加兰他敏介导Nrf2/HO-1信号通路对慢性高眼压大鼠视网膜神经节细胞损伤的保护作用

目的 探讨加兰他敏介导核因子E2相关因子2(Nrf2)/血红素氧合酶-1(HO-1)信号通路对慢性高眼压大鼠视网膜神经节细胞（RGCs）损伤的影响及其机制。方法 48只SD大鼠随机分为假手术组、模型组、加兰他敏组、加兰他敏+ML385组,每组12只。假手术组大鼠进行手术操作但不烙闭巩膜静脉,其余各组大鼠采用巩膜上静脉烙闭法建立慢性高眼压大鼠模型。加兰他敏组大鼠ip 4 mg/kg加兰他敏溶液;加兰他敏+ML385组大鼠ip 4 mg/kg加兰他敏溶液和30 mg/kg ML385。采用便携式动物眼压计测定眼压;苏木精–伊红（HE）染色观察视网膜组织形态及RGCs数量;TUNEL染色检测RGCs凋亡情况;试剂盒法检测视网膜组织中丙二醛（MDA）、超氧化物歧化酶（SOD）和过氧化氢酶（CAT）水平;Western blotting法检测视网膜组织中Nrf2、HO-1蛋白表达。结果 与模型组相比,加兰他敏组大鼠眼压降低,视网膜组织病理损伤得到改善,RGCs数量增多,RGCs凋亡率降低,视网膜组织中SOD、CAT活性以及Nrf2、HO-1蛋白表达水平均升高,MDA含量降低（P<0.05）...     

Nogo受体对糖尿病大鼠视网膜神经节细胞凋亡的影响

目的 探讨Nogo受体对糖尿病大鼠视网膜神经节细胞凋亡的影响及机制。方法 SD大鼠腹腔注射链脲佐菌素诱导糖尿病模型,分为对照组,糖尿病组,siNgR组,siRNA空白组,每组10只。siNgR组糖尿病大鼠玻璃体内给予Nogo受体反义核苷酸序列;siRNA空白组糖尿病大鼠玻璃体内注射阴性核苷酸序列。1个月后,免疫荧光组化检测Nogo受体与视网膜神经节细胞（RGC）标志物Brn3a的共存;以TUNEL染色观察RGC凋亡;试剂盒检测视网膜丙二醛（MDA）含量;Western blot检测视网膜Nogo受体及半胱氨酸天冬氨酸蛋白酶3（caspase-3）表达。结果 Nogo受体大量表达于视网膜RGC内,对照组、糖尿病组、siRNA空白组及siNgR组MDA含量分别为（3.68±0.47）、（8.07±1.24）、（7.54±1.53）及（5.12±0.62）μmol/g 蛋白。与对照组相比,糖尿病组及siRNA空白组视网膜RGC凋亡明显,Nogo受体及caspase-3表达上调,MDA含量增加（P<0.05）。与糖尿病组相比siNgR组视网膜RGC凋亡减少、Nogo受体及caspase-3表达下调、MDA含量降低（P<0.05）。结论 Nogo受体表达上调参与了糖尿病视网膜RGC凋亡,其机制可能与Nogo受体上调caspase-3表达、诱发视网膜氧化应激有关。     

神经生长因子β对兔实验性青光眼视网膜神经节细胞的保护作用

目的 探讨神经生长因子β（NGF-β）对青光眼性视网膜神经节细胞（RGCs）损害的保护作用。方法 建立家兔青光眼模型,并动态观察眼压。于术后14d、21d随机选择一眼玻璃体腔注射NGF-β 1μg和细胞色素C 0．1mg（实验组）,对侧眼仅注射细胞色素C 0．1mg作对照（对照组）,共10只兔。术后28d处死实验兔摘取眼球及视神经作组织学观察,并用核苷酸末端转移酶介导的dUTP缺口末端标记法（TUNEL）检测RGCs凋亡状况。结果 组织学检查显示术后28d时视神经神经纤维丢失程度实验组有8例轻于对照组,1例相似,1例较对照组为重。对照组视神经节细胞层凋亡阳性率13．46％,明显高于实验组的10．60％（P〈0．01）。结论 NGF-β对青光眼性视网膜神经节细胞损害具有一定的保护作用。     

