Morphometric analysis of rat superior cervical ganglion after axotomy and nerve growth factor treatment

Summary The changes in neuronal number, cell body size and nuclear size have been followed for 12 weeks after postganglionic axotomy of the rat superior cervical ganglion. Axotomy was carried out at 6 days post partum and treatment with nerve growth factor (NGF) was from 6–21 days.During normal development there is a 30% decrease in the number of neurons in the superior cervical ganglion; axotomy increases the loss of cells resulting in a 90% decrease by 28 days post partum. The normal decrease is prevented and the enhanced loss of cells after axotomy is decreased by administration of NGF. Thus the increased number of cells observed after NGF administration appears to be due to the survival of cells that otherwise would have degenerated.NGF causes a rapid enlargement of both the cell bodies and the nucleus in the normal and axotomized ganglia. This increase in size rapidly reverses after cessation of treatment. These changes in cell size may account for the previously observed changes in cell profile number with NGF.There is a large increase in the number of non-neuronal cells during normal development and axotomy prevents this increase. NGF treatment results in a 6 fold increase in the number of non-neuronal cells and it is suggested that these are required to support the massive fibre outgrowth that occurs in NGF treated ganglia.It is concluded that these results are consistent with a physiological role for NGF as the trophic substance supporting adrenergic neurons making the appropriate contact with their target cell.     

霍乱毒素及外周神经对视神经远端切断后视网膜谷氨酸能节细胞再生的作用

目的：探讨霍乱毒素(CTx)及外周神经对成年金黄地鼠远端视神经受损后视网膜谷氨酸((Glu)能节细胞(RGCs)再生的作用。方法：远端切断视神经并缝接自体坐骨神经(AG),玻璃体内注射CTx及／或植入小段坐骨神经分支(SN)。动物分为AG CTx组;AG SN组;AG SN CTx组,分别存活4W、5W,荧光金和免疫荧光组织化学双标法标记再生的RGCs。结果：术后5W,AG CTx组;AG SN组;AG SN CTx组Glu免疫反应阳性RGCs再生数分别占再生总数的4.25％、2.50％及6.00％,AG SN组与AG SN CTx组间差异显著。结论：CTx与SN能协同促进视神经远端切断后Glu能节细胞的再生．     

Differential effects of intravitreal optic nerve and sciatic nerve grafts on the survival of retinal ganglion cells and the regeneration of their axons

We have investigated the effects of intravitreal sciatic nerve (SN) and/or optic nerve (ON) grafts on the survival and the axonal regeneration of retinal ganglion cells (RGCs). Following transection of the ON, approximately 40% RGCs survived at 7 days post-axotomy (dpa). Results showed that the intravitreal ON graft significantly promoted the survival of RGCs at 7 dpa (39,063 vs 28,246). Intravitreal SN graft, however, did not rescue axotomized RGCs at 5, 7 or 14 dpa. Axotomized RGCs could be induced to regenerate axons along a segment of SN graft attached to the proximal stump of ON. On average, 608 axotomized RGCs were induced to regenerate axons along the attached SN graft. The presence of intravitreal SN graft promoted about 100% increase in the number of regenerating RGCs (1,227) relative to the control groups. The intravitreal ON graft, surprisingly, also induced about 100% more regenerating RGCs (1220) than in the control group. When SN and ON grafts were co-transplanted into the vitreous, about 200% more regenerating RGCs (1916) were observed than in the control group. These findings illustrated that the intravitreal ON graft rescued axotomized RGCs and enhanced the regeneration of retinal axons. This is the first report to show that ON promotes RGC axonal regeneration. The intravitreal SN graft did not rescue RGCs but promoted axonal regeneration. The differential effects of intravitreal ON and SN grafts on the survival and the RGC regeneration suggest that these might be two independently operating events.     

Opiate and histamine H1 receptors are present on some substance P-containing dorsal root ganglion cells

Using combined immunohistochemical and receptor binding techniques, substance P-containing sensory neurones of rhesus monkey cervical dorsal root ganglia were examined for the presence of opiate or histamine (H1) receptors. Serial sets of three sections were examined sequentially for substance P-containing neurones, opiate receptors using [3H]etorphine binding and histamine (H1) receptors using [3H]mepyramine binding. Of 3484 dorsal root ganglion cells, 513 contained substance P. Of 30 randomly chosen substance P-positive neurones, 6 possessed opiate receptors and 7 histamine receptors, including 4 neurones with both sets of binding sites. The results are interpreted to suggest that both nociceptors and non-nociceptive sensory inputs may be biochemically heterogeneous and that a simple correlation between substance P content and a particular receptive field profile is unlikely.     

