外源性BDNF对急性高眼压后大鼠视网膜EIK-1磷酸化的影响

为了研究脑源性神经营养因子(BDNF)干预对急性高眼压(HIOP)后大鼠视网膜EIK-1磷酸化的影响,本实验将72只成年大鼠随机分为单纯高眼压组、BDNF预处理高眼压组和溶媒预处理高眼压组。BDNF预处理高眼压组和溶媒预处理高眼压组动物左眼于加压前2d分别给予BDNF预处理或溶媒,右眼设为正常对照。各组动物左眼眼压升高至闪光视网膜电图b波消失的临界眼压并维持60min,动物分别存活1、3、7、14d后处死,冰冻切片行p-EIK-1的免疫组织化学染色。结果显示:单纯高眼压组急性高眼压后大鼠视网膜节细胞层p-EIK-1阳性细胞数较正常对照组下调(P<0.05);溶媒预处理高眼压组实验结果与单纯急性高眼压组相似。BDNF预处理高眼压组急性高眼压后1、3、7d组大鼠视网膜节细胞层p-EIK-1阳性细胞数与正常对照组相似,14d组下调(P<0.05)。此结果提示外源性BDNF促进了急性高眼压后视网膜节细胞层EIK-1的活化,节细胞层EIK-1的磷酸化对受损的视网膜有保护作用。     

脑源性神经营养因子对急性高眼压后大鼠视网膜Müller细胞的影响

目的：检测脑源性神经营养因子(BDNF)干预急性高眼压后大鼠视网膜 Müller 细胞谷氨酰胺合成酶(GS) 和谷氨酸/天冬氨酸转运体(GLAST)表达的变化,探讨 BDNF 保护节细胞(RGCs)的可能机制。方法：成年大鼠分为3个 BDNF 干预组和3个溶媒对照组并进行玻璃体内注射。2 d 后将预处理动物左眼眼压升高至闪光视网膜电图 b 波消失的临界眼压且维持缺血60 min。实验动物分别存活1、3或7 d 后通过免疫组织化学检测大鼠视网膜 GS 和 GLAST 的表达变化。结果：与正常对照组比较,溶媒对照组 GS 和 GLAST 在存活1 d 和3 d 时表达上调,7d 时下降;BDNF 干预组未出现表达明显变化。结论：BDNF 可能不是通过 Müller 细胞上调 GS 和 GLAST,降低胞外 Glu 来保护 RGCs。     

脑源性神经营养因子对实验性大鼠高眼压视网膜节细胞EIK-1磷酸化水平的作用

目的:观察脑源性神经营养因子(BDNF)干预对急性高眼压(HIOP)后大鼠视网膜EIK-1磷酸化水平的影响,以了解BDNF抗高眼压损伤的机制。方法:成年大鼠72只随机分为HIOP组、BDNF预处理高眼压组和溶媒预处理高眼压组(n=24),每组又根据4个时间点细分为4个亚组(n=6)。BDNF预处理高眼压组和溶媒预处理高眼压组大鼠左眼于加压前2 d分别给予BDNF预处理或等量溶媒剂,右眼作为正常对照。用生理盐水升高各组大鼠左侧眼压,至眼压升高至闪光视网膜电图b波消失的临界眼压时停止注射,并维持60 min。大鼠分别存活1、3、7、14 d后处死,冰冻切片行p-EIK-1的免疫组织化学染色。结果:与正常对照比较,急性高眼压后,HIOP和溶媒预处理高眼压视网膜节细胞层p-EIK-1阳性细胞数明显减少(P0.01);BDNF预处理高眼压组急性高眼压后1d和3d节细胞层p-EIK-1阳性细胞数与正常对照组相似,7d和14 d时有轻度减少(P0.05)。结论:外源性BDNF可以促进实验大鼠急性高眼压后视网膜节细胞层EIK-1活化,提示节细胞层EIK-1的磷酸化介导了BDNF对受损的视网膜的保护作用。     

