John D. Houle

Treatment of the chronically injured spinal cord with neurotrophic factors can promote axonal regeneration from supraspinal neurons

Axonal regeneration has been demonstrated by supraspinal neurons long after a spinal cord injury, although this potential seems limited to a few neurons in specific nuclear groups. Whether the regenerative response could be enhanced by exposure to neurotrophic factors was examined in this study. Neurons injured during a cervical spinal cord hemisection lesion were labeled with true blue (TB). Four weeks after spinal cord injury, gel foam saturated with brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), ciliary neurotrophic factor (CNTF), or saline as a control was placed into the lesion cavity. The gel foam was replaced with fresh factor after 3 days, and 4 days later a peripheral nerve (PN) graft was apposed to the rostral cavity wall. Four weeks later neurons that grew an axon into the PN graft were labeled with nuclear yellow (NY). Cells that were double labeled (TB and NY) represented chronically injured neurons capable of axon regeneration. Cells labeled with NY only were either acutely injured neurons capable of axonal regrowth or uninjured neurons that had sprouted into the PN graft. The total number of TB/NY-labeled neurons was significantly increased following exposure to BDNF, NT-3, or CNTF. Specific regions most influenced by NT-3 and BDNF were the reticular formation and red nucleus. Treatment with CNTF resulted in a significant increase in most brain regions with a major contribution to descending pathways in the spinal cord, the motor cortex being the exception, with no evidence of axonal regeneration by neurons forming the corticospinal tract. The total number of NY-only labeled neurons also was significantly greater after treatment with BDNF or CNTF. These results demonstrate the potential to increase the regenerative response of specific chronically injured supraspinal neurons by application of neurotrophic factors to the injury site.

Activated Macrophage/Microglial Cells Can Promote the Regeneration of Sensory Axons into the Injured Spinal Cord

A prominent role for phagocytic cells in the regenerative response to CNS or PNS injury has been suggested by numerous studies. In the present work we tested whether increasing the presence of phagocytic cells at a spinal cord injury site could enhance the regeneration of sensory axons from cut dorsal roots. Nitrocellulose membranes treated with TGF-beta or coated with microglial cells were cotransplanted with fetal spinal cord tissue into an injured adult rat spinal cord. Cut dorsal roots were apposed to both sides of the nitrocellulose. Four weeks later, animals were sacrificed and spinal cord tissue sections were processed for immunocytochemical detection of calcitonin gene-related peptide (CGRP-ir) to identify regenerated sensory axons. Adjacent sections were processed with the antibody ED-1 or the lectin GSA-B4 for detection of macrophage/microglial cells in association with the regrowing axons. Qualitative and quantitative data indicate a correlation between the pattern and extent of axonal regeneration and the presence of phagocytic cells along the nitrocellulose implant. Axonal regeneration could be experimentally limited by implanting a nitrocellulose strip treated with macrophage inhibitory factor. These results indicate that increasing the presence of activated macrophage/microglial cells at a spinal cord injury site can provide an environment beneficial to the promotion of regeneration of sensory axons, possibly by the release of cytokines and interaction with other nonneuronal cells in the immediate vicinity.

Transplants of fibroblasts genetically modified to express BDNF promote axonal regeneration from supraspinal neurons following chronic spinal cord injury.

Transplants of fibroblasts genetically modified to express BDNF (Fb/BDNF) have been shown to promote regeneration of rubrospinal axons and recovery of forelimb function when placed acutely into the injured cervical spinal cord of adult rats. Here we investigated whether Fb/BDNF cells could stimulate supraspinal axon regeneration and recovery after chronic (4 week) injury. Adult female Sprague-Dawley rats received a complete unilateral hemisection injury at the third cervical spinal cord segment (C3). Four-five weeks later the injury site was exposed and rats received transplants of unmodified fibroblasts (Fb/UM) or Fb/BDNF. Four-five weeks after transplantation, locomotor recovery was examined on a test of forelimb usage and regeneration of supraspinal axons was studied following injection of the anterograde tracer biotin dextran amine (BDA). Rubrospinal tract (RST), reticulospinal tract (ReST), and vestibulospinal tract (VST) axons regenerated into transplants of either Fb/UM or Fb/BDNF but the length of axonal growth was significantly different in the two groups. The absolute distance of ReST growth was 1.8-fold greater in Fb/BDNF than in Fb/UM and the absolute distance of growth of RST and VST axons showed a statistically significant 4-fold increase. All three types of regenerated axons occupied a greater proportional length of Fb/BDNF transplants than of Fb/UM transplants. Only VST axons extended into the host spinal cord caudal to the Fb/BDNF grafts, but these axons were sparse. Rats receiving Fb/BDNF used both forelimbs together to explore walls of a cylinder more often than rats receiving Fb/UM, indicating partial recovery of forelimb usage. These results demonstrate that fibroblasts genetically modified to express BDNF promote axon regeneration from supraspinal neurons in the chronically injured spinal cord with accompanying partial recovery of locomotor performance.

