HRP法对异种神经移植后再生纤维恢复的形态学研究

目的用辣根过氧化酶(HRP)逆行追踪技术探讨异种神经移植后神经纤维的再生.方法将多次冻融处理后的兔胫神经移植于大鼠坐骨神经,术后第2、4、6、8和10周,将HRP注人大鼠坐骨神经吻合部远侧端.结果移植术后第4周起在L4～5脊神经节见到HRP标记细胞,从第6周在腰段脊髓前角内见到标记细胞,其数量随术后存活期延长而增多.术后4周在移植神经内见少量再生神经纤维,6周后再生神经纤维穿过异种移植神经进入大鼠坐骨神经远侧端.结论自移植术后4周起,移植神经内已有再生纤维并部分恢复了轴浆流,证实了用HRP法可反映移植后神经纤维的再生情况.     

OMgp不同结构域在抑制神经突起生长中的作用

OMgp(oligodendrocyte-myelin glycoprotein)是一种在中枢神经系统表达的GPI连接的糖蛋白。最新发现,它具有诱使生长锥溃变和抑制神经突起再生的作用,这一作用是通过与nogo-66等神经再生抑制因子竞争结合同一受体NgR而实现的。但其相互作用的确切部位尚不能肯定。利用GST融合蛋白表达系统,分段表达了含有不同OMgp结构域的片段,对其与NgR作用的结构域进行了研究。结果表明,在OMgp与NgR的黏附结合过程中,OMgp的亮氨酸富含重复序列结构域是必需的,只有含该结构域的OMgp蛋白片段才能黏附表达有NgR的CHO细胞,并抑制神经突起的生长;在体外,含有丝／苏氨酸富含重复序列结构域的OMgp蛋白片段虽然具有微弱的沉降NgR的功能,但并不能抑制神经突起的生长。该结果将有助于中枢神经系统损伤后神经再生的理论与治疗研究。     

再生神经中微管,神经丝与轴突截面积的变化

用电镜及图象分析的方法研究了再生轴突中微管、神经丝与轴突截面积的变化,发现神经再生过程中微管及神经丝的密度增加,并与轴突截面积呈相关关系,而且微管的变化更早,更明显。由于微管参与了轴浆转运的机制,微管的增加提示其在神经再生中起了重要的作用。     

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 。     

黄瓜去顶苗直接成花的形态学研究

本文对黄瓜0—7天幼苗及去顶后0—8天诱导花芽分化苗与诱导营养芽分化苗的子叶节进行了系统石蜡切片观察,未发现0—7天幼苗的子叶叶腋存在潜伏芽。去顶后1—2天在子叶叶柄基部与切口之间的表皮下细胞分裂形成突起,去顶后6天诱花苗与诱芽苗的突起表现出形态差异,诱花苗突起的上端变钝,而诱芽苗突起的上端成尖锥状。去顶后8天诱花苗在子叶叶柄与切口之间形成完整的花芽。另发现有少量花芽起源于切口处细胞。对去顶后0—6天诱花苗与诱芽苗的子叶节还进行了电镜扫描观察,观察结果与石蜡切片基本一致。     

Tropic 1808基因在大鼠损伤神经组织中的表达

目的观察Tropic 1808基因在大鼠正常和损伤坐骨神经组织中的表达,探讨Tropic 1808基因在周围神经损伤与再生过程中的作用.方法采用地高辛标记的Tropic 1808 cDNA探针、抗大鼠S-100蛋白抗体,以原位杂交和免疫组织化学双重染色法,观察Tropic 1808基因在正常和损伤大鼠坐骨神经组织中的表达.结果免疫组化结果显示,大鼠正常坐骨神经可表达S-100蛋白,但表达量较低;神经损伤后,其远侧端S-100蛋白的表达量明显增加.原位杂交结果显示,大鼠正常坐骨神经组织未见Tropic 1808 mRNA杂交信号;损伤神经的远侧端呈现较强的阳性信号,而且在部分S-100强阳性反应区可见Tropic 1808 mRNA杂交信号.结论 Tropic 1808基因在正常坐骨神经组织中未见表达;坐骨神经损伤后,其远侧端增殖的雪旺氏细胞可表达Tropic 1808 mRNA.提示,Tropic 1808是一种周围神经损伤后特异表达的基因.     

