CDK11P58和Cyclin D3在损伤后大鼠坐骨神经中的表达变化

为了探讨大鼠坐骨神经损伤对CDKll^p58和Cyclin D3表达的影响，本实验将成年SD大鼠随机分为假手术组和夹伤组，运用Western blot结合免疫荧光组织化学双标技术，观察了坐骨神经损伤后坐骨神经夹伤段、脊髓腰膨大前角和伤侧腓肠肌内CDKll^P58和Cyclin D3的表达变化。结果显示：（1）坐骨神经夹伤后，坐骨神经夹伤段及脊髓腰膨大前角、伤侧腓肠肌中的CDKll^58蛋白的表达先逐渐下降，后又逐渐上升；而Cyclin D3的表达无明显变化；（2）在假手术组的坐骨神经、脊髓腰嘭大前角和腓肠肌内都可观察到CDKll^P58和Cyclin D3的表达；而坐骨神经夹伤后，在上述部位可检测到CDKll^P58的表达强度明显减弱，但Cyclin D3无明显变化；（3）免疫荧光双标结果显示CDKll^P58和Cyclin D3在假手术组的坐骨神经、脊髓腰膨大前角和伤侧腓肠肌内都有共存；而在坐骨神经损伤后这种共存强度明显降低。以上结果提示，坐骨神经损伤可影响坐骨神经及脊髓腰膨大、伤侧腓肠肌内CDKll^P58的表达，但对Cyclin D3无明显影响，表明CDKll^P58可能在周围神经损伤和修复中发挥重要作用。     

CDK11P58和Cyclin D3在损伤后大鼠坐骨神经中的表达变化

为了探讨大鼠坐骨神经损伤对CDK11P58和Cyclin D3表达的影响,本实验将成年SD大鼠随机分为假手术组和夹伤组,运用Western blot结合免疫荧光组织化学双标技术,观察了坐骨神经损伤后坐骨神经夹伤段、脊髓腰膨大前角和伤侧腓肠肌内CDK11P58和Cyclin D3的表达变化.结果显示:(1)坐骨神经夹伤后,坐骨神经夹伤段及脊髓腰膨大前角、伤侧腓肠肌中的CDK11P58蛋白的表达先逐渐下降,后又逐渐上升;而Cyclin D3的表达无明显变化;(2)在假手术组的坐骨神经、脊髓腰膨大前角和腓肠肌内都可观察到CDK11P58和Cyclin D3的表达;而坐骨神经夹伤后,在上述部位可检测到CDK11P58的表达强度明显减弱,但Cyclin D3无明显变化;(3)免疫荧光双标结果显示CDK11P58和Cyclin D3在假手术组的坐骨神经、脊髓腰膨大前角和伤侧腓肠肌内都有共存;而在坐骨神经损伤后这种共存强度明显降低.以上结果提示,坐骨神经损伤可影响坐骨神经及脊髓腰膨大、伤侧腓肠肌内CDK11P58的表达,但对Cyclin D3无明显影响,表明CDK11P58可能在周围神经损伤和修复中发挥重要作用.     

大鼠坐骨神经夹伤后SSeCKS的表达变化

为了探讨大鼠坐骨神经夹伤后SSeCKS(Src抑制的蛋白激酶C底物)在神经中的表达变化及其意义,本研究制备了成年SD大鼠坐骨神经夹伤模型,通过Western blotting和免疫组织化学方法检测坐骨神经夹伤后SSeCKS表达的时空变化。Western blotting结果显示:大鼠坐骨神经夹伤后,SSeCKS的表达迅速升高,伤后6h即达到高峰,之后逐渐下降。免疫组织化学染色结果表明SSeCKS在神经夹伤两端高表达,以近端明显,呈簇状聚集。夹伤远端表达较近端稍低。远离夹伤处及相应对侧,SSeCKS的表达水平有所下降且分布较为均一。免疫荧光组织化学双标记结果显示:夹伤两端SSeCKS与S100和NF200的共存不明显,但可见部分SSeCKS与GAP43双标纤维。本研究提示大鼠坐骨神经夹伤可引起SSeCKS的表达变化,该变化可能参与周围神经损伤后某些伤害性刺激信号分子的转导并与损伤后神经的再生及功能修复有关。     