脑源性神经营养因子预处理后急性高眼压下大鼠视网膜TrkB的表达变化

目的：检测脑源性神经营养因子(BDNF)干预后大鼠视网膜TrkB的表达变化。为受损后视网膜节细胞的保护及外源性BDNF的应用提供一定的理论基础。方法：大鼠左眼分为急性眼高压及BDNF预处理组。使左眼眼压升高至闪光视网膜电图b波消失的临界眼压并维持60 min,分别存活1～14 d后处死,冰冻切片行尼氏染色及TrkB的免疫组织化学。结果：急性高眼压组各时间点节细胞层细胞数目均显著少于BDNF预处理组;1、3 d时TrkB的表达明显增加,7、14 d时则明显减少;BDNF预处理组在存活1、3 d时TrkB的表达明显增加,7 d时则下降至正常对照组水平,14 d时再次明显增加。结论：急性高眼压后大鼠视网膜内TrkB的表达存在时空变化,TrkB表达的上调提示视网膜对BDNF的需求增加。     

脑源性神经营养因子对急性高眼压后大鼠视网膜Mueller细胞的影响

目的：检测脑源性神经营养因子（BDNF）干预急性高眼压后大鼠视网膜Mueller细胞谷氨酰胺合成酶（GS）和谷氨酸／天冬氨酸转运体（GLAST）表达的变化，探讨BDNF保护节细胞（RGCs）的可能机制。方法：成年大鼠分为3个BDNF干预组和3个溶媒对照组并进行玻璃体内注射。2d后将预处理动物左眼眼压升高至闪光视网膜电图b波消失的临界眼压且维持缺血60min。实验动物分别存活1、3或7d后通过免疫组织化学检测大鼠视网膜GS和GLAST的表达变化。结果：与正常对照组比较，溶媒对照组GS和GLAST在存活1d和3d时表达上调，7d时下降；BDNF干预组未出现表达明显变化。结论：BDNF可能不是通过Mueller细胞上调GS和GLAST，降低胞外Glu来保护RGCs。     

外源性BDNF对急性高眼压后大鼠视网膜ERK1/2磷酸化的影响

为研究脑源性神经生长因子(BDNF)干预对急性高眼压后大鼠视网膜磷酸化的细胞外信号调节激酶(p-ERK1/2)表达变化的影响,本实验将成年大鼠随机分为单纯高眼压组、BDNF预处理高眼压组和溶媒预处理高眼压组,BDNF预处理高眼压组和溶媒预处理高眼压组动物左眼于加压前2d分别给予BDNF或溶媒预处理,右眼设为正常对照。各组动物左眼眼压升高至闪光视网膜电图b波消失的临界眼压并维持60min,动物分别存活1、3、7、14d后处死,冰冻切片行p-ERK的免疫组织化学染色。结果显示与正常对照相比,单纯高眼压组急性高眼压后p-ERK表达下调(P<0.05),1、3、7d组内核层出现p-ERK阳性细胞;溶媒预处理高眼压组实验结果与单纯急性高眼压组相似;BDNF预处理高眼压组急性高眼压后1、3、7d时p-ERK的表达与正常对照组相似,7d和14d出现了重新分布。此结果提示外源性BDNF可能通过促进急性高眼压后视网膜ERK1/2的活化对受损的视网膜起保护作用。     

BDNF对小鼠高眼压后视网膜CB-IR神经元的影响

<正> 前期研究显示:BDNF可促进高眼压后视网膜电图b波的恢复,并抑制高眼压后谷氨酸过度生成.钙结合素(CB)能缓冲胞内过量Ca~(2+),被认为具有抗兴奋毒性作用.本实验以免疫组织化学方法研究BDNF对高眼压后视网膜CB-IR神经元的影响,以期进一步阐明BDNF抗高眼压损伤作用.实验组、对照组Wstar大鼠各15只,分别于玻璃体内注射脑源性神经营养因子(BDNF)(3μg/kg)或等量小牛白蛋白,另3只大鼠作正常组.注射后两天,升高各鼠眼内压至各自视网膜电图b波消失的临界眼内压,维持90分钟后降至正常,动物分别存活4小时、1天、3天,经主动脉灌注固定,取视网膜冰冻切片,ABC法行CB免疫组织化学反应.光镜观察结果:①正常组:视网膜CB-IR神经元主要分布于节细胞层与内核层.节细胞数量多且着色深,水平细胞与无长突细胞也有较多呈CB-IR,中等着色,部分双极细胞呈较浅着色.②对照组:存活4小时者,CB-IR节细胞明显少于正常组,且着色较浅;存活1天者,CB-IR神经元少于存活4小时者,但着色较之略深;存活3天者,CB-IR神经元略多于存活1天者,水平细胞着色接近正常,其他神经元着色与前两者比较变化不大.③BDNF组:存活4小时者,内核层CB-IR神经元少于正常组,各类神经元着色稍浅于正常组;存活1天、3天者,各类神经元的数目与着色近似于正常组.高眼压使     