黄瓜去顶苗直接成花的形态学研究

本文对黄瓜０－７天幼苗及去顶后０－８天诱导花芽分化苗与诱导营养芽分化苗的子叶节进行了系统石蜡切片观察,未发现０－７天幼苗的子叶叶腋存在潜伏芽。去顶后１－２天在子叶叶柄基部与切口之间的表皮下细胞分裂形成突起,去顶后６天诱花苗与诱芽苗的突起表现出形态差异,诱花苗突起的上端变钝,而诱芽苗突起的上端成尖锥状。去顶１后８天诱花苗在子叶叶柄与切口之间形成完整的花芽。另发现有少量花芽起源于切口外细胞。对去顶后０     

免疫酶双重染色法显示再生神经中的神经生长因子与睫状节神经营养因子

本研究以成人正中神经切割伤后２～３个月的神经干为材料,冰冻切片,用免疫双重染色技术显示了神经生长因子与睫状节神经营养（诱向）因子在再生的周围神经组织中的表达与分布。神经生长因子选用ＡＰＡＡＰ法．其阳性产物呈红色;睫状节神经营养（诱向）因子选用ＡＢＣ系统,４氯－１－萘酚显色,阳性产物为褐色。光镜下观察：神经生长因子的阳性反应产物出现在正中神经切割伤后再生的神经纤维中,高倍镜下可见其阳性产物分布在轴索,而在雪旺氏细胞中没能见到呈红色的阳性反应产物;睫状节神经营养（诱向）因子分布在一些细胞体积大、核大呈增生活跃状态的雪旺氏细胞中。红与褐双色反应产物色调清晰,效果较好。研究结果提示：睫状节神经营养（诱向）因子与神经生长因子在人周围神经再生过程中起着十分重要的作用。     

神经突再生抑制因子Nogo研究进展

髓磷脂所表达的Nogo蛋白可能是阻止中枢神经再生的关键因素。nogo基因的克隆成功是近年神经再生研究的一个重要进展。nogo基因至少编码三种蛋白质,分别称为Nogo-A、Nogo-B和Nogo-C。Nogo-A即以前所指的NI-250。Nogo-A的单克隆抗体IN-1,能中和Nogo对神经突起再生的抑制作用,促使受损的神经纤维再生,并使神经功能得到部分恢复。本文介绍Nogo的研究概况、生物学作用及其在中枢神经损伤修复方面可能的应用前景。     

大鼠损伤坐骨神经远侧端CNTF表达的免疫组织化学研究

目的：探讨大鼠坐骨神经损伤后CNTF的表达和变化。方法：采用抗CNTF抗免疫组织化学方法和计算机图像处理系统定量观察大鼠正常坐骨神经与坐骨神经横断损伤后1周、2周、4周神经远侧端CNTF的表达。结果：正常大鼠坐骨神经具有高水平的CNTF免疫阳性反应,坐骨神经损伤后1周、2周、4周远侧端神经中CNTF免疫阳性反应均低于正常坐骨神经,免疫阳性反应强度为正常＞伤后1周＞伤后2周＞伤后4周,呈逐渐减弱趋势。结论：大鼠坐骨神经损伤后远侧端神经中CNTF的表达呈下调性变化。     

The influence of peripheral nerve graft's predegeneration stage on the regrowth of hippocampal injured neurites and concomitant changes in submicrosomal fraction proteins of grafts.

