Expression of neurotrophins in the adult spinal cord in vivo

Potential roles of trophins in the normal and injured spinal cord are largely undefined. However, a number of recent studies suggest that adult spinal cord expresses neurotrophin receptors and responds to the neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3), particularly after injury. The data indicate that trophins may enhance regrowth after damage and may represent a new therapeutic approach to injury. Neurotrophins are reportedly present in the spinal cord, but the cellular localization is unknown. This information is critical to begin delineating mechanisms of actions. To approach this problem, we examined whether spinal cord glia express BDNF and NT3 in vivo and have begun to define cellular distribution. Specific antibodies directed against the neurotrophins were utilized to visualize neurotrophin protein. Initial studies indicated that small cells in the white matter of adult rat spinal cord express BDNF and NT3. Large neurotrophin-positive neurons were also identified in the ventral cord. To identify the neurotrophin-positive cells, co-localization studies were performed utilizing neurotrophin polyclonal antisera together with monoclonal antibodies directed against the astrocyte marker, glial fibrillary acidic protein (GFAP). In the white matter of adult spinal cord, GFAP-positive and GFAP-negative cells expressed BDNF and NT3. Our study suggests that astrocyte and non-astrocyte cells provide trophic support to the adult spinal cord.     

Lidase treatment of spinal cord transected rats

Russian investigators have recently reported clinical recovery of enzyme treated, spinal cord transected rats. Using the exact protocols outlined by Matinian and Andreasian, we repeated a portion of their experiment using a Lidase preparation manufactured in the USSR. Animals were evaluated for return of bladder function, clinical evidence of hind limb motor function, cortical evoked response after sciatic nerve stimulation, and axonal transport of cortically injected tritiated proline by regenerated corticospinal axons. The only difference between treated and control animals was that the mean total body weight of the treated animals increased more than that of controls (p < 0.05). No animal walked, had return of voluntary motor activity, showed cortical evoked response, or had evidence for transport of tritiated proline over regenerated corticospinal axons.     

Regeneration of transected spinal cord in young adult rats using freeze-dried alginate gel.

We have recently reported that freeze-dried alginate gel, which was developed in our laboratory, enhanced peripheral nerve regeneration. The purpose of this study was to examine whether alginate gel is capable of promoting nerve regeneration in the severed spinal cord of adult mammals. Using Wistar rats at 30 days of age (P30), the T9-T10 spinal cord was totally resected and alginate gel was implanted across the gap. Forty-five days after surgery myelinated and unmyelinated axons regenerated throughout the gap with remaining alginate gel. The elongated axons established electrophysiologically functional projections across the gap. In conclusion, freeze-dried alginate gel could be a promising material as an artificial nerve guide for repair of injured central nervous system.     

Enzyme treatment of spinal cord transected rats.

Russian investigators have recently reported clinical recovery of enzyme treated, spinal cord transected rats. Using the exact protocols of Matinian and Andreasian's two most successful treatment regimens, we repeated their experiments using United States produced pure hyaluronidase and trypsin or trypsin and elastase. Animals were evaluated for return of bladder function, clinical evidence of hind limb motor function, cortical evoked response after sciatic nerve stimulation, and axonal transport of cortically injected tritiated proline by regenerated corticospinal axons. None of the treated animals differed from control animals treated only with the enzyme vehicle. No animals walked, had return of voluntary motor activity, showed cortical evoked response, or had evidence for transport of tritiated proline over regenerated corticospinal axons.     

大鼠脊髓全横断损伤模型的建立

目的建立一种实用、可靠的大鼠脊髓全横断损伤模型。方法55只SD大鼠随机分为假手术组及脊髓全横断损伤组,横断组选择T12节段横断大鼠脊髓。分别于术后24h、7d及21d取L2节段制成20μm冰冻切片行HE染色,并记录其后肢运动功能评分(BBB评分)。结果脊髓全横断大鼠双下肢运动功能BBB评分在同一时间点之间相比明显低于假手术组,差别有统计学意义(P<0.01)。而且,脊髓横断以下节段出现大量空泡,神经元数量明显减少,胶质细胞明显增生,以腹角为甚;全横断组腹角神经元计数与假手术组在同一时间点比较有明显差异(P<0.01)。大鼠的30天存活率达72%。结论本方法为一种稳定可靠,操作简单的大鼠脊髓全横断模型制作法。     

