FGF对周围神经损伤后脊髓前角运动神经元的保护作用

目的：研究周围神经损伤后,局应用碱性成纤维细胞生长因子（basic fibroblast growth factor ,bFGF）对脊髓前角运动神经元的保护作用。方法：切为SD大鼠右侧坐骨神经,近端套接单盲硅胶管后,管内立好注入20μlbFGF（浓度为4000u/ml）或等量生理盐水,术后2周,观察L3－L5脊央前角外侧群大、中小型神经元乙酰胆碱酯酶（acetyl cholinesterase,AChE）,酸性磷酸酶（acid phosphatas,Acp)和一氧化氮合酶（nitric oxide synthase,NOS)的阳性细胞数、反应强度和平均灰度。结果：坐骨神经损伤后,脊髓腰节段运动神经元因受损伤的影响发明明显改变。表现为AChE降低,Acp,NOS活性升高,局部应用bFGF后,大型运动神经元(α运动神经元）阳性细胞数,反应强度和平均灰度接近正常侧,与对照组比较具有显性意义（P＜0．01）。结论：bFGF可以有效地防止周围神经损伤引起的脊髓前角α运动神经元退变,促进其恢复。     

神经生长因子对兔坐骨神经损伤后脊髓前角运动神经元乙酰胆碱酯酶的影响

钳夹损伤兔右坐骨神经,于损伤处注射蛇毒ＮＧＦ４００Ｂｕ／ｋｇ／日,损伤术后１,３,７天和２,３,４,６,８周动态观察脊髓腰段伤侧第Ⅸ板层外侧群的大型运动神经元的ＡＣｈＥ活性改变。结果表明术后１,３天实验组（指损伤给药组）和对照组（指损伤对照组）ＡＣｈＥ活性均下降（Ｐ＞００５）;术后１,２,３周对照组ＡＣｈＥ活性明显下降,而实验组ＡＣｈＥ活性逐渐趋于恢复（Ｐ＜００１）;术后６周实验组ＡＣｈＥ活性恢复至正常水平（Ｐ＜００１）。本研究显示蛇毒ＮＧＦ对坐骨神经损伤后脊髓前角运动神经元ＡＣｈＥ活性恢复有促进作用,从而对运动神经元可起一定的保护作用和促进恢复的作用     

周围神经损伤后外源性GKNF对神经元的保护作用

采用硅管套接大鼠切断的坐骨神经模型,局部给予胶质细胞源性神经营养因子（ＧＤＮＦ）,应用尼氏染色、酶组织化学染色方法,观察到外源性ＧＤＮＦ能减少脊髓修复侧前角运动神经元死亡的数目,降低脊髓前角运动神经元及脊神经节感觉神经元中胆碱酯酶（ＣＨＥ）及酸性磷酸酶（ＡＣＰ）变化的幅度。这表明外源性ＧＤＮＦ能保护周围神经切断后引起的神经元损伤．     

地塞米松对大鼠坐骨神经损伤后运动神经元的恢复作用

本实验使用120只Wistar系大鼠,采用定位、定量、定时的方法压挫坐骨神经后。给予治疗剂量的地塞米松,动态观察脊髓腰段伤侧前角运动神经元的SDH、AChE和ACP的酶组织化学及细胞超微结构的变化。结果提示,地塞米松对受伤害神经元具有稳定细胞酶活性和细胞超微结构的作用,可以减轻继发性损害,对神经元的恢复有积极作用。     

移植神经营养素-4基因工程细胞对脊髓前角神经元的保护作用

用重组逆转录病毒介导的体外神经营养素－４（ＮＴ－４）基因转移的方法,制备高表达ＮＴ－４基因工程细胞并移植大鼠坐骨神经离断处,尼氏染色和ＣｈＥ染色的结果表明,移植ＮＴ－４基因工程细胞对外周神经损伤所造成的运动神经元的退变有显著的改善作用,而且这种作用可以维持三个月。     