Early changes in muscle fiber size and gene expression in response to spinal cord transection and exercise

Muscles of spinal cord-transected rats exhibit severe atrophy and a shift toward a faster phenotype. Exercise can partially prevent these changes. The goal of this study was to investigate early events involved in regulating the muscle response to spinal transection and passive hindlimb exercise. Adult female Sprague-Dawley rats were anesthetized, and a complete spinal cord transection lesion (T10) was created in all rats except controls. Rats were killed 5 or 10 days after transection or they were exercised daily on motor-driven bicycles starting at 5 days after transection and were killed 0.5, 1, or 5 days after the first bout of exercise. Structural and biochemical features of soleus and extensor digitorum longus (EDL) muscles were studied. Atrophy was decreased in all fiber types of soleus and in type 2a and type 2x fibers of EDL after 5 days of exercise. However, exercise did not appear to affect fiber type that was altered within 5 days of spinal cord transection: fibers expressing myosin heavy chain 2x increased in soleus and EDL, and extensive coexpression of myosin heavy chain in soleus was apparent. Activation of satellite cells was observed in both muscles of transected rats regardless of exercise status, evidenced by increased accumulation of MyoD and myogenin. Increased expression was transient, except for MyoD, which remained elevated in soleus. MyoD and myogenin were detected both in myofiber and in satellite cell nuclei in both muscles, but in soleus, MyoD was preferentially expressed in satellite cell nuclei, and in EDL, MyoD was more readily detectable in myofiber nuclei, suggesting that MyoD and myogenin have different functions in different muscles. Exercise did not affect the level or localization of MyoD and myogenin expression. Similarly, Id-1 expression was transiently increased in soleus and EDL upon spinal cord transection, and no effect of exercise was observed. These results indicate that passive exercise can ameliorate muscle atrophy after spinal cord transection and that satellite cell activation may play a role in muscle plasticity in response to spinal cord transection and exercise. Finally, the mechanisms underlying maintenance of muscle mass are likely distinct from those controlling myosin heavy chain expression.Muscles of spinal cord-transected rats exhibit severe atrophy and a shift toward a faster phenotype. Exercise can partially prevent these changes. The goal of this study was to investigate early events involved in regulating the muscle response to spinal transection and passive hindlimb exercise. Adult female Sprague-Dawley rats were anesthetized, and a complete spinal cord transection lesion (T10) was created in all rats except controls. Rats were killed 5 or 10 days after transection or they were exercised daily on motor-driven bicycles starting at 5 days after transection and were killed 0.5, 1, or 5 days after the first bout of exercise. Structural and biochemical features of soleus and extensor digitorum longus (EDL) muscles were studied. Atrophy was decreased in all fiber types of soleus and in type 2a and type 2x fibers of EDL after 5 days of exercise. However, exercise did not appear to affect fiber type that was altered within 5 days of spinal cord transection: fibers expressing myosin heavy chain 2x increased in soleus and EDL, and extensive coexpression of myosin heavy chain in soleus was apparent. Activation of satellite cells was observed in both muscles of transected rats regardless of exercise status, evidenced by increased accumulation of MyoD and myogenin. Increased expression was transient, except for MyoD, which remained elevated in soleus. MyoD and myogenin were detected both in myofiber and in satellite cell nuclei in both muscles, but in soleus, MyoD was preferentially expressed in satellite cell nuclei, and in EDL, MyoD was more readily detectable in myofiber nuclei, suggesting that MyoD and myogenin have different functions in different muscles. Exercise did not affect the level or localization of MyoD and myogenin expression. Similarly, Id-1 expression was transiently increased in soleus and EDL upon spinal cord transection, and no effect of exercise was observed. These results indicate that passive exercise can ameliorate muscle atrophy after spinal cord transection and that satellite cell activation may play a role in muscle plasticity in response to spinal cord transection and exercise. Finally, the mechanisms underlying maintenance of muscle mass are likely distinct from those controlling myosin heavy chain expression.

