Transient retinal ischemia-reperfusion in rats

Objective To investigate the effect of transient ischemia-reperfusion on the retina in rats. Methods Retinal ischemia-reperfusion was induced in rats by increasing the intraocular pressure.After 1 or 5 minutes of ischemia, retinal neuronal cell death at diffesent periods of reperfusion was studied using the TdT-deoxynucleotide terminal nick-end labeling (TUNEL) method and light microscopy.Retinal IL-1β and TNFα were quantified by an enzyme-linked immunosorbent assay (ELISA). Results A few migrating leukocytes were noticed in the retina after transient retinal ischemia-reperfusion. Rare TUNEL-positive (T+) cells were noticed in the outer granular layer or the rod and cone layer, and not in ganglion cell layer in control eyes, but they were significantly increased in the outer granular layer, the inner granular layer, and ganglion cell layer in the eyes treated with 1 or 5 minutes of retinal ischemia-reperfusion (P<0.05).Retinal IL-1β was significantly increased at 6 hours after reperfusion in the eyes treated with 1 or 5 minutes ischemia over the control eyes (P<0.05), but retinal TNFα was not significantly increased (P>0.05 ). Conclusion Transient retinal ischemia-reperfusion for only 1 or 5 minutes of ischemia can induce the upregulation of retinal IL-1β and apoptosis of retinal neuronal cells.This kind of apoptosis in individual cells, however, was not sufficient to affect the whole retinal function.     

Therapeutic effect of bFGF on retina ischemia-reperfusion injury

Background Basic fibroblast growth factor (bFGF) plays important roles in retina degeneration, light injury, mechanical injury, especially in retina ischemia-reperfusion injury (RIRI). This study was to investigate the therapeutical effect of bFGF on RIRI and its mechanisms.Methods Experimental RIRI was induced by increasing intraocular pressure (IOP) in the eyes of 48 rats. These rats were divided into normal control, ischemia-reperfusion and bFGF-treated groups.Histological and ultrastructural changes of in the retina of different groups were observed, and the number of retinal ganglion cells (RGCs) was quantitatively analyzed under microscopy. Apoptotic cells were detected using the TdT-dUTP terminal nick-end labeling (TUNEL) method. The expression of caspase-3 was determined by streptavidin peroxidase (SP) immunohistochemistry. Atomic absorption spectrum method was used to evaluate the intracellular calcium changes.Results At the early stage of retinal ischemia-reperfusion injury, retina edema in the treated group was significantly eliminated compared with the untreated ischemic animals. RGCs in the bFGF-treated group was more than those in the untreated ischemic group during the post-reperfusion stages. In ischemic group, apoptotic cells could be found at 6th hours after reperfusion and reached the peak at 24th hours. At 72th hours no apoptotic cells could be found. The changes in caspase-3 expression had a similar manner. The intracellular calcium of rat retina began to increase at lth hour, reached the peak at 24 hours, and began to decease at 72th hours. The change of the three markers in the treatment group showed a similar pattern, but they were all relatively less obvious.Conclusion Apoptosis may play a vital role in RIRI. bFGF may has therapeutical effects on RIRI by inhibiting the increase of intracellular calciums and caspase-3 expression.     

Caspase-dependent retinal ganglion cell apoptosis in the rat model of acute diabetes

Background Neural apoptosis is generally believed to be mediated by two distinct pathways, caspase-dependant and caspase-independent pathways. This study investigated the apoptotic pathways involved in retinal ganglion cells in acute diabetes in rats. Methods Diabetes was induced in male Wistar rats by a peritoneal injection of streptozotocin （STZ）. Expression and localization of caspase-3 and apoptosis-inducing factor （AIF） proteins in the retina of diabetic rats was examined by Western blotting and immunohistochemistry analyses. Terminal transferase dUTP nick end labeling （TUNEL） assay and immunofluorescent staining specific for caspase-3 and AIF were applied to analyze for apoptosis of retinal ganglion cells. In addition, a caspase-3 inhibitor DEVD-CHO was injected intravitreally to further determine the apoptotic pathways of retinal ganglion cells triggered in acute diabetes. Results Two weeks after induction of diabetes, a significant increase in caspase-3 protein expression and localization occurred in the nerve fiber layer, ganglion cell layer, and inner plexiform layer of the retina. Four weeks after the onset of diabetes, the increase in caspase-3 expression was profound eight weeks postinduction of diabetes （P 〈0.05）. Meanwhile, no AIF protein expression was detected in this study. In addition, intravitreal administration of the caspase-3 inhibitor DEVD-CHO reduced apoptosis of retinal ganglion cells by its direct inhibitory action on caspase-3. Conclusion Caspase-dependent apoptotic pathways may be the main stimulant of STZ-induced retinal ganglion cell apoptosis in acute diabetes.     