实验性大鼠颅眶联合伤后依达拉奉对视神经的保护作用

目的：探讨实验性大鼠颅眶联合损伤（cranio-orbital injury,COI）后视神经损伤机制及依达拉奉的保护作用。方法：144只清洁级SD大鼠,雌雄各半,随机分为对照组、损伤组和治疗组,每组48只。损伤组和治疗组大鼠利用液压冲击颅脑损伤仪建立颅眶联合伤模型,治疗组造模以后腹腔注射依达拉奉30 mg·kg-1,每日2次,共14日。3组分别在造模后3、6、10、14 d取视网膜组织,检测视网膜神经节细胞（retinal ganglion cells,RGCs）活性氧（reactive oxygen species, ROS）含量及不同时间点RGCs凋亡率,光镜下观察视网膜HE染色微观结构,共聚焦显微镜下观察RGCs的TUNEL荧光标记凋亡情况。结果：造模后损伤组RGCs的ROS含量逐渐升高,第10天达到高峰,RGCs凋亡率变化趋势与此相一致,与对照组及治疗组差异有统计学意义（P＜0.05）,治疗组与对照组相比较无明显差异。光镜下损伤组视网膜结构紊乱,损伤组RGCs的TUNEL荧光标记阳性率明显高于其余两组。结论：COI后RGCs的ROS含量明显升高,导致细胞出现凋亡失调。依达拉奉通过清除ROS逆转这一病理过程,具有明确的视神经保护作用,值得临床推广。     

促红细胞生成素对急性高眼压大鼠视网膜凋亡细胞的影响

目的:观察重组人促红细胞生成素(rhEPO)对急性高眼压大鼠视网膜凋亡细胞的影响,探讨rhEPO对视网膜细胞的保护作用。方法:选用36只健康成年雄性Wistar大鼠,随机分为正常组4只,生理盐水组16只,rhEPO组16只,再根据造模成功后观察时间的不同将生理盐水组和rhEPO组分为四组:即6h组、24h组、48h组和72h组,每组4只。采用生理盐水前房加压灌注法制作大鼠急性高眼压模型,利用原位末端标记法(TUNEL)法检测各组动物视网膜在不同时间点的凋亡细胞。结果:正常组大鼠视网膜基本未见凋亡细胞,生理盐水组和rhEPO组在神经节细胞层、内核层均可见凋亡细胞,但rhEPO组与生理盐水组比较,凋亡细胞减少,差异有统计学意义(P<0.01)。结论:急性高眼压对大鼠视网膜的损伤主要由神经节细胞和内核层细胞的凋亡引起,rhEPO预处理可减少高眼压视网膜的凋亡细胞,发挥对视网膜的保护作用。     

当归补血汤抑制慢性青光眼大鼠视网膜神经节细胞凋亡

目的观察当归补血汤对慢性高眼压模型大鼠视网膜神经节细胞(retinalganglioncells,RGCs)凋亡的抑制作用。方法应用烧灼巩膜静脉的方法制作大鼠慢性高眼压模型。50只SD大鼠分入正常组、Timolol组(T)、Timolol加中药组(TC)和高眼压未治疗组(EP)。观察4、6、8周后处死,制作病理切片,计数凋亡视网膜神经节细胞数。结果TC组凋亡RGCs数为1.01±0.55(个/张),明显低于T组5.1±0.74(个/张)和EP组7.28±0.44(个/张,P<0.01);TUNEL-N末端标记凋亡的RGCs数在正常组和给药组之间无显著性差异,但明显低于EP组(P<0.01)。结论当归补血汤对实验性高眼压所致的RGCs的凋亡具有抑制作用。     

