Intraspinal transplantation of embryonic spinal cord tissue in neonatal and adult rats

Fetal rat spinal cord tissue was obtained on gestational day 14 (E14) and transplanted into 2-4-mm-long intraspinal cavities produced by partial spinal cord lesions in adult and neonatal rats. At regular post-transplantation intervals, light and electron microscopy, autoradiographic demonstration of tritiated thymidine labelling, and immunocytochemical localization of glial fibrillary acidic protein (GFAP) were used to identify surviving donor tissues and to study their differentiation and extent of fusion with recipient spinal cords. In some experiments, wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was also employed to examine whether neurons within the grafts projected axons into the host spinal cord and vice versa. Lastly, immunocytochemistry was used to determine whether any supraspinal serotoninergic (5-HT) axons from the host extended into the transplants. Over 80% of the grafts survived in lesions of both the neonatal and adult rat spinal cord for periods of 1-16 months (duration of experiment), and considerable maturation of donor tissue was evidenced, which even included the appearance of some topographical features of the normal spinal cord. Many of the transplants extended the entire length of the lesion, and were often closely apposed to the injured surfaces of the recipient spinal cords without an intervening dense glial scar. At post-transplantation intervals of 2-4 months, injection of WGA-HRP into the host spinal cord (5 mm from the transplant in adult animals or as much as 20 mm in neonatal recipients) demonstrated retrogradely labelled neurons and anterogradely labelled axons in the grafts. Likewise, injecting WGA-HRP into transplants in adult recipients resulted in labelling of neurons in adjacent segments of the host spinal cord; some labelled axons, derived from donor neurons, were also present in neighboring spinal gray matter. Finally, immunocytochemistry revealed 5-HT-like immunoreactive fibers in transplants that had been prelabelled with tritiated thymidine. These observations demonstrate the potential of embryonic spinal cord transplants to replace damaged intraspinal neuronal populations and to restore some degree of anatomical continuity between the isolated rostral and caudal stumps of the injured mammalian spinal cord.     

Altered transcription of glutamatergic and glycinergic receptors in spinal cord dorsal horn following spinal cord transection is minimally affected by passive exercise of the hindlimbs

Gene expression is altered following a spinal transection (STx) in both motor and sensory systems. Exercise has been shown to influence gene expression in both systems post‐STx. Gene expression alterations have also been shown in the dorsal root ganglia and nociceptive laminae of the spinal cord following either an incomplete spinal cord injury (SCI) or a contusive SCI. However, the effect of STx and exercise on gene expression in spinal cord laminae I‐III has not fully been examined. Therefore, the purpose of this study was to determine whether gene expression in laminae I‐III is altered following STx and determine whether superimposed passive exercise of the hindlimbs would influence gene expression post‐STx in laminae I‐III. Laser capture microdissection was used to selectively harvest laminae I‐III of lumbar spinal cord sections, and quantitative RT‐PCR was used to examine relative expression of 23 selected genes in samples collected from control, STx and STx plus exercise rats. We demonstrate that post‐STx, gene expression for metabotropic glutamate receptors 1, 5 and 8 were up‐regulated, whereas ionotropic glutamatergic receptor (Glur2) and glycinergic subunit GLRA1 expression was down‐regulated. Daily exercise attenuated the down‐regulation of Glur2 gene expression in laminae I‐III. Our results demonstrate that in a STx model, gene expression is altered in laminae I‐III and that although passive exercise influences gene expression in both the motor and sensory systems, it had a minimal effect on gene expression in laminae I‐III post‐STx.     

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. © 2014 Wiley Periodicals, Inc.     

用免疫细胞化学方法观察胚胎脊髓组织移植后的细胞构筑特征

本文应用生长抑素、5-羟色胺、P物质,胶质酸性蛋白的抗体,通过免疫细胞化学反应,对胚胎脊髓组织移植后的细胞构筑特征进行了观察。结果发现,移植后2—6个月的胚胎脊髓组织内分布有与上述抗体相应的免疫阳性神经元及神经纤维。在移植组织与宿主脊髓融合差的情况下,移植界面可见胶质酸性蛋白免疫反应明显增强。研究表明,免疫细胞化学方法可反映移植组织的生长分化程度,并可显示移植部位的细胞构筑特征。     