GDNF在坐骨神经损伤后大鼠L4—6背根节神经元的表达及变化

目的 观察坐骨神经夹伤后不同时间点,胶质细胞源性神经营养因子（ＧＤＮＦ）在成年大鼠Ｌ４～６背根节（ＤＲＧ）中的分布,探讨ＧＤＮＦ对感觉神经元损伤的反应。方法 成年大鼠右侧坐骨神经夹伤后,分别取伤后１、５、７、１０、１４、２８和５６ｄＬ４～６背根节,行抗ＧＤＮＦ多克隆抗体免疫组织化学ＡＢＣ法染色,之后对染色结果进行图像分析。结果 ＧＤＮＦ免疫反应存在于坐骨神经夹伤后１、５、７、１０、１４、２８和５６ｄ成年大鼠Ｌ４～６背根节神经元中,图像分析结果表明,伤后１、３、５和７ｄ的损伤侧背根节神经元的平均光密度大于对照侧背根节神经元。结论 大鼠坐骨神经夹伤后,ＧＤＮＦ在背根节Ｌ４～６感觉神经元中有一定量的变化,且伤后１周内ＧＤＮＦ在伤侧背根节神经元中的表达增强。     

Nogo(N-18)在正常大鼠脊髓和坐骨神经的分布及损伤后变化的免疫组织化学研究

目的　研究Nogo蛋白在大鼠脊髓的正常分布及损伤后的变化 ,探索其在脊髓运动神经元表达的意义和作用。　方法　大鼠分脊髓夹伤组、坐骨神经夹伤组及正常对照组。损伤动物存活 1d和 3d后 ,分别进行Nogo(N 18)抗体的免疫组织化学染色。　结果　正常大鼠脊髓灰质腹角的运动神经元出现强烈的Nogo(N 18)免疫反应 ;而在脊髓白质呈阴性反应 ;寡突胶质细胞呈明显的阳性反应 ;在脊髓全段未发现Nogo染色的星形胶质细胞。在脊髓夹伤区域以外的上下部分 ,灰质和白质对Nogo(N 18)的表达与对照组的脊髓相同 ,在损伤部位的白质 ,出现明显的Nogo(N 18)免疫反应物。在正常和损伤的大鼠坐骨神经均未发现阳性反应的施万细胞。　结论　Nogo蛋白在正常大鼠脊髓运动神经元的分布和在损伤部位的积聚 ,提示人们要重新认识Nogo的功能     

大鼠坐骨神经损伤后Src抑制的蛋白激酶C的底物在背根神经节中的表达变化

目的:探讨大鼠坐骨神经损伤后,Src抑制的蛋白激酶C的底物(SSeCKS)在背根神经节(DRG)中的表达变化及其意义。方法:制备成年SD(Sprague-Dawley)大鼠坐骨神经夹伤及切断模型。通过Western印迹法、Real-time PCR及免疫组织化学方法检测坐骨神经损伤后SSeCKS在DRG中表达的时空变化。结果:大鼠坐骨神经夹伤后6h,DRG中可检测到SSeCKS的表达并逐步升高,伤后12h达到高峰,2d后逐渐下降;而大鼠坐骨神经切断后DRG中SSeCKS的表达高峰发生在伤后2周,1d时最低;SSeCKS主要分布于DRG大、中、小神经元胞质;SSeCKS与NeuN、NF200以及GAP43存在共定位。结论:大鼠坐骨神经损伤后,引起DRG中SSeCKS的表达变化,其可能参与疼痛信号转导通路并与周围神经损伤后的再生有关。     

外周神经损伤大鼠背根神经节中Ephrin B1及RYK 受体表达的变化

目的研究外周神经损伤后背根神经节细胞中Ephrin B1及其相关受体的表达变化。方法建立一侧坐骨神经夹伤的大鼠动物模型,通过免疫荧光组织化学方法检测受损侧背根神经节细胞中Ephrin B1及其相关受体Eph B1、Eph B2、Eph B3和Eph A4、RYK等的表达,并分析阳性细胞数和不同大小阳性细胞的构成比例。结果外周坐骨神经受损侧背根神经节细胞中Ephrin B1的表达明显减弱,而Eph B1、Eph B2、Eph B3和Eph A4受体的表达无明显变化,但RYK受体的表达则明显加强。结论Ephrin B1和RYK受体在一侧外周坐骨神经夹伤后的大鼠背根神经节细胞中表达的变化,说明它很有可能参与了损伤后的功能活动。     