The present work has a twofold aim: 1. To ascertain whether the stimulative influence of peripheral nerve grafts on injured hippocampal neurons depends on the time lapse after transection and; 2. To examine whether the mentioned effect runs parallel to the time-dependent changes of proteins contents and composition in the submicrosomal fraction from transected rat sciatic nerves. Fluorescence microscope examination revealed that FITC-HRP labeled cells extending their neurites into the implanted peripheral nerve segments were particularly numerous among the hippocampal neurons when 7- and 35-day-old predegenegated distal stumps were used as grafts. Discontinuous SDS-slab polyacrylamide gel electrophoresis of submicrosomal fraction proteins obtained from distal stumps of rat sciatic nerves was performed at the 7, 14, 21 and 35 days after transection. Among the obtained protein fractions the most interesting seem to be the ones of 47 and 54 kDa, which reached maximal levels at the 7th day and the 50 kDa fraction with a maximum at the 35th experimental day. It is possible that the growth promoting power of the employed grafts depends on the presence of proper proteins.     

The origin and possible significance of substance P immunoreactive networks in the prevertebral ganglia and related structures in the guinea-pig

The distribution and origin of substance P immunoreactive nerve elements have been studied in the guinea-pig prevertebral ganglia by the indirect immunohistochemical technique, using a monoclonal antibody to substance P. Non-varicose substance P immunoreactive nerve fibres enter or leave the ganglia in all nerves associated with them, traversing the ganglia in larger or smaller bundles. Networks, mainly single-stranded, of varicose substance P immunoreactive nerve fibres also permeate the ganglia, forming a loose meshwork among the neurons. Similar networks are present in the lumbar paravertebral ganglia. In all these ganglia, neuronal somata do not in general show substance P immunoreactivity. The various nerves connected with the inferior mesenteric ganglion have been cut, in single categories and in various combinations, and the ganglion examined, after intervals of up to six days. Cutting the colonic or hypogastric nerves, which connect the ganglion with the hindgut and pelvic organs, leads to accumulation of substance P immunoreactive material in their ganglionic stumps, extending retrogradely to intraganglionic non-varicose fibres traceable through into the intermesenteric and lumbar splanchnic nerves. There is some local depletion of intraganglionic varicose networks. Cutting the intermesenteric nerve, which connects the coeliac-superior mesenteric ganglion complex with the ganglion, leads to accumulation of substance P immunoreactive material in its cranial stump and depletion of its distal stump; a minimal depletion is detectable in the inferior mesenteric ganglion itself. Cutting the lumbar splanchnic nerves, which connect the ganglion with the upper lumbar spinal cord and dorsal root ganglia, leads to accumulation of substance P immunoreactive material in their proximal stumps and total depletion of their distal, ganglionic stumps; in the ganglion there is subtotal loss of non-varicose substance P immunoreactive fibres and of varicose nerve networks, and the few surviving non-varicose fibres are traceable across the ganglion from the intermesenteric nerve to the colonic and hypogastric nerves. Cutting the intermesenteric and lumbar splanchnic nerves virtually abolishes substance P immunoreactive elements from the ganglion within three days postoperatively. It is concluded that these arise centrally to the ganglion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of proteins transported at different rates by axoplasmic flow in the dorsal root afferents of rats.

Proteins synthesized by soma located in L4 dorsal root ganglia and supplied to the axonal branches extending centrally in the dorsal root and peripherally towards the sciatic nerve were analyzed for radioactivity following injections of [3H] leucine into the L4 dorsal root ganglia. All proteins located in the dorsal root and sciatic nerve were analyzed by SDS acrylamide gel electrophoresis at various times post injection. The differences in radioactivity between the dorsal root and sciatic nerve proteins were mainly quantitative and not qualitative, with many proteins of various molecular weight ranges being transported into both segments. Generally, it appears that in both axonal branches the high molecular weight proteins are transported at the highest rate, medium weights slower and low molecular weight proteins slowest. More proteins of high and low molecular weights are transported into the dorsal root whereas more of those of medium molecular weight are transported towards the sciatic nerve.     

