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BDNF infusion into the sensorimotor cortex promotes sprouting of inact corticospinal fibers within the… Khodarahmi, Kourosh 2008

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BDNF INFUSION INTO THE SENSORIMOTOR CORTEX PROMOTES SPROUTING OF INTACT CORTICOSPINAL FIBERS WITHIN THE SPINAL CORD AFTER A UNILATERAL PYRAMIDAL LESION by Kourosh Khodarahmi B.Sc., The University of British Columbia, 2000  A THESIS SUMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDES (Neuroscience)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) October 2008 © Kourosh Khodarahmi, 2008  ABSTRACT  More than half of all spinal cord injuries are anatomically incomplete, yet many of these result in complete loss of motor function below the level of injury. One approach to enhance functional recovery is to exploit spared CNS axons (that extend past the point of injury) to sprout and connect to potential targets. We have previously found that application of the neurotrophin; BDNF, to the sensory-motor cortex stimulates expression of regeneration associated genes such as GAP-43, and Tal tubulin, and results in enhanced sprouting of injured corticospinal fibers rostral to the site of injury. Here, we investigated whether infusion of BDNF into the intact sensorimotor cortex induces sprouting of undamaged corticospinal fibers into denervated cervical spinal cord. We also studied the effect of this treatment using several behavioral tasks: gait analysis, forelimb inhibition during swimming, and food pellet reaching task. The results show that BDNF infusion into the intact sensorimotor cortex subsequent to a unilateral pyramidal lesion increases (3.2 fold) the sprouting of intact corticospinal fibers into the denervated, contralateral grey matter at the lumbar level of the spinal cord when compared with vehicle treated rats. This effect was not seen at the cervical level of the spinal cord. Functionally, unilateral pyramidal injury of corticospinal axons significantly increased toe spread of the contralateral denervated forelimb and hindlimb when compared to the uninjured side. BDNF treatment showed a recovery to presurgical levels. Testing of fine motor control with a food pellet reaching task demonstrated deficits in the impaired forelimb but did not show any improvement due to BDNF treatment.  III  Table of Contents Abstract  .  ii  Table of Contents  iii  List of Tables  ix  List of Figures  x  Acknowledgements  xiii  1. Introduction  1  1.1 The Anatomy of Movement  1  1.2 Recovery after Spinal Cord Injury  5  1.3 Challenges to Regeneration/Plasticity  7  1.3.1  1.3.2  Extrinsic Inhibitory Influences  7  1.3.1.1 The Glial Scar  8  1.3.1.2 Chontroitin Sulphate Proteoglycan  9  1.3.1.3 Myelin  10  1.3.1.4 Semaphorins  11  Intrinsic Control of Axonal Sprouting/Regeneration  11  1.4 Spontaneous Regeneration and Plasticity of the Corticospinal Tract  12  1.5 Promoting The Ability Of Axons To Overcome Inhibition  15  1.5.1  Enhanced RegenerationlPlasticity  15  1.6 Functional Testing of the Corticospinal System  16  1.7 Rationale for Current Study  18  iv 1.8 Hypothesis  .20  2. Methods  21  2.1 Animal Husbandry  21  2.2 Experimental Studies  21  2.2.1 Fourteen Day Experimental Study  21  2.2.2 Forty-Two Day Experimental Study  22  2.3 Surgical Preparation  —  BDNF and Vehicle Osmotic Pump Preparation  2.4 Surgical Procedure  23 24  2.4.1 Pyramidotomy  24  2.4.2 Osmotic Pump Implantation  25  2.4.3 Axonal Labeling  26  2.5 Perfusion and Tissue Harvest  28  2.6 Tissue Preparation and Histological Techniques  28  2.6.1 Cryoprotection and Tissue Storage  28  2.6.2 Cryostat Sectioning  30  2.6.3 Immunohistochemistry  30  2.6.3.1 Determination of Lesion Size (Anti-Neurofilament)  30  2.6.3.2Visualization of BDA Labeled Corticospinal Axon Profiles in Cervical and Lumbar Spinal Cord  31  2.7 Quantifications  31  2.7.1 Quantification of Lesion Size  31  2.7.2 Quantification of Sprouting  33  2.8. Behavioural Evaluation 2.8.1 Pretest Procedure  35 35  V  2.8.2 Food Pellet Reaching Task 2.9 Gait Analysis  3. Results  .36 40  42  3.1 Weight of Rats Increased with a Restricted Diet  42  3.2 BDNF Induces Seizures  42  3.3 Surgical Observations  43  3.3.1  3.3.2  Cannula Implantation into the Intact Left Sensorimotor Cortex Causes Damage  43  Injury of the Corticospinal Tract at the Level of the Pyramids Leaves Spared Tissue  44  3.4 Anterograde BDA Labels Dorsal, Lateral and Ventral CST axons, and Fibers Within the Grey Matter 3.4.1  BDA Labeling of CS axons at the Cervical Level in the 14 Day Study  3.4.2  3.4.7  51  BDA Labeling of CS Axons at the Lumbar Level in the l4DayStudy  3.4.6  50  Normalization of Corticospinal Axon Profiles in Cervical Grey Matter (42 Day Study)  3.4.5  48  BDA Labeling of CS Axons at the Cervical Level in the 42 Day Study  3.4.4  45  Normalization of Corticospinal Axon Profiles in Cervical Grey Matter (l4DayStudy)  3.4.3  45  53  Normalization of Corticospinal Axon Profiles in Lumbar Grey Matter (14 Day Study)  54  Choice of Normalization Factor  55  vi  3.5 Axonal Profile Qutification  .56  3.5.1 Intact dorsal corticospinal tract encroaches into the contralateral denervated white matter after pyramidal lesion  56  3.5.2 A unilateral pyramidal lesion significantly induced sprouting of intact corticospinal axons into the contralateral side of the cervical spinal cord 14 days after injury, however, BDNF or vehicle did not significantly induce further sprouting  56  3.5.3 A unilateral pyramidal lesion significantly induced sprouting of intact corticospinal axons into the contralateral side of the cervical spinal cord 42 days after injury, however, BDNF or vehicle did not significantly induce further sprouting  58  3.5.4 Application of BDNF following a unilateral pyramidal lesion significantly induced sprouting of intact corticospinal axons into the contralateral side of the Lumbar spinal cord when compared to vehicle treated, and unlesioned sham operated animals 14 days after injury  3.6 Behavioral Quantification 3.6.1 Food Pellet Reach Task  59  61 61  6.6.1 .1 Training success rate plateaus but does not increase or decrease with further practice or testing  61  3.6.1.2 Functional Testing  62  Thirteen Point Analysis  62  Success Rate  64  Single Attempt Success Rate  65  VII  3.6.2 Gait Analysis  .67  3.6.2.1 A Unilateral left pyramidal lesion produces an increase in toe spread in the forepaw and hind paw on the right denervated side compared to the left side  67  3.6.2.2 Infusion of vehicle into the intact right sensorimotor cortex reduces the increased toe spread in the denervated right hindlimb caused by pyramidal injury  68  3.6.2.3 Infusion of BDNF into the intact right sensorimotor cortex reduces the increased toe spread in the denervated right hindlimb earlier when compared with vehicle infusion  70  3.6.2.4 Comparison of toe spread in the right denervated hind paw of BDNF treated, vehicle treated, and pyramidal lesion alone groups  4. Discussion  71  73  4.1 Possible Effect of Food Restriction  73  4.2 BDNF Induces Seizures  74  4.3 BDNF Treatment Reaches all the BDA Labeled Corticospinal Neurons  75  4.4 The Site of Camiula Implantation Might Have Preferentially Expose BDNF to Hindlimb Corticospinal Neurons Over Forelimb Corticospinal Neurons  76  4.5 BDNF Did Not Increase Uptake of BDA Into Corticospinal Neurons  77  4.6 Cannula Implantation Into the Intact Left Sensorimotor Cortex Causes Damage  79  4.7 Pyramidal Lesions of the Corticospinal Tract Leaves Spared Tissue  79  4.8 Damage to Surrounding Systems After a Pyramidal Lesion Confounds Behavioral Assessment 4.9 Sprouting in The Spinal Cord After Unilateral Pyramidal Lesion  81 82  VIII  4.10 Sprouting in the Spinal Cord After Unilateral Pyramidal Lesion and BDNF Treatment (Possible Explanation)  87  4.11 Sprouting in the Spinal Cord After Unilateral Pyramidal Lesion and BDNF Treatment (Alternate Explanation) 4.12  88  Behavioral Testing  89  4.12.1 Reach Test and Gait Analysis  89  4.12.2 Forelimb Function After a Unilateral Pyramidal Lesion  90  4.12.2.1  Reach Training  90  4.12.2.2  Reaching Behavior After Pyramidal Injury  92  4.12.2.3  Reaching Behavior After a Unilateral Pyramidal Lesion and BDNF Treatment  4.12.3 Hindlimb Function After a Unilateral Pyramidal Lesion  94 95  5. Conclusions  98  6. References  100  7. Appendices  116  Appendix A  —  Animal Care Certificate  116  ix List of Tables Table 2.1 Thirteen point ordinal scale for the food pellet reaching task  38  x List of Figures  Fig 1.1 Schematic diagram of spinal cord cross section with major and minor components of corticospinal tract  4  Fig 2.1 Experimental Study (14 day time course)  22  Fig 2.2 Experimental Study (42 day time course)  23  Fig 2.3 Illustration of treatment pump implantation and BDA injection sites overlaid a map of topographical organization of forelimb and hindlimb sensorimotor cortex  26  Fig 2.4 Topographical relations of spinal cord, dorsal roots and vertebral column in the rat  29  Fig 2.5 Neurofilament immuno-labelled axons above the level of the pyramids to approximate the size of the pyramidal lesion  32  Fig 2.6 BDA labelled corticospinal axons above the level of the pyramids (left pyramid)  33  Fig 2.7 Quantification of sprouting. Cy3 labelling of BDA in the cervical spinal cord  34  Fig 2.8 (A) Reach to grasp food pellet reach task training and testing apparatus schematic. (B and C) Pictures taken of rats during reach to grasp training  36  Fig 2.9 Toe spread measurement by functional testing of gait  41  Fig 3.1 Weight of rats increase with a restricted diet  42  Fig 3.2 Percent Area of Spared Axons in Injured Left Pyramid (14 Day Experimental Study)  44  Fig 3.3 Percent area of spared axons in injured left pyramid (42 Day Experimental Study)  45  Fig 3.4 Fibres innervating the gray matter in uninjured and pyramidal injured rats  46  Fig 3.5 Average Intact Axons in the denervated dorsal CST and denervated ventral CST labeled with Cy3  47  Fig 3.6 Topographical Cy3 labelling of BDA in the cervical enlargement (C6) of rats in the 14 day experimental study  48  xi  Fig. 3.7 Selection of normalizing factor for the cervical enlargement of rats in the 14 day experimental study  49  Fig. 3.8 Topographical Cy3 labelling of BDA in the cervical enlargement (C6) of rats in the 42 day experimental study  51  Fig 3.9 Selection of normalizing factor for the cervical enlargement of rats in the 42 day experimental study  53  Fig 3.10 Labelling of Corticospinal axons at the Lumbar enlargement (L4) in the 14 Day Study  54  Fig 3.11 Selection of normalizing factor for the lumbar enlargement of rats in the 14 day experimental study  55  Fig 3.12 (A) Number of pixels drawn in the denervated half of the cervical (C6) spinal cord in 14 day experimental study rats. (B) Normalized number of pixels drawn in the denervated half of the cervical (C6) spinal cord  57  Fig 3.13 (A) Number of pixels drawn in the denervated half of the cervical (C6) spinal cord in 42 day experimental study rats. (B) Normalized number of pixels drawn in the denervated half of the cervical (C6) spinal cord  59  Fig 3.14 (A) Number of pixels drawn in the denervated half of the cervical (L4-5) spinal cord in 14 day experimental study rats. (B) Normalized number of pixels drawn in the denervated half of the cervical (L4-5) spinal cord  60  Fig 3.15 Training success rate in the reach to grasp food pellet task  62  Fig 3.16 Thirteen point analysis of the reach to grasp food pellet task in the 42 day study  63  Fig 3.17 Success rate of retrieval in the reach to grasp food pellet task (42 day study)  65  Fig 3.18 Single attempt success rate retrieval in the reach to grasp food pellet task (42 day study)  66  Fig 3.19 Forelimb and hindlimb toe spread before and after a left unilateral pyramidal lesion (14 day study)  68  Fig 3.20 Forelimb and Hindlimb toe spread before and after a left unilateral pyramidal lesion with either Vehicle or BDNF treatment (14 day study)  70  XII  Fig 3.21 Comparison of toe spread in the right denervated hind paw of BDNF treated, vehicle treated, and pyramidal lesion alone  72  Fig 4.1 Sites of origin of corticospinal neurons in the rat with reference to 6 different coronal distances from the frontal pole  76  Fig 4.2 Summary schematic diagram of the patterns of axonal labeling in the white matter of the brain after injections of biotinylated dextran into various areas of neocortex  80  Fig 4.3 Images of the rat brain in cross section at the level of the pyramidal decussation showing structures lying dorsal to the pyramid  81  XIII  Acknowledgements I look back on my time in the Tetzlaff Lab with an immense fondness. At the time I began my studies, I did not know how much of a family our supervisor, and my lab mates and others would become. I loved getting to know each and every one of you. Our Father Wolf, our Uncle Jie, our brothers David, Egidio, Brian and Ward, our sisters Clairie, and Carmen, and our cousins Cheryl, Jeremy, and Emily. Our relationships may be tested by distance and time, however, my memories our friendships will not fade. Special thanks to my supervisor. Thank you, Wolf, for seeing the potential in me and placing no limits on what direction I would take. Thank you to Jie. Your energetic and encouraging nature is a great comfort to all the people who know you. Thank you to my defence committee, John Steeves, Matt Ramer, Brian Kwon, and Tim O’Connor for your input and constructive comments. I would also like to acknowledge the Rick Hansen Institute, and ICORD for their support in my research. During the process of conducting my experiments and writing this thesis, I experienced many trials and tribulations, as well as moments of pure love and lasting joy. I wish to thank my family and friends for their support, and encouragement. To my wife, I love you. You give me the ambition to be a better person, and achieve the dreams that we share in our lives. I look forward with anticipation on our future path together.  1  Introduction A spinal cord injury usually occurs when a sudden, traumatic blow to the spine fractures, dislocates or distracts vertebrae. Primary spinal cord damage occurs at the moment of impact which typically compresses or tears the spinal cord tissue and produces characteristic damage of the gray and white matter depending on the primary injury mechanisms. The ultimate tissue damage exceeds this primary injury due to the additional secondary injury triggered by a cascade of metabolic and inflammatory events which includes the apoptotic death of neurons and oligodendrocytes as well as the further demise of initially spared axons. These destructive processes start within minutes of injury and last for days to weeks (Anderson & Hall, 1993; McBride and Rodts, 1994; Nolan, 1994; Tator, 1995; Chiles & Cooper, 1996; Faden, 1996; Schwab & Bartholdi, 1996; Medana & Esiri, 2003). There are several pertinent issues regarding the current state of experimental treatments and interventions after CNS injury. They include attempts to promote neuroprotection (For review: Faden & Stoica, 2007), plasticity of spared neuronal circuits (For review: Raineteau & Schwab, 2001), axonal regeneration (For review: Kwon et al., 2005), and rehabilitation (For review: Dietz & Harkema, 2004). In this thesis, special emphasis will be placed on anatomical plasticity and functional recovery/compensation after partial injury of the corticospinal system.  1.] The Anatomy ofMovement Complex movements require integration of sensory information with motor output through descending tracts (such as the corticospinal, rubrospinal and reticulospinal systems). In humans, locomotion is initiated by activity in reticulospinal neurons (RS) of the brainstem locomotor centres, and modulated by input from other processing centers (Steeves and Jordan, 1980). The pontine reticulospinal tract descends uncrossed into the spinal cord, terminating onto  2  interneurons. These, in turn, bilaterally terminate onto excitatory medial extensor motoneurons. The medullary reticulospinal tract descends bilaterally to provide inhibitory inputs to motoneurons supplying the proximal limbs (Steeves & Jordan, 1980). The reticulospinal system closely interacts with sensory feedback to initiate central pattern generation in the spinal cord (Grillner 1985, 1996). With increased activation of the locomotor centre, the speed of locomotion increases and interlimb coordination can change from a walk to a run. Other processing centers influence their control by modulating movements. The cerebellum receives sensory information through the spinal cord, as well as information about motor output from the cerebral cortex and red nucleus. It functions to integrate information and provide feedback to ongoing movement. Afferent sensory fibers into the cerebellum arise from the cervical or lumbar/thoracic spinal cord via the cuneocerebellar/rostral spinocerebellar pathway and spinal cerebellar tracts respectively. The cerebellum interacts with the descending pathways to organize limb movements, and maintain balance. The cerebellar cortex sends signals to three cerebellar nuclei; the fastigial nucleus, dentate nucleus, and interposed nucleus. The fastigial nucleus sends projections to the pontine reticular formation, and vestibular nucleus, and subsequently to the pontine reticulospinal tract, and lateral vestibulospinal tract. The dentate nucleus projects to the ventrolateral nucleus of the thalamus, and subsequently to the corticospinal tract motor cortex. The interposed nucleus sends a projection to the red nucleus and another to the thalamus (Morton & Bastion, 2004; Thach et al., 1992). The basal ganglia are a grouping of nuclei in the forebrain that include the caudate nucleus, the putamen and the globus pallidus. The substantia nigra and the subthalamic nucleus are functionally associated with the basal ganglia. All these structures relay information to, and  3  are interconnected with the sensorimotor cortex and the corticospinal system. The basal ganglia exert a tonic inhibitory influence on motor centres that is released when a voluntary behavior is initiated. Damage to any of these structures can result in a resting tumor, rigidity and bradykinesia (Dubois et al., 2005; Alexander et al., 1986). The lateral vestibulospinal tract originates from cells of the lateral vestibular nucleus in the brainstem. The tract descends uncrossed into the spinal cord innervating interneurons and motoneurons responsible for control of postural muscles and regulation of extensor tone. The medial vestibulospinal tract originates from cells of the medial vestibular nucleus, which receives inputs from the semicircular canals and from stretch receptors in neck muscles. This tract descends uncrossed and provides output for postural adjustments of the head and upper limbs (Drew et al., 2002). The rubrospinal tract originates in the red nucleus of the midbrain and decussates before descending the spinal cord. The red nucleus receives input from the motor cortex and the cerebellum. A lesion of the red nucleus causes impairments in the skilled forelimb reaching that recovers with time (Whishaw et al., 1998). This recovery is thought to occur because the rubrospinal system functions as a parallel tract to the corticospinal system. In primates, injuries directly to the rubrospinal system result in minimal deficits. However, when combined with injuries to the corticospinal system, severe deficits occur that do not recover with time (Whishaw et al., 1998; Kennedy, 1990). For the purpose of this thesis, I will now focus on the corticospinal tract. In the rat, the corticospinal cell bodies are located in layer V of the sensorimotor cortex. As the axons descend into the spinal cord, the tight fascicle divides into four distinct tracts at the level of the medulla and runs the entire length of the spinal cord (Brosamle & Schwab, 1997). The major component  4  (1) decussates to the contralateral ventral portion of the dorsal funiculus, and a minor component (2) runs parallel in the contralateral dorsolateral funiculus. Corticospinal axons also descend as two other minor tracts in the uncrossed (3) ipsilateral dorsal funiculus and the (4) ipsilateral ventral funiculus of the cord (Figure 1.1; Alisky et a!., 1992; Brosamle & Schwab, 2000; Cabana & Martin, 1985; Goodmann et al., 1966; Vahising & Feringa, 1980; Joosten et al., 1987, 1992; Rouiller et al., 1991).  Dorsal  3)  0  U  Ventral  Fig 1.1 Schematic diagram of spinal cord cross section showing major and minor components of corticospinal tracts running from the right half of the rat brain (Contralateral = Left; Ipsilateral  Right).  (1) contralateral ventral portion of the dorsal funiculus; (2) contralateral dorsolateral (3) ipsilateral dorsal funiculus and the (4) ipsilateral ventral funiculus of the cord.  The corticospinal axons in the ventral tract have similar diameters and myelination as CST fibers of the dorsal crossed tract (Brosamle & Schwab, 1997; 2000). Ventral corticospinal axons send axon collaterals into different spinal segments in the ipsilateral grey matter and have  5  dense terminal arbors (Brosamle & Schwab, 1997). Although the ipsilateral corticospinal tracts comprise 10-15% of the total of the total corticospinal axons, they have not been studied in detail in the rat (Guth et al., 1980; Blight and Young, 1989; Bregman et al., 1995; Brosamle & Schwab, 1997; 2000; Whishaw & Metz, 2002). Only a small fraction of corticospinal axons in the rat terminate directly onto motoneurons, while most synapse with intemeurons. They can be organized into two groupings depending on whether they control the postural/proximal muscles of the arms and trunk or the distal muscles responsible for fine motor control (Lawrence & Kuypers, 1968; Weidner et al., 2001; Whishaw & Metz, 2002; Hyland & Jordan, 1997; McKenna et al., 2000; Whishaw & Pellis, 1990, Whishaw et al., 1993). The dorsal crossed corticospinal tract is thought to be involved in control of fine motor movement involving distal muscles such as suppinationlpronation and arpeggio of the digits (Whishaw et al., 1993; Weidner et al., 2001). The ventral uncrossed corticospinal tract is thought to be involved in the control of postural and proximal muscles (Lawrence & Kuypers, 1968; Weidner et al., 2001; Whishaw & Metz, 2002). These muscles are crucial for the proper execution of coordinated skilled movements in the rat, including correct aiming of the limb in reaching for food (Hyland & Jordan, 1997; McKenna et al., 2000; Whishaw & Pellis, 1990, Whishaw et al., 1993).  