Galβ-1,4-GlcNAc在钳夹伤后大鼠坐骨神经中的表达变化

目的:分析Galβ-1,4-GlcNAc在正常和钳夹伤后大鼠坐骨神经中的表达变化.方法:采用免疫荧光和凝集素组织化学的方法检测Galβ-1,4-GlcNAc在正常和夹伤的大鼠坐骨神经中的表达定位.然后用图像分析的方法分析Galβ-1,4-GlcNAc在正常和夹伤的大鼠坐骨神经中的表达变化.结果:运用免疫荧光、凝集素组织化学方法和图像分析的方法检测Galβ-1,4-GlcNAc在夹伤后的坐骨神经中的表达定位及变化,发现其表达在外周神经中的雪旺细胞中并于夹伤后的一周达到高峰,于夹伤后两周恢复至正常水平.结论:提示Galβ-1,4-GlcNAc在坐骨神经夹伤后发生表达变化,并且主要表达在坐骨神经的雪旺细胞中,说明Galβ-1,4-GlcNAc在周围神经再生的早期发挥着重要的作用.     

Ga1β-1，4-G1cNAc在钳夹伤后大鼠坐骨神经中的表达变化

目的：分析Ga1β-1，4-G1cNAc在正常和钳夹伤后大鼠坐骨神经中的表达变化。方法：采用免疫荧光和凝集素组织化学的方法检测Ga1β-1，4-G1cNAc在正常和夹伤的大鼠坐骨神经中的表达定位。然后用图像分析的方法分析Ga1β-1，4-G1cNAc在正常和夹伤的大鼠坐骨神经中的表达变化。结果：运用免疫荧光、凝集素组织化学方法和图像分析的方法检测Ga1β-1，4-G1cNAc在夹伤后的坐骨神经中的表达定位及变化，发现其表达在外周神经中的雪旺细胞中并于夹伤后的一周达到高峰，于夹伤后两周恢复至正常水平。结论：提示Ga1β-1，4-G1cNAc在坐骨神经夹伤后发生表达变化，并且主要表达在坐骨神经的雪旺细胞中，说明Ga1β-1，4-G1cNAc在周围神经再生的早期发挥着重要的作用。     

p27kip1和Skp2在损伤后大鼠坐骨神经中的表达变化

为了探讨损伤后周围神经p27kip1和S期激酶相关蛋白2(Skp2)的定位表达和变化,本实验将成年SD大鼠随机分为正常对照组、夹伤组和切断组,运用Western blot结合免疫组织化学及免疫荧光双标,在大鼠坐骨神经损伤时,对p27kip1和Skp2表达的影响进行了研究。结果表明:(1)坐骨神经夹伤后,p27kip1蛋白表达先逐渐下降,后又逐渐上升;坐骨神经切断后,远侧段p27kip1蛋白表达持续下降,而近侧段p27kip1蛋白表达在切断后6h明显下降,后又逐渐升高至正常水平,而Skp2表达变化与之相反;(2)免疫组织化学染色结果显示,坐骨神经切断后1w,远侧段从断端到末端,p27kip1阳性信号逐渐增加,而Skp2阳性信号逐渐减弱;(3)免疫荧光双标显示,正常和损伤坐骨神经的雪旺氏细胞中都有p27kip1和Skp2表达。以上结果提示:周围神经损伤后影响雪旺氏细胞中p27kip1和Skp2的表达变化,为进一步研究它们在周围神经损伤和修复中的作用机制奠定基础。     

大鼠中枢神经系统衰老的形态学研究—大鼠舌下神经逆行轴浆运输的年龄性变化

本文用HRP作为神经元标记的示踪剂,研究了不同年龄组大鼠舌下神经逆行轴浆运输的速度以及相同时间内不同年龄组大鼠舌下神经元中HRP的消逝程度。结果表明:随着大鼠的增龄,舌下神经中HRP逆行轴浆运输速度下降,在成年组为122.32mm/日,而老年组仅为73.34mm/日;同时,舌下神经元对HRP的清除能力也下降,在HRP标记细胞数达到最高峰后的60小时,幼年组舌下神经元对HRP的清除率为90%。成年组为87%,而老年组则降为68%。对上述发现所产生的衰老学方面的意义进行了探讨。     

Cervical cord neurons labeled by horseradish peroxidase application to the sciatic nerve in the rat

After horseradish peroxidase (HRP) application to the proximal cut end of the sciatic nerve in rats aged 3-10 days, HRP-labeled neuronal cell bodies were found ipsilaterally in the ventrolateral region of the ventral horn in the cervical enlargements of the spinal cord. Such labeled neurons were occasionally seen in rats aged 15 days, but not seen at all in rats aged 60-90 days. The labeling was presumably the result of a retrograde transneuronal axonal transport of HRP applied to the sciatic nerve.     