Nonvisual ganglion cells, circuits and nonvisual pigments

To the editor： WANG and his colleagues provided the evidence that ＂both melanopsin-containing and super/or collicular retinal ganglion cells were damaged by chronic ocular hypertension, indicating that glaucomatous neural degeneration involves the non-image-forming visual pathway＂） These cells as the authors have beautifully described dealing with a variety of functions including biological clock; pupillary light reaction and they are affected by glaucoma. The report is scientific and quite interesting. I would like to congratulate the authors for their superb studies.     

Effects of Atorvastatin on Warfarin-induced Aortic Medial Calcification and Systolic Blood Pressure in Rats

The effect of atorvastatin on warfarin-induced aortic medial calcification and systolic blood pressure （SBP） of rats induced by warfarin was studied. Thirty healthy and adult rats were randomly divided into Warfarin group （n=10）, Atorvastatin group （n=10） and normal control group （n=10）. Caudal arterial pressure of rats was measured once a week, and 4 weeks later, aorta was obtained. Elastic fiber, collagen fiber and calcium accumulation in tunica media of cells were measured by Von Kossa staining. The results showed that warfarin treatment led to elevation of systolic blood pressure and aortic medial calcification. The chronic treatment also increased collagen, but decreased elastin in the aorta. However, the atorvastatin treatment had adverse effects. It was concluded that treatment with atorvastatin presented evidence of blood pressure lowing and calcification reducing. These data demonstrate that atorvastatin protected aortic media from warfarin-induced calcification and elevation of systolic blood pressure.     

Culture of rat retinal ganglion cells

This study aimed to modify the mixed and purified culture of rat retinal ganglion cells(RGCs) in vitro.The retinae of 1-3 day old Sprague-Dawley(SD) rats were separated bluntly into two layers:inner layer and outer layer,under a surgical microscope.Retinal cells isolated from different layers(inner layer,outer layer and whole retinal tissue) by using enzyme dissociation method were cultured in F12/DMEM medium containing 15% FBS.After 3-day culture,the RGCs in the retinal cells obtained from mixed culture of inner,outer,and whole retinal tissue were identified by immunocytochemical staining of Thy-1.1,and the rate of RGCs to retinal cells(RGCs%) was calculated.Two monoclonal antibodies,anti-macrophages/granulocytes(OX-41) against rat macrophage and antibody against rat Thy-1.1(OX-7),were used to purify RGCs by either a conventional or modified two-stepped immunopanning procedure(purification in situ).Purified RGCs were seeded at different cell density and cultured in F12/DMEM medium containing 15% FBS.Immunocytochemical staining for Thy-1.1,MTT,and PI-Hoechst33342 fluorescence imaging were used to identify the purity and the viability of RGCs in purified culture of RGCs.The results showed:(1) Immunocytochemistry of different retinal tissue layers culture revealed that the RGCs% was(19.9±1.2)%,(0.5±0.2)%,and(6.2±1.7)% respectively in the mixed culture of inner,outer,and whole retinal tissue,with differences being significant(P<0.05);(2) fluorescent double staining of Hoechst33342 and PI indicated that with the same RGCs%,RGCs obtained from purification in situ grew well with more neurite outgrowth than those by the conventional two-stepped immunopanning method;(3) the viability of purified RGCs seeded at high density was in-creased and the cells developed complex intercellular networks.The viability of RGCs was declined with the decreasing seeding density,and most cells presented round or oval in shape with thin neurites.It was concluded that:(1) RGCs% in the inner layer retina was higher than that in the outer layer retina;(2) RGCs obtained by in situ purification had more neurite outgrowth and lower mortality than those by conventional two-stepped immunopanning procedure;(3) the viability of purified RGCs could be increased by increasing cell seeding density to some extent.     

