内源性信号通路在神经元轴突再生中的功能和机制研究

神经元内源性再生能力是神经元再生和功能恢复的关键。近年研究发现了多个调控轴突再生的内源性信号通路,其中丝裂原活化蛋白激酶（MAPK）信号通路和磷脂酰肌醇3-激酶/蛋白激酶B（PI3K/Akt）信号通路最具代表性。MAPK信号通路参与轴突损伤的感知、再生的启动和维持等过程,通过调节蛋白合成和细胞骨架来调控轴突再生;PI3K/Akt信号通路则通过调节损伤后神经元内相关基因的转录和翻译调控轴突再生。多种再生促进信号的共同作用能进一步提升轴突再生能力。本文综述了MAPK和PI3K/Akt信号通路在不同模式生物中调控轴突再生的研究进展,并展望了其在促进体内神经功能恢复上的重要作用。     

大鼠腓总神经“π”式桥接于胫神经后再生神经的电生理溯源

目的 研究大鼠胫神经原位桥接切断的腓总神经，观察腓总神经再生程度、神经纤维的来源等。方法 将断裂的腓总神经近端和远端分别就近与胫神经施行端侧吻合，存活18个月后，电生理检测再生神经纤维的动作电位传导，取腓总神经远段行光镜及电镜观察神经纤维再生数量及状态。结果 远段腓总神经有明显的神经纤维再生，远段腓总神经通过邻近神经的桥接与近段腓总神经之间有动作电位传导。结论 断裂腓总神经“π”式桥接于胫神经，部分再生神经纤维可能来源于原腓总神经近段，部分来自胫神经。     

大鼠腓总神经端侧吻合于胫神经再生神经的逆行追踪

 【目的】 研究大鼠腓总神经被切断端侧吻合于胫神经后, 再生的腓总神经纤维的来源。【方法】 将大鼠腓总神经切断,将其远端与胫神经施行端侧吻合,存活24个月后,再次手术暴露并将HRP（辣根过氧化物酶）注入远段腓总神经干内,72h后取大鼠脊髓L3-6节段和L4、L5脊神经节冰冻切片,用四甲基联苯胺显色显示神经细胞;并取腓总神经远段、腓总神经近段中部行电镜观察神经纤维超微结构,正常腓总神经作对照。【结果】 模型鼠远段腓总神经再生神经纤维清晰、明显,神经纤维略细;脊髓L3-6节段双侧灰质前角和双侧L4、L5脊神经节均见到蓝染细胞,均以手术侧为少;腓总神经近段与正常腓总神经比较无差异、未见华勒氏变迹象。【结论】 断裂腓总神经端侧吻合于胫神经,再生神经纤维不支持因腓总神经近段溃变轴突退缩形成生长锥引导所致,来源于胫神经可能性大。     

奥氮平连续处理后大鼠中缝背核组织差异表达蛋白的生物信息学分析

目的 分析奥氮平连续处理的大鼠中缝背核蛋白的差异表达,探究奥氮平使用早期导致代谢障碍可能的中枢5-HT机制。方法 将40只SD大鼠随机分配到奥氮平组[灌胃奥氮平1.2 mg/(kg·d）]和对照组（灌胃等量0.9%氯化钠溶液）,两组各分配10只雌性大鼠、10只雄性大鼠。给药1次/d,连续28 d。最后一次给药1 h后处理大鼠,并取大脑中缝背核样本。利用绝对和相对定量同位素标记技术联合液相色谱-串联质谱技术对大鼠中缝背核组织进行蛋白质组学分析,进一步对差异表达蛋白进行GO、KEGG通路、COG、蛋白互作网络分析。另将24只大鼠随机分为4组：2个奥氮平组和2个对照组（6只/组）,类似方法得到大鼠中缝背核样本,根据蛋白质组学数据选择目标基因的表达进行qRT-PCR和Western blot验证。结果 筛选出奥氮平组与对照组大鼠中缝背核差异表达蛋白有72种上调、142种下调。GO注释分析显示,涉及奥氮平的差异表达蛋白参与细胞过程、生物调节、代谢过程、应激反应、多细胞生物过程以及结合、催化活性、分子功能调节、转录调节活性等分子功能。KEGG富集分析显示,涉及奥氮平的差异表达蛋白主要参与流体剪切应...     

