Immunocytochemical analysis of the dopamine system in the brain and spinal cord of the European eel, Anguilla anguilla

Summary The distribution of dopamine-containing perikarya and fibres in the central nervous system of the eel, Anguilla anguilla, was determined by using a specific dopamine antiserum. Telencephalic dopamine-immunoreactive somato are located in the external cell layer of the olfactory bulb and throughout the rostrocaudal extent of the subpallium; immunoreactive fibres are located primarily in the bulb and in ventral and lateral portions of the hemispheres. Diencephalic dopamine-immunoreactive neurons are associated with the ventricles in the preoptic area and hypothalamus and in the posterior tubercle. Many of the neurons in the hypothalamus are liquor-contacting. Very few immunoreactive neurons are located in the mesencephalon, and no dopamine-containing cells are found in regions that can be homologized with the ventral tegmental area and substantia nigra of amniotes. There is a rich innervation of the medial octavolateralis nucleus and certain layers of the torus semicircularis and of the tectum. dopamine-containing neurons are located in the vagal lobe, by the vagal motor nucleus and in the area postrema, which provides a rich dopaminergic innervation of the brainstem motor column and of the reticular formation. Immunoreactive liquor-contacting neurons line the central canal and another type of labelled neuron lies dorsally in the spinal cord.     

Simvastatin treatment improves functional recovery after experimental spinal cord injury by upregulating the expression of BDNF and GDNF

The aim of this study was to determine the therapeutic efficacy of simvastatin treatment starting 1 day after spinal cord injury (SCI) in rat and to investigate the underlying mechanism. Spinal cord injury was induced in adult female Sprague–Dawley rats after laminectomy at T9-T10. Then additionally with sham group (laminectomy only) the SCI animals were randomly divided into 3 groups: vehicle-treated group; 5-mg/kg simvastatin-treated group; and 10-mg/kg simvastatin-treated group. Simvastatin or vehicle was administered orally at 1 day after SCI and then daily for 5 weeks. Locomotor functional recovery was assessed during 8 weeks postoperation by performing open-field locomotor test and inclined-plane test. At the end of study, motor evoked potentials (MEPs) and somatosensory evoked potentials (SEPs) were assessed to evaluate the integrity of spinal cord pathways. Then, the animals were killed, and 1-cm segments of spinal cord encompassing the injury site were removed for histopathological analysis. Immunohistochemistry was performed to observe the expression of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) in the spinal cord. Results show that the simvastatin-treated animals showed significantly better locomotor function recovery, better electrophysiological outcome, less myelin loss, and higher expression of BDNF and GDNF. These findings suggest that simvastatin treatment starting 1 day after SCI can significantly improve locomotor recovery, and this neuroprotective effect may be related to the upregulation of BDNF and GDNF. Therefore, simvastatin may be useful as a promising therapeutic agent for SCI.     

Role of IL-6 in spinal cord injury in a mouse model

In recent years, various studies have been conducted toward the goal of achieving regeneration of the central nervous system using neural stem cells. However, various complex factors are involved in the regulation of neural stem cell differentiation, and many unresolved questions remain. It has been reported that after spinal cord injury, the intrinsic neural stem cells do not differentiate into neurons but, rather, into astrocytes, resulting in the formation of glial scars. Based on reports that the expression of interleukin (IL)-6 and the IL-6 receptor (IL-6R) is sharply increased in the acute stages after spinal cord injury and that IL-6 may serve as a factor strongly inducing the differentiation of neural stem cells into astrocytes, we examined the effects of an antibody to IL-6R in cases of spinal cord injury and found that the antibody suppressed secondary injury (caused by inflammatory reactions) and glial scar formation, facilitating functional recovery. This article presents the data from this investigation and discusses the relationship between IL-6 signals and spinal cord injury.     

