GDNF及BDNF对受损运动神经元的长期修复

为了研究胶质细胞源神经营养因子（ＧＤＮＦ） 及脑源神经营养因子（ＢＤＮＦ） 对切断轴突的新生运动神经元的长期维持存活及促进神经再生的作用, 我们选用出生时单侧切断坐骨神经的雏鸡模型, 用裸ＤＮＡ 转染方法, 在损伤神经附近的肌肉中转染ＧＤＮＦ ｃＤＮＡ 和ＢＤＮＦ ｃＤＮＡ 的真核表达载体,观察在体表达的神经营养因子对损伤的修复作用。结果显示,在体表达的ＧＤＮＦ 在８ 周内能使切断坐骨神经的腰脊髓运动神经元近９０ ％ 维持存活。切断的坐骨神经从断端向远体端再生,最长再生达９ ．５ｍ ｍ 。表达两个因子比单独表达ＧＤＮＦ 对运动神经元的存活无显著性差异。而两个因子协同作用对坐骨神经的再生更为有效,坐骨神经再生最长的可达１５ ．４ｍ ｍ 。     

脑源神经营养因子基因转染神经断端促进切断坐骨神经再生

为了研究体内表达的脑源神经营养因子（Ｂｒａｉｎ－ｄｅｒｉｖｅｄＮｅｕｒｏｔｒｐｈｉｃｆａｃｔｏｒ,ＢＤＮＦ）对脊髓运动神经元存活及切断神经再生的作用,我们将人ＢＤＮＦｃＤＮＡ克隆到真核表达载体ｐＣＢ６中,使ＢＤＮＦ基因在ＣＭＶ启动子控制下表达。在雏鸡出生后３小时内及第二天直接将ｐＣＢ－ＢＤＮＦｃＤＮＡ·ｌｉｐｏｆｅｃｔｉｎ混合物注射到坐骨神经预切断位点附近肌肉内。第二天切断坐骨神经,神经切断１０天后进行实验检测,观察到,ＢＤＮＦｃＤＮＡ转染阻止了切断神经一侧的腰脊髓内（Ｌ４－Ｌ６）运动神经元的大量死亡。并显著的促进了切断坐骨神经的再生。这些结果表明直接注射含ＢＤＮＦｃＤＮＡ的质粒对损伤的神经进行基因治疗,具有良好的前景;ｌｉｐｏｆｅｃｔｉｎ介导重组质粒进行基因转染是导入外源基因到体内的一个可行方法。     

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。     

胶质细胞源神经营养因子cDNA全序列的克隆及其生物学功能的研究

为了研究ＧＤＮＦ在神经系统中的生物学功能,通过ＲＴ－ＰＣＲ方法从大鼠睾丸总ＲＮＡ中扩增出ＧＤＮＦｃＤＮＡ全序列,序列分析表明与ＧｅｎＢａｎｋ中的顺序完全相同．将ＧＤＮＦｃＤＮＡ以非融合方式连接在真核表达载体ｐＥＧＦＰ－ＮⅠ的绿色荧光蛋白的上游,在ＣＭＶ启动子控制下表达．通过绿色荧光蛋白报告基因的表达表明ＧＤＮＦｃＤＮＡ能在真核细胞ＨｅＬａ中很好表达．采用裸ＤＮＡ转染方法研究ＧＤＮＦ对损伤的坐骨神经的修复作用,在雏鸡出生后３ｈ切断其右侧坐骨神经,将ｐＥＧＦＰ－ＧＤＮＦ与Ｌｉｐｏｆｅｃｔｉｎ的混合物注射到坐骨神经切断位点附近肌肉内,５ｄ后追补一次．２０ｄ后进行实验检测,观察到ＧＤＮＦｃＤＮＡ的转染阻止了切断神经侧的腰脊髓内［Ｌ４－Ｌ６］运动神经元的大量死亡,并显著促进了切断坐骨神经的再生．     

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     

大鼠坐骨神经切断后外源性bcl-2对脊髓运动神经元的保护作用

采用大鼠坐骨神经切断损伤模型,行神经外膜端端对线缝合,术中依不同组别,动物于神经缝合处远端0．5cm处分别注射人的正义和反义bcl-2重组腺病毒(Ad／s-bcl-2、Ad／as-bcl-2),报道基因重组腺病毒(Ad／lacZ)和生理盐水。术后48h,7d,15d和30d常规灌注固定大鼠,取L4-L6脊髓节段,应用X-gal染色、bel-2原位杂交和免疫组化染色、TUNEL染色以及乙酰胆碱酯酶(AChE)组织化学染色方法,观察到外源基因能在脊髓中表达,同时外源性Ad／s-bcl-2能显著减少L4到L6节段脊髓前角运动神经元凋亡的数目,减少脊髓前角运动神经元中因坐骨神经切断导致的AChE活性的降低幅度,并加快其恢复。而Ad／as-bcl-2可显著增加坐骨神经切断诱导的脊髓前角运动神经元凋亡数目以及AChE活性降低幅度,并延缓其恢复。这些观察结果表明,外源性bcl-2能保护周围神经切断后引起的脊髓运动神经元损伤。     