基因重组碱性成纤维细胞生长因子对大鼠视网膜神经节细胞的影响

目的:研究基因重组碱性成纤维细胞生长因子(rbFGF)对大鼠视网膜神经节细胞(RGCs)的影响。方法:大鼠在视神经部分损伤后,球后注射生理盐水、维生素B₁₂、rbFGF,伤后4周进行轴突定量、视网膜神经节细胞定量以及RGCs凋亡的检测,观察视神经损伤修复情况。结果:伤后4周时,生理盐水和维生素B₁₂对RGCs无挽救作用,800U、1600U和2400U的rbFGF对RGCs挽救率分别为24.5%、27.3%、28.5%,800UrbFGF组、1600UrbFGF组和2400UrbFGF组未发生溃变的轴突数分别是损伤未治疗组的2.03、2.43、2.31倍。流式细胞仪检测结果显示:rbFGF治疗7d后,RGCs凋亡率显著减少。结论:rbFGF可提高视网膜神经节细胞的存活率,减少轴突溃变,有抗凋亡作用,对视神经损伤有显著的促功能修复作用。     

H89和wortmannin对霍乱毒素促进远端视神经损伤后视网膜节细胞再生的影响

目的：探讨H89和wortmannin对霍乱毒素(CTx)促进成年金黄地鼠远端视神经受损后视网膜节细胞(RGCs)再生的影响。方法：远端切断视神经并对接一段自体坐骨神经(AG)，玻璃体内注射CTx及H89、wortmannin。动物随机分为AG；AG CTx组；AG CWx H89组；AG CTx wortmannin组，4w后粒蓝逆行标记再生的RGCs，荧光显微镜下观察计数。结果：AG CTx组再生的视网膜节细胞平均数(43．2)明显高出AG组(2.6)，AG CTx H89组平均再生数为3．2，与AG组相近，明显低于AG CTx组。AG CTx W组平均再生数为9.6，明显高于AG，低于AG CTx组。结论：H89和wortmannin可部分抑制CTx对视神经远端切断后视网膜节细胞再生的促进作用。     

Differential effects of nicotinic acetylcholine receptor stimulation on negative occasion setting

We have previously shown that nicotine enhances learning in a negative occasion setting task in which rats are trained to distinguish between two different trial types. During reinforced trials, a target stimulus (a tone) is presented and immediately followed by food reward. On nonreinforced trials, a feature stimulus (a light) is presented prior to the tone and indicates the absence of reward following presentation of the tone. The goal of the present study was to identify the behavioral mechanism through which nicotine affects this form of learning, and to determine which subtype(s) of nicotinic acetylcholine receptors mediate the effects of nicotine. Consistent with our prior findings, nicotine administration enhanced the ability of rats to discriminate between the two trial types. Nicotine enhanced the magnitude of the discrimination by decreasing responding to the tone on nonreinforced trials. Nicotine-treated rats also learned the discrimination in fewer sessions than control rats. A significant new finding was that nicotine also increased the orienting response to the light, suggesting that nicotine may enhance learning the serial feature negative discrimination by increasing attention to the visual feature. In addition, we found that RJR-2403, a selective α4β2 nicotinic receptor agonist, also enhanced discrimination. However, RJR-2403 did not affect responding on nonreinforced trials, nor did RJR-2403 affect orienting to the light. Together these data indicate that nicotine may enhance discrimination by enhancing tone-reward associability through α4β2 nicotinic receptors and by enhancing attention to the light through non-α4β2 receptor subtypes.     

The effects of postganglionic axotomy and nerve growth factor on the superior cervical ganglia of developing mice

Summary Sectioning of the two major outflows from the superior cervical ganglia in two week mice results in a marked drop in the number of neurons within one week of operation and a smaller drop over the following two weeks. In animals receiving daily injections of nerve growth factor (NGF), the effect of axotomy is modified. One week after axotomy, the number of neurons in the axotomized ganglia is approxima the same in NGF treated animals as in the control, sham operated ganglia. Over the next two weeks, however, the cell death that results from axotomy is no longer prevented by treatment with NGF. The normal, hyperplastic response to NGF appears to occur independently of the cell reaction caused by axotomy.     

Effects of nerve growth factor and cAMP on expression of acetylcholine receptor in PC12 cells.