周围神经损伤后外源性GDNF对神经元的保护作用

采用硅管套接大鼠切断的坐骨神经模型 ,局部给予胶质细胞源性神经营养因子 (GDNF) ,应用尼氏染色、酶组织化学染色方法 ,观察到外源性GDNF能减少脊髓修复侧前角运动神经元死亡的数目 ,降低脊髓前角运动神经元及脊神经节感觉神经元中胆碱酯酶 (CHE)及酸性磷酸酶 (ACP)变化的幅度。这表明外源性GDNF能保护周围神经切断后引起的神经元损伤。     

重组RA—538及反义c—myc腺病毒对人胃癌,食管癌和bcl—2高表达细胞系的作用

本课题研究ＲＡ５３８、反义c-ymc重组腺病毒对人胃癌（ＳＧＣ７９０１）、食管癌（Ｅ Ｃ１０９、ＥＣ８７１２）、正常人胚肺２ＢＳ（２ＢＳ）及ｂｃｌ－２高表达细胞第的体仙外生物学作用及其分子机制。结果显示Ａｄ－ＲＡ５３８及Ａｄ－ＡＳｃ－ｍｙｃ对ＳＧＣ７９０１细胞体内外均具有明显的生长抑制及凋亡诱导作用,并能抑制其ｃ－ｍｙｃ、ｂｃｌ－２、ｃｙｃｌｉｎＤ１基因的表达及刺激ｂａｘ基因的表达。对ＥＣ１０９、ＥＣ８     

慢性缺氧大鼠肺血管c-myc及bcl-2基因表达研究

应用免疫组织化学和Ｎｏｒｔｈｅｒｎ杂交方法,对慢性缺氧大鼠肺组织（主要是肺血管壁）原癌基因ｃ－ｍｙｃ和抗凋亡基因ｂｃｌ－２表达进行了研究。免疫组织化学观察提示,正常对照组大鼠肺血壁Ｃ－ｍｙｃ和Ｂｃｌ－２蛋白仅为弱阳性或不表达,慢性缺氧１、２周组大鼠肺血管壁这两种抗原表达比对照组显著增强,呈强阳性,Ｎｏｒｔｈｅｒｎ杂交结果表明：慢性缺氧１、２周组大鼠肺组织内ｃ－ｍｙｃ和ｂｃｌ－２的ｍＲＮＡ表达比对照组显著增加,以上结果提示,ｃ－ｍｙｃ及ｂｃｌ－２两种基因调节的细胞增殖与凋亡可能参与了慢性缺氧性肺动脉高压的发病进程。     

bcl-2与FAS在NO介导的肝细胞凋亡中的作用

目的　探讨bcl 2与FAS在NO介导的重型肝炎肝细胞凋亡中的作用。方法　雌性Balb/C小鼠 2 4只随机分为A、B、C、D 4组 ,每组 6只 ,分别为正常对照组、模型组、L Arg干预组 (NO供体干预 )、L NAME干预组 (NOS抑制剂干预 )。各组动物均于用药后 6h处死 ,留取血清、肝组织。用Griess法测定血清NO代谢产物NO-2 ,TUNEL原位检测肝细胞凋亡状况并计算凋亡指数 ,免疫组织化学法检测各组肝组织Fas表达及原位杂交技术检测各组肝组织bcl 2mRNA的表达。结果　 1.应用NO合成干预因素后 ,C组及D组NO-2 水平分别较B组升高及降低。 2 .正常肝组织经TUNEL检测未见凋亡细胞 ,其凋亡指数为 0。B组存在肝细胞凋亡现象 ,凋亡指数为 16 2 7% ,C组凋亡指数上升 (2 4 5 2 % ) ,而D组凋亡指数下降 (7 92 ) % ,组间比较有显著性差异 ,P值 <0 0 1。 3.正常肝细胞内未见Fas蛋白及bcl 2mRNA表达。B组Fas表达较广泛 ,C及D组Fas表达呈不同程度的增强和减弱 ;组间比较差异有显著性 ,P值 <0 0 1。B组及C组均未见bcl 2mRNA杂交信号 ,仅D组见肝细胞内出现杂交信号。结论　高水平的NO具有明显的促进肝细胞凋亡的作用。NO对于肝细胞的损伤可以通过介导凋亡的方式实现。NO在介导肝细胞凋亡的过程中 ,启动了FAS凋亡途径 ,而抑制NO的过量产生可     