前角运动神经元向脊神经节投射的探讨——CB-HRP示踪及电镜研究

本实验将１％ＣＢ－ＨＲＰ注入大鼠左侧腰４节段脊神经节（ＤＲＧ）两天后,观察到同侧相应节段脊髓前角第Ⅷ层和Ⅸ层内有ＨＲＰ标记胞体。电镜观察显示,在切断大鼠左侧Ｌ４和Ｌ５前根后５～７天,在相应节段的ＤＲＧ内见到变性纤维或终末。上述结果提示,前角运动神经元可发出纤维经前根到ＤＲＧ,及可能参与调节一级感觉信息的传入。     

神经生长因子对神经损伤后运动终板再生影响的研究——酶组织化学和超微结构观察

本研究采用定位、定量方式钳夹兔右侧坐骨神经,ＮＧＦ组动物于神经损伤处局部喷布蛇毒神经生长因子（ｎｅｒｖｅｇｒｏｗｔｈｆａｃｔｏｒ,ＮＧＦ）,对照组动物于同部位给予等量生理盐水。对胫骨前肌中的红肌、白肌和中间型肌纤维运动终板乙酰胆碱酯酶（ＡＣｈＥ）活性和超微结构改变进行观察。结果表明,神经损伤早期,ＡＣｈＥ活性持续下降,超微结构显示逐渐变性。第４周末,ＮＧＦ组运动终板ＡＣｈＥ活性和超微结构恢复正常。第８周末,对照组运动终板酶活性和超微结构才基本恢复正常。本研究结果提示,周围神经损伤后,外源性ＮＧＦ能促进运动神经元轴突再生,最终促进运动终板结构和功能恢复     

低氧对肺动脉内皮细胞PDGF—B链释放及平滑肌细胞生长的影响

应用蛋白ｄｏｔｂｌｏｔ技术检测了低氧内皮细胞条件培养液（ＨＥＣＣＭ）和常氧内皮细胞条件培养液（ＮＥＣＣＭ）内ＰＤＧＦ相对含量,并利用［３Ｈ］－ＴｄＲ掺入法和流式细胞术观察了ＨＥＣＣＭ和ＮＥＣＣＭ及加入特异ＰＤＧＦ抗体对肺动脉平滑肌细胞（ＰＡＳＭＣ）生长的影响。结果表明,ＨＥＣＣＭ中的ＰＤＧＦ含量明显高于ＮＥＣＣＭ;ＨＥＣＣＭ能明显增强ＰＡＳＭＣ内ＤＮＡ合成,促进ＰＡＳＭＣ从Ｇｏ／Ｇ１期进入Ｓ期;当预先加入ＰＤＧＦ－Ｂ链抗体时,则会明显地抑制ＨＥＣＣＭ对ＰＡＳＭＣ的ＤＮＡ合成,阻止ＰＡＳＭＣ从Ｇｏ／Ｇ１期进入Ｓ期。结果提示,低氧时ＰＡＳＭＣ增殖与肺动脉内皮细胞分泌释放ＰＤＧＦ增加有关     

人GDNF结构与功能关系

胶质细胞源性神经营养茵子（ＧＤＮＦ）在神经系统操作修复中具有重要作用。根据大鼠ＧＤＮＦ晶体结构结果,用ＰＣＲ方法改造人ＧＤＮＦ编码基因,在大肠杆菌中表达并纯化获得了一系列２ ＧＤＮＦ片段缺失及插入突变体,通过对脊髓神经元存活 测定来观察结构改造对人ＧＤＮＦ神经营养活性的影响。结果表明,ＧＤＮＦ分子内部的”胱氨酸结“结构对于ＧＤＮＦ分子构象的维持十分重要,ＧＤＮＦ分子中α螺旋、指状结构１区、指状结构     

Motor neuron degeneration after sciatic nerve avulsion in adult rat evolves with oxidative stress and is apoptosis.