The pattern of axonal degeneration in the peripheral nervous system varies with different types of lesion

Degeneration of axons in the mouse sciatic nerve was examined using conventional silver staining and by noting the presence or absence of a compound action potential on stimulating the nerve distal to the point of crush or cut. The presence of myeloperoxidase-positive cells was also examined in frozen sections of the nerve. In all experiments the distal, disconnected segment was studied. Degeneration after crushing with fine watchmakers forceps always began in the most distal part of the nerve and proceeded in a distoproximal direction, from the nerve entry point into a muscle back to the crush site. Myeloperoxidase-positive cells were also recruited into the nerve starting at the distal rather than proximal (the originally injured) end. This result favours the view that degeneration is triggered by lack of trophic support from the cell body rather than entry of deleterious substances at the site of the injury, for the terminals furthest from both the source of supply and the injury are affected first. Degeneration following nerve section was always more rapid than after crushing. The rate of axonal sealing at the injury was, however, no slower than after crushing, so it does not seem likely that greater entry of possible degeneration-triggering material at the injury site is the explanation for this. Crush lesions in which the perineurium was also cut open and the blood supply at the site was damaged, degenerated at the slower rate characteristic of simple crushes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Motor innervation of the mole and shrew snout muscles by means of horseradish peroxidase technique

The axonal transport method of the horseradish peroxidase (HRP) was used for defining the anatomical background of the snout movements in 54 Japanese shrew-moles, Urotrichus talpoides, 4 Temminck's moles, Mogera wogura, and 21 big-clawed shrews, Sorex unguiculatus. Either snout muscle or proximal stump of the facial nerve on one side was exposed to HRP. The results obtained from this experimental study were as follows: HRP-containing cells were found either ipsilaterally or bilaterally in the facial motor nucleus in both the moles and shrews. The HRP-containing cells were grouped into 4 subnuclei: ventrolateral, ventromedial, dorsal and accessory. In case of the snout muscle being exposed to the HRP were the HRP-containing cells found in the ventral groups. A HRP-positive fiber bundle was found at the genu of the facial motor root extending toward the median raphe, when the proximal stump of the facial nerve was exposed to the HRP.     

Localization of neurons giving rise to the oculomotor parasympathetic outflow: A HRP study in cat

Localization of neurons giving rise to preganglionic fibers to the ciliary ganglion was attempted in the cat, utilizing retrograde axonal transport of horseradish peroxidase (HRP). After injection of HRP into the oculomotor nerve root at the level of the interpeduncular fossa, a few neurons of the Edinger-Westphal nucleus (EW) were labeled with HRP rostrally within the anteromedian nucleus (AM);HRP-labeled EW-neurons were rarely seen caudally within the visceral nucleus (VN). Other possible preganglionic neurons labeled with HRP were distributed mainly in rostromedial tegmental areas close to the lateral border of the AM, and in rostroventral areas of the mesencephalic central gray.     

Axonal transport of ribonucleoprotein particles (vaults).