Temporal progressive antigen expression in radial glia after contusive spinal cord injury in adult rats

In the development of the CNS, radial glial cells are among the first cells derived from neuroepithelial cells. Recent studies have reported that radial glia possess properties of neural stem cells. We analyzed the antigen expression and distribution of radial glia after spinal cord injury (SCI). Sprague-Dawley rats had a laminectomy at Th11-12, and spinal cord contusion was created by compression with 30 g of force for 10 min. In the injury group, rats were examined at 24 h and 1, 4, and 12 weeks after injury. Frozen sections of 20-microm thickness were prepared from regions 5 and 10 mm rostral and caudal to the injury epicenter. Immunohistochemical staining was performed using antibodies to 3CB2 (a specific marker for radial glia), nestin, and glial fibrillary acidic protein (GFAP). At 1 week after injury, radial glia that bound anti-3CB2 MAb had spread throughout the white matter from below the pial surface. From 4 weeks after injury, 3CB2 expression was also observed in the gray matter around the central canal, and was especially strong around the ependymal cells and around blood vessels. In double-immunohistochemical assays for 3CB2 and GFAP or 3CB2 and nestin, coexpression was observed in subpial structures that extended into the white matter as arborizing processes and around blood vessels in the gray matter. The present study demonstrated the emergence of radial glia after SCI in adult mammals. Radial glia derived from subpial astrocytes most likely play an important role in neural repair and regeneration after SCI.     

Enteric glia promote regeneration of transected dorsal root axons into spinal cord of adult rats

After spinal cord injury axonal regeneration is poor, but may be enhanced by the implantation of olfactory ensheathing glia (OEG). Enteric glia (EG) share many properties of OEG. Transected dorsal root axons normally do not regenerate through the central nervous system myelin into the spinal cord. We tested whether EG, like OEG, could promote regeneration in this paradigm. Three weeks after EG implantation, numerous regenerating dorsal root axons reentered the spinal cord. Ingrowth of dorsal root axons was observed using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate. Primary sensory afferents invaded laminae 1, 2, and 3, grew through laminae 4 and 5, and reached the dorsal gray commissure. No axonal ingrowth was observed in control animals, indicating that transplanted EG enabled regeneration of the injured dorsal root axons into the adult spinal cord. Thus, EG implantation may be beneficial in promoting axonal growth after central nervous system injury.     

Fetal locus coeruleus transplanted into the transected spinal cord of the adult rat

Locus coeruleus tissue from 16-day-old fetal rats has been successfully transplanted into the transected spinal cord of the young adult female rat. During development, the solid tissue implant (approximately equal to 1.2 mm3) always breaks up into smaller cell clusters. Fluorescent axons from the fetal neurons invaded the host tissue, and grew for several millimeters away from the cell-body region. The damaged rostral noradrenergic axons in the ventral horn (projecting from the locus coeruleus) exhibited an intense proliferation in response to a neurotrophic factor produced by the fetal implant. Finally, in 2/8 cases, the axons of the fetal, noradrenergic locus coeruleus neurons remained confined to the fetal nuclear tissue. The results are discussed in the context of recent attempts to reconstruct the damaged mammalian spinal cord.     

Differential effects of neurotrophins on neuronal survival and axonal regeneration after spinal cord injury in adult rats