三七皂苷对大鼠高眼压RGCL神经元的保护作用

目的 探讨三七皂苷（panax notoginseng saponins，PNS）是否对持续性高眼压导致的大鼠视网膜神经元损伤有保护作用。方法 健康SD大鼠42只，其中6只为正常对照组，其余36只采用AKIRA法，烙闭大鼠右跟上巩膜静脉，制作大鼠持续性高跟压模型，随机分为生理盐水组（A组），PNS治疗组（B组）分别予生理盐水及PNS150mg／kg进行治疗，按观察时间随机分为3个亚组。治疗1、2、4周处死大鼠剥取视网膜作全层铺片行（retinalganglion cell slayer、RGCL）神经元计数分析。结果 正常对照组双眼RGCL神经元计数比较差异无统计学意义（P〉0．05），A组及B组实验眼在以上各时段RGCL神经元计数与正常比较差异均有统计学意义（P〈0．05）．自身对照眼与正常组比较差异无统计学意义（P〉0．05）、第4周时B组RGCL神经元计数高于A组．差异有统计学意义（P〈0．05）。结论 tPNS可对持续性高眼压大鼠RGCL神经元损伤提供部分保护作用。     

银杏叶提取物对糖尿病大鼠视网膜神经节细胞凋亡的作用观察

目的 观察银杏叶提取物（EGB）对糖尿病大鼠视网膜神经节细胞凋亡的影响,探讨EGB防治糖尿病性视网膜病变的作用机制. 方法 选择健康成年Wistar 大鼠50只, 随机分成正常对照组、模型组、低剂量EGB组、中剂量EGB组、高剂量EGB组.腹腔内注射链脲佐菌素（STZ） 诱发大鼠糖尿病.低、中和高剂量EGB组分别给予EGB溶液50 ,100和200 mg·kg^-1·d^-1,灌胃给药,模型组和正常组均灌胃等体积0.9%氯化钠溶液,均连续12周.制备大鼠视网膜石蜡切片行末端脱氧核苷酸转移酶介导的dUTP缺口末端标记 （TUNEL） 法, 检测视网膜神经节细胞凋亡指数（AI）,并对染色结果行计算机图像分析.电镜观察各组神经节细胞的超微结构变化. 结果 模型组大鼠神经节细胞AI比正常对照组显著增加（P＜0.01), 中、高剂量EGB组神经节细胞AI与模型组比较明显减少（P＜0.01),低剂量EGB组神经节细胞AI与模型组比较差异无显著性（P＞0.05）.中、高剂量EGB组神经节细胞超微结构的损害明显减轻. 结论 EGB可以通过抑制视网膜神经节细胞的凋亡来减轻糖尿病性视网膜病变.     

丹七散提取物抗N-甲基-D-天冬氨酸诱导的视神经节细胞凋亡机制

目的:观察丹七散提取物(DQSE)对N-甲基-D-天冬氨酸(NMDA)诱导损伤的大鼠视网膜神经节(RGCL)细胞抗凋亡作用,并探讨其作用机制。方法:Wistar大鼠随机分为对照组、NMDA组、DQSE(3.9,7.8,15.6 g·kg^-1)组和阳性对照组(MK801,NMDA抑制剂)。给药7 d后,对照组大鼠玻璃体注射生理盐水,其余组玻璃体注射NMDA制备视网膜损伤模型。给药14 d后,TUNEL法观察RGCL凋亡;免疫组织染色法检测视网膜casepase-3表达分布;Western-blotting法测定视网膜Cleaved casepase-3,-8和-9表达水平。结果:中、高剂量DQSE能显著减少大鼠RGCL的TUNEL阳性表达率(P<0.01,P<0.001)和casepase-3阳性表达率(P<0.01,P<0.001)。DQSE(3.9,7.8,15.6 g·kg^-1)剂量依赖性地降低视网膜Cleaved casepase-3,-8,和-9表达水平。结论:DQSE通过下调Cleaved caspase-3蛋白及其上游的Cleaved caspase-8、-9蛋白表达水平产生抗凋亡作用,进而减少RGCL细胞丢失,实现视网膜保护作用。     

