). Myelin-associated glycoprotein inhibits neurite/axon growth and causes growth cone collapse. Journal of Neuroscience Research, 46(4), 404-414. doi: 10.1002/(SICI)1097-4547(19961115) 148  Liang, P., Moret, V., Wiesendanger, M., & M., R. E. (1991). Corticomotoneuronal connections in the rat: Evidence from double-labeling of motoneurons and corticospinal axon arborizations. The Journal of Comparative Neurology, 311(3), 356-366. doi: 10.1002/cne.903110306 Liepinsh, E., Ilag, L. L., Otting, G., & Ibanez, C. F. (1997). NMR structure of the death domain of the p75 neurotrophin receptor. EMBO J, 16(16), 4999-5005. doi: 10.1093/emboj/16.16.4999 Liu, Y., Kim, D., Himes, B. T., Chow, S. Y., Schallert, T., Murray, M., Tessler, A., & Fischer, I. (1999). Transplants of fibroblasts genetically modified to express BDNF promote regeneration of adult rat rubrospinal axons and recovery of forelimb function. J Neurosci, 19(11), 4370-4387.  Lohmann, S. M., DeCamilli, P., Einig, I., & Walter, U. (1984). High-affinity binding of the regulatory subunit (RII) of cAMP-dependent protein kinase to microtubule-associated and other cellular proteins. Proc Natl Acad Sci U S A, 81(21), 6723-6727.  Lu, B., Pang, P. T., & Woo, N. H. (2005). The yin and yang of neurotrophin action. Nat Rev Neurosci, 6(8), 603-614. doi: nrn1726 [pii] 10.1038/nrn1726 [doi] Lu, L., Stanley, B. A., & Pegg, A. E. (1991). Identification of residues in ornithine decarboxylase essential for enzymic activity and for rapid protein turnover. Biochem J, 277 ( Pt 3), 671-675.  Lundblad, M., Andersson, M., Winkler, C., Kirik, D., Wierup, N., & Cenci, M. A. (2002). Pharmacological validation of behavioural measures of akinesia and dyskinesia in a rat model of Parkinson's disease. Eur J Neurosci, 15(1), 120-132. doi: 1843 [pii] MacLellan, C., Shuaib, A., & Colbourne, F. (2002). Failure of delayed and prolonged hypothermia to favorably affect hemorrhagic stroke in rats. Brain Res, 958(1), 192-200. doi: S0006899302037022 [pii] Martinez, A., Castro, A., Gil, C., Miralpeix, M., Segarra, V., Domenech, T., Beleta, J., Palacios, J. M., Ryder, H., Miro, X., Bonet, C., Casacuberta, J. M., Azorin, F., Pina, B., & Puigdomenech, P. (2000). Benzyl derivatives of 2,1,3-benzo- and benzothieno[3,2-a]thiadiazine 2,2-dioxides: first phosphodiesterase 7 inhibitors. J Med Chem, 43(4), 683-689. doi: jm990382n [pii] Massion, J. (1967). The mammalian red nucleus. Physiol Rev, 47(3), 383-436.  Massion, J. (1988). Red nucleus: past and future. Behavioural Brain Research, 28(1-2), 1-8. doi: 10.1016/0166-4328(88)90071-X May, R. M. (Ed.). (1928). Degeneration & regeneration of the nervous system, translated and edited by Raoul M. May. (Vol. 2nd). London, England: Oxford University Press, 1928. 149  Mayr, B., & Montminy, M. (2001). Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol, 2(8), 599-609. doi: 35085068 [pii] 10.1038/35085068 [doi]  Mayr, B. M., Canettieri, G., & Montminy, M. R. (2001). Distinct effects of cAMP and mitogenic signals on CREB-binding protein recruitment impart specificity to target gene activation via CREB. Proc Natl Acad Sci U S A, 98(19), 10936-10941. doi: 10.1073/pnas.191152098 [doi] 191152098 [pii] McKenna, J. E., & Whishaw, I. Q. (1999). Complete compensation in skilled reaching success with associated impairments in limb synergies, after dorsal column lesion in the rat. J Neurosci, 19(5), 1885-1894.  McKerracher, L., David, S., Jackson, D. L., Kottis, V., Dunn, R. J., & Braun, P. E. (1994). Identification of myelin-associated glycoprotein as a major myelin-derived inhibitor of neurite growth. Neuron, 13(4), 805-811.  McQuarrie, I. G., & Grafstein, B. (1973). Axon outgrowth enhanced by a previous nerve injury. Arch Neurol, 29(1), 53-55.  McQuarrie, I. G., & Grafstein, B. (1981). Effect of a conditioning lesion on optic nerve regeneration in goldfish. Brain Res, 216(2), 253-264.  McQuarrie, I. G., & Grafstein, B. (1983). Effect of acetoxycycloheximide and dibutyryladenosine cyclic 3':5'-monophosphate on axonal regeneration in the goldfish optic nerve. Brain Res, 279(1-2), 377-381.  McQuarrie, I. G., Grafstein, B., Dreyfus, C. F., & Gershon, M. D. (1978). Regeneration of adrenergic axons in rat sciatic nerve: effect of a conditioning lesion. Brain Res, 141(1), 21-34.  McQuarrie, I. G., Grafstein, B., & Gershon, M. D. (1977). Axonal regeneration in the rat sciatic nerve: effect of a conditioning lesion and of dbcAMP. Brain Res, 132(3), 443-453.  Medina, V., Edmonds, B., Young, G. P., James, R., Appleton, S., & Zalewski, P. D. (1997). Induction of Caspase-3 Protease Activity and Apoptosis by Butyrate and Trichostatin A (Inhibitors of Histone Deacetylase): Dependence on Protein Synthesis and Synergy with a Mitochondrial/Cytochrome c-dependent Pathway. Cancer Research, 57(17), 3697-3707.  Meller, K. (1992). Axoplasmic transport of horseradish peroxidase in single neurons of the dorsal root ganglion studied in vitro by microinjection. Cell Tissue Res, 270(1), 139-148.  Menahan, L. A., Hepp, K. D., & Wieland, O. (1969). Liver 3': 5'-Nucleotide Phosphodiesterase and its Activity in Rat Livers Perfused with Insulin. European Journal of Biochemistry, 8(3), 435-443.  150  Meyer-Franke, A., Wilkinson, G. A., Kruttgen, A., Hu, M., Munro, E., Hanson, M. G., Jr., Reichardt, L. F., & Barres, B. A. (1998). Depolarization and cAMP elevation rapidly recruit TrkB to the plasma membrane of CNS neurons. Neuron, 21(4), 681-693.  Mi, S. (2008). Troy/Taj and its role in CNS axon regeneration. Cytokine Growth Factor Rev, 19(3-4), 245-251. doi: S1359-6101(08)00040-3 [pii] 10.1016/j.cytogfr.2008.04.007 [doi] Mi, S., Lee, X., Shao, Z., Thill, G., Ji, B., Relton, J., Levesque, M., Allaire, N., Perrin, S., Sands, B., Crowell, T., Cate, R. L., McCoy, J. M., & Pepinsky, R. B. (2004). LINGO-1 is a component of the Nogo-66 receptor/p75 signaling complex. Nature Neuroscience, 7(3), 221-228. doi: Doi 10.1038/Nn1188 Michel, J. J. C., & Scott, J. D. (2002). AKAP Mediated Signal Transduction. Annual Review of Pharmacology and Toxicology, 42(1), 235-257. doi: doi:10.1146/annurev.pharmtox.42.083101.135801 Michel, P. P., Vyas, S., & Agid, Y. (1995). Synergistic Differentiation by Chronic Exposure to Cyclic AMP and Nerve Growth Factor Renders Rat Phaeochromocytoma PC12 Cells Totally Dependent upon Trophic Support for Survival. European Journal of Neuroscience, 7(2), 251-260.  Miller, F. D., & Kaplan, D. R. (2001). Neurotrophin signalling pathways regulating neuronal apoptosis. Cell Mol Life Sci, 58(8), 1045-1053.  Miller, F. D., Naus, C. C., Durand, M., Bloom, F. E., & Milner, R. J. (1987). Isotypes of alpha-tubulin are differentially regulated during neuronal maturation. J Cell Biol, 105(6 Pt 2), 3065-3073.  Miller, F. D., Tetzlaff, W., Bisby, M. A., Fawcett, J. W., & Milner, R. J. (1989). Rapid induction of the major embryonic alpha-tubulin mRNA, T alpha 1, during nerve regeneration in adult rats. J Neurosci, 9(4), 1452-1463.  Mills, I., Moreno, F. J., & Fain, J. N. (1984). Forskolin inhibition of glucose metabolism in rat adipocytes independent of adenosine 3',5'-monophosphate accumulation and lipolysis. Endocrinology, 115(3), 1066-1069.  Miro, X., Perez-Torres, S., Artigas, F., Puigdomenech, P., Palacios, J. M., & Mengod, G. (2002). Regulation of cAMP phosphodiesterase mRNAs expression in rat brain by acute and chronic fluoxetine treatment. An in situ hybridization study. Neuropharmacology, 43(7), 1148-1157.  Miro, X., Perez-Torres, S., Palacios, J. M., Puigdomenech, P., & Mengod, G. (2001). Differential distribution of cAMP-specific phosphodiesterase 7A mRNA in rat brain and peripheral organs. Synapse, 40(3), 201-214. doi: 10.1002/syn.1043 Miyamoto, M. D., & Breckenridge, B. M. (1974). A Cyclic Adenosine Monophosphate Link in the Catecholamine Enhancement of Transmitter Release at the Neuromuscular Junction. J. Gen. Physiol., 63(5), 609-624. doi: 10.1085/jgp.63.5.609 151  Molnar-Kimber, K., Yonno, L., Heaslip, R., & Weichman, B. (1993). Modulation of TNF alpha and IL-1 beta from endotoxin-stimulated monocytes by selective PDE isozyme inhibitors. Agents Actions, 39 Spec No, C77-79.  Molnar-Kimber, K. L., Yonno, L., Heaslip, R. J., & Weichman, B. M. (1992). Differential regulation of TNF-alpha and IL-1beta production from endotoxin stimulated human monocytes by phosphodiesterase inhibitors. Mediators Inflamm, 1(6), 411-417. doi: 10.1155/S0962935192000620 Monaghan, T. K., Mackenzie, C. J., Plevin, R., & Lutz, E. M. (2008). PACAP-38 induces neuronal differentiation of human SH-SY5Y neuroblastoma cells via cAMP-mediated activation of ERK and p38 MAP kinases. J Neurochem, 104(1), 74-88. doi: JNC5018 [pii] 10.1111/j.1471-4159.2007.05018.x [doi] Monnier, P. P., Sierra, A., Schwab, J. M., Henke-Fahle, S., & Mueller, B. K. (2003). The Rho/ROCK pathway mediates neurite growth-inhibitory activity associated with the chondroitin sulfate proteoglycans of the CNS glial scar. Molecular and Cellular Neuroscience, 22(3), 319-330.  Monsul, N. T., Geisendorfer, A. R., Han, P. J., Banik, R., Pease, M. E., Skolasky, R. L., Jr., & Hoffman, P. N. (2004). Intraocular injection of dibutyryl cyclic AMP promotes axon regeneration in rat optic nerve. Exp Neurol, 186(2), 124-133. doi: 10.1016/S0014-4886(03)00311-X Montminy, M. R., Sevarino, K. A., Wagner, J. A., Mandel, G., & Goodman, R. H. (1986). Identification of a cyclic-AMP-responsive element within the rat somatostatin gene. Proc Natl Acad Sci U S A, 83(18), 6682-6686.  Moon, A., & Drubin, D. G. (1995). The ADF/cofilin proteins: stimulus-responsive modulators of actin dynamics. Mol Biol Cell, 6(11), 1423-1431.  Moore, P. F., Iorio, L. C., & McManus, J. M. (1968). Relaxation of the guinea-pig tracheal chain preparation by N6,2'-O-dibutyryl 3',5'-cyclic adenosine monophosphate. J Pharm Pharmacol, 20(5), 368-372.  Moreau-Fauvarque, C., Kumanogoh, A., Camand, E., Jaillard, C., Barbin, G., Boquet, I., Love, C., Jones, E. Y., Kikutani, H., Lubetzki, C., Dusart, I., & Chedotal, A. (2003). The Transmembrane Semaphorin Sema4D/CD100, an Inhibitor of Axonal Growth, Is Expressed on Oligodendrocytes and Upregulated after CNS Lesion. J. Neurosci., 23(27), 9229-9239.  