1.2 Recovery after Spinal Cord Injury Spontaneous recovery of sensory and motor function does occur over the first few weeks following incomplete injuries to the CNS. During this time, the remaining intact tracts begin a process of repair to compensate for the losses in sensory and motor function.  6  This recovery may be attributed to several mechanisms. From hours to days, and even weeks after the injury, there is a re-establishment of transmission in uninjured axons during a period called spinal shock (Ditunno et aL, 2004; Hiermemenzel et al., 2000). Second, a later phase of recovery involves the reestablishment of efficient transmission of impulses along axons, which maybe attributed to remyelination of axons by oligodendrocytes (Keirstead & Blakemore, 1999; Beattie et al., 2002). Finally, axonal regeneration/plasticity and functional recovery/compensation in rats and humans has shown the programmed ability for the CNS to spontaneously recover after injury. The time course of these mechanisms may overlap as the CNS recovers from injury. Recovery may be enhanced by intervention after injury via four major avenues. 1.  Neuroprotection is of greatest importance in preserving local nervous architecture. As mentioned previously, spinal injuries substantially increase in severity due to secondary damage following injury. If long distance axons, local intemeurons, and supporting glia are conserved, there is less loss of function, and a clearly broader potential for intrinsic functional rehabilitation. However, recent studies into the clinical application of neuroprotective agents that hold promise experimentally, have failed to translate into successful clinical trials, and illustrate the complexity of traumatic spinal cord injury (Schouten, 2007; For review: Faden & Stoica, 2007).  2. Axonal collateral sprouting is the short distance (millimeters) growth of axonal branches which reorganize neural signaling to compensate for the loss of function after injury. Sprouting increases spinal cord plasticity in at least two ways. First, injured axons may grow to form synapses onto other descending spinal systems bridging the injury (ie. Propriospinal axons)(Bayere et al. 2004; Vavrek et al. 2007). Second, sprouting  7  collaterals may originate from intact uninjured axons below the level of injury to facilitate the loss of function after partial spinal cord injury (Weidner et al., 2001). 3. Axonal regeneration is the long distance (millimeters or centimeters) axonal regrowth of injured axons directed by correct environmental cues, around or through the lesion site, ultimately making appropriate and viable synaptic connections distal to the injury. A successful therapy for the recovery of function through axonal sprouting and regeneration will no doubt involve the understanding of the processes and mechanisms responsible for the weak regenerative capacity of adult neurons (Raivich & Makwana, 2007). 4. After rehabilitation, the spinal cord can regenerate stereotypic locomotor movements, sometimes even when there are no connections to the brainstem (Edgerton & Roy, 2002). After physically ‘training’ the limbs to move in a stereotypic fashion, sensory feedback to the central pattern generators in the spinal cord, signal memorized movements to the muscles (Jordan et al., 2008; Rossignol et al., 2007). With daily practice, the training is continuous, which maintains the learned movements. There are several extrinsic and intrinsic factors in the CNS that inhibit recovery after spinal cord injury. It is important to understand these factors in order to improve functional outcome after CNS injury.  1.3 Challenges to Regeneration/Plasticity 1.3.1 Extrinsic Inhibitory Influences The environment of injured axons hinders regeneration. These include inhibitory molecules found in proximity to degenerate neurons, the physical barrier formed by scar tissue and the inhibitory molecules on the scar tissue itself. The inhibitory environment is composed of  8  oligodentrocyte precursors, miningeal fibroblasts, vascular endothelial cells, reactive astrocytes and microglia (Sandvig et al., 2004; Stichel and Muller 1998). Generally, axons attempting to penetrate the scar tissue are blocked by inhibitory molecules on these cells, including chondroitin sulphate proeoglycans (CSPGs), Nogo, and Semaphorins (He and Koprivica, 2004; Schweigreiter et al., 2004; Properzi et al., 2005; Morgenstem et al., 2002; Kaneko et al., 2006; Kikuchi et al., 2003).  1.3.1.1 The Glial Scar Glial scars demarcate the area of damage and its formation maybe an attempt by the body to re-establish the blood brain barrier compromised by the events of the injury (Shearer et al., 2003). After injury, meningeal cells proliferate and migrate into the lesion site in order to restore the compromised glial limitans, the outermost layer of nervous tissue composed of astrocytes, just under the pia matter (Shearer et al., 2003). In the process, a fibrous scar forms composed primarily of collagen IV, which is thought to serve as a scaffold for fibroblasts and blood vessels, and aids macrophage movement in the acute lesion (Maxwell et al., 1984; Berry et al., 1983). Collagen is produced by invading miningeal cells, astrocytes, and endothelial cells with the aid of an enzyme that utilizes iron as a cofactor (Liesi and Kauppila, 2002; Berry et al., 1983). However, it may become a serious physical barrier to axonal growth. Recent studies have used iron chelators in order to suppress scarring. Following transection of the dorsal corticospinal tract at T-8 in adult rats, animals treated with a chelator displayed axonal growth through the lesion area into both grey and white matter, extending up to 1.5-2 cm below the level of injury. When examined for behavioral recovery, injured animals receiving treatment improved significantly in the open field, and in the horizontal ladder and catwalk locomotor tests when compared to  9  control animals (Klapka et al., 2005). Although neutralizing the physical barrier of the glial scar is important in promoting a less inhibitory environment in the CNS, it may be in itself not sufficient in promoting the regeneration needed to recover from a significant injury.  1.3.1.2 Chonfroitin Sulphate Proteoglycan Chondroitin Sulphate Proteoglycan (CSPG) is a general term describing a number of proteins (aggrecan, biglycan, brevican, neurocan, NG2, phosphacan, and versican) with repeating suiphated disaccharide chondroitin sulphate chains attached to them. After spinal cord injury, brevican, neurocan, NG2, and versican are upregulated in astrocytes, miningeal cells, and oligodendrocyte precursors (Properzi et al., 2005). Experimental evidence shows that the glycosaminoglycan (GAG) chains give CSPGs their major inhibitory character. Protein interactions are also important in their function (Morgenstem et al., 2002). It is difficult to remove the inhibitory influence of CSPGs is difficult because of the diverse nature of their structure and origin. One approach has been to digest the glycosaminoglycan (GAG) chain, a common feature of all CSPGs. This component can be degraded into disaccharides using chondroitinases rendering the inhibitory substrate more permissive to growth. Treatment with Chondroitinase ABC after spinal cord injury has been shown to induce sprouting of corticospinal axons into grey matter at and below a C4 crush (Barritt et al., 2006; Bradbury et al., 2002). Some cortical connections have also been restored to the spinal cord possibly influencing a recovery of beam walking, stride length and width, and to a lesser extent, grid walking (Bradbury et al., 2002). Although CSPGs promote a less inhibitory environment in the CNS, it alone is not sufficient in promoting the axonal regeneration needed to recover from a significant injury.  10  1.3.1.3 Myelin Factors found within myelin also inhibit axon regeneration. This electrically insulating layer surrounds the axonal shaft and aids in transmitting electrical impulses through saltatory conduction. After CNS injury, myelin often degenerates and exposes inhibitory molecules. A number of inhibitory molecules have been identified on myelin: MAG, NogoA, Omgp, and Chondroitin Sulphate Proteoglycan (CSPG), Netrins and Ephrins (Ramer et al., 2005; He and Koprivica, 2004; Schweigreiter et al., 2004). Inhibition of Nogo-A using antibodies (i.e. EN-i) after a unilateral pyramidal injury model, enhanced collateral sprouting of intact corticospinal fibers into the denervated side of the spinal cord (Thalimair et al., 1998). This treatment also induced corticospinal axons to send collaterals into the gracile, cuneate and red nuclei (Thalimair et al., 1998; Z’Graggen et al., 1998). This treatment has been tested in the marmoset monkey after a unilateral thoracic corticospinal injury. The monkeys that did not receive EN-i treatment showed labeled corticospinal tract fibers that stopped rostra! to the lesion site, and many retraction bulbs. Three out of four monkeys treated with iN-i showed BDA labeled fibers that traveled from the proximal stump of the lesioned CST through and around the lesion site in a manner resembling regenerating axons (Fouad et a!., 2004; Steward et al., 2003). Even though myelin has inhibitory components in the mature CNS, the architecture of white matter was proposed to be conducive to the growth of axons if appropriate receptors are expressed. White matter contains astrocytic processes that determine the normal direction of growth and guide the growing axon (Li & Raisman, 1993). Although neutralizing the inhibitory effects of NogoA is important in promoting  11  a less inhibitory environment in the CNS, it has in itself not been sufficient in promoting the regeneration needed to recover from a significant injury.  1.3.1.4 Semaphorins The majority of research into the semaphorins and their receptors has been focused on understanding their roles in the CNS in development and dysfunction. Until recently, there were no in vivo studies aimed at inhibiting semaphorins since function blocking antibodies specific for semaphorins or their receptors, plexins and neuropilins have been difficult to produce. Recently, a selective inhibitor of Sema3A, SM-2 16289 has been identified in vitro and when applied for 4 weeks with an osmotic pump to the site of full T8 transection, there was enhanced growth of ascending dorsal column fibers but no corticospinal regrowth through the site of injury. Hindlimb movement was abolished after transection. Although hindlimb paralysis showed minimal recovery in control rats (average BBB score: 0.55 at 14 weeks after injury), hindlimb movement improved significantly in treated rats from 5 to 14 weeks (average BBB score: 5.13 at 14 weeks after injury). Retransection of the lesion subsequently, suggested that the significant functional recovery observed was most likely caused by regeneration across the lesion site. (Kaneko et al., 2006; Kikuchi et a!., 2003).  1.3.2 Intrinsic Control ofAxonal Sprouting/Regeneration After axonal injury, peripheral motor and sensory neurons exhibit a significant and prolonged increase in regeneration associated genes, such as tubulin and GAP-43. Although PNS neurons regenerate after injury their response differs significantly from that of CNS neurons (Filbin, 2006; Kwon et al., 2004; Plunet et al., 2002; Fernandes et al., 1999; Tetzlaffet al., 1991,  12  1994). Gene profiling after acute spinal cord injury in the adult rat has indicated that growthassociated genes are upregulated following injury (Plunet et al., 2002). However, this cell body response is intrinsically weaker and fails to persist for more than two weeks even when presented with a permissive growth environment such as a peripheral nerve graft (Hiebert et al., 2002; Song et al., 2001; Fernandes et al., 1999; Kobayashi et al., 1997). It is hypothesized that CNS neurons would have a much greater ability to overcome the inhibitory environment of the CNS if the growth properties were stimulated (Hammond et al., 1999; Steeves and Tetzlaff, 1998; Giehl and Tetzlaff, 1994).  1.4 Spontaneous Regeneration and Plasticity ofthe Corticospinal Tract Neonatal animals have a remarkable propensity for growth and plasticity after CNS injury (Kolb et al., 1998; Kolb & Whishaw, 1989). After a unilateral pyramidal lesion in young hamsters, intact uninjured corticospinal fibers send out arbors into the denervated contralateral cervical spinal cord (Kuang & Kalil, 1990). Although some plasticity remains into adulthood, the regenerative and compensatory contingencies available to young neurons are maximal at earlier stages. In adult rats, growth of collaterals from injured as well as spared CST fibers occurs spontaneously following spinal cord injury rostral and caudal to the lesion (Weidner at al., 2001; Hiebert et a!., 2002; Bareyre et al., 2004; Vavrek et a!., 2006). Sprouting of corticospinal fibers after partial injury has been shown to functionally compensate for losses in fine motor control. To demonstrate this, Weidner et al (2001), trained rats in the food pellet reach to grasp task, to successfully retrieve food pellets 70.2% of the time. After dorsal (C3) and ventral (C2) column lesion to remove  97% of the corticospinal tract bilaterally, there were significant disturbances  13  of forelimb function. Immediately after injury, the rats were only capable of retrieving pellets in 3.5% of the trials, and their success did not improve with time. In another group of rats, after dorsal column lesion to remove 90- 95% of the corticospinal tract bilaterally at C3, the rats showed significant disturbances of forelimb function up to two weeks postoperatively. However, at four weeks, food pellets were retrieved successfully in 58.5% of all trials, which was not significantly different from uninjured rats. In a separate group of animals, which received a bilateral pyramidal injury to remove  85% of corticospinal fibers in both dorsal and ventral  tracts, the animals showed retrieval of pellets in 24.7% of trials, four weeks after injury. Although this was significantly different from uninjured rats, it is important to note that this success rate shows a trend towards the control success rate. Underlying this spontaneous compensation of function, it was found that after injury, intact axons from the ventral and lateral tracts below the level of injury, sprout and tenninate onto neurons of the medial motor neuron pool (Weidner et al., 2001). This assumption was made due to the abolishment of reaching behavior upon transection of the ventral corticospinal tract. The corticospinal tract also sprouts onto propriospinal neurons to develop new circuits to re-establish function. After a dorsal hemisection at thoracic level 8, the severed corticospinal axons sprouted onto cervical grey matter and made connections with short and long propriospinal neurons. The connections with short propriospinal neurons were subsequently lost. However, those connections to long propriospinal neurons were retained, bridging the lesion site even 12 weeks after injury. These long propriospinal neurons arborize onto lumbar motor neurons, creating a new functional circuit (Bareyre et al, 2004). If there is reorganization of the spinal circuitry, it would cause there to be a reorganization of cortical maps following injury. A retrogradely transported pseudorabies virus labelled with green fluorescent protein was injected  14  into the muscles of the leg. The extent of the corticospinal cell body localization was visualized in the sensorimotor cortex, which showed ectopic localization in the forelimb sensorimotor cortex (Bareyre et al., 2004). Functionally, it was found that animals that received a thoracic dorsal hemisection abolished any hindlimb placing reflex. This reflex recovered between 1 and 3 weeks post injury, which is consistent with the timeframe needed for the reorganization of spinal circuitry. This recovery was again abolished when the rats received a pyramidal lesion, lending more evidence that sprouting of the corticospinal tract contributed to the improved function (Bareyre et al., 2004). For these anatomical changes in the spinal cord to be effective, the corticospinal system must form synapses on appropriate interneurons and/or motoneurons. Motoneurons in rats that innervate the arms and upper body are somatotopically organized in the ventral grey matter of the cervical spinal cord. Those that innervate the proximal muscles concerned with posture, and arm movement are found more medial in the gray matter compared to those that innervate smaller, more distal muscles involved in fine motor control of the hands and fingers (Weidner et al., 2001; Iwaniuk & Whishaw, 2000; Hostege, 1991). The recruitment of motoneurons in such a manner to produce fine coordinated motor control is brought about by integration of higher centers and sensory feedback through intemeurons (Liang et al., 1991; Kuang & Kalil, 1990). The integration of signals for the proper output of motoneurons illustrates the importance of appropriate synapse formation by descending systems after injury not only onto the correct intemeurons or motoneurons but also in respect to a specific relative location on a particular dendrite or region on a cell body. After corticospinal injury for example, synaptic input onto interneurons or motoneurons controlling fine motor movements is disrupted. Spontaneously,  15  other descending systems or spared axons of the corticospinal system sprout; however, they must make appropriate connections onto interneurons and motoneurons to compensate for any loss of function (Weidner et al., 2001; Vavrek et a!., 2006; Bareyre et al., 2004). One of the goals in sprouting and regeneration research is to induce sprouting of many collaterals, because the activity dependent plasticity and pruning of aberrant connections seen in development will select for appropriate connections for functional recovery (Wolpaw, 2006, Bregman et al., 1997).  1.5 Promoting the Ability ofAxons to Overcome Inhibition 1.5.] Enhanced Regeneration/Plasticity The promotion of the regenerationlplasticity of the corticospinal tract has been the target of a lot of effort due to its influence in locomotion in humans. In one study, rats received a unilateral pyramidal injury above the level of the decussation and subsequently, the purine nucleoside inosine was applied via an osmotic pump for 14 days to the intact sensorimotor cortex. After labeling the corticospinal axons with BDA, sections taken from the cervical enlargement showed a significant increase in sprouting of axon collaterals into the contralateral grey and white matter (Benowitz et al., 1999). Building on this finding, after unilateral sensorimotor contusion, and application of inosine to the lateral ventricles for 28 days, a 4-5 fold increase of intact cervical corticospinal axon profiles was observed crossing into the denervated contralateral spinal cord. The ability of mosine treated rats to recover a food pellet was significantly better with treatment. In testing forelimb asymmetry, and walking on a horizontal ladder, all groups recovered to baseline levels with no significant difference in recovery time (Smith et al., 2007).  16  The applications of neurotrophins have similar abilities to elicit growth. Neurotrophic factors are naturally found in the CNS during development where they function to regulate the survival and differentiation of neurons during development as well as plasticity in the normal brain (Kuipers & Bramham, 2006; Lu et al., 2005; Lush & Parada, 2005; Raineteau et al., 2001). Corticospinal neurons express trkB receptors for the neurotrophin, Brain Derived Neurotrophic Factor (BDNF) (Giehi & Tetzlaff 1996). The expression of trkB receptors is maintained in the neuronal cell body after injury; however they are lost from the axonal shaft after a few days. This phenomenon does not completely eliminate treatment effects with BDNF at the site of spinal cord injury, as it does promote some regeneration (Tobias et al., 2003; Kobayashi et a!., 1996). Application of BDNF at the cell body seems to be more effective, as it has been demonstrated that the application of BDNF to the motor cortex results in an increase in regeneration associated gene expression for GAP-43 and tubulin (Giehi & Tetzlaff 1996; Giehl et al., 2001). The induction of a cell body response in corticospinal neurons via treatment of the sensorimotor motor cortex with BDNF resulted in a significant increase in sprouting rostral to the site of thoracic injury, although it does not facilitate long distance axonal regeneration of axons into a peripheral nerve graft (Hiebert et a!., 2002). The induced collateral sprouting of injured corticospinal axons by BDNF was again recently described after a dorsal overhemisection in the thoracic cord. These fibers were shown to make a number of contacts with proprio spinal intemeurons that bridge the lesion site and promote functional recovery (Vavrek et al., 2006).  