NG2 expression in rats with acute T10 spinal cord injury

Rat models of T10 spinal cord injury were established with a clamp method.NG2 expression was detected with immunohistochemical staining and western blot.Ten days after spinal cord injury,the number of NG2-positive cells in the damaged areas and NG2 absorbance were both significantly increased.The findings indicate that acute T10 spinal cord injury in rats can lead to upregulation of NG2 protein expression in damaged areas.     

Peptide accumulations in proximal endbulbs of transected axons

Axons proximal to a transection develop into enlarged, but presumed 'passive' endbulb structures. In previous studies, we observed that proximal stumps of transected sciatic nerves accumulate discrete and striking deposits of calcitonin gene-related peptide (CGRP) that have apparent direct and local actions on nearby microvessels. In this work, we provide evidence that CGRP, in the company of several additional peptides, are deposited through 'arrested' anterograde transport into axon endbulbs that develop after transection. In proximal stump tips of rat sciatic nerves transected 48 h earlier, CGRP accumulation colocalized with a label for neurofilament that was accentuated at axon tips, but was prevented by a concurrent more proximal sciatic section. Similarly, interruption of CGRP deposition eliminated its apparent actions on local microvessels following injury. CGRP accumulation was also observed in sural nerve proximal stump tips, indicating its presence in sensory axons despite the known declines in the sensory neuronal synthesis of CGRP that occur following axotomy. Peptide accumulation was not unique to CGRP, with a similar pattern of anterograde accumulation observed for substance P (SP), neuropeptide Y (NPY) and galanin. Deposited peptides and perhaps other axonal constituents in the milieu of a peripheral nerve injury may be associated with important local physiological actions in the regenerative microenvironment.     

Hyperemia of injured peripheral nerve: sensitivity to CGRP antagonism

Intrinsic mechanisms of vasodilatation may prevent injury-related ischemia in peripheral nerve endoneurium. We examined local perfusion up to 10 days following local crush, partial injury or simple exposure of the rat sciatic nerve. By employing epineurial hCGRP(8-37), a receptor antagonist of CGRP, and serial hydrogen clearance measurements, we estimated the component of post-injury blood flow related to local CGRP action. Injury-related ischemia was not observed at any of the time points studied at or proximal to injury. Instead, endoneurial blood flow (EBF) increased at 24 h proximal to crush or partial injury, and at 48 h within the crush zone when compared to sham operated controls or to a pooled reference range of EBF. Composite blood flow (F) was also elevated at 48 h and 5 days within the crush zone suggesting hyperemia involving the epineurial plexus, perineurial vessels and AV shunts. hCGRP(8-37) constricted vasa nervorum at most time points but its effect on EBF was maximum and exceeded controls within the crush zone at 48 h. The findings indicate that certain types of nerve injury, including focal crush, are associated with hyperemia, not ischemia. CGRP vasodilatation may account for part of this response, implying a local peptidergic afferent fiber response to nerve trunk injury.     

Sprouting of CGRP primary afferents in lumbosacral spinal cord precedes emergence of bladder activity after spinal injury

Spinal cord injury (SCI) severely disrupts bladder function. What mediates bladder dysfunction after SCI is currently unknown. We investigated the role that primary afferent sprouting in lumbosacral cord may play in emergence of bladder activity after complete spinal cord transection. Rats had a bladder cannula chronically implanted. They were then subjected to complete surgical spinal cord transection at T9/T10. Cystometrographic analysis (0.1 ml/min) after injury revealed that bladder activity emerged in the form of nonvoiding contractions in all rats at approximately 5 days post transection. At 10-14 days after transection nonvoiding contractions remained and voiding contractions emerged that had increased maximal pressures (12-41 vs. 24-57 cmH(2)O) but were less efficient (6-15% vs. 79-100%) when compared to control implanted rats. We looked for sprouting 3 days and 8 days post transection, timepoints preceding the emergence of nonvoiding and voiding contractions respectively. Increases in CGRP density and distribution were seen in L6 and S1 spinal cord within lamina groupings of II-IV, V and VI, as well as lamina X at 8 days post transection. This increase remained in most lamina at 21 days post transection. Colocalization with the growth cone marker Gap-43 3 days and 5 days post transection at the level of the lumbosacral preganglionic nucleus verified that CGRP positive afferents were sprouting in L6/S1 spinal cord prior to emergence in bladder activity. These data provide support for the hypothesis that primary afferent sprouting contributes to emergence of bladder activity after spinal cord transection.     