Morgan, D. M. L. (1998). Polyamines: An Introduction. In D. M. L. Morgan (Ed.), Methods in Molecular Biology: Polyamine Protocols (Vol. 79, pp. 3-30). Totowa, New Jersey: Humana Press. Muir, G. D., Webb, A. A., Kanagal, S., & Taylor, L. (2007). Dorsolateral cervical spinal injury differentially affects forelimb and hindlimb action in rats. Eur J Neurosci, 25(5), 1501-1510. doi: EJN5411 [pii] 10.1111/j.1460-9568.2007.05411.x [doi] 152  Muir, G. D., & Whishaw, I. Q. (1999a). Complete locomotor recovery following corticospinal tract lesions: measurement of ground reaction forces during overground locomotion in rats. Behav Brain Res, 103(1), 45-53.  Muir, G. D., & Whishaw, I. Q. (1999b). Ground reaction forces in locomoting hemi-parkinsonian rats: a definitive test for impairments and compensations. Exp Brain Res, 126(3), 307-314.  Muir, G. D., & Whishaw, I. Q. (2000). Red nucleus lesions impair overground locomotion in rats: a kinetic analysis. Eur J Neurosci, 12(3), 1113-1122. doi: ejn987 [pii] Mukhopadhyay, G., Doherty, P., Walsh, F. S., Crocker, P. R., & Filbin, M. T. (1994). A novel role for myelin-associated glycoprotein as an inhibitor of axonal regeneration. Neuron, 13(3), 757-767.  Murray, A. J., Peace, A. G., & Shewan, D. A. (2009). cGMP promotes neurite outgrowth and growth cone turning and improves axon regeneration on spinal cord tissue in combination with cAMP. Brain Res, 1294, 12-21. doi: S0006-8993(09)01534-0 [pii] 10.1016/j.brainres.2009.07.071 [doi] Murray, A. J., & Shewan, D. A. (2008). Epac mediates cyclic AMP-dependent axon growth, guidance and regeneration. Mol Cell Neurosci, 38(4), 578-588. doi: S1044-7431(08)00129-2 [pii] 10.1016/j.mcn.2008.05.006 [doi] Murray, H. M., & Gurule, M. E. (1979). Origin of the rubrospinal tract of the rat. Neurosci Lett, 14(1), 19-23.  Murray, M., Kim, D., Liu, Y., Tobias, C., Tessler, A., & Fischer, I. (2002). Transplantation of genetically modified cells contributes to repair and recovery from spinal injury. Brain Res Brain Res Rev, 40(1-3), 292-300. doi: S0165017302002114 [pii] Nance, D. M., & Burns, J. (1990). Fluorescent dextrans as sensitive anterograde neuroanatomical tracers: applications and pitfalls. Brain Res Bull, 25(1), 139-145. doi: 0361-9230(90)90264-Z [pii] Nathan, P. W., & Smith, M. C. (1982). The rubrospinal and central tegmental tracts in man. Brain, 105(Pt 2), 223-269.  Neelon, F. A., & Birch, B. M. (1973). Cyclic Adenosine 3':5'-Monophosphate-dependent Protein Kinase. Interaction with Butyrlated Analogues of Cyclic Adenosine 3':5'-Monophosphate. J. Biol. Chem., 248(24), 8361-8365.  Neumann, S., Bradke, F., Tessier-Lavigne, M., & Basbaum, A. I. (2002). Regeneration of sensory axons within the injured spinal cord induced by intraganglionic cAMP elevation. Neuron, 34(6), 885-893.  Neumann, S., & Woolf, C. J. (1999). Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury. Neuron, 23(1), 83-91.  153  Newlon, M. G., Roy, M., Morikis, D., Carr, D. W., Westphal, R., Scott, J. D., & Jennings, P. A. (2001). A novel mechanism of PKA anchoring revealed by solution structures of anchoring complexes. EMBO J, 20(7), 1651-1662. doi: 10.1093/emboj/20.7.1651 Nicholson, C. D., Challiss, R. A., & Shahid, M. (1991). Differential modulation of tissue function and therapeutic potential of selective inhibitors of cyclic nucleotide phosphodiesterase isoenzymes. Trends Pharmacol Sci, 12(1), 19-27.  Nicol, X., Hong, K. P., & Spitzer, N. C. (2011). Spatial and temporal second messenger codes for growth cone turning. Proc Natl Acad Sci U S A, 108(33), 13776-13781. doi: 1100247108 [pii] 10.1073/pnas.1100247108 [doi] Nieder?st, B., Oertle, T., Fritsche, J., McKinney, R. A., & Bandtlow, C. E. (2002). Nogo-A and myelin-associated glycoprotein mediate neurite growth inhibition by antagonistic regulation of RhoA and Rac1. J Neurosci, 22(23), 10368-10376.  Nikulina, E., Tidwell, J. L., Dai, H. N., Bregman, B. S., & Filbin, M. T. (2004). The phosphodiesterase inhibitor rolipram delivered after a spinal cord lesion promotes axonal regeneration and functional recovery. Proc Natl Acad Sci U S A, 101(23), 8786-8790. doi: 10.1073/pnas.0402595101 NSCISC. (2008). Spinal Cord Injury - Facts and Figures at a Glance: National Spinal Cord Injury Statistical Center. O'Neill, J. P., Schroder, C. H., & Hsle, A. W. (1975). Hudrolysis of butyryl derivatives of adenosine cyclic 3':5'-monophosphate by Chinese hamster ovary cell extracts and characterization fo the products. J Biol Chem, 250(3), 990-995.  Ochs, S. (1972). Fast transport of materials in mammalian nerve fibers. Science, 176(32), 252-260.  Ochs, S., Sabri, M. I., & Johnson, J. (1969). Fast transport system of materials in mammalian nerve fibers. Science, 163(868), 686-687.  Oertle, T., van der Haar, M. E., Bandtlow, C. E., Robeva, A., Burfeind, P., Buss, A., Huber, A. B., Simonen, M., Schnell, L., Brosamle, C., Kaupmann, K., Vallon, R., & Schwab, M. E. (2003). Nogo-A inhibits neurite outgrowth and cell spreading with three discrete regions. J Neurosci, 23(13), 5393-5406.  ?greid, D., Ekanger, R., Suva, R. H., Miller, J. P., Sturm, P., Corbin, J. D., & D?skeland, S. O. (1985). Activation of protein kinase isozymes by cyclic nucleotide analogs used singly or in combination. European Journal of Biochemistry, 150(1), 219-227.  Onodera, S. (1984). Olivary projections from the mesodiencephalic structures in the cat studied by means of axonal transport of horseradish peroxidase and tritiated amino acids. J Comp Neurol, 227(1), 37-49. doi: 10.1002/cne.902270106 154  Onodera, S., & Hicks, T. P. (1999). Review : Evolution of the Motor System: Why the Elephant's Trunk Works Like a Human's Hand. The Neuroscientist, 5(4), 217-226. doi: 10.1177/107385849900500411 Onodera, S., & Hicks, T. P. (2009). A comparative neuroanatomical study of the red nucleus of the cat, macaque and human. PLoS One, 4(8), e6623. doi: 10.1371/journal.pone.0006623 Onodera, S., & Hicks, T. P. (2010). Carbocyanine dye usage in demarcating boundaries of the aged human red nucleus. PLoS One, 5(12), e14430. doi: 10.1371/journal.pone.0014430 Onodera, S., Nitatori, T., & Hicks, T. P. (2004). Olivary projection from the rostral part of the nucleus of Darkschewitsch in the postnatal rat as revealed through the use of a carbocyanine dye. Brain Res, 1015(1-2), 194-197. doi: 10.1016/j.brainres.2004.04.042 Orsal, D., Perret, C., & Cabelguen, J. M. (1988). Comparison between ventral spinocerebellar and rubrospinal activities during locomotion in the cat. Behav Brain Res, 28(1-2), 159-162.  Ozaki, N., Shibasaki, T., Kashima, Y., Miki, T., Takahashi, K., Ueno, H., Sunaga, Y., Yano, H., Matsuura, Y., Iwanaga, T., Takai, Y., & Seino, S. (2000). cAMP-GEFII is a direct target of cAMP in regulated exocytosis. Nat Cell Biol, 2(11), 805-811. doi: 10.1038/35041046 Papaioannou, J. N. (1971). Rubral functions in the rat: a lesion study. Neuropsychologia, 9(3), 345-349.  Park, J., Yiu, G., Kaneko, S., Wang, J., Chang, J., He, X., Garcia, K., & He, Z. (2005). A TNF receptor family member, TROY, is a coreceptor with Nogo receptor in mediating the inhibitory activity of myelin inhibitors. Neuron, 45, 345 - 351. doi: 10.1016/j.neuron.2004.12.040 Park, J. B., Yiu, G., Kaneko, S., Wang, J., Chang, J., & He, Z. (2005). A TNF Receptor Family Member, TROY, Is a Coreceptor with Nogo Receptor in Mediating the Inhibitory Activity of Myelin Inhibitors. Neuron, 45(3), 345-351.  Park, K. K., Hu, Y., Muhling, J., Pollett, M. A., Dallimore, E. J., Turnley, A. M., Cui, Q., & Harvey, A. R. (2009). Cytokine-induced SOCS expression is inhibited by cAMP analogue: impact on regeneration in injured retina. Mol Cell Neurosci, 41(3), 313-324. doi: S1044-7431(09)00080-3 [pii] 10.1016/j.mcn.2009.04.002 [doi] Paterson, J. M., Smith, S. M., Harmar, A. J., & Antoni, F. A. (1995). Control of a novel adenylyl cyclase by calcineurin. Biochem Biophys Res Commun, 214(3), 1000-1008. doi: S0006-291X(85)72385-6 [pii] 10.1006/bbrc.1995.2385 [doi] Pearse, D. D., Pereira, F. C., Marcillo, A. E., Bates, M. L., Berrocal, Y. A., Filbin, M. T., & Bunge, M. B. (2004). cAMP and Schwann cells promote axonal growth and functional recovery after spinal cord injury. Nat Med, 10(6), 610-616. doi: 10.1038/nm1056 155  Pegg, A. E. (1986). Recent advances in the biochemistry of polyamines in eukaryotes. Biochem J, 234(2), 249-262.  Pegg, A. E., & Casero, R. A., Jr. (2011). Current status of the polyamine research field. Methods Mol Biol, 720, 3-35. doi: 10.1007/978-1-61779-034-8_1 Pestronk, A., Watson, D. F., & Yuan, C. M. (1990). Neurofilament phosphorylation in peripheral nerve: changes with axonal length and growth state. J Neurochem, 54(3), 977-982.  Pichichero, M., Beer, B., & Clody, D. E. (1973). Effects of dibutyryl cyclic AMP on restoration of function of damaged sciatic nerve in rats. Science, 182(113), 724-725.  Pieroni, J. P., Harry, A., Chen, J., Jacobowitz, O., Magnusson, R. P., & Iyengar, R. (1995). Distinct characteristics of the basal activities of adenylyl cyclases 2 and 6. J Biol Chem, 270(36), 21368-21373.  Pohjanpelto, P., & Knuutila, S. (1982). Polyamine deprivation causes major chromosome aberrations in a polyamine-dependent Chinese hamster ovary cell line. Exp Cell Res, 141(2), 333-339.  Pohjanpelto, P., & Knuutila, S. (1984). Induction of major chromosome aberrations in Chinese hamster ovary cells by alpha-difluoromethylornithine. Cancer Res, 44(10), 4535-4539.  Pohjanpelto, P., Virtanen, I., & Holtta, E. (1981). Polyamine starvation causes disappearance of actin filaments and microtubules in polyamine-auxotrophic CHO cells. Nature, 293(5832), 475-477.  Pollard, J. D., McLeod, J. G., & Gye, R. S. (1973). Regeneration through peripheral nerve allografts. An electrophysiological and histological study following the use of immunosuppressive therapy. Arch Neurol, 28(1), 31-37.  Posternak, T., Sutherland, E. W., & Henion, W. F. (1962). Derivatives of cyclic 3',5'-adenosine monophosphate. Biochim Biophys Acta, 65, 558-560.  Posternak, T., & Weimann, G. (1974). The preparation of acylated derivatives of cyclic nucleotides. Methods Enzymol, 38, 399-409.  Premont, R. T., Matsuoka, I., Mattei, M.