1.6 Functional Testing ofthe Corticospinal System The motor system has long been viewed as highly organized in a definite hierarchy; the motor cortex is at the top and controls every movement of the body. This original view has  17  evolved now to a concept of a highly distributed system. In this model, both the sensory input and the motor output follow different and parallel paths, and motor commands are initiated at distinct levels. In such a system, reorganizations of the motor system after lesion occur not only at the cortical level but also at any subcortical level and even within the spinal cord. The sensorimotor cortex projects to various subcortical targets, such as the pontine nuclei, nucleus raphe magnus, the reticular formation, the inferior olive, the trigeminal nucleus and the dorsal column nuclei. Partial lesions therefore impair but not to eliminate particular functions; this is indeed frequently observed in animal models. In humans, corticospinal injury has been thought to impair locomotion. This traditional thinking was a result of patients with lesions to the internal capsule due to stroke which affected more than the corticospinal system (Weisendanger, 1981). More and more evidence from primate models suggest that the corticospinal system in higher mammals is responsible for fine motor skill as well (Metz et al., 1998; Weisendanger, 1981). In the rat, discrete corticospinal lesions produce focal deficits of fine motor skills (Whishaw et a!., 1993; Weidner et a!., 2001). It has been shown that the motor cortex and corticospinal system are active when precise positioning of the limb is necessary during locomotion just as if it is involved during intentional reaching (Georgopoulos & Griliner, 1989). A number of different functional tests have been developed that exist to breakdown aspects of voluntary movement. Voluntary movement can propel the rat during locomotion, or help stabilize the body in exploration, or manipulate the environment in a useful manner. Injury to the corticospinal system by a pyramidal lesion results in deficits in voluntary motor function of the forepaws (Whishaw et al., 1993; Z’Graggen et al., 1998).  18  In this thesis, I will focus on two quantitative tests of forelimb and/or hindlimb function, the food pellet reach task, and gait analysis. Injury to the corticospinal system results in deficits in locomotion and gait in rats (Metz et al., 1998 and Fanardjian et al., 2000). The cat-walk is a novel test of gait which has the benefit of measuring a number of locomotor-related assessments simultaneously (ie. toe spread, base of support, stride length, angle of rotation of paws, etc.) (Hamers et al., 2006; van Meeteren et al., 2003; and Vrinten and Hamers, 2003). The forelimb reaching test assesses accurate forelimb fine motor control in the distal part of the limb as well as in the paw (Montoya et al., 1991; Whishaw et al., 1997; Nikkhah et al., 1998; MacLellan et al., 2002; Samsam et al., 2004; Whishaw et al., 1993; Thallmair et al., 1998; Z’Graggen et al., 1998; and Weidner et al., 2001). The food pellet-retrieval task requires the rat to reach through an opening in a wall for a rounded food pellet placed on a stage. This movement is driven by both proximal (advance of forelimb/pronation of limb) and distal muscles of the forelimb (arpeggio/suppination of wrist) which work in concert to execute accurate reaching and retrieval of a food pellet (Whishaw, 1996; and Whishaw & Pellis, 1990). The fine motor control that is necessary is facilitated by the corticospinal tract, which is modified in the sensorimotor cortex with input from propriospinal and olfactory afferents (Hermer-Vazquez et al., 2007; Piecharka et al., 2005; Whishaw et al., 1993; Whishaw & Tomie, 1989).  1.7 Rationale for Current Study The majority of people that sustain an injury to the spinal cord are in their early thirties (McColl et al., 1997) although a bimodal distribution has recently emerged with a second peak in elderly people (>60). It is striking to note that although ambulation after spinal cord injury is important in the eyes of one who is afflicted, it does not rate highest in their minds. Once  19  injured, living with the reality of SCI, bladder and kidney problems, sexual function, and bowel control, are foremost on their list of factors that would make life easier (White et al., 1992; 1993; Hart et al., 1996; Widerstrom-Noga et al., 1999; Cox et al., 200; Pentland et al., 2002; Anderson, 2004).  Recovery of function, of even one spinal level may make the biggest difference in daily life for these individuals. One approach to enhance functional recovery is to exploit undamaged CNS neurons (that extend past the point of injury) to sprout and connect to potential targets. Here, I will investigate whether infusion of BDNF into the intact sensorimotor cortex induces sprouting of undamaged corticospinal fibers into denervated spinal cord. The corticospinal system is a very good model used to understand possible induction of sprouting after partial central nervous system injury. The cell bodies of corticospinal neurons are superficially located in the sensorimotor cortex making treatment of these neurons very amenable. Their axons run through the internal capsule, and converge into a tight fascicle over the ventral surface of the medulla on its way to the spinal cord. This exposure of the corticospinal tract at the level of the medulla allows for the precise lesion known as a pyramidotomy, severing  90% of the axons (Whishaw et al., 1993; Whishaw & Metz, 2002).  We have previously found that application of the neurotrophin; BDNF, to the sensory motor cortex stimulates expression of regeneration associated genes such as GAP-43, and Tcd tubulin, and results in enhanced sprouting of injured corticospinal fibers rostral to the site of thoracic injury (Giehl and Tetzlaff, 1996; Giehi et al., 2001; Hiebert et al., 2002). While trophic factor therapy clearly could be used to stimulate sprouting/regeneration of injured CNS fibers, we will focus on inducing sprouting of intact or uninjured corticospinal axons (axons that were spared, not lesioned as the result of an injury). The main reason for this selection is that these  20  intact fibers extend past the point of injury and therefore will not require long distance regeneration to reach potential targets. Rats were infused with BDNF and injected with the anterograde tracer BDA for 14 days, after which they were killed to harvest CNS tissue. A 42 day experimental study was used to lengthen the amount of time between treatment after pyramidal injury and tissue harvest. This prolonged timepoint allowed for any increase in sprouting over time, and/or detect if these sprouts are retained. In addition, if BDA is injected at the same time BDNF treatment is applied, it may induce greater uptake of the anterograde tracer, therefore skewing the results to show a false increase in sprouting. This study will avoid this possibility by applying the tracer later. The time course of experimentation is also important, as longer periods of time are necessary to follow recovery processes such as rerouting of signals, synapse formation, and remyelination of reinforced connections. Here we will assess functional recovery with the reach to grasp task, and gait analysis.  1.8 Hypothesis It is my hypothesis that Application ofthe neurotrophic factor, BDNF to the intact corticospinal neurons, following a unilateral pyramidal injury, can facilitate sprouting ofthe intact corticospinalfIbers into the denervated halfofthe spinal cord, and improve the functional outcome.  21  Methods 2.1. Animal Husbandry The rats used in this experiment were purchased from rat breeding facilities at the University of British Columbia (UBC) South Campus. All experiments were conducted in accordance with the UBC Animal Care Ethics Committee, and adhered to guidelines of the Canadian Council on Animal Care. Sprague-Dawley rats weighing 175-225 grams and between 6 and 8 weeks of age were used. At this age, the rats have a mature CNS. The rats were randomly grouped four to each 50.5cm x 40.5cm x 21cm plastic cage. Once grouped, the animals remained with the same cage mates for the entirety of the experiment under a 12 hour light/dark cycle. The rats had free access to fresh water from their cage and were fed ad libitum with regular large rodent food pellets (LabDiet 5001, Missoula, MT) until the beginning of the experiment. Surgical procedures and behavioral testing were conducted during the light cycle period. 2.2 Experimental Studies Two experimental studies were conducted. One was a fourteen day experimental design beginning from the time BDNF was infused into the sensorimotor cortex to the time the rat was killed. The other was a 28 day design that allowed a greater amount of time from infusion of BDNF to kill. 2.2.1 Fourteen Day Experimental Study On day zero (Fig 2.1), all rats received a left pyramidal lesion, injections of the anterograde tracer Biotinylated Dextran Amine (BDA, Molecular Probes, Eugene, OR) into the cortex as well as an infusion of BDNF (500 ng/ 0.5 mi/br; n=4) or vehicle (n=4) for 14 days via a  22  cannula implanted 1.5 mm below the non-axonotmized cortex. To understand the normal extent of corticospinal innervation to the contralateral grey matter, some animals received a sham operation without lesion (n=5). The corticospinal tract was labeled using 200n1 of (25%) BDA injected with a Hamilton syringe into each of 8 sites. Two weeks was allowed for the BDA to label corticospinal axons in the gray matter before the animals were killed and perfused in order to harvest the brain and spinal cord. BDA Injection (Day 0) (8x200 nI %)  BDNFor Vehicle (Day 0) (500nglO.5mlIhr for 14 Days) Unilateral pyramidal lesion (Day 0) Assess growth of uninjured corticospinal fibers into the denervated cervical spinal cord (Day 14)  Fig 2.1 Experimental study (14 day time course). At Day 0, a left unilateral pyramidal lesion was perfonned. During the same procedure, BDA was injected to the contralateral intact sensorimotor cortex and the cannula delivering BDNF for 14 days was implanted. Rats survived a total of 14 days.  2.2.2 Forty-Two Day Experimental Study The 42 day design (Fig 2.2), contained three groups of rats that received a left unilateral pyramidal lesion. The first group received application of BDNF (n6) at the time of surgery, while the second received vehicle (n6). The BDNF/vehicle was administered for 14 days using the same procedures as the previous study. A third group received a lesion without the placement  23  of a camiula or pump (n5). Four weeks after the initial injury, and two weeks after the end of treatments, BDA was injected into the non-axotomized cortex. The animals survived another two weeks to allow the tracer to label corticospinal axons and their collaterals in the cervical and lumbar gray matter. BOA Injection (Day 28) (8x200 nI %)  BDNFor Vehicle (Day 0) (500ngIO.5miIhr for 14 Days) Unilateral pyramidal lesion (Day 0) Assess growth of uninjured corticospinal fibers into the denervated cervical spinal cord (Day 42)  Fig 2.2 Experimental Study (42 day time course). At Day 0, a left unilateral pyramidal lesion was  performed. During the same procedure, the cannula delivering BDNF for 14 days was implanted into the contralateral intact sensorimotor cortex. Twenty-eight days after injury, the cannula was removed from the cortex, and BDA was injected. Rats survived a total of 42 days.  2.3 Surgical Preparation BDNF and Vehicle Osmotic Pump Preparation -  BDNF or vehicle treatment to the intact right sensorimotor cortex of the rat was applied via an infusion cannula connected with Silastic tubing (VWR) to an Aizet osmotic minipump (#2002, Durect Corp., Cupertino, CA) according to the manufacturer’s instructions. The osmotic minipump delivered its contents at a rate of 0.5 i1/hr for 14 days. BDNF and vehicle solutions were prepared freshly in a sterile workbench under aseptic conditions. Stock vehicle solution was prepared for each minipump by supplementing 225pL  24  20mM sterile phosphate buffered saline (PBS) with 75pL 2% Rat Serum Albumin (RSA; Sigma), and 6 iL (100 U) PenicillinlStreptomycin. This solution was filtered with a 0.2iim filter to remove contaminants. For each BDNF minipump used, 285 iL of vehicle was removed from the stock vehicle solution, and 15 .tl of 20 tg/jiL BDNF was added, for a final concentration of 1p.g/IL BDNF. Each minipump delivered a maximum of 168 1 iL of solution over 14 days. BDNF was delivered at a rate of—j 500 ng/0.5pL/hr. BDNF or vehicle solution was injected into a 2-3 cm piece of Silastic tubing and attached to the respective minipump. The minipump was then pre-incubated overnight in sterile 20mM PBS at 37°C prior to surgery. The experimenter was blind as to the identity of the BDNF/vehicle group.  2.4 Presurgical procedure On the day of surgery, the rats were moved into a surgery preparation room separate from the surgical suite. This was done to reduce the anxiety and alarm reaction of the rats due to the smell of blood from surgery (Stevens & Gerzog-Thomas, 1977; Stevens & Saplikoski, 1973).  2.4.1 Pyramidotomy Adult male Sprague—Dawley rats were anesthetized with an intraperitoneal injection of a mixture of ketamine hydrochloride (72 mg/kg; Bimeda-MTC, Cambridge, ON) and xylazine hydrochloride (9 mg/kg; Bayer Inc., Etobicoke, ON). Additional doses of anesthesia were given throughout the surgery if needed. A rectal thermometer was used to monitor core temperature, while temperature was maintained with a heating pad under the rat. The ventral surface of the neck area of the rat was shaved and the rat was placed in a supine position on a surgical plate  25  above a heating pad and secured for surgery. The skin of the neck was cut with a single 5 cm vertical incision and the muscle layers were pried apart with a blunt probe. The base of the skull was exposed, by retracting the trachea. A window was opened through the base of the skull using a Dremel rotating drill to access the CST rostral to the pyramidal decussation. The dura was opened and a left unilateral pyramidotomy at the level of the caudal medulla oblongata was performed by selectively transecting the fibers of the CST using fine microscissors to a depth of 1mm. Any bleeding was stopped with Gelfoam. The retractors were removed after surgery and the muscles replaced with a single suture before the skin was closed with metal wound clips. Sham operated animals underwent the same surgical procedure, including the incision of the dura, except that no lesion was performed.  2.4.2 Osmotic Pump Implantation The rat was secured into a stereotaxic frame in a prone position and the scalp was shaved. An 4cm incision was made along the longitudinal axis of the head with a scalpel. The cranium was exposed and dried with a sterile cotton swab to allow for visualization of the cranial sutures. A window was made in the cranium with a Dremel rotary drill to expose the dura overlying the right sensorimotor cortex. The dura was cut open and a 27 gauge steel cannula (3280P/Spc; Plastics One Inc.), was inserted 1.5 mm into the parenchyma of the sensorimotor cortex: 1 mm posterior to Bregma and 2.0 mm lateral to the midline (Fig 2.3). The cannula was anchored to the cranium with two stainless steel watchmaker screws and covered with dental cement. The cannula was attached to an Alzet osmotic minipump with 2-3 cm piece of Silastic tubing and the minipump was placed into a subcutaneous pocket above the shoulders of the rat.  26  The incision was closed with metal wound clips and the rats were placed in a heated recovery chamber until they woke up from anesthesia.  Pu.nip and BDA Injection Sites • BDAlnjection Sites: (B (Bre)OL(Latera1)2.O);  /  ‘  Bf)L3O)(BO3L3O’(B1OL1j 10L2 )B 1L.O(B DL’ I (B-25L25)  (B  1  OPumpP)acernent:flrena—1.O Lateral2M  Bregma  Fig 2.3 Illustration of treatment pump and BDA injection sites overlaid a map oftopographical organization of forelimb/hindlimb sensorimotor cortex (Adapted from Fundamental Neuroscience, 1999).  2.4.3 Axonal Labeling The 10,000 Da form of Biotinylated Dextran Amine (BDA) was used to visualize the corticospinal tract and corticospinal axon profiles within the spinal cord. This tracer is highly soluble, readily internalized within the cell, and preferentially transported anterogradely (Veenman et al., 1992). BDA is advantageous for use in labeling neurons for several reasons. First, its application is easy because BDA can be pressure injected into the nervous system. Second, BDA can be deposited into defined sites that label specific projections like the corticospinal tract. Third, BDA labels corticospinal axons within the grey matter with a high degree of detail (Brosamle & Schwab, 1997). Fourth, paraformaldehyde fixes BDA by its lysine residues to surrounding proteins (Reiner et al., 2000). This allows for effective post fixed immunohistochemical procedures without affecting labeling.  27  In the 42 day experimental study, the rats were anesthetized 28 days after injury and implantation of the minipump/cannula. The metal wound clips on the scalp were removed and the scalp resected to expose the cannula embedded in dental cement. The cannula was carefully pried apart from the cranium in order to expose the brain. To visualize any growth of corticospinal axon profiles in the gray matter of both cervical and lumbar enlargements of the spinal cord, the 10,000 MW anterograde tracer biotin Dextran amine (BDA; Molecular Probes, Eugene, OR) was pressure injected into the right sensorimotor cortex surrounding the cannula site. 200 nl of 25% BDA was injected into each of eight sites, using a Hamilton syringe with a pulled glass micropipette tip (40-60 jim). The sites of injection were: (B (Bregma) 0.5, L (Lateral) 2.0); (B 0.5, L 3.0); (B -0.3, L 3.0); (B -1.0, L 1.5); (B -1.0, L 2.5); (B -1.8, L 2.0); (B  -  2.5, L 1.5); (B -2.5, L 2.5) (Fig 2.3). These sites covered both the forelimb and hindlimb areas of the sensorimotor cortex (Akintunde & Buxton, 1992). Extra care was taken not to puncture any cortical blood vessels by adjusting the injection site. The micropipette was inserted through the dura mater to a depth of 1.5 mm from the surface of the cortex. The BDA was injected after two minutes to allow for the brain tissue to make a seal with the micropipette tip. The BDA was injected in five equal aliquots of 40 nl over a period of two minutes. Once all 200 nl of BDA was injected, the micropipette left in place for another two minutes to prevent drawing out any BDA from the injection site. The wound was closed with metal wound clips, and the animals were allowed to recover from anesthesia in a heated chamber. Two weeks was allowed for the BDA to travel along and label corticospinal axons before the animals were killed and perfused. In the 14 day group, the cortex was therefore injected with BDA as outlined above, just prior to cannula implantation.  28  2.5 Perfusion and Tissue Harvest To harvest brain and spinal tissue, animals were overdosed with an intraperitoneal injection of chloral hydrate (BDH Chemicals, Toronto, Ontario, Canada). Once the respiration was very deep and slow, and there was no longer any reflex in response to pinching the hindlimb, or touching the eyeball, the rats were transcardially perfused with room temperature phosphate buffered saline (PBS) followed by 200 mL of cold 4% paraformaldehyde (PFA) in phosphate buffer. The forebrain and hindbrain, as well as the cervical and lumbar enlargements of the spinal cord, were dissected and post fixed in 4% PFA at 4°C overnight.  2.6 Tissue Preparation and Histological Techniques 2.6.1 Cryoprotection and Tissue Storage The brain and spinal cord sections were cryoprotected by transferring the tissue into increasing concentrations of sucrose solution (12%, 18%, and 24%). After immersion in 24% sucrose solution overnight, the hindbrain containing the lesion site was dissected and mounted onto filter paper. A code was written onto the filter paper to blind the observer for future histological analysis. The mounted tissue was frozen with isopentane cooled with dry ice. Spinal sections were treated in a similar manner. The cervical enlargement was sectioned at C-4, C-5, C-6, C-7, and the lumbar enlargement was cross sectioned at L3, L4, L5, and L6 as described by Anatomy of the Laboratory Rat (Hebel & Stromberg, 1976; Fig. 2.4). The sections were individually mounted onto coded paper and then frozen with the rostral cross plane of the section exposed. These sections were used to quantify the corticospinal axon profiles in the gray matter. All the tissue harvested was stored in a -80°C freezer until they were removed for sectioning on the cryostat.  