糖尿病神经病理性痛大鼠脊髓背角凋亡细胞与凋亡蛋白酶caspase-3的表达变化

目的探讨糖尿病神经病理性痛（DNP）大鼠脊髓背角凋亡细胞及与之相联系的凋亡蛋白酶caspase-3的表达变化。方法健康雄性sD大鼠60只,随机分为DNP组和正常对照组,每组30例。DNP组：腹腔注射链脲霉素（STZ）50mg／kg制备成DNP模型;正常对照组给予等容量0．9％氯化钠溶液。分别给予注射STZ或0．9％氯化钠溶液后3、5、7周时取10只大鼠,采集静脉血样,测定空腹血糖浓度,然后测定机械缩足阈值,取2组大鼠L1-5脊髓组织,采用原位末端标记法（TUNEL）检测2组大鼠脊髓背角神经细胞凋亡的变化。采用免疫组织化学染色法观察凋亡蛋白酶caspase-3的表达变化。结果与正常对照组比较,DNP组血糖升高,机械缩足阈值降低（P〈0．05）,凋亡细胞与凋亡蛋白酶caspase-3的表达逐渐增加（P〈0．05）。结论DNP大鼠脊髓背角凋亡细胞与凋亡蛋白酶caspase-3的表达上调。     

KR-31378, a Potassium-Channel Opener,Induces the Protection of Retinal Ganglion Cells in Rat Retinal Ischemic Models

KR-31378 is a newly developed K_ATP-channel opener. To investigate the ability of KR-31378 to protect retinal ganglion cells (RGC), experiments were conducted using two retinal ischemia models. Retinal ischemia was induced by transient high intraocular pressure (IOP) for acute ischemia and by three episcleral vein occlusion for chronic retinal ischemia. KR-31378 was injected intraperitoneally and administered orally in the acute and chronic ischemia models, respectively. Under the condition of chronic ischemia, RGC density in the KR-31378–treated group was statistically higher than that in the non-treated group, and IOP was reduced. In the acute retinal ischemia model, 90% of RGC were degenerated after one week in non-treated retina, but, RGC in KR-31378–treated retina were protected from ischemic damage in a dose-dependent manner and showed inhibited glial fibrillary acidic protein (GFAP) expression. Furthermore, the KR-31378 protective effect was inhibited by glibenclamide treatment in acute ischemia. These findings indicate that systemic KR-31378 treatment may protect against ischemic injury–induced ganglion cell loss in glaucoma.     

Suppressive effect of astaxanthin on retinal injury induced by elevated intraocular pressure

The aim of this study was to clarify the possible protective effect of astaxanthin (ASX) on the retina in rats with elevated intraocular pressure (EIOP). Rats were randomly divided into two groups which received olive oil or 5 mg/kg/day ASX for a period of 8 weeks. Elevated intraocular pressure was induced by unilaterally cauterizing three episcleral vessels and the unoperated eye served as control. At the end of the experimental period, neuroprotective effect of ASX was determined via electrophysiological measurements of visual evoked potentials (VEP) and rats were subsequently sacrificed to obtain enucleated globes which were divided into four groups including control, ASX treated, EIOP, EIOP + ASX treated. Retinoprotective properties of ASX were determined by evaluating retinal apoptosis, protein carbonyl levels and nitric oxide synthase-2 (NOS-2) expression. Latencies of all VEP components were significantly prolonged in EIOP and returned to control levels following ASX administration. When compared to controls, EIOP significantly increased retinal protein oxidation which returned to baseline levels in ASX treated EIOP group. NOS-2 expression determined by Western blot analysis and immunohistochemical staining was significantly greater in rats with EIOP compared to ASX and control groups. Retinal TUNEL staining showed apoptosis in all EIOP groups; however ASX treatment significantly decreased the percent of apoptotic cells when compared to non treated ocular hypertensive controls. The presented data confirm the role of oxidative injury in EIOP and highlight the protective effect of ASX in ocular hypertension.     

