视网膜神经节细胞的发生和视神经的再生

<正> 视网膜是间脑的衍生物。视网膜神经节细胞在发生过程中,是视杯神经上皮最早分化的细胞。在80年代以前,认为成年哺乳动物的视神经象中枢神经系统中某些神经纤维一样,当其受损伤后,只有再生的反应,却无再生的能力。随着对成体哺乳动物中枢神经系统再生研究的突破,大大推动了对视神经再生的研究。从1985年以来,对成体哺乳动物用外周神经段植入自体视网膜或视神经,诱发视网膜神经节细胞轴突再生的成功,激励着学者们对其再生的可能性和条件性进行了广泛深入的探索。在此仅就视网膜神经节细胞的发生和视神经的再生,主要是哺乳动物视神经再生的问题做一综述。     

外伤性视神经损伤后视神经及视网膜形态的动态观察

目的:了解外伤性视神经损伤后的病理变化、溃变特点与时相间的关系。方法:参照Allen脊髓损伤法,造成视神经眶尖段间接600gcm力冲击、挤压伤。伤后对视神经和视网膜行形态学动态观察。结果:①伤后48h,视神经轻度肿胀和空泡反应;1周时损伤处视神经出现溃变,神经胶质细胞增生,视网膜神经节细胞(retinalganglioncells,RGCs)形态改变不明显;2周时神经纤维轴束间空泡样改变,局灶性坏死,RGCs核固缩和细胞数量减少。术后3月,视神经损伤部位直径缩小,形成胶质疤痕,RGCs数量明显减少,核固缩细胞增多。②RGCs数量于术后48h、1周、2周、1月和3月分别比正常对照组低3.35%、13.23%、19.74%、23.20%、29.28%。③视网膜细胞在48h内出现凋亡。结论:本实验模型可造成明确的视神经和视网膜损伤,神经元的损伤程度从节细胞、中间神经元、感光细胞的次序依次递减。视网膜和视神经损伤的严重程度与时间呈相关性。RGCs数量在48h至1周时下降速率最快。     

不同剂量氯化锂对成年大鼠视神经切断后视网膜神经节细胞的保护作用

目的:研究不同剂量氯化锂(LiCl)对成年大鼠视神经切断后视网膜神经节细胞(RGCs)存活的作用。方法:眶内切断72只成年雌性SD大鼠左侧视神经且残端留置荧光金(FG)后,随机分为生理盐水对照组和低剂量(30 mg/kg/d)、中剂量(60 mg/kg/d)、高剂量(85 mg/kg/d)氯化锂实验组。术前1 d及术后每天腹腔注射生理盐水或不同剂量的氯化锂溶液,直至术后2 d、7 d或14 d处死动物。平铺视网膜后取样计数FG逆行标记的存活节细胞,并由此计算出每一视网膜内节细胞的平均密度。结果:术后2 d各剂量氯化锂组节细胞平均密度与对照组相比无显著性差异(P>0.05)。当存活时间增至7 d时,各剂量氯化锂组节细胞密度均明显高于对照组(P<0.01),且中剂量组节细胞密度增高最为显著。术后14 d时,低剂量与中剂量组节细胞密度仍显著高于对照组(P<0.01),但高剂量组节细胞密度与对照组相比无统计学差异(P>0.05)。结论:腹腔内注射氯化锂可显著促进成年大鼠视神经切断后节细胞的存活,这种神经保护作用为剂量依赖性。     