大鼠脊髓挤压伤后NT-3、NT-4在腹角运动神经元表达的变化

我们前面的研究已证实,神经生长因子和脑源性神经营养因子与成年大鼠挤压性脊髓损伤修复有关。在本研究中,通过免疫组织化学ABC法,我们探讨了挤压伤后不同时间脊髓腹角神经元NT-3和NT-4的表达。结果显示,在对照组,NT-3和NT-4的阳性反应主要分布在脊髓腹角神经元,挤压性脊髓损伤后7天和21天,NT-3阳性神经元的数量较对照组和24小时组明显增加,比较之,损伤后24小时和7天,NT-4阳性神经元的数量已较正常者增多,且NT-T的反应强度21天者较24小时和7天者有增多。结果表明NT-3和NT-4的表达在挤压性损伤后的脊髓腹角神经元被不同程度地上调,提示NT-3和NT-4可能与挤压性脊髓损伤修复有关。     

Short- and long-term effects of ciliary neurotrophic factor on androgen-sensitive motoneurons in the lumbar spinal cord

Motoneuron death in the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN) of the lumbar spinal cord is androgen regulated. As a result, many more SNB and DLN motoneurons die in perinatal female rats than in males, whereas treatment of newborn females with androgen results in a permanent sparing of the motoneurons and their target muscles. We previously observed that a neurotrophic molecule, ciliary neurotrophic factor (CNTF), also arrests the death of SNB motoneurons and their target musculature, at least in the short term. The present study compares the short- and long-term consequences of perinatal CNTF treatment on motoneuron number in the SNB, the DLN, and the retrodorsolateral nucleus (RDLN), a motor pool in the lower lumbar cord that does not exhibit hormone-regulated cell death. Female pups were treated with CNTF or vehicle alone from embryonic day 22 through postnatal day 6 (P6). Motoneuron number in each nucleus was then determined immediately after treatment on P7, or 10 weeks later (P77). CNTF treatment significantly elevated motoneuron number in the SNB and DLN on P7; the volume of SNB target muscles on P7 was also greater in the CNTF-treated group. These effects were transient, however, as motoneuron number and ratings of muscle size were not different in CNTF- and vehicle-treated females on P77. Perinatal CNTF treatment did not alter cell number in the RDLN at either age. The finding that effects of CNTF on SNB and DLN motoneuron number are short lived contrasts with the permanent effects of early androgen treatment, and has implications for molecular models of the actions of androgen and neurotrophic factors on the developing spinal cord. © 1996 John Wiley & Sons, Inc.     

Vascular endothelial growth factor protects spinal cord motoneurons against glutamate-induced excitotoxicity via phosphatidylinositol 3-kinase

