骨髓间充质干细胞与施万细胞联合移植对大鼠周围神经 损伤端侧吻合的修复效果研究

目的:探究骨髓间充质干细胞(MSCs)与施万细胞(SCs)联合移植对大鼠周围神经损伤端侧吻合的修复效果。方法:选取SD雌性大鼠60只均制作成坐骨神经损伤端侧吻合模型,并将其随机分为联合移植组、MSCs组和SCs组,分别对吻合端进行骨髓间充质干细胞与SCs联合移植、MSCs移植、SCs移植。观察分析三组大鼠的神经电生理学指标和腓神经功能指数(PFI)和神经传导速度(NCV)。结果:三组大鼠的PFI和NCV均有所改善,且联合移植组的PFI和NCV均优于其他两组,并随着时间推移损伤坐骨神经功能恢复越来越好。结论:MSCs与SCs均具有促进大鼠周围神经身上修复的功能,且两种细胞联合移植效果更加明显。     

太鼠坐骨神经、胫神经、腓总神经在脊髓胶状质定位投射的定量分析—FRAP 法

本实验按照跨神经节溃变的原理,用抗氟化物酸性磷酸酶(FRAP)法和显微测量,对大鼠坐骨神经,胫神经和腓总神经感觉纤维在脊髓胶状质的定位投射进行了定量分析。大鼠坐骨神经和胫神经向胶状质的纵向投射为 L_(2～3);腓总神经为 L_(2.6)及 S_1的上中部。水平向投射,坐骨神经:L_(2～3)主要为胶状质的最内侧和中间区的部分区域,L_4～S_1,主要为内侧,中间和部分外侧区的全部胶状质,但未见向胶状质最外侧区投射;胫神经:L_2～S_1主要为内侧区,中间及部分外侧区仅部分实验动物有投射;腓总神经:仅向 L_2～S_1的中间和部分外侧区一处投射。     

游离锌离子轴突运输的实验研究

目的：研究大鼠坐骨神经结扎后游离锌离子在轴突内的定位分布,探讨锌离子在含锌神经元内的轴突运输。方法：应用轴流阻滞／神经结扎术结合光、电镜锌金属自显影技术,检测含锌神经元轴突内的游离锌离子子。结果：锌阳性反应产物主要分布在靠近结扎点的坐骨神经近端和远端轴突内,并且随着结扎时间的延长锌离子在轴突近端和远端的积累逐渐增加。此外,电镜结果表明锌离子主要定位于无髓神经纤维以及薄髓鞘的有髓神经纤维轴突内。结论：游离锌离子在含锌神经元轴突内进行双向轴突运输,即顺行运输和逆行运输。     

周围神经或其组织成分移植修复成年哺乳动物视网膜节细胞的损伤

本综述重点阐述了移植周围神经或其组织成分雪旺细胞、成纤维细胞和神经营养因子,改善成年哺乳动物中枢神经系统抑制神经再生的微环境、增强受损神经元的内在再生潜力,以促进细胞损伤后的存活和轴突再生。     

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.     

关于健康成人左右运动神经传导参数的比较

目的:研究正常人左、右侧的末梢神经传导特点及易损伤性,探讨生活习惯与末梢神经潜在性损伤的内在关联,提高电生理诊断准确率。方法:100名志愿者为对象,检测正中、尺、胫和腓神经的复合肌肉动作电位(CMAP)、F波,观察左、右侧的神经传导参数及左右差值与生活习惯之间的联系。结果:左侧尺、胫运动神经传导速度(MCV)慢于右侧(P值各为0.013、0.011)。MCV≤X-1S尺神经组的远端潜伏期(D Lat)、F波最短潜伏期(F-Lat)延长于MCV>X-1S组(P值均为0.000)。MCV≤X-1S胫神经组的近端波幅(P Amp)低于MCV>X-1S组(P=0.000)。右侧腓神经D Lat延长于左侧(P=0.007),D Lat≥X+1S腓神经组的MCV、F-Lat平均值慢或延长于D Lat相似文献   