不同穴位和频率的电针刺激对成年大鼠视神经切断后视网膜神经节细胞存活的作用

目的:研究不同穴位和频率的电针刺激对成年大鼠视神经切断后视网膜神经节细胞(节细胞)存活的作用。方法:54只成年SD大鼠接受右侧视神经眶内切断术及节细胞荧光金(Fluoro Gold,FG)逆行性标记后,随机分为9组,对照组(含2、7、14 d组)及不同穴位、频率和伤后存活时间组合的电针组(含假穴4/20 Hz电针7 d组、百会穴4/20 Hz电针7 d组、睛明穴2 Hz电针7 d组、睛明穴4/20 Hz电针2、7、14 d组),每组6只动物。结果:(1)睛明穴4/20 Hz电针7 d组存活节细胞平均密度显著高于对照、假穴和百会穴组(P<0.01),而假穴和百会穴组与对照组间无显著性差异;(2)睛明穴2 Hz与睛明穴4/20 Hz电针7 d组节细胞密度虽无显著性差异,但均显著高于对照7 d组(P<0.01);(3)分别以4/20 Hz电针刺激睛明穴2、7、14 d,与对照组相同时间点相比,仅在伤后7 d出现节细胞密度的显著增高(P<0.01)。结论:以4/20 Hz与2 Hz两种不同频率的电针刺激睛明穴,均能在成年大鼠视神经切断后7 d延缓节细胞的死亡。就本研究所观察的不同穴位和频率而言,电针的神经保护作用取决于针刺穴位而非电针频率。     

醛糖还原酶基因敲除促进视神经损伤后巨噬细胞向M2方向极化并促进视神经功能恢复

目的观察小鼠视神经夹伤(ONC)后醛糖还原酶(AR)对视神经损伤后功能恢复的影响及可能机制。方法分别采用C57BL/6-AR+/+(B6野生型)、C57BL/6-AR-/-(AR基因敲除)、Thy1-YFP/AR+/+和Thy1-YFP/AR-/-小鼠,建立ONC模型。行视觉电生理F-VEP检查,观察ONC后AR基因敲除对小鼠视神经转导功能的影响;通过视网膜组织冰冻切片,观察AR基因敲除对Thy1-YFP转基因小鼠ONC后存活视网膜神经节细胞数目的影响;玻璃体内注射Alexa Fluor488标记的霍乱毒素B(CTB)顺行标记,观察AR基因敲除对小鼠ONC后神经纤维生长的影响;Western blot法检测野生型小鼠ONC后AR基因的表达变化,及AR基因敲除对M1、M2型巨噬细胞特异性分子诱导型一氧化氮合酶(iNOS)、精氨酸酶1(Arg1)表达的影响。结果AR基因敲除可促进小鼠ONC后视神经转导功能的恢复,增加存活的视神经节细胞数量;AR基因敲除可促进小鼠ONC后视神经纤维生长;AR基因敲除可促使小鼠ONC后巨噬细胞向M2方向极化。结论 AR可能通过调节视神经损伤后巨噬细胞极化影响视神经功能恢复。     

人类肾上皮细胞氧化损伤模型的建立

目的:采用过氧化氢(H2O2)对人类肾脏近端小管上皮细胞系(HKC)进行氧化损伤,建立损伤的细胞模型。方法:通过检测细胞活力、培养基中的丙二醛(MDA)含量和细胞内超氧化物歧化酶(SOD)活性的变化研究细胞损伤的程度;利用激光共聚焦显微镜观察细胞损伤前后骨桥蛋白(OPN)表达水平的变化;采用扫描电子显微镜(SEM)观察细胞损伤前后形貌和膜表面微结构的变化。结果:用1mmol/LH2O2作用HKC不同时间后,细胞活力和SOD活性逐渐下降,MDA释放量增加,OPN表达量显著增加并在作用时间为1h时达到最大值;经损伤后的细胞变得干瘪收缩,且膜表面粗糙,鞭毛、突触大部分断裂脱落。结论:H2O2能够明显损伤HKC,细胞损伤后,其活力和膜表面超微结构发生变化,并在膜上表达能黏附草酸钙晶体的OPN。因此,可用1mmol/LH2O2作用HKC1h建立损伤模型。     