The mechanisms for motor neuron degeneration and regeneration in adult spinal cord following axotomy and target deprivation are not fully understood. We used a unilateral sciatic nerve avulsion model in adult rats to test the hypothesis that retrograde degeneration of motor neurons resembles apoptosis. By 21 days postlesion, the number of large motor neurons in lumbar spinal cord was reduced by approximately 30%. The death of motor neurons was confirmed using the terminal transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling method for detecting fragmentation of nuclear DNA. Motor neuron degeneration was characterized by aberrant accumulation of perikaryal phosphorylated neurofilaments. Structurally, motor neuron death was apoptosis. Apoptotic motor neurons undergo chromatolysis followed by progressive cytoplasmic and nuclear condensation with chromatin compaction into uniformly large round clumps. Prior to apoptosis, functionally active mitochondria accumulate within chromatolytic motor neurons, as determined by cytochrome c oxidase activity. These dying motor neurons sustain oxidative damage to proteins and nucleic acids within the first 7 days after injury during the progression of apoptosis, as identified by immunodetection of nitrotyrosine and hydroxyl-modified deoxyguanosine and guanosine. We conclude that the retrograde death of motor neurons in the adult spinal cord after sciatic nerve avulsion is apoptosis. Accumulation of active mitochondria within the perikaryon and oxidative damage to nucleic acids and proteins may contribute to the mechanisms for apoptosis of motor neurons in the adult spinal cord.     

Comparison of three neuro-tracing techniques for identification of the sciatic spinal nerve origin in mice

To identify sensory and motor neurons associated with the sciatic nerve in adult mice, three methods for applying fluorescent tracers (Fluorogold and Dil) were investigated: direct application, intraneural injection and impregnation of a sectioned nerve in a silicone chamber. Most accurate localization of the neurons on the dorsal root ganglia and spinal cord was accomplished by introducing the proximal stump of a transected sciatic nerve into a silicone chamber, filled with tracers and then decalcifying the tissue. Fluorogold was an effective tracing agent, in contrast to Dil, which was not. In addition to associations with cephalic ganglia L4, L5 and L6, as seen in rats, contributory neurons to the sciatic nerve were located in other ganglia in the mouse. These findings show that the silicone chamber-tissue decalcification technique is a viable tool for obtaining comparative neuroanatomical information in the mouse model.     

Motor neuron degeneration after sciatic nerve avulsion in adult rat evolves with oxidative stress and is apoptosis

The mechanisms for motor neuron degeneration and regeneration in adult spinal cord following axotomy and target deprivation are not fully understood. We used a unilateral sciatic nerve avulsion model in adult rats to test the hypothesis that retrograde degeneration of motor neurons resembles apoptosis. By 21 days postlesion, the number of large motor neurons in lumbar spinal cord was reduced by ∼30%. The death of motor neurons was confirmed using the terminal transferase‐mediated deoxyuridine triphosphate‐biotin nick‐end labeling method for detecting fragmentation of nuclear DNA. Motor neuron degeneration was characterized by aberrant accumulation of perikaryal phosphorylated neurofilaments. Structurally, motor neuron death was apoptosis. Apoptotic motor neurons undergo chromatolysis followed by progressive cytoplasmic and nuclear condensation with chromatin compaction into uniformly large round clumps. Prior to apoptosis, functionally active mitochondria accumulate within chromatolytic motor neurons, as determined by cytochrome c oxidase activity. These dying motor neurons sustain oxidative damage to proteins and nucleic acids within the first 7 days after injury during the progression of apoptosis, as identified by immunodetection of nitrotyrosine and hydroxyl‐modified deoxyguanosine and guanosine. We conclude that the retrograde death of motor neurons in the adult spinal cord after sciatic nerve avulsion is apoptosis. Accumulation of active mitochondria within the perikaryon and oxidative damage to nucleic acids and proteins may contribute to the mechanisms for apoptosis of motor neurons in the adult spinal cord. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 185–201, 1999     

Progranulin contributes to endogenous mechanisms of pain defense after nerve injury in mice

Progranulin haploinsufficiency is associated with frontotemporal dementia in humans. Deficiency of progranulin led to exaggerated inflammation and premature aging in mice. The role of progranulin in adaptations to nerve injury and neuropathic pain are still unknown. Here we found that progranulin is up-regulated after injury of the sciatic nerve in the mouse ipsilateral dorsal root ganglia and spinal cord, most prominently in the microglia surrounding injured motor neurons. Progranulin knockdown by continuous intrathecal spinal delivery of small interfering RNA after sciatic nerve injury intensified neuropathic pain-like behaviour and delayed the recovery of motor functions. Compared to wild-type mice, progranulin-deficient mice developed more intense nociceptive hypersensitivity after nerve injury. The differences escalated with aging. Knockdown of progranulin reduced the survival of dissociated primary neurons and neurite outgrowth, whereas addition of recombinant progranulin rescued primary dorsal root ganglia neurons from cell death induced by nerve growth factor withdrawal. Thus, up-regulation of progranulin after neuronal injury may reduce neuropathic pain and help motor function recovery, at least in part, by promoting survival of injured neurons and supporting regrowth. A deficiency in this mechanism may increase the risk for injury-associated chronic pain.     