Demonstration of the potential for chronically injured neurons to regenerate axons into intraspinal peripheral nerve grafts

Experiments were carried out to determine if neurons damaged by injury to the spinal cord retain the ability to regenerate their axonal process for a prolonged period of time after the initial response to injury and if peripheral nerve (PN) grafts could support the regrowth of these processes. True blue (TB) was injected into one side of the adult rat lumbar spinal cord to label neurons with axons coursing through this region. Seven days later spinal cord tissue surrounding the injection sites was removed by aspiration to create a hemisection cavity 3-4 mm in length. Four weeks later scar tissue lining the lesion cavity was removed prior to grafting 1 cm segments of autologous tibial nerve to the rostral and the caudal surfaces of the cavity wall. The distal end of each graft was ligated and left unapposed to spinal cord tissue. Four weeks later the distal end of each PN graft was exposed to nuclear yellow (NY) to retrogradely label neurons that had grown an axon into the graft. Neurons containing both TB and NY were deemed capable of axonal regeneration while in a chronically injured state. Double-labeled (TB/NY) neurons were found in the ipsilateral spinal cord in laminae IV through X, excluding IX, and in Laminae VI and VII contralateral to the lesion. Most neurons were located within 10 mm of the lesion, with the majority caudal to the lesion. Nearly 50% (range 24-74%) of lumbar dorsal root ganglion neurons containing TB also were labeled with NY.(ABSTRACT TRUNCATED AT 250 WORDS)

Exercise-induced gene expression in soleus muscle is dependent on time after spinal cord injury in rats.

Cycling exercise attenuates atrophy in hindlimb muscles and causes changes in spinal cord properties after spinal cord injury in rats. We hypothesized that exercising soleus muscle expresses genes that are potentially beneficial to the injured spinal cord. Rats underwent spinal cord injury at T10 and were exercised on a motor‐driven bicycle. Soleus muscle and lumbar spinal cord tissue were used for messenger RNA (mRNA) analysis. Gene expression of brain‐derived neurotrophic factor (BDNF) and glial cell line‐derived neurotrophic factor (GDNF) was elevated 11‐ and 14‐fold, respectively, in soleus muscle after one bout of exercise performed 5 days after spinal cord transection. Also, c‐fos and heat shock protein‐27 (HSP27) mRNA abundance were increased 11‐ and 7‐fold, respectively. When exercise was started 2 days after the injury, the changes in gene expression were not observed. By contrast, at 2 but not at 5 days after transection, expression of the HSP27 gene was elevated sixfold in the lumbar spinal cord, independent of exercise. Electromyographic activity in soleus muscles was also decreased at 2 days, indicating that the spinal cord was less permissive to exercise at this early time. Long‐term exercise for 4 weeks attenuated muscle atrophy equally well in rats started at 2 days or 5 days after injury. We conclude that BDNF and GDNF released from exercising muscle may be involved in exercise‐induced plasticity of the spinal cord. Furthermore, the data suggest that the lumbar spinal cord undergoes time‐dependent changes that temporarily impede the ability of the muscle to respond to exercise. Muscle Nerve 29: 73–81, 2004

Passive exercise and fetal spinal cord transplant both help to restore motoneuronal properties after spinal cord transection in rats

Spinal cord transection influences the properties of motoneurons and muscles below the lesion, but the effects of interventions that conserve muscle mass of the paralyzed limbs on these motoneuronal changes are unknown. We examined the electrophysiological properties of rat lumbar motoneurons following spinal cord transection, and the effects of two interventions shown previously to significantly attenuate the associated hindlimb muscle atrophy. Adult rats receiving a complete thoracic spinal cord transection (T‐10) were divided into three groups receiving: (1) no further treatment; (2) passive cycling exercise for 5 days/week; or (3) acute transplantation of fetal spinal cord tissue. Intracellular recording of motoneurons was carried out 4–5 weeks following transection. Transection led to a significant change in the rhythmic firing patterns of motoneurons in response to injected currents, as well as a decrease in the resting membrane potential and spike trigger level. Transplants of fetal tissue and cycling exercise each attenuated these changes, the latter having a stronger effect on maintenance of motoneuron properties, coinciding with the reported maintenance of structural and biochemical features of hindlimb muscles. The mechanisms by which these distinct treatments affect motoneuron properties remain to be uncovered, but these changes in motoneuron excitability are consistent with influences on ion conductances at or near the initial segment. The results may support a therapeutic role for passive limb manipulation and transplant of stem cells in slowing the deleterious responses of motoneurons to spinal cord injury, such that they remain more viable for subsequent alternative strategies. Muscle Nerve 29: 234–242, 2004

Chronically injured supraspinal neurons exhibit only modest axonal dieback in response to a cervical hemisection lesion.