Transplantation of fetal spinal cord tissue into the chronically injured adult rat spinal cord

Transplants of fetal central nervous system (CNS) tissue into the acutely injured rat spinal cord have been demonstrated to differentiate and partially integrate with the adjacent host neuropil. In the present study, we examined the potential for applying a transplantation approach to chronic spinal cord lesions. In particular, we were interested in learning whether host-graft fusion would be adversely affected by an advanced histopathology characterized in part by glial scar formation. Hemisection cavities were prepared at lumbar levels of the adult rat spinal cord 2-7 weeks prior to the transplantation of spinal cord tissue obtained from 14-day rat fetuses. Graft survival, differentiation, and integration with the host spinal cord were subsequently evaluated by light microscopic techniques at post-transplantation intervals of 1-6 months. Immunocytochemistry was also employed to examine the extent of astrocytic scar formation at the host-graft interface and serotoninergic innervation of the grafts. In some other cases, anterograde and retrograde transport of wheat germ agglutinin-conjugated horseradish peroxidase was used to determine whether axonal projections were formed between the host spinal cords and grafts. By 2 weeks after injury the initial lesion cavities were surrounded by a continuous astrocytic scar which remained intact for at least 7 weeks after injury in nongrafted control animals. In other animals, transplantation into these advanced lesions resulted in well-differentiated grafts with a 90% long-term survival rate. Although dense gliosis was still present along the lesion surfaces of the recipient spinal cord, foci of confluent host-graft neuropil were observed where interruptions in the scar had occurred. Donor tissue integrated most often with the host spinal cord at interfaces with host gray matter; however, some implants also exhibited sites of fusion with damaged host white matter. Thus, some regions of confluent graft and host neuropil could be routinely identified, despite the presence of a dense glial scar along the walls of the chronic lesion site at the time of transplantation. Anterograde and retrograde tract-tracing results suggested that some axonal projections into these grafts had originated from host neurons located immediately adjacent to the donor-recipient interface. In addition, immunocytochemistry revealed some host serotoninergic axons (presumably of supraspinal origin) traversing nongliotic interfaces. The results of this study raise the possibility that grafted fetal CNS tissue has a capacity for stimulating partial regression of an established glial scar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of exercise and fetal spinal cord implants on the H-reflex in chronically spinalized adult rats

This study investigated the modulation of hindlimb reflex excitability after transection of the spinal cord in adult rats. After transection, the H-reflex exhibited decreased depression at high stimulation frequencies compared to intact animals. Groups of animals which received a spinal cord transection followed by either an exercise regimen for the hindlimbs or a fetal spinal cord implant, showed high stimulation frequency depression similar to controls. This suggests that each of these palliative strategies helped to ‘normalize’ the excitability of specific spinal reflexes.     

Effects of limb exercise after spinal cord injury on motor neuron dendrite structure

An integration center subserving locomotor leg movements resides in the upper lumbar spinal cord. If this neuronal network is preserved after a spinal cord injury, it is possible to stimulate this circuitry to initiate and promote walking. The several effective approaches (electrical stimulation, pharmacologic agents, physical therapy training programs) may all share a common modus operandi of altering synaptic activity within segmental spinal cord. To understand the neural substrate for the use-dependent behavioral improvement, we studied the dendritic architecture of spinal motor neurons. In the first experiment, we compared three groups of animals: animals with an intact spinal cord, animals that had a complete spinal cord transection (SCT) and animals with SCT who engaged in a daily exercise program of actively moving paralyzed hindlimbs through the motions of walking. When compared with animals with an intact spinal cord, the motor neurons from animals with SCT displayed marked atrophy, with loss of dendritic membrane and elimination of branching throughout the visible tree within transverse tissue slices. None of these regressive changes were found in the motor neurons from SCT animals that underwent exercise. In a second experiment, we inquired whether exercise of animals with an intact spinal cord influenced dendrite structure. Increased exercise had very modest effects on dendrite morphology, indicating an upper limit of use-dependent dendrite growth. Our findings suggest that the dendritic tree of motor neurons deprived of descending influences is rapidly pruned, and this finding is not observed in motor neurons after SCT if hindlimbs are exercised. The functional benefits of exercise after SCT injury may be subserved, in part, by stabilizing or remodeling the dendritic tree of motor neurons below the injury site.     