-G., Pouille, Y., Defer, N., & Hanoune, J. (1996). Identification and Characterization of a Widely Expressed Form of Adenylyl Cyclase. J. Biol. Chem., 271(23), 13900-13907. doi: 10.1074/jbc.271.23.13900 Prendergast, J., & Misantone, L. J. (1980). Sprouting by tracts descending from the midbrain to the spinal cord: The result of thoracic funiculotomy in the newborn, 21-day-old, and adult rat. Experimental Neurology, 69(3), 458-480. doi: 10.1016/0014-4886(80)90045-X Qiu, J., Cai, D., Dai, H., McAtee, M., Hoffman, P. N., Bregman, B. S., & Filbin, M. T. (2002). Spinal axon regeneration induced by elevation of cyclic AMP. Neuron, 34(6), 895-903.  156  Raineteau, O., Fouad, K., Bareyre, F. M., & Schwab, M. E. (2002). Reorganization of descending motor tracts in the rat spinal cord. Eur J Neurosci, 16(9), 1761-1771.  Rall, T. W., & Sutherland, E. W. (1958). Formation of a cyclic adenine ribonucleotide by tissue particles. J Biol Chem, 232(2), 1065-1076.  Ram?n y Cajal, S. (1991). Cajal's Degeneration and Regeneration of the Nervous System (R. May, M., Trans. Vol. 5). New York: Oxford University Press. Rangarajan, S., Enserink, J. M., Kuiperij, H. B., de Rooij, J., Price, L. S., Schwede, F., & Bos, J. L. (2003). Cyclic AMP induces integrin-mediated cell adhesion through Epac and Rap1 upon stimulation of the beta 2-adrenergic receptor. J Cell Biol, 160(4), 487-493. doi: jcb.200209105 [pii]10.1083/jcb.200209105 [doi] Reich, J. B., Burmeister, D. W., Schmidt, J. T., & Grafstein, B. (1990). Effect of conditioning lesions on regeneration of goldfish optic axons: time course of the cell body reaction to axotomy. Brain Research, 515(1-2), 256-260.  Reichardt, L. F. (2006). Neurotrophin-regulated signalling pathways. Philos Trans R Soc Lond B Biol Sci, 361(1473), 1545-1564. doi: G280147415872711 [pii] 10.1098/rstb.2006.1894 [doi] Reiner, A., Veenman, C. L., Medina, L., Jiao, Y., Del Mar, N., & Honig, M. G. (2000). Pathway tracing using biotinylated dextran amines. Journal of Neuroscience Methods, 103(1), 23-37.  Reyes-Irisarri, E., Perez-Torres, S., & Mengod, G. (2005). Neuronal expression of cAMP-specific phosphodiesterase 7B mRNA in the rat brain. Neuroscience, 132(4), 1173-1185. doi: 10.1016/j.neuroscience.2005.01.050 Rho, M. J., Lavoie, S., & Drew, T. (1999). Effects of red nucleus microstimulation on the locomotor pattern and timing in the intact cat: a comparison with the motor cortex. J Neurophysiol, 81(5), 2297-2315.  Richardson, P. M., & Issa, V. M. (1984). Peripheral injury enhances central regeneration of primary sensory neurones. Nature, 309(5971), 791-793.  Richardson, P. M., Issa, V. M., & Aguayo, A. J. (1984). Regeneration of long spinal axons in the rat. J Neurocytol, 13(1), 165-182.  Richardson, P. M., McGuinness, U. M., & Aguayo, A. J. (1980). Axons from CNS neurons regenerate into PNS grafts. Nature, 284(5753), 264-265.  Richter-Landsberg, C., & Jastorff, B. (1985). In vitro phosphorylation of microtubule-associated protein 2: differential effects of cyclic AMP analogues. J Neurochem, 45(4), 1218-1222.  157  Rick Hansen Foundation. (2009). About Spinal Cord Injury, from http://www.rickhansen.com/index.php?option=com_content&task=view&id=67&Itemid=67 Rivero, J. A., & Adunyah, S. E. (1998). Sodium Butyrate Stimulates PKC Activation and Induces Differential Expression of Certain PKC Isoforms during Erythroid Differentiation. Biochemical and Biophysical Research Communications, 248(3), 664-668. doi: 10.1006/bbrc.1998.9041 Robichaud, A., Savoie, C., Stamatiou, P. B., Tattersall, F. D., & Chan, C. C. (2001). PDE4 inhibitors induce emesis in ferrets via a noradrenergic pathway. Neuropharmacology, 40(2), 262-269. doi: S0028390800001428 [pii] Rodriguez-Tebar, A., Dechant, G., & Barde, Y. A. (1990). Binding of brain-derived neurotrophic factor to the nerve growth factor receptor. Neuron, 4(4), 487-492. doi: 0896-6273(90)90107-Q [pii] Rodriguez-Tebar, A., Dechant, G., & Barde, Y. A. (1991). Neurotrophins: structural relatedness and receptor interactions. Philos Trans R Soc Lond B Biol Sci, 331(1261), 255-258. doi: 10.1098/rstb.1991.0013 Rodriguez-Tebar, A., Dechant, G., Gotz, R., & Barde, Y. A. (1992). Binding of neurotrophin-3 to its neuronal receptors and interactions with nerve growth factor and brain-derived neurotrophic factor. EMBO J, 11(3), 917-922.  Roisen, F. J., Murphy, R. A., Pichichero, M. E., & Braden, W. G. (1972). Cyclic adenosine monophosphate stimulation of axonal elongation. Science, 175(17), 73-74.  Roscioni, S. S., Elzinga, C. R., & Schmidt, M. (2008). Epac: effectors and biological functions. Naunyn Schmiedebergs Arch Pharmacol, 377(4-6), 345-357. doi: 10.1007/s00210-007-0246-7 Rowland, J. W., Hawryluk, G. W., Kwon, B., & Fehlings, M. G. (2008). Current status of acute spinal cord injury pathophysiology and emerging therapies: promise on the horizon. Neurosurg Focus, 25(5), E2. doi: 10.3171/FOC.2008.25.11.E2 Ruff, R. L., McKerracher, L., & Selzer, M. E. (2008). Repair and Neurorehabilitation Strategies for Spinal Cord Injury. Annals of the New York Academy of Sciences, 1142(The Year in Neurology 2008), 1-20. doi: 10.1196/annals.1444.004 Rydel, R. E., & Greene, L. A. (1988). cAMP analogs promote survival and neurite outgrowth in cultures of rat sympathetic and sensory neurons independently of nerve growth factor. Proc Natl Acad Sci U S A, 85(4), 1257-1261.  Sadana, R., & Dessauer, C. W. (2009). Physiological Roles for G Protein-Regulated Adenylyl Cyclase Isoforms: Insights from Knockout and Overexpression Studies. Neurosignals, 17(1), 5-22. doi: 10.1159/000166277 158  Saling, M., Sitarova, T., Vejsada, R., & Hnik, P. (1992). Reaching behavior in the rat: absence of forelimb peripheral input. Physiol Behav, 51(6), 1151-1156.  Salminen, A., Tapiola, T., Korhonen, P., & Suuronen, T. (1998). Neuronal apoptosis induced by histone deacetylase inhibitors. Brain Res Mol Brain Res, 61(1-2), 203-206.  Samarzija, M., Zuljevic, E., Jakopovic, M., Sever, B., Knezevic, A., Dumija, Z., Vidjak, V., & Samija, M. (2009). One year efficacy and safety of oral sildenafil treatment in severe pulmonary hypertension. Coll Antropol, 33(3), 799-803.  Sands, W. A., & Palmer, T. M. (2008). Regulating gene transcription in response to cyclic AMP elevation. Cell Signal, 20(3), 460-466. doi: S0898-6568(07)00316-6 [pii] 10.1016/j.cellsig.2007.10.005 [doi] Sarkar, D., Erlichman, J., & Rubin, C. (1984). Identification of a calmodulin-binding protein that co-purifies with the regulatory subunit of brain protein kinase II. J. Biol. Chem., 259(15), 9840-9846.  Sasaki, T., & Takai, Y. (1998). The Rho Small G Protein Family-Rho GDI System as a Temporal and Spatial Determinant for Cytoskeletal Control. Biochemical and Biophysical Research Communications, 245(3), 641-645.  Schallert, T., Fleming, S. M., Leasure, J. L., Tillerson, J. L., & Bland, S. T. (2000). CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury. Neuropharmacology, 39(5), 777-787. doi: S0028390800000058 [pii] Schallert, T., Hernandez, T. D., & Barth, T. M. (1989). Preoperative intermittent feeding or drinking regimes enhance post-lesion sensorimotor function. In J. Schulkin (Ed.), Preoperative Events : Their Effects on Behavior Following Bain Damage Hillsdale, N.J.: L. Erlbaum Associates, 1989. Schallert, T., & Jones, T. A. (1993). "Exuberant" neuronal growth after brain damage in adult rats: the essential role of behavioral experience. J Neural Transplant Plast, 4(3), 193-198. doi: 10.1155/NP.1993.193 Schallert, T., Kozlowski, D. A., Humm, J. L., & Cocke, R. R. (1997). Use-dependent structural events in recovery of function. Adv Neurol, 73, 229-238.  Schallert, T., & Lindner, M. D. (1990). Rescuing neurons from trans-synaptic degeneration after brain damage: helpful, harmful, or neutral in recovery of function? Can J Psychol, 44(2), 276-292.  Schallert, T., Pendergrass, M., & Farrar, S. B. (1982). Cholecystokinin-octapeptide effects on eating elicited by "external" versus "internal" cues in rats. Appetite, 3(2), 81-90.  Schiwy, N., Brazda, N., & Muller, H. W. (2009). Enhanced regenerative axon growth of multiple fibre populations in traumatic spinal cord injury following scar-suppressing treatment. 159  Eur J Neurosci, 30(8), 1544-1553. doi: EJN6929 [pii] 10.1111/j.1460-9568.2009.06929.x [doi] Schmued, L., Kyriakidis, K., & Heimer, L. (1990). In vivo anterograde and retrograde axonal transport of the fluorescent rhodamine-dextran-amine, Fluoro-Ruby, within the CNS. Brain Res, 526(1), 127-134.  Schreyer, D. J., & Jones, E. G. (1982). Growth and target finding by axons of the corticospinal tract in prenatal and postnatal rats. Neuroscience, 7(8), 1837-1853.  Schrimsher, G. W., & Reier, P. J. (1993). Forelimb motor performance following dorsal column, dorsolateral funiculi, or ventrolateral funiculi lesions of the cervical spinal cord in the rat. Exp Neurol, 120(2), 264-276. doi: S0014-4886(83)71060-5 [pii] 10.1006/exnr.1993.1060 [doi] Schwab, J. M., Brechtel, K., Mueller, C. A., Failli, V., Kaps, H. P., Tuli, S. K., & Schluesener, H. J. (2006). Experimental strategies to promote spinal cord regeneration--an integrative perspective. Prog Neurobiol, 78(2), 91-116. doi: S0301-0082(05)00196-6 [pii] 10.1016/j.pneurobio.2005.12.004 [doi] Schwab, M. E., & Caroni, P. (1988). Oligodendrocytes and CNS myelin are nonpermissive substrates for neurite growth and fibroblast spreading in vitro. J Neurosci, 8(7), 2381-2393.  Schwede, F., Christensen, A., Liauw, S., Hippe, T., Kopperud, R., Jastorff, B., & Doskeland, S. O. (2000). 8-Substituted cAMP analogues reveal marked differences in adaptability, hydrogen bonding, and charge accommodation between homologous binding sites (AI/AII and BI/BII) in cAMP kinase I and II. Biochemistry, 39(30), 8803-8812. doi: bi000304y [pii] Schweigreiter, R., Walmsley, A. R., Niederost, B., Zimmermann, D. R., Oertle, T., Casademunt, E., Frentzel, S., Dechant, G., Mir, A., & Bandtlow, C. E. (2004). Versican V2 and the central inhibitory domain of Nogo-A inhibit neurite growth via p75NTR/NgR-independent pathways that converge at RhoA. Mol Cell Neurosci, 27(2), 163-174. doi: S1044-7431(04)00130-7 [pii] 10.1016/j.mcn.2004.06.004 [doi] Seiler, N., Sarhan, S., & Roth-Schechter, B. F. (1984). Polyamines and the development of isolated neurons in cell culture. Neurochem Res, 9(7), 871-886.  Seino, S., & Shibasaki, T. (2005). PKA-Dependent and PKA-Independent Pathways for cAMP-Regulated Exocytosis. Physiol. Rev., 85(4), 1303-1342. doi: 10.1152/physrev.00001.2005 Semmler, J., Wachtel, H., & Endres, S. (1993). The specific type IV phosphodiesterase inhibitor rolipram suppresses tumor necrosis factor-alpha production by human mononuclear cells. Int J Immunopharmacol, 15(3), 409-413.  