RNA was previously shown to be transported into both dendritic and axonal compartments of nerve cells, presumably involving a ribonucleoprotein particle. In order to reveal potential mechanisms of transport we investigated the axonal transport of the major vault protein of the electric ray Torpedo marmorata. This protein is the major protein component of a ribonucleoprotein particle (vault) carrying a non-translatable RNA and has a wide distribution in the animal kingdom. It is highly enriched in the cholinergic electromotor neurons and similar in size to synaptic vesicles. The axonal transport of vaults was investigated by immunofluorescence, using the anti-vault protein antibody as marker, and cytofluorimetric scanning, and was compared to that of the synaptic vesicle membrane protein SV2 and of the beta-subunit of the F1-ATPase as a marker for mitochondria. Following a crush significant axonal accumulation of SV2 proximal to the crush could first be observed after 1 h, that of mitochondria after 3 h and that of vaults after 6 h, although weekly fluorescent traces of accumulations of vault protein were observed in the confocal microscope as early as 3 h. Within the time-period investigated (up to 72 h) the accumulation of all markers increased continuously. Retrograde accumulations also occurred, and the immunofluorescence for the retrograde component, indicating recycling, was weaker than that for the anterograde component, suggesting that more than half of the vaults are degraded within the nerve terminal. High resolution immunofluorescence revealed a granular structure-in accordance with the biochemical characteristics of vaults. Of interest was the observation that the increase of vault immunoreactivity proximal to the crush accelerated with time after crushing, while that of SV2-containing particles appeared to decelerate, indicating that the crush procedure with time may have induced perikaryal alterations in the production and subsequent export to the axon of synaptic vesicles and vault protein. Our data show that ribonucleoprotein-immunoreactive particles can be actively transported within axons in situ from the soma to the nerve terminal and back. The results suggest that the transport of vaults is driven by fast axonal transport motors like the SV2-containing vesicles and mitochondria. Vaults exhibit an anterograde and a retrograde transport component, similar to that observed for the vesicular organelles carrying SV2 and for mitochondria. Although the function of vaults is still unknown studies of the axonal transport of this organelle may reveal insights into the mechanisms of cellular transport of ribonucleoprotein particles in general.     

大鼠中脑导水管周围灰质－中缝大核－三叉神经脊束核尾侧亚核通路的电镜研究

用ＰＨＡ－Ｌ顺行追踪和ＨＲＰ逆行追踪结合的方法，在电镜下观察到来自中脑导水管周围灰质的ＰＨＡ－Ｌ顺行标记轴突终末与中缝大核向三叉神经脊束核尾侧亚核投射的ＨＲＰ逆行标记神经元形成以非对称性为主的轴树突触。另外，发现顺行标记的轴突终末也与中缝大核内其它神经元之间形成多种突触联系。本研究结果首次在突触水平证明了中脑导水管周围灰质中缝大核－三叉神经脊束核尾侧亚核的间接通路，同时又发现中脑导水管周围灰质下行投射的终末与中缝大核内的神经元成分之间还形成比较复杂的突触联系，并对之进行了讨论。     

On the noradrenaline loading in axonal amine storage granules in rat crushed sciated nerves.

The increase of NA in rat sciatic nerve above a crush was investigated. The transported amounts of NA did not increase quite linearly with time, but more NA was found above the crush at 6 and 12 h than would be expected from the 3 h value. One possible reason for this phenomenon--an increased NA loading of the accumulated axonal granules--was investigated by 2 types of double crush experiments. One type involved simultaneous double crushes 1-1.5 cm apart. The increase in NA in the isolated segment 6 h after crushing indicated that the axonal amine storage granules had increased their NA load by about 70%. In the second type ("delayed double crushes") a distal crush was made 6 h before a second crush, 1-1.5 cm proximal to the first crush. 1-12 h after the second high crush the NA content of the isolated segment was assayed. The results indicated an increased NA content in the axonal granules of 75% already 3-4 h after the isolation of the segment, remaining constant up to 9 h after the second crush. The results indicate that axonal storage granules may increase their NA content by a factor of about 2 (1.7) while being transported distally in the axons. This information together with the information from the preceding article of a mobile NA fraction of 45%, was used to calculate the rate of transport of NA granules proximal to a crush. The value obtained was 9 mm/h which is in good agreement with the value obtained for transport distal to a crush (8 mm/h) in the preceding article.     

Disconnected optic axons persist in the visual pathway during regeneration of the retino-tectal projection in the frog

Summary In this study, we crushed one optic nerve in the frog Litoria (Hyla) moorei and at intervals thereafter anterogradely labelled optic axons with horseradish peroxidase (HRP). For one series, HRP was applied between the eye and the crush site and in a second series between the crush site and the chiasm. A tectal projection of regenerating axons was seen in both series but, in addition, up to 12 weeks post-crush, the second series displayed an additional projection. Its appearance matched that of the disconnected, but persisting, optic axon terminals which are found after enucleation or optic nerve ligation. We conclude that, in the frog, many disconnected optic axons persist throughout the period of optic nerve regeneration and of restoration of an orderly retino-tectal map.Abbreviation HRP horseradish peroxidase