Early activation of caspase-1 after retinal ischemia and reperfusion injury in mice

Background Caspases are important in the signaling pathway of cellular apoptosis. Caspase-3 protein expression has been shown to increase and parallel to neuronal apoptosis in retinal ischemia injury. This study was to determine whether caspase-1 is involved in neuronal cell death or in retinal ischemia and repeffusion injury.Methods In twenty-one adult mice, ischemia was induced by increasing the intraocular pressure.The animals were sacrificed at 1 hour, 3 hours, 6 hours, 1 day, 3 days and 7 days after reperfusion.Frozen sections were used for caspase-1 immunostaining and TUNEL labeling.Results In normal retina, no caspase-1 positive cells were seen. One hour after ischemia,numerous positive cells were noted in the ganglion cell layer (GCL) and inner side of inner nuclear layer (INL). At 3 hours, caspase-1 positive cells continued to increase and peaked at 6 hours, then decreased significantly at 1 day. TUNEL positive cells were detected at 3 hours and peaked at 1 day after ischemia. Double labeling of caspase-1 and TUNEL only showed few cells with co-localization after ischemia.Conclusion Caspase-1 immunoreactivity preceds to the TUNEL labeling in the GCL and INL after retinal ischemia and reperfusion injury and its early activation may play an important role in the initiation of neuronal apoptosis.     

A new rat model of glaucoma induced by intracameral injection of silicone oil and electrocoagulation of limbal vessels

Background  A satisfied glaucoma model is absent now. The aim of this study was to evaluate the effect of a combination of intracameral injection of silicone oil and electrocoagulation of corneal limbal vessels and episcleral veins in the rats to establish glaucoma model.

Methods  Operation was performed in each of the left eyes of 90 adult male rats. Right eyes were used as controls. Measurement of intraocular pressure (IOP) was performed with an applanation tonometer (Tono-Pen). Retinal ganglion cells (RGCs) were retrogradely labeled by applying FluoroGold onto the bilateral superior colliculus.

Results  During the follow-up (24 weeks), the IOP of the study eyes was significantly higher (P <0.05) than the control eyes (at final examination, IOP of control eyes was (13.4±1.0) mmHg and IOP of study eyes was (16.1±1.8) mmHg). Correspondingly, at 24 weeks after operation, the RGCs density of the study eyes (2286.11±290.45/mm²) was significantly lower than the control eyes (2626.46±164.85/mm², P <0.01). In the operated eyes, histological examination showed excavation of optic disc and increased neuroglial cells in the optic nerve, reduced thickness of retina and diminution of retinal ganglion cells, and atrophy of ciliary body and iris. Notably, the anterior chamber angle of the operated eye remained open.

Conclusions  A combination of intracameral injection of silicone oil and electrocoagulation of corneal limbal vessels and episcleral veins may establish a reliable glaucoma model for further research.

     

Validation of glaucoma-like features in the rat episcleral vein cauterization model

Background Glaucoma,an irreversible optic nerve neuropathy,always results in blindness.This study aimed to evaluate glaucoma-like features in the rat episcleral vein cauterization （EVC） model by multiple in vivo and in vitro evidences.Methods Wistar rat was used in this study.The elevated intraocular pressure （IOP） was induced by cauterization of three episcleral veins.lOP was monitored with Tono-Pen XL tonometer.Time-dependent changes to the neuronal retinal layers were quantified by Fourier domain-optical coherence tomography.The function of retina was evaluated by electroretinogram （ERG）.Survival of retinal ganglion cells （RGCs） was quantified by retrograde labeling.Histology study was performed with retinal sections stained with hematoxylin-eosin,glial fibrillary acidic protein,and neuronal nuclear antigen.Retina and aqueous humor protein were extracted and cytotoxic protein tumor necrosis factor alpha （TNF-α） and alpha-2 macroglobulin （α2m） were measured with Western blotting.Results EVC is a relatively facile intervention,with low failure rates （＜5％）.After surgical intervention,chronic mild lOP elevation （about 1.6-fold over normal,P ＜0.05） was induced for at least 6 weeks without requiring a second intervention.High lOP causes chronic and progressive loss of RGCs （averaging about 4％ per week）,progressive thinning of neuronal retinal layers （3-5 μm per week）,and reduction of a-and b-wave in ERG.EVC method can also induce glial cell activation and alterations of inflammation proteins,such as TNF-α and α2m.Conclusion EVC method can establish a robust,reliable,economic and highly reproducible glaucomatous animal model.     