三维取向PLGA神经导管促进坐骨神经再生的实验研究

目的:探讨应用改进静电纺丝技术一次成型制备三维(3D)取向聚乳酸与聚羟基乙酸共聚物(PLGA)纳米神经导管的可行性,检测其对坐骨神经再生的促进作用。方法:应用改进的静电纺丝技术制备无缝取向PLGA纳米神经导管,通过扫描电镜和透射电镜检测支架的纳米结构;分别制备取向和非取向纳米纤维支架修复13mm坐骨神经缺损模型。36只成年SD大鼠随机分为3组(每组12只),A组:非取向PLGA神经导管组(阴性对照);B组:取向PLGA神经导管组,C组:自体神经移植组(阳性对照),于术后3月通过大体观察、行走足印分析、腓肠肌萎缩率、电生理检测、组织形态学检测、透射电镜检测及图像分析,评价无缝取向PLGA纳米神经导管修复坐骨神经缺损的效果。结果:神经导管修复神经缺损三月后,大体观察显示神经导管结构完整,无坍塌和断裂;各组再生神经均有通过神经导管长入远端。B组与C组的腓肠肌萎缩率和神经电传导速度无统计学差异(P0.05),均优于A组。B组与C组再生神经纤维数量及成熟程度均要明显优于A组。结论:无缝取向PLGA纳米神经导管能够诱导并促进神经再生,提高坐骨神经再生的质量,有望成为自体神经移植的替代物。     

植入血管束的血管化人工神经导管对受损神经功能恢复影响的实验研究

目的:研究植入血管束的血管化人工神经导管修复SD大鼠长段坐骨神经缺损对神经功能恢复的影响。方法:将18只成年雌性SD大鼠制成14mm的大鼠坐骨神经缺损模型后,随机分为3组(每组12条神经),分别采用不同的修复方法。A组:自体神经移植组(自体组);B组:普通PGLA神经导管移植组(导管组);C组:植入自体血管束的普通PGLA神经导管移植组(血管化导管组)。观察术后大鼠后肢皮肤溃疡面积;检测术后6周、12周时步态变化和肌电图。结果:术后各组SD大鼠均出现后肢溃疡,血管化导管组SD大鼠后肢溃疡愈合较导管组早2周。血管化导管组步态检测SFI明显优于导管组,与自体神经移植组无明显差异。肌电图检测表明血管化导管组无论是神经传导速度,还是动作电位振幅均明显大于导管组(P<0.05),与自体神经移植组无明显差异(P>0.05)。结论:植入血管束的血管化人工神经导管能有效地促进受损神经的功能恢复。     

周围神经损伤后神经组织中Par-3蛋白表达和分布的变化

目的观察极性蛋白Par-3在损伤后神经组织中的表达和分布,探讨Par-3蛋白在周围神经损伤后髓鞘再生中的作用。方法 32只Sprague Dawley大鼠随机分为正常对照组、损伤组(坐骨神经损伤后第1、2、4、8周)。制备坐骨神经挤压伤模型,分别于损伤后各时间点,采用免疫组织化学法检测坐骨神经损伤远端Par-3蛋白的表达和分布。结果正常大鼠坐骨神经组织中即存在Par-3蛋白,但表达量少,且仅分布于Schwann细胞核内。坐骨神经损伤后,Par-3蛋白的表达和分布发生变化。损伤后1周,Par-3蛋白表达开始升高,Par-3散在分布于Schwann细胞核和细胞浆内。损伤后2周,神经组织中的Par-3蛋白达峰值,在Schwann细胞浆内呈不对称性分布似包绕轴突,呈新月形或C形。损伤后4周和8周,Par-3蛋白表达显著降低,神经组织中Par-3蛋白主要分布于Schwann细胞核内,胞浆内很少。结果 极性蛋白Par-3可能参与周围神经损伤后Schwann细胞的髓鞘再生。     

聚己内酯/壳聚糖神经导管复合骨髓间充质干细胞促进大鼠坐骨神经损伤修复的研究

目的:观察聚己内酯/壳聚糖神经导管复合骨髓间充质干细胞修复大鼠坐骨神经缺损的效果。方法:将24只SD大鼠随机分为4组,制备右侧坐骨神经5mm缺损模型,A组聚己内酯/壳聚糖神经导管复合骨髓间充质干细胞移植组;B组聚己内酯神经导管复合骨髓间充质干细胞移植组;C组壳聚糖神经导管复合骨髓间充质干细胞移植组;D组自体神经移植组。术后每2周进行坐骨神经功能指数检测,12周时行电生理、腓肠肌湿重恢复率、组织学观察和免疫组织化学检测。结果:坐骨神经功能指数显示,A组运动功能恢复速度较B、C组快,但比D组慢。A组电生理和腓肠肌湿重恢复率的检测结果与C、D组相比无统计学意义(P0.05),但优于B组(P0.05)。组织学观察,A组再生神经纤维排列密集。S-100免疫组织化学结果表明A组有大量雪旺细胞增生。结论:聚己内酯/壳聚糖神经导管复合骨髓间充质干细胞能够促进周围神经损伤修复,效果与壳聚糖神经导管、自体神经相同,优于聚己内酯神经导管。     