A型肉毒杆菌神经毒素重链干预大鼠脊髓损伤后蛋白表达的初步研究

目的:初步观察大鼠脊髓损伤模型基础上局部注射小剂量A型肉毒杆菌神经毒素重链(BoNT/A HC)后对局部蛋白表达谱的影响,为探讨BoNT/A HC干预在体神经损伤后相关蛋白表达及其干预神经再生机制提供实验基础。方法:复制大鼠单侧腰段脊髓损伤模型;采用SDS-PAGE及双向电泳观察不同剂量BoNT/A HC(2μg、4μg、6μg和8μg)对脊髓损伤后局部(包括损伤部位及其近头端部分脊髓组织)蛋白表达谱的干预作用。结果:大鼠单侧腰段脊髓损伤2 d时局部脊髓组织结构明显破坏崩解,损伤波及左侧脊髓灰质及白质;脊髓损伤局部SDS-PAGE及考马斯亮蓝染色显示,于损伤同时局部一次性注射不同剂量BoNT/A HC后,某些蛋白表达与单纯损伤组相比明显不同,而与正常组基本一致;双向电泳结果进一步显示,损伤局部注射6μg BoNT/A HC后2 d和20 d时,在不同等电点及不同蛋白分子量水平上,有10余种蛋白表达与单纯损伤组明显不同,呈向正常转化的趋势。结论:大鼠脊髓损伤局部注射BoNT/A HC一定时间可影响损伤局部蛋白表达谱的变化,这种变化呈现由损伤造成的蛋白表达变化被转向正常的趋势。     

神经营养因子对脊髓损伤修复作用研究进展

脊髓损伤（SCI）是一种严重的神经系统创伤,后果严重常导致患者不同程度地瘫痪和大小便障碍,其致残率与耗费高给家庭及社会造成严重的负担.因此研究脊髓组织损伤后的再生和修复具有重要的现实意义.大量实验研究表明神经营养因子对脊髓损伤后的神经组织修复过程有重要作用.就这一领域新的研究进展作一综述.     

原位胶原凝胶对大鼠脊髓损伤后功能恢复的影响

目的研究脊髓损伤后原位形成胶原凝胶对神经功能恢复的影响。方法15只健康雌性SD大鼠随机分为胶原组、对照组和假手术组．Allen撞击法制作脊髓损伤模型。用微量注射器将胶原溶液注入损伤部位,在体温作用下让其原位形成凝胶,对照组注入PBS溶液,每周进行运动评分,6周后取脊髓组织进行免疫荧光染色,观察损伤部位的胶质瘢痕和轴突生长情况。结果胶原组大鼠第5周起BBB评分显著高于对照组,免疫荧光显示损伤部位胶质瘢痕少于对照组,且长入损伤部位的轴突也多于对照组。结论原位形成的胶原凝胶能抑制损伤部位胶质瘢痕的形成,并能促进轴突再生长入损伤部位。证明胶原是一种较好的能用于修复脊髓损伤的可注射材料。     

c-fos expression in bladder-specific spinal neurons after spinal cord injury using pseudorabies virus

PURPOSE: c-fos expression in spinal neurons that are activated by lower urinary tract stimulation are not organ specific. In this experiment, we demonstrated changes of c-fos expression in bladder-specific preganglionic neurons (PGNs) and interneurons using pseudorabies virus (PRV). MATERIALS AND METHODS: Forty Sprague-Dawley rats were used. We identified the neuronal pathway associated with the bladder by injecting PRV into the detrusor. An immunohistochemical method was used to stain Fos-protein encoded by the c-fos gene. Immunofluorescent staining for PRV was performed to evaluate changes in bladder-specific spinal neurons. RESULTS: Immunofluorescent staining with choline acetyltransferase (ChAT) revealed that the sacral parasympathetic nucleus (SPN) regions contained 9.8 PGNs/section. In rats with chronic spinal cord injury by intravesical saline instillation, 82.4+/-10.3% of PGNs in SPN exhibited Fos-immunoreactive (IR). Two and a half days after PRV infection, PRV-IR PGNs were observed at 5.4 PGNs/section, and 2.7+/-1.6% of them exhibited Fos-IR. Unlike ChAT-IR PGNs, PRV-IR PGNs are bladder-specific neurons and PRV-IR and Fos-IR cells found in the back of PRV-IR PGNs are bladder-specific interneurons. Three days after PRV infection, we observed many PRV-IR and Fos-IR cells in the dorsal commissure. These neurons are interneurons distributed in the bladder. CONCLUSION: We confirmed that in chronic spinal cord injury, the patterns of c-fos expression in bladder-specific spinal neurons were similar to those in voiding-reflex related spinal neurons, which had already been demonstrated earlier. We believe that our methodology can be applied to study interactions between voiding and other organs as well, such as the urethra and prostate.     