Induction of axon and dendrite formation during early RGC-5 cell differentiation

The retinal ganglion cell (RGC)-like RGC-5 line can be differentiated with staurosporine to stop dividing, extend neurites, and increase levels of several ganglion cell markers. This allows study of regulation of neurite development on a single cell basis. However, it is unclear whether the neurites induced by differentiation have features characteristic of dendrites or axons. To address this question, RGC-5 cells were differentiated with staurosporine and then immunoblotted for microtubule-associated protein 2 (MAP2) and actin, or stained immunocytochemically for different MAP2 isoforms, tau, growth-associated protein 43 (GAP-43), or the neuronal marker beta-III-tubulin. We found that staurosporine-induced differentiation led to an upregulation of MAP2c, a MAP2 isoform expressed in developing neurons. Some neurites expressed MAP2c but not the dendritic markers MAP2a and MAP2b, consistent with an axonal phenotype. Some neurites expressed the axonal marker tau in a characteristic proximal-to-distal gradient, and had GAP-43 labeling characteristic of axonal growth cones. The presence of MAP2c in differentiated RGC-5 cells is indicative of RGC-like neurite development, and the pattern of staining for the different MAP2 isoforms, as well as positivity for tau and GAP-43, indicates that differentiation induces axon-like and dendrite-like neurites.     

The mechanism of astragaloside IV promoting sciatic nerve regeneration

3-O-beta-D-xylopyranosyl-6-O-beta-D-glucopyranosyl-cycloastragenol (astragaloside Ⅳ), the main active component of the traditional Chinese medicine astragalus membranaceus, has been shown to be neuroprotective. This study investigated whether astragaloside Ⅳ could promote the repair of injured sciatic nerve. Denervated sciatic nerve of mice was subjected to anastomosis. The mice were intraperitoneally injected with 10, 5, 2.5 mg/kg astragaloside Ⅳ per day for 8 consecutive days. Western blot assay and real-time PCR results demonstrated that growth-associated protein-43 expression was upregulated in mouse spinal cord segments L4-6 after intervention with 10, 5, 2.5 mg/kg astragaloside Ⅳ per day in a dose-dependent manner. Luxol fast blue staining and electrophysiological detection suggested that astragaloside Ⅳ elevated the number and diameter of myelinated nerve fibers, and simultaneously increased motor nerve conduction velocity and action potential amplitude in the sciatic nerve of mice. These results indicated that astragaloside Ⅳ contributed to sciatic nerve regeneration and functional recovery in mice. The mechanism underlying this effect may be associated with the upregulation of growth-associated protein-43 expression.     

Influence of sex and estrous cycle on the effects of acute tryptophan depletion induced by a gelatin-based mixture in adult Wistar rats

Women are more vulnerable to develop depression and anxiety disorders than men. This may be related to higher serotonergic vulnerability in women. Serotonergic vulnerability entails that differences between people in the regulation of serotonin (5-HT) determine the vulnerability of an individual to develop depression or other 5-HT-related disorders. The aim of the present experiment was to evaluate whether male and female Wistar rats differ in serotonergic vulnerability. Here, a stronger behavioral response to acute tryptophan (TRP) depletion was assumed to reflect serotonergic vulnerability. Twenty-four male and 48 female rats were repeatedly subjected to treatment with a gelatin-based protein-carbohydrate mixture, either with or without L-tryptophan. Female estrous cycle phase was determined by means of vaginal smears and the females were divided into two groups based on their estrous cycle phase: pro-estrus/estrus and met-estrus/di-estrus. Blood samples showed stronger TRP depletion in males than females. There was no effect of estrous cycle on plasma TRP concentrations. In contrast, treatment effects on some brain TRP concentrations were influenced by estrous cycle phase, females in pro-estrus/estrus showed the strongest response to TRP depletion. In the open field test and home cage emergence test, females in pro-estrus/estrus also showed the strongest behavioral response to acute TRP depletion. In general, females showed more activity than males in anxiety-related situations and this effect appeared to be enhanced by TRP depletion. In the social interaction test, passive body contact in males and females in pro-estrus/estrus was decreased after TRP depletion whereas it was increased in females in the met-estrus/di-estrus phase. Acute TRP depletion affected object recognition, but did not affect behavior in the forced swimming test and a reaction time task. It is concluded that sex and estrous cycle phase can influence the behavioral response to TRP depletion, and that females in pro-estrus/estrus show the strongest behavioral response to acute TRP depletion.     

Anaglyph of Retinal Stem Cells And Developing Axons: Selective Volume Enhancement In Microscopy Images

Retinal stem cell culture has become a powerful research tool, but it requires reliable methods to obtain high‐quality images of living and fixed cells. This study describes a procedure for using phase contrast microscopy to obtain three‐dimensional (3‐D) images for the study of living cells by photographing a living cell in a culture dish from bottom to top, as well as a procedure to increase the quality of scanning electron micrographs and laser confocal images. The procedure may also be used to photograph clusters of neural stem cells, and retinal explants with vigorous axonal growth. In the case of scanning electron microscopy and laser confocal images, a Gaussian procedure is applied to the original images. The methodology allows for the creation of anaglyphs and video reconstructions, and provides high‐quality images for characterizing living cells or tissues, fixed cells or tissues, or organs observed with scanning electron and laser confocal microscopy. Its greatest advantage is that it is easy to obtain good results without expensive equipment. The procedure is fast, precise, simple, and offers a strategic tool for obtaining 3‐D reconstructions of cells and axons suitable for easily determining the orientation and polarity of a specimen. It also enables video reconstructions to be created, even of specimens parallel to the plastic base of a tissue culture dish, It is also helpful for studying the distribution and organization of living cells in a culture, as it provides the same powerful information as optical tomography, which most confocal microscopes cannot do on sterile living cells. Anat Rec, 297:770–780, 2014. © 2014 Wiley Periodicals, Inc.     