荧光金逆行标记乳鼠视网膜神经节细胞的实验研究

目的建立荧光金逆行标记乳鼠视网膜神经节细胞的方法。方法选取出生1 d的SD大鼠乳鼠,腹腔注射水合氯醛麻醉后,剪开头部皮肤,向双侧上丘各注射4%荧光金注射液1μl,缝合皮肤,4 d后处死,提取视网膜,胰酶消化后培养,显微镜下观察被标记的视网膜神经节细胞。结果混合培养的视网膜细胞中,可见被荧光金标记的视网膜神经节细胞,大约占细胞总数的0.34%,视网膜神经节细胞可存活3周。结论荧光金逆行标记乳鼠视网膜神经节细胞是一种可行的方法,可用于视网膜神经节细胞的鉴定。     

大鼠慢性高眼压诱导视神经损伤的研究

目的建立大鼠慢性高眼压模型,对其眼压、视网膜节细胞(RGCs)和视神经轴突损伤进行研究。方法对40只大鼠行慢性高眼压模型手术,分为4组:术后即刻、5 d、2周和4周,每组10只,分别测定眼压。采用全视网膜-RGCs Cresyl Violet染色,术后2周和4周对大鼠视网膜进行RGCs计数,并对视神经进行对位苯二胺(PPD)染色。对照组20只大鼠,分为2组,每组10只。结果31只大鼠眼压升高,眼压升高率为77.5%,且4周内眼压维持稳定。眼压升高2周时中心和周边RGCs分别减少11.0%和11.3%,眼压升高4周时中心和周边视网膜分别丢失RGCs达17%和24.6%,与正常对照组比较差异均有统计学意义(P<0.05);而眼压未升高的RGCs未见明显丢失,与正常对照组相比差异无统计学意义(P>0.05)。高眼压组大鼠视神经轴突在颞上方有大约5.3%损伤,球后1 mm的视神经在光镜下可以看到视神经轴突水肿和髓鞘碎屑。结论慢性高眼压动物模型是一种可以重复且有效的青光眼模型,它模拟了人类慢性眼压升高所导致的RGCs丢失和视神经的损伤。     

The effect of melatonin on retinal ganglion cell survival in ischemic retina

Our objective was to determine whether melatonin increases retinal ganglion cell (RGC) survival in ischemic mouse retina. Transient retinal ischemia was induced by an acute elevation of intraocular pressure in C57BL/6 mice. To evaluate the effect of melatonin on retinal ischemia, an equal amount of either melatonin or vehicle was intraperitoneally injected into the mice 1 hour before ischemia, at the time of ischemia, and 1 hour after ischemia. Hypoxia inducible factor 1α (HIF-1α) and glial fibrillary acidic protein (GFAP) expression were assessed 6, 12, and 24 hours after ischemia-reperfusion by Western blot. RGC survival was measured 2 weeks after ischemia-reperfusion. The expression of HIF-1α and GFAP peaked 24 hours after ischemia-reperfusion in ischemic retina. The treatment of ischemic retina with melatonin resulted in the inhibition of increased expression of HIF-1α and GFAP. RGC survival was greater in retinas treated with melatonin than in retinas treated with vehicle 2 weeks after ischemia-reperfusion. On the basis of our results, we suggest that melatonin treatment increased RGC survival in ischemic mouse retina. The neuroprotective effect of melatonin is mediated by the inhibition of HIF-1α stabilization and reduced activity of glial cells in ischemic mouse retina.     

肝细胞生长因子在原发性开角型青光眼视神经损伤中的作用及机制研究

目的 探讨肝细胞生长因子( HGF)的受体间质表皮转化因子(c-met)激活是否能促进视网膜神经节细胞存活和再生,以及HGF在慢性青光眼大鼠模型中可能的神经保护作用机制.方法 SD大鼠随机分成3组:正常对照组、慢性高眼压组、慢性高眼压HGF注射组;灼烧3条巩膜上静脉制备慢性青光眼模型;烧灼巩膜上静脉术后立即在玻璃腔内注射4 μl HGF(1 μg/μl);iCare眼压计检查大鼠的眼内压变化;ELISA法测定前房内HGF水平;免疫染色检查c-met在视网膜细胞中的分布;使用末端脱氧核苷酸转移酶dUTP缺口末端标记( TUNEL)染色观察视网膜神经节细胞的凋亡;Western blot法检测蛋白激酶B(Akt)、磷酸化蛋白激酶B( p-Akt)、B细胞淋巴瘤基因-2(Bcl-2)和抗凋亡蛋白B细胞淋巴瘤/白血病-xl(Bcl-xl)蛋白的表达水平.结果 术后早期眼压显著升高,并维持2周;与正常组相比,慢性高眼压大鼠组前房HGF明显升高;与慢性高眼压组相比,慢性高眼压注射HGF组TUNEL标记的RGC减少,差异有统计学意义( P＜0. 05);在视网膜中,c-met主要在神经节细胞(RGC)层的细胞中表达;在视网膜样本中,与正常对照组比较,慢性高眼压组p-Akt、Bcl-2和Bcl-xl蛋白表达显著下调(P＜0. 05);与慢 性高眼压组相比,慢性高眼压 HGF 注射组 p-Akt、Bcl-2 和Bcl-xl蛋白表达显著上调(P＜0. 05).结论 HGF可能通过激活Akt信号通路以及增加其下游的相关抗凋亡蛋白表达而发挥对慢性高眼压视神经的保护作用.     