160  Serezani, C. H., Ballinger, M. N., Aronoff, D. M., & Peters-Golden, M. (2008). Cyclic AMP: master regulator of innate immune cell function. Am J Respir Cell Mol Biol, 39(2), 127-132. doi: 2008-0091TR [pii] 10.1165/rcmb.2008-0091TR [doi] Sette, C., & Conti, M. (1996). Phosphorylation and activation of a cAMP-specific phosphodiesterase by the cAMP-dependent protein kinase. Involvement of serine 54 in the enzyme activation. J Biol Chem, 271(28), 16526-16534.  Shao, Z., Browning, J., Lee, X., Scott, M., Shulga-Morskaya, S., Allaire, N., Thill, G., Levesque, M., Sah, D., & McCoy, J. (2005). TAJ/TROY, an orphan TNF receptor family member, binds Nogo-66 receptor 1 and regulates axonal regeneration. Neuron, 45(3), 353 - 359. doi: 10.1016/j.neuron.2004.12.050 Shaywitz, A. J., & Greenberg, M. E. (1999). CREB: A Stimulus-Induced Transcription Factor Activated by A Diverse Array of Extracellular Signals. Annual Review of Biochemistry, 68(1), 821-861. doi: 10.1146/annurev.biochem.68.1.821 Shelly, M., Cancedda, L., Lim, Byung K., Popescu, A. T., Cheng, P.-l., Gao, H., & Poo, M.-m. (2011). Semaphorin3A Regulates Neuronal Polarization by Suppressing Axon Formation and Promoting Dendrite Growth. Neuron, 71(3), 433-446. doi: 10.1016/j.neuron.2011.06.041 Shi, G.-X., Rehmann, H., & Andres, D. A. (2006). A Novel Cyclic AMP-Dependent Epac-Rit Signaling Pathway Contributes to PACAP38-Mediated Neuronal Differentiation. Mol. Cell. Biol., 26(23), 9136-9147. doi: 10.1128/mcb.00332-06 Shumsky, J. S., Tobias, C. A., Tumolo, M., Long, W. D., Giszter, S. F., & Murray, M. (2003). Delayed transplantation of fibroblasts genetically modified to secrete BDNF and NT-3 into a spinal cord injury site is associated with limited recovery of function. Exp Neurol, 184(1), 114-130. doi: S0014488603003984 [pii] Silver, J., & Miller, J. H. (2004). Regeneration beyond the glial scar. Nat Rev Neurosci, 5(2), 146-156. doi: 10.1038/nrn1326 nrn1326 [pii] Skalhegg, B. S., Johansen, A. K., Levy, F. O., Andersson, K. B., Aandahl, E. M., Blomhoff, H. K., Hansson, V., & Tasken, K. (1998). Isozymes of cyclic AMP-dependent protein kinases (PKA) in human lymphoid cell lines: levels of endogenous cAMP influence levels of PKA subunits and growth in lymphoid cell lines. J Cell Physiol, 177(1), 85-93. doi: 10.1002/(SICI)1097-4652(199810)177 Skalhegg, B. S., & Tasken, K. (2000). Specificity in the cAMP/PKA signaling pathway. Differential expression,regulation, and subcellular localization of subunits of PKA. Front Biosci, 5, D678-693.  Skene, J. H. (1989). Axonal growth-associated proteins. Annu Rev Neurosci, 12, 127-156. doi: 10.1146/annurev.ne.12.030189.001015 161  Skene, J. H., & Willard, M. (1981a). Axonally transported proteins associated with axon growth in rabbit central and peripheral nervous systems. J Cell Biol, 89(1), 96-103.  Skene, J. H., & Willard, M. (1981b). Electrophoretic analysis of axonally transported proteins in toad retinal ganglion cells. J Neurochem, 37(1), 79-87.  Sklar, P., Anholt, R., & Snyder, S. (1986). The odorant-sensitive adenylate cyclase of olfactory receptor cells. Differential stimulation by distinct classes of odorants. J. Biol. Chem., 261(33), 15538-15543.  Soblosky, J. S., Song, J.-H., & Dinh, D. H. (2001). Graded unilateral cervical spinal cord injury in the rat: evaluation of forelimb recovery and histological effects. Behavioural Brain Research, 119(1), 1-13. doi: 10.1016/S0166-4328(00)00328-4 Soderling, S. H., Bayuga, S. J., & Beavo, J. A. (1998). Identification and characterization of a novel family of cyclic nucleotide phosphodiesterases. J Biol Chem, 273(25), 15553-15558.  Soldatenkov, V. A., Prasad, S., Voloshin, Y., & Dritschilo, A. (1998). Sodium butyrate induces apoptosis and accumulation of ubiquitinated proteins in human breast carcinoma cells. Cell Death Differ, 5(4), 307-312. doi: 10.1038/sj.cdd.4400345 Solomon, S. S., Brush, J. S., & Kitabchi, A. E. (1970). Divergent Biological Effects of Adenosine and Dibutyryl Adenosine 3',5'-Monophosphate on the Isolated Fat Cell. Science, 169(3943), 387-388.  Somers, M. F. (2001). Spinal Cord Injury: Functional Rehabilitation (2nd ed.). Upper Saddle River, New Jersey: Prentice Hall. Spaulding, S. W. (1993). The ways in which hormones change cyclic adenosine 3',5'-monophosphate-dependent protein kinase subunits, and how such changes affect cell behavior. Endocr Rev, 14(5), 632-650.  Stackhouse, S. K., Murray, M., & Shumsky, J. S. (2008). Effect of Cervical Dorsolateral Funiculotomy on Reach-to-Grasp Function in the Rat. Journal of Neurotrauma, 25(8), 1039-1047. doi: 10.1089/neu.2007.0419 Sunahara, R. K., Dessauer, C. W., & Gilman, A. G. (1996). Complexity and diversity of mammalian adenylyl cyclases. Annu Rev Pharmacol Toxicol, 36, 461-480. doi: 10.1146/annurev.pa.36.040196.002333 Sunahara, R. K., & Taussig, R. (2002). Isoforms of Mammalian Adenylyl Cyclase: Multiplicities of Signaling. Mol. Interv., 2(3), 168-184. doi: 10.1124/mi.2.3.168 Sutherland, E. W. (1971). [Nobel prize in physiology or medicine 1971: the action of hormones outlined]. Lakartidningen, 68(44), 4991-4995.  162  Sutherland, E. W., & Rall, T. W. (1958). Fractionation and characterization of a cyclic adenine ribonucleotide formed by tissue particles. J Biol Chem, 232(2), 1077-1091.  Sutkowski, E. M., Tang, W. J., Broome, C. W., Robbins, J. D., & Seamon, K. B. (1994). Regulation of forskolin interactions with type I, II, V, and VI adenylyl cyclases by Gs alpha. Biochemistry, 33(43), 12852-12859.  Svenningsson, P., Nishi, A., Fisone, G., Girault, J.-A., Nairn, A. C., & Greengard, P. (2004). DARPP-32: An Integrator of Neurotransmission. Annual Review of Pharmacology and Toxicology, 44(1), 269-296. doi: 10.1146/annurev.pharmtox.44.101802.121415 Swislocki, N. I. (1970). Decomposition of dibutyryl cyclic AMP in aqueous buffers. Anal Biochem, 38(1), 260-269.  Tanaka, S., Ishii, K., Kasai, K., Yoon, S. O., & Saeki, Y. (2007). Neural expression of G protein-coupled receptors GPR3, GPR6, and GPR12 up-regulates cyclic AMP levels and promotes neurite outgrowth. J Biol Chem, 282(14), 10506-10515. doi: M700911200 [pii] 10.1074/jbc.M700911200 [doi] Tasken, K., & Aandahl, E. M. (2004). Localized effects of cAMP mediated by distinct routes of protein kinase A. Physiol Rev, 84(1), 137-167. doi: 10.1152/physrev.00021.2003 Tasken, K., Skalhegg, B. S., Tasken, K. A., Solberg, R., Knutsen, H. K., Levy, F. O., Sandberg, M., Orstavik, S., Larsen, T., Johansen, A. K., Vang, T., Schrader, H. P., Reinton, N. T., Torgersen, K. M., Hansson, V., & Jahnsen, T. (1997). Structure, function, and regulation of human cAMP-dependent protein kinases. Adv Second Messenger Phosphoprotein Res, 31, 191-204.  Taussig, R., Iniguez-Lluhi, J. A., & Gilman, A. G. (1993). Inhibition of adenylyl cyclase by Gi alpha. Science, 261(5118), 218-221.  Taussig, R., Tang, W., Hepler, J., & Gilman, A. (1994). Distinct patterns of bidirectional regulation of mammalian adenylyl cyclases. J. Biol. Chem., 269(8), 6093-6100.  Taylor, S. S., Kim, C., Cheng, C. Y., Brown, S. H., Wu, J., & Kannan, N. (2008). Signaling through cAMP and cAMP-dependent protein kinase: diverse strategies for drug design. Biochim Biophys Acta, 1784(1), 16-26. doi: S1570-9639(07)00256-7 [pii] 10.1016/j.bbapap.2007.10.002 [doi] Taylor, S. S., Radzio-Andzelm, E., Madhusudan, Cheng, X., Ten Eyck, L., & Narayana, N. (1999). Catalytic subunit of cyclic AMP-dependent protein kinase: structure and dynamics of the active site cleft. Pharmacol Ther, 82(2-3), 133-141.  Tegenge, M. A., Roloff, F., & Bicker, G. (2011). Rapid differentiation of human embryonal carcinoma stem cells (NT2) into neurons for neurite outgrowth analysis. Cell Mol Neurobiol, 31(4), 635-643. doi: 10.1007/s10571-011-9659-4 163  Tello, J. F. (1911). La influencia del neurotropismo en la regenerati?n de los centros nerviosos. Trab. Lab. Invest. Biol. Univ. Madr., 9, 123-159.  Tello, J. F. (1923). Gegenw?rtige Anschauugen ?ber den Neurotropismus. Vortr. EntwMech. Org., 33, 1-73.  ten Donkelaar, H. J. (1988). Evolution of the red nucleus and rubrospinal tract. Behavioural Brain Research, 28(1-2), 9-20. doi: 10.1016/0166-4328(88)90072-1 Tesmer, J. J. G., Sunahara, R. K., Gilman, A. G., & Sprang, S. R. (1997). Crystal Structure of the Catalytic Domains of Adenylyl Cyclase in a Complex with Gs alpha GTPS. Science, 278(5345), 1907-1916. doi: 10.1126/science.278.5345.1907 Tetzlaff, W., Alexander, S. W., Miller, F. D., & Bisby, M. A. (1991). Response of facial and rubrospinal neurons to axotomy: changes in mRNA expression for cytoskeletal proteins and GAP-43. J Neurosci, 11(8), 2528-2544.  Tetzlaff, W., & Bisby, M. A. (1989). Neurofilament elongation into regenerating facial nerve axons. Neuroscience, 29(3), 659-666.  Tetzlaff, W., Bisby, M. A., & Kreutzberg, G. W. (1988). Changes in cytoskeletal proteins in the rat facial nucleus following axotomy. J Neurosci, 8(9), 3181-3189.  Tetzlaff, W., Kobayashi, N. R., Giehl, K. M., Tsui, B. J., Cassar, S. L., & Bedard, A. M. (1994). Response of rubrospinal and corticospinal neurons to injury and neurotrophins. Prog Brain Res, 103, 271-286.  Tetzlaff, W., & Kreutzberg, G. W. (1985). Ornithine decarboxylase in motoneurons during regeneration. Exp Neurol, 89(3), 679-688.  Tetzlaff, W., Leonard, C., Krekoski, C. A., Parhad, I. M., & Bisby, M. A. (1996). Reductions in motoneuronal neurofilament synthesis by successive axotomies: a possible explanation for the conditioning lesion effect on axon regeneration. Exp Neurol, 139(1), 95-106. doi: 10.1006/exnr.1996.0084 Thallmair, M., Metz, G. A., Z'Graggen, W. J., Raineteau, O., Kartje, G. L., & Schwab, M. E. (1998). Neurite growth inhibitors restrict plasticity and functional recovery following corticospinal tract lesions. Nat Neurosci, 1(2), 124-131. doi: 10.1038/373 The Rick Hansen Institute. (2011). Spinal Cord Injury: Progress in Care & Outcomes in the Last 25 Years. In H. K. A. Inc. (Ed.), Health Economics, Planning and Information (pp. 73). Delta, BC. Tillerson, J. L., Cohen, A. D., Caudle, W. M., Zigmond, M. J., Schallert, T., & Miller, G. W. (2002). Forced nonuse in unilateral parkinsonian rats exacerbates injury. J Neurosci, 22(15), 6790-6799. doi: 22/15/6790 [pii] 20026651 [doi] 164  Tillerson, J. L., Cohen, A. D., Philhower, J., Miller, G. W., Zigmond, M. J., & Schallert, T. (2001). Forced limb-use effects on the behavioral and neurochemical effects of 6-hydroxydopamine. J Neurosci, 21(12), 4427-4435. doi: 21/12/4427 [pii] Treistman, S. N., & Levitan, I. B. (1976). Alteration of electrical activity in molluscan neurones by cyclic nucleotides and peptide factors. Nature, 261(5555), 62-64.  