Myelinated axons and the pyramidal cell modules in monkey primary visual cortex

In addition to the horizontal bands of myelinated axons that produce the line of Gennari and the inner band of Baillarger, the macaque primary visual cortex contains prominent vertical bundles of myelinated axons. In tangential sections through layer IVC, these axon bundles are regularly arranged. They have a mean center-to-center spacing of about 23 μm, and each one contains an average of 34 (S.D. ± 13) myelinated axons. These bundles seem to be largely composed of efferent fibers, because in material in which pyramidal cells have been labelled in layer II/III and in layers IVA and IVB the axons of these neurons descend towards the white matter in bundles. However, it is doubtful whether all of the descending myelinated axons from the superficial layers emerge from the cortex, since counts show that the bundles contain maximum numbers of myelinated axons at the level of layer IVC, and that in layers V and VI their number is reduced by about 30%. Perhaps some of the axons enter the line of Baillarger, in layer V. When the bundles of myelinated axons and the clusters of apical dendrites of the layer V pyramidal cells are visualized simultaneously within layer IVC in electron microscopic preparations, it is apparent that their center-to-center spacing is similar, namely, about 23 μm and that a bundle of axons has a cluster of apical dendrites lying adjacent to it. Because of this association, and because axons from layer III pyramidal cells have been shown to enter the bundles, it is suggested that the myelinated axon bundles contain the efferent axons from the projection neurons in the individual pyramidal cell modules. However, in addition to the myelinated axons, the bundles contain unmyelinated axons, so that they also probably serve as the conduits for axons forming connections between layers. It is proposed that the pyramidal cell modules are the basic, functional neuronal units of the visual cortex, and since the neurons within a particular module can be expected to have slightly different inputs and response properties from those in neighboring modules, the individual axon bundles that emerge from each module would be expected to carry a unique set of efferent information. © 1996 Wiley-Liss, Inc.     

Neurofilament spacing,phosphorylation, and axon diameter in regenerating and uninjured lamprey axons

It has been postulated that phosphorylation of the carboxy terminus sidearms of neurofilaments (NFs) increases axon diameter through repulsive electrostatic forces that increase sidearm extension and interfilament spacing. To evaluate this hypothesis, the relationships among NF phosphorylation, NF spacing, and axon diameter were examined in uninjured and spinal cord-transected larval sea lampreys (Petromyzon marinus). In untransected animals, axon diameters in the spinal cord varied from 0.5 to 50 μm. Antibodies specific for highly phosphorylated NFs labeled only large axons (>10 μm), whereas antibodies for lightly phosphorylated NFs labeled medium-sized and small axons more darkly than large axons. For most axons in untransected animals, diameter was inversely related to NF packing density, but the interfilament distances of the largest axons were only 1.5 times those of the smallest axons. In addition, the lightly phosphorylated NFs of the small axons in the dorsal columns were widely spaced, suggesting that phosphorylation of NFs does not rigidly determine their spacing and that NF spacing does not rigidly determine axon diameter. Regenerating neurites of giant reticulospinal axons (GRAs) have diameters only 5–10% of those of their parent axons. If axon caliber is controlled by NF phosphorylation via mutual electrostatic repulsion, then NFs in the slender regenerating neurites should be lightly phosphorylated and densely packed (similar to NFs in uninjured small caliber axons), whereas NFs in the parent GRAs should be highly phosphorylated and loosely packed. However, although linear density of NFs (the number of NFs per micrometer) in these slender regenerating neurites was twice that in their parent axons, they were highly phosphorylated. Following sectioning of these same axons close to the cell body, axon-like neurites regenerated ectopically from dendritic tips. These ectopically regenerating neurites had NF linear densities 2.5 times those of uncut GRAs but were also highly phosphorylated. Thus, in the lamprey, NF phosphorylation may not control axon diameter directly through electrorepulsive charges that increase NF sidearm extension and NF spacing. It is possible that phosphorylation of NFs normally influences axon diameter through indirect mechanisms, such as the slowing of NF transport and the formation of a stationary cytoskeletal lattice, as has been proposed by others. Such a mechanism could be overridden during regeneration, when a more compact, phosphorylated NF backbone might add mechanical stiffness that promotes the advance of the neurite tip within a restricted central nervous system environment. © 1996 Wiley-Liss, Inc.