Ultrastructure of fetal spinal cord and cortex implants into adult rat spinal cord

The ultrastructure of cortex and spinal cord from 11-, 12-, and 15-day-old fetuses implanted into the spinal cord of adult rats was studied over 3 months. Under deep Chloropent anesthesia, a 0.5 × 1.0-mm square of fetal cortex or a 1.0-mm segment of fetal spinal cord was implanted subpially between the left dorsal column and the dorsal horn of 70 adult rats. Implants grew toward gray matter, usually interfacing with the host at the isthmus between the horns of the spinal cord. However, implants were observed that occupied the entire left dorsal and ventral horns of the left half of the host spinal cord. Implants had concentric zones: A central zone with basal lamina lined joined channels and subjacent neuroglia; a zone of differentiating implant nervous system; a zone with basal lamina lined implant with overlying pial cells on the dorsal and lateral surfaces of the implant; a zone that interfaced with the host with overlapping neuropil on the lateral and ventral surfaces of the implant. Neuron types were typical for cortical or spinal implants. Implants survived for 3 months and reached stages of neuronal and neuroglial maturation similar to controls. Both fetal spinal cord and brain were successful as implants, had delayed differentiation, and formed complex neuropils. The zone of overlapping interface of the donor and host is an anatomical indication of physiological and functional integration.     

Effect of methylprednisolone on motor function and spinal cord blood flow after spinal cord compression in rats

The effect of methylprednisolone (MP) on neurologic recovery and spinal cord blood flow (SCBF) was investigated up to 4 days after a spinal cord compression injury in rats. The injury was produced at midthoracic level by applying a load of 35 g on a 2.2 x 5.0 mm compression plate for 5 min, which resulted in transient paraparesis. MP was given as a bolus dose of 30 mg/kg i.v. 60 min after injury (n = 20) and controls were given saline (n = 10). The motor performance was assessed daily as the capacity angle on the inclined plane and SCBF was measured by 14C-iodoantipyrine autoradiography on Days 1 or 4. On Day 1 the capacity angle was reduced from about 63 degrees preoperatively to 33 +/- 2 degrees (mean +/- SEM) in the control group and to 50 +/- 1 degrees in the group treated with MP (p less than 0.05). Thereafter there was a slight improvement in both groups, but the difference persisted throughout the observation period. On Day 4 both gray and white matter SCBF was better preserved in MP-treated animals than in the control group (59 +/- 4 versus 49 +/- 3 ml/min/100 g tissue for gray matter and 13.6 +/- 0.6 versus 10.7 +/- 0.8 ml/min/100 g tissue for white matter). Posttraumatic treatment with MP, thus, improved both the neurologic recovery during the first 4 days and SCBF as measured on Day 4. It is speculated that the effect of MP is at least partly exerted on the vascular bed.     

Synaptic development in the injured spinal cord cavity following co-transplantation of fetal spinal cord cells and autologous activated Schwann cells

Transplantation of activated transgenic Schwann cells or a fetal spinal cord cell suspension has been widely used to treat spinal cord injury. However, little is known regarding the effects of co-transplantation. In the present study, autologous Schwann cells in combination with a fetal spinal cord cell suspension were transplanted into adult Wistar rats with spinal cord injury, and newly generated axonal connections were observed ultrastructurally. Transmission electron microscopic observations showed that...     

成年大鼠脊髓全横断损伤后酪氨酸激酶受体C在脊髓和大脑皮层的表达

目的 研究神经营养因子3(neurotrophin-3,NT-3)的受体-酪氨酸激酶受体C(tyrosine kinase receptor C,TrkC)在脊髓损伤(spinal cord injury,SCI)后神经重塑中的作用.方法 研究脊髓全横断损伤大鼠手术后第1、3、7和14 d时,低位胸髓节段和大脑中央前回...     

Neural plasticity after spinal cord injury

Plasticity changes of uninjured nerves can result in a novel neural circuit after spinal cord injury, which can restore sensory and motor functions to different degrees. Although processes of neural plasticity have been studied, the mechanism and treatment to effectively improve neural plasticity changes remain controversial. The present study reviewed studies regarding plasticity of the central nervous system and methods for promoting plasticity to improve repair of injured central nerves. The results showed that synaptic reorganization, axonal sprouting, and neurogenesis are critical factors for neural circuit reconstruction. Directed functional exercise, neurotrophic factor and transplantation of nerve-derived and non-nerve-derived tissues and cells can effectively ameliorate functional disturbances caused by spinal cord injury and improve quality of life for patients.     