Tresguerres, M., Levin, L. R., & Buck, J. (2011). Intracellular cAMP signaling by soluble adenylyl cyclase. Kidney Int, 79(12), 1277-1288. doi: 10.1038/ki.2011.95 Udina, E., Ladak, A., Furey, M., Brushart, T., Tyreman, N., & Gordon, T. (2010). Rolipram-induced elevation of cAMP or chondroitinase ABC breakdown of inhibitory proteoglycans in the extracellular matrix promotes peripheral nerve regeneration. Exp Neurol, 223(1), 143-152. doi: S0014-4886(09)00365-3 [pii] 10.1016/j.expneurol.2009.08.026 [doi] Vahlsing, H. L., & Feringa, E. R. (1980). A ventral uncrossed corticospinal tract in the rat. Experimental Neurology, 70(2), 282-287. doi: 10.1016/0014-4886(80)90027-8 van Kan, P. L., & McCurdy, M. L. (2001). Role of primate magnocellular red nucleus neurons in controlling hand preshaping during reaching to grasp. J Neurophysiol, 85(4), 1461-1478.  van Kan, P. L., & McCurdy, M. L. (2002a). Contribution of primate magnocellular red nucleus to timing of hand preshaping during reaching to grasp. J Neurophysiol, 87(3), 1473-1487.  van Kan, P. L., & McCurdy, M. L. (2002b). Discharge of primate magnocellular red nucleus neurons during reaching to grasp in different spatial locations. Exp Brain Res, 142(1), 151-157. doi: 10.1007/s00221-001-0924-5 Vargas-Origel, A., Gomez-Rodriguez, G., Aldana-Valenzuela, C., Vela-Huerta, M. M., Alarcon-Santos, S. B., & Amador-Licona, N. (2009). The Use of Sildenafil in Persistent Pulmonary Hypertension of the Newborn. Am J Perinatol, 27(3), 225-230. doi: 10.1055/s-0029-1239496 Veenman, C. L., Reiner, A., & Honig, M. G. (1992). Biotinylated dextran amine as an anterograde tracer for single- and double-labeling studies. Journal of Neuroscience Methods, 41(3), 239-254.  Vergara-Aragon, P., Gonzalez, C. L., & Whishaw, I. Q. (2003). A novel skilled-reaching impairment in paw supination on the "good" side of the hemi-Parkinson rat improved with rehabilitation. J Neurosci, 23(2), 579-586. doi: 23/2/579 [pii] Vintermyr, O., Boe, R., Brustugun, O., Maronde, E., Aakvaag, A., & Doskeland, S. (1995). Cyclic adenosine monophosphate (cAMP) analogs 8-Cl- and 8-NH2-cAMP induce cell death independently of cAMP kinase-mediated inhibition of the G1/S transition in mammary carcinoma cells (MCF-7). Endocrinology, 136(6), 2513-2520. doi: 10.1210/en.136.6.2513 165  Visel, A., Alvarez-Bolado, G., Thaller, C., & Eichele, G. (2006). Comprehensive analysis of the expression patterns of the adenylate cyclase gene family in the developing and adult mouse brain. The Journal of Comparative Neurology, 496(5), 684-697. doi: 10.1002/cne.20953 Vlachopoulos, C., Terentes-Printzios, D., Ioakeimidis, N., Rokkas, K., & Stefanadis, C. (2009). PDE5 inhibitors in non-urological conditions. Curr Pharm Des, 15(30), 3521-3539.  Voorhies, A. C., & Jones, T. A. (2002). The behavioral and dendritic growth effects of focal sensorimotor cortical damage depend on the method of lesion induction. Behav Brain Res, 133(2), 237-246. doi: S0166432802000293 [pii] Wahl, S., Barth, H., Ciossek, T., Aktories, K., & Mueller, B. K. (2000). Ephrin-A5 Induces Collapse of Growth Cones by Activating Rho and Rho Kinase. J. Cell Biol., 149(2), 263-270. doi: 10.1083/jcb.149.2.263 Walsh, D. A., Perkins, J. P., & Krebs, E. G. (1968). An Adenosine 3',5'-Monophosphate-dependant Protein Kinase from Rabbit Skeletal Muscle. J. Biol. Chem., 243(13), 3763-3765.  Wan, G., Zhou, L., Lim, Q. E., Wong, Y. H., & Too, H.-P. (2011). Cyclic AMP signalling through PKA but not Epac is essential for neurturin-induced biphasic ERK1/2 activation and neurite outgrowths through GFR?2 isoforms. Cellular Signalling, 23(11), 1727-1737. doi: 10.1016/j.cellsig.2011.06.007 Wang, K. C., Kim, J. A., Sivasankaran, R., Segal, R., & He, Z. (2002). P75 interacts with the Nogo receptor as a co-receptor for Nogo, MAG and OMgp. Nature, 420(6911), 74-78. doi: Doi 10.1038/Nature01176 Wang, K. C., Koprivica, V., Kim, J. A., Sivasankaran, R., Guo, Y., Neve, R. L., & He, Z. (2002). Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature, 417(6892), 941-944. doi: 10.1038/nature00867 Weidner, N., Ner, A., Salimi, N., & Tuszynski, M. H. (2001). Spontaneous corticospinal axonal plasticity and functional recovery after adult central nervous system injury. Proc Natl Acad Sci U S A, 98(6), 3513-3518. doi: 10.1073/pnas.051626798 Westphal, R. S., Soderling, S. H., Alto, N. M., Langeberg, L. K., & Scott, J. D. (2000). Scar/WAVE-1, a Wiskott-Aldrich syndrome protein, assembles an actin-associated multi-kinase scaffold. EMBO J, 19(17), 4589-4600. doi: 10.1093/emboj/19.17.4589 Whishaw, I. Q., & Gorny, B. (1996). Does the red nucleus provide the tonic support against which fractionated movements occur? A study on forepaw movements used in skilled reaching by the rat. Behav Brain Res, 74(1-2), 79-90.  Whishaw, I. Q., Gorny, B., & Sarna, J. (1998). Paw and limb use in skilled and spontaneous reaching after pyramidal tract, red nucleus and combined lesions in the rat: behavioral and anatomical dissociations. Behav Brain Res, 93(1-2), 167-183.  166  Whishaw, I. Q., Pellis, S. M., Gorny, B. P., & Pellis, V. C. (1991). The impairments in reaching and the movements of compensation in rats with motor cortex lesions: an endpoint, videorecording, and movement notation analysis. Behav Brain Res, 42(1), 77-91.  Whishaw, I. Q., Tomie, J. A., & Ladowsky, R. L. (1990). Red nucleus lesions do not affect limb preference or use, but exacerbate the effects of motor cortex lesions on grasping in the rat. Behav Brain Res, 40(2), 131-144.  Whishaw, I. Q., Woodward, N. C., Miklyaeva, E., & Pellis, S. M. (1997). Analysis of limb use by control rats and unilateral DA-depleted rats in the Montoya staircase test: movements, impairments and compensatory strategies. Behav Brain Res, 89(1-2), 167-177.  Wilkins, R. H. (1964). NEUROSURGICAL CLASSIC. XVII. [Historical Article]. J Neurosurg, 21, 240-244. doi: 10.3171/jns.1964.21.3.0240 Wilkins, R. H. (2011). Neurosurgical Classic-XVII Edwin Smith Surgical Papyrus. Cyber Museum of Neurosurgery, 2011, from http://www.neurosurgery.org/cybermuseum/pre20th/epapyrus.html Will, B., & Kelche, C. (1992). Environmental approaches to recovery of function from brain damage: a review of animal studies (1981 to 1991). Adv Exp Med Biol, 325, 79-103.  Willoughby, D., & Cooper, D. M. F. (2007). Organization and Ca2+ Regulation of Adenylyl Cyclases in cAMP Microdomains. Physiol. Rev., 87(3), 965-1010. doi: 10.1152/physrev.00049.2006 Wong, B. J., & Mattox, D. E. (1991). The effects of polyamines and polyamine inhibitors on rat sciatic and facial nerve regeneration. Exp Neurol, 111(2), 263-266. doi: 0014-4886(91)90014-4 [pii] Wong, S. T., Henley, J. R., Kanning, K. C., Huang, K. H., Bothwell, M., & Poo, M. M. (2002). A p75(NTR) and Nogo receptor complex mediates repulsive signaling by myelin-associated glycoprotein. Nat Neurosci, 5(12), 1302-1308. doi: nn975 [pii]10.1038/nn975 [doi] Wong, S. T., Trinh, K., Hacker, B., Chan, G. C. K., Lowe, G., Gaggar, A., Xia, Z., Gold, G. H., & Storm, D. R. (2000). Disruption of the Type III Adenylyl Cyclase Gene Leads to Peripheral and Behavioral Anosmia in Transgenic Mice. Neuron, 27(3), 487-497.  Wouterlood, F. G., & Jorritsma-Byham, B. (1993). The anterograde neuroanatomical tracer biotinylated dextran-amine: comparison with the tracer Phaseolus vulgaris-leucoagglutinin in preparations for electron microscopy. Journal of Neuroscience Methods, 48(1-2), 75-87.  Wu, D., Yang, P., Zhang, X., Luo, J., Haque, M. E., Yeh, J., Richardson, P. M., Zhang, Y., & Bo, X. (2009). Targeting a dominant negative rho kinase to neurons promotes axonal outgrowth and partial functional recovery after rat rubrospinal tract lesion. Mol Ther, 17(12), 2020-2030. doi: mt2009168 [pii] 10.1038/mt.2009.168 [doi] 167  Wu, D., Zhang, Y., Bo, X., Huang, W., Xiao, F., Zhang, X., Miao, T., Magoulas, C., Subang, M. C., & Richardson, P. M. (2007). Actions of neuropoietic cytokines and cyclic AMP in regenerative conditioning of rat primary sensory neurons. Exp Neurol, 204(1), 66-76. doi: S0014-4886(06)00555-3 [pii] 10.1016/j.expneurol.2006.09.017 [doi] Wu, K. Y., Zippin, J. H., Huron, D. R., Kamenetsky, M., Hengst, U., Buck, J., Levin, L. R., & Jaffrey, S. R. (2006). Soluble adenylyl cyclase is required for netrin-1 signaling in nerve growth cones. Nat Neurosci, 9(10), 1257-1264. doi: 10.1038/nn1767 Xia, Z., & Storm, D. R. (1997). Calmodulin-regulated adenylyl cyclases and neuromodulation. Current Opinion in Neurobiology, 7(3), 391-396.  Xu, N., Engbers, J., Khaja, S., Xu, L., Clark, J. J., & Hansen, M. R. (2012). Influence of cAMP and protein kinase A on neurite length from spiral ganglion neurons. Hearing Research, 283(1?2), 33-44. doi: 10.1016/j.heares.2011.11.010 Yakovlev, A. G., Knoblach, S. M., Fan, L., Fox, G. B., Goodnight, R., & Faden, A. I. (1997). Activation of CPP32-like caspases contributes to neuronal apoptosis and neurological dysfunction after traumatic brain injury. J Neurosci, 17(19), 7415-7424.  Yamaguchi, K., & Goto, N. (2006). Development of the human magnocellular red nucleus: A morphological study. Brain and Development, 28(7), 431-435. doi: 10.1016/j.braindev.2006.01.001 Yamashita, T., Higuchi, H., & Tohyama, M. (2002). The p75 receptor transduces the signal from myelin-associated glycoprotein to Rho. J. Cell Biol., 157(4), 565-570. doi: 10.1083/jcb.200202010 Yamashita, T., & Tohyama, M. (2003). The p75 receptor acts as a displacement factor that releases Rho from Rho-GDI. Nat Neurosci, 6(5), 461-467. doi: nn1045 [pii]10.1038/nn1045 [doi] Yamashita, T., Tucker, K. L., & Barde, Y.-A. (1999). Neurotrophin Binding to the p75 Receptor Modulates Rho Activity and Axonal Outgrowth. Neuron, 24(3), 585-593.  Yan, S.-Z., Huang, Z.-H., Andrews, R. K., & Tang, W.