Fetal Spinal Cord Transplants Research Articles

The Therapeutic Effectiveness of Delayed Fetal Spinal Cord Tissue Transplantation on Respiratory Function Following Mid-Cervical Spinal Cord Injury.

Respiratory impairment due to damage of the spinal respiratory motoneurons and interruption of the descending drives from brainstem premotor neurons to spinal respiratory motoneurons is the leading cause of morbidity and mortality following cervical spinal cord injury. The present study was designed to evaluate the therapeutic effectiveness of delayed transplantation of fetal spinal cord (FSC) tissue on respiratory function in rats with mid-cervical spinal cord injury. Embryonic day-14 rat FSC tissue was transplanted into a C4 spinal cord hemilesion cavity in adult male rats at 1week postinjury. The histological results showed that FSC-derived grafts can survive, fill the lesion cavity, and differentiate into neurons and astrocytes at 8weeks post-transplantation. Some FSC-derived graft neurons exhibited specific neurochemical markers of neurotransmitter (e.g., serotonin, noradrenalin, or acetylcholine). Moreover, a robust expression of glutamatergic and γ-aminobutyric acid-ergic fibers was observed within FSC-derived grafts. Retrograde tracing results indicated that there was a connection between FSC-derived grafts and host phrenic nucleus. Neurophysiological recording of the phrenic nerve demonstrated that phrenic burst amplitude ipsilateral to the lesion was significantly greater in injured animals that received FSC transplantation than in those that received buffer transplantation under high respiratory drives. These results suggest that delayed FSC transplantation may have the potential to repair the injured spinal cord and promote respiratory functional recovery after mid-cervical spinal cord injury.

N-methyl-D-aspartate receptor antagonist MK-801 prevents apoptosis in rats that have undergone fetal spinal cord transplantation following spinal hemisection.

Spinal cord injury is the main cause of paraplegia, but effective therapies for it are lacking. Embryonic spinal cord transplantation is able to repair spinal cord injury, albeit with a large amount of neuronal apoptosis remaining in the spinal cord. MK-801, an N-methyl-D-aspartate (NMDA) receptor antagonist, is able to reduce cell death by decreasing the concentration of excitatory amino acids and preventing extracellular calcium ion influx. In this study, the effect of MK-801 on the apoptosis of spinal cord neurons in rats that have received a fetal spinal cord (FSC) transplant following spinal hemisection was investigated. Wistar rats were divided into three groups: Spinal cord hemisection injury with a combination of FSC transplantation and MK-801 treatment (group A); spinal cord hemisection injury with FSC transplantation (group B); and spinal cord injury with insertion of a Gelfoam pledget (group C). The rats were sacrificed 1, 3, 7 and 14 days after the surgery. Apoptosis in spinal slices from the injured spinal cord was examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling reaction, and the expression of B-cell lymphoma-2 (Bcl-2) was measured by immunohistochemistry. The positive cells were quantitatively analyzed using a computer image analysis system. The rate of apoptosis and the positive expression of Bcl-2 protein in the spinal cord neurons in the three groups decreased in the following order: C>B>A (P<0.05) and A>B>C (P<0.05), respectively. This indicates that treatment with the NMDA receptor antagonist MK-801 prevents apoptosis in the spinal cord neurons of rats that have undergone FSC transplantation following spinal hemisection.

Transplanting neural progenitors into a complete transection model of spinal cord injury

Neural progenitor cell (NPC) transplantation is a promising therapeutic strategy for spinal cord injury (SCI) because of the potential for cell replacement and restoration of connectivity. Our previous studies have shown that transplants of NPC, composed of neuron- and glia-restricted progenitors derived from the embryonic spinal cord, survived well in partial lesion models and generated graft-derived neurons, which could be used to form a functional relay. We have now examined the properties of a similar NPC transplant using a complete transection model in juvenile and adult rats. We found poor survival of grafted cells despite using a variety of lesion methods, matrices, and delays of transplantation. If, instead of cultured progenitor cells, the transplants were composed of segmental or dissociated segments of fetal spinal cord (FSC) derived from similar-staged embryos, grafted cells survived and integrated well with host tissue in juvenile and adult rats. FSC transplants differentiated into neurons and glial cells, including astrocytes and oligodendrocytes. Graft-derived neurons expressed glutaminergic and GABAergic markers. Grafted cells also migrated and extended processes into host tissue. Analysis of axon growth from the host spinal cord showed serotonin-positive fibers and biotinylated dextran amine-traced propriospinal axons growing into the transplants. These results suggest that in treating severe SCI, such as complete transection, NPC grafting faces major challenges related to cell survival and formation of a functional relay. Lessons learned from the efficacy of FSC transplants could be used to develop a therapeutic strategy based on neural progenitor cells for severe SCI.