29  Fig 2.4 Topographical relations of spinal cord, dorsal roots and vertebral column in the rat Figure 2.4 has been removed due to copyright restrictions. The image removed showed topographical relations of spinal cord, dorsal roots and vertebral column in the rat. Measurements for each spinal level included length, width, ratio of width to height, and length of spinal nerve roots. Image was taken from Hebel R, Stromberg MW (1976) Anatomy of the laboratory rat. Williams and Wilkins, Baltimore  30  2.6.2 Cryostat Sectioning The hindbrain was cryostat sectioned in a coronal plane at a thickness of 20 jim. The hindbrain and forebrain sections were collected onto Superfrost Plus slides (Fisher Scientific, Houston, TX) and stored at -80°C freezer until they were removed for analysis. C6 and L4/5 sections were cryostat sectioned and transferred into a 24 well culture plate filled with 0.01 M PBS to wash the cross sections. Cryostat sections were cut as needed and immediately processed for histochemical analysis. The remaining mounted spinal cord was stored at -80°C.  2.6.3 Immunohistochemistry 2.6.3.1 Determination ofLesion Size (Anti-Neurofilament) Corticospinal axons at the level of the pyramids are easily distinguishable from the surrounding tissue with Alexa 488 anti-rabbit anti-Neurofilament immunoreactivity. Slides containing hindbrain cryostat sections taken at the level of injury were washed two times in a bath of 0.01 M PBS for 10 minutes each time and transferred into a bath containing a solution of 0.01 M PBS with 0.1% Triton-X. The slide was moved to a level incubation chamber, and 250p.L of a primary antibody solution of 0.01 M PBS with 0.1% Triton-X, 1% normal goat serum, and 1/300 rabbit anti-neurofilament was pipetted onto the tissue sections and left for three hours at room temperature. The slides were washed two times with 0.01 M PBS for ten minutes, and placed back into the incubation chamber, where 300 jiL of 10% normal goat serum block in 0.01 M PBS with 0.1% Triton-X was added for 30 minutes at room temperature. To visualize the primary antibody under fluorescence, 200 jiL of a secondary antibody solution of 0.01 M PBS with 0.1% Triton-X, 1% normal goat serum, and 1/200 goat-anti-rabbit Alexa 488 was applied for 2 hours at room temperature under darkness to prevent bleaching of the fluorophore. The  31  slides were washed two times in a bath of 0.01 M PBS with 0.1% Triton-X for 10 minutes and transferred into a bath containing a solution of 0.01 M PBS. The slides were mounted and stored in the dark at 4°C.  2.6.3.2 Visualization ofBDA labeled Corticospinal Axon Profiles in Cervical and Lumbar Spinal Cord Corticospinal axons in both white matter and gray matter were visualized by incubating floating spinal cord cryostat sections with a Streptavidin conjugated Cy3 (Jackson ImmunoResearch, West Grove, PA). After sectioning, the spinal cord sections were washed three times in 0.01 M PBS with 0.1% Triton-X. The sections were incubated with 4999 iL of 0.01 M PBS with 0.1% Triton-X, and 1/500 Streptavidin conjugated Cy3 for 1 hour at room temperature, under darkness. The sections were washed three times after incubation with 0.01 M PBS with 0.1% Triton-X to remove any excess Cy3. The sections were placed into a deep Petri dish filled with 0.01 M PBS. The floating sections were placed onto Superfrost Plus slides (Fisher Scientific, Houston, TX) coded to double blind the experimenter. This method eliminated any air bubbles under the sections, or buckling of the sections when placed on slides. The slides were stored in the dark at 4°C.  2.7 Quantifications 2.7.1 QuantUlcation ofLesion Size To approximate the size of the pyramidal lesion, the area of intact neurofilament immuno-labeled axons in the injured left pyramid was compared with the total area of the left pyramid. Sections were visualized with a Zeiss fluorescent microscope (Thornwood, NY) and digital images were captured with a Qimaging camera (Bumaby, BC) using the Northern Eclipse  32 7.0 software (Empix Imaging, Mississauga, ON). The digital images were imported into Photoshop 6.0 and the perimeter of the intact portions of the injured left pyramid was manually traced on a new layer using a pencil two pixels in diameter. The area was then filled with the paint bucket tool and measured. The same procedure was followed when outlining the perimeter of the entire injured pyramid (Fig 2.5).  Fig 2.5 Neurofilament immuno-labelled axons below the level of injury and above the level of the pyramids (A). To approximate the size of the pyramidal lesion, the area of intact neurofilament immuno labeled axons in the injured left pyramid was compared with the total area of the left pyramid (C and D).  33  The BDA labeled pyramidal tract of uninjured rats was first measured in order to more accurately quantify lesion size in injured rats. Details of pyramidal shape and outline demarcation were studied (Fig 2.6).  Fig 2.6 BDA labelled corticospinal axons above the level of the pyramids (left pyramid).  The traced layer was imported into Eclipse 7.0, and the number of pixels in the layer was quantified. The number of pixels in the injured left pyramid was divided by the number of pixels in the entire pyramid to calculate the percentage of lesion area remaining.  2.7.2 QuantfIcation ofSprouting To quantify sprouting of intact corticospinal axons into the right denervated half of the spinal cord gray matter, cervical and lumbar spinal cord cross sections were visualized with a Zeiss fluorescent microscope (Thornwood, NY) and digital images were captured with a Qimaging camera (Burnaby, BC) using the Northern Eclipse 7.0 software (Empix Imaging, Mississauga, ON). Multiple images (l 5) were captured to reconstruct the entire gray matter under higher magnification. An image montage was produced using Adobe Photoshop 6.0 by overlapping landmarks on the images, and using the opacity tool to ensure complete overlap. On  34  a new layer, the labeled corticospinal axon profiles in the right half of the spinal cord gray matter demarcated at the midline by the central canal were manually traced at 200x magnification with a digital pencil two pixels in diameter (Fig 2.7). The traced layer was imported into Northern Eclipse 7.0 software and the number of pixels drawn was quantified.  Fig 2.7 Quantification of sprouting. (A, D) Cy3 labeling of BDA in the cervical spinal cord. (B, E) Magnified views of contralateral grey matter. (C, F) Labeled corticospinal axon profiles in the right half of the spinal cord gray matter demarcated at the midline by the central canal, were manually traced at 200x magnification with a digital pencil two pixels in diameter.  35  To account for differences in BDA labeling between animals, the quantified data was normalized with the number of corticospinal axons present in the ventral corticospinal tract in the most rostral section.  Normalized Sprouting =  Number of pixels in denervated half of spinal cord Number of axons in the ventral CST  2.8 Behavioural Evaluation 2.8.1 Pretest Procedure All Sprague-Dawley rats brought into the animal facility were habituated to manipulation and human contact by holding the rats in latex gloved hands for 10-15 minutes each for 2-3 days, or as needed. Habituation occurred when the rat no longer shivered when held, and freely explored the experimenter’s hands. The rats were diet restricted in order to modify behaviors, to allow for functional testing after injury. When rats had free access to a regular large rodent food pellet (LabDiet 5001, Missoula, MT), each rat ate approximately 20 to 26g of food daily depending on their original body weight. Upon food restriction, each rat was fed 13 g of large food pellets per day in addition to grain based dustless food pellets (Bioserv, New Jersey) used during the food pellet reaching test (approximately 1 Og per day). Post-surgery, the rats were fed ad libitum for two days and then placed back on the restricted diet. Weight was measured weekly throughout the experiment. The rats were tested for handedness to select for only right handed rats. Handedness (lateralization) is an intrinsic characteristic of rats and not a result of learning (Miklyaeva et a!., 1991; Stashkevich & Kulikov, 2001). To determine the rat’s preferred forelimb, the rats were  36  subject to the pre-training procedure for the reach task. Rats that preferred their left forelimb or were deemed ambidextrous were excluded for this study. This ensured that all rats in the study would receive a left pyramidal injury to affect the contralateral preferred right forelimb in the reach task.  2.8.2 Food Pellet Reaching Task Rats used in the 42 day experimental study were trained one month prior to surgery and tested in the week before surgery. Each rat was placed into a 25x30x35 cm Plexiglas box which was open at the top to enable easy cleaning and exchange of animals during testing (Fig 2.8, Whishaw et al., 1993). On one side of the box, a 12 cm long by 1cm wide slit was cut into the Plexiglas wall from the base. A 4 cm long by 2 cm wide stage was mounted horizontally on the exterior, 3cm from the base of the box. 190mg grain based dustless food pellets (Bioserv, New Jersey) placed in a dimple on the stage, 2cm outside the opening. A 190mg food pellet was chosen as it allows for greatest retrieval success (Metz & Whishaw, 2000).  I Fig 2.8 (A) Reach to grasp food pellet reach task training and testing apparatus schematic. (B and C) Pictures taken of rats during reach to grasp training.  37  In order to record the rodents perfonning the task, a 100 watt lamp was used to illuminate the testing. Two Cannon ZR35 mini digital video cameras were used, operating at 30 frames per second. One camera was placed directly in front of the stage, while the other was angled to view the side of the stage. All the equipment was in place during both training and testing sessions; however video was only recorded for testing sessions. During training sessions, rats were trained to reach for a 190 mg food pellet. The rat was placed in the testing box and a food pellet was placed on the floor of the testing box by inserting it into the box through the opening above the presentation stage. As the rat explored its environment, it would find and eat the pellet. The length of time it took the rat to eat a pellet ranged from 5 seconds to 20 seconds. After 3-5 food pellets were presented in this manner, food pellets were then held by the experimenter on the stage at the opening of the test box with a gloved hand. The experimenter released the pellet once the rat grasped the pellet with its mouth. When the rat was comfortable taking food from the experimenter in this fashion, the experimenter would not release the food pellet until the rat inevitably touched the pellet with its forepaw. After 2-3 pellets, the rat learned to consistently touch the pellet with its forepaw before grasping the pellet with its mouth (Hermer-Vazquez et al., 2007). During the next stage, the experimenter only released the pellet when the rat grasped the pellet with its paw. With more success, the pellet was presented at a greater and greater distance from the opening to the test box until the pellet was left in the dimple on the presenting stage. In order for the rat to grasp the food pellet, it must pronate the paw 90°. The rat’s anatomy does not permit a rotatory movement at the wrist. To compensate for the lack of movement, the rat moves its forearm across the midline of the body to pronate the paw  38 (Whishaw & Pellis, 1990). To accommodate this movement, the food pellets were placed in a dimple on the stage opposite to the preferred forearm. Once the rat was able to reach for the food pellet, a 45mg food pellet was placed in the back of the test box after each reach attempt. This was done to encourage the rat to move away from the opening. This forced the rat to approach the opening with a new stance for each successive trial. This was important because an unfavorable stance (ie. bad angle to the opening in the wall) increased the probability of an unsuccessful reach. Prior to surgery, the rats completed a series of training sessions until a 60-70% successful retrieval rate was maintained. This was achieved through approximately 6-7 training sessions where 50 reaches were performed per session. The rats were tested after training for a pre-surgical baseline, as well as 7, 16, 28 and 42 days after injury. Each testing session consisted of 25 reaches, and was video recorded. Each recording was digitally transferred onto a computer and reviewed in slow motion replay. The reaching behavior was scored using a modified 13-point non-linear scale developed in the lab and previously published (Table 2.1; Chan et al., 2005).  Table 2.1 Thirteen Point Ordinal Scale 0. Rat unable to lift or move hand. (Is only able to sniff the food). 1. Rat lifts paw vertically, but is unable to advance it towards the opening in the wall. 2. Rat lifts paw vertically, advances it towards the opening in the wall, but is unable to advance the paw through the opening (i.e. Paw hits the wall). 3. Rat lifts paw vertically, advances it towards the opening in the wall, advances the paw though the opening, makes contact with the stage, but is unable to touch the pellet, or touches the pellet lightly without knocking the pellet out of the dimple (does not grasp the pellet, and aiming is impaiid). 4. Rat lifts paw vertically, advances it towards the opening in the wall, advances the paw though the opening, makes several corrective movements aiming with difficulty, then touches and knocks the pellet out of the dimple (no grasp).  39 5. Rat lifts paw vertically, advances it towards the opening in the wall, advances the paw though the opening, immediately makes contact with pellet, (i.e. Aim is successful) and knocks the pellet out of the dimple (no grasp). 6. Rat lifts paw vertically, advances it towards the opening in the wall, and advances the paw though the opening, makes several corrective movements aiming with difficulty, grasps the pellet (flexing digits around pellet), moves the pellet, while still in its grasp, but drops it before presenting it to the mouth. 7. Rat lifts paw vertically, advances it towards the opening in the wall, advances the paw though the opening, immediately makes contact with pellet, (i.e. Aim is successful) grasps the pellet (flexing digits around pellet), moves the pellet, while still in its grasp, but drops it before presenting it to the mouth. 7b. Same as 7 above, however, the rat uses his uninjured paw to help retract the paw. 8. Rat lifts paw vertically, advances it towards the opening in the wall, advances the paw though the opening, makes several corrective movements aiming with difficulty, grasps the pellet (flexing digits around pellet), and moves the pellet, while still in its grasp, dragging the pellet on the stage before presenting it to the mouth. 8b. Same as 8 above, however, the rat uses his uninjured paw to help retract the paw grasping the pellet towards the mouth. 9. Rat lifts paw vertically, advances it towards the opening in the wall, advances the paw though the opening, makes a single corrective movement, grasps the pellet (flexing digits around pellet), and moves the pellet, while still in its grasp, dragging the pellet on the stage before presenting it to the mouth. 9b. Same as 9 above, however, the rat uses his uninjured paw to help retract the paw grasping the pellet towards the mouth. 10. Rat lifts paw vertically, advances it towards the opening in the wall, advances the paw though the opening, immediately makes contact with pellet, (i.e. Aim is successful) grasps the pellet (flexing digits around pellet), moves the pellet, while still in its grasp, dragging the pellet on the stage before presenting it to the mouth. 1 Ob. Same as 10 above, however, the rat uses his uninjured paw to help retract the paw grasping the pellet towards the mouth. 11. Rat lifts paw vertically, advances it towards the opening in the wall, advances the paw though the opening, makes several corrective movements aiming with difficulty, grasps the pellet (flexing digits around pellet), moves the pellet, while still in its grasp, lifting the pellet off the stage (i.e. By suppination, or by straight lift) before presenting it to the mouth. 1 lb. Same as 11 above, however, the rat uses his uninjured paw to help retract the paw grasping the pellet towards the mouth. 12. Rat lifts paw vertically, advances it towards the opening in the wall, advances the paw though the opening, makes a single corrective movement, grasps the pellet (flexing digits around pellet), moves the pellet, while still in its grasp, lifting the pellet off the stage (i.e. By suppination, or by straight lift) before presenting it to the mouth. 12b. Same as 12 above, however, the rat uses his uninjured paw to help retract the paw grasping the pellet towards the mouth. 13. Rat lifts paw vertically, advances it towards the opening in the wall, advances the paw though the opening, immediately makes contact with pellet, (i.e. Aim is successful) grasps the pellet (flexing digits around pellet), moves the pellet, while still in its grasp, lifting the pellet off the stage (i.e. By supination, or by straight lift) before presenting it to the mouth.  40  2.9 Gait Analysis The gait analysis was conducted on rats in the 14 day experimental study and was modified from de Medinaceli et al. (1982). The apparatus included a “holding” box (50.5cmx4O.5cmx2lcm) and a “home” box of the same size, separated from each other by 100cm. A plank (l20cmx8cm) was placed connecting the “holding” box to the “home” box, to ensure that 10cm of the plank was over the edge of the boxes. Rats were trained to traverse the plank from the “holding” box to the “home” box without hesitating. In order to facilitate this, a 100W light bulb was illuminated over the end of the plank above the “holding” box. The intense light worked as a deterrent against resting on the plank. In the “home” box, a smaller cardboard box (33cmx28cmx36cm) with its opening facing the plank was filled with bedding to the level of the plank. This created a dark space that was familiar and comfortable. All four of the rats housed together in a cage were trained and tested at one time. During training, each rat was placed in the “holding” box for 5 seconds and then raised to the plank above the “holding” box. The rat then walked across the plank to the “home” box. This was repeated a total of three times in each of two different training sessions. Presurgical testing occurred during the third session. The plank was covered with newsprint that was cut to fit the dimensions. To visualize footprints on the paper, the rat’s forepaws were placed in a thick mixture of sugarless Lemon-Lime (green) Kool-Aid drink mix and water. The rat’s hind paws were placed into sugarless Strawberry (red) Kool-Aid drink mix and water. This method resulted in dark footprints, with clearly visible foot pads and toes. Each testing session consisted of three trials. Each trial was timed to control for speed. The rats were tested for a pre-surgical baseline, as well as 3, 7, and 14 days after injury. Once testing was complete, the strips of paper were scanned into the computer and measurements were  41  taken using Adobe Photoshop 6.0. The mean values of “base of support,” “stride length,” and “toe spread,” from three consecutive step cycles (left and right) in each trial was measured. Base of support was measured as the center to center distance between the left and right forepaws or hind paws. Stride length was measured as the distance between the forelimb or hindlimb in one step cycle with the same forelimb or hindlimb of the next consecutive step cycle. Toe spread was measured as the distance between the first and fourth toe in the forepaw, and the first and fifth toe in the hind paw (Fig 2.9).  Forelimb Hindlimb  I  H V  Fig 2.9 Toe spread was measured by functional testing of gait. Forelimb toespread was measured from the first toe to the fourth toe. Hindlimb toe spread was measured from the first toe to the fifth toe.  42  Results 3.1 Weight ofRats Increased with a Restricted Diet To increase motivation to perform the food pellet (reach to grasp) test, the rats were placed on a restricted diet. The weight of rats under experimentation was recorded over the entire duration of experimentation. Rats on the restricted diet began experimentation with an average weight of 266+/-4.2g, and weighed 330+/-11.9g upon the conclusion of testing (Fig 3.1).  Weight of Rats with Time 400 350  -  300 250 200 4)  150 100  -  50 0 Pre-surgica  I Week  2 Week  3 Week  4 Week  5 Week  6Weck  Surgical Week  Fig 3.1 Weight of rats increase with a restricted diet  3.2  BDNF Induces Seizures  In both experimental designs, the neurotrophin BDNF was infused into the rat sensorimotor cortex via a cannula connected to an osmotic minipump. These rats exhibited seizures isolated to the right forelimb and hindlimb (corresponding to the infusion of BDNF into the left  43  sensorimotor cortex). The number of rats that experienced seizures varied in the two experimental designs. In the 14 day experimental study, 3 out of 4 BDNF treated rats were observed having seizures; whereas, in the 42 day experimental design, 2 out of 6 rats were observed having seizures. The seizures appeared to occur after the rats performed intentional movements involving the sensorimotor cortex during behavioral testing. Seizures were also induced at times of stress such as when the rats were handled and manipulated. The rats did not display any seizure activity when observed in their home cages in the animal storage facility. Each bout of convulsions lasted 2-3 minutes, and incapacitated the rats onto their nonconvulsing right side. The right forelimb and hindlimb alternated between being maximally extended and relaxed every few seconds. These convulsions were more pronounced in the hindlimb. Once a bout fmished, the rat would resume the normal activity of exploring its environment. The seizures would return periodically every 5 minutes. If the rat experienced seizures, it was returned to its home cage with its cage mates and tested at a later time.  3.3 Surgical Observations 3.3.1 Cannula Implantation into the Intact Left Sensorimotor Cortex Causes Damage The tip of the cannula delivering BDNF or vehicle was placed 1.5 mm into the right intact sensorimotor cortex for 14 days in the 14 day experimental study and 28 days for the 42 day experimental study. Upon removing the cannula, a hole approximately a quarter of a millimeter in diameter was visible and little to no hemorrhage was seen at the site. However, upon inspection under the microscope, damage could be seen around the site of cannula infusion. The extent of damage did not visually differ between vehicle and BDNF treated animals.  44  3.3.2 Injury ofthe Corticospinal Tract at the Level ofthe Pyramids Leaves Spared Tissue In both studies, the left corticospinal tract was transected immediately above the pyramidal decussation. At this level, the medial lemniscus and olivary complex are located dorsally to the corticospinal tract (Paxinos & Watson, 1998; Whishaw et al., 1993). Since both of these structures are involved in motor performance, it is important that they are not damaged by the unilateral pyramidotomy procedure. Hindbrain cross sections were analyzed to determine the extent of corticospinal transection (Fig 2.5). Some corticospinal tract fibers were spared in most rats, either medially or laterally. Typically, there was isolated sparing of 10-15 % in the left pyramidal tract. There was no evidence of damage to underlying medullary structures. The area of spared tissue was quantified below the level of pyramidal lesion. There was no significant difference in the percentage of spared axons between BDNF, vehicle, and lesioned only rat groups in the 14 or 42 day experimental study. There was also no significant difference in lesion size when any treatment group was compared in either of the two studies (14 Day ANOVA p=O.95; 42 day ANOVA p=O.93; Fig 3.2 and 3.3).  