The developmental change in acetylcholine-induced current (IACh) was investigated in PC12 cells cultured in the presence or absence of either nerve growth factor (NGF) or 8-Br-cAMP for 10 days. Currents were recorded in the whole-cell mode of the conventional patch-clamp technique. Morphological observations revealed that control cells do not undergo a change in morphology during culture. NGF-treated cells developed long neurites and had dense connections with other cells. 8-Br-cAMP-treated cells developed short neurites, and did not have dense connections. Control cells did not show a change in the mean density of IACh (VH = -80 mV) during the culture period. NGF-treated cells had a maximal increase in the current density of IACh at 5 days after the beginning of treatment, and did not show any further increase until 10 days. 8-Br-cAMP-treated cells had a decrease in current density during the first 6 days, but recovered to the control level after 7 days of treatment. It is concluded that maximal expression of ACh receptors occurs in the relatively early days of development, and that intracellular cAMP does not contribute greatly to their expression.     

Differential activation of dendritic cells by nerve growth factor and brain-derived neurotrophic factor

BACKGROUND: Neurotrophins are involved in inflammatory reactions influencing several cells in health and disease including allergy and asthma. Dendritic cells (DCs) play a major role in the induction of inflammatory processes with an increasing role in allergic diseases as well. OBJECTIVE: The aim of this study was to investigate the influence of neurotrophins on DC function. METHODS: Monocyte-derived dendritic cells were generated from allergic and non-allergic donors. Neurotrophin receptors were demonstrated by western blotting, flow cytometry and fluorescence microscopy. Activation of small GTPases was evaluated by pull-down assays. DCs were incubated with nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and supernatants were collected for measurement of IL-4, IL-6, IL-10, IL-12p70, TNF-alpha and TGF-beta. RESULTS: Receptor proteins were detectable by western blot, fluorescence activated cell sorting analysis and fluorescence microscopy. Signalling after neurotrophin stimulation occurred in a ligand-specific pattern. NGF led to decreased RhoA and increased Rac activation, while BDNF affected RhoA and Rac activity in a reciprocal fashion. Cells of allergics released a significantly increased amount of IL-6, while for healthy subjects a significantly higher amount of IL-10 was found. CONCLUSION: These data indicate that DCs are activated by the neurotrophins NGF and BDNF by different pathways in a receptor-dependant manner. These cells then may initiate inflammatory responses based on allergic sensitization releasing preferred cytokines inducing tolerance or a T-helper type 2 response.     

感光细胞缺失对视网膜节细胞melanopsin表达的影响

目的：研究感光细胞缺失对视网膜节细胞 melanopsin 表达的影响。方法：SD 大鼠腹腔注射60 mg/kg N- 甲基-N-亚硝基脲建立感光细胞变性缺失的动物模型,用免疫荧光方法观察视网膜铺片及切片中 melanopsin 阳性细胞的分布及特征。结果：在药物注射12 h 后动物视网膜感光细胞层开始凋亡,细胞数目随时相减少,13 d 后完全消失。在正视网膜节细胞表达 melanopsin,呈串珠样分布在胞体及突起的胞膜上,其树突穿过节细胞层, 在内网层的两侧分叉形成网状结构。在感光细胞缺失动物模型组,12 h 时,节细胞表达 melanopsin 更广泛;而后突起上的 melanopsin 表达随时减少。28 d 时,melanopsin 的表达主要局限于节细胞的胞体。结论：感光细胞缺失影响节细胞突起 melanopsin 的表达。     

Effects of minocycline and tetracycline on retinal ganglion cell survival after axotomy

In the present study, we compared the in vivo neuroprotective efficacy of intraperitoneally administered tetracycline and minocycline to enhance the survival of retinal ganglion cells (RGCs) following unilateral axotomy of the adult rat optic nerve. We also examined the effects of the tetracycline drugs on the activation of retinal microglia. RGCs in retinal whole-mounts were visualized by retrograde labeling with fluorogold. The presence of activated microglia was confirmed immunohistochemically using OX-42 monoclonal antibodies. Optic nerve axotomy produced RGC death and increased activation of microglia. No significant RGC loss was seen prior to 5 days and approximately 50% and 80-90% cell loss occurred at 7 and 14 days, respectively. Examination of the effects of tetracycline and minocycline on RGC survival at 7 days post-axotomy, revealed increased numbers of RGCs in minocycline-treated animals (75% of non-axotomized control) compared with vehicle-only (52% of control) and tetracycline-treated (58% of control) animals. The densities of RGCs (RGCs/mm2+/-S.D.) for control, vehicle-, tetracycline- and minocycline-treated axotomized animals were 1996+/-81, 1029+/-186, 1158+/-190 and 1497+/-312, respectively. The neuroprotective effect of minocycline seen at 7 days was transient, since RGCs present in minocycline-treated animals at 14 days post-axotomy (281+/-43, 14% of control) were not significantly different to vehicle-treated animals (225+/-47, 11% of control). OX-42 staining of activated retinal microglia was reduced in tetracycline- and minocycline-treated axotomized animals compared with axotomized animals receiving vehicle-only. These results demonstrate that systemic administration of the second-generation tetracycline derivative, minocycline, delays the death of axotomized RGCs by a mechanism that may be associated with inhibition of microglia activation. The neuroprotective efficacy of minocycline following optic nerve axotomy was superior to that of tetracycline.     

