Rehabilitation Strategies after Spinal Cord Injury: Inquiry into the Mechanisms of Success and Failure.

Body-weight supported locomotor training (BWST) promotes recovery of load-bearing stepping in lower mammals, but its efficacy in individuals with a spinal cord injury (SCI) is limited and highly dependent on injury severity. While animal models with complete spinal transections recover stepping with step-training, motor complete SCI individuals do not, despite similarly intensive training. In this review, we examine the significant differences between humans and animal models that may explain this discrepancy in the results obtained with BWST. We also summarize the known effects of SCI and locomotor training on the muscular, motoneuronal, interneuronal, and supraspinal systems in human and non-human models of SCI and address the potential causes for failure to translate to the clinic. The evidence points to a deficiency in neuronal activation as the mechanism of failure, rather than muscular insufficiency. While motoneuronal and interneuronal systems cannot be directly probed in humans, the changes brought upon by step-training in SCI animal models suggest a beneficial re-organization of the systems' responsiveness to descending and afferent feedback that support locomotor recovery. The literature on partial lesions in humans and animal models clearly demonstrate a greater dependency on supraspinal input to the lumbar cord in humans than in non-human mammals for locomotion. Recent results with epidural stimulation that activates the lumbar interneuronal networks and/or increases the overall excitability of the locomotor centers suggest that these centers are much more dependent on the supraspinal tonic drive in humans. Sensory feedback shapes the locomotor output in animal models but does not appear to be sufficient to drive it in humans.

The implications of injury in the developing nervous system on upper extremity function.

STUDY DESIGN: Literature review. PURPOSE: The corticospinal system (CS) and peripheral nervous system (PNS) are common sites of damage during the early stages of life. The prenatal or immediately prenatal period is the most common time for damage to occur. Here we briefly review the basic features of the development of the CS and the PNS and the clinical consequences of injury to or improper development of these systems on upper extremity (UE) function. RESULTS: The proper development of both the CS and PNS is necessary to achieve adequate function of the (UE). Injury or improper development of these systems can lead to upper extremity dysfunction and limit participation in activities of daily living. CONCLUSIONS: Both the PNS and CS play major roles in the proper functioning of the UE. A better understanding of their roles and common developmental disorders is needed to move rehabilitation of motor impairments forward.

Forelimb locomotor rating scale for behavioral assessment of recovery after unilateral cervical spinal cord injury in rats.

BACKGROUND: Cervical spinal cord injury (SCI) models in rats have become increasingly useful because of their translational potential. The goal of this study was to design, develop and validate a quick and reliable forelimb locomotor rating scale for adult rats with unilateral cervical SCI injury. NEW METHOD: Adult female rats were subjected to a C5 unilateral mild contusion (n=10), moderate contusion (n=10) or hemisection injury (n=9). Forelimb locomotion was evaluated before injury, four times during the first week (Days 2, 3, 4 and 7) and weekly for up to 8 weeks post-injury. Scoring categories were identified and animals were ranked based on their performance in these categories. The scale was validated for its usefulness by comparing animals with different injury models (dorsolateral funiculotomy C3/4), levels of injury (moderate contusion C4) and sex (male - moderate contusion C3/4) and also by correlating FLS scores with other established behavioral tests (grid walking and kinetic tests). RESULTS AND COMPARISON WITH EXISTING METHODS: Forelimb performance on both the grid-walking and kinetic tests was positively correlated with the forelimb locomotor rating scale (FLS). Histological analysis established a positive correlation between the spared tissue and the observed FLS score. Our results show that the new rating scale can reliably detect forelimb deficits and recovery predicted by other behavioral tests. Furthermore, the new method provides reproducible data between trained and naïve examiners. CONCLUSION: In summary, the proposed rating scale is a useful tool for assessment of injury and treatments designed to enhance recovery after unilateral cervical SCI.

Acute and prolonged hindlimb exercise elicits different gene expression in motoneurons than sensory neurons after spinal cord injury.