醛糖还原酶在小鼠视神经损伤后的表达及作用

目的 观察视神经中醛糖还原酶(AR)在小鼠视神经横断(ONT)损伤后的表达变化及其对视网膜神经节细胞存活和再生的影响.方法 采用C57BL/6-AR+/+(野生型)小鼠、C57BL/6-AR-/-(AR基因敲除)小鼠、Thy1-YFP/AR+/+(AR+/+ YFP 小鼠)小鼠和Thy1-YFP/AR-/-小鼠(AR-/-YFP小鼠),建立视神经横断模型.通过Western blot方法,检测横断损伤后的视网膜中AR分子的表达变化;利用视网膜组织冰冻切片计数观察比较AR基因敲除小鼠和野生型小鼠视神经横断损伤后存活的视网膜神经节细胞数目;通过Western blot法,观察比较神经生长相关蛋白43(GAP-43)在AR基因敲除小鼠和野生型小鼠视神经横断后的表达变化.结果 Western blot检测发现AR分子在野生型小鼠视神经横断后表达随时间延长逐渐升高.AR敲除小鼠在视神经横断损伤后其存活的视网膜神经节细胞数目多于野生型小鼠.AR敲除小鼠在视神经横断损伤后GAP-43的表达量高于野生型小鼠组(P＜0.05).结论 AR参与了视神经横断损伤后的视网膜神经节细胞存活的调节,AR缺失会促进视神经横断损伤后的再生修复.     

荧光金逆行标记观察正常大鼠视网膜神经节细胞及轴突

使用荧光金通过立体定位仪逆行标记对正常大鼠视网膜神经节细胞及其轴突进行观察研究。成年SD大鼠6只(12眼),体重250±20g,按照标记后1周,2周分为2组,每组3只(n=6眼)。将荧光金注射到大鼠的外侧膝状体和上丘,观察视网膜神经节细胞(RGCs)的数量和视网膜神经节细胞及其轴突的形态。结果显示:视网膜铺片的节细胞边界清楚,易于观察和计数。荧光金标记1周后,RGCs密度为2210±128个/mm2;标记后2周,RGCs密度为2164±117个/mm2。视网膜神经节细胞的轴突内荧光分布均匀,呈线性。结论是使用荧光金逆行标记能够可靠、有效地研究视网膜神经节细胞及其轴突。     

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。     

Neuroprotective effects of Epigallocatechin-3-gallate (EGCG) in optic nerve crush model in rats

Epigallocatechin-3-gallate (EGCG), the major catechin found in green tea, is a powerful antioxidant and has anti-inflammatory with neuroprotective potential. This study aims to investigate the neuroprotective effects of EGCG in an optic nerve crush (ONC) model in rats. Seventy-two Wistar rats were randomly divided into four groups: normal control (group A), sham operation + EGCG (group B), ONC + vehicle (group C), and ONC + EGCG (group D). The rats were treated intraperitoneally and orally with either vehicle or EGCG (25 mg/kg, injected daily for 5 days and 2 mg/kg orally daily afterwards). Two days after the first injection, an ONC injury was performed by using a micro optic nerve clipper with 40 g power at approximately 2 mm from the optic nerve head for 60 s. Fluorogold was injected into the bilateral superior colliculi 5 days before sacrifice and fluorescent gold-labelled retinal ganglion cells (RGCs) were counted under fluorescence microscopy on days 7, 14 and 28 after ONC. The expression of Neurofilament triplet L (NF-L) was measured via immunohistochemical and Western blotting analysis. In group C, a progressive loss of RGCs was observed after ONC. In contrast, the density of RGCs was significantly higher in group D (p = 0.009, independent samples t-test) on day 7 after ONC, and statistical differences were obtained on days 14 and 28 (p = 0.026 and p = 0.019, respectively, independent samples t-test). The results of immunohistochemical and Western blotting analysis showed significantly higher NF-L protein expression in group D in comparison with group C on days 7, 14 and 28 after ONC. These findings suggest that there are protective effects of EGCG on RGCs after ONC, indicating EGCG might be a potential therapeutic agent for optic nerve diseases.     