Spinal cord injury (SCI) induces retrograde cell death in descending pathways, which can be prevented by long-term intrathecal infusion of neurotrophins (Novikova et al. [2000] Eur J Neurosci 12:776-780). The present study investigates whether the same treatment also leads to improved regeneration of the injured tracts. After cervical SCI in adult rats, a peripheral nerve graft was attached to the rostral wall of the lesion cavity. The animals were treated by local application into the cavity of Gelfoam soaked in (1) phosphate buffered saline (untreated controls) or (2) a mixture of the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) (local treatment), or by intrathecal infusion of BDNF + NT-3 for (3) 2 weeks (short-term treatment) or (4) 5-8 weeks (long-term treatment). Despite a very strong survival effect, long-term treatment failed to stimulate ingrowth of descending tracts into the nerve graft. In comparison with untreated controls, the latter treatment also caused 35% reduction in axonal sprouting of descending pathways rostral to the lesion site and 72% reduction in the number of spinal cord neurons extending axons into the nerve graft. Local and short-term treatments neither prevented retrograde cell death nor enhanced regeneration of descending tracts, but induced robust regeneration of spinal cord neurons into the nerve graft. These results indicate that the signal pathways promoting neuronal survival and axonal regeneration, respectively, in descending tracts after SCI respond differently to neurotrophic stimuli and that efficient rescue of axotomized tract neurons is not a sufficient prerequisite for regeneration.     

Evaluation of transected spinal cord regeneration in the rat

Rat spinal cords were subjected to a 200 g/cm force acceleration injury at T10. Ten days later, the cords were totally transected at T10 and the rats separated into two groups: group C (controls) had the spinal cord realigned end-to-end; group X had 3 mm trimmed from proximal and distal cord stumps and a semifluid collagen' matrix (CM) bioimplant was inserted in the gap. The CM polymerized to a firm gel at body temperature within 2 h. All rats were maintained 90 days posttransection (dpt). At 90 dpt, they were examined for local spinal cord blood flows, somatosensory evoked potentials, and a neurological evaluation. After killing, the cords were processed for electron and light microscopy and monoamine histofluorescence. The results indicated that CM can support the growth of central neurites, fibroblasts, and an adequate anastomotic network of blood vessels. Control scar tissue does not promote the presence of never fibers and blood vessels to the extent observed in the CM. Somatosensory evoked potential early waveforms were present in CM-bioimplanted rats but not in controls. No rat regained walking ability at 90 dpt but muscle tone and strength appeared better in CM-implanted than in control rats. We conclude that a CM bridge can provide a well vascularized, relatively nonhostile environment for central neurites and catecholaminergic axons extending from the proximal spinal cord tissue across the CM bridge and into the distal stump.     

慢性压迫性脊髓损伤后巢蛋白mRNA表达的观察

目的观察成年大鼠慢性压迫性脊髓损伤后及减压后早期巢蛋白与巢蛋白mRNA的相关性表达。方法选用健康wistar大鼠50只，体重280～320g，制备慢性压迫性脊髓损伤中度、重度及重度损伤减压后3d、10d模型，取自距胜迫边缘5mm段脊髓组织切片。正常成年大鼠作为对照组。行巢蛋白免疫组织化学染色，巢蛋白mRNA原位杂交实验，计算机罔像分析仪定量分析，观察巢蛋白、巢蛋白mRNA在脊髓中央管、灰质和白质中表达的变化，探讨巢蛋白与巢蛋白mRNA表达的相关性。结果成年大鼠慢性膻迫性脊髓损伤中、重度及重度压迫损伤减压后3d，巢蛋白在自、灰质及脊髓中央管室管膜细胞中均有明显表达（P〈0．05），以重度压迫组最为显著（P〈0．01）。减压后10d组灰质与正常对照组比较，差异无显著性意义（P=0．483）。重度压迫组及减压后3d组，巢蛋白mRNA在脊髓灰质、白质及中央管室管膜细胞中均有显著性表达（P〈0．05），以灰质前角第Ⅸ板层、后角和室管膜下区最为显著。中度压迫组，巢蛋白mRNA在灰质前角第Ⅸ板层及中央管室管膜细胞中有显著性表达（P〈0．05），其余区域仅有微弱表达，而白质内巢蛋白mRNA表达于软脊膜下星形胶质细胞的足突中。减压后10d组灰质内巢蛋白mRNA的表达与正常对照组比较，无显著性差异（P=0．375）。正常对照组中无表达。结论成年大鼠慢性压迫性脊髓损伤及减压后早期存在神经前体细胞的增殖。增殖的神经前体细胞巢蛋白与巢蛋白mRNA表达的相关性具有与胚胎发育期脊髓相似的特征。     