Uptake of Endoneurial Lipoprotein into Schwann Cells and Sensory Neurons Is Mediated by Low Density Lipoprotein Receptors and Stimulated After Axonal Injury

During Wallerian degeneration of rat sciatic nerve, the expression of apolipoprotein E increases and apolipoprotein E-containing endoneurial lipoproteins accumulate in the distal nerve segment. In established primary cultures dissociated from dorsal root ganglia, Schwann cells and sensory neurons internalized rhodamine-labeled lipoproteins isolated from crushed rat sciatic nerve as well as low density lipoprotein (LDL) from human serum. The uptake of endoneurial lipoproteins could be inhibited by an excess of LDL or at low temperature (4 degrees C). After transection of nerve fibers in dorsal root ganglia explant cultures, the uptake of lipoproteins was markedly stimulated in Schwann cells that were in close proximity to degenerating neurites. A specific monoclonal antibody directed to the bovine LDL receptor (clone C7) was shown to cross-react with LDL receptor preparations of rat endoneurial cells. LDL receptor immunoreactivity was expressed by cell bodies and processes of cultured Schwann cells, sensory neurons, and fibroblasts from dorsal root ganglia. Incubation of Schwann cells and neurons with the LDL receptor antibody strongly inhibited the uptake of endoneurial lipoproteins. Our results provide direct evidence for the important role of the LDL receptor-mediated pathway to internalize endoneurial lipoproteins into Schwann cells and peripheral neurons required for reuse of cholesterol and other lipids in myelin and plasma membrane biogenesis during nerve repair.     

Characterization of proteins transported at different rates by axoplasmic flow in the dorsal root afferents of rats

Proteins synthesized by soma located in L₄ dorsal root ganglia and supplied to the axonal branches extending centrally in the dorsal root and peripherally towards the sciatic nerve were analyzed for radioactivity following injections of [³H] leucine into the L₄ dorsal root ganglia. All proteins located in the dorsal root and sciatic nerve were analyzed by SDS acrylamide gel electrophoresis at various times post injection. The differences in radioactivity between the dorsal root and sciatic nerve proteins were mainly quantitative and not qualitative, with many proteins of various molecular weight ranges being transported into both segments. Generally, it appears that in both axonal branches the high molecular weight proteins are transported at the highest rate, medium weights slower and low molecular weight proteins slowest. More proteins of high and low molecular weights are transported into the dorsal root whereas more of those of medium molecular weight are transported towards the sciatic nerve.     

Regeneration of central and peripheral synaptic connections in the locomotor system of the pteropod molluscClione limacina

The neural network underlying rhythmic wing movements in the molluscClione limacina is well-studied. Two different groups of motoneurons innervate two distinct groups of wing muscles. The locomotor rhythm generated in the left and right pedal ganglia is synchronized by interneurons. When the axons of the locomotor motoneurons are crushed, numerous fine neurites sprout towards the denervated muscles and reach them in 8–15 days. At this stage motoneurons project to and synapse on not only correct but equally incorrect muscle targets. After 2 weeks of regeneration the number of incorrect neurites and synaptic connections begins to decrease and following 1.5–2 months all incorrect connections are eliminated, incorrect axons are withdrawn and the behavioral deficit is compensated. In this study the regeneration of interneurons and the growth profiles of inter- and motoneurons were also studiedin vitro. Two individually isolated pedal ganglia were co-cultured in three different configurations: a) the wing nerve stump from one ganglion was fixed against the commissural stump from another ganglion; b) the wing nerve stumps were fixed against each other; c) the commissural stumps were fixed against each other. Under the above experimental conditions we found that the interneurons were able to cross only the contact between two commissural stumps, and in this case found their original targets, restored correct connections and synchronized the rhythm in two pedal ganglia. In contrast, motoneurons were able to cross all types of contacts.     

Intermediate Filaments of Schwann Cells

Abstract: Intermediate filaments were prepared from distal stumps of rabbit sciatic nerve 5 weeks after nerve section, at which time Schwann cells account for 85–90% of the cell area. A polypeptide of molecular weight 58,000 was the main component of this fraction. An antiserum raised in guinea pig against this polypeptide stained all cells present in the distal stump, as well as Schwann cells and 3T3 cells in culture. The identity of the molecular weight 58,000 polypeptide obtained from distal stumps with vimentin was proved with one and two-dimensional sodium dodecyl sulfate pol yacrylamide gel electrophoresis and with immunoautoradiography. It is concluded that the intermediate filament subunit of undifferentiated Schwann cells is vimentin. The possibility that Schwann cells in normal nerve may have another type of intermediate filament besides vimentin cannot be ruled out.     