Regrowth of retinal ganglion cell axons into a peripheral nerve graft in the adult hamster is enhanced by a concurrent optic nerve crush

Summary Transplantation of a segment of peripheral nerve to the retina of the adult hamster resulted in regrowth of damaged ganglion cell axons into the graft, with the fastest regenerating axons extending at 2 mm/day after an initial delay of 4.5 days (Cho and So 1987b). In this study, the effect of making 2 lesions on the same axon (the conditioning lesion effect) on the regrowth of ganglion cell axons into the peripheral nerve graft was examined. When a conditioning lesion (first lesion) was made by crushing the optic nerve 7 or 14 days before the peripheral nerve grafting (the second lesion) to the retina, the distance of regrowth achieved by the fastest regenerating axons in the graft, measured at the 7th post-grafting day, was lower than in animals with a peripheral nerve grafted to a normal eye. This indicated that in contrast to the situation in peripheral nerve axons (Forman et al. 1980) and goldfish optic axons (Edwards et al. 1981), the conditioning lesion was unable to enhance the regrowth of mammalian retinal ganglion cell axons. However, when crushing of the optic nerve was followed immediately by peripheral nerve grafting, an enhancement in axonal regrowth could be observed. The initial delay time before the axons extended into the peripheral nerve graft was reduced by 1 day while the rate of elongation of the fastest regrowing axons in the graft apparently remained unchanged. Moreover, the shortening of the initial delay could still be observed even when the sequence of performing the 2 lesions was reversed. From these data, it was concluded that the classical conditioning lesion effect was not responsible for the enhancement observed. Rather it was suggested that changes in the intra-retinal environment brought about by crushing of the optic nerve might account for it.     

Long term survival of cat retinal ganglion cells after intracranial optic nerve transection

Summary After intracranial transection of the optic nerve in cats the retinal stump of the nerve was injected with HRP. Surviving retinal ganglion cells can be retrogradely labeled at least up to 15.5 months of postoperative survival.     

Parameters of optic nerve electrical stimulation affecting neuroprotection of axotomized retinal ganglion cells in adult rats

We previously showed the enhancement of survival of retinal ganglion cells (RGCs) by electrical stimulation (ES) of the optic nerve (ON) stump in adult rats. To elucidate the mechanisms underlying the survival enhancement, we determined whether the neuroprotective effect of ES is affected by the following parameters: stimulation time, frequency of current pulses and starting of ES. ES for 10 min immediately after ON transection was not effective in increasing the number of surviving RGCs 7 days after the transection, but that for 30 min was effective. ES at 20 Hz was the most effective, when applied just after axotomy. When the starting of ES to the ON was shifted either 3 h after or 4 h before the axotomy, the neuroprotective effect of ES was not observed. These results suggest that the electrical activation of RGCs and/or the transected ON interfere with early events after axotomy that leads to RGC death.     

大鼠骶神经钳夹伤致神经源性膀胱模型的建立及评估

目的建立并评估大鼠骶神经钳夹伤后神经源性膀胱模型。方法将25只健康雌性大鼠随机分为对照组(n=10)和钳夹伤组(n=15)。钳夹伤组对大鼠双侧骶2神经进行钳夹伤,对照组仅暴露双侧骶2神经。造模后观察大鼠自主排尿情况,4周后检测尿流动力学指标,并对膀胱和骶神经进行组织形态学观察。结果造模48 h后钳夹伤组大鼠下腹部膀胱充盈,自主排尿受限,对照组大鼠可自主排尿。造模4周后钳夹伤组大鼠的最大膀胱容量大于对照组,差异具有统计学意义(P<0.05);钳夹伤组大鼠漏尿点压及膀胱内压高于对照组,差异具有统计学意义(P<0.05)。造模4周后钳夹伤组大鼠的膀胱明显胀大,HE染色膀胱壁肌层厚度小于对照组,差异具有统计学意义(P<0.05)。结论大鼠双侧骶2神经钳夹伤可建立神经源性膀胱模型,造模方法简单易行且模型稳定。     