大鼠脊髓损伤后轴突病理变化与胶质瘢痕的关系

目的 观察脊髓损伤(SCI)后轴突变化及其与胶质瘢痕的关系.方法 应用Allen's法建立大鼠脊髓损伤模型,通过行为学评分、免疫荧光及神经束路示踪等观察SCI后轴突的病理变化,及其与胶质瘢痕的关系,并测量胶质瘢痕的厚度.结果 SCI后损伤处的轴突呈断裂、扭曲状,SCI后1 周损伤轴突呈再生趋势,2周时再生明显,与此相应动物运动功能逐渐恢复,4周时胶质瘢痕形成,再生的轴突被瘢痕阻挡.头尾侧胶质瘢痕厚度(107.00±20.12)μm大于两侧边厚度(69.92±24.37)μm.结论 SCI后轴突仍具有再生能力,但被胶质瘢痕所阻挡,瘢痕厚度的测量为将来去除胶质瘢痕提供了实验依据.     

实验性糖尿病周围神经病大鼠脊髓及背根神经节CGRP、VIP表达变化

目的通过观察糖尿病大鼠周围神经形态学的变化以及脊髓、背根神经节中降钙素基因相关肽(CGRP)、血管活性肠肽(VIP)表达变化,以探讨神经肽在糖尿病性神经病中所起的作用。方法Wistar大鼠模型的建立:采用腹腔一次性注射pH4.4枸橼酸钠缓冲液配制的1.25%链脲佐菌素(Streptozotocin STZ)制成糖尿病周围神经病大鼠。用ABC免疫组化方法观察8周和12周大鼠降钙素基因相关肽(CGRP)、血管活性肠肽(VIP)在脊髓及背根神经节的表达。结果糖尿病大鼠8周和12周时背根神经节、12周时脊髓CGRP、VIP表达下降(P<0.05),糖尿病大鼠脊髓8周时CGRP、VIP表达与正常大鼠比无明显差别。结论CGRP和VIP参与了糖尿病周围神经病的发病,糖尿病对大鼠背根神经节CGRP、VIP的表达影响更早。     

Interaction of NG2+ glial progenitors and microglia/macrophages from the injured spinal cord

Spinal cord contusion produces a central lesion surrounded by a peripheral rim of residual white matter. Despite stimulation of NG2⁺ progenitor cell proliferation, the lesion remains devoid of normal glia chronically after spinal cord injury (SCI). To investigate potential cell–cell interactions of the predominant cells in the lesion at 3 days after injury, we used magnetic activated cell sorting to purify NG2⁺ progenitors and OX42⁺ microglia/macrophages from contused rat spinal cord. Purified NG2⁺ cells from the injured cord grew into spherical masses when cultured in defined medium with FGF2 plus GGF2. The purified OX42⁺ cells did not form spheroids and significantly reduced sphere growth by NG2⁺ cells in co‐cultures. Conditioned medium from these OX42⁺ cells, unlike that from normal peritoneal macrophages or astrocytes also inhibited growth of NG2⁺ cells, suggesting inhibition by secreted factors. Expression analysis of freshly purified OX42⁺ cells for a panel of six genes for secreted factors showed expression of several that could contribute to inhibition of NG2⁺ cells. Further, the pattern of expression of four of these, TNFα, TSP1, TIMP1, MMP9, in sequential coronal tissue segments from a 2 cm length of cord centered on the injury epicenter correlated with the expression of Iba1, a marker gene for OX42⁺ cells, strongly suggesting a potential regional influence by activated microglia/macrophages on NG2⁺ cells in vivo after SCI. Thus, the nonreplacement of lost glial cells in the central lesion zone may involve, at least in part, inhibitory factors produced by microglia/macrophages that are concentrated within the lesion. © 2009 Wiley‐Liss, Inc.     