High-Frequency Electrical Stimulation Can Be a Complementary Therapy to Promote Nerve Regeneration in Diabetic Rats

The purpose of this study was to evaluate whether 1 mA of percutaneous electrical stimulation (ES) at 0, 2, 20, or 200 Hz augments regeneration between the proximal and distal nerve stumps in streptozotocin diabetic rats. A10-mm gap was made in the diabetic rat sciatic nerve by suturing the stumps into silicone rubber tubes. Normal animals were used as the controls. Starting 1 week after transection, ES was applied between the cathode placed at the distal stump and the anode at the proximal stump every other day for 3 weeks. At 4 weeks after surgery, the normal controls and the groups receiving ES at 20, and 200 Hz had a higher success percentage of regeneration compared to the ES groups at 0 and 2 Hz. In addition, quantitative histology of the successfully regenerated nerves revealed that the groups receiving ES at a higher frequency, especially at 200 Hz, had a more mature structure with more myelinated fibers compared to those in the lower-frequency ES groups. Similarly, electrophysiology in the ES group at 200 Hz showed significantly shorter latency, larger amplitude, larger area of evoked muscle action potentials and faster conduction velocity compared to other groups. Immunohistochemical staining showed that ES at a higher frequency could significantly promote calcitonin gene-related peptide expression in lamina I-II regions in the dorsal horn and recruit a higher number of macrophages in the diabetic distal sciatic nerve. The macrophages were found that they could stimulate the secretion of nerve growth factor, platelet-derived growth factor, and transforming growth factor-β in dissected sciatic nerve segments. The ES at a higher frequency could also increase cutaneous blood flow in the ipsilateral hindpaw to the injury. These results indicated that a high-frequency ES could be necessary to heal severed diabetic peripheral nerve with a long gap to be repaired.     

Quantitative Evaluation of Neurite Outgrowth in Cultures of Human Foetal Brain and Dorsal Root Ganglion Cells Using an Enzyme-Linked Immunoadsorbent Assay for Human Neurofilament Protein

An enzyme-linked immunoadsorbent assay has been developed to evaluate comparative levels of neurofilament protein in developing primary cultures of human foetal dorsal root ganglion and brain tissue. The quantitative parameters of the assay, relating linearity of response with varying levels of neurofilament protein, were verified by comparing the relative binding of human species-specific (BF10) and cross-species-reactive (RT97) monoclonal antibodies to mixtures of human and baboon spinal cord homogenates that had been passively adsorbed onto microtitre wells. In human neural cultures, the localisation of neurofilament protein to growing neurites was determined by indirect immunofluorescence staining with anti-neurofilament antibodies and, using the immunoadsorbent assay, a time-dependent increase in the level of neurofilament protein was detected that correlated with the morphological time course of neurite development. In the case of dorsal root ganglion cells over 6 days in vitro, a seven- to ninefold greater increase in neurofilament protein levels was observed in cultures treated with nerve growth factor when compared with control unstimulated preparations. The quantitative responsiveness of dorsal root ganglion neurones to nerve growth factor detected by the neurofilament assay indicates its potential usefulness in the identification and analysis of neurotrophic and neurotoxic factors or cellular interactions operating in vitro.     

Involvement of gicerin, a cell adhesion molecule, in development and regeneration of chick sciatic nerve

We have examined the role of gicerin, an immunoglobulin superfamily cell adhesion molecule, in chick sciatic nerves during development and regeneration. Gicerin was expressed in the spinal cord, dorsal root ganglion (DRG) and sciatic nerves in embryos, but declined after hatching. Neurite extensions from explant cultures of the DRG were promoted on gicerin's ligands, which were inhibited by an anti-gicerin antibody. Furthermore, gicerin expression was upregulated in the regenerating sciatic nerves, DRG and dorsal horn of the spinal cord after injury to the sciatic nerve. These results indicate that gicerin might participate in the development and regeneration of sciatic nerves.