We examined gene expression in the lumbar spinal cord and the specific response of motoneurons, intermediate gray and proprioceptive sensory neurons after spinal cord injury and exercise of hindlimbs to identify potential molecular processes involved in activity dependent plasticity. Adult female rats received a low thoracic transection and passive cycling exercise for 1 or 4weeks. Gene expression analysis focused on the neurotrophic factors: brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and their receptors because of their potential roles in neural plasticity. We also examined expression of genes involved in the cellular response to injury: heat shock proteins (HSP) -27 and -70, glial fibrillary acidic protein (GFAP) and caspases -3, -7, and -9. In lumbar cord samples, injury increased the expression of mRNA for TrkB, all three caspases and the HSPs. Acute and prolonged exercise increased expression of mRNA for the neurotrophic factors BDNF and GDNF, but not their receptors. It also increased HSP expression and decreased caspase-7 expression, with changes in protein levels complimentary to these changes in mRNA expression. Motoneurons and intermediate gray displayed little change in mRNA expression following injury, but acute and prolonged exercise increased levels of mRNA for BDNF, GDNF and NT-4. In large DRG neurons, mRNA for neurotrophic factors and their receptors were largely unaffected by either injury or exercise. However, caspase mRNA expression was increased by injury and decreased by exercise. Our results demonstrate that exercise affects expression of genes involved in plasticity and apoptosis in a cell specific manner and that these change with increased post-injury intervals and/or prolonged periods of exercise.

Amphetamine-enhanced motor training after cervical contusion injury.

Individually, motor training, pharmacological interventions, and housing animals in an enriched environment (EE) following spinal cord injury (SCI) result in limited functional improvement but, when combined, may enhance motor function. Here, we tested amphetamine (AMPH)-enhanced skilled motor training following a unilateral C3-C4 contusion injury on the qualitative components of reaching and on skilled forelimb function, as assessed using single-pellet and staircase reaching tasks. Kinematic analysis evaluated the quality of the reach, and unskilled locomotor function was also tested. Animals receiving AMPH and skilled forelimb training performed better than operated control animals on qualitative reaching, but not on skilled reaching. Those that received the combination treatment and were housed in EE cages showed significantly less improvement in qualitative reaching and grasping. Kinematic analysis revealed a decrease in digit abduction during skilled reaching among all groups, with no differences among groups. Kinematics provided no evidence that improved function was related to improved quality of reach. There was no evidence of neuroprotection in the cervical spinal cord. The absence of evidence for kinematic improvement or neuroprotection suggested that AMPH-enhanced motor training is due primarily to supraspinal effects, an enhancement of attention during skilled motor training, or plasticity in supraspinal circuitry involved with motor control.

Peripheral nerve graft with immunosuppression modifies gene expression in axotomized CNS neurons.

Adult central nervous system (CNS) neurons do not regenerate severed axons unaided but may regenerate axons into apposed predegenerated peripheral nerve grafts (PNGs). We examined gene expression by using microarray technology in laser-dissected lateral vestibular (LV) neurons whose axons were severed by a lateral hemisection at C3 (HX) and in lateral vestibular nucleus (LVN) neurons that were hemisected at C3 and that received immunosuppression with cyclosporine A (CsA) and a predegenerated PNG (termed I-PNG) into the lesion site. The results provide an expression analysis of temporal changes that occur in LVN neurons in nonregenerative and potentially regenerative states and over a period of 42 days. Axotomy alone resulted in a prolonged change in regulation of probe sets, with more being upregulated than downregulated. Apposition of a PNG with immunosuppression muted gene expression overall. Axotomized neurons (HX) upregulated genes commonly associated with axonal growth, whereas axotomized neurons whose axons were apposed to the PNG showed diminished expression of many of these genes but greater expression of genes related to energy production. The results suggest that axotomized LVN neurons express many genes thought to be associated with regeneration to a greater extent than LVN neurons that are apposed to a PNG. Thus the LVN neurons remain in a regenerative state following axotomy but the conditions provided by the I-PNG appear to be neuroprotective, preserving or enhancing mitochondrial activity, which may provide required energy for regeneration. We speculate that the graft also enables sufficient axonal synthesis of cytoskeletal components to allow axonal growth without marked increase in expression of genes normally associated with regeneration.