急性高眼压后大鼠视网膜BDNF和TrkB的表达变化

研究急性高眼压后大鼠视网膜BDNF及其高亲和力受体TrkB的表达变化规律,为视网膜损伤后的治疗提供理论依据。本实验采用急性高眼压大鼠模型(成年SD大鼠左眼眼压升高至闪光视网膜电图b波消失的临界眼压并维持60min,分别存活1、3、7、14d后处死),运用免疫组织化学染色方法观察了急性高眼压后大鼠视网膜BDNF及TrkB的表达情况。结果显示急性眼高后1d、3d视网膜BDNF的表达变化不明显,TrkB的表达增强,7d、14d时BDNF、TrkB的表达均明显减少。这种急性高眼压后早期大鼠视网膜内BDNF和TrkB表达变化存在的时空差异提示促进内源性BDNF表达或补充外源性BNDF有可能缓解急性高眼压早期视网膜的损伤。     

外源性脑源性神经营养因子对急性高眼压后大鼠视网膜磷酸化TrkB表达的影响

目的:检测脑源性神经营养因子(BDNF)干预对急性高眼压后大鼠视网膜磷酸化TrkB(p-TrkB)表达变化的影响。方法:成年大鼠随机分为单纯高眼压组、BDNF预处理高眼压组和溶媒预处理高眼压组,BDNF预处理高眼压组和溶媒预处理高眼压组动物左眼于加压前2d分别给予BDNF预处理或溶媒,右眼设为正常对照。各组动物左眼眼压升高至闪光视网膜电图b波消失的临界眼压并维持60min,动物分别存活1、3、7、14d后处死,冷冻切片行p-TrkB的免疫组织化学显色。结果:单纯高眼压组急性高眼压后p-TrkB的表达显著下调;溶媒预处理高眼压组实验结果与单纯急性高眼压组相似;BDNF预处理高眼压组p-TrkB的表达随再灌时间的延长进行性下调,但在各时间点的表达均显著高于单纯高眼压组。结论:外源性BDNF干预部分缓解了急性高眼压后视网膜p-TrkB表达的下调,对急性高眼压后的大鼠视网膜起到一定的保护作用。     

急性眼高压大鼠视网膜谷氨酸/天冬氨酸转运体和谷氨酰胺合成酶的表达

目的：通过检测急性眼高压后大鼠视网膜谷氨酸/天冬氨酸转运体(GLAST)和谷氨酰胺合成酶(GS)的表达变化,探讨急性青光眼视网膜节细胞(RGCs)损伤的可能机制。方法：成年大鼠,根据不同存活时间分组。左眼眼压升高至闪光视网膜电图b波消失的临界眼压且维持缺血60min。实验动物分别存活1、3、7、14d后通过免疫组织化学检测大鼠视网膜GLAST和GS的表达变化。结果：GLAST和GS在存活1d和3d时表达上调然后下调,GS还存在重新分布。结论：GLAST和GS的表达与存活时间密切相关,两者表达的相似变化是急性青光眼RGCs损伤的重要原因。     