Percent Area of Spared Axons in Injured Left Pyramid (14 Day Paradigm) 20 c  18  <  16  0 X  0  14 0.  LI  12  -  6 4 2.  0  .--......—.  BDNF Lesioned  Vehicle Lesioned  Untreated Lesioned  Treatment  Fig 3.2 Percent Area of Spared Axons in Injured Left Pyramid (14 Day Experimental Study)  45  Percentage Area of Spared Axons in Injured left Pyramid (42 Day Paradigm) 20 18 C  o 16  0.  4.. 2 BDNF Lesoned  Vehide Lesoned  Untreated Lcsoned  Treatment  Fig 3.3 Percent area of spared axons in injured left pyramid (42 Day Experimental Study)  3.4 Anterograde BDA Labels Dorsal, Lateral, and Ventral CSTAxons, and Fibers Within the Grey Matter 3.4.1 BDA Labeling of CS Axons at the Cervical Level in the 14 Day Study Corticospinal fibers were labeled at the level of the C6 spinal cord with BDA. Using Cy3-coupled Streptavidin, this allowed for robust visualization of the dorsal, ventral and lateral components of the corticospinal tract. Fibers innervating the gray matter on both the ipsilateral and contralateral sides relative to injury and injection were labeled with Cy3, as seen in Fig 3.4.  46  Fig 3.4 Fibers innervating the grey matter on both the ipsilateral and contralateral sides at this level were also strongly labeled with Cy3. A and B show BDA labelling in the contralateral grey matter of an uninjured rat. C and D show BDA labelling in a left unilateral pyramidal injured rat.  There was, however, a high level of variability in the extent of labeling seen when comparing rats within a group. Some rats showed significantly more traced axons in the dorsal and ventral components of the corticospinal tracts than others. The number of traced corticospinal axons located in the dorsal and ventral funiculi of the denervated half of the spinal cord was quantified. Figure 3.5 shows the average number of intact axons counted in the denervated dorsal component (Fig 1.1-3) of the corticospinal tract was 33 +1- 3 SEM. On average, there were 102 +1- 13 corticospinal axons found in the ventral component (Fig 1.1-4).  47  Average Intact Axons in Cervical Spinal Cord 140 120 •Denervated Dorsal CST (Fig. 1.1-3)  100 80 60  SDenervated Ventral CST (Fig. 1.1-4)  40 20 0 14 Day Study  42 Day Study  Fig 3.5 Average Intact Axons in the denervated dorsal CST and denervated ventral CST labeled with Cy3.  The areas of the grey matter innerved by the intact corticospinal tract were variable as revealed in cross sections of the spinal cord. Some animals showed labeling in Rexed laminae I VI, X of the dorsal horn (Fig 3.6 A), while others showed labeling of fibers within Rexed laminaes V-Vu, X of the ventral horn (Fig 3.6 B). The majority of animals however, showed labeling intermediate between these extremes. Corticospinal fibers in the contralateral denervated grey matter were sparse and localized in laminae III-X.  48  Fig 3.6 Cy3 labelling of BDA in the cervical enlargement (C6) of rats in the 14 day experimental study. (A) Some rats showed topographical labeling into the dorsal horn. (B) Labeling in some rats showed topographical labeling into the ventral horn.  3.4.2 Normalization ofCorticospinal Axon Profiles in Cervical Grey Matter (14 Day Study) In order to allow for effective comparison of corticospinal growth between rats, and treatment groups, the quantification of labeled axon profiles in the denervated half of the spinal  49  cord was normalized with a number of different approaches. This was done to choose the most appropriate method. The number of pixels drawn in the denervated grey matter (quantification of labeled axon profiles) was calculated. This was plotted against the total number of corticospinal axons located in the denervated dorsal and ventral funiculi. The degree of labeling of corticospinal axon profiles in the denervated grey matter was directly proportional to the number of corticospinal axons counted in the denervated dorsal funiculus (Fig 3.7 A), ventral funiculus (Fig 3.7 B), and both dorsal + ventral funiculi (Fig 3.7 C).  A  B  Number of Pixels Drawn in the Denervated Grey Matter Vs. Corticospinal Axon Count in the Denervated Dorsal Funiculus 100  y 03014*15.40  Number of Pixels Drawn in the Denervated Grey Matter Vs. Corticospiaal Axon Count in the Denervated Ventral Funiculus 400 y 00025 25.01 R0.600  t*0.460 350  300 10  n  .4 CO  50  4 4,  4  251  + 200  4  4+  :  10•  5000  10000  10000  20000  25000  00000  05000  40000  45000  50000  +  0  0000  Nun,berefPhceI Drawn n the Danerated Grey Mntter  C  4  • •••  150  10000  04000  .  20000  25024  30000  33000  40000  45004  50000  N,rn,b.r ef Pieá Drawn hi the Denervated Grey Mat.r  Number of Pixels Drawn in the Denervated Grey Matter Vs. Corticospinal Axon Count in the Denervated Dorsal ÷ Ventral Funiculus 450. 400 350•  . .  300 250  C> —  000150 100 SO-  0  •• 5000  10300  15030  20000  25000  30000  05000  40000  45500  50000  Nambaref PiIe Drawn ho the Deneroatad Gray Matter  Fig. 3.7 Selection of normalizing factor. For rats in the 14 day experimental study, the number of pixels drawn in the denervated grey matter was plotted against the total number of corticospinal axons located in the denervated dorsal funiculus (A), ventral funiculus (B), and both dorsal + ventral funiculi (C).  50  Regression analysis of the corticospinal axon profiles in the grey matter versus the number of CS axons in the dorsal, ventral, and dorsal + ventral funiculi, showed R2 values of 0.465, 0.6004, and 0.5923 respectively. The corticospinal axon count in the ventral and dorsal+ventral funiculi were found to best normalize for the variability in corticospinal labeling at the cervical level. The Y-intercepts for these regressions were 15, 25, and 45 axons respectively, suggesting that a critical number of axons in the respective funiculi might be required to detect grey matter collaterals. 3.4.3 BDA Labcling of CS axons at the Cervical Level in the 42 Day Study Similar to the 14 day study, corticospinal axons in the white matter and their fibers innervating both the intact and denervated halves of the spinal grey matter were labeled in the 42 day study. There was some variability in the grey matter projections between rats (most extreme cases shown in Fig 3.8 A, B), however, the majority of animals exhibited consistent tracing of both ventral and dorsal projections. In this group of rats, the average axon count in the denervated dorsal corticospinal tract was 20 +1- 2. The average ventral corticospinal axon count was 72+!- 8.  51  Fig. 3.8 Cy3 labelling of BDA in the cervical enlargement (C6) of rats in the 42 day experimental study. (A) Some rats showed topographical labeling into the dorsal horn of the grey matter. (B) Labeling in some rats showed topographical labeling of fibers into the ventral horn.  3.4.4 Normalization ofCorticospinal Axon Profiles in Cervical Grey Matter (42 Day Study) The amount of corticospinal fibers in the denervated half of the spinal cord was again compared with the number of corticospinal axons found in the denervated dorsal (Fig. 3.9 A),  52  ventral (Fig. 3.9 B) and both funiculi together (Fig. 3.9 C). As with the animals in the 14 day study, the degree of corticospinal tract labeling at the cervical level was found to be directly proportional to the amount of corticospinal axon profiles seen within the denervated grey matter of the spinal cord. Linear regression analysis comparing the corticospinal axon profiles seen within the denervated grey matter of the spinal cord to the corticospinal axon count in the dorsal, ventral, and dorsal+ventral funiculus, revealed R 2 values of 0.1166, 0.2862, and 0.2655 respectively. In this group of rats, the axon profiles drawn in the denervated grey matter were also compared to the number of corticospinal axons visualized at the level of the pyramid (Fig 3.9 D). The R 2 value for this comparison was 0.28 19. The corticospinal axon count in the ventral, dorsal  +  ventral funiculi, and at the pyramids best predicted the amount of label in the denervated grey matter. The Y-intercepts for these regressions were 9, 1, 11, and 775 axons using the dorsal, ventral, dorsal  +  ventral, and pyramidal corticospinal axon counts respectively.  53  A  B  Number of Pixels Drawn in the Denervated Grey Matter Vs. Corticospinal Axon Count in the Denervated Dorsal Funiculus  y0401fl9.132 50.0.114  70  61:  Number of Pixels Drawn in the Denervated Grey Matter Vs. Corticospinal Axon Count in the Denervated Ventral Funiculus 300  40.000.+0S95 500.246  .  2601  +  ___—c,  ‘ •.___ ••  :1  107  4  21  10  .  0  2000  4000  *  6200  4  .4,  10000  0003  12000  14000  0  15000  2000  D  Number of Pixels Drawn In the Denervated Grey Matter Vs. Corticospinal Axon Count In the Denervated Dorsal + Ventral Funiculus  310  .  2000  01,4.26S  H. I  1  •  4000  5000  0000  4  10000  02000  14020  15001  Nomborof Pin.Isdrewn In th. D.n.ro.twIG..y Matter  Nsmbee,fPtaels Dwrn, lethe Denar07tsd Grey MetIer  C  4  4  4  Number of Pixels Drawn in the Denervated Grey Matter Vs. Corticospinal Axon Count in the PyramidalTract y0.070117310  1040  HI  1507;  Q 2200  4  .  —  500.221  4  1400  4  °°1 1 :ii  •  4  ‘__‘  :  ..  9  i 0  0101  0040  5044  0000  10004  12000  Numtserof Ptxelottwwn tn the Deoereated Gray Matter  14400  100083  0  2030  4004  5000  0000  10004  12040  14000  10000  NamberofPixelo Drawn Is the Denersated Gray Matter  Fig 3.9 Selection of normalizing factor. For rats in the 42 day experimental study, the number of pixels drawn in the denervated grey matter was plotted against the total number of corticospinal axons located in  the denervated (A) dorsal funiculus, (B) ventral funiculus, both (C) dorsal + ventral funiculi and at the(D) level of the pyramids.  3.4.5 BDA Labeling ofCS Axons at the Lumbar Level in the 14 Day Study In cross sections taken from the lumbar enlargement (L4) of the spinal cord of rats in our 14 day study, the dorsal corticospinal tract was robustly labeled with Cy3 (Fig 3.10). At this level, our labeling method was not successful in labeling the ventral component of the corticospinal tract. Fibers innervating the grey matter were strongly labeled. Cy3 labeling of BDA in the corticospinal tract, and fibers within the grey matter was variable between rats similar to the  54  cervical enlargement. The average number of corticospinal axons in the dorsal tract at this lumbar level was 19 +1- 6 axons.  Fig 3.10 Labeling of Corticospinal axons at the Lumbar enlargement (L4) in the 14 Day Study  3.4.6 Normalization ofCorticospinal Axon Profiles in Lumbar Grey Matter (14 Day Study)  Since only the dorsal component of the corticospinal tract was labeled in our animals, the amount of corticospinal fibers in the denervated half of the spinal cord was only compared with the number of BDA labeled corticospinal axons found in the denervated dorsal funiculus. Similar to comparisons made at the cervical level, the number of axons in the denervated dorsal  55  corticospinal tract was found to be directly proportional to the amount of corticospinal axon profiles seen within the grey matter (Fig 3.11). A linear regression was plotted and the R 2 value equaled 0.400. The Y-intercept for the regression line was 5 axons. Number of Pixels Drawn in the Denervated Grey Mailer Vs. Corticospinal Axon Count in the Denervated Dorsal Funiculus (14) 14 Day Paradigm 70 y= 0.OOIx-f 4.541 R=0.400  $  60  j  °  40  0  zzr.zEZ.Z  2000  4000  6000  8000  10000  12000  14000  16000  18000  20000  Number of Pixels in the Denervated Grey Mttcr  Fig 3.11 Selection of normalizing factor. For the lumbar enlargement of rats in the 14 day experimental study, the number of pixels drawn in the denervated grey matter was plotted against the total number of corticospinal axons located in the denervated dorsal funiculus.  3.4.7 Choice ofNormalization Factor In order to directly compare or normalize quantifications of growth or lack thereof between rats and treatment groups a factor needed to be chosen. The number of axons labeled in the various components of the cortico spinal tract itself was used as the normalization factor. To best select the normalization factor, the highest R 2 value and lowest Y-intercept of the regression line in the comparison of axon count with axon profile quantification in the denervated spinal  56  cord was taken into consideration. A combination of the uncrossed dorsal and ventral corticospinal axon counts was chosen since both these components of the corticospinal tract were major contributors to the innervation evident in the denervated half of the spinal cord.  3.5 Axonal Profile QuantfIcation 3.5.1. Intact dorsal corticospinal tract encroaches into the contralateral denervated white matter after pyramidal lesion  The cross section taken from the spinal cord of an unlesioned animal (Fig 3.4 A) showed the normal state of the dorsal corticospinal tract. The interface between the left and the right aspects of the tract was clearly demarcated by a perpendicular border just above the midline of the central canal. After a pyramidal lesion, (Fig 3.4 C) this demarcation was not as sharp and shifted to encroach into the space occupied by the lesioned tract. This resulted in the dorsal and ventral edges of the labeled tract becoming rounded at the interface between the two tracts.  3.5.2 A unilateral pyramidal lesion significantly induced sprouting of intact corticospinal axons into the contralateral side ofthe cervical spinal cord 14 days after injury, however, BDNF or vehicle did not sign flcantly induce further sprouting. Normally in the cervical spinal cord, there is a substantial innervation of corticospinal axon profiles from the intact sensorimotor cortex into the denervated half of the C6 spinal cord (Fig 3.12 A). In unlesioned sham operated animals (n=5; 2 sections/animal), 7470 +\- 1213 pixels were drawn. Unlesioned sham operated rats displayed an average of 56 +1- 8 pixels/section after normalization with dorsal + ventral corticospinal axons. In rats with BDNF or vehicle treatment, after a unilateral pyramidal lesion (n4; 2 sections/animal), an average of  57  10958 +1- 2270 pixels and 18071 +1- 5139 pixels were drawn per section respectively, and after nonnalization, 82 +1- 12 and 130 +1- 27 pixels/section was calculated respectively (Fig 3.12 B). Using a one way ANOVA, normalized data were compared among treatment groups and was found to be significant (p=0.042). Further to an analysis of variance, a two sample Student’s t-test assuming equal variance was used to compare the averages of each treatment group. BDNF  and vehicle treatment groups were significantly different from the unlesioned sham operated group (p=O.O , 0.0057 respectively). Infusion of BDNF did not significantly induce further 44 sprouting in the cervical spinal cord when compared with vehicle treatment (Fig 3.12 B).  A  Denervated Grey Matter Axon Profiles (C6) -14 Day Paradigm 25000  20000  -  15030 0  I 5003  -  BDNF+Px  Vehde+Px  Unesioned  Treatment  B  Denervated Grey Matter Axon Profiles (C6) after Dorsal and Ventral CST Normalization 14 Day Paradigm -  *  +  *+  Vehde + Px  Unlesioned  160 140 120 100 80 50 40 20 0  BDNF + Ps  Tratm.nt  Fig 3.12 (A) Number of pixels drawn in the denervated half of the cervical (C6) spinal cord in 14 day experimental study rats. (B) Normalized number of pixels drawn in the denervated half of the cervical (C6) spinal cord. The symbols * and + show significance from unlesioned rats. Numbers of rats BDNF +Px n=4; Vehicle + Px n4; Unlesioned n5.  58  3.5.3 A unilateral pyramidal lesion signflcantly induced sprouting of intact corticospinal axons into the contralateral side ofthe cervical spinal cord 42 days after injury, however, BDNF or vehicle did not sign fIcantly induce further sprouting In the 42 day study, a greater amount of time was allowed for growth of corticospinal fibers into the denervated half of the spinal cord. The same methods were used to collect data from this group, allowing for comparison of the treatment groups to the unlesioned sham operated animals. After a unilateral pyramidal lesion (n5; 2 sections/animal), an average of 7965 +1- 772 pixels was quantified and resulted in 85 +1- 10 pixels/section after normalization. In animals with BDNF or vehicle treatment after a unilateral pyramidal lesion (n=6; 2 sections/animal), an average of 6432 +/- 770 pixels and 6742 +/- 668 pixels were drawn in the denervated grey matter, which resulted in 95 +/- 9 and 142 +/- 35 pixels/section after normalization (Fig 3.13 A, B). Using a one way ANOVA, normalized data were compared among treatment groups and found to be significant (p=0.0396). Further to an analysis of variance, a two sample Student’s t test assuming equal variance was used to compare the averages of each treatment group. BDNF, vehicle, and pyramidal lesion alone treatment groups were significantly different from the unlesioned sham operated group (P0.0019; 0.020; and 0.0079 respectively). Infusion of BDNF or vehicle did not significantly induce further sprouting at the level of the cervical spinal cord when compared with pyramidal lesion alone. As with our 14 day study, infusion of BDNF did not significantly induce further sprouting at the level of the cervical spinal cord when compared with vehicle treatment.  59  A  Contralateral Grey Matter Axon Profiles (C6) 42 Day Paradigm  9000 8000 1 7000 6000 5000 0 .0  4003  E z  z  2000 1000 0—  BONF+Px  Vehide+Px  Px  Un1esoned  Tretmcrtt  B  Contralateral Grey Matter Axon Profiles (C6) after Dorsal and Ventral CST Normalization- 42 Day Paradigm A  *  180  *+A  150 140 120 100  -  0  E 0  z  so-’ &o-4 40 20 0.  VeNde ÷ Ps  n  UnIesoned Treotment  Fig 3.13 (A) Number of pixels drawn in the denervated half of the cervical (C6) spinal cord in 42 day experimental study rats. (B) Normalized number of pixels drawn in the denervated half of the cervical (C6) spinal cord. The symbols *, + and A show significance from unlesioned rats. Numbers of rats BDNF +Px n6; Vehicle + Px n=6; Px n=5; Unlesioned n=5.  3.5.4 Application ofBDNFfollowing a unilateral pyramidal lesion signicantly induced sprouting ofintact corticospinal axons into the contralateral side ofthe lumbar spinal cord when compared to vehicle treated, and unlesioned sham operated animals 14 days after injury Sections taken from the L4-5 level of the lumbar enlargement were also analyzed for potential sprouting. Quantifications of corticospinal axon profiles within the denervated half of the lumbar grey matter after pyramidal injury showed an average of 3286 +1- 1119 pixels (Fig  60  3.13 A). The vehicle treatment group showed an average of 7809 +1- 2552 pixels, while the BDNF treatment groups showed an average of 8350 +1- 3416 pixels. The raw quantifications were normalized with the number of BDA labeled corticospinal axons found in the denervated dorsal funiculus. Normalized quantifications of BDNF, vehicle, and unlesioned sham operated groups were 1166 +1- 265; 362 +1- 100; and 227 +1- 62 pixels/section, respectively (Fig 3.14 B).  A  CorticospinalAxon Profiles in Denervated Grey Matter {Lumbar4-5) 14 Day Paradigm -  14000  12000  10000 0.  0 e  8000  .0  E  5080-  4000  2000  Vehic’e ÷  6  Treatment  B  Corticospina Ixon Profiles in Denervated Grey Matter (Iumbar4-5) with Denervated DorsaL CST NormaLization 14 Day Paradigm 1600  *  +  1400  1200 C 0  1008 80t-  a  z  600 400 200  --  -  I I  -  SDNF * Ps  Vahde, Ps  I  U,teso÷ed  Treatment  Fig 3.14 (A) Number of pixels drawn in the denervated half of the cervical (L4-5) spinal cord in 14 day experimental study rats. (B) Normalized number of pixels drawn in the denervated half of the cervical (L4-5) spinal cord. The symbols * and + show significance from unlesioned rats.  61  Using a one way ANOVA, normalized data of the CST branches at L4/5 were compared among treatment groups and found to be significant (p0.0035). Further to an analysis of variance, a two sample Student’s t-test assuming equal variance was used to compare the averages of each treatment group. The BDNF treatment group was significantly different from both the vehicle and the unlesioned sham operated groups (p=0.0015 and 0.0031 respectively). Vehicle treatment was not significantly different when compared to the unlesioned sham operated group (p=0.13). Infusion of BDNF significantly induced further sprouting in the lumbar enlargement when compared with vehicle and unlesioned sham operated groups (Fig 3.14).  3.6 Behavioral Quantfication 3.6.1 Food Pellet Reach Task 3.6.1.1 Training success rate plateaus but does not increase or decrease with further practice or testing Once the rats learned how to reach for food pellets (for training procedure, see methods) in the reach to grasp task, the rats were monitored to assess any gain in successful skilled reaching over time. Twenty-one rats were tracked in each session. Every session, consisted of 25 reach attempts and were conducted for 13 consecutive days. A percentage was calculated by dividing the number of successful retrievals by the total of 25 attempts (Fig 3.15). A one-way Analysis of Variance revealed that there was no difference in the average reaching success of the rats at any testing time (p = 0.41).  62  Training Success Rate 0..  OJ 0.5  03 WI  0.4 Li,  0.3 0.2 0.1 0 1  2  3  4  5  6  7  8  9  10  11  12  13  Session Number Fig 3.15 Training success rate in the reach to grasp food pellet task. Training success rate plateaus but does not increase or decrease with further practice or testing.  