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective death of motoneurons. Recently, vascular endothelial growth factor (VEGF) has been identified as a neurotrophic factor and has been implicated in the mechanisms of pathogenesis of ALS and other neurological diseases. The potential neuroprotective effects of VEGF in a rat spinal cord organotypic culture were studied in a model of chronic glutamate excitotoxicity in which glutamate transporters are inhibited by threohydroxyaspartate (THA). Particularly, we focused on the effects of VEGF in the survival and vulnerability to excitotoxicity of spinal cord motoneurons. VEGF receptor-2 was present on spinal cord neurons, including motoneurons. Chronic (3 weeks) treatment with THA induced a significant loss of motoneurons that was inhibited by co-exposure to VEGF (50 ng/mL). VEGF activated the phosphatidylinositol 3-kinase/Akt (PI3-K/Akt) signal transduction pathway in the spinal cord cultures, and the effect on motoneuron survival was fully reversed by the specific PI3-K inhibitor, LY294002. VEGF also prevented the down-regulation of Bcl-2 and survivin, two proteins implicated in anti-apoptotic and/or anti-excitotoxic effects, after THA exposure. Together, these findings indicate that VEGF has neuroprotective effects in rat spinal cord against chronic glutamate excitotoxicity by activating the PI3-K/Akt signal transduction pathway and also reinforce the hypothesis of the potential therapeutic effects of VEGF in the prevention of motoneuron degeneration in human ALS.     

P0 and myelin basic protein-like immunoreactivities following ligation of the sciatic nerve in the rat

In this work we analyzed variations in the expression of MBPs and P₀ in ligated sciatic nerves of young and adult rats at 3, 7, and 14 days postligation (PL), by immunohistochemistry and SDS-PAGE of isolated myelin. A protein redistribution was seen in the distal stump of ligated nerves with the appearance of immunoreactive clusters. Using the KS400 image analyzer, immunostained area values were obtained from the different nerves dissected. In adult rats, there was an increase of the immunostained area for MBP from 3 to 7 days PL, coincident with a reorganization of the marker in clusters, followed by a marked decrease at 14 days. P₀ immunolabeling gave similar results without, however, a decrease of the immunostained area at the longer survival time tested. Young animals showed an acceleration in the process of protein redistribution and digestion within ligated nerves, which followed a similar pattern as that of adult animals. Analysis by electrophoresis showed a marked decrease in P₀ and MBP at 7 days PL in young rats and 14 days PL in adult rats. The functional significance of protein clustering within myelin in injured nerves deserves further analysis.     

Effect of axotomy on cholinergic enzyme activities in the spinal cord and sciatic nerve of the dog

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity measured in the ventral and dorsal part of the dog spinal cord (L6-S2) and in the stumps of the sciatic nerve 5, 10, 15 and 21 days after its transection were compared with the corresponding activities in the intact contralateral nerve and in sham-operated animals. AChE was also examined histochemically. Changes in the enzyme activities in the central nerve stump were correlated with activity changes in the spinal cord. In the central nerve stump, a marked (25%) increase in AChE activity was found on the fifth day after transection, but by the 21st day it fell below control value levels; up to the 15th day it showed good correlation with AChE activity in the ventral spinal cord. Histochemically, pronounced reduction of enzymatic activity was found in the ipsilateral part of the spinal cord. On the 15th day, ChAT activity in the ventral spinal cord was also significantly decreased and the accumulation of the enzyme in the central nerve stump was negligible. On the contrary, at the last 21-day interval examined, a significant increase in ChAT activity and a nonsignificant increase in AChE activity was found in the spinal cord, but their activities in the central nerve stump were decreased. In the degenerated peripheral nerve stump ChAT activity dropped by an average of 99% and AChE activity by 48% during the first 15 days after transection but, on the 21st day, AChE activity was 22% higher than at the preceding interval.     