-J. (1998). Conversion of Forskolin-Insensitive to Forskolin-Sensitive (Mouse-Type IX) Adenylyl Cyclase. Mol Pharmacol, 53(2), 182-187.  Yin, Y., Cui, Q., Gilbert, H. Y., Yang, Y., Yang, Z., Berlinicke, C., Li, Z., Zaverucha-do-Valle, C., He, H., Petkova, V., Zack, D. J., & Benowitz, L. I. (2009). Oncomodulin links inflammation to optic nerve regeneration. Proc Natl Acad Sci U S A, 106(46), 19587-19592. doi: 10.1073/pnas.0907085106 Yin, Y., Henzl, M. T., Lorber, B., Nakazawa, T., Thomas, T. T., Jiang, F., Langer, R., & Benowitz, L. I. (2006). Oncomodulin is a macrophage-derived signal for axon regeneration in retinal ganglion cells. Nat Neurosci, 9(6), 843-852. doi: 10.1038/nn1701 168  Yiu, G., & He, Z. (2006). Glial inhibition of CNS axon regeneration. Nature Reviews. Neuroscience, 7(8), 617-627. doi: 10.1038/nrn1956 Yusta, B., Ortiz-Caro, J., Pascual, A., & Aranda, A. (1988). Comparison of the Effects of Forskolin and Dibutyryl Cyclic AMP in Neuroblastoma Cells: Evidence that Some of the Actions of Dibutyryl Cyclic AMP Are Mediated by Butyrate. Journal Of Neurochemistry, 51(6), 1808-1818.  Zelenin, P. V., Beloozerova, I. N., Sirota, M. G., Orlovsky, G. N., & Deliagina, T. G. (2010). Activity of red nucleus neurons in the cat during postural corrections. J Neurosci, 30(43), 14533-14542. doi: 30/43/14533 [pii] 10.1523/JNEUROSCI.2991-10.2010 [doi]     169  Appendix  Table I: Summary of notable neuronal studies involving axon regeneration or survival using altered levels of cAMP Studies listed here involve changing cAMP levels by various means such as with the use of PDE inhibitors, AC activators or with cAMP analogues etc. in order to: stimulate or augment a regenerative axonal response after injury or damage, overcome axonal inhibitors to regeneration (in-vitro or in-vivo), increase axonal recovery after injury or increase survival etc.  The table is ordered chronologically.  Abbreviations for the table: N.S.:Nervous System, Tx:Treatment, Ctl:Control, N/A: Not Applicable/Available, CL: Conditioning Lesion, SC: Schwann Cell, TG: Transgenic, GC: Growth Cone, inh: Inhibitor, inc: Increase, prcd: Produced, phos: Phosphorylated.  170      Source Model and Tests Paradigm Outcome In-vitro N.S. In-vivo N.S. In-vitro In-vivo In-vitro In-vivo Roisen et al., (1972) - Cell Model: 8.5 day old cultured DRGs (White Leghorn Chick) PNS N/A N/A - Tx: (5 mM) db-cAMP or cAMP - Ctl: Base medium or 5?-AMP (5 mM) - Applied to culture - 48hrs- assessment N/A - Db-cAMP produced longer and more numerous axons than the other test groups N/A Pichichero et al., (1973) N/A N/A - Animal model: Holtzman rat (adult) - Injury: sciatic nerve crush - Behavioural Tests: sensorimotor foot flick test PNS N/A - Tx: (50mg/kg) db-cAMP - Ctl: Saline - Daily injection of Tx or Ctl in biceps leg muscle  N/A - Sensorimotor function returned much quicker for the db-cAMP treated animals McQuarrie et al., (1977) N/A N/A - Animal Model: Rat (Adult) - Injury: sciatic nerve crush - Behavioural Tests: Pinch test (6 or 9days after) PNS N/A - Tx: (50mg/kg) db-cAMP - Ctl: Saline - Daily injection of Tx or Ctl in biceps leg muscle N/A - Pinch test results were no different between saline or db-cAMP treated groups. Gershenbaum et al.,(1980) N/A N/A - Animal Model: Rat (Adult) - Injury: sciatic nerve crush - Behavioural Tests: Sensorimotor heat test PNS N/A - Tx: (50mg/kg) db-cAMP - Ctl: Saline - Daily injection of Tx or Ctl in biceps leg muscle N/A - Sensorimotor function returned much quicker for the db-cAMP treated animals (16d versus 26d) Gunning et al., (1981) - Cell model: PC12 PNS N/A N/A - Tx: db-cAMP (1 mM) or NGF (ng/ml) or both - 7 day assessment N/A - db-cAMP changes RNA and protein levels - db-cAMP cannot prime cells - initiates but does not maintain neurite growth - effects are additive with NGF N/A Carlsen et al., (1982b) N/A N/A - Animal model: Sprague-Dawley rat (adult) - Injury: sciatic nerve crush - Behavioural Tests: Pinch test (1,3,5 days) PNS N/A - Conditioning lesion 7 days prior to crush - Measurement of AC along the axon N/A - AC accumulated proximal to injury site - cAMP accumulation correlated with sprouting response 171      Source Model and Tests Paradigm Outcome In-vitro N.S. In-vivo N.S. In-vitro In-vivo In-vitro In-vivo Kilmer et al. (1984) N/A N/A - Animal model: Rana pipiens - Injury: sciatic nerve crush - Behavioural Tests: none PNS N/A - Tx: 0.5ml of 10-4M forskolin (AC stimulant) via mini-pump to dorsal lymph sac - Measurement: rate of nerve regeneration N/A - Forskolin treated frogs experienced a 40% increase in the rate of sensory nerve regeneration Vs controls Kilmer et al., (1987) N/A N/A - Animal model: Hamsters (adult) - Injury: sciatic nerve crush - Behavioural Test: pinch test PNS N/A - Tx: 21day pellet with: db-cAMP (.23mg/day), forskolin (.23mg/day), 8-Br-cAMP (.71mg/day), caffeine (1.19mg/day), placebo - Measurement: rate of nerve regeneration  N/A - 8-Br-cAMP had the greatest reduction in growth initiation time and fastest regeneration rates followed by forskolin - Db-cAMP decreased initiation time, not rate - Caffeine reduced initiation rate and regeneration rate but did not increase cAMP levels Rydel et al., (1988) - Cell model: rat superior cervical ganglion (P1-3), rat DRG (E15) PNS N/A N/A - Tx: cultures treated with either NGF, CPT-cAMP, 8Br-cAMP, db-cAMP, Oco2-cAMP, forskolin - Measurement: survival and neurite outgrowth N/A - CPT-cAMP and 8Br-cAMP resulted in the greatest survival times and neurite outgrowth in comparison to the other treatments N/A Hanson et al., (1998) - Cell model: rat SMN (spinal motor neurons; E15) CNS N/A N/A - Tx: cultures treated with combinations of  BDNF, CTNF, GDNF, FGF, BMP-7, HGF (10ng/ml) and/or NT-3 (50ng/ml) versus IMBX (10 mM) and/or forskolin (10?M) - Measurement: MTT survival assay  N/A - Added effects of trophic factors alone are transient, SMNs die < 1 week - Increases in cAMP without trophic factors allow most SMNs to survive ? 1 week - Multiple factors + increases in cAMP produce survival times > 3 weeks N/A 172      Source Model and Tests Paradigm Outcome In-vitro N.S. In-vivo N.S. In-vitro In-vivo In-vitro In-vivo Cai et al., (1999) - Cell model: rat cerebellar neurons and DRGs (>P7-9)  CNSPNS N/A N/A - Tx: cultures treated with either db-cAMP (1mM), BNDF, GDNF or NGF (200ng/ml) and plated on MAG expressing CHO cells N/A - Cultures primed with neurotrophins (not NGF) overcome MAG via cAMP pathway - Cultures exposed to db-cAMP overcame MAG inhibition N/A Cai et al., (2001) - Cell model: rat retinal ganglion neurons (E18 or P5) and DRGs (P1 or P5) CNSPNS - Animal model: rat (P2-3) - Injury: T6 overhemisection - Behavioural Tests: none CNS - Tx: cultures treated with db-cAMP (1mM), H89 (2?M), KT2750 (200nM), KT5820(1 ?M), Rp-cAMP (20?M) or Sp-cAMP (50?M) - Tx: transplant with H-89 or saline placed on spinal cord - Measurement: growth of fibres through transplant  - Neurite growth prevented in cultures with either PKA or cAMP blockers in P1 cultures suggesting cAMP mechanism necessary for plasticity and growth - CST fibres failed to grow into transplants with PKA blockers Neumann et al., (2002) - Cell model: rat DRGs (adult) PNS - Animal model: rat (adult) - Injury: T6-7 bilateral dorsal column lesion - Behavioural Tests: none CNS - DRGs cultured on PDL coated with/without forskolin (48hrs) - Preinjected L4-5 DRGs with db-cAMP (1.5mL 10?M db-cAMP) grown on permissive and inhibitory (myelin based) substrates - Ctl: saline  - Tx: db-cAMP (1.5mL 10?M db-cAMP) injected into L4-5 DRGs - Measurement: growth of fibres through to, through or around injury  - Forskolin or preinjected db-cAMP enhanced neurite outgrowth over controls - Preinjected db-cAMP allows neurite outgrowth on inhibitory substrate - Sciatic dorsal column axons grew into, around or to the surface of the lesion site in db-cAMP injected rats - In non-injected animals sciatic dorsal column axons did not grow into the lesion Cai et al.,(2002) - Cell model: rat DRGs (P0-P7), cerebellar neurons (P5) PNSCNS N/A N/A - Tx: cultures treated with either db-cAMP (1 mM) or preprimed with BNDF (200ng/ml) 1 day before with or without DRB (5?M), and plated on MAG expressing CHO cells N/A - Ability of elevated cAMP cerebellar cells to extend over neurites is transcription dependent - Db-cAMP increases Arg-1 levels - Blocking polyamine synthesis prevents cAMP effect on neurite outgrowth N/A 173      Source Model and Tests Paradigm Outcome In-vitro N.S. In-vivo N.S. In-vitro In-vivo In-vitro In-vivo Qiu et al.,(2002) - Cell model: rat DRGs (P18) PNS - Animal model: rat (adult) - Injury: T7 bilateral dorsal column lesion - Behavioural Tests: none CNS - Tx: L4-5 DRG cultures treated with db-cAMP (1 mM), H89 (400nM), KT2750 (200nM) KT5823(1 mM) - Grown on myelin expressing CHO cells - Tx: 2 ?L of db-cAMP (50 mM), or saline injected into L4-5, or L5 DRGs - Ctl: saline injection - Measurement: fibre length  - Peripheral nerve lesions performed 1d prior to injury increase cAMP levels 3 fold - Growth on MAG inhibited neurite growth of DRGs injected with PKA inhibitors 1d or 1wk post lesion - DRGs that were injected with db-cAMP regenerated their dorsal column fibres significantly more than the saline injected controls Monsul et al., (2004) N/A N/A - Animal model: rat (adult) - Injury: optic nerve crush  - Behavioural Tests: none CNS N/A - Tx: db-cAMP (5 or 10 ?L of 139, 48, 17, 6 or 2 x  10-2 ?M db-cAMP) injected into superior orbit of eye - Ctl: 10 ?L sodium chloride - Measurement: count of axons past crush site  N/A - Single ocular injection of db-cAMP increased regeneration of optic nerves - Maximal efficacy dose is 6x10-2 ?M - Survival of RGCs not increased Nikulina et al., (2004) - Cell model: rat DRGs (30day old) PNS - Animal model: rat (adult) - Injury: spinal cord hemisection C3/4 - Behavioural Tests: vertical exploration CNS - Tx: DRG cultures treated 0.1, 0.25, 0.5, 1.0 or 2.0?M rolipram grown on myelin expressing CHO cells - Dissociated DRGs from in vivo studies grown on myelin expressing CHO cells - Tx: rolipram 0.4, 0.5 or 0.7?mol/kg and 0.4 ?mol/kg for 24, 36, and 72hrs + transplant. Rolipram delivered via osmotic mini-pump. - Ctl: vehicle 10 ?L/hr - Measurement: number of 5-HT fibres in transplant, GFAP reactivity - DRGs extended neurites on inhibitory myelin; 0.5?M