脊髓损伤后硫酸软骨素蛋白多糖及GFAP表达的变化

目的探讨脊髓损伤后NG2、Neurocan、GFAP表达的变化。方法将32只雌性SD大鼠随机分为空白对照组和模型组。模型组采用脊髓横切法制作脊髓损伤模型,空白对照组仅切除T10全椎板及T9、T11部分椎板,对脊髓未作任何处理。分别在大鼠脊髓损伤制作后3、7、14及28 d时取材,利用免疫组织化学染色方法检测NG2、Neurocan、GFAP的表达情况。结果模型组脊髓损伤后3 d时NG2的表达出现明显升高,7 d时NG2的表达达到最高点,14 d及28 d时NG2仍然维持在较高水平,但与7 d时的表达相比有下降,空白对照组NG2各时间点均呈低表达。模型组脊髓损伤后7 d时Neurocan的表达显著增加,于14 d时达到最高点,从14 d开始Neurocan的表达开始逐步下降,空白对照组Neurocan各时间点均呈低表达。模型组脊髓损伤后3 d时GFAP的表达明显升高,14 d时GFAP的表达达到顶点,28 d时损伤部位GFAP的表达均较空白对照组明显升高,两组比较差异有统计学意义(均P<0.05)。结论脊髓损伤后NG2、Neurocan、GFAP表达升高,可能是脊髓损伤后抑制轴突再生的因素之一。     

Swimbladder gas gland cells of the European eel cultured in a superfusion system

Swimbladder gas gland cells are polar epithelial cells which release acidic metabolites through the membranes of an extensive basolateral labyrinth, and secret surfactant via exocytosis at apical membranes. We have developed a method to establish primary cell cultures of gas gland cells in order to establish a model system for physiological analysis of gas gland cell function in vitro. Isolated gas gland cells attach to collagen S coated surfaces. Cells cultured in collagen S coated petri dishes were flat and showed no histological polarity. Cells cultured on Anodisc membranes in a superfusion system, in which the apical and basal side of the cells was supplied with a saline solution and with glucose containing DMEM cell culture medium, respectively, showed a clear polarity similar to the in vivo situation. Measurement of lactate release at the apical side and at the basal side revealed that these cells were functionally polar and secreted at least 70% of the lactate at their basal membranes. Gas gland cells could also be cultured in an air/liquid system, in which the apical membrane was exposed to humidified air. Cells cultured under these conditions released lactate only on the basal side and histologically were similar to cells cultured in the superfusion system.     

Effect of pollen typhae on inhibiting autophagy in spinal cord injury of rats and its mechanisms

Purpose: To investigate the effects and mechanism of pollen typhae on spinal cord injury (SCI) in rats. Methods: The SCI model was built and animals were randomly divided into three groups according to different concentrations of pollen typhae. Protein, mRNA, and fluorescence expression levels of light-chain-3 (LC-3) and Beclin-1 were determined by western blotting (WB), real-time PCR and immunofluorescence, along as Akt and mammalian target of rapamycin (mROT) by WB. The demyelination area and integrated optical density (IOD) were analyzed by luxol fast blue (LFB) and Nissl staining, respectively; Behavioral assessments were assessed by Basso Beattie Bresnahan (BBB) scale. Results: Protein, mRNA, and fluorescence expression levels of LC-3 and Beclin-1 were significantly increased after SCI, while were obviously decreased by administration of pollen typhae, along with protein level of Akt and mROT. The demyelination area was significantly reduced, while IOD and BBB were significantly increased compared with the model group. Conclusion: Autophagic activity increased in damaged neural tissue after SCI, and pollen typhae have certain therapeutic effect on SCI, the higher concentration of pollen typhae, the more effective. Besides, pollen typhae also provided neuroprotective effect and improved locomotor function. The effects may be produced by blockade of Akt/mTOR pathway.     

Regulatory expression of Neurensin-1 in the spinal motor neurons after mouse sciatic nerve injury

Axonal regeneration after crush injury of the sciatic nerve has been intensely studied for the elucidation of molecular and cellular mechanisms. Neurite extension factor1 (Nrsn1) is a unique membranous protein that has a microtubule-binding domain and is specifically expressed in neurons. Our studies have shown that Nrsn1 is localized particularly in actively extending neurites, thus playing a role in membrane transport to the growing distal ends of extending neurites. To elucidate the possible role of Nrsn1 during peripheral axonal regeneration, we examined the expression of Nrsn1 mRNA by in situ hybridization and Nrsn1 localization by immunocytochemistry, using a mouse model. The results revealed that during the early phase of axonal regeneration of motor nerves, Nrsn1 mRNA is upregulated in the injured motor neuron. Nrsn1 is localized in the cell bodies of motor neurons and at the growing distal ends of regenerating axons. These results indicate that Nrsn1 plays an active role in axonal regeneration as well as in embryonic development.     