Intraspinal transplantation and modulation of donor neuron electrophysiological activity

Rat fetal spinal cord (FSC) tissue, naturally enriched with interneuronal progenitors, was introduced into high cervical, hemi-resection (Hx) lesions. Electrophysiological analyses were conducted to determine if such grafts exhibit physiologically-patterned neuronal activity and if stimuli which increase respiratory motor output also alter donor neuron bursting. Three months following transplantation, the bursting activity of FSC neurons and the contralateral phrenic nerve were recorded in anesthetized rats during a normoxic baseline period and brief respiratory challenges. Spontaneous neuronal activity was detected in 80% of the FSC transplants, and autocorrelation of action potential spikes revealed distinct correlogram peaks in 87% of neurons. At baseline, the average discharge frequency of graft neurons was 13.0±1.7Hz, and discharge frequency increased during a hypoxic respiratory challenge (p<0.001). Parallel studies in unanesthetized rats showed that FSC tissue recipients had larger inspiratory tidal volumes during brief hypoxic exposures (p<0.05 vs. C2Hx rats). Anatomical connectivity was explored in additional graft recipients by injecting a transsynaptic retrograde viral tracer (pseudorabies virus, PRV) directly into matured transplants. Neuronal labeling occurred throughout graft tissues and also in the host spinal cord and brainstem nuclei, including those associated with respiratory control. These results underscore the neuroplastic potential of host–graft interactions and training approaches to enhance functional integration within targeted spinal circuitry.

Delayed Intervention with Transplants and Neurotrophic Factors Supports Recovery of Forelimb Function after Cervical Spinal Cord Injury in Adult Rats

The adult central nervous system is capable of considerable anatomical reorganization and functional recovery after injury. Functional outcomes, however, vary greatly, depending upon size and location of injury, type and timing of intervention, and type of recovery and plasticity evaluated. The present study was undertaken to assess the recovery of skilled and unskilled forelimb function in adult rats after a C5/C6 spinal cord over-hemisection and delayed intervention with fetal spinal cord transplants and neurotrophins. Recovery of forelimb function was evaluated during both target reaching (a skilled behavior) and vertical exploration (an unskilled behavior). Anatomical tracing and immunohistochemistry were used to assess the growth of descending raphespinal, corticospinal, and rubrospinal fibers at the injury site, tracts that normally confer forelimb function. Delayed intervention with transplants and either brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3) restored skilled left forelimb reaching to pre-injury levels. Animals showed recovery of normal reaching movements rather than compensation with abnormal movements. Transplants and NT-3 also improved right forelimb use during an unskilled vertical exploration, but not skilled right reaching. Intervention with fetal transplant tissue supported the growth of descending serotonergic, corticospinal, and rubrospinal fibers into the transplant at the lesion site. The addition of neurotrophins, however, did not significantly increase axonal growth at the lesion site. These studies suggest that the recovery of skilled and unskilled forelimb use is possible after a large cervical spinal cord injury following delayed intervention with fetal spinal cord and neurotrophins. Plasticity of both spared and axotomized descending pathways likely contributes to the functional recovery observed.

Differences in cytokine gene expression profile between acute and secondary injury in adult rat spinal cord

It is likely that the environment within the injured spinal cord influences the capacity of fetal spinal cord transplants to support axonal growth. We have recently demonstrated that fetal spinal cord transplants and neurotrophin administration support axonal regeneration after spinal cord transection, and that the distance and amount of axonal growth is greater when these treatments are delayed by several weeks after injury (Coumans et al., 2001). In this study, we sought to determine whether differences in inflammatory mediators exist between the acutely injured spinal cord and the spinal cord after a second injury and re-section, which could provide a more favorable environment for the axonal re-growth. The results of this study show a more rapid induction of transforming growth factor (TGF) β1 mRNA expression in the re-injured spinal cord than the acutely injured spinal cord and an attenuation of proinflammatory cytokine mRNA expression. Furthermore, there was a rapid recruitment of activated microglia/macrophages in the degenerating white matter rostral and caudal to the injury but fewer within the lesion site itself. These findings suggest that the augmentation of TGFβ-1 gene expression and the attenuation of pro-inflammatory cytokine gene expression combined with an altered distribution of activated microglia/macrophages in the re-injured spinal cord might create a more favorable milieu for transplants and axonal regrowth as compared to the acutely injured spinal cord.