rolipram most effective - Dissociated DRGs treated with rolipram overcame MAG inhibition in culture; 0.4?mol/kg/hr most effective     - Numbers of 5-HT fibres in transplant much greater than vehicle treated animals - Most effective dose of rolipram is 0.4 ?mol/kg/hr - GFAP reactivity decreased the most with 0.4 ?mol/kg/hr  rolipram treated group - Functional recovery improved for rolipram treated group as indicated by vertical exploration  test 174      Source Model and Tests Paradigm Outcome In-vitro N.S. In-vivo N.S. In-vitro In-vivo In-vitro In-vivo Pearse et al., (2004) N/A N/A - Animal model: rat (adult Fisher) - Injury: spinal cord moderate contusion (NYU) at T8 - Behavioural Tests: BBB CNS N/A - Tx: rolipram, Schwann Cell (transplant, 2x106 cells), SC+db-cAMP (0.25?l, 50mM), rolipram (0.5?l/hr, 0.5 mg/kg/day) +SC, rolipram+db-cAMP+SC  - Drugs via mini osmotic-pump - Ctl: injury alone N/A - cAMP levels decline after SCI contusions - db-cAMP and rolipram prevent cAMP levels from dropping - increased cAMP levels increase regeneration, SC myelination, myelinated axon preservation and axon sparing - rolipram+db-cAMP +SC most effective treatment combination   - elevated cAMP improved fine and gross locomotor function Gao et al., (2004) - Cell model: rat DRGs (P5-7), cerebellar neurons (P5-7) - Cell model:mouse (bitransgenic), DRGs , hippocampal neurons (1mth or 4mth) PNS CNS - Animal model: rat - Injury: dorsal column lesion T6-7 - Behavioural Tests: none CNS - Tx: DRG and cerebellar cultures grown on myelin expressing CHO cells with 1 mM db-cAMP and with or without KT-5720(PKA-I) (200nM), PI3K-I (20?M), MEK-I (5 ?M), CaMK-I (10 ?M) - Tx: Recombinant adenovirus expressing VP-CREB (constitutively active) injected into L4 DRGs - Ctl: GFP adenovirus injected into L4 DRGS  - Increases in cAMP cannot overcome MAG inhibition when DN CREB is induced - Constitutively active CREB overcame MAG inhibition - TG mice DRGS or hippocampal neurons with constitutive CREB overcame MAG inhibition - Arg-1 regulated by CREB - Db-cAMP prevented myelin from reducing CREB activation  - Activation of CREB by DRGs was sufficient to induce regeneration in vivo 175     Source Model and Tests Paradigm Outcome In-vitro N.S. In-vivo N.S. In-vitro In-vivo In-vitro In-vivo Han et al., (2004) N/A N/A - Animal model: rat - Injury: Sciatic nerve crush (for CL), dorsal column lesion, peripheral nerve graft - Behavioural Tests: none CNS N/A - Tx: L5 DRG injected with 2 ?L db-cAMP (50 mM), sciatic nerve crush (DRG untouched) - Ctl: L5 DRG injected with saline - Measurement: ?1 and ?III tubulin levels after injury, number of sensory axons in peripheral nerve graft N/A - Db-cAMP increased  ?1 and ?III tubulin levels much more and for longer periods than CL - Rate of regeneration not increased for db-cAMP treated groups - Db-cAMP did not increase velocity of tubulin towards axon tips Lane et al., (2004) N/A N/A - Animal model: rat (adult) - Injury: crush of dorsolateral funiculus at level C3/4 - Behavioural Tests: none CNS N/A - Tx: db-cAMP (25 mM) delivered at 12 ?L/day (via 2wk mini osmotic pump) proximal to RN. - Ctl: vehicle solution delivered at 12 ?L/day (via 2wk mini osmotic pump) proximal to RN. - Measurement: in situ hybridization of T?1 and GAP43 (RAGs) gene expression levels. Size of RN cells measured against uninjured contralateral side N/A - Db-cAMP maintained T?1 and GAP43 gene expression in the RN whereas the vehicle controls did not - Db-cAMP prevented atrophy of axotomized rubrospinal neurons in comparison to the vehicle controls 176     Source Model and Tests Paradigm Outcome In-vitro N.S. In-vivo N.S. In-vitro In-vivo In-vitro In-vivo Wu et al., (2006) - Cell model: rat  E15-16 DRG, E15 DSC (Dorsal Spinal Cord) explants, E18 Hippocampal  PNSCNS N/A N/A - Tx: DRGs & Hippocampal cells plated on PDL, fibronectin (1?g/ml); DSC (mimimal media) - plus: Netrin-1 (75ng/ml), KH7 (3?M) [sAC blocker], sAC lentivirus, KT5720 (1?M) [PKA blocker] - Ctl: saline, LacZ lentivirus, 8-Br-cAMP (3?M), KH7.15 (3?M) N/A - sAC found in peripheral & central neurons - sAC infected neurons had longer axons than LacZ ctls - Netrin-1 inc. cAMP levels 140% vs saline ctls, inc filopodia and GC size, activated sAC - KH7 blocked Netrin-1 effects - siRNA sAC (KO) reduced GC & filopodia elaboration - 8-Br-cAMP rescued KH7 tx cells - HT5720 blocked Netrin-1 effects N/A Yin et al., (2006) - Cell model: rat  (adult Sprague- Dawley) L4-5 DRG CNS - Animal model: rat (adult Fisher) - Injury: optic nerve crush - Behavioural Tests: N/A CNS - Tx: DRGs injected with saline (5?L; Ctl) or oncomodulin (200ng/ ?L) were cultured on laminin + poly-D-lysine with or w/o CSPGS for 7 days - Additional cultures included: mannose, forskolin and/or oncomodulin in RPMI-1640 - Tx: intraocular injections  of microspheres containing oncomodulin and/or sp-8-Br-cAMP in 10 ?L saline - Ctl: intraocular injections of blank microspheres in 10 ?L saline - Measurement: regenerating axon  length - Oncomodulin bound strongly to RGC when cAMP levels were increased by forskolin - DRGs grown on substrate with CSPGs did not grow neurites - Pretreated DRGS with oncomodulin grown on substrate with CSPGs did grow neurites (enhanced further with ChABC) - Oncomodulin did not affect axonal regeneration of the optic nerve - Sp-8-Br-cAMP increased axonal regeneration 2 fold - Sp-8-Br-cAMP + oncomodulin increased axonal regeneration 5-7 fold over baseline 177     Source Model and Tests Paradigm Outcome In-vitro N.S. In-vivo N.S. In-vitro In-vivo In-vitro In-vivo Tanaka et al., (2007) - Cell model: rat  (Sprague- Dawley) P7 cerebellar granule neurons (CGN)  CNS N/A N/A - Tx: CGN plated on PDL/laminin treated with MAG-Human IgG Fc chimera. CGNs were either infected with constitutively active GPCRs (GPR3,6, or 12) or grown with either 1 mM db-cAMP or 200ng/mL BDNF - Ctl: CGNs plated on PDL/laminin treated with Human IgG Fc chimera N/A - Constitutively active GPRs (3,6,12) increase native levels of cAMP and enhanced  neurite outgrowth - cAMP levels correspond to degrees of neurite extension - GPRs (3,6,12) overcame MAG inhibition - Db-cAMP treated CGNs produced the longest neurite lengths while BDNF produced the shortest N/A Wu et al., (2007) - Cell model: rat (Sprague-Dawley) female 6-8wks old L4-5 DRG PNS - Animal model: rat (Sprague-Dawley) female 6-8wks old L4-5 DRG - Injury: crush injury proximal to DRG ganglia - Behavioural Tests: None PNS - Tx: 1-1.5 ?L of saline or db-cAMP (33 mM) injected into DRGs 2 days before removal and incubation with or w/o AG490 (JAK2 inhibitor) and plated on poly-D-lysine (N2 serum free) - Ctl: Sham, saline or sciatic nerve injury - Tx: 1-1.5 ?L of either lentivirus containing pRRL-srCNTF-IRES-GFP and/or db-cAMP (33 mM) injected into DRG before crush injury Ctl: PBS injections Measurement: numbers and length of regenerating axons - Sciatic nerve injury had the highest percentage of neurons with growing neurites followed by db-cAMP neurons. Ctls had minimal neurite outgrowth - AG490 inhibited neurite outgrowth in sciatic nerve and db-cAMP groups - Db-cAMP injection into DRGs increased LIF and IL6 mRNA levels - Db-cAMP and/or LV-srCNTF had more regenerating axons 10 and 15mm from the crush site than Ctl groups - Db-cAMP and LV-srCNTF did not produce synergistic effects - LV srCNTF mimics a conditioning inj.   178   Source Model and Tests Paradigm Outcome In-vitro N.S. In-vivo N.S. In-vitro In-vivo In-vitro In-vivo Murray et al.,  (2008) - Cell model: rat (Sprague-Dawley) at E14-16, P1-3, adult DRG  PNS N/A N/A - Tx: DRGs plated on poly-D-lysine (10?g/mL) and laminin-1 (2 ?g/mL) with rolipram (100nM) or 8-Br-Sp-cAMPs (2 or 20?M) or 8-CPT-2'-O-Me-cAMP (2 or 20?M) - Ctl: saline N/A - Epac developmentally regulated. Epac1appears neonatally. Epac2 appears in adulthood - 8-CPT-2'-O-Me-cAMP (2 ?M) selectively activated Epac activity (not PKA) and increased neurite outgrowth - siRNAs against Epac abolished outgrowth - Epacs required for endogenous growth N/A Ahmed et al., (2009) - Cell model: rat (Sprague-Dawley) female, 6-8 wks old, retinal ganglion cells CNS N/A N/A - Tx: RGCs plated on poly-D-lysine  (100 ?g/mL) and laminin (20?g/mL) and/or CNS myelin extract (10?g/mL).   Cells treated with CNTF (10ng/mL) and/or forskolin (50?M) and/or Y27632 (1-40?M) [ROCK inhibitor] - Ctl: no CNTF or Y27632 or forskolin N/A - Y27632 only provides neurite outgrowth when paired with CNTF - Y27632 promotes neurite outgrowth when cAMP levels are increased - Y27632 did not allow neurite outgrowth by itself on when grown on inhibitory myelin substrates - Synergism b/w Y27632 + forskolin + CNTF produced the greatest numbers of neurites and length when grown on myelin extracts N/A Gordon et al., (2009) N/A N/A - Animal model: rat - Injury: femoral nerve axotomy - Measurement: muscle force PNS N/A - Tx: 20Hz electrical stimulation (1hr) or rolipram (0.5 ?L/hr, 0.5mg/kg/day) - Ctl: saline N/A - More motor neurons grew across gap versus Ctls - Rolipram more effective than electrical stimulation - Greater numbers of motor units in Tx versus Ctl  179     Source Model and Tests Paradigm Outcome In-vitro N.S. In-vivo N.S. In-vitro In-vivo In-vitro In-vivo Murray et al., (2009) - Cell model: rat (Sprague-Dawley) P1-4, adult DRGs  PNS N/A N/A - Tx: DRGs plated on poly-D-lysine with 8-Br-cGMP (2,20,200?M) and/or sp-cAMP (20?M) and/or NEP1-40 (100nM) and/or rolipram (100nM) - Ctl: Rp-8-Br-cAMP, NEP1-40 Scrambled N/A - cGMP promotes growth cone turning - cGMP by itself does not promote adult neurite regeneration - cGMP requires cAMP for neurite regeneration - synergistic neurite outgrowth when cAMP, cGMP, NEP1-40 are combined N/A Park et al., (2009) N/A N/A - Animal model: rat (Fisher 344) young adult (8-10 wks old) - Injury: optic nerve transection (1.5mm away) - Measurement: SOCs mRNA,  CNS N/A - Tx: Intraocular injections of  CNTF (1.5?g) and/or CPT-cAMP (0.1 mM) - Ctl: saline (3 ?L) and/or LY294002 (2?M; Akt inhibitor) and/or AG490 (2 mM; JAK inh.) and/or KT5720 (10?M; PKA inh.) N/A - CNTF raises SOCs (1,2,3) levels - SOCs are negative cytokine regulators. Reduces CNTF effectiveness - CPT-cAMP reduces SOCs 1 & 3 levels thereby increasing regenerative capacity Bretzner et al., (2010) N/A N/A - Animal model: rat (Sprague-Dawley) adult, male - Injury: dorsolateral funiculus crush at C3/4 - Behavioural Tests: vertical exploration test, food pellet reaching test, plantar heat sensory test  CNS N/A - Tx:  mouse (YFP) LP-OEC (150-180K cells) infusion and/or db-cAMP (0.5 ?g/ ?L/hr) and/or rolipram (0.4?g/kg/hr) - Ctl: vehicle solution  N/A - Rolipram increased axonal density in the lesion site; db-cAMP had no effect - Neither db-cAMP nor rolipram promoted regeneration through OEC transplanted cells - Rolipram increased OEC density - Rolipram + OECs improved performance of cylinder test - Food pellet reaching test unaffected by all Txs. 180  Source Model and Tests Paradigm Outcome In-vitro N.S. In-vivo N.S. In-vitro In-vivo In-vitro In-vivo Joset et al., (2010) - Cell model: rat (primary hippocampal neurons; E19, DRGs (E19), CGNs (P1-4) CNS PNS N/A N/A - Tx: cells plated on poly-D-lysine  and laminin with Nogo?20 (300nM) [GC collapse]or Sema3A (40nM) with db-cAMP (1 mM) and/or NEP1-40 (1?M) - Ctl:  Nogo?21 (300nM) N/A - Nogo?20 is internalized and transported to the cell body. Activates Rho, reduces p-CREB, causes GC collapse. - Db-cAMP elevated p-CREB and overcame  Nogo?20 neurite growth inhibition and GC collapse N/A Kurimoto et al., (2010) N/A N/A - Animal model: mouse (C57BL/6J PTENflx/flx) 6-8 wks old - Injury: optic nerve crush - Behavioural Tests: N/A CNS N/A - Tx: intraocular injections of Zymosan  (1.5-12.5 ?L) and/or CPT-cAMP (4-500?M) and/or rOcm (50ng) and/or P1 (Ocm antagonist; 2.3?g/ ?L)  and/or AAV-Cre2 (PTEN deletion) - Ctl:  Rp-cAMPs (100?M) and/or P? (2.3?g/ ?L) N/A - cAMP enables Ocm to bind to the inner retina in a dose dependent manner - Ocm requires cAMP in order for enhanced regeneration to occur - Maximal regeneration occurs with maximum synergism of Zymosan, CPT-cAMP andAAV-Cre Udina et al., (2010) N/A N/A - Animal model: rat (Sprague-Dawley) adult female - Injury: right common peroneal nerve transection 10mm proximal to flexor muscle entrance - Behavioural Tests: none PNS N/A - Tx: rolipram (0.4 ?M /kg/hr) delivered via mini osmotic pump and/or 20U/mL chABC (single application) for 1hr - Ctl: saline filled pump N/A - Rolipram accelerated the onset of regeneration of sensory and motor axons compared with saline controls - Rolipram increased the number of sensory axons travelling across the injury site - Rolipram did not increase the rate of axon regeneration - chABC and Rolipram did not produce a synergistic increase in axon regeneration 181     Source Model and Tests Paradigm Outcome In-vitro N.S. In-vivo N.S. In-vitro In-vivo In-vitro In-vivo Tegenge et al., (2011) - Cell model: human (Ntera2)NT1/D2 precursor cells differentiated to resemble neurons N/A N/A N/A - Tx: cells plated on poly-D-lysine with 8-Br-cAMP (100-1000?M) or forskolin (10 or 50 ?M)  - Ctl: DMSO (0-0.5%) or Rp-cAMP (10 ?M; PKA inhibitor) and/or H89 (5 ?M; PKA inhibitor) N/A - Elevated cAMP via forskolin or by cell permeable analogue 8-Br-cAMP increased the number cells with neurites and overall neurite length - 8-Br-cAMP increased the number of neurites and neurite length in a dose dependent manner - PKA inhibitors (H89 and Rp-cAMP) prevented neurite outgrowth N/A Wan et al., (2011) - Cell model: Human Neuroblastoma BE(2)-C cells, PC12 CNS N/A N/A - Tx: GDNF, NTN(Neurturin, a NTF), NGF (50ng/ml), FSK (10?M), db-cAMP (100?M), PACAP [regulates GC via Gprot+tmAC] (100nM), 2-Me-cAMP [Epac], 50,100,200 ?M, 6-Bnz-cAMP [PKA] (50,100,200 ?M), U0126 [MEK inh] (10?M), Rp-8-Br-cAMPs [PKA inh] (200?M) N/A - Cells plated with NGF produced neurite outgrowth all cells - GDNF&NTN prcd outgrowth on GFR?2a/c not b cells [ GFR?2 = GDNF family receptor]. - Only  GFR?2a/c phos CREB using NTN.  Co treatment with GDNF or NTN plus db-cAMP or FSK or PACAP inc neurite outgrowth - NTN+FSK worked with 6-Bnz-cAMP  (PKA) not 2-Me-cAMP (Epac) means PKA is necessary for neurite outgrowth not Epac  N/A 182  Source Model and Tests Paradigm Outcome In-vitro N.S. In-vivo N.S. In-vitro In-vivo In-vitro In-vivo Shelly et al., (2011) - Cell Model: Rat dissociated hippocampal and cortical neurons (E18) CNS N/A N/A - Tx: Plated with Sema3A (stripes) (0.5/0.05?g/ml), netrin-1 (0.5/0.05ng/ml), KT5720 (PKA inh) or KT5823 (PKG inh) 2nM, rolipram (1?M), BDNF (0.5ng/ml), NGF (0.5ng/ml).  BDNF [bath] (50ng/ml), NGF [bath] (50ng/ml) N/A - Axons formed off of Sema3A stripes (dendrites on) - Axons differentiated off the stripe (no difference for BSA or NGF stripes) - Sema3A upregulated cGMP/PKG which down regulated cAMP/PKA - cAMP or BDNF requires LKB1 (ser/thr kinase) to be phos for axon formation - Sema3A activates PDE4 - NGF [bath] has no affect on 2nd messenger systems N/A Nicol et al., (2011) - Cell Model: Xenopus laevis (frog), spinal cord cells from stage 21-22 embryos CNS N/A N/A - Tx: Netrin-1 (gradient), FSK (10?M), SQ22536 [AC blocker] (10?M N/A - Spinal neurons grew towards gradients of Netrin-1 when cAMP+Ca2+ were present; did not when AC was inhibited - cAMP inc in filopodia when Netrin-1 was present - SQ22536 blocked ACs reduced  Ca2+ frequency; FSK rescued it - Ca2+ environment did not affect cAMP response - Netrin-1 applied to GC centres inc cAMP in the centre of the GC - mCherry-PAC? (Netrin-1 mimic) creates gradients of cAMP - cAMP required for axon guidance  N/A 183     Source Model and Tests Paradigm Outcome In-vitro N.S. In-vivo N.S. In-vitro In-vivo In-vitro In-vivo Xu et al., (2012) - Cell Model: Rat P5 spiral ganglion neurons (SGN)  CNS N/A N/A - Tx: plated on laminin (20?g/ml), fibronectin (20?g/ml) or human tenscin C (20?g/ml) + NT-3 (50ng/ml) and/or cpt-cAMP [PKA] (0.1-10mM), FSK (0.1-10?M), 8-pCPT-2?-O-Me-cAMP [Epac] (200?M), Rp-cAMPs [PKA inh] (1mM) N/A - cpt-cAMP produced a biphasic response: 1mM = increased neurite length, 10mM decreased neurite length - cpt-cAMP reduced branching with increasing concentration - independent of substrate (fibronectin or tenascin C) - FSK increased neurite length at all concentrations - Epac agonist did not increase neurite length - PKA blocker did not block neurite growth - Overexpressed PKA fails to produce dominant neurite - Cytoplasmic PKA (phos) required for survival N/A 184     Source Model and Tests Paradigm Outcome In-vitro N.S. In-vivo N.S. In-vitro In-vivo In-vitro In-vivo Blesch et al., (2012) - Cell Model: Rat, female Fisher, 3 month old, L4-6 DRGs; Cerebellar granule neurons (CGN) from P7 rats PNSCNS - Animal model: rat (Fisher) adult female - Injury: bilateral transaction of the dorsal funiculi at C3; preconditioning crush of the sciatic nerve for 15s - Behavioural Tests: none PNSCNS - Tx:Adult DRGs removed 3 or 7 days after CL or mesopram (PDE4 inhibitor) - Cultivation included db-cAMP (2mM)  - Tx: syngenic marrow stromal cells (2?l; 75,000cells/ ?l) coated with NT-3 (1?g/?l)+Lenti-NT-3 and Lenti-GFP injected into DC lesion - Rolipram or mesopram (2.6mg/kg/day) via pump (10?l/hr) given 1wk prior to C3 injury - db-cAMP (50mM) injected 1day after C3 lesion - Conditioning lesions (CL) where given -7,+1,+7 days before or after the injury - Tracing: CTB - Ctl: PBS injections, naive animals, Lenti-GFP  - CL lesioned DRGs produced a 2x increase in neurite length - Mesopram did not increase neurite length - db-cAMP (2mM) did not increase neurite length - db-cAMP did increase neurite length in postnatal DRGs - CLs increased endogenous cAMP by 2x (within 1day) and remained elevated 1wk later - Mesopram increases cAMP to the same magnitude as that of CLs at 3days but declines to baseline levels by day 7 regardless of continuous infusion - Lenti-NT-3 w/o CL resulted in small number of axons crossing the injury interface - Lenti-NT-3 + CL increased number of axons 8x crossing injury interface; distance travelled by axons were 6x greater as well -  CLs at -7 or +1 day have the same growth effect; +7 CLs are without effect - db-cAMP injected 5 days before injury resulted in a 2x increase in the number of bridging axons - Mesopram injections alone did not result in axon growth across rostral border - Microarray analysis at 1,3,7,14 days resulted in significantly more activated genes in CL treated animals than those treated with mesopram alone 185      Source Model and Tests Paradigm Outcome In-vitro N.S. In-vivo N.S. In-vitro In-vivo In-vitro In-vivo Harel et al., (2012) - Cell Model: Rat, adult female Sprague-Dawley DRGs (thoracic, lumbar, cervical) PNS - Animal model: rat (Sprague-Dawley) adult female - Injury: right side hemilaminectomy and partial facetectomy exposing C8 DRG with root. 1wk later, root crush injury half way between DREZ and DRG for 10s - Behavioural Tests: none PNSCNS - Tx: DRGs (cervical, thoracic and lumbar) plated on poly-L-lysine, laminin (5?g/ml), aggrecan (0.7mg/ml). - Two groups: unconditioned and conditioned - Unconditioned: Ocm [oncomodulin] (200ng/?l) or db-cAMP (50mM) or both added to cultures on day1 - (Pre)conditioned L4, L5, L6 DRGs injected with Ocm (200ng/?l) or db-cAMP (50mM) 7 days before extraction and plating - Plating included an inhibitory proteoglycan rim - Tx: Ocm (200ng/?l) or db-cAMP (50mM) or both injected into the DRG (C8) [preconditioning] - 1wk later, root crush injury - 3wks later ? dextran-Texas red tracing applied - 4wks after axon regeneration measured preconditioning - Ctl: ChABC (1?l ? 20U/ml)  injection (no preconditioning drugs); saline - Ocm or db-cAMP alone resulted in minimal neurite extension - Ocm+db-cAMP produced a 3x increase in the length of neurites of unconditioned DRGs across inhibitory rim - Preconditioning with Ocm or db-cAMP alone produces no significant increases of processes extending across the proteoglycan rim - Ocm+db-cAMP produced significantly more neurites across the inhibitory rim vs either treatment alone - Preconditioned Ocm+db-cAMP resulted in significantly more axonal growth compared with direct exogenous treatment - Saline treated DRGs extended up to the DREZ but did not cross it - ChABC alone produced minimal regeneration across the DREZ - Ocm+ChABC resulted in minimal axons across the DREZ - db-cAMP+ChABC also resulted in minimal axons across the DREZ - db-cAMP+Ocm+ ChABC resulted in a greater degree of regeneration than either treatment alone 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.24.1-0073272/manifest

Comment

Related Items