3.6.1.2 Functional Testing Thirteen Point Analysis In the food pellet reach to grasp task, rats in the 42 day experimental study were scored on a 13 point ordinal scale (Fig 3.16). This 13 point ordinal scale was developed in the lab specifically for the evaluation of reaching behavior in rats (Table 2.1). Pre-surgical baseline testing indicated that the scores for the four groups (unlesioned sham operated, n=5; pyramidal lesion alone, n=5; vehicle treatment with pyramidal lesion, n6; and BDNF treated with pyramidal lesion, n=6) were not significantly different from each other (score  =  9.57 +1- 0.38,  10.62 +1- 0.52, 9.95 +1- 0.44, and 10.4 +1- 0.51 respectively; p= 488 Kruskal-Wallis ANOVA . 0 on ranks). Testing one week after injury showed a significant decrease in average score of the  63  pyramidal lesion alone (7.36 +1- 0.50), vehicle (6.76 +1- 0.41), and BDNF (6.94 +1- 0.58) treatment groups when compared to unlesioned sham operated animals (9.52 +1- 0.39) (p<0.OOl,  p<O.OOl, p<0.00i respectively; Mann-Whitney rank sum test). The vehicle (6.76 +1- 0.41) and BDNF (6.94 +1- 0.58) treatment groups did not show any significant difference when compared to each other, or to the pyramidal lesion alone (7.36 +1- 0.50) groups (p=0.90 Kruskal-Wallis ANOVA on ranks). On day 42, the reaching scores for the pyramidal lesion alone, vehicle, and BDNF treatment groups were significantly different from the unlesioned sham operated group (p=O.O l 3 , p=O.OO2, and p<0.00i respectively). When all the reaching scores for the lesioned groups were compared at this time point, there was no significant difference (p0.274).  Reach Task 13 Point Scale -  12 A  A  +  +  A  ** 10  8  ‘U  6 0 0. -I  4  2  0 Day 16  Day 28  Timepoint  Unlesioned  U Px  U Vehicie + Px  U BDNF + Px  Fig 3.16 Thirteen point analysis of the reach to grasp food pellet task in the 42 day study. The symbols A + and show significance from unlesioned rats.  ,  64  Success Rate Information gathered from the 13 point ordinal scale showed the success rates for reaching and retrieving a food pellet in the reach to grasp task. Success was defined by the rat’s ability to extend the preferred forelimb through the opening of the reach task box, retrieve the food pellet and present it to the mouth. In the 13 point scale, only score levels 8, 9, 10, 11, 12, and 13 were considered to be successful. Pre-Surgical baseline testing affirmed that all the treatment groups began the experimental phase with a statistically similar success rate (Unlesioned sham operated 58 +1- 4.9 %; Pyramidal lesion alone 72 +1- 6.4%; vehicle and pyramidal lesion 64 +1- 5.6%; BDNF and pyramidal lesion 72 +7- 6.4%; p0.231 Kruskal-Wallis ANOVA on ranks). In general, success rates for lesioned groups were significantly less than unlesioned sham operated rats in the first two weeks after injury. On the second week of testing, there was a significant difference between groups (p=0.038 Kruskal-Wallis ANOVA on ranks). The reaching success of rats in the BDNF treatment group (24 +7- 6.2%) was significantly less than the vehicle treated group (47 +7-5.8; pO.Ol2). At four and six weeks after injury, there was again a significant difference between groups (p0.028 and p= 023 respectively Kruskal-Wallis . 0 ANOVA on ranks). Pyramidal lesioned rats (58 +7-7% and 56 +7- 7% respectively) as well as vehicle treated + lesion rats (55 +7- 5.8% and 56 +1-5.8% respectively) showed no difference in reaching success from uninjured rats (62 +7-4.9 and 53 +7- 5; p=O.756; and p=O.’733). However, rats in the BDNF treatment group (42 +7- 7% and 36 +7- 6.9% respectively) were significantly less successful in reaching when compared to unlesioned (p=0.0 156 and p”O.O 198 respectively) and pyramidal injured rats (p=0.0258 and pO.O 169 respectively). At six weeks, BDNF treated rats were also significantly less successful at reaching when compared to vehicle treated rats (p0.0127; Fig 3.17).  65  Reach Task Percent Success Rate -  90  A  + *  A  A  +  + *  *  A  A  A  +  +  A  A  +  *  + *  + *  ##  80-  70  60  50 C  400  30  20  10  0  -  —  Pre-Surgical Baseline  —,---  —.-  Day7  Day 16  Day 28  Day 42  Timepont  • Unesioned  U  Px  a Vehicle+ Px  • BDNF + Px  Fig 3.17 Success rate of retrieval in the reach to grasp food pellet task (42 day study). The symbols and # show significance in rats.  *,  Single Attempt Success Rate Single attempt success rate is a more stringent analysis of reaching behavior gathered from the 13 point ordinal scale. It isolated and described the rat’s ability to successfully retrieve a food pellet on the very first attempt of each trial without any corrective movements. On our 13 point scale, only score levels 7, 10, and 13 were considered to fulfill the single attempt requirement. Pre-surgical baseline testing showed that unlesioned sham operated (48 +1- 5%), pyramidal lesion only (62 +1- 6.9%), vehicle treatment/pyramidal lesion (53 +1- 5.8%), and BDNF treatment/pyramidal lesion (44 +1- 7%) groups began experimentation with a statistically similar single attempt success (p0.272). One week after injury, the success rates of pyramidal  A  66  lesion only (12 +7- 4.6%), vehicle treatment/pyramidal lesion (15 +1- 4.1%), and BDNF treatment/pyramidal lesion (22 +7- 5.9%) groups were significantly lower than unlesioned sham operated controls (p< 0.001, p<O.OOl, pO.O22 respectively). The single attempt success of both the pyramidal lesion alone and vehicle treated/pyramidal lesion groups showed some recovery 3 weeks after injury and treatment. However, the single attempt success rate of the BDNF treated group continued to decrease. Four and six weeks after injury, the BDNF group (6+/-2.4%; 18 +/- 5.5% respectively) displayed a significantly lower single attempt success when compared with the unlesioned sham operated (52 +7- 5%) group (p<0.001). The vehicle treated group (45 +7- 5.8%) however, showed marked improvements in single attempt success rate and was not statistically different from the unlesioned sham operated animals (52 +1- 5%; p=O.452; Fig 3.18).  Reach Task Single Attempt Success -  80 A  A  + **  70  +  A  A  A  + **  A  *  *  x 60  A  A  +  -  *  *  x ##  w 50a  E  40  30-  20  I0  I  0  PreSurgca 8ase8ne  Oay7  DaylE  Oay2S  Dey42  11mepont  •Unesioned  IPx  •Vehicle+Px  IBDNF+Px  Fig 3.18 Single attempt success rate retrieval in the reach to grasp food pellet task (42 day study). The symbols * + A, # and x show significance in rats.  67  3.6.2 Gait Analysis Gait analysis was performed as decribed in the methods section. The paw prints left behind in the tracks were strongly marked and the features of the paws easily recognizable. The toes were individually marked and the areas of the paw pads that support the weight of the rat were evident (Fig 2.9). This made measurement of certain parameters clear and simple. The toe spread between the first and fourth toe of the forepaw, and the first and fifth toe of the hind paw, was measured during behavioral testing in the 14 day study. Measurements in stride length and base of support (results not shown), did not significantly change after pyramidal injury.  3.6.2.1 A Unilateral left pyramidal lesion produces an increase in toe spread in the forepaw and hindpaw on the right denervated side compared to the left side In the 14 day experimental study, pre-surgical baseline testing showed that toe spread between the left intact (1.71 +1- 0.02 cm) and right denervated (1.74 +1- 0.01 cm) forepaws, as well as between the left intact (1.73 +- 0.01 cm) and right denervated (1.73 +1- 0.01 cm) hind paws was statistically similar (forepaws p=O.l ; hind paws p=O.41 Student’s t-test assuming 2 unequal variances). However, beginning 3 days after a left pyramidal lesion (n=4), there were marked differences in toe spread between the left intact (1.79 +1- 0.02 cm) and right denervated (1.86 +1- 0.02 cm) forepaws (p=0.005 1) and left intact (1.88 +1- 0.02 cm) and right denervated (1.93 +1- 0.02) hind paws (p=0.023). The rats were tested again 7 days after injury, and showed significant differences in toe spread between the left intact (1.81 +1- 0.02 cm) and right denervated (1.85 +1- 0.02 cm) forepaws (p0.024) and left intact (1.97 +1- 0.02 cm) and right denervated (2.06 +1- 0.02) hind paws (p0.002l) Two weeks after injury, there was a significant difference in toe spread found in the left intact (1.88 +1- 0.02 cm) and right denervated (1.95 +1-  68  (j=rO.OO however, the left intact (2.00 +1- 0.02 cm) and right denervated ); 0.01 cm) forepaws 21 (2.03 +1- 0.02) hind paws were not significantly different (p=O.l ; Fig 3.19) 5  A  Toespread Forelimb Left Unilateral Pyramidal Lesion -  2.1 E  *  *  *  2 1.9  -  —Left Foreltmb Px  a 1.8  -  1.7  -—Right Denerveted Forelimb- Px Pre-Surgic& Baseline  Day3  Day?  Day 14  Tiniepoint  B  Toespread Hindlimb Left Unilateral Pyramidal Lesion -  *  *  2.112 a .  1.9 —Left Hindlimb  1.8 1.7 —---------—--———-—-—-——----—  —  -  Rx  —Right Denervated Hindlimb  1.6  Pe-5urgical Baseline  Day3  Day7  Day 14  limepoint  Fig 3.19 Forelimb and hindlimb toespread before and after a left unilateral pyramidal lesion (14 day study). A Unilateral left pyramidal lesion produces an increase in toe spread in the forepaw and hind paw on the right denervated side compared to the left side. The symbol * shows significance between the left and right limb in the timepoint shown. N=4  3.6.2.2 Infusion ofvehicle into the intact right sensorimotor cortex reduces the increased toe spread in the denervated right hindlimb caused by pyramidal injuly  Rats in the 14 day experimental study were treated with vehicle as a control to compare to BDNF treatment. Pre-surgical baseline testing of this control group (n=4) showed that toe  spread between the left and right forepaws (1.65 +1- 0.01 cm; 1.66 +1- 0.02 cm respectively), as  69  well, the toe spread between the left and right hind paws (1.63 +1- 0.02 cm and 1.65 +1- 0.01 cm) was statistically similar when using a Students t-test assuming unequal variances (forepaws p=O.3l; hind paws pO.22). Three days after a left pyramidal lesion and vehicle treatment, the toe spread in the intact left (1.79 +1- 0.01 cm) and denervated right (1.81 +1- 0.02 cm) forepaws were not significantly different (p=0.20) similar to the pyramidal lesion alone group. However, the left intact (1.84 +1- 0.02 cm) and right denervated (1.89 +1- 0.02) hind paws were significantly different (p=0.044). When the rats were tested again one week after injury, the difference between left intact (1.84 +1- 0.01 cm) and right denervated (1.87 +1- 0.01 cm) forepaws (p=0.037) and left intact (1.84 +1- 0.02 cm) and right denervated (1.90 +1- 0.02) hind paws (p=0.022) were both significant. The final test, two weeks after injury showed a significant difference in toe spread found in the left (1.84 +1- 0.01 cm) and right (1.90 +1- 0.01 cm) forepaws ). For the first time, the left intact hind paw toe spread (1.96 +1- 0.02 cm) was more 00097 (p=O. than the toe spread of the right denervated (1.94 +1- 0.02) hind paw (Fig 3.20 A, C). Normally, the left intact toe spread is less than the right denervated toe spread after a left pyramidal lesion. Although the difference in toe spread was not significant (p=0.17), there was a change in trend when compared with the pyramidal lesion alone group.  70  A  Toespread Forelimb (Left Pyramidal 1.esion/Vehicle Treatment) -  2.I  Toespread Forelimb (Left Pyramidal Lesion/BDNFTreatnient) -  2.1  *  *  E  *  E 1.9  . —  •  B  1.9  LeftForeiimb  —LftForlimt,  1.8 ——‘RightDenervated Forehrnb  1.7 1.8 4—— Pe-S4gicaI  Dy3  D7  PrStgicaI  4 Dvl  Timpoint  C  -  *  Dy7  Dy14  litnepoint  Toespread Hind limb (Left Pyramidal Lesion!Vehicle Treatment) 2.1  D3  D  Toespread Hindlimb (Left Pyramidal Lesion/BDNFTreatment) -  2.1  *  2  -  2-  a  a 1.8  1.9 —LftHindIirnb  —  Left Hindlimb  1.8  1.8  •1  2  —Right enervated Hindlimb  13  —  1.7  -  Right Oenrvted Hindlin,b  1.6  1.6 Pc-5urgai 8ejine  Day3  l.9y7  Timepoint  Dy14  Pre-S.gic1 6eTne  DatS  Dyl  Oy14  Timepoint  Fig 3.20 Forelimb and Hindlimb toespread before and after a left unilateral pyramidal lesion with either Vehicle or BDNF treatment (14 day study). Infusion of vehicle into the intact right sensorimotor cortex reduces the increased toe spread in the denervated right hindlimb caused by pyramidal injury. Infusion of BDNF into the intact right sensorimotor cortex reduces the increased toe spread in the denervated right hindlimb earlier when compared with vehicle infusion. The symbol * shows significance between the left and right limb in the timepoint shown. Numbers of rats BDNF +Px n=4; Vehicle + Px n=4.  3.6.2.3 Infusion ofBDNF into the intact right sensorimotor cortex reduces the increased toe spread in the denervated right hindlimb earlier when compared with vehicle infusion  In the 14 day experimental study pre-surgical baseline testing of rats treated with BDNF (n=4) after sustaining a pyramidal lesion showed statistically similar toe spread between the left intact and right denervated forepaws (1.64 +1- 0.01 cm; 1.62 +1- 0.02 cm respectively), as well as, the toe spread between the left intact and right denervated hind paws (1.68 +1- 0.02 cm and 1.67 +1- 0.02 cm) (forepaws p=O.l6; hind paws p=O.25 Student’s t-test assuming unequal variances). Three days after a left pyramidal lesion and BDNF treatment, the toe spread in the intact left (1.77 +1- 0.01 cm) and denervated right (1.80 +7- 0.01 cm) forepaws was significantly  71  different (p=0.029). The left intact (1.82 +1- 0.02 cm) and right denervated (1.89 +7- 0.02) hind paws were also significantly different (p=0.0 12) at this time point. When the rats were tested again one week after injury, the difference between left intact (1.75 +1- 0.01 cm) and right denervated (1.80 +7- 0.02 cm) forepaws was found to be significantly different (p=0.010). However, the left intact (1.83 +7- 0.02 cm) hind paw, which was nonnally less than the right denervated hind paw in the pyramidal lesion group at this time point, displayed a greater toe spread (1.79 +1- 0.02). The difference in these toe spreads was however not significant (p=0.060). The rat’s final test two weeks after injury did not show a significant difference in toe spread between the left (1.79 +7- 0.01 cm) and right (1.81 +1- 0.01 cm) forepaws (p:0.070). The toe spread in the left intact hind paw (1.95 +1- 0.02 cm) at the two week time point in BDNF treated rats was again less than the toe spread of the right denervated (1.85 +7- 0.02) hind paw (Fig 3.20 B, D). This difference in the hind paw was significant (p<0.001) and much greater than that seen at the same time point in vehicle treated rats.  3.6.2.4 Comparison oftoe spread in the right denervated hindpaw ofBDNF treated, vehicle treated, andpyramidal lesion alone groups  At the pre-surgical time point, statistical comparisons were made of the toe spread of the right denervated hind paw between BDNF treated, vehicle treated, and pyramidal lesion alone groups (Fig 3.21). A one-way ANOVA showed the differences to be significant. Further analysis using a Student’s t-test assuming unequal variances showed that both the BDNF treated and vehicle treated group were significantly different from the pyramidal lesion alone group (p<0.001 and <0.00 1 respectively) but were not significantly different from each other (p0.24). Three days after injury, all the injury groups showed increased toes spread in the denervated  72  hindlimb, presumably due to pyramidal lesion. There was again no difference in average toe spread between the BDNF and vehicle treated groups (p=O.50); however, toe spread in the pyramidal lesion only group was still significantly greater than the BDNF but not the vehicle treated group (BDNF/Px pO.O48 and vehicle/Px p=O.O54). Seven days after injury, there were some significant differences seen in toe spread. Even though toe spread continued to increase in the pyramidal lesion only group, the toe spread of the vehicle treated group remained at the same level as on the third day after injury. The toe spread of the BDNF treatment group began to recover back towards pre-surgical levels. All groups were significantly different from one another (ANOVA p<<O.OO1; Student’s t-test, BDNF/Px p<<O.OO1; Vehicle/Px p<O.OO1; BDNF/Vehicle p<O.OO1). Two weeks after injury, hind paw toe spread in the pyramidal alone lesion group stabilized and did not increase, while the BDNF and vehicle treated groups remained significantly reduced in comparison (ANOVA p<<O.OO1; Student’s t-test, BDNF/Px p<<O.OO1; Vehicle/Px pO.0001 5; BDNF/Vehicle pO.000 1)(Fig. 3.21).  Toes pr:ead Comparison of Groups Right Denervated Hindflmb *  21 —  E  *  2 —Right Denevtd Hir,dlirnb-Px  19 .  1g  -  Right enervtd Hindlimb -Vehicle  16 Pre-Surgical Baseline  DayS  Day?  Day 14  —RlghtDenervated Hindlimh-BDNF  limepoint  Fig 3.21 Comparison of toe spread in the right denervated hind paw of BDNF treated, vehicle treated, and pyramidal lesion alone groups shows a graded recovery of toe spread, depending on treatment, towards pre-surgical baseline levels. Pyramidal lesioned animals showed a significant difference in toe spread of hindlimbs from other treatment groups pre-surgically and 3 days after lesion. The symbol * shows a significant difference of either treatment group from the Px only group at the timepoint shown.  73  Discussion In this thesis, I tested the hypothesis that infusion of BDNF into the intact sensorimotor cortex induces sprouting of undamaged corticospinal axons into the denervated spinal cord after transection of the contralateral tract at the level of the pyramids. I studied the effect of this treatment using the reach to grasp food pellet test, and gait analysis. A significant increase in BDNF-induced sprouting was found in lumbar but not cervical spinal cord. This correlated with enhanced hindlimb function while no improvements were seen in forelimb performance. This study has several shortcomings that will be discussed below.  4.1 Possible Effects ofFood Restriction To aid in training and testing of the “reach to grasp task”, the rats were placed on a restricted food diet to enhance motivation for learning the desired tasks. This might have affected the outcome data as our lab recently discovered that fasting on alternate days (every other day fasting “EODF”) promoted forelimb recovery from cervical spinal cord injury and sprouting of corticospinal axons (Plunet et al., 2006). Over a period of approximately 45 days, the food restricted rats gained an average of 36 grams of body weight. By comparison, in the studies by Plunet et a!., (2008) adult Sprague Dawley rats on EODF gained 44g over a period of 35 days, while rats on the ad libitum food access regimen gained an average of 90g. Given that a restricted diet may be beneficial in aiding recovery of forelimb function after cervical spinal cord injury and promotes sprouting my results may have been affected in a similar manner. This might have promoted sprouting in the BDNF, vehicle treated, and lesion alone rat groups as well. Since these EODF studies were conducted years after my experimental work, I  74  was not aware of this possibility and no ad libitum fed controls were tested to permit any conclusions. It is important to note that behavioral testing with dietary restriction does cause the rats to become more frantic when attempting the reach to grasp food pellet reach task for example. The time taken to grasp food pellets as well as overall reaching success, decreases with dietary restriction (Smith & Metz, 2005).  4.2 BDNF Induces Seizures In my experiments, animals treated with BDNF exhibited focal seizures affecting the left forelimb and hindlimb. These seizures occurred only during the first 2 weeks after implantation of the cannula and minipump delivering BDNF. The seizures were not seen in the vehicle treated rats, and hence unlikely due to any disturbance from vehicle infusion. In the rat, systemic kainate application has been shown to induce seizure activity, and significantly increases BDNF in the superficial layers of the cortex (Dugich-Djordjevic et a!., 1992). Elevated levels of BDNF have also been shown in the hippocampal and temporal lobes of human epileptic brains (Takahashi et at., 1999). As well, BDNF has been demonstrated to increase the amplitude and the frequency of excitatory postsynaptic currents (EPSCs) shortly after application in vitro (Levine et at., 1995; Kafitz et a!., 1999). However, not all rats treated with BDNF exhibited seizure activity. This could mean that some rats inadvertently did not receive the BDNF infusion. There are multiple reasons why this is most likely not the case. The lack of seizure response to BDNF infusion can be a result of variances in cortical maps of forelimb and hindlimb areas between rats, as well as inter-individual variations in  75  excitability (Akintunde & Buxton, 1992; Leergaard et al., 2004). Previous studies from our lab and others have shown that BDNF diffused at least 1.5mm around the tip of the cannula and that rescue effects of axotomized corticospinal neurons were seen for over 2.5 mm (Giehi & Tetzlaff, 1996; Kobayahsi et al., 1997). Therefore, minute variances in cannula depth and position should not greatly influence what populations of neurons are exposed to BDNF. A control to ensure that the cortex was infused with BDNF would be to conduct a histochemical assessment of BDNF distribution within the infusion site of the sensorimotor cortex (Anderson et al., 1995). Earlier studies in our lab performed such controls in the midbrain with consistent positive outcome, i.e. no evidence of pump failures (Kobayashi et al., 1997).  