Axonal Regeneration and Functional Recovery after Complete Spinal Cord Transection in Rats by Delayed Treatment with Transplants and Neurotrophins

Little axonal regeneration occurs after spinal cord injury in adult mammals. Regrowth of mature CNS axons can be induced, however, by altering the intrinsic capacity of the neurons for growth or by providing a permissive environment at the injury site. Fetal spinal cord transplants and neurotrophins were used to influence axonal regeneration in the adult rat after complete spinal cord transection at a midthoracic level. Transplants were placed into the lesion cavity either immediately after transection (acute injury) or after a 2-4 week delay (delayed or chronic transplants), and either vehicle or neurotrophic factors were administered exogenously via an implanted minipump. Host axons grew into the transplant in all groups. Surprisingly, regeneration from supraspinal pathways and recovery of motor function were dramatically increased when transplants and neurotrophins were delayed until 2-4 weeks after transection rather than applied acutely. Axonal growth back into the spinal cord below the lesion and transplants was seen only in the presence of neurotrophic factors. Furthermore, the restoration of anatomical connections across the injury site was associated with recovery of function with animals exhibiting plantar foot placement and weight-supported stepping. These findings suggest that the opportunity for intervention after spinal cord injury may be greater than originally envisioned and that CNS neurons with long-standing injuries can reinitiate growth, leading to improvement in motor function.

Reinnervation of the Biceps Brachii Muscle Following Cotransplantation of Fetal Spinal Cord and Autologous Peripheral Nerve into the Injured Cervical Spinal Cord of the Adult Rat

In order to compensate the loss of motoneurons resulting from severe spinal cord injury and to reestablish peripheral motor connectivity, solid pieces of fetal spinal cord, taken from embryonic day 14 rat embryos, were transplanted into unilateral aspiration lesions of the cervical spinal cord of adult rats. Concomitantly, one end of a 3.5-cm autologous peripheral nerve graft was put in close contact with the embryonic graft; the other end was sutured to the distal stump of the musculocutaneous nerve which innervate the biceps brachii muscle. The animals were examined 3 and 6 months after surgery. Following intramuscular injection of horseradish peroxidase, retrograde axonal labeling studies indicated that both transplanted and host spinal neurons were able to extend axons all the way through the peripheral nerve graft and nerve stump, up to the reconnected muscles. The labeled cells in the transplant were generally observed close to the intraspinal tip of the peripheral nerve graft. Retrograde axonal tracing, as well as electrophysiological and histological data, demonstrated the sensory and motor reinnervation of the reconnected muscles. This muscular reinnervation was able to reverse the atrophic changes observed in the denervated muscle. In control experiments, the extraspinal end of the peripheral nerve graft was ligatured in order to compare the differentiation of the transplanted neurons and the survival of their growing axons with or without their muscular targets. Six months after both types of surgery, large-size grafted neurons, identified as motoneurons by immunocytochemistry for peripherine and calcitonin gene-related peptide, were only observed in fetal spinal cord transplants which were connected to denervated muscles, thus demonstrating the trophic influence of the muscle target on the survival and differentiation of the transplanted neurons and on the maintenance of the axons they had grown into the peripheral nerve graft.

Mechanisms leading to restoration of muscle size with exercise and transplantation after spinal cord injury.