双荧光逆向追踪法研究大鼠视网膜节细胞的中枢投射

目的：观察向上丘投射的大鼠视网膜神经节细胞(RGCs)在视网膜内的分布、密度及细胞类型上的差异。方法：将 DiI 与荧光金(FG)注射入 SD 大鼠的同侧和(或)对侧上丘,不同时相荧光显微镜下观察两种荧光标记 RGCs 的分布情况。结果：视网膜与视神经内均可见荧光标记;视网膜内标记 RGCs 的数目随时间延长略有增加。DiI 与 FG 的标记效率无明显差异;RGCs 大多数为对侧投射;视网膜颞背侧区域可见双侧投射细胞;视网膜颞腹侧周边区集中存在同侧投射细胞,多数胞体较大;视网膜其它区域可见零星同侧投射细胞。结论：DiI 与 FG 可高效率逆行标记 RGCs 及部分突起;SD 大鼠视网膜内并存着向对侧、双侧及同侧脑区投射的 RGCs,后二者数量较少,分布局限;同侧投射以大胞体细胞为多。     

Müller细胞反应性胶质化在急性高眼压致大鼠视网膜损伤中作用

 目的 观察Müller细胞反应性胶质化在急性高眼压(AOH)大鼠视网膜中变化及其抑制对视网膜损伤的影响。方法 建立大鼠AOH青光眼模型,分为正常对照(Ctrl)、AOH和AOH+玻璃体内注射胶质毒素α-氨基己二酸(AAA)后再灌注1、3和5d组,以及单纯AAA和AOH+PBS对照组。TUNEL染色检测细胞凋亡,GFAP免疫荧光染色反应Müller细胞反应性胶质化程度,Thy-1染色标记视网膜神经节细胞(RGCs)。结果 AOH可致大鼠视网膜内丛状层和内核层明显变薄、神经节细胞层内细胞排列紊乱和数量减少,并诱发Müller细胞反应性胶质化(GFAP表达增加)。同时,AAA抑制Müller细胞反应性胶质化可明显缓解AOH所致RGCs丢失和凋亡发生。结论 Müller细胞反应性胶质化参与AOH所致视网膜损伤,抑制其反应性胶质化可能是改善高眼压性青光眼视网膜病变的一种有效治疗方法。     

Expression of N-methyl-d-aspartate receptor 1 in rats with chronic ocular hypertension

High levels of glutamate can be toxic to retinal ganglion cells (RGCs). This study investigated the relationship between the N-methyl-d-aspartate receptor 1 (NR) and RGC death in a rat model of chronic ocular hypertension (COHT). COHT was induced in one eye of each rat by episcleral vein cauterization. Retinal protein expression was evaluated at 1, 3, 5 and 9 weeks after cauterization. Quantitative real-time polymerase chain reaction and Western blot analysis showed that NR1 expression was significantly increased in cauterized retinae. NR1 immunoreactivity was observed in the inner nuclear layer (INL) and ganglion cell layer (GCL) in the retina of rats with COHT. RGC density was evaluated after retrograde labeling with fluoro-gold (FG) and 4-di-10-ASP (DiA). A significant decrease in RGC density was observed in ocular hypertensive eyes, and NR1 expression in the GCL suggested an important role of NR1 in the death of RGCs. Memantine (10 mg/kg), an N-methyl-d-aspartate receptor antagonist, was administered orally once daily for up to 5 weeks, while rats in the control group received vehicle phosphate-buffered saline only. Treatment with memantine resulted in a significant reduction in RGC loss and NR1 expression in the eyes of rats COHT. These findings suggest that excessive expression of NR1 is involved in RGC death in glaucoma.     

持续高眼压对大鼠视网膜神经节细胞的损伤

目的 观察持续高眼压对大鼠视网膜神经节细胞（RGCs）的损害。方法 烙闭大鼠上巩膜静脉,制作大鼠持续性高眼压模型,分时点摘取眼球,光镜及TUNEL法观察大鼠视网膜各层厚度变化和RGCs凋亡率,电镜观察RGCs超微结构变化。结果 随着高眼压时间持续,视网膜神经节细胞及纤维层逐渐变薄,节细胞及轴突的超微结构呈进行性损害,神经节细胞的细胞凋亡率增加。结论 持续高眼压导致大鼠RGCs的病理改变过程及其结果符合青光眼视神经损伤的病理特点。     

Immunohistochemical localization of sortilin and p75 in normal and ischemic rat retina