Phagocytic microglial phenotype induced by glibenclamide improves functional recovery but worsens hyperalgesia after spinal cord injury in adult rats

Microglial cell plays a crucial role in the development and establishment of chronic neuropathic pain after spinal cord injuries. As neuropathic pain is refractory to many treatments and some drugs only present partial efficacy, it is essential to study new targets and mechanisms to ameliorate pain signs. For this reason we have used glibenclamide (GB), a blocker of K_ATP channels that are over expressed in microglia under activation conditions. GB has already been used to trigger the early scavenger activity of microglia, so we administer it to promote a better removal of dead cells and myelin debris and support the microglia neuroprotective phenotype. Our results indicate that a single dose of GB (1 μg) injected after spinal cord injury is sufficient to promote long‐lasting functional improvements in locomotion and coordination. Nevertheless, the Randall–Selitto test measurements indicate that these improvements are accompanied by enhanced mechanical hyperalgesia. In vitro results indicate that GB may influence microglial phagocytosis and therefore this action may be at the basis of the results obtained in vivo.     

Behavior of the H-reflex in humans following mechanical perturbation or injury to rostral spinal cord

In humans, H-reflexes are suppressed during early spinal shock. In animals, rostral cord injury results in loss of segmental reflexes within seconds. If H-reflexes persist under general anesthesia, can they be used to monitor the integrity of the rostral cord? In part I of this study, we recorded H-reflexes intraoperatively in 25 patients to elucidate general anesthesia effect. In 23 subjects, H-reflexes were consistently elicited, and within ±13% of the normalized group mean amplitude. In part II, we recorded H-reflexes in 31 patients during spinal cord surgery to elucidate H-reflex behavior immediately following rostral spinal cord injury. In 6, abrupt suppression of the H-reflex coincided with cord injury. In 4 of 6, suppression was transient and less than 50% of baseline; none of these patients developed neurological deficits. In 2, suppression exceeded 90% and persisted throughout surgery; both patients developed profound deficits. We conclude that (1) the H-reflex can be consistently elicited under general anesthesia in most patients, (2) rostral cord injury rapidly suppresses the H-reflex, and (3) the degree and duration of H-reflex suppression reflects the severity of the injury. © 1996 John Wiley & Sons, Inc.     

BD PuraMatrix peptide hydrogel as a culture system for human fetal Schwann cells in spinal cord regeneration

BD PuraMatrix peptide hydrogel, a three‐dimensional cell culture model of nanofiber scaffold derived from the self‐assembling peptide RADA16, has been applied to regenerative tissue repair in order to develop novel nanomedicine systems. In this study with PuraMatrix, self‐assembling nanofiber scaffold (SAPNS) and Schwann cells (SCs) were isolated from human fetal sciatic nerves, cultured within SAPNS, and then transplanted into the spinal cord after injury (SCI) in rats. First, the peptide nanofiber scaffold was evaluated via scanning electron microscopy and atomic force microscopy. With phase‐contrast microscopy, the appearance of representative human fetal SCs encapsulated in PuraMatrix on days 3, 5, and 7 in 12‐well plates was revealed. The Schwann cells in PuraMatrix were cultured for 2 days, and the SCs had active proliferative potential. Spinal cord injury was induced by placing a 35‐g weight on the dura of T9–T10 segments for 15 min, followed by in vivo treatment with SAPNS and human fetal SCs (100,000 cells/10 μl/injection) grafted into spinal cord 7 days after SCI. After treatment, the recovery of motor function was assessed periodically using the Basso, Beattie, and Bresnahan scoring system. Eight weeks after grafting, animals were perfusion fixed, and the survival of implanted cells was analyzed with antibody recognizing SCs. Immunohistochemical analysis of grafted lumber segments at 8 weeks after grafting revealed reduced asterogliosis and considerably increased infiltration of endogenous S100⁺ cells into the injury site, suggesting that PuraMatrix may play an important role in the repair observed after SAPNS and human fetal SC transplantation. © 2012 Wiley Periodicals, Inc.     

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.