The effects of remote retinal stimulation on the responses of cat retinal ganglion cells.

1. Action potentials were recorded from optic nerve fibres of lightly anaesthetized cats while parts of the retina remote from the receptive field were stimulated by a shifting grating. 2. Vigorous responses can be obtained under these conditions, confirming McIlwain (1966), Krüger & Fischer (1973), and others. 3. These 'shift responses' are not caused by fluctuations of stray light because (a) they cannot be reduced by deliberately increasing or decreasing the light falling on the receptive field synchronously with the shifting grating; (b) a steady adapting light applied to the receptive field does not raise the threshold for the responses, whereas adapting light on the peripheral retina does, and (c) the threshold for the responses is elevated more following bleaching adaptation of the periphery than following bleaching adaptation of the centre. 4. Shift responses are strong, of short latency, and brief in duration in brisk-transient (Y-type) neurones. With few exceptions they are weak but long-lasting in brisk-sustained (X-type) neurones. 5. Shift responses are unlike responses from the main receptive field in having a distinct threshold; the magnitude of the response to weak gratings is not simply proportional to contrast, as is the case with weak stimuli applied to the receptive field. 6. It is thought that the excitatory pathway may involve amacrine cells, and that this mechanism may be concerned with the detection of the shifts of the image that occur with saccadic eye movements.     

Distribution and possible origins of substance P-containing nerve fibers in the rat liver.

The distribution and possible origins of substance P-containing nerve fibers in the rat liver were investigated by immunohistochemistry and nerve transection. Nerve fibers with substance P-like immunoreactivity formed a more complex network than previously known in the walls of portal vein branches. Substance P-immunoreactive fibers were seen not only in and around the walls of the hepatic artery, but also in close association with the hepatic veins and bile ducts. Transection of the greater splanchnic nerves and/or the vagus nerves indicated that substance P-immunoreactive fibers in the walls of the portal and hepatic veins enter the liver via both nerves, and that those associated with the hepatic artery and bile ducts stem from the greater splanchnic nerves. The widespread distribution of hepatic substance P and its complex innervation pattern within the liver suggest that it is involved in a variety of physiological processes in this organ.     

Administration of brain-derived neurotrophic factor suppresses the expression of heat shock protein 27 in rat retinal ganglion cells following axotomy

Optic nerve transection results in the apoptotic cell death of the majority of retinal ganglion cells by 14 days. The neurotrophin brain-derived neurotrophic factor (BDNF) enhances survival of retinal ganglion cells. In addition, the small heat shock protein Hsp27, with its anti-apoptotic effects, may be important for neuron survival following axotomy or trophic factor withdrawal. We recently reported the induction and expression of Hsp27 in a subset of retinal ganglion cells following axotomy. Here we have examined the effect of BDNF administration on the expression of Hsp27 in axotomized adult rodent retinal ganglion cells. Retinal ganglion cells were pre-labeled with Fluorogold prior to optic nerve transection and concomitant intraocular injection of BDNF or vehicle. Hsp27 immunofluorescence was examined in retinal sections from 4 to 28 days following injury. Consistent with previous survival studies, the number of Fluorogold-labeled retinal ganglion cells declined from 100% at 4 days to approximately 15% by 14 days following axotomy and vehicle injection. In contrast, with BDNF administration, retinal ganglion cell survival was maintained at 100% to 7 days following axotomy. We report that the number of Hsp27-positive injured retinal ganglion cells, as detected by immunohistochemical staining, was decreased by 50% in BDNF-treated retinas, when compared with vehicle-treated controls. This decreased expression of Hsp27 in response to BDNF treatment was seen both at early (4 days) and delayed (14 days) times. BDNF following optic nerve transection significantly reduced the expression of Hsp27 in retinal ganglion cells. These results indicate that BDNF may down-regulate alternate cell survival pathways, including the stress-induced expression of Hsp27, and may help to explain the failure of chronic neurotrophin treatment to maintain long-term retinal ganglion cell survival.