Peripherally-derived BDNF promotes regeneration of ascending sensory neurons after spinal cord injury

Background

The blood brain barrier (BBB) and truncated trkB receptor on astrocytes prevent the penetration of brain derived neurotrophic factor (BDNF) applied into the peripheral (PNS) and central nervous system (CNS) thus restrict its application in the treatment of nervous diseases. As BDNF is anterogradely transported by axons, we propose that peripherally derived and/or applied BDNF may act on the regeneration of central axons of ascending sensory neurons.

Methodology/Principal Findings

The present study aimed to test the hypothesis by using conditioning lesion of the sciatic nerve as a model to increase the expression of endogenous BDNF in sensory neurons and by injecting exogenous BDNF into the peripheral nerve or tissues. Here we showed that most of regenerating sensory neurons expressed BDNF and p-CREB but not p75NTR. Conditioning-lesion induced regeneration of ascending sensory neuron and the increase in the number of p-Erk positive and GAP-43 positive neurons was blocked by the injection of the BDNF antiserum in the periphery. Enhanced neurite outgrowth of dorsal root ganglia (DRG) neurons in vitro by conditioning lesion was also inhibited by the neutralization with the BDNF antiserum. The delivery of exogenous BDNF into the sciatic nerve or the footpad significantly increased the number of regenerating DRG neurons and regenerating sensory axons in the injured spinal cord. In a contusion injury model, an injection of BDNF into the footpad promoted recovery of motor functions.

Conclusions/Significance

Our data suggest that endogenous BDNF in DRG and spinal cord is required for the enhanced regeneration of ascending sensory neurons after conditioning lesion of sciatic nerve and peripherally applied BDNF may have therapeutic effects on the spinal cord injury.     

Exogenous heat shock cognate protein Hsc70 prevents axotomy-induced death of spinal sensory neurons

Elevation of intracellular heat shock protein (Hsp)70 increases resistance of cells to many physical and metabolic insults. We tested the hypothesis that treatment with Hsc70 can also produce that effect, using the model of axotomy-induced neuronal death in the neonatal mouse. The sciatic nerve was sectioned and in some animals purified bovine brain Hsc70 was applied to the proximal end of the nerve immediately thereafter and again 3 days later. Seven days postaxotomy, the surviving sensory neurons of the lumbar dorsal root ganglion (DRG) and motoneurons of the lumbar ventral spinal cord were counted to assess cell death. Axotomy induced the death of approximately 33% of DRG neurons and 50% of motoneurons, when examined 7 days postinjury. Application of exogenous Hsc70 prevented axotomy-induced death of virtually all sensory neurons, but did not singificantly alter motoneuron death. Thus, Hsc70 may prove to be useful in the repair of peripheral sensory nerve damage.     

T-588 Protects Motor Neuron Death Following Axotomy

R(-)-1-(benzo [b] thiophen-5-yl)-2-[2-(N,N-diethylamino)ethoxy] ethanol hydrochloride) (T-588) enhances acetylcholine release. This compound slows the motor deterioration of wobbler mouse motor neuron disease and enhances neurite outgrowth and choline acetyltransferase activity in cultured rat spinal motor neurons. We examined the ability of T-588 on axotomized spinal motor neuron death in the rat spinal cord. After the postnatal unilateral section of sciatic nerve, there was approximately a 50% survival of motor neurons in the fourth lumbar segment. In comparison with vehicle, intraperitoneal injection of T-588 for 14 consecutive days rescued spinal motor neuron death. Our results showing in vivo neurotrophic activity of T-588 for motor neurons support the applicability of T-588 for the treatment of motor neuron diseases, such as amyotrophic lateral sclerosis and motor neuropathies.     

低温保存许旺细胞对周围神经再生的作用

目的：比较原代培养许旺细胞（Schwann cells,SCs)和冷冻保存的SCs移植对损伤后坐骨神经再生的作用。方法：原代培养和液氮保存的SCs分别移植到桥接缺损坐骨神经的硅胶管内。在移植后不同时间（第6和8周末）,硅胶管远端神经干内注射HRP,逆行追踪背根神经节和脊髓前角的标记神经元数量;测量再生神经纤维的复合动作电位传导速度;电镜观察再生神经纤维的髓鞘形成。结果：原代培养和冷冻保存SCs在移植后不同时间其背根神经节和脊髓前角神经元HRP标记细胞数量、再生神经纤维的复合动作电位传导速度基本一致,再生神经纤维髓鞘的形成未见明显差别。结论：冷冻保存的SCs仍具有促进损伤后周围神经再生的能力。