Role of spared pathways in locomotor recovery after body-weight-supported treadmill training in contused rats.

Body-weight-supported treadmill training (BWSTT)-related locomotor recovery has been shown in spinalized animals. Only a few animal studies have demonstrated locomotor recovery after BWSTT in an incomplete spinal cord injury (SCI) model, such as contusion injury. The contribution of spared descending pathways after BWSTT to behavioral recovery is unclear. Our goal was to evaluate locomotor recovery in contused rats after BWSTT, and to study the role of spared pathways in spinal plasticity after BWSTT. Forty-eight rats received a contusion, a transection, or a contusion followed at 9 weeks by a second transection injury. Half of the animals in the three injury groups were given BWSTT for up to 8 weeks. Kinematics and the Basso-Beattie-Bresnahan (BBB) test assessed behavioral improvements. Changes in Hoffmann-reflex (H-reflex) rate depression property, soleus muscle mass, and sprouting of primary afferent fibers were also evaluated. BWSTT-contused animals showed accelerated locomotor recovery, improved H-reflex properties, reduced muscle atrophy, and decreased sprouting of small caliber afferent fibers. BBB scores were not improved by BWSTT. Untrained contused rats that received a transection exhibited a decrease in kinematic parameters immediately after the transection; in contrast, trained contused rats did not show an immediate decrease in kinematic parameters after transection. This suggests that BWSTT with spared descending pathways leads to neuroplasticity at the lumbar spinal level that is capable of maintaining locomotor activity. Discontinuing training after the transection in the trained contused rats abolished the improved kinematics within 2 weeks and led to a reversal of the improved H-reflex response, increased muscle atrophy, and an increase in primary afferent fiber sprouting. Thus continued training may be required for maintenance of the recovery. Transected animals had no effect of BWSTT, indicating that in the absence of spared pathways this training paradigm did not improve function.

Post-stroke depression: the case for augmented, individually tailored cognitive behavioural therapy.

In this review, we begin by considering why post-stroke depression (PSD) is so prevalent. We then examine the current evidence base to support cognitive behavioural therapy (CBT) as a treatment approach for the condition. While there is limited evidence currently, we demonstrate that much remains to be established with regard to PSD and the efficacy of CBT. We argue there is every reason to believe CBT should be an effective treatment, but that clinicians must augment and individually tailor this approach to ensure effectiveness. We set out our rationale for a novel augmented, individually tailored CBT protocol, and describe five key components that we believe once incorporated, and tested using randomized controlled methods, should enhance treatment outcome of PSD.

A training paradigm to enhance motor recovery in contused rats: effects of staircase training.

BACKGROUND: Ambulating on stairs is an important aspect of daily activities for many individuals with incomplete spinal cord injury (SCI), and little is known about the effect of training for this specific task. OBJECTIVE: The goal of this study was to determine whether staircase ascent training enhances motor recovery in animals with contusion injury. METHODS: Rats received a midthoracic contusion lesion of moderate severity and were randomly divided into 2 groups, with one group receiving staircase ascent training for up to 8 weeks and the other receiving no training. To assess the direct effect of training, a task-specific staircase climbing test was performed. Open field test (BBB) and gait analysis (CatWalk) assessed overground recovery, and a grid test was used to assess improvement in sensorimotor tasks. Changes in muscle mass of the forelimb and hindlimb muscles were also measured, and the extent of spared white matter was determined for lesion verification and anatomical correlations. RESULTS: Staircase training improved the task-specific performance of ascent. Gait parameters, including base of support, stride length, regularity index (RI), and step sequence, also improved. Overground locomotion and the grid test, both showed a trend of improved performance. Finally, hindlimb muscle mass was maintained with training. CONCLUSIONS: Staircase ascent training after incomplete SCI has beneficial effects on task-specific as well as nonspecific motor and sensorimotor activities.

Forelimb locomotor assessment scale (FLAS): novel assessment of forelimb dysfunction after cervical spinal cord injury.