硫化氢对青光眼大鼠视网膜细胞的保护作用及机制研究

目的研究硫化氢(H2S)对青光眼大鼠视网膜细胞的保护作用及作用机制。方法雄性SD大鼠随机分为对照组、模型组和低、高剂量实验组,每组25只。模型组及低、高剂量实验组大鼠均用光凝法建立青光眼大鼠模型,造模成功后,低、高剂量实验组大鼠分别腹腔注射50,100μmol·kg^-1·d^-1硫氢化钠(NaHS)溶液(H2S供体),对照组与模型组腹腔注射等量生理盐水,连续干预2周。用动物眼压计测量各组大鼠干预后眼内压,以原位末端标记染色法检测视网膜神经节细胞(RGCs)凋亡情况,用蛋白质印迹法检测各组大鼠视网膜组织中磷酸化原癌基因蛋白Jun氨基末端激酶(p-JNK)、磷酸化细胞外调节蛋白激酶(p-ERK)及磷酸化丝裂原活化蛋白激酶p38(p-p38 MAPK)蛋白相对表达量。结果对照组、模型组和低、高剂量实验组大鼠干预后眼内压分别为(18.55±3.02),(37.11±2.62),(29.34±3.22),(23.87±1.68)mmHg,RGCs凋亡指数分别为(3.24±1.12)%,(59.03±5.92)%,(23.26±3.87)%,(12.52±2.33)%,视网膜组织中p-JNK蛋白相对表达量分别为0.10±0.04,0.81±0.13,0.19±0.05,0.15±0.06,p-ERK蛋白相对表达量分别为0.22±0.05,0.89±0.08,0.32±0.09,0.27±0.06,p-p38 MAPK蛋白相对表达量分别为0.40±0.06,0.94±0.06,0.51±0.11,0.45±0.08。模型组及低、高剂量实验组以上各指标与对照组比较,差异均有统计学意义(均P<0.05);低、高剂量实验组以上各指标与模型组相比,差异均有统计学意义(均P<0.05)。结论 H2S具有降低青光眼大鼠眼内压,改善视网膜组织病变,保护RGCs的作用,其作用机制可能与抑制MAPK信号转导通路有关。     

The role of TLR4/NF-κB signaling pathway in activated microglia of rats with chronic high intraocular pressure and vitro scratch injury-induced microglia

Glaucoma is a kind of blind-causing disease with structural damages of optic nerve and defection of visual field. It is believed that the death of retinal ganglion cell (RGC) is a consequential event of over-reactive immune orchestral cells such as microglia. Previous evidences in animal and clinical studies show the innate immunity plays a pivotal role in neuro-inflammation of glaucoma. Toll-like receptor 4 (TLR4) is expressed on microglia and mediates many neuroinflammatory diseases. We aimed to explore the impacts of high intraocular pressure (IOP) on rat microglia in retina and the regulation of TLR4/NF-κB signaling pathway in scratched microglia cells. In our study, we successfully established chronic high IOP rat model by episcleral vein cauterization (EVC) which behaved like the chronic glaucoma. Besides, we set up an in vitro scratch-induced injury model in rat microglia cells. We found the level of activated microglia cells were significantly increased in the retina of chronic high IOP groups. Moreover, the inhibition of TLR4/NF-κB signaling pathway suppressed the expression of TLR4 protein and mRNA levels of P50, IL-6 and TNF-α. Our original study provided a theoretical basis on targeting TLR4/NF-κB to suppress pro-inflammatory factors releasing in activated microglia and it might be a good treatment target to prevent glaucoma from progressing.     

Effect of botulinum toxin A on the intraocular pressure and the retina in an animal model

Objective: The purpose of our study was to investigate the effect of an inadvertent intravitreal injection of botulinum toxin A (BTA) on the intraocular pressure (IOP) and the retina in an animal model.

Methods: BTA was injected intravitreally in normotensive rats. IOP was measured preoperatively as well as 1, 2, and 4 weeks postoperatively. Retinas were stained in vivo using a retrograde labelling technique and the density of retinal ganglion cells (RGCs) was determined. Immunohistochemistry was performed for rhodopsin and retinal glial fibrillary acidic protein (GFAP).

Results: Significant temporary IOP elevation occurred in all groups in the immediate postoperative period (ANOVA, p < 0.05). IOP changes in the intermediate period were not statistically significant (ANOVA, p > 0.05). The differences in the density of RGCs after BTA injection were not statistically significant (ANOVA, p > 0.05). All retinas displayed the same immunostaining pattern for rhodopsin and GFAP.

Conclusion: Our findings indicate that BTA has probably no severe impact on IOP and the retina after an inadvertent intravitreal injection. However, temporary rise of IOP may possibly occur in the immediate postoperative period due to a volume-effect.     