Changes in Rapid Transport of Phospholipids in the Rat Sciatic Nerve During Axonal Regeneration

Abstract: Axonal transport of phospholipids in normal and regenerating sciatic nerve of the rat was studied. At various intervals after axotomy of the right sciatic nerve in the midthigh region and subsequent perineurial sutures of the transected fascicles, a mixture of 60 μCi [Me-^HC]choline and 15 μCi [2-³H]glycerol in the region of the spinal motor neurons of the L5 and L6 segments was injected bilaterally. The amount of radioactive lipid (and in certain cases its distribution in various lipid classes) along the nerve was determined as a function of time. Three days after fascicular suture and 6 h after spinal cord injection of precursors, there was an accumulation of labeled phospholipids and sphingolipids in the transected sciatic nerve in the region immediately proximal to the site of suture. Nine days after, there was a marked increase in the accumulation of radioactivity in the distal segments of the injured nerve, which increased up to 14 days after cutting and disappeared as regeneration proceeded (21–45 days). In all segments of both normal and regenerating nerve fibers, as well as in L5 and L6 spinal cord segments, only phosphatidylcholine and sphingomyelin were labeled with [¹⁴C]choline. These results suggest that the regeneration process in a distal segment of a peripheral neuron, following cutting and fascicular repairing by surgical sutures, is sustained in the first 3 weeks by changes in the amount of phospholipids rapidly transported along the axon towards the site of nerve fiber outgrowth.     

Protein Synthesis and Axonal Transport During Nerve Regeneration

Abstract— Protein synthesis and axonal transport have been studied in regenerating peripheral nerves. Sciatic nerves of bullfrogs were unilaterally crushed or cut. The animals were killed 1, 2, or 4 weeks later, and 8th and 9th dorsal root ganglia removed together with sciatic nerves and dorsal roots. The ganglia were selectively labeled in vitro with [³⁵S]-methionine. Labeled proteins, in dorsal root ganglia and rapidly transported to ligatures placed on the sciatic nerves and dorsal roots, were analyzed by two-dimensional polyacryl-amide gel electrophoresis. Qualitative analysis of protein patterns revealed no totally new proteins synthesized or rapidly transported in regenerating nerves. However, quantitative comparison of regenerating and contralateral control nerves revealed significant differences in abundance for some of the proteins synthesized in dorsal root ganglia, and for a few of the rapidly transported proteins. Quantitative analysis of rapidly transported proteins in both the peripheral processes (spinal nerves) and central processes (dorsal roots) revealed similar changes despite the fact that the roots were undamaged. The overall lack of drastic changes seen in protein synthesis and transport suggests that the neuron in its program of normal maintenance synthesizes and supplies most of the materials required for axon regrowth.     

Phosphorylation of Proteins in Normal and Regenerating Goldfish Optic Nerve

Within 6 h after radiolabeled phosphate was injected into the eye of goldfish, labeled acid-soluble and acid-precipitable material began to appear in the optic nerve and subsequently also in the lobe of the optic tectum, to which the optic axons project. From the rate of appearance of the acid-precipitable material, a maximal velocity of axonal transport of 13-21 mm/day could be calculated, consistent with fast axonal transport group II. Examination of individual proteins by two-dimensional gel electrophoresis revealed that approximately 20 proteins were phosphorylated in normal and regenerating nerves. These ranged in molecular weight from approximately 18,000 to 180,000 and in pI from 4.4 to 6.9. Among them were several fast transported proteins, including protein 4, which is the equivalent of the growth-associated protein GAP-43. In addition, there was phosphorylation of some recognizable constituents of slow axonal transport, including alpha-tubulin, a neurofilament constituent (NF), and another intermediate filament protein characteristic of goldfish optic axons (ON2). At least some axonal proteins, therefore, may become phosphorylated as a result of the axonal transport of a phosphate carrier. Some of the proteins labeled by intraocular injection of 32P showed changes in phosphorylation during regeneration of the optic axons. By 3-4 weeks after an optic tract lesion, five proteins, including protein 4, showed a significant increase in labeling in the intact segment of nerve between the eye and the lesion, whereas at least four others (including ON2) showed a significant decrease. When local incorporation of radiolabeled phosphate into the nerve was examined by incubating nerve segments in 32P-containing medium, there was little or no labeling of the proteins that showed changes in phosphorylation during regeneration. Segments of either normal or regenerating nerves showed strong labeling of several other proteins, particularly a group ranging in molecular weight from 46,000 to 58,000 and in pI from 4.9 to 6.4. These proteins were presumably primarily of nonneuronal origin. Nevertheless, if degeneration of the axons had been caused by removal of the eye 1 week earlier, most of the labeling of these proteins was abolished. This suggests that phosphorylation of these proteins depends on the integrity of the optic axons.     