We describe here a novel forelimb locomotor assessment scale (FLAS) that assesses forelimb use during locomotion in rats injured at the cervical level. A quantitative scale was developed that measures movements of shoulder, elbow, and wrist joints, forepaw position and digit placement, forelimb-hindlimb coordination, compensatory behaviors adopted while walking, and balance. Female Sprague-Dawley rats received graded cervical contusions ranging from 200 to 230 ("mild," n=11) and 250-290 kdyn ("moderate," n=13) between C5 and C8. Rats were videotaped post-injury as they walked along an alley to determine deficits and recovery of forelimb function. Recovery of shoulder and elbow joint movement occurred rapidly (within 1-7 days post-injury), whereas recovery of wrist joint movement was slower and more variable. Most rats in all groups displayed persistent deficits in forepaw and digit movement, but developed compensatory behaviors to allow functional forward locomotion within 1-2 weeks post-injury. Recovery of forelimb function as measured by the FLAS reached a plateau by 3 weeks post-injury in all groups. Rats with mild contusions displayed greater locomotor recovery than rats with moderate contusions, but exhibited persistent deficits compared to sham controls. Reliability was tested by having seven raters (three internal, four external) from different laboratories, independently and blindly score videos of all rats. The multivariate correlation between all raters, all animals, and all time points ranged from r(2)=0.88-0.96 (p<0.0001), indicating a high inter-rater reliability. Thus, the FLAS is a simple, inexpensive, sensitive, and reliable measure of forelimb function during locomotion following cervical SCI.

Exercise induces cortical plasticity after neonatal spinal cord injury in the rat.

Exercise-induced cortical plasticity is associated with improved functional outcome after brain or nerve injury. Exercise also improves functional outcomes after spinal cord injury, but its effects on cortical plasticity are not known. The goal of this investigation was to study the effect of moderate exercise (treadmill locomotion, 3 min/d, 5 d/week) on the somatotopic organization of forelimb and hindlimb somatosensory cortex (SI) after neonatal thoracic transection. We used adult rats spinalized as neonates because some of these animals develop weight-supported stepping, and, therefore, the relationship between cortical plasticity and stepping could also be examined. Acute, single-neuron mapping was used to determine the percentage of cortical cells responding to cutaneous forelimb stimulation in normal, spinalized, and exercised spinalized rats. Multiple single-neuron recording from arrays of chronically implanted microwires examined the magnitude of response of these cells in normal and exercised spinalized rats. Our results show that exercise not only increased the percentage of responding cells in the hindlimb SI but also increased the magnitude of the response of these cells. This increase in response magnitude was correlated with behavioral outcome measures. In the forelimb SI, neonatal transection reduced the percentage of responding cells to forelimb stimulation, but exercise reversed this loss. This restoration in the percentage of responding cells after exercise was accompanied by an increase in their response magnitude. Therefore, the increase in responsiveness of hindlimb SI to forelimb stimulation after neonatal transection and exercise may be due, in part, to the effect of exercise on the forelimb SI.

Effect of cervical dorsolateral funiculotomy on reach-to-grasp function in the rat.

Cervical spinal cord injury (SCI) can severely impair reaching and grasping ability, and several descending systems, including the rubrospinal tract and corticospinal tract, have been implicated in the control of reach-to-grasp movements. The primary aim of this study was to characterize further the forelimb deficits associated with a cervical dorsolateral funiculotomy, which ablates the rubrospinal tract but spares the dorsal and ventral corticospinal tract in the rat. Adult female rats that preferred to use their right forelimb to reach for single pellets received a lesion to the right cervical dorsolateral funiculus between the C3-4 dorsal roots. Gross forelimb motor function was assessed by measuring spontaneous forelimb usage during exploration in a cylinder, and fine motor function was assessed using staircase and single pellet reaching tests. Single pellet reaching was further evaluated by qualitative and quantitative kinematic scoring of the movement components. Histological analysis included the quantification of spared white matter. Cervical dorsolateral funiculotomy produced marked deficits in reaching performance on both the single pellet and staircase reaching tests, with transient deficits in gross forelimb usage in the cylinder. Quantitative kinematics also revealed a reduction in digit abduction during the reach, which persisted throughout the 8-week post-SCI period. Tests of reach-to-grasp function, therefore, were more sensitive than a test of gross forelimb usage after cervical dorsolateral funiculotomy and did not show recovery over the 8-week survival period. We suggest that the staircase test is a useful screening tool for intervention studies because of its ease of implementation, and that the single pellet test is valuable for examining reaching accuracy and detailed kinematics.