Induction of neuronal phenotypes from NG2+ glial progenitors by inhibiting epidermal growth factor receptor in mouse spinal cord injury

Besides neural stem cells, some glial cells, such as GFAP+ cells, radial glia, and oligodendrocyte progenitor cells can produce neuronal cells. Attractively, NG2+ glial progenitors exhibit lineage plasticity, and they rapidly proliferate and differentiate in response to central nervous system (CNS) injuries. These attributes of NG2+ glial progenitors make them a promising source of neurons. However, the potential of neuronal regeneration from NG2+ glial progenitors in CNS pathologies remains to be investigated. In this study, we showed that antagonizing epidermal growth factor receptor (EGFR) function with EGFR inhibitor caused a significant number of proliferative NG2+ glial progenitors to acquire neuronal phenotypes in contusive spinal cord injury (SCI), which presumably led to an accumulation of newly generated neurons and contributed to the improved neural behavioral performance of animals. In addition, the neuronal differentiation of glial progenitors induced by EGFR inhibitor was further confirmed with two different cell lines either in vitro or through ex vivo transplantation experiment. The inhibition of EGFR signaling pathway under the gliogenic conditions could induce these cells to acquire neuronal phenotypes. Furthermore, we find that the Ras‐ERK axis played a key role in neuronal differentiation of NG2+ glial progenitors upon EGFR inhibition. Taken together, our studies suggest that the EGFR inhibitor could promote neurogenesis post SCI, mainly from the NG2+ glial progenitors. These findings support the possibility of evoking endogenous neuronal replacement from NG2+ glial progenitors and suggest that EGFR inhibition may be beneficial to CNS trauma. © 2012 Wiley Periodicals, Inc.     

从成年大鼠中枢神经系统不同区域分离NG2蛋白聚糖阳性神经祖细胞

目的探索从成年大鼠中枢神经系统(CNS)不同区域是否能分离培养出NG2蛋白聚糖阳性神经祖细胞(NG2细胞)。方法从成年雌性大鼠解剖出CNS的9个不同区域,分别经木瓜蛋白酶消化和Optiprep不连续梯度离心,从离心产生的组织细胞密集带,用含B27添加剂和碱性成纤维细胞生长因子2(FGF2)的NeurobasalA培养液,分离培养出增殖性细胞,再以免疫荧光双重染色法鉴定细胞性质。结果应用上述方法,可从成年大鼠CNS的9个不同区域分离出具神经干细胞(NSCs)潜能的NG2细胞。结论成年大鼠CNS的非神经发生区域同样存在NSCs样细胞,且可通过适当方法体外培养。     

Regulation of chondroitin sulphate proteoglycan and reactive gliosis after spinal cord transection: effects of peripheral nerve graft and fibroblast growth factor 1

M‐J. Lee, C. J. Chen, W‐C. Huang, M‐C. Huang, W‐C. Chang, H‐S. Kuo, M‐J. Tsai, Y‐L. Lin and H. Cheng (2011) Neuropathology and Applied Neurobiology 37, 585–599 Regulation of chondroitin sulphate proteoglycan and reactive gliosis after spinal cord transection: effects of peripheral nerve graft and fibroblast growth factor 1 Aims: The combined treatment of peripheral nerve (PN) graft and fibroblast growth factor (FGF)‐1 for spinal cord injury produces functional recovery, but how it affects injury events is still unknown. This project studied the effect of PN graft and FGF‐1 on white matter degeneration following spinal cord injury. Methods: Rats were divided into four groups: (i) complete spinal cord transection and T8 segment removed; the remaining three groups underwent transection followed by (ii) PN grafting; (iii) supply of exogenous FGF‐1; and (iv) PN grafting plus FGF‐1 treatment. Chondroitin sulphate proteoglycan (CSPG) deposition, astrocytes and macrophage activation, cavity size, and calcitonin gene‐related peptide and synaptophysin immunoreactivity were compared. Results: Peripheral nerve grafting increased CSPG levels compared to transection surgery alone. This CSPG was associated with the proximity to the PN graft. FGF‐1 reduced CSPG deposition in grafted animals regardless of the proximity to the graft. The CSPG reduction was accompanied by reduced GFAP expression and macrophage activation. The amount of CSPG with dissociated glycosaminoglycan did not differ between groups. FGF‐1 in Schwann cell–astrocyte coculture did not reduce CSPG deposition. Furthermore, the PN graft increased the calcitonin gene‐related peptide immunoreactivity and altered the distribution of synaptophysin‐positive axons. Conclusion: Peripheral nerve graft supported sensory re‐innervation and partial protection of the grey matter, but up‐regulated CSPG in the graft–stump junction compared to non‐grafted rats. The reduction of CSPG was caused by FGF‐1–PN synergy, and did not involve dissociation of CSPG or the suppression of a general immune response.     