We have shown that cycling exercise combined with fetal spinal cord transplantation restored muscle mass reduced as a result of complete transection of the spinal cord. In this study, mechanisms whereby this combined intervention increased the size of atrophied soleus and plantaris muscles were investigated. Rats were divided into five groups (n = 4, per group): control, nontransected; spinal cord transected at T10 for 8 wk (Tx); spinal cord transected for 8 wk and exercised for the last 4 wk (TxEx); spinal cord transected for 8 wk with transplantation of fetal spinal cord tissue into the lesion site 4 wk prior to death (TxTp); and spinal cord transected for 8 wk, exercised for the last 4 wk combined with transplantation 4 wk prior to death (TxExTp). Tx soleus and plantaris muscles were decreased in size compared with control. Exercise and transplantation alone did not restore muscle size in soleus, but exercise alone minimized atrophy in plantaris. However, the combination of exercise and transplantation resulted in a significant increase in muscle size in soleus and plantaris compared with transection alone. Furthermore, myofiber nuclear number of soleus was decreased by 40% in Tx and was not affected in TxEx or TxTp but was restored in TxExTp. A strong correlation (r = 0.85) between myofiber cross-sectional area and myofiber nuclear number was observed in soleus, but not in plantaris muscle, in which myonuclear number did not change with any of the experimental manipulations. 5'-Bromo-2'-deoxyuridine-positive nuclei inside the myofiber membrane were observed in TxExTp soleus muscles, indicating that satellite cells had divided and subsequently fused into myofibers, contributing to the increase in myonuclear number. The increase in satellite cell activity did not appear to be controlled by the insulin-like growth factors (IGF), as IGF-I and IGF-II mRNA abundance was decreased in Tx soleus and plantaris, and was not restored with the interventions. These results indicate that, following a relatively long postinjury interval, exercise and transplantation combined restore muscle size. Satellite cell fusion and restoration of myofiber nuclear number contributed to increased muscle size in the soleus, but not in plantaris, suggesting that cellular mechanisms regulating muscle size differ between muscles with different fiber type composition.

Cycling exercise and fetal spinal cord transplantation act synergistically on atrophied muscle following chronic spinal cord injury in rats.

The potential of two interventions, alone or in combination, to restore chronic spinal cord transection-induced changes in skeletal muscles of adult Sprague-Dawley rats was studied. Hind limb skeletal muscles were examined in the following groups of animals: rats with a complete spinal cord transection (Tx) for 8 weeks; Tx with a 4-week delay before initiation of a 4-week motor-assisted cycling exercise (Ex) program; Tx with a 4-week delay before transplantation (Tp) of fetal spinal cord tissue into the lesion cavity; Tx with a 4-week delay before Tp and Ex; and uninjured control animals. Muscle mass, muscle to body mass ratios, and mean myofiber cross-sectional areas were significantly reduced 8 weeks after transection. Whereas transplantation of fetal spinal cord tissue did not reverse this atrophy and exercise alone had only a modest effect in restoring lost muscle mass, the combination of exercise and transplantation significantly increased muscle mass, muscle to body mass ratios, and mean myofiber cross-sectional areas in both soleus and plantaris muscles. Spinal cord injury (SCI) also caused changes in myosin heavy chain (MyHC) expression toward faster isoforms in both soleus and plantaris and increased soleus myofiber succinate dehydrogenase (SDH) activity. Combined exercise and transplantation led to a change in the expression of the fastest MyHC isoform in soleus but had no effect in the plantaris. Exercise alone and in combination with transplantation reduced SDH activity to control levels in the soleus. These results suggest a synergistic action of exercise and transplantation of fetal spinal cord tissue on skeletal muscle properties following SCI, even after an extended post-injury period before intervention.

Direct Agonists for Serotonin Receptors Enhance Locomotor Function in Rats that Received Neural Transplants after Neonatal Spinal Transection

We analyzed whether acute treatment with serotonergic agonists would improve motor function in rats with transected spinal cords (spinal rats) and in rats that received transplants of fetal spinal cord into the transection site (transplant rats). Neonates received midthoracic spinal transections within 48 hr of birth; transplant rats received fetal (embryonic day 14) spinal cord grafts at the time of transection. At 3 weeks, rats began 1-2 months of training in treadmill locomotion. Rats in the transplant group developed better weight-supported stepping than spinal rats. Systemic administration of two directly acting agonists for serotonergic 5-HT(2) receptor subtypes, quipazine and (+/-)-1-[2, 5]-dimethoxy-4-iodophenyl-2-aminopropane), further increased weight-supported stepping in transplant rats. The improvement was dose-dependent and greatest in rats with poor to moderate baseline weight support. In contrast, indirectly acting serotonergic agonists, which block reuptake of 5-HT (sertraline) or release 5-HT and block its reuptake (D-fenfluramine), failed to enhance motor function. Neither direct nor indirect agonists significantly improved locomotion in spinal rats as a group, despite equivalent upregulation of 5-HT(2) receptors in the lumbar ventral horn of lesioned rats with and without transplants. The distribution of immunoreactive serotonergic fibers within and caudal to the transplant did not appear to correspond to restoration of motor function. Our results confirm our previous demonstration that transplants improve motor performance in spinal rats. Additional stimulation with agonists at subtypes of 5-HT receptors produces a beneficial interaction with transplants that further improves motor competence.