In our previous study, we found the increased expression of p75^NTR and sortilin by Western blotting in ischemic retina induced by elevated intraocular pressure (IOP). Cell specific expression of sortilin and p75 neurotrophin receptor (p75^NTR) was now characterized in normal and ischemic rat retina induced by elevated IOP by double-labeling immunochemistry. Two patterns of sortilin staining in normal retina were identified: punctate and consistent. The former was seen in the retinal ganglion cell (RGC) bodies, probably in Golgi apparatus. The latter was found in astrocytes and Müller glial cells (MGCs). The expression pattern of sortilin did not change in ischemic state induced by elevated IOP. p75^NTR was not found in RGCs, but in MGCs of most of the retinal layers, especially the inner plexiform layer (IPL), and outer plexiform layer (OPL) of normal retina. Taken together, the enhanced expression of sortilin in MGCs might be involved in the neuro–glial interactions in ischemic retina, but may not directly contribute to RGCs death through proNGF binding to complex receptor composed of sortilin and p75^NTR in ischemic retina.     

大鼠视神经切断后视网膜双极细胞PKC-α和recoverin的表达

为了探讨视神经切断后视网膜内部是否存在突触可塑性改变,本实验采用大鼠视神经切断模型,通过免疫组织化学方法检测视神经切断后视网膜双极细胞PKC-α和recoverin的表达变化。结果显示:正常视网膜中,PKC-α和recoverin阳性产物主要见于视网膜内核层、内网层及节细胞层,另外外核层也可见少量recoverin阳性细胞。视神经切断后3d,大鼠视网膜内网层高倍镜下可见PKC-α和recoverin免疫阳性终末的数量开始增加,14d时增至最高,21d、28d呈现逐渐减少的趋势。本研究结果提示视神经切断后视网膜双极细胞与节细胞之间的突触可能存在早期增生,后期溃变的可塑性变化。     

Complement mediated apoptosis leads to the loss of retinal ganglion cells in animal model of glaucoma

This study investigated the role of complement in the protection of retinal ganglion cells (RGCs) in chronic ocular hypertension model of glaucoma. Intraocular pressure (IOP) was elevated in the right eye of Lewis rats by laser photocoagulation (two treatments, 7days apart) of episcleral and limbal veins. Left eye did not receive laser treatment and served as control. Animals were injected with cobra venom factor every fifth day starting day 7 after first laser, to deplete the complement system. Animals were sacrificed at 6-week post-laser. Levels of C3 split products and membrane attack complex (MAC) were elevated in the retina of eyes with increased IOP and complement depletion reduced the loss of Brn3a(+) RGCs accompanied by decreased expression of GFAP and reduced MAC deposition. In complement depleted rats with increased IOP, reduced TUNEL(+) cells in ganglion cell layer, and decreased levels of active caspase-8 and active caspase-9 was observed compared to PBS treated complement sufficient rats with increased IOP. Interestingly, complement depletion also resulted in reduction of calcium influx and levels of BAD in the retinal cells of the eyes with increased IOP. Together, our results provide evidence that complement mediated apoptosis plays a pivotal role in the loss of RGCs in chronic ocular hypertension model of glaucoma.     

The projection of the temporal retina in rats,studied by retrograde transport of horseradish peroxidase

Summary Horseradish peroxidase (HRP) was injected unilaterally into the lateral geniculate nucleus or tectum, or both, in 26 hooded rats in order to mark the exact extent of the retina from which uncrossed optic axons arise. This region occupied about a quarter of the retina, in the temporal periphery, following thalamic injections, but a much smaller region following tectal injections. By comparing the proportions of HRP positive neurones in nasal and temporal retinae of both eyes it was shown that: (1) within the region supplying uncrossed axons the majority of the ganglion cells nevertheless project contralaterally, (2) a large proportion of the ganglion cells from the temporal crescent project bilaterally, which does not occur from the remainder of the retina, (3) ganglion cells of all sizes contribute to both ipsilateral and contralateral projections. The results also support earlier suggestions that the smallest neurones in the ganglion cell layer do not send an axon into the brain, and are therefore not ganglion cells