Noradrenergic innervation of the rat spinal cord caudal to a complete spinal cord transection: Effects of olfactory ensheathing glia

Transplantation of olfactory bulb-derived olfactory ensheathing glia (OEG) combined with step training improves hindlimb locomotion in adult rats with a complete spinal cord transection. Spinal cord injury studies use the presence of noradrenergic (NA) axons caudal to the injury site as evidence of axonal regeneration and we previously found more NA axons just caudal to the transection in OEG- than media-injected spinal rats. We therefore hypothesized that OEG transplantation promotes descending coeruleospinal regeneration that contributes to the recovery of hindlimb locomotion. Now we report that NA axons are present throughout the caudal stump of both media- and OEG-injected spinal rats and they enter the spinal cord from the periphery via dorsal and ventral roots and along large penetrating blood vessels. These results indicate that the presence of NA fibers in the caudal spinal cord is not a reliable indicator of coeruleospinal regeneration. We then asked if NA axons appose cholinergic neurons associated with motor functions, i.e., central canal cluster and partition cells (active during fictive locomotion) and somatic motor neurons (SMNs). We found more NA varicosities adjacent to central canal cluster cells, partition cells, and SMNs in the lumbar enlargement of OEG- than media-injected rats. As non-synaptic release of NA is common in the spinal cord, more associations between NA varicosities and motor-associated cholinergic neurons in the lumbar spinal cord may contribute to the improved treadmill stepping observed in OEG-injected spinal rats. This effect could be mediated through direct association with SMNs and/or indirectly via cholinergic interneurons.     

神经生长因子及其结合肌基膜管移植修复脊髓横断性损伤的组织学观察

目的 对应用神经生长因子（ＮＧＦ）及其结合肌基膜管（ＭＢＬ）修复脊髓横断性损伤进行组织学评价。方法 横断雌性家犬脊髓后,分为三组：（Ａ）ＭＢＬ移植结合注射ＮＧＦ组７只。（Ｂ）脊髓单纯横断注射ＮＧＦ组６只。（Ｃ）对照组切除脊髓０．５ｃｍ４只。六个月后用免疫且化学方法对神经轴突和胶质细胞网络框架结构,进行特异染色,并用图像分析方法对脊髓横断处的远近端横截面进行神经纤维数量对比。结果 各组远端神经纤维数     

Ultrastructural organization of regenerated adult dorsal root axons within transplants of fetal spinal cord.

It has previously been demonstrated that the severed central branches of adult mammalian dorsal root ganglion cells regenerate into transplants of fetal spinal cord. The aim of this study was to determine whether these regenerating axons form synapses, and, if they do, to characterize them morphologically. Embryonic day 14 or 15 spinal cord was transplanted into the lumbar enlargement of adult Sprague-Dawley rats, and the L4 or L5 dorsal root was cut and then juxtaposed to the transplant. One to 3 months later the regenerated dorsal roots were labeled by anterograde filling with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) or by immunocytochemistry for calcitonin gene-related peptide (CGRP). Dorsal root labeling with WGA-HRP demonstrated that regenerated axon terminals made synaptic contacts within transplants, and stereological electron microscopic analysis demonstrated that CGRP-immunoreactive axon terminals occupied an average of 9% of the neuropil within 2 mm of the dorsal root-transplant interface. The majority of synapses were axodendritic, but a significant percentage were axosomatic or axoaxonic. Since axoaxonic synapses were observed in transplants in which both pre- and postsynaptic profiles of axoaxonic synapses were labeled for CGRP, some regenerated axons apparently form synapses with each other. Approximately 90% of synaptic contacts were simple, 9% were complex, and 25% of the complex terminals were immunopositive for CGRP. Glia occupied 25% of the neuropil within 1 mm of the dorsal root-transplant interface, but only 6% of the neuropil 1-2 mm from the interface. We also performed a stereological analysis of the neuropil in lamina I. The area fractions of neuropil occupied by myelinated axons, perikarya, and dendrites were similar in transplants and in lamina I. However, the area fraction occupied by unmyelinated axons was significantly smaller in transplants, and the area fraction occupied by axon terminals was significantly larger in transplants compared with lamina I. Regenerated CGRP-immunoreactive synaptic terminals in transplants were significantly larger than in normal lamina I, and their synaptic contact length was also increased, suggesting that a compensatory mechanism for increasing synaptic efficiency might occur within the transplants. Synaptic density, however, was significantly reduced in transplants, indicating a smaller number of synaptic terminals per unit area. In lamina I, as in the transplant, most synapses were axodendritic, but the percentage of axosomatic and axoaxonic terminals was lower in lamina I than in the transplants.(ABSTRACT TRUNCATED AT 400 WORDS)     