4.3 BDNF Treatment Reaches Most ofthe BDA Labeled Corticospinal Neurons The results showed that BDNF did not significantly further induce sprouting of corticospinal fibers within the cervical enlargement when compared with the pyramidal lesion alone group. It is possible that the quantifications of the sprouting within the cervical spinal cord were not accurate because corticospinal axon profiles may have been labeled with BDA that were not exposed to BDNF treatment in the cortex. This would wash out any growth effects from BDNF-treated corticospinal neurons. The BDA sites were distributed throughout the forelimb and hindlimb areas of the sensorimotor cortex (Fig 2.3). The cannula delivering either BDNF or vehicle was implanted 1.0 mm posterior to Bregma and 2.0 mm lateral to the midline. The placement of the pump ensured that all of the labeled corticospinal neurons lay within the range of BDNF infusion (as described in the methods). Furthermore, sprouting of corticospinal neurons within the lumbar enlargement was found to be significant when compared with the pyramidal  81  4.8 Damage to Surrounding Systems After a Pyramidal Lesion Confounds Behavioral Assessment It is difficult to avoid some damage of the olivary complex and medial lemniscus located dorsal to the pyramids (Whishaw et al., 1993, Fig 4.3). Injury to the medial lemniscus would further exacerbate the deficits incurred in the contralateral half of the body and head (gracile, cuneate, and trigeminal nuclei) after pyramidal lesion due to improper processing of touch and proprioception. Injury to the inferior olivary complex would result in deficits similar to the deficits after destroying the entire contralateral cerebellum resulting in ataxia of the contralateral side of the body (Bozhilova-Pastirova & Ovtscharoff, 2000).  Fig 4.3 Images of the rat brain in cross section at the level of the pyramidal decussation showing structures lying dorsal to the pyramid. Figure 4.3 has been removed due to copyright restrictions. The image removed showed images of the human brain in cross section. Focus was placed to the region of the pyramidal decussation. Image was taken from Anatomical Foundations of Neuroscience http://publish.uwo.ca/-jkiernan/anfound.htm.  82  My observations of the rats were not sensitive enough to detect any motor deficits as a result of injury to medial lemniscus or the inferior olive. There was also no anatomical evidence of apparent injury to either of these structures; however, the surgery to transect the pyramid more than likely affected these structures transiently, if not permanently. This is likely since vascular supply of these structures stems from small arteries branching from the basilar artery. Small blood vessels are not visible to the surgeon and they may have been damaged to varying degrees, leading to degeneration of more dorsal structures. Even the occurrence of a slight edema within the medulla might temporarily disturb the function of other motor and sensory systems in the close vicinity of the pyramids such as the olivary nucleus and the medial lemniscus (Metz et al., 1998).  4.9 Sprouting in the Spinal Cord After Unilateral Pyramidal Lesion The phenomenon of spontaneous sprouting to compensate for the loss of innervation has been very well documented from injured as well as intact corticospinal axons (Aoki et al., 1986; Goldstein et al., 1997; Hiebert et al., 2002; Kuang & Kalil, 1990; Vavrek et al., 2006; Weidner et al., 2001). It is important however, to distinguish the origins of sprouting from intact corticospinal axons as it will help to understand the behavioral implications. In this section, I briefly remind the reader of some of the literature shining light onto the origins and dynamics of the sprouting seen in the spinal cord. I apologize for reiterating some of the concepts presented in the introduction. As outlined in the introduction, 10-15% of corticospinal axons in the uninjured rat spinal cord run in the uncrossed ipsilateral dorsal and ventral funiculus of the cord (Alisky et al., 1992; Brosamle & Schwab, 2000; Cabana & Martin, 1985; Goodmann et al., 1966; Vahlsing&Feringa,  83  1980; Joosten et al., 1987, 1992; Rouiller et a!., 1991). Along with the crossed dorsal and dorsolateral components, there are four different components of the cortico spinal tract (Brosamle & Schwab, 1997). The ipsilateral components of the corticospinal tract run the entire length of the spinal cord all the way to the lumbar enlargement and represent a complete corticospinal projection with similar axon diameters and myelination as CST fibres of the dorsal crossed tract (Brosamle & Schwab, 1997; 2000). Ventral corticospinal axons send multiple collaterals into the ipsilateral grey matter that massively arborize, extending fibres into different spinal segments (Brosamle & Schwab, 1997). In cross sections taken from animals in the present study, the mean number of ipsilateral ventral axons in the cervical enlargement of 42 day, and 14 day study groups was 102+1-13, and 7 1+1-8 axons respectively. It has been found that BDA tracing of the ventral component greatly underestimates the number of detected fibres, and that most, if not all of the small diameter axons in the area of the ventral midline are uncrossed ventral corticospinal axons (Brosamle & Schwab, 2000). In contrast, the mean number of axons in the uncrossed dorsal funiculus of the cervical enlargement of 42 and 14 day study groups was 20+1-2, and 33+1-3 axons respectively. Little is known about either group of axons in terms of their function in the ipsilateral grey matter, however the ipsilateral components are significant projections from the sensorimotor cortex. Although it may seem that this group of neurons/axons may not have a large role in function or recovery after spinal cord injury, their extensive arborisation and the fact that just a few fibres can have significant effects on locomotion are important factors to focus attention on this group of neurons (Guth et a!., 1980; Blight and Young, 1989; Bregman et al., 1995; Brosamle & Schwab, 1997; 2000).  84  Normally in response to injury, very few fibers originating from the crossed dorsal corticospinal tract cross back through the dorsal funiculus to innervate the contralateral grey matter in the cervical spinal cord (Bareyre et aL, 2002; Hiebert et a!., 2002; Kuang & Kalil, 1990; 1994; Weidner et al., 2001; Theriault & Tatton, 1989; Alinsky et al., 1991). When corticospinal fibers sprout from this component, they preferentially grow through grey matter rather than through white matter to potentially innervate the denervated grey matter (Hiebert et al., 2002; Vavrek et al., 2006). In the present study, in all treatment groups, there were very few corticospinal fibers seen crossing through the bridge of grey matter (lamina X) around the central canal between the left and right halves of the spinal cord. Behaviourly, if such growth were to occur, it would possibly cause bilateral innervation from a single cortex that may functionally result in mirror movements of the limbs. Examples of mirror movements exist after mutations in the mouse ephrin-B3 or EphA4 locus, which cause the corticospinal tract to bilaterally innervate both contralateral and ipsilateral motor neuron pools. This type of innervation caused these rats to exhibit a hopping locomotion (Yokoyama et al., 2001). Mirror movements were not observed in any of the animals tested in the present study. Since there is significant sprouting of intact corticospinal fibers into the denervated spinal cord, the question remains; where do they sprout from? The observations above do not indicate sprouting from collaterals of the crossed dorsal corticospinal tract. It is therefore my hypothesis that the majority of sprouting quantified in the denervated grey matter after a pyramidal injury, arise from the uncrossed ipsilateral dorsal and ventral corticospinal tracts. The ipsilateral cortico spinal tracts have been implicated in sprouting of corticospinal fibers into the partially injured spinal cord in a number of studies. After incomplete midthoracic  85  spinal cord injury, which spared the left ventral funiculus, growth cones of sprouting corticospinal axons within the lumbar spinal cord were observed three days after injury (Goldstein et al., 1997). In another study, after midthoracic hemisection in the monkey, the CST projection pattern in the lumbar spinal cord displayed a significant increase in newly formed projections from the ipsilateral ventral CST into the denervated side of the spinal cord below the level of injury (Aoki et al., 1986). As described in the introduction, innervation of spinal cord grey matter by corticospinal axons follows a highly ordered and topographical order (ie. somatosensory vs. motor) during development as well as after CNS injury (Bareyre et al., 2002). A set of experiments conducted by Weidner et al. (2001), illustrated the selective changes in the corticospinal axons as it related to the potential mechanisms that underlie recovery. These authors made lesions of defined components of the corticospinal tracts in the rostral cervical spinal cord. Either of the dorsal or ventral corticospinal tract, was removed and the mean number of BDA-labeled CST axons contacting ChAT-labeled motoneurons in the medial and lateral motoneuron columns in C4 was counted. Motoneurons that innervate the proximal muscles (involved in control of stance, and gross arm movement) are found more medial in the gray matter than those that innervate more distal muscles (mediate fine motor control of the paws and fingers) (Whishaw et al., 1993; Puskar & Antal, 1997). It is acknowledged that only 1-2% of corticospinal axons make connections directly onto motoneurons, however, many more branch onto pools that are functionally related (Liang et al., 1991; Kuang & Kalil, 1990). The results from Weidner’s studies showed that if the ventral corticospinal tract was transected leaving the dorsal CST intact, the mean number of CST terminals/neuron in the medial motor column was reduced (no longer innervated from the  76 lesion only group, reinforcing the interpretation that sprouting can be detected is a result of treatment.  Fig 4.1 Sites of origin of corticospinal neurons in the rat with reference to 6 different coronal distances from the frontal pole. Figure 4.1 has been removed due to copyright restrictions. The image removed showed images of cross sections of rat brain in which the frontal pole is 3.75mm rostral to Bregma, which would make section A (B + 2.35mm), B (B +0.75mm), C (B -0.65mm), D (B -1.65mm), E (B 2.85mm), F (B 4.25mm). Image was taken from Akintunde A, Buxton DF. (1992) Differential sites of origin and collateralization of corticospinal neurons in the rat: a multiple fluorescent retrograde tracer study. Brain Res. 575(1): 86-92. -  -  4.4 The site of Cannula Implantation Might Have Preferentially Exposed BDNF to Hindlimb Corticospinal Neurons Over Forelimb Corticospinal Neurons In this study, there was no detectable increase in sprouting within the cervical enlargement after BDNF treatment when compared with rats that were applied with vehicle. However, there was a 3.2 fold increase in sprouting in the lumbar enlargement with the same comparison. It is important to rule out any possible variation in BDNF exposure of forelimb and hindlimb corticospinal neurons based on cannula placement. In my study the hindlimb area of  77  the sensorimotor cortex appeared to be preferentially infused with BDNF based on its proximity to the infusion cannula. Upon further study of cortical maps, it was found that the cannula was placed in the hindlimb sensorimotor cortex (Akintunde & Buxton, 1992, Fig. 4.1). Fig.4.1 shows the sites of origin of corticospinal neurons in the rat with reference to 6 different coronal distances from the frontal pole. The frontal pole is 3.75mm rostral to Bregma, which would make section A (B  +  2.35mm), B (B +0.75mm), C (B -0.65mm), D (B -1.65mm), E (B 2.85mm), F (B 4.25mm). -  -  According to this study, the majority of C6 terminating corticospinal neurons are located frontal to Bregma -0.65mm and occipital to B +2.35. The majority of Li terminating corticospinal neurons are occipital to Bregma -0.65mm (Akintunde & Buxton, 1992; Leergaard et al., 2004). The placement of the cannula in our animals, at Bregma -1.0, and lateral 2.0mm clearly placed the site of infusion of BDNF in the lumbar region of the sensorimotor cortex, however, the infusion diameter of BDNF being applied should have exposed a great percentage of forelimb corticospinal neurons as well. Indeed, the injection sites for BDA produced well labeled corticospinal fibers in both the cervical and lumbar enlargements of the spinal cord, and all of the BDA injection sites fell within the BDNF infusion diameter (Giehl & Tetzlaff, 1996; Kobayahsi et al., 1997). Yet, the size of BDA is smaller than BDNF (10,000 vs 26,000) and BDNF tends to be restricted in its diffusion by binding to several of its receptors (Anderson et al 1995). Therefore a treatment of hindlimb versus forelimb neurons might have occurred in this study.  4.5 BDNF Did Not Increase Uptake ofBDA Into Corticospinal Neurons BDA is a dextran-lysine (Dextran amine) conjugated to a biotin (Vitamin B7) group that is biologically inert, has a low toxicity, is resistant to degradation, and highly water soluble  78  allowing for easy transport into neurons (Reiner et al., 2000; Haugland, 1996). The mechanism of BDA uptake appears to be by pinocytosis for intact neurons (Reiner et a!., 2000; Jiang et al., 1993). Endocytotic vesicles are continuously formed at the plasma membranes to take up neurotransmitters, and with them other substances in the extracellular fluid. BDA is taken up non-specifically during this process. For all the advantages BDA has to offer, uptake of BDA can be variable depending on factors other than the concentration of BDA injected. For example, the rate of pinocytosis can be increased by co-application of the glutamate receptor agonist, NMDA (Jiang et al., 1993). In the same way, BDNF might cause increased uptake of BDA by activating ion channels, and modulating endocytosis of synaptic vesicles (Reichardt, 2006). In slices of cortex, BDNF binds to TrkB receptors, and opens Na ion channels as immediately as glutamate administration (Kafitz et al., 1999). BDNF also has been shown to induce entry of Ca 2 into dentate granule cells (Canossa et al., 2001). This could have potentially confounded my results in the 14 day experimental study since BDA was injected into the sensorimotor cortex just before implantation of the cannula delivering BDNF for 14 days. There was no significant difference in the mean number of pixels drawn in the denervated cervical grey matter in both BDNF and vehicle treated rats; in fact on average, there were more pixels drawn in the vehicle treated rats (Fig 3.12 A). BDNF therefore did not appear to increase uptake of BDA into corticospinal neurons. This was further reinforced with our 42 day study where BDA was injected into the intact sensorimotor cortex two weeks after BDNF infusion had stopped. Again, the mean number of pixels drawn in the cervical enlargement in BDNF treated rats was less than that seen in vehicle treated rats (Fig 3.13A).  79  4.6 Cannula Implantation Into the Intact Left Sensorimotor Cortex Causes Damage The cannula delivering BDNF or Vehicle was implanted into the right intact sensorimotor cortex at a depth of 1.5mm. In our studies, the cannula was in place for 14 or 28 days (14 and 42 day studies respectively). This produced a localized minor injury, to the intact cortex. Hence, the corticospinal neurons treated to induce sprouting into the denervated half of the spinal cord are themselves injured or are in the vicinity of injured neurons. This focal ‘injury’ to the cortex could induce plasticity and sprouting within the brain and the spinal cord (Emerick AJ, Kartje GL., 2004; Dancause et al., 2005). Our Vehicle treatment control attempted to address this variable of our method. To prevent damage to the cortex due to a cannula in future studies, there are other less invasive methods of delivering BDNF such as using adeno-associated viral vectors, or marrow stromal cells expressing BDNF (Kwon et a!., 2007; Lu et al, 2005). It has also been possible to increase endogenous BDNF levels within the cortex through exercise, as well as transcranial magnetic stimulation (Neeper et a!., 1996; Muller et al., 2000).  4.7 A Pyramidal Lesion ofthe Corticospinal Tract Leaves Spared Tissue In my experimental study, the left corticospinal tract was transected just above the pyramidal decussation. At the level of injury, the basilar artery runs along the midline between the pyramids. In order to avoid damage to this artery, which is typically fatal, approximately 510% of the corticospinal tract was spared. In the present study, some corticospinal tract fibers were spared in the majority of rats. Although this sparing of tissue was variable between rats, the percentage of spared axons in the injured left pyramid did not significantly differ between groups in either of the studies. There was typically isolated medial sparing of— 12% of the pyramidal  80  tract. The degree to which the pyramid was lesioned was similar to previous studies in rats (Bareyre et al., 2004; Weidner et al., 2001; Muir and Whishaw, 1999; Whishaw et al., 1993). The sparing should not have affected behavioral testing as it has been shown that rats with pyramidal lesions that removed greater than 90% of the tract exhibited the same behavioral deficits as fully lesioned rats (Muir & Whishaw, 1999). Rats with greater than 70% of the pyramidal tract lesioned, also exhibit deficits in the reach to grasp food pellet test (Whishaw et al., 1993). The predisposed sparing of the medial aspect of the pyramidal tract does not selectively leave any particular group of axons intact. There is no topographic organization of corticospinal axons within the pyramidal tract corresponding to any region of the sensorimotor cortex (Coleman et al., 1997; Fig 4.2). Behavioural and electrophysiological impairments also have not been shown to vary with lesion location within the pyramidal tract (Piecharka et al., 2005).  Fig 4.2 Summary schematic diagram of the patterns of axonal labelling in the white matter of the brain after injections of biotinylated dextran into various areas of neocortex. Figure 4.2 has been removed due to copyright restrictions. The image removed showed that each map was constructed by noting the precise location of the labelling seen after injections into each of the cortical areas examined. The different symbols in the white matter represent the location of labelled axons from the different neocortical areas. Image was taken from Coleman KA, Baker GE, Mitrofanis J. (1997) Topography of fibre organisation in the corticofugal pathways of rats. J Comp Neurol. 381(2): 143-57.  86  ventral CST), and the number of CST terminals/neuron in the lateral motor column remained the same (ventral CST did not innervate this motoneuron column) when compared to uninjured animals. This showed that the ventral corticospinal tract normally innervates the medial motoneurons because after transection, the number of contacts was reduced. However, more importantly, there was no increase in the number of contacts into either the medial or lateral motoneuron column from the intact dorsal corticospinal tract to compensate for the loss of ventral input (Weidner et al., 2001). Furthermore, the Weidner (2001) study described a significant increase of contacts in the medial motoneuron pool, and fewer contacts in the lateral motoneuron pool after the dorsal corticospinal tract was transected leaving the ventral CST intact when compared with uninjured rats. This result indicates that the dorsal CST innervated both the medial and lateral motoneurons, since there was a reduction in contacts in the lateral motoneuron column, and there were an increased number of contacts counted in the medial motoneuron column to compensate for the loss of dorsal innervation. More importantly, although there was a significant decrease in the number of contacts with the lateral motoneuron colunm after denervation of the dorsal CST, the ventral corticospinal tract did not increase the number of contacts to this group of motoneurons. Instead, there was a significant and specific increase in formation of contacts from the ventral corticospinal tract selectively onto the medial motoneuron pool. Also, bilateral lesions of the pyramidal tracts, or combined lesions of the dorsal and ventral CST did not show any compensatory increase in contacts to either motoneuron pool (Weidner et al., 2001). The evidence in the Weidner study suggests that the increase in contacts to motoneurons seen after dorsal corticospinal tract injury was exclusively mediated by the ventral corticospinal tract, and that they preferentially contacted the medial motoneuron column. Indeed, in support of  87  this idea, the ipsilateral ventral corticospinal tract has also been anatomically shown to innervate interneurons in the ventromedial region of the spinal cord (Joosten et al., 1992). Since the medial motoneuron group is involved in control of axial and proximal muscles, the ventral corticospinal innervation of the medial motoneuron column can be inferred to influence stance, posture, and gross movements of the arm (i.e. proximal limb muscles discussed later). Moreover, the ventral corticospinal tract has little influence on the lateral motor column, and control of fine motor, (i.e. distal limb muscles) movements (Weidner et al., 2001).  