In Vivo Phosphorylation of Axonal Proteins in Goldfish Optic Nerve During Regeneration

In vivo phosphorylation of axonal proteins was investigated in normal and regenerating optic nerves of goldfish by two-dimensional gel electrophoresis. By 6-24 h after intraocular injection of H3(32)PO4, approximately 20 optic nerve proteins ranging in size from 19 to 180 kilodaltons and in pI from 4.4 to 6.8 were seen to have incorporated radiolabel. Five of these proteins showed a robust increase in incorporation of phosphate during regeneration. Among the latter was an acidic (pI 4.5) 45-kilodalton protein, which has previously been shown to be conveyed by fast axonal transport and to increase dramatically in its rate of synthesis during regeneration of goldfish optic axons.     

Changes in Protein Content of Goldfish Optic Nerve During Degeneration and Regeneration Following Nerve Crush

Abstract: After the goldfish optic nerve was crushed, the total amount of protein in the nerve decreased by about 45% within 1 week as the axons degenerated, began to recover between 2 and 5 weeks as axonal regeneration occurred, and had returned to nearly normal by 12 weeks. Corresponding changes in the relative amounts of some individual proteins were investigated by separating the proteins by two-dimensional gel electrophoresis and performing a quantitative analysis of the Coomassie Brilliant Blue staining patterns of the gels. In addition, labelling patterns showing incorporation of [³H]proline into individual proteins were examined to differentiate between locally synthesized proteins (presumably produced mainly by the glial cells) and axonal proteins carried by fast or slow axonal transport. Some prominent nerve proteins, ON1 and ON2 (50–55 kD, pI ～6), decreased to almost undetectable levels and then reappeared with a time course corresponding to the changes in total protein content of the nerve. Similar changes were seen in a protein we have designated NF (～130 kD, pI ～5.2). These three proteins, which were labelled in association with slow axonal transport, may be neurofilament constituents. Large decreases following optic nerve crush were also seen in the relative amounts of α- and β-tubulin, which suggests that they are localized mainly in the optic axons rather than the glial cells. Another group of proteins, W2, W3, and W4 (35–45 kD, pI 6.5–7.0), which showed a somewhat slower time course of disappearance and were intensely labelled in the local synthesis pattern, may be associated with myelin. A small number of proteins increased in relative amount following nerve crush. These included some, P1 and P2 (35–40 kD, pIs 6.1–6.2) and NT (～50 kD, pI ～5.5), that appeared to be synthesized by the glial cells. Increases were also seen in one axonal protein, B (～45 kD, pI ～4.5), that is carried by fast axonal transport, as well as in two axonal proteins, HA1 and HA2 (～60 and 65 kD respectively, pIs 4.5–5.0), that are carried mainly by slow axonal transport. Other proteins, including actin, that showed no net changes in relative amount (but presumably changed in absolute amount in direct proportion to the changes in total protein content of the nerve), are apparently distributed in both the neuronal and nonneuronal compartments of the nerve.     

Axonal Transport of Choline Lipids in Normal and Regenerating Rat Sciatic Nerve

Abstract: Biochemical methods were used to study the time course of transport of choline phospholipids (labeled by the injection of [³H]choline into the ventral horn of the lumbar spinal cord) in rat sciatic nerve. Autoradiographic methods were used to localize the transported lipid within motor axons. Transported phospholipid, primarily phosphatidylcholine, present in the nerve at 6 h, continued to accumulate over the following 12 days. No discrete waves of transported lipid were observed (a small wave of radioactive phospholipid moving at the high rate would have been missed); the amounts of radioactive lipid increased uniformly along the entire sciatic nerve. In light-microscope autoradiographs, a class of large-caliber axons, presumably motor axons, retained the labeled lipid. Some lipid, even at 6 h, was seen within the myelin sheaths. Later, the labeling of the myelin relative to axon increased. The continued accumulation of choline phospholipids in the axons probably signifies their prolonged release from cell bodies and their retention in various axonal membranes, including the axolemma. The build-up of these phospholipids in myelin probably represents their transfer from the axons to the myelin sheaths surrounding them. When nerves are crushed and allowed to regenerate for 6 or 12 days, choline phospholipids transported during these times enter the regenerating nerve. In light and electron microscope autoradiographs, transported lipid was seen to be localized primarily in the regenerating axons. However, grains overlay the adjacent Schwann cell cytoplasm, indicating transported lipids were transferred from the regenerating axons to the associated Schwann cells. In addition, some cells not associated with growing axons were labeled, suggesting that phosphatidylcholine and possibly acetylcholine, carried to the regenerating axons by axonal transport, were actively metabolized in the terminal, with released choline label being used by other cells. These results demonstrate that axonal transport supplies mature and growing axons and their glial cells with choline phospholipids.     