Fetal Spinal Cord Transplants and Exogenous Neurotrophic Support Enhance c-Jun Expression in Mature Axotomized Neurons after Spinal Cord Injury

The responses of the central (CNS) and peripheral (PNS) nervous system to axotomy differ in a number of ways; these differences can be observed in both the cell body responses to injury and in the extent of regeneration that occurs in each system. The cell body responses to injury in the PNS involves the upregulation of genes that are not upregulated following comparable injuries to CNS neurons. The expression of particular genes following injury may be essential for regeneration to occur. In the present study, we have evaluated the hypothesis that expression of the inducible transcription factor c-Jun is associated with regrowth of axotomized CNS neurons. In these experiments, we compared c-Jun expression in axotomized brainstem neurons after thoracic spinal cord hemisection alone (a condition in which no regrowth occurs) and in groups of animals where hemisections were combined with treatments such as transplants of fetal spinal cord tissue and/or application of neurotrophic factors to the lesion site. The latter conditions enhance the capacity of the CNS for regrowth. We have demonstrated that hemisections alone do not upregulate expression of c-Jun, indicating that this particular cell body response is not a direct result of axotomy. However, c-Jun expression is upregulated in animals that received application of transplants and neurotrophins. Because these interventions also promote sprouting and regrowth of CNS axons after spinal cord lesions, we suggest that transplants and exogenous neurotrophic factor application activate a cell body response consistent with a role for c-Jun in axonal growth.

Effects of osteogenic protein-1 (OP-1) treatment on fetal spinal cord transplants to the anterior chamber of the eye.

Spinal cord injury represents a serious medical problem, and leads to chronic conditions that cannot be reversed at present. It has been suggested that trophic factor treatment may reduce the extent of damage and restore damaged neurons following the injury. We have tested the effects of osteogenic protein-1 (OP-1, also known as BMP-7), a member of the transforming growth factor-beta superfamily of growth factors, on developing spinal cord motor neurons in an intraocular transplantation model. Embryonic day 13 or 18 spinal cord tissue was dissected, incubated with OP-1 or vehicle, and injected into the anterior chamber of the eye of adult rats. Injections of additional doses of OP-1 were performed weekly, and the overall growth of the grafted tissue was assessed noninvasively. Four to 6 weeks postgrafting, animals were sacrificed and the tissue was processed for immunohistochemistry using antibodies directed against choline acetyltransferase, neurofilament, and the dendritic marker MAP-II. We found that OP-1 treatment stimulated overall growth of spinal cord tissue when dissected from embryonic day 18, but not from embryonic day 13. OP-1 treatment increased cell size and extent of cholinergic markers in motor neurons from both embryonic stages. The neurons also appeared to have a more extensive dendritic network in OP-1-treated grafts compared to controls. These findings indicate that OP-1 treatment may reduce the extent of axotomy-induced cell death of motor neurons, at least in the developing spinal cord.

Fetal Spinal Cord Transplants Support the Development of Target Reaching and Coordinated Postural Adjustments after Neonatal Cervical Spinal Cord Injury

Neonatal midthoracic spinal cord injury disrupts the development of postural reflexes and hindlimb locomotion. The recovery of rhythmical alternating movements, such as locomotion, is enhanced in injured animals receiving fetal spinal cord transplants. Neonatal cervical spinal cord injury disrupts not only locomotion but also skilled forelimb movement. The aims of this study were to determine the consequences of cervical spinal cord injury on forelimb motor function and to determine whether transplants of fetal spinal cord support normal development of skilled forelimb use after this injury. Three-day-old rats received a cervical spinal cord lesion at C3, with or without a transplant of fetal cervical spinal cord (embryonic day 14); unoperated pups served as controls. Animals were examined daily during the first month of life using a behavioral protocol that assessed reflexes, postural reactions, and forelimb motor skills. They also were trained and tested as adults to assess performance in goal-directed reaching tasks. The onset of postural reflexes was delayed in the lesion-only group, and goal-directed reaching and associated postural adjustments failed to develop. The transplant group developed reflex responses and skilled forelimb activity that resembled normal movement patterns. Transplant animals developed both target reaching and accompanying postural adjustments. Target reaching requires integration of segmental, intersegmental, and supraspinal input to propriospinal and motor neurons over many spinal cord levels. Transplants may support the reestablishment of input onto these neurons, permitting the development of skilled forelimb activity after neonatal cervical spinal cord injury. The neuroanatomical reorganization of descending and propriospinal input was examined in the companion paper (Diener and Bregman, 1998).