Neurotrophic factors promote and enhance locomotor recovery in untrained spinalized cats.

In spinal cats, locomotor recovery without rehabilitation is limited, but weight-bearing stepping returns with treadmill training. We studied whether neurotrophins administered to the injury site also restores locomotion in untrained spinal cats and whether combining both neurotrophins and training further improves recovery. Ordinary rat fibroblasts or a mixture of fibroblasts secreting brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) (Fb-NTF) were grafted into T12 spinal transection sites. Cats with each type of transplant were divided into two groups: one receiving daily training and the other receiving no training. As expected, trained cats with/without neurotrophin-producing transplants could step on the treadmill. Untrained cats without neurotrophin-producing transplants could not locomote. However, untrained cats with neurotrophin-secreting transplants performed plantar weight-bearing stepping at speeds up to 0.8 m/s as early as 2 wk after transection. Locomotor capability and stance lengths in these animals were similar to those in animals receiving training alone, suggesting that administration of BDNF/NT-3 was equivalent to treadmill training in restoring locomotion in chronically spinalized cats. Cats receiving both interventions showed the greatest improvement in step length. Anatomical evaluation indicated that all transections were complete and that axons did not enter the cord caudal to the graft. Thus BDNF/NT-3 secreting fibroblasts were equivalent to training in their ability to engage the locomotor circuitry in chronic spinal cats. Furthermore, the rapid time-course of recovery and the absence of axonal growth through the transplants indicate that the restorative mechanisms were not related to supraspinal axonal growth. Finally, the results show that transplants beneficial in rodents are applicable to larger mammals.

Paralysis elicited by spinal cord injury evokes selective disassembly of neuromuscular synapses with and without terminal sprouting in ankle flexors of the adult rat.

Neuromuscular junctions (NMJs) innervated by motor neurons below spinal cord injury (SCI) have been reported to remain intact despite the interruption of supraspinal pathways and the resultant loss of activity. Here we report notably heterogeneous NMJ responses to SCI that include overt synapse disassembly. Complete transection of the thoracic spinal cord of adult rats evoked massive sprouting of nerve terminals in a subset of NMJs in ankle flexors, extensor digitorum longus, and tibialis anterior. Many of these synapses were extensively disassembled 2 weeks after spinal transection but by 2 months had reestablished synaptic organization despite continuous sprouting of their nerve terminals. In contrast, uniform and persistent loss of acetylcholine receptors (AChRs) was evident in another subset of NMJs in the same flexors, which apparently lacked terminal sprouting and largely maintained terminal arbors. Other synapses in the flexors, and almost all the synapses in the ankle extensors, medial gastrocnemius, and soleus, remained intact, with little pre- or postsynaptic alteration. Additional deafferentation of the transected animals did not alter the incidence or regional distribution of either type of the unstable synapses, whereas cycling exercise diminished their incidence. The muscle- and synapse-specific responses of NMJs therefore reflected differential sensitivity of the NMJs to inactivity rather than to differences in residual activity. These observations demonstrate the existence of multiple subpopulations of NMJs that differ distinctly in pre- and postsynaptic vulnerability to the loss of activity and highlight the anatomical instability of NMJs caudal to SCI, which may influence motor deficit and recovery after SCI.

Combining motor training with transplantation of rat bone marrow stromal cells does not improve repair or recovery in rats with thoracic contusion injuries.