4.10 Sprouting in the Spinal Cord After Unilateral Pyramidal Lesion and BDNF Treatment (Possible Explanation)  Although there was significant spontaneous sprouting as a response to pyramidal lesion, there was no further increase in sprouting with the application of BDNF. This result was contrary to my original hypothesis. It is possible to speculate why this result occurred. One possibility involves the importance of appropriate synapse formation and reinforcement after sprouting as well as the ideas expressed previously about specificity of motoneuron pool innervation (Glover, 2000; Weidner et al., 2001). After pyramidal injury and spontaneous recovery via sprouting, there may not have been enough viable contacts available within the medial motoneuron pooi for the ipsilateral corticospinal system to innervate. Therefore, there would not be any further induction of sprouting over control levels. Unlike the cervical spinal cord, BDNF applied to the sensorimotor cortex significantly induced greater sprouting of intact corticospinal fibers into the denervated half of the lumbar enlargement after a pyramidal lesion, when compared to the pyramidal lesioned only group. If  88  the ipsilateral corticospinal tract is the major contributor to sprouting into the denervated half of the spinal cord, and BDNF does not induce this tract to sprout in the cervical spinal cord, why is there induced sprouting in the lumbar enlargement? The effects of lesions of the dorsal and ventral corticospinal tracts have not yet been studied as it relates to influences and changes in the number of contacts to motoneuron pools in the lumbar cord. However, it is known that relative to the forelimb, rats do not possess an extensive repertoire of skilled distal hindlimb movements or fine motor control (lateral motoneuron pools) (Metz et al., 1998). Also, many of the muscles in the hindlimb are proximally driven and devoted to postural control, stance, and locomotion (Metz et al., 1998). This evidence would suggest a greater potential for sprouting of corticospinal axons onto both medial and lateral motoneuron columns. The similarity of function and location between motoneuron poois may have induced ipsilateral corticospinal axons to sprout after application of BDNF, to compensate for the loss of dorsal innervation.  4.11 Sprouting in the Spinal Cord After Unilateral Pyramidal Lesion And BDNF Treatment (Alternate Explanation)  Alternatively BDNF or neurotrophic factors can act variably to induce sprouting or even prevent death of CNS neurons in a number of different systems (Hagg, 1998; Ramer et a!., 2007). For example, the BDNF dose response curve showing the ability of different concentrations of BDNF to prevent death of axotomized nigrostriatal neurons follows a bell shaped curve (Hagg, 1998). Either too high or low a concentration is not as potent in rescuing the neurons as an optimal intermediate concentration. Although survival of corticospinal neurons may require a different concentration than what is needed for outgrowth, the same idea can be applied to sprouting efficiency. The concentration of BDNF seen in the environment of the  89  hindlimb corticospinal neurons may have been more conducive to sprouting, and the lower concentration seen by the environment of the forelimb corticospinal neurons may not have been sufficient to induce sprouting, since the tip of the cannula was inserted into the hindlimb sensorimotor cortex. In fact, it was observed that the application of BDNF to the forelimb sensorimotor cortex in this model slightly reduced sprout formation in the cervical spinal cord.  4.12 Behavioral Testing 4.12.1 Reach Test and Gait Analysis Since experimentation on rats is for the sole benefit of humans, functional testing should also be readily relatable to human movements. The proximal musculature attached to the scapula in the rat used for reaching, allows for the same degree of physical movements made by the ball and socket joint in the shoulders of primates (Wishaw & Miklyaeva, 1996). Reaching ability was tested using a 13 point ordinal scale developed in the lab (Table 2.1). In this scale, I strived to define hierarchical levels of recovery seen after CNS injury. The verbal descriptions used in the 13 point scale tried to decrease variability from experiment to experiment, observer to observer, and laboratory to laboratory. This is a major factor when proposing a new scale to measure functional changes. Much of the scale described changes in the number of attempts the rats made before grasping the food pellet and if the pellet was dragged along the stage or lifted before it was retrieved into the testing box. I believed that successful retrieval of a food pellet after a single attempt should be placed higher on the scale (highest being normal) than retrieval after a number of attempts. As well, lifting the pellet was placed higher on the scale than dragging it on the  90  stage. These divisions helped to compile information about endpoint results such as percent success, or single attempt success. Very little of the scale used described individual component movements involved in reaching to grasp, and retrieving a pellet. Dissociating changes in component movements is just as important and give more insight into the progress of injury, and recovery after spinal cord injury as changes in endpoint success (Whishaw et al., 1997). For example, studies have shown that rats are able to achieve presurgical reach success after CNS injury while retaining impairments in motor control (Whishaw et al., 1997). This recovery of success rate was a result of compensatory movements that although successful, does nothing to describe anatomical recovery after a lesion of the motor cortex. Albeit time consuming, the component movements involved in Sprague-Dawley rat reaching have not been elucidated. Gait analysis after a pyramidal injury is a proven, well tested quantitative test to assess any potential deficits, and recovery after treatment. This test however, is not a generalized behavioral test that can be used to assess most injury models. Gait analysis requires that the rats to be able to consistently step, to produce a distinct footprint. A major question that arises when reviewing the results is whether the modest amount of collateral sprouting in all the treatment groups after pyramidal injury has useful functional consequences.  4.12.2 Forelimb Function After a Unilateral Pyramidal Lesion 4.12.2.1 Reach Training Once reaching ability was acquired, in the reach to grasp task, the rats did not increase or decrease in their success rate in reaching over 13 training sessions. The training success rate  91  remained at around 60% which was similar to presurgical baseline testing in reaching success in both studies. This level of reaching was similar to previous research (Whishaw et a!., 2003; Weidner et al., 2001; Whishaw et a!., 1998; Whishaw et a!., 1993). However, the low success rate begs the answer to why the rats do not achieve perfect success prior to surgery. It was observed that some of the rats had near perfect reaching success, while others were relatively “impaired.” This depression in success rate could be a result of variation in forearm length (naturally, or because of differences in size/age of the rats) or variation in proximal control of limb muscles. Since the rats must reach past their head to grasp the food pellet, variation in reaching distance could also occur because of differences in the ability to retract the head to elongate the reach. The rats were selected for handedness in order to reduce variations. One must be cautious about moving the food pellet closer to the opening in the reaching apparatus to increase success rate. This stretch to grasp a pellet may be beneficial in making this test sensitive to detecting fine motor deficits after corticospinal tract lesion. Overextending the reach may recruit more of its fine motor capabilities facilitated by the corticospinal system. The idea of increased sensitivity was illustrated by the difference in rates between the success rate and single attempt success rate graphs (Fig 3.17 & 3.18). For example, the mean difference in success rate between pre-surgical baseline testing, and testing on Day 7 or Day 16 was much less pronounced in retrieval success rate than that seen in single attempt success rate. Also, the more stringent analysis of single attempt success rate graph at 42 days after injury more closely mirrors the relative amounts of sprouting seen in the cervical spinal cord. It is important to test the effect of overextension on the sensitivity of reaching success rate because it may elucidate the true effects of CST transection by activating and stressing the muscles that were denervated.  92  4.12.2.2 Reaching Behavior After Pyramidal Injury Essentially, after unilateral pyramidal injury, the dorsal corticospinal tract on the contralateral side of the spinal cord is removed, leaving sole innervation from the ipsilateral dorsal, dorsolateral and ventral corticospinal tracts. It has been shown that the dorsal corticospinal tract is involved in control of both distal muscles (fine motor control of grasping, pronation, suppination) and proximal muscles (including movements of lifting, aiming, advancing, and withdrawing the limb) (Whishaw et al., 1993; Metz & Whishaw, 2002). The ipsilateral ventral corticospinal tract has not been implicated in the control of detectable movement during functional testing nor does it mediate compensation from impairments following its lesion (Weidner et al., 2001; Metz et al., 1998; Metz & Whishaw, 2002). Here I proposed that the ipsilateral corticospinal tract is responsible for the significant increase in sprouting after pyramidotomy, and it is my contention that some functional recovery of forelimb reaching is a result of this sprouting. However, I found no recovery of forelimb reaching after pyramidal lesion, to presurgical levels in the 13 point dissociated ordinal scale. The significant sprouting of corticospinal axons that occurred in all the rats in groups with pyramidal lesion, was not sufficient to cause functional recovery. As well, endpoint analysis of single attempt success rate did not show recovery of function either; although, in both cases there was a trend towards recovery. In the least stringent endpoint analysis of retrieval success rate, there was a recovery of function to levels achieved by unlesioned animals. The recovery of successful pellet retrieval rate after pyramidal lesion has been shown to be similar to recovery of function correlating with sprouting of the ventral CST after dorsal CST lesion (Weidner et al., 2001). In that study, transection of the dorsal CST caused a significant impairment in retrieval rate that recovered to uninjured levels 4 weeks after injury. If the dorsal  93  and ventral CST was lesioned in the spinal cord, or both the medullary pyramids were transected to remove the dorsal and ventral corticospinal innervation of spinal cord, little to no recovery of pellet retrieval rate was seen. This result reinforces the idea that the ventral CST is a component source of recovery after pyramidal lesion since removal of input from this tract abolishes any chance of recovery from dorsal corticospinal tract injury. How can there be a recovery of retrieval success rate while the 13 point analysis and single attempt success does not? The answer lays in the specificity of each of these analyses in assessing corticospinal dysfunction. These approaches to deciphering reaching success describe different aspects of reaching behaviour. The least stringent retrieval success rate only describes the rat’s capability in retrieving a food pellet. The reaching success seen after pyramidal lesion was not solely a result of sprouting of corticospinal axon profiles within the denervated cervical spinal cord. The rat may be able to accomplish this endpoint in a number of ways: a.  Compensatory sprouting from corticospinal neurons spared after pyramidal lesion. This possibility must always be foremost when reviewing behavioural outcomes after CNS injury because even though testing immediately after CNS injury may show the abolishment of a certain functional ability, minimal sparing of neurons can cause astonishing recovery of function not caused by growth of injured, or as in this study, spared fibres (Kakulas, 1999; Fehlings & Tator, 1995). In humans, it has been observed that as little as 7% of the total number of axons below the level of injury can sustain a paralyzed patients ability to recover motor control, and sensory feedback (Bunge et al., 1993; Kakulas, 1999). One must be reminded that spared dorsal CST fibers still innervate the grey matter and cause a recovery of function. An anterograde tracer such as Cholera Toxin B (CTB) could be injected into the  94  injured cortex to quantify the amount of corticospinal axon sparing, and growth in the denervated half of the spinal cord. b.  After an injury to the CNS, there are many loci where reorganization and growth can occur such as the sensorimotor cortex, the red nucleus, reticular formation, inferior olivary complex, pontine nuclei and the cuneate and gracile nuclei (Kuang & Kalil, 1990; Antal, 1984; Steeves & Jordan, 1980). There have been numerous studies that have identified corticospinal sprouting onto these nuclei either spontaneously or after therapeutic treatment (Li & Stritmatter, 2003; Wenk et al., 1999; Kuang & Kalil, 1990; 1994; Schnell & Schwab, 1990; Lawrence & Kuypers, 1968 a; b;). For example, CST collaterals have been shown to contact long descending PrI and therefore bypassing the lesion in order to connect to the distal cord (Bareyre et a!., 2004; Vavrek et al., 2006).  4.12.2.3 Reaching Behavior After a Unilateral Pyramidal Lesion and BDNF Treatment The same amount of corticospinal axon sprouts were detected in the cervical spinal cord with BDNF treatment when compared to the pyramidal lesion alone group, therefore, little change in forelimb reaching would be expected as a result of corticospinal control. However, the possibility still exists that there could have been increased sprouting of corticofugal projections to the subcortical structures after infusion with BDNF to the intact cortex over pyramidal injury alone (Iarikov et a!., 2007; Tobias et al., 2003). These sprouts could then control motor output through secondary projections to the contralateral denervated spinal cord to compensate for the loss of pyramidal function. Experiments with double lesions of the cortico and rubrospinal tracts at C5/C6 in cats, for example have shown that the recovery of ‘food-taking’ followed during 2 months of behavioural testing depends on a take-over via ipsilateral ventral systems; most  95  probably through the cortico-reticulospinal pathway, with a command via C3-C4 propriospinal neurones (Pettersson et al., 2007; Aistermark et al., 1981; Blagovechtchenski et al., 2000). Although this possibility exists, little improvement was seen in the rats tested.  4.12.3 Hindlimb Function After a Unilateral Pyramidal Lesion A unilateral pyramidal lesion affects the corticospinal tract that runs the entire length of the spinal cord. We have described the deficits in the forelimbs with the use of the reach to grasp food pellet test, however, currently, there are very few behavioral tests specific to the assessment of corticospinal function of the hindlimb. As mentioned earlier, the corticospinal tract in the rat functions to resolve fme motor control, however, the rat’s hindlimb has not been assessed to dissociate this control from postural control, weight support, and locomotion (Metz et al., 1998). In fact a number of functional tests show a transient impairment of the denervated hindlimb after a unilateral pyramidal injury, which recovers to normal motor function four weeks after injury (Metz et al., 1998). Specifically, rats showed a rapid recovery to baseline levels for narrow beam performance, trunk posture, and weight support, and toe dragging (described by open field testing). The only long term deficit in the hindlimb was a lasting hypermetria caused by a hyperfiexion of the proximal joints resulting in a higher lifting of the denervated hindlimb in the swing phase (described by open field and kinetic analysis). There was also a persistent tendency towards an increased toe spread after pyramidal lesion, however, this difference was not significant (described by gait analysis; Metz et al., 1998). To detect any possible functional changes after unilateral pyramidal lesion, we utilized an analysis of gait because it is easily administered, and it is a quantitative behavioural test that measures a number of parameters in locomotion in both forelimbs and hindlimbs. There were no  96  significant changes found in measures of base of support, and stride length after injury; however there was a significant difference in toe spread in the denervated forelimb and hindlimb when compared to the contralateral limbs. By the second week after injury, the difference in toe spread in the hindlimbs was not significant, although this difference was sustained in the forelimbs. This result, albeit weak, likely reflects a real effect, as Metz et a!. (1998) also described, rather a clear difference after injury. In the Metz study, inner toe spread was defined as the distance between the prints of the second and third hind toes while in the present study, we measured from the first toe to the fifth toe. The significance found in the present study appears most likely because of the accumulated toe spread between all five toes. In BDNF treated rats, there was again a significant increase in the toe spread of the denervated hindlimb 3 days after injury, however, at 7 days there was a significant decrease in toe spread toward baseline levels when compared to the contralateral hindlimb. This relative difference was more pronounced two weeks after injury. Control vehicle treatedlpyramidal injured rats showed a very similar recovery of hindlimb toe spread when compared to the toe spread of the pyramidal lesioned rats. Concomitant with the decrease in toe spread in the BDNF treated rats, there was a significant 3.2 fold increase in corticospinal axon profiles in the denervated lumbar grey matter when compared to vehicle treated rats. Control vehicle treated rats showed a significant 1.6 fold increase in sprouts when compared to sham operated unlesioned rats. This 1.6 fold increase however, was not accompanied by significant recovery in function. If the sprouting of corticospinal fibers causes the changes seen in toe spread, the answer to when this threshold amount is reached, is more straightforward. The comparison of toe spread between treatment groups in the denervated hindlimb (Fig 3.21), illustrated that a behavioral  97  effect presumably due to sprouting occurred between 3 and 7 days. Previous research has shown that sprouting in rats was maximal as early as 1 week after injury (Bareyre et al., 2002). Quantification of lumbar sprouting between these time points would further address this question.  98  Conclusions In conclusion, the results of the present study demonstrated that after a unilateral pyramidal lesion, the ipsilateral corticospinal tract from the intact sensorimotor cortex may significantly sprout to increase corticospinal innervation to the denervated grey matter by 1.6 fold. This spontaneous sprouting occurs quickly, within two weeks after injury, and is sustained even 6 weeks following injury. Following a unilateral pyramidal injury, I found that the application of BDNF to the intact corticospinal neurons can further induce significant sprouting of lumbar corticospinal axons up to 3.2 fold when compared to vehicle treated controls. However, this treatment did not cause any further sprouting in the cervical enlargement. The highly regulated topographic nature of corticospinal innervation and its specific role in fine motor control of distal forelimb muscles may have limited the growth of the ipsilateral corticospinal axons responsible for proximal limb control in the cervical spinal cord. The same restrictions may not exist in the lumbar spinal cord since much of the function involved in hindlimb movement is controlled by proximal muscles. After a unilateral pyramidal injury, the denervated forelimb sustained a significant deficit in reaching ability as described in the 13 point ordinal scale, single attempt success rate, and success rate. The sprouting of cervical corticospinal axons was not sufficient to recover the 13 point analysis of reaching ability and single attempt success rate to unlesioned sham operated levels, even 6 weeks after injury. However, four weeks after injury, the less stringent analysis of food pellet retrieval success rate recovered to unlesioned sham operated levels. BDNF did not induce recovery of forelimb reaching as analyzed by the 13 point scale and single attempt success rate. In fact, it significantly depressed retrieval success rate when compared to rats 6 weeks after pyramidal lesion.  99  Analysis of toe spread during walking revealed an increase in distance between the first and fifth toe in the denervated hindlimb after unilateral pyramidal injury that recovered toward presurgical baseline levels with BDNF treatment. The time course of recovery coincided with corticospinal sprouting in the lumbar spinal cord. Although sprouting of intact descending axons contributes significantly to recovery of function after partial injury, it in itself has not been sufficient in promoting the desired recovery after severe spinal cord injuries. It appears that there is still a need for enhanced axonal regeneration to gain meaningful recovery after severe injury of the spinal cord.  100  References Akintunde A, Buxton DF. 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J Neurosci. 23(4): 1424-3 1.  Appiiidix  Ac  The University of British Columbia  ANIMAL CARE CERTIFICATE  PROTOCOL NUMBER:  A03-0112  INVESTIGATOR OR COURSE DIRECTOR: DEPARTMENT:  CORD  PROJECT OR COURSE TITLE:  regeneration ANIMALS:  Tetzlaff, W.G.  Cell body treatment for the promotion of spinal cord  Rats 240  START DATE:  03-10-01  FUNDING AGENCY:  APPROVAL DATE: June  28, 2004  Christopher Reeve Paralysis Foundation  The Animal Care Committee has examined and approved the use of animals for the above experimental project or teaching course, and have been given an assurance that the animals involved will be cared for in accordance with the principles contained in Care of Experimental Animals A Guide for Canada, published by the Canadian Council on Animal Care. -  -I—_ 4  Approval of the UBC on Animal Care by one of: Dr. W.K. Milsom, Chair Dr. J. Love, Director, Animal Care Centre Ms. L. Macdonald, Manager, Animal Care Committee This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies. A copy of this certificate must be displayed in your animal facility.  Office of Research Services and Administration 102, Agronomy Road, Vancouver, V6T 1Z3 Phone: 604-827-5111 FAX: 604-822-5093  

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