Fetal Spinal Cord Transplants Support Growth of Supraspinal and Segmental Projections after Cervical Spinal Cord Hemisection in the Neonatal Rat

Cervical spinal cord injury at birth permanently disrupts forelimb function in goal-directed reaching. Transplants of fetal spinal cord tissue permit the development of skilled forelimb use and associated postural adjustments (, companion article). The aim of this study was to determine whether transplants of fetal spinal cord tissue support the remodeling of supraspinal and segmental pathways that may underlie recovery of postural reflexes and forelimb movements. Although brainstem-spinal and segmental projections to the cervical spinal cord are present at birth, skilled forelimb reaching has not yet developed. Three-day-old rats received a cervical spinal cord overhemisection with or without transplantation of fetal spinal cord tissue (embryonic day 14); unoperated pups served as normal controls. Neuroanatomical tracing techniques were used to examine the organization of CNS pathways that may influence target-directed reaching. In animals with hemisections only, corticospinal, brainstem-spinal, and dorsal root projections within the spinal cord were decreased in number and extent. In contrast, animals receiving hemisections plus transplants exhibited growth of these projections throughout the transplant and over long distances within the host spinal cord caudal to the transplant. Raphespinal axons were apposed to numerous propriospinal neurons in control and transplant animals; these associations were greatly reduced in the lesion-only animals. These observations suggest that after neonatal cervical spinal cord injury, embryonic transplants support axonal growth of CNS pathways and specifically supraspinal input to propriospinal neurons. We suggest that after neonatal spinal injury in the rat, the transplant-mediated reestablishment of supraspinal input to spinal circuitry is the mechanism underlying the development of target-directed reaching and associated postural adjustments.

Axotomized rubrospinal neurons rescued by fetal spinal cord transplants maintain axon collaterals to rostral CNS targets.

Neurons that maintain extensive axon collaterals proximal to the site of axotomy may be better able to survive injury. Early lesions of the rubrospinal tract lead to retrograde cell death of the majority of axotomized immature neurons. Transplants of fetal spinal cord tissue rescue axotomized rubrospinal neurons and promote their axonal regeneration. Rubrospinal neurons develop many of their axon collaterals postnatally. The present study tests the hypothesis that the axotomized rubrospinal neurons that are rescued by transplants and regenerate their axons are those neurons that have established axon collaterals to targets rostral to the lesion. Neonatal rats received a transplant of fetal spinal cord tissue placed into a midthoracic spinal cord hemisection. One month after transplantation, the retrogradely transported fluorescent tracers fast blue (FB) and diamidino yellow (DY) were used to identify rubrospinal neurons with collaterals to particular targets. FB was injected either into the interpositus nucleus of the cerebellum or into the gray matter of the cervical enlargement to identify collaterals to these targets, and DY was injected into the spinal cord approximately 5 mm caudal to the transplant and lesion site to label retrogradely the neurons that regenerated their axons. Double labeling was observed in the axotomized neurons of the red nucleus after tracer injections into the cervical spinal cord but not after injections into the cerebellum. This labeling pattern indicates that axotomized rubrospinal neurons that are rescued and regenerate axons caudal to the transplant maintain axon collaterals at cervical spinal cord levels. Cerebellar collaterals do not appear to play a role in the survival and regrowth of axotomized rubrospinal neurons.