bFGF对高眼压下兔视网膜神经节细胞的保护作用

目的：研究碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)对高眼压下兔视网膜神经节细胞的保护作用。方法：每周1次前房注入甲基纤维素制成慢性高眼压动物模型，随机将动物分为高眼压对照组、外科对照组和bFGF治疗组，另加1组正常对照组。治疗组每周1次玻璃体内注射bFGF，高眼压对照组不作任何处理，外科对照组仅于玻璃体内注入与治疗组等量的磷酸缓冲生理盐水(plosphate buffered saline，PBS)，实验持续4wk。每周测定各组动物的图像视网膜电图(pattern electroretinogram，PERG)，观察各组动物b波变化趋势，最后光镜、电镜观察动物视网膜神经节细胞的超微结构变化。结果：治疗组PERGb波振幅仅见轻微下降趋势，与正常组接近，两对照组则下降趋势显著，结果经方差分析有统计学意义；形态学上可见治疗组节细胞超微结构变化轻微，对照组则变化明显。结论：bFGF作为一种神经营养因子，对高眼压下兔视网膜神经节细胞具有保护作用。     

多聚物1诱导自身免疫对大鼠高眼压模型视神经的保护作用

目的 探讨多聚物1诱导自身免疫对大鼠高眼压模型视神经的保护作用及其机制.方法 高眼压大鼠随机分为2组,分别皮下注射200μg多聚物1和同等剂量的磷酸盐缓冲液(PBS).荧光金逆行标记视网膜神经节细胞(RGC),在荧光显微镜下进行计数.免疫组织化学方法 观察T细胞在视神经及视网膜的聚集情况.结果 多聚物1组免疫后17、24、31 d RGC存活数目(个/mm2:2617±17、2588±206、2394±15)均多于PBS组(个/mm2:2357±37、2277±340、2129±17,P<0.01、0.05、0.01).免疫后10、17、24、31 d视网膜上聚集的T细胞数多聚物1组(个/mm2:11.3±2.8、36.7±5.2、33.9±3.0、21.4±5.9)也均多于PBS组(个/mm2:4.7±3.6、19.7±2.4、15.3±4.0、13.3±4.4,均P<0.01).结论 多聚物1对大鼠高眼压模型的视网膜神经节细胞RGC有保护作用;高眼压造成的损害引起视网膜处T细胞的聚集,多聚物1能增加聚集于视网膜上的T细胞数量,这可能与多聚物1的视神经保护作用有关.     

多聚物-1免疫高眼压大鼠视网膜中助性T细胞的变化

目的 探讨辅助性T细胞(Th细胞)在多聚物-1( Cop-1)对高眼压大鼠视网膜神经节细胞保护中可能发挥的作用.方法 Wistar大鼠分别结扎3根巩膜上静脉,建立高眼压模型96只,完全随机法分入Cop-1免疫组48只,磷酸盐缓冲液(PBS)免疫组48只,另10只正常大鼠作对照.于免疫后3、7、10、17、24、31 d...     

利鲁唑对急性高眼压致大鼠视网膜损伤的保护作用

目的 探讨腹腔内注射利鲁唑对急性高眼压所致的大鼠视网膜损伤的保护作用.方法 将120只大鼠分为正常对照组、高眼压对照组及利鲁唑干预组,每组40只.采用前房灌注的方式建立急性高眼压模型,免疫组化及末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记法(TUNEL) 分别测定3组大鼠再灌注后12、24、48、72 h的Caspase-3蛋白表达情况及大鼠视网膜各层细胞的凋亡水平,HE染色观察各组大鼠视网膜结构改变.结果 与高眼压对照组相比,利鲁唑干预组视网膜增厚及结构紊乱情况明显减轻,不同时间点Caspase-3的表达水平及凋亡细胞数明显降低(P< 0.05).结论 利鲁唑可减少大鼠急性高眼压损伤后视网膜神经节细胞凋亡,对视网膜具有一定的保护作用.