川芎嗪注射液对高眼压大鼠视网膜神经节细胞凋亡的作用机制研究

目的 观察川芎嗪注射液通过磷脂酰肌醇-3激酶/丝氨酸-苏氨酸蛋白激酶/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/mTOR)通路对高眼压大鼠模型视网膜神经节细胞凋亡的影响.方法 用前房注射羟丙基甲基纤维素的方法建立高眼压大鼠模型.按照体重将造模成功大鼠随机分为4组:模型组、川芎嗪组、激动剂组和抑制剂组;另取10只正常大鼠...     

藏红花酸对链脲佐菌素诱导的糖尿病大鼠视网膜神经上皮的保护作用

目的研究藏红花酸对链脲佐菌素(Streptozotocin,STZ)诱导的糖尿病大鼠视网膜神经上皮的保护作用及机制。方法♂SD大鼠40只,随机选取30只腹腔注入STZ(60 mg·kg^-1)将大鼠随机分为3组:糖尿病对照组、藏红花酸低剂量组(50 mg·kg^-1·d^-1,p.o.)、藏红花酸高剂量组(100 mg·kg^-1·d^-1,p.o.)。空白对照组及糖尿病组每天给予3 mL 0.5%CMC-Na。第8周末取各组大鼠眼球检测视网膜神经上皮caspase-3、蛋白激酶C(protein kinase C,PKC)表达量,实时荧光定量PCR检测大鼠视网膜神经上皮Bcl-2、TNF-α、Bax、PKC-βmRNA表达水平。结果caspase-3、TNF-α、PKC及Bax表达量DM组表达最高,DM+CRO组比DM组明显下降(P<0.05),Bcl-2表达量DM+CRO处理组较DM组明显升高(P<0.01)。结论藏红花酸对STZ诱导的糖尿病大鼠视网膜神经上皮具有保护作用,其可能机制是抑制TNF-α、Bax、caspase-3表达,提高Bcl-2的表达,并且藏红花酸可以减少视网膜神经上皮PKC蛋白和基因的表达,保护糖尿病大鼠视网膜。     

色素上皮衍生因子对大鼠视神经夹伤模型视网膜组织中Caspase-3和Bax表达的影响

目的 探讨色素上皮衍生因子对大鼠视神经夹伤模型视网膜组织中天冬氨酸特异性半胱氨酸蛋白酶3(Caspase-3)、Bax表达的影响.方法 90只SD大鼠随机分为空白对照组、模型组、色素上皮衍生因子(PEDF)组,每组30只.除空白对照组外,均建立视神经夹伤大鼠模型,均取左侧眼球为标本,造模成功后,模型组玻璃体腔内注射平衡盐溶液5μl,PEDF组玻璃体内注射PEDF 5μl(浓度0.2μg/μl),模型组和PEDF组1周、2周再分2次分别向玻璃体腔注射平衡盐溶液5μl和PEDF 5μl.3组大鼠均在第3周时取视网膜组织,电镜下观察视网膜组织超微结构的改变,采用免疫组化染色法观察Caspase-3、Bax表达情况,以及视网膜神经节细胞凋亡情况.结果 透射电镜观察模型组与PEDF组同空白对照组相比,均存在线粒体水肿、胞质浓缩轴突肿胀.同模型组相比,PEDF组胞质浓缩轴突肿胀轻,线粒体结构较完整.TUNEL法细胞凋亡监测:空白对照组染色阴性,模型组和PEDF组均可检测到染色阳性的RGCs,PEDF组TUNEL染色阳性的RGCs表达较模型组显著降低(P<0.05).PEDF组Caspase-3呈弱阳性表达,模型组呈强阳性表达,PEDF组Caspase-3光密度值显著低于模型组(P<0.05);模型组、PEDF组Bax表达高于空白对照组(P<0.05),PEDF组Bax表达又低于模型组,差异有统计学意义(P<0.05).结论 Caspase-3、Bax是诱导RGCs细胞凋亡的重要因子,采用PEDF干预治疗能够降低Caspase-3、Bax表达,减轻RGCs损伤.