Previous studies have demonstrated that either transplantation of bone marrow stromal cells (MSC) or physical exercise regimens can elicit limited functional recovery following spinal cord injury, presumably through different mechanisms. The present study examined whether transplantation of MSC derived from transgenic Fischer alkaline phosphatase (AP) rats, in combination with exercise, would have synergistic effects leading to recovery of function that is greater than either alone. Adult female Sprague-Dawley rats received a moderate thoracic contusion injury and were divided into three groups: operated controls (Op-Control), MSC transplant recipients (MSC), and MSC transplant recipients plus exercise (MSC+Ex). Nine days after contusion, a Vitrogen matrix +/-one million MSC was injected into the lesion site in all animals. Immunosuppression with high doses of Cyclosporine A, required for MSC survival, was provided for all animals. Passive hindlimb exercise on motorized bicycles was applied 1 h/day, 3 days/week to the MSC+Ex group. A battery of behavioral tests was performed weekly to assess motor and sensory functions in all 3 groups for 12 weeks. Morphological evaluation included MSC survival, evidence of axonal growth into grafts, phenotypic analysis of MSC, and lesion/transplant size. The weight of the medial gastrocnemius muscle, a hindlimb muscle activated during stance, was used to identify extent of atrophy. No differences in motor recovery were found among the three groups. MSC survived 3 months after transplantation, indicating that the immunosuppression treatment was successful. The extent of survival was variable, and there was no correlation between MSC survival and behavioral scores. The matrix persisted, filling the lesion cavity, and some axons grew into the lesion/matrix but to a similar extent in all groups. There was no difference in lesion/matrix size among groups, indicating no neuroprotective effect on the host provided by the treatments. Immunocytochemical analysis provided no evidence that MSC differentiated into neurons, astrocytes or oligodendrocytes. Muscle mass of the medial gastrocnemius was diminished in the Op-Control group indicating significant atrophy, but was partially preserved in both the MSC and MSC+Ex groups. Our results indicate that combining the beneficial effects of rat MSC and this exercise protocol was not sufficient to enhance behavioral recovery.

Role of the 5-HT2C receptor in improving weight-supported stepping in adult rats spinalized as neonates.

Loss of descending serotonergic (5-HT) projections after spinal cord injury (SCI) contributes to motor deficits and upregulation of receptors on partially denervated serotonergic targets in the spinal cord. Serotonergic agonists acting on these upregulated receptors are potential therapeutic agents that could ameliorate motor deficits. However, modification of 5-HT receptors following complete spinal cord injury results in different effects by 5-HT2C receptor agonists and antagonists. For example, administration of 5-HT2C receptor agonists suppresses locomotor activity in normal animals, but enhances it in spinalized animals. In addition, administration of 5-HT2C receptor agonists does not induce activity-dependent hindlimb tremors in normal animals, but does induce them in spinalized animals. We therefore extended our previous work with the 5-HT2C receptor agonist 1-(m-chlorophenyl)-piperazine hydrochloride (mCPP), which enhances weight-supported stepping when administered to adult rats spinalized as neonates, to identify the optimal dose for improved weight-supported stepping with minimal side effects. In order to determine whether mCPP enhances weight-supported stepping after SCI is through activation of the 5-HT2C receptor, we performed the following experiments. We determined that stimulation of the 5-HT1A receptor did not contribute to this improvement in weight-support. We reversed the increase in mCPP-induced weight-supported stepping with SB 206,553, a 5-HT2C receptor antagonist. We also provide evidence for denervation-induced upregulation of 5-HT2C receptors in the injured spinal cord. Since mCPP does not have the behavioral toxicity associated with non-selective 5-HT2 receptor agonists, targeting the 5-HT2C receptor may have clinical relevance for the treatment of SCI.

Immunosuppression with either cyclosporine a or FK506 supports survival of transplanted fibroblasts and promotes growth of host axons into the transplant after spinal cord injury.