Maternal prenatal stress in rats influences c-fos expression in the spinal cord of the offspring

Previous studies in humans have reported a link between maternal stress and disturbed infant physiological behavior. The objective of our study was to examine in experimental rats how maternal prenatal stress induced by a forced swim test affects offspring afferent spinal responses mediated by stimulation of vaginocervical receptors. The activation of spinal cord neurons showing c-fos expression was examined following vaginocervical mechanical stimulation in adult rats, which were the offspring of dams exposed to gestational stress from E10 until delivery. Vaginocervical stimulation of both prenatal-stressed and non-prenatal-stressed rats induced an increase in immunoreactive protein in the spinal cord ranging from T12 to S1 segmental levels. However, a significantly higher (40%) increase in the expression of Fos-immunoreactive neurons was observed in vaginocervical stimulated prenatally stressed rats than in non-stimulated prenatally stressed ones. This increase was higher in L5-S1 levels than in T12-L4. When the regional distribution was examined, results showed that up to 80% of activated neurons were located in the dorsal horn in both non-stimulated prenatally stressed and stimulated prenatally stressed groups, with a significantly higher density in the latter. Our results demonstrate that maternal prenatal stress can have consequences on vaginocervical responses conveyed to the spinal cord. The increase in Fos labeled neurons in T12-S1 in prenatally stressed rats induced by vaginocervical stimulation suggests the hypersensitivity of the genital tract associated with activation of spinal circuits spanning multiple segments.     

诱导型一氧化氮合酶在强啡肽致脊髓损伤中的作用

目的：探讨诱导型一氧化氮合酶（ｉＮＯＳ）在强啡肽致脊髓损伤中的作用。方法：［３Ｈ］－左旋精氨酸转化法测定腹侧和背侧脊髓ｉＮＯＳ活性,原位杂交法观测脊髓ｉＮＯＳｍＲＮＡ表达及其细胞分布。结果：大鼠蛛网膜下腔注射（ＩｎＩ）强啡肽Ａ１－１７（Ｄｙｎ）２０ｎｍｏｌ引起持久性截瘫和迟发性神经元死亡;在Ｄｙｎ致瘫后２～３ｈｉＮＯＳｍＲＮＡ表达开始增多增强,４ｈ达高峰,２４ｈ和４８ｈ仍见广泛表达,其分布以胶质细胞和大运动神经元为主;腹侧脊髓ｉＮＯＳ活性在Ｄｙｎ致瘫后４ｈ显著升高,并持续至２４ｈ和４８ｈ;提前１０ｍｉｎＩｎＩ选择性ｉＮＯＳ抑制剂氨基胍１μｍｏｌ可显著对抗Ｄｙｎ２０ｎｍｏｌ引起的持久瘫及伤后４ｈ腹侧脊髓ｉＮＯＳ活性升高。结论：ｉＮＯＳ持续性高表达与Ｄｙｎ致脊髓损伤机制有关     

冠状动脉球囊损伤后管壁原癌基因c-myc表达的变化

目的：探讨原癌基因c-myc的表达与冠状动脉球囊损伤后内膜增殖的关系。方法：建立猪的冠状动脉球囊损伤模型，观察形态学变化，并用RT-PCR的方法检测损伤冠状动脉壁内c-myc mRNA的表达。结果：冠状动脉球囊损伤后6周管壁内膜明显增生，管腔狭窄，c-myc mRNA的相对表达强度明显高于正常冠状动脉壁。结 论：c-myc基因与冠状动脉球囊损伤后内膜增殖密切相关。     

Brain-derived neurotrophic factor reduces necrotic zone and supports neuronal survival after spinal cord hemisection in adult rats

Spinal cord injury (SCI) often results in necrotic changes leading to cavity formation and glial scar tissue in the lesion zone. We have examined the effects of continuous topical administration of brain-derived neurotrophic factor (BDNF) on cavity formation and neuronal death after SCI. Following retrograde prelabeling of the tibial motoneurons in the L4–L6 spinal cord segments with the fluorescent dye Fast blue, a spinal hemisection was performed in the L5 segment. At 4 weeks postoperatively, only 66% of the labeled motoneurons remained in the untreated animals, while BDNF treatment resulted in a significant reduction in size of the lesion cavity and 92% motoneuron survival. A therapeutic potential of BDNF in the early treatment of SCI is suggested.     