Reinnervation of denervated muscle by transplantation of fetal spinal cord to transected sciatic nerve in the rat

When motor neurons in the spinal cord are destroyed, regeneration of motor axons and muscle reinnervation cannot be expected. We attempted reinnervation of the denervated muscle, i.e. motor unit reconstruction, using transplantation of the fetal spinal cord to the peripheral nerve. The sciatic nerve of an adult rat was resected for 20 mm, and a cavity was prepared using an autologous femoral vein at the distal stump of the nerve. The fetal spinal cord was then transplanted into the venous cavity. After 3–6 months, no voluntary muscle contraction was observed due to the absence of communication with the central nervous system. However, reinnervation of the muscles via the sciatic nerve by the transplanted spinal neurons was demonstrated electrophysiologically and histochemically. This suggested that a motor unit can be reconstructed by fetal spinal cord transplantation even if the original motor neurons in the spinal cord are not available.

Fetal Spinal Cord Transplants Rescue Some Axotomized Rubrospinal Neurons from Retrograde Cell Death in Adult Rats

Intraspinal transplants of fetal spinal cord may contribute to recovery after spinal cord injury by keeping axotomized neurons alive. In this study we examined whether transplants rescued axotomized red nucleus (RN) neurons from retrograde cell death in adult rats. RN neurons were labeled by retrograde transport of Fluorogold (FG); 1 week later right-sided RN neurons were axotomized by left-sided hemisection at C3–4 vertebral level, and Embryonic Day 14 spinal cord or gelfoam was introduced into the cavity. Additional rats received hemisection and a transplant of fetal spinal cord or gelfoam without FG injection. At 2 and 4 months, the number of neurons in the magnocellular portion of the RN contralateral to the hemisection decreased 35–40% in rats that received gelfoam; mean soma area of surviving neurons decreased 40%. RN cell loss was reduced to 20% in rats that received fetal spinal cord transplants, but the decrease in mean soma area was unchanged. Transplants therefore rescued about half of the axotomized RN neurons that otherwise would have died but did not prevent perikaryal atrophy. Anterograde transport of WGA–HRP injected into RN 2 months after transplantation showed that rubrospinal axons reached the site of injury but rarely entered transplants; FG injections caudal to transplants showed that axons of transplant neurons extended at least two segments into host spinal cord. Fetal spinal cord transplants may therefore contribute to locomotor recovery in adults with spinal cord injuries both by preventing retrograde cell death and by establishing novel circuits across the site of injury.

1132 Early development of fetal spinal cord transplants in adult rat dorsal column

1132 Early development of fetal spinal cord transplants in adult rat dorsal column

Early development of fetal spinal cord transplants in adult rat dorsal column

In order to understand the mechanism of the muscular regenerative process which occurs in mdx mice, the expression of neural cell adhesion molecule (NCAM) isoforms and their polysialylated (PSA) derivatives were studied during the postnatal development of normal and mdx mice in relation to the stage of the regeneration of muscle fibres, in the quadriceps. NCAM expression was also examined during in vitro differentiation of satellite cells isolated from both mdx and normal muscles. The immunohistochemical and biochemical analyses were done using antibodies for the different isoforms. The data presented here suggest that before the onset of necrosis and regeneration, the expression of NCAM isoforms in the quadriceps of mdx mice was similar to normal mice. Later, NCAM and PSA-NCAM expression in mdx mice increased and was related to the muscular regenerative process, and the overall level of NCAM expression can be considered as a good index of muscle regeneration. Young regenerative fibres expressed NCAM and PSA-NCAM, while mature regenerative fibres, in which myonuclei remained centrally located, did not express either NCAM or the PSA isoforms. Therefore, in terms of NCAM expression, the fibres in mdx muscle with centrally located nuclei appeared similar to mature fibres found in normal adult muscle. A major form of 145 kDa and a minor form of 115 kDa were detected in mdx regenerative muscle. The 145 kDa NCAM was sialylated, as demonstrated by its sensibility to exoneuraminidase which generates a desialoform of 125 kDa, but not polysialylated since it was not recognized by the anti-MenB antibody, specific for PSA-NCAM. In contrast, the molecular forms of NCAM migrating as a broad band from 160 kDa to 220 kDa were identified as PSA-NCAM. The comparison of in vitro differentiation of normal and mdx satellite cells showed that the expression of NCAM isoforms by mdx cells was similar to that expressed by normal cells. Both our in vivo and in vitro data concerning NCAM expression show that regeneration in mdx mice does not differ from that observed in other necrotic diseases. In other words, NCAM is unlikely to be a dystrophin-associated molecule since lack of dystrophin does not affect its expression.