Fibroblasts that have been genetically modified to secrete neurotrophins can stimulate axonal regeneration, rescue injured neurons, and improve function when grafted into a spinal cord injury site. These grafts are usually allografts that require immunosuppression to prevent rejection. In this study, we compared the effects of two immunophilin-ligands (cyclosporine A [CsA] and FK506) that are used clinically to prevent transplant rejection on protection of grafted fibroblasts. As there are risks associated with prolonged immunosuppression, we compared the effects of 2 or 8 weeks of administration of these drugs, in combination with our standard methylprednisolone protocol, in animals that survived for 8 weeks, to determine whether a shorter course of immunosuppression would be effective. Outcome measures included fibroblast survival, infiltration of activated macrophages and microglia into the graft, final lesion size, and growth of host axons into the graft. The graft consisted of a Vitrogen matrix into which fibroblasts were suspended; the graft was placed into a C3/C4 lateral funiculus lesion. The fibroblasts were isolated from a transgenic strain of Fischer rats that produce the marker alkaline phosphatase (Fb/AP). This enabled us to track the grafted fibroblasts and to evaluate the extent of their survival. The grafted matrix filled the lesion cavity. The density of fibroblasts within the matrix differed according to treatment. Fibroblast survival was most robust in animals that received 8 weeks of immunophilin-ligand treatment. FK506 supported greater Fb/AP survival than CsA. ED-1 immunostaining for activated microglia and macrophages showed an inverse correlation between AP immunoreactivity and the density of immune cells within the graft. Thus, prolonged administration of either FK506 or CsA was necessary for maximal fibroblast survival and for limiting the macrophage invasion of the graft. None of the FK506 or CsA protocols modified the size of the lesion, indicating that these immunophilin-ligands had little effect on secondary enlargement of the lesion and therefore little neuroprotective effect. Because immunophilin-ligands have been shown to be neurotrophic, we used RT-97 immunostaining for neurofilaments and calcitonin gene related protein (CGRP) staining for dorsal root axons to visualize axons that grew into the graft. Some axons grew into the matrix even in the absence of immunophilin-ligand treatment, suggesting that the Vitrogen matrix itself is permissive, but all of the immunophilin-ligand protocols were much more effective in eliciting axonal growth. Growth of axons into the transplants was equally increased by drug treatment for 2 or 8 weeks. Thus, both treatments improved fibroblast survival, diminished immune cell invasion, and promoted axonal growth, and a 2-week course of treatment with either immunophilin-ligand was as effective as 8 weeks in stimulating axonal growth.

Transplantation of neuronal and glial restricted precursors into contused spinal cord improves bladder and motor functions, decreases thermal hypersensitivity, and modifies intraspinal circuitry.

Transplanting neuronal and glial restricted precursors (NRP/GRP) into a midthoracic injury 9 d after contusion improved bladder and motor function, diminished thermal hypersensitivity, and modified lumbosacral circuitry compared with operated controls (OP-controls). Histological analysis showed that NRP/GRP survived, filled the lesion site, differentiated into neurons and glia, and migrated selectively. Volume of spinal cord spared was increased in NRP/GRP recipients, suggesting local protection. Bladder areflexia developed in both operated groups, but NRP/GRP recipients exhibited an accelerated recovery, with decreased micturition pressure and fewer episodes of detrusor hyperreflexia. Because noradrenergic receptors proliferate after spinal injury and descending noradrenergic pathways contribute to regulation of bladder control, we examined the effects of administering an alpha-1A-adrenergic antagonist, Tamsulosin, on urodynamics. This improved all cystometric parameters in both operated groups, and micturition pressure in NRP/GRP rats recovered to normal levels. Both operated groups initially showed increased sensitivity to a thermal stimulus applied to the tail; the NRP/GRP rats showed significant improvement over time. NRP/GRP grafts also produced greater recovery of hindlimb function in several tests, although both groups showed persistent and similar deficits in locomotion on a grid. Because bladder, hindlimb, and tail sensory and motor functions are organized through lumbosacral cord, we examined descending and primary afferent projections at L6-S1. The density of serotonergic, noradrenergic, and corticotrophin releasing factor-positive fibers increased in the NRP/GRP group compared with OP-controls, suggesting some sparing and/or sprouting of these modulatory pathways. Immunocytochemical staining density of dorsal root axons in the dorsal horn increased in the OP-controls but appeared normal in the NRP/GRP group. Synaptophysin immunoreactivity in the lumbosacral dorsal horn was similar among groups, consistent with restoration of synaptic density in both groups of operated animals but by different pathways. We suggest that local protection provided by NRP/GRP resulted in increased sparing/sprouting of descending pathways, which prevented sprouting by dorsal root axons, and that this modification in lumbosacral circuitry contributes to the recovery of function.