细胞移植治疗脊髓损伤的最新进展

一般认为脊髓损伤(spinal cord injury,SCI)造成患者局部甚至全身的瘫痪.大小便失禁和损伤平面以下的感觉消失等功能缺失是永久性的.     

Assessment of axonal dysfunction in an in vitro model of acute compressive injury to adult rat spinal cord axons

An in vitro model of spinal cord injury was developed to study the pathophysiology of posttraumatic axonal dysfunction. A 25 mm length of thoracic spinal cord was removed from the adult male rat (n = 27). A dorsal column segment was isolated and pinned in a recording chamber and superfused with oxygenated (95%O₂/5% CO₂) Ringer. The cord was stimulated with a bipolar electrode, while two point responses were recorded extracellularly. Injury was accomplished by compression with a modified aneurysm clip which applied a 2 g force for 15 s. With injury the compound action potential (CAP) amplitude decreased to 53.7 ± 5.4% (P < 0.001), while the latency increased to 115.6 ± 3.1% (P < 0.0025) of control values. The absolute refractory period increased with injury from 1.7 ± 0.1 ms to 2.1 ± 0.1 ms (P < 0.001). With train stimulation (200 and 400 Hz), injured axons showed evidence of high frequency conduction failure (P < 0.05). The infusion of 5 mM 4-aminopyridine (4-AP), a blocker of voltage-sensitive ‘fast’ K channels confined to internodal regions, resulted in broadening of the CAP of injured axons to 114.9 ± 3.1% of control (P < 0.05). Ultrastructural analysis of the injured dorsal column segments revealed marked axonal and myelin pathology, including considerable myelin disruption.In conclusion, we have developed and characterized an in vitro model of mammalian spinal cord injury which simulates many of the features of in vivo trauma. Injured axons display characteristic changes in physiological function including a shift in refractory period and high frequency conduction failure. The ultrastructural data and response of injured axons to 4-AP suggest that myelin disruption with exposure of ‘fast’ K⁺ channels contributes to posttraumatic axonal dysfunction.     

The expression of nerve growth factor receptor on Schwann cells and the effect of these cells on the regeneration of axons in traumatically injured human spinal cord

To investigate the effects of Schwann cells and nerve growth factor receptor (NGFR) on the regeneration of axons, autopsy specimens of spinal cord from 21 patients with a survival time of 2 h to 54 years after spinal cord trauma were studied using immunohistochemistry and electron microscopy. Regenerating sprouts of axons could be observed as early as 4 days after trauma. At 4.5 months after trauma, many regenerating nests of axons appeared in the injured spinal cord. The regeneration nests contained directionally arranged axons and Schwann cells. Some axons were myelinated. In injured levels of the spinal cord, the Schwann cells exhibited an increased expression of NGFR within spinal roots. These results show that an active regeneration process occurs in traumatically injured human spinal cord. The NGFR expressed on Schwann cells could mediate NGF to support and induce the axon regeneration in the central nervous system. Received: 20 June 1995 / Revised, accepted: 18 September 1995     

Metamorphosis and the regenerative capacity of spinal cord axons in Xenopus laevis