Acceleration of axonal outgrowth in rat sciatic nerve at one week after axotomy

Following injury of sciatic motor axons in the rat, the rate of axonal outgrowth is faster if there has been a prior “conditioning” axotomy. The acceleration of outgrowth is due to an acceleration of SCb, the rate [slow (SC)] component of axonal transport that carries cytomatrix proteins; this occurs throughout the axon by 7 days after the conditioning axotomy (Jacob and McQuarrie, 1991a, J. Neurobiol. 22:570–583). To further characterize the conditioning lesion effect (CLE), it is important to know (1) the minimum effective conditioning interval (time between conditioning and testing lesions), (2) whether the cell body reaction is required, and (3) whether outgrowth accelerates after a single axotomy. Outgrowth distances were measured by radiolabeling all newly synthesized neuronal proteins and detecting those carried to growth cones by fast axonal transport. When the conditioning and testing lesions were made simultaneously (0 day conditioning interval), there was no CLE. With a conditioning interval of 3 days, there was a shortening of the initial delay (before the onset of outgrowth) without a change in outgrowth rate. With conditioning intervals of 7, 14, and 21 days, the rates of outgrowth were increased by 8%, 22%, and 11%, respectively. To determine whether the cell body reaction to axotomy is necessary for the CLE, a nonaxotomizing stimulus to axonal growth (partial denervation) was used in place of a conditioning axotomy. This had no effect on the rate of outgrowth from a testing lesion made 14 days later. Finally, we examined the possibility that outgrowth accelerates after a single lesion. Outgrowth was faster at 6–9 days after axotomy than at 3–6 days (p < 0.001), and accelerated further at 9–12 days (p < 0.001). We conclude that (1) the shortest effective conditioning interval is 3 days; (2) the cell body reaction is necessary for the CLE; (3) axonal outgrowth from a single axotomy accelerates in concert with the anabolic phase of the cell body reaction. The SCb motor is, in turn, upregulated by this reaction. This suggests that the SCb motor responds to a fast-transported signal that is a product of the cell body reaction. © 1993 John Wiley & Sons, Inc.     

Protein S-nitrosylation and denitrosylation in the mouse spinal cord upon injury of the sciatic nerve

Nitric oxide is a pain signaling molecule and exerts its influence through two primary pathways: by stimulation of soluble guanylylcyclase and by direct S-nitrosylation (SNO) of target proteins. We assessed in the spinal cord the SNO-proteome with two methods, two-dimensional S-nitrosothiol difference gel electrophoresis (2D SNO-DIGE) and SNO-site identification (SNOSID) at baseline and 24h after sciatic nerve injury with/without pretreatment with the nitric oxide synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME). After nerve injury, SNO-DIGE revealed 30 proteins with increased and 23 proteins with decreased S-nitrosylation. SNO-sites were identified for 17 proteins. After sham surgery only 3 proteins were up-nitrosylated. L-NAME pretreatment substantially reduced both constitutive and nerve injury evoked up-S-nitrosylation. For the top candidates S-nitrosylation was confirmed with the biotin switch technique and time course analyses at 1 and 7days showed that SNO modifications of protein disulfide isomerase, glutathione synthase and peroxiredoxin-6 had returned to baseline within 7days whereas S-nitrosylation of mitochondrial aconitase 2 was further increased. The identified SNO modified proteins are involved in mitochondrial function, protein folding and transport, synaptic signaling and redox control. The data show that nitric oxide mediated S-nitrosylation contributes to the nerve injury-evoked pathology in nociceptive signaling pathways.     

Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motoneurons

In situ hybridization histochemistry and RNA blot analysis were used to study expression of nerve growth factor receptor (NGF-R) mRNA in rat spinal cord motoneurons. The results show that NGF-R mRNA is expressed at high levels in rat spinal cord motoneurons at the time of naturally occurring cell death. This expression is sustained, but reduced, during synapse formation and is subsequently greatly reduced in the adult spinal cord. A unilateral crush lesion of the sciatic nerve resulted in an 8-fold increase in NGF-R mRNA in adult rat spinal cord motoneurons 3 days after lesion, compared with the nonlesioned side. NGF-R mRNA induction was even more pronounced 7 and 14 days after lesion, reaching levels 12 times higher than those on the nonlesioned side. However, 6 weeks after lesion, when the motor function of the leg was largely restored, NGF-R expression had decreased to levels similar to those on the contralateral side. We therefore suggest that NGF-R mediates a trophic or axonal guidance function for developing and regenerating spinal cord motoneurons.