Cholesterol Synthesis and Nerve Regeneration

Abstract: In this report, we examine the requirement of cholesterol biosynthesis and its axonal transport for goldfish optic nerve regeneration. Cholesterol, labeled by intraocular injection of [³H]mevalonolactone. exhibited a delayed appearance in the optic tectum. Squalene and other minor components were labeled but not transported. Following optic nerve crush, the amount of labeled cholesterol transport was elevated, while retinal labeling was not altered relative to control fish. A requirement for cholesterol biosynthesis is inferred from the inhibition of neurite outgrowth in retinal explants caused by the cholesterol synthesis inhibitor, 20, 25-diazacholes-terol. The inhibition of growth could be overcome by addition of mevalonolactone, but not cholesterol, to the medium. Intraperitoneal administration of 200 nmol of dia-zacholesterol resulted in 92-98% inhibition of retinal cholesterol synthesis and accumulation of labeled des-mosterol and other lipids in fish retina and brain which persisted for 2 weeks. Diazacholesterol-treated fish showed no reduction in the amount of lipid-soluble radioactivity transported following intraocular injection of [³H]mevalonolactone, but there were alterations in the chromatographic pattern of the transported labeled lipids. In contrast to its effects on neurite outgrowth in vitro , diazacholesterol did not inhibit optic nerve regeneration in vivo , as measured both by arrival of labeled rapidly transported protein at the tectum and by time required for the return of visual function.     

Regeneration of motor axons in crayfish limbs: Distal stump activation followed by synaptic reformation

Summary Servered distal stumps of limb motor axons in the crayfish Procambarus clarkii remain ultrastructurally intact for at least 2–3 ms after being severed from their cell body. Initial regeneration of a motor axon is associated with the appearance of up to 200 small profiles (satellite axons) having no glial sheath adjacent to the large surviving stump for about 1 cm distal to the lesion at 4–5 wks postoperatively. These satellite axons are seen 2–4 cm distally at the target muscles 3–4 ms postoperatively. By 14–15 ms postoperative, the motor sheaths from the lesion site to the target muscles contain small axonal processes having thick glial sheaths. Behavioral tests show that some axons that are reconnected to the CNS at 4–5 wks may not be connected at 14–15 ms, whereas other axons not connected by 3–4 ms may be connected at 14–15 ms when the original distal stumps have degenerated.We suggest that all these data can best be explained by the view that motor axons in crayfish limbs initially regenerate via activation of the surviving distal stump by satellite axons which grow out from proximal stump. In most cases, these satellite axons continue to activate the surviving distal stump as they slowly grow to the target muscle. Eventually the satellite axons reform synapses on the target muscle and the original distal stump degenerates.This work was supported by NSF grants BNS 77-27678 and 80-22248 and an NIH RCDA 00070 to GDB. The authors would like to thank Mr. Martis Ballinger, Mr. Robert Reiss, and Mrs. Mary Raymond for their excellent technical assistance. We would also like to thank Dr. Wesley Thompson and Mr. Douglas Baxter for helpful discussions.     

Quantitative Analysis of Axonal Transport of Cytoskeletal Proteins in Chicken Oculomotor Nerve

We studied the axonal transport characteristics of major cytoskeletal proteins: tubulin, the 69,000 molecular weight protein of chicken neurofilaments, and actin. After intracerebral injection of [35S]methionine, we monitored the specific radioactivity of these proteins as they passed through a very short nerve segment of the chicken oculomotor nerve. Specific radioactivities were assessed by quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The transport patterns obtained for tubulin and the neurofilament protein were very similar, corresponding to transport rate ranges of 1-15 and 1-10 mm/day, respectively. A narrower velocity range of 3 to 4.3 mm/day was found for actin. Tubulin and the neurofilament protein appeared to be largely dispersed during the course of their transit along the nerve. The radioactivity associated with the proteins studied persisted in the nerve segment for a long time after the bulk of the labeled molecules had swept down. Finally, none of these proteins was observed to be transported with the fast axonal transport.