Throughout the vertebrate subphylum, the regenerative potential of central nervous system axons is greatest in embryonic stages and declines as development progresses. For example, Xenopus laevis can functionally recover from complete transection of the spinal cord as a tadpole but is unable to do so after metamorphosing into a frog. Neurons of the reticular formation and raphe nucleus are among those that regenerate axons most reliably in tadpole and that lose this ability after metamorphosis. To identify molecular factors associated with the success and failure of spinal cord axon regeneration, we pharmacologically manipulated thyroid hormone (TH) levels using methimazole or triiodothyronine, to either keep tadpoles in a permanently larval state or induce precocious metamorphosis, respectively. Following complete spinal cord transection, serotonergic axons crossed the lesion site and tadpole swimming ability was restored when metamorphosis was inhibited, but these events failed to occur when metamorphosis was prematurely induced. Thus, the metamorphic events controlled by TH led directly to the loss of regenerative potential. Microarray analysis identified changes in hindbrain gene expression that accompanied regeneration-permissive and -inhibitory conditions, including many genes in the permissive condition that have been previously associated with axon outgrowth and neuroprotection. These data demonstrate that changes in gene expression occur within regenerating neurons in response to axotomy under regeneration-permissive conditions in which normal development has been suspended, and they identify candidate genes for future studies of how central nervous system axons can successfully regenerate in some vertebrates.     

Carbon filaments direct the growth of postlesional plastic axons after spinal cord injury

The effect of implantation of carbon filaments and fetal tissues on the axonal regeneration following contusion injury in a rat model was investigated by in situ immunofluorescence. Female Sprague-Dawley rats were subjected to severe contusion injury to the spinal cord at T9-T10. All animals were divided into 5 groups (N = 5/group): normal controls. surgical controls, with carbon filament implants, with fetal tissue implants and with implants consisting of fetal tissue cocultured with carbon filaments. After a 10-week survival period, the astroglial response was assessed by immunoreactive glial fibrillary acidic protein and the neuro-axonal profile by immunoreactive phosphorylated and nonphosphorylated neurofilament proteins. The contusion injury resulted in: (a) dramatically increased immunoreactivity of glial fibrillary acidic protein indicating injury-associated reactive astrogliosis, (b) increase in immunoreactive phosphorylated neurofilament protein indicating upregulated phosphorylation of neurofilament protein, (c) with no change in the highly differentiated nonphosphorylated neurofilament protein which normally occur in the nonregenerating mature neurons. Implantation of fetal tissues alone following contusion injury did not show any appreciable change with regard to the immunoreactivities for the glial and neuronal markers studied, compared to the injury controls. However, the implantation of carbon filaments alone or together with fetal tissues directed the growth of glial fibrillary acidic protein-positive astroglia and phosphoneurofilament-positive neurites along the carbon fibers, with no effect on nonphosphoneurofilament protein. In conclusion, implantation of carbon filaments appears to be critical for facilitating the attachment of astroglia forming a substrate and scaffolding that can further support and direct the growth of postlesional plastic axons across the lesion. In addition, carbon filament prostheses in combination with fetal tissue implants provides an improved combinational approach to promote regrowth of injured neurons following injury.     

Calcitonin gene-related peptide-like immunoreactivity in spinal motoneurons of the male mouse is affected by castration and genotype

Calcitonin gene-related peptide (CGRP) is found in motoneurons of the mammalian spinal cord, including motoneurons of the androgen-dependent spinal nucleus of the bulbocavernosus (SNB) of the mouse. Motoneurons of the SNB innervate the bulbocavernosus (BC), a striated muscle involved in penile reflexes. CGRP is thought to be a trophic factor produced by motoneurons to regulate the expression of the acetylcholine receptor at the neuromuscular junction. In rats, the number of SNB motoneurons containing CGRP is increased by gonadal steroids. This regulation appears to rely on an activity-dependent factor produced by the BC muscle. The purpose of the present study was to examine, using immunohistochemistry, the steroid dependence of CGRP in the SNB of male house mice. Genotypic differences in the steroid regulation of CGRP immunoreactivity were examined in three strains of mice that differ in their behavioral sensitivity to castration. The results demonstrate that castration reduces the number of CGRP-positive SNB motoneurons in mice. The magnitude of the change in CGRP in response to castration and the length of time required following castration to alter CGRP were dependent on genotype. Interestingly, the effect of castration in mice, to reduce the number of CGRP-immunoreactive SNB motoneurons, is opposite in direction from the increase in CGRP SNB motoneurons observed in rats observed following castration. These experiments suggest that androgens may alter neuromuscular junction function of mouse SNB by regulating the production of CGRP in a species-specific, genotypically dependent fashion.