This study examined the extent of axon retraction (dieback) exhibited by injured brain stem neurons in a chronic spinal cord injury (SCI) condition. Adult female rats subjected to a cervical (C3) hemisection lesion were sacrificed 1, 4, 8, or 14 weeks after injury and the spinal cord from C1 to the lesion cavity was removed. One week prior to sacrifice, a microinjection of biotinylated dextran amine (BDA, 0.5 microliter) was made into the red nucleus, lateral vestibular nucleus, or medullary reticular formation of each animal. Horizontal cryostat sections were processed with avidin-HRP to detect supraspinal axons anterogradely labeled with BDA. Terminal end bulbs of axons were identified and their distance from the lesion site was measured by a computerized image analysis program. At all postinjury intervals, numerous rubrospinal, vestibulospinal, and reticulospinal tract axons were found immediately adjacent to the lesion site and over 60% of all terminals were within 500 micrometer at 1 and 4 weeks. The mean axonal distance of 450-500 micrometer from the lesion indicated that many injured axons had retracted farther than 500 micrometer from the lesion site; however, long-term maintenance of the mean axonal distance from the lesion at less than 500 micrometer indicated the absence of progressive dieback after SCI. While some modest changes occur in specific supraspinal pathways following SCI, axonal retraction does not appear to be a contributing factor to the diminished regenerative effort by certain brain stem neurons that has been observed at long postinjury intervals.

Nerve growth factor (NGF)-treated nitrocellulose enhances and directs the regeneration of adult rat dorsal root axons through intraspinal neural tissue transplants

Severed adult rat dorsal roots were apposed to an intraspinal transplant of fetal spinal cord (FSC) tissue co-grafted with nerve growth factor (NGF)-treated nitrocellulose strips. Axonal regrowth from the injured roots was assessed by calcitonin gene-related peptide immunoreactivity (CGRP-IR). Dense fascicles of regenerating CGRP-IR axons lined the entire length of NGF-treated nitrocellulose, with many crossing the graft-host interface ventrally to extend into the host neuropil. In contrast, CGRP-IR axon regrowth was not promoted by untreated nitrocellulose implants. These results indicate that substrate bound NGF can promote and direct the intraspinal regeneration of a specific population of dorsal root axons.

Effects of fetal spinal cord tissue transplants and cycling exercise on the soleus muscle in spinalized rats

Studies were carried out to determine if an intraspinal transplant (Trpl) of fetal spinal cord tissue or hind limb exercise (Ex) affected the changes in myosin heavy chain (MyHC) composition or myofiber size that occur following a complete transection (Tx) of the lower thoracic spinal cord of the adult rat. In one group of animals, transplants were made acutely, whereas in a second group, daily cycling exercise was initiated 5 days after injury, with animals in both groups being sacrificed 90 days after injury. The soleus muscle is normally composed of myofibers expressing either type I (90%) or type IIa (10%) MyHC. Following a spinal transection, expression of type I MyHC isoform decreased (18% of myofibers), type IIa MyHC expression increased (65% of myofibers), and the majority of myofibers (80%) expressed type IIx MyHC. Most myofibers coexpressed multiple MyHC isoforms. Compared with Tx only, with Ex or with Trpl, there was a decrease in the number of myofibers expressing type I or IIa isoforms but little change in expression of IIx MyHC. Myofibers expressing the IIb isoform appeared in several transplant recipients but not after exercise. Transection resulted in atrophy of type I myofibers to approximately 50% of normal size, whereas myofibers were significantly larger after exercise (74% of control) and in Trpl recipients (77% of control). Type IIa myofibers also were significantly larger in Trpl recipients compared with the Tx only group. Overall, the mean myofiber size was significantly greater after exercise and in Trpl recipients compared with myofibers in Tx only animals. Thus, although neither strategy shifted the MyHC profile towards the control, both interventions influenced the extent of atrophy observed after spinalization. These data suggest that palliative strategies can be developed to modulate some of the changes in hind limb muscles that occur following a spinal cord injury.