MicroRNA-223 expression in neutrophils in the early phase of secondary damage after spinal cord injury

MicroRNA (miR)s are short non-coding RNAs that suppress the translation of target genes, and play an important role in gene regulation. Despite this prominence, there are few reports that refer to the expression of miRs after spinal cord injury (SCI). Previously, we reported on miR-223 expression after SCI in mice. The purpose of this study is to reveal the distribution of miR-223 and identify the cells that express miR-223 in the injured spinal cord. Quantitative polymerase chain reaction analysis revealed high expression of miR-223 at 12 h after SCI. Double staining of in situ hybridization and immunohistochemistry showed that the signals of miR-223 merged with Gr-1 positive neutrophils. Our data indicate that miR-223 might regulate neutrophils in the early phase after SCI.     

脂肪源性干细胞促进大鼠受损坐骨神经传导及脊髓脑源性神经营养因子和睫状神经营养因子的表达

目的:探讨脂肪源性干细胞(ADSCs)对坐骨神经损伤大鼠神经传导功能以及脊髓脑源性神经营养因子(BDNF)和睫状神经营养因子(CNTF)表达的影响。方法:将第4代ADSCs移植入脱细胞神经移植物(ANA)中,构建组织工程神经。大鼠随机分为正常组、杜氏改良Eagle培养基营养混合物F12(DMEM)组和ADSC组。DMEM组和ADSC组均建立坐骨神经损伤模型,后用相应的组织工程神经桥接损伤神经的断端。术后6周采用神经电生理记录仪检测各组大鼠坐骨神经传导速度和波幅,采用免疫荧光和Real-time PCR检测各组大鼠脊髓脑源性神经营养因子(BDNF)、睫状神经营养因子(CNTF)蛋白和mRNA的表达。结果:ADSC组大鼠坐骨神经传导速度、波幅和脊髓BDNF和CNTF蛋白及mRNA表达均显著高于DMEM组。结论:ADSCs可增加坐骨神经传导速度和波幅、上调脊髓BDNF和CNTF的表达。     

白藜芦醇对大鼠脊髓损伤后氧化与抗氧化过程的影响

目的探讨白藜芦醇﹙RES﹚对脊髓损伤﹙SCI﹚后髓过氧化物酶﹙MPO﹚和超氧化物歧化酶﹙SOD﹚活性的影响。方法 2008年2月~2010年8月在武汉大学人民医院动物实验中心将96只SD健康成年雄性大鼠随机分成4组,根据Allen’s法制成中度脊髓损伤模型,术后立即管饲白藜芦醇100mg/kg或甲基强的松龙﹙MPSS﹚100mg/kg;通过比色法观察脊髓损伤8小时、1天、3天及7天后白藜芦醇组脊髓MPO及SOD活性的变化,并与MPSS﹙甲基强的松龙﹚组进行疗效对比。结果在脊髓损伤后8小时、1天、3天及7天白藜芦醇组MPO及SOD活性与损伤组差异均有统计学意义,显示白藜芦醇对脊髓损伤具有明显神经细胞保护作用,而且白藜芦醇组与MPSS组间损伤后1天差异具有统计学意义﹙<0.05﹚。结论白藜芦醇在脊髓损伤后能够有效抑制MPO活性的升高幅度,并提高SOD活性,对损伤后脊髓起保护作用。     

生物材料支架在治疗脊髓损伤中的应用

背景:由于脊髓损伤后神经神经再生能力弱,修复受损脊髓组织并使其实现功能正常化仍是目前医学难题。生物组织材料学的快速发展以及其在医学中广泛应用为脊髓损伤修复提供了新的治疗理念和方法。目的:总结生物材料支架对脊髓损伤后神经组织再生修复研究并对其发展趋势进行展望,以探讨修复脊髓损伤的方法并总结经验。方法:应用PubMed数据库高级检索功能,检索2011年1月至2021年1月的文献,检索词为“Spinal cord injury;Biomaterials;Nerve regeneration;Material”;应用知网、万方、维普等数据库高级检索功能,检索2011年1月至2021年1月的相关文献,检索词为“脊髓损伤;生物材料;支架”。结果与结论:随着生物工程研究和医学结合的进一步深入,生物材料支架已被广泛应用于脊髓损伤修复的研究,生物材料的组织相容性、降解性等方面均有了改善;生物材料种类较多,各有其利弊,取其优点制备成复合支架并负载种子细胞、细胞因子或药物对神经再生效果更佳。但复合支架如何选择材料组合,如何选择种子细胞、细胞因子或药物,使生物材料支架联合种子细胞、细胞因子或药物成为最佳组合值得深入研究。总之,生物材料修复脊髓损伤是一个新思路,可能成为促进脊髓损伤修复的突破点。