Effects of Governor Vessel electroacupuncture on the systematic expressions of NTFs in spinal cord transected rats

This study evaluated the effects Governor Vessel electroacupuncture (GVEA) on the systematic regulation of neurotrophic factors (NTFs) in the spinal segments caudal (CSS) to the site of transection in rats subjected to spinal cord transection (SCT). Using RT-PCR, we amazingly found the gene expressions of NGF, IGF-1, FGF-2, CNTF, PDGF, TGF-β1, TrkA, TrkB and TrkC were downregulated following GVEA treatment. However, the number of GAP-43 and Synaptophysin profiles in the CSS in the GVEA rats showed a significant increase, compared with non-EA animals, although both the 5-HT and corticospinal fibers have no statistical differences in the CSS. Simultaneously, there was significant recovery in hindlimb locomotor and sensory functions after GVEA treatment. Therefore, these findings challenge the past view that GVEA promotes functional restoration, which is linking to the up-regulation of NTFs in rats subjected to SCT. The present findings may give some novel indication on the mechanism of acupuncture for the treatment of SCI.     

Functional repair of transected spinal cord in embryonic chick

The purpose of this study was to determine the developmental stage of the chick embryo when descending spinal tracts lose the capacity for anatomical and functional repair after complete transection of the thoracic spinal cord. Previous studies have demonstrated that the first reticulospinal projections descend to the lumbar cord by embryonic day (E) 5. A comparison of the distribution and density of retrogradely labelled brainstem-spinal neurons in embryos versus hatchling chicks suggests that the descent of all brainstem-spinal projections is essentially complete to lumbar levels between E10 and El2. Transections and control sham operations were performed on different embryos from E3 through E14 of development. After a recovery period of 5-18 days, the extent of anatomical repair was assessed by injecting a small volume of a retrograde tract-tracing chemical into the upper lumbar spinal cord, caudal to the transection site. The brainstem nuclei were then examined for the number and distribution of retrogradely labelled brainstem-spinal neurons. In comparison to control animals, anatomical recovery appeared to be complete for embryos transected as late as E12, whereas thoracic cord transections conducted on E13-E14 resulted in reduced labelling of most brainstem-spinal nuclei. In addition, a number of E3-E6 transected embryos were allowed to hatch and with some assistance a few E7-E14 transected embryos also hatched. Functional recovery was assessed by behavioral observations and by focal electrical stimulation of brainstem locomotor regions (known to have direct projections to the lumbar spinal cord). Brainstem stimulation experiments were undertaken on transected and control embryos, either in ovo on E18-E20 or after hatching. Leg and wing muscle electromyographic recordings were used to monitor any brainstem evoked motor activity. Voluntary open-field locomotion (hatchling chicks) or brainstem evoked locomotion (embryonic or hatchling) in animals transected on or before E12 was indistinguishable from that observed in control (i.e. sham-operated or unoperated) chicks, indicating that complete functional recovery had occurred. In contrast, chicks transected on or after El3 showed reduced functional recovery. Since a previous study has shown that neurogenesis in chick brainstem-spinal neurons is complete prior to E5, the possible intrinsic neuronal mechanisms underlying the repair of descending supraspinal pathways are: (1) subsequent projections from later developing (undamaged) neurons, or (2) regrowth of previously axotomized projections (regeneration). For the E5-E12 chick embryos examined in this study, significant descending supraspinal fibers are present within the thoracic cord at the time of transection. Even if the transection is made at E12, when descending projections have completed their development to the lumbar cord, there is still a similar number and distribution of brainstem-spinal neurons labelled afterward (when compared to controls). This suggests that regeneration of previously axotomized projections may account for some of the observed anatomical and functional repair of brainstem-spinal pathways.     