Transplants of fibroblasts expressing BDNF and NT-3 promote recovery of bladder and hindlimb function following spinal contusion injury in rats.

We examined whether fibroblasts, genetically modified to express BDNF and NT-3 (Fb-BDNF/NT3) and transplanted into a thoracic spinal injury site, would enhance recovery of bladder function and whether this treatment would be associated with reorganization of lumbosacral spinal circuits implicated in bladder function. Rats received modified-moderate contusion injuries at T8/9, and 9 days later, Fb-BDNF/NT3 or unmodified fibroblasts (OP-controls) were delivered into the cord. Fb-BDNF/NT3 rats recovered from areflexic bladder earlier, showed decreased micturition pressure and fewer episodes of detrusor hyperreflexia, compared to OP-controls. There were also improvements in hindlimb function in the Fb-BDNF/NT3 group although locomotion on a more challenging substrate (grid) and tail withdrawal latency in response to a thermal stimulus showed persisting deficits, little recovery, and no differences between the groups. Immunocytochemistry at L6-S1 revealed changes in density of afferent and descending projections to L6-S1 cord. The density of small dorsal root axons increased in the superficial layers of the dorsal horn in OP-controls but not in Fb-BDNF/NT3, suggesting sprouting of primary afferents following injury that was inhibited by Fb-BDNF/NT-3. In contrast, the trophic factor secreting transplants stimulated sprouting and/or sparing of descending modulatory pathways projecting to the lumbosacral spinal cord. No differences in synaptophysin immunoreactivity were seen in the dorsal horn which suggested that synaptic density was similar but achieved by sprouting of different systems in the two operated groups. Fb-BDNF/NT3 transplanted into injured spinal cord thus improved both bladder and hindlimb function, and this was associated with reorganization of spinal circuitry.

Alginate encapsulated BDNF-producing fibroblast grafts permit recovery of function after spinal cord injury in the absence of immune suppression.

Encapsulation of cells has the potential to provide a protective barrier against host immune cell interactions after grafting. Previously we have shown that alginate encapsulated BDNF-producing fibroblasts (Fb/BDNF) survived for one month in culture, made bioactive neurotrophins, survived transplantation into the injured spinal cord in the absence of immune suppression, and provided a permissive environment for host axon growth. We extend these studies by examining the effects of grafting encapsulated Fb/BDNF into a subtotal cervical hemisection on recovery of forelimb and hindlimb function and axonal growth in the absence of immune suppression. Grafting of encapsulated Fb/BDNF resulted in partial recovery of forelimb usage in a test of vertical exploration and of hindlimb function while crossing a horizontal rope. Recovery was significantly greater compared to animals that received unencapsulated Fb/BDNF without immune suppression, but similar to that of immune suppressed animals receiving unencapsulated Fb/BDNF. Immunocytochemical examination revealed neurofilament (RT-97), 5-HT, CGRP and GAP-43 containing axons surrounding encapsulated Fb/BDNF within the injury site, indicating axonal growth. BDA labeling however showed no evidence of regeneration of rubrospinal axons in recipients of encapsulated Fb/BDNF, presumably because the amounts of BDNF available from the encapsulated grafts are substantially less than those provided by the much larger numbers of Fb/BDNF grafted in a gelfoam matrix in the presence of immune suppression. These results suggest that plasticity elicited by the BDNF released from the encapsulated cells contributed to reorganization that led to behavioral recovery in these animals and that the behavioral recovery could proceed in the absence of rubrospinal tract regeneration. Alginate encapsulation is therefore a feasible strategy for delivery of therapeutic products produced by non-autologous engineered fibroblasts and provides an environment suitable for recovery of lost function in the injured spinal cord.