大鼠视神经吸断伤后相关分子表达的变化

目的：研究视神经损伤后神经胶质细胞去分化的程度及相关分子的表达。方法：成年雄性大鼠视神经吸断伤后,采用免疫组织化学、原位杂交组织化学结合计算机图像分析,检测视神经巢蛋白、胶质纤维酸性蛋白 (GFAP)、髓鞘碱性蛋白(MBP)、神经纤维丝(NF)以及 Nogo-A mRNA 在伤后3、7、14和28 d 4个不同时相点的表达。结果：视神经伤后,巢蛋白表达上调,在28 d 时表达最高;GFAP 表达先下调,7 d 时最低,随后逐渐上调; MBP 表达上调,呈先上升后降低的趋势;NF 表达呈明显下降趋势;Nogo-A mRNA 表达呈上升趋势,3 d 到7 d 的变化最显著。结论：视神经损伤后相关分子表达提示其神经胶质细胞可去分化为神经前体细胞或神经干细胞,但这些前体细胞或干细胞呈不利于神经再生的状态。     

Histological and immunohistochemical study of the potential therapeutic impacts of bone marrow mesenchymal stem cells and exosomes for sciatic nerve crush injury model in rats

ABSTRACT

The aim was to evaluate the potential effect of bone marrow-derived mesenchymal stem cells (BMSC) and BMSC-EX in the sciatic nerve injury. Forty-five rats were divided into four groups, i.e. Group I control no surgery or treatment, Group II sciatic nerve crush surgery and 1 week post-surgery, Group III sciatic nerve crush injury was treated with BMSC, and Group IV sciatic nerve crush injury was treated with BMSC-EX. All animal groups were euthanized 2 weeks after treatment. Sciatic nerves were examined histologically and immunohistochemically. Group II showed various histological changes, i.e. vacuolation, degeneration and loss of nerve fibers. Group II showed a significant increase in collagen fibers, but with significant reduction in osmium tetroxide (OsO₄)-stained myelin sheaths and neurofilament immunostaining compared to Group I. Group II also revealed distortion and disruption of myelinated nerve fibers. Groups III and IV showed significant improvement of both histological and immunohistochemical changes in Group II. BMSC and BMSC-EX were shown to improve axonal regeneration.     

Neuroprotective effect on retinal ganglion cells by transpupillary laser irradiation of the optic nerve head

This study demonstrates that subthreshold transpupillary thermotherapy (TTT) laser irradiation on optic nerve head protects retinal ganglion cells (RGCs) in an optic nerve crush (ONC) model. TTT was performed in right eyes with an 810-nm diode laser aimed at the center of the optic nerve head, using the following protocol: power 60 mW, duration 60 s, spot size 500 μm. Fluoro-Gold was injected into bilateral superior colliculi 5 days before sacrifice and fluorescent gold labeled RGCs were counted under fluorescence microscopy. In the ONC group, a progressive loss of RGCs was observed; however, in comparison with the ONC group, RGCs density was significantly higher (P = 0.001, independent samples t-test) at day 7 postoperative and only borderline significances were obtained at days 14 and 28 postoperative (P = 0.044 and P = 0.045, respectively, independent samples t-test) in ONC + TTT group, which implies the potential neuroprotective role of TTT. This protective effect seems to be heat shock proteins (HSPs) related, because intraperitoneal Quercetin (an inhibitor of HSPs, 4 mg/kg/day for 7 days) could completely abolish this protective effect at days 7, 14 and 28 postoperative (P = 0.012, P = 0.002, and P = 0.000, respectively, independent samples t-test). Minimal collateral damage of TTT on optic nerve head tissue, peripapillary RGCs and the myelin sheath of the optic nerve were observed under transmission electron microscopy. These findings suggested that subthreshold TTT might be a safe and practical approach to protect RGCs. The underlying mechanisms may involve TTT-induced HSPs in RGCs.