Chemokine expression in the white matter spinal cord precursor niche after force‐defined spinal cord contusion injuries in adult rats

Inflammatory cascades induced by spinal cord injuries (SCI) are localized in the white matter, a recognized neural stem‐ and progenitor‐cell (NSPC) niche of the adult spinal cord. Chemokines, as integrators of these processes, might also be important determinants of this NSPC niche. CCL3/CCR1, CCL2/CCR2, and SDF‐1α/CXCR4 were analyzed in the ventrolateral white matter after force defined thoracic SCI: Immunoreactivity (IR) density levels were measured 2 d, 7 d, 14 d, and 42 d on cervical (C 5), thoracic (T 5), and lumbar (L 5) levels. On day post operation (DPO) 42, chemokine inductions were further evaluated by real‐time RT‐PCR and Western blot analyses. Cellular phenotypes were confirmed by double labeling with markers for major cell types and NSPCs (nestin, Musashi‐1, NG2, 3CB2, BLBP). Mitotic profiles were investigated in parallel by BrdU labeling. After lesion, chemokines were induced in the ventrolateral white matter on IR‐, mRNA‐, and protein‐level. IR was generally more pronounced after severe lesions, with soaring increases of CCL2/CCR2 and continuous elevations of CCL3/CCR1. SDF‐1α and CXCR4 IR induction was focused on thoracic levels. Chemokines/‐receptors were co‐expressed with astroglial, oligodendroglial markers, nestin, 3CB2 and BLBP by cells morphologically resembling radial glia on DPO 7 to DPO 42, and NG2 or Musashi‐1 on DPO 2 and 7. In the white matter BrdU positive cells were significantly elevated after lesion compared with sham controls on all investigated time points peaking in the early time course on thoracic level: Here, chemokines were co‐expressed by subsets of BrdU‐labeled cells. These findings suggest an important role of chemokines/‐receptors in the subpial white matter NSPC niche after SCI. © 2010 Wiley‐Liss, Inc.     

大鼠脊髓损伤后Nogo-A表达变化的免疫组织化学研究

目的观察大鼠脊髓损伤后不同时间点Nogo—A蛋白在脊髓的表达变化。方法大鼠分脊髓损伤组、假手术组和正常对照组。损伤动物存活1d、3d、7d后，分别进行Nogo—A抗体的免疫组织化学染色。结果损伤部位的灰质神经元和白质少突胶质细胞均呈明显的Nogo—A免疫阳性反应，随着损伤后存活时间的延长，两者的阳性反应细胞相对染色强度及数量均逐渐下降。结论脊髓损伤后，Nogo—A在灰质神经元有表达，其表达相对强度和表达细胞数量先明显增加，随后逐渐减少，与少突胶质细胞的表达变化相似。     

Temporal changes in the expression of TGF-beta 1 and EGF in the ventral horn of the spinal cord and associated precentral gyrus in adult Rhesus monkeys subjected to cord hemisection

It is well known that some growth factors can not only rescue neurons from death, but also improve motor functions following spinal cord injury. However, their cellular distribution in situ and temporal expressions following spinal cord injury have not been determined, especially in primates. This study investigated the temporal changes in the expression of two growth factors--epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta1) in the injured motoneurons of the spinal cord and the associated precentral gyrus in adult Rhesus monkeys subjected to spinal cord hemisection. Animals were allowed to survive 7, 14, 30 and 90 days post operation (dpo). Functional recovery of the hindlimbs was assessed using Tarlov scale. The immunohistological expressions of EGF and TGF-beta1 in the ventral horn motoneurons decreased sharply at 7 dpo in the cord segments caudal to the lesion site, which was followed by an increase and a peak between 14 and 30 dpo for EGF and at 90 dpo for TGF-beta1. Changes in the expression of EGF in the precentral gyrus were similar to that in the spinal cord. No TGF-beta1 immunoreactive neurons were detected in the precentral gyrus. In the spinal segments rostral to the lesion, the expressions of EGF and TGF-beta1 peaked at 30 dpo. The mRNA of EGF was detected in both spinal motoneurons and the precentral gyrus, while that of TGF-beta1, only in the spinal motoneuons, suggesting that the spinal motoneurons themselves could synthesize both the growth factors. Partial locomotor recovery in hindlimbs was seen, especially after 14 dpo. It was concluded that a possible association existed between the modulation of EGF and TGF-beta1 and the recovery of locomotor function, and their roles differed somewhat in the neuroplasticity observed after spinal cord injury in primates.