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。     

外源性脑源性神经营养因子对急性高眼压后大鼠视网膜谷氨酸转运体1表达的影响

目的:检测外源性脑源性神经营养因子(BDNF)对急性高眼压后大鼠视网膜谷氨酸转运体1(GLT-1)表达的影响。方法:72只SD大鼠随机分成急性高眼压组、溶媒预处理急性高眼压组和BDNF预处理急性高眼压组,每组动物左眼制作急性高眼压模型,右眼为正常对照,每组动物分别存活1、3、7或14d。用免疫组织化学方法检测外源性BDNF对视网膜GLT-1表达的影响。结果:溶媒预处理急性高眼压组中GLT-1的表达与急性高眼压组相同。与正常视网膜比,急性高眼压组GLT-1的表达在再灌1d时上调,3d时达到高峰,7、14d时表达下调,但仍高于正常对照组。在BDNF预处理急性高眼压组,再灌1d时GLT-1表达明显高于急性高眼压组,3、7、14d时GLT-1表达与急性高眼压组无明显差别。结论:外源性BDNF对急性高眼压后大鼠视网膜的保护作用可能与再灌早期提前上调GLT-1的表达有关。     

BDNF对急性高眼压后大鼠视网膜三种钙结合蛋白表达的影响

目的：检测外源性BDNF对急性高眼压后大鼠视网膜Parvalbumin（PV），calbindin D-28k（CB）和calretinin（CR）表达的影响。方法：72只SD大鼠随机分成高眼压组、溶媒预处理高眼压组和BDNF预处理高眼压组，每组动物左眼制作急性高眼压模型，右眼为正常对照，动物分别存活1、3、7或14d。用免疫组化方法检测视网膜PV、CB和CR的表达变化。结果：溶媒预处理高眼压组中PV、CB和CR的表达与高眼压组相似。高眼压组中，随着再灌时间延长，内层视网膜中PV和CB标记的神经元数先下调再逐渐恢复，而CR标记的神经元数逐渐减少；再灌1d时PV和CR具有从内核层到内网层的重新分布。在BDNF预处理高眼压组，PV、CB和CR标记的神经元数在再灌1d、7d和14d时与高眼压组比较无明显差异，但在再灌3d时，明显高于后者（P〈0．05）。结论：BDNF预处理对急性高眼压后视网膜的保护作用可能与其再灌早期上调钙结合蛋白的表达有关。     

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。     

急性大鼠眼高压诱导的不同缺血/再灌内层视网膜的变化

目的：探讨急性眼高压诱导的不同缺血／再灌条件下内层视网膜形态与功能的变化。方法：成年大鼠,根据不同加压与再灌时间分组。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30～90min。实验动物分别再灌1、3、7或14d复测b波,尼氏染色检测节细胞(RGCs)密度、内层视网膜厚度。结果：缺血60或90min组RGCs密度和内层视网膜厚度显著下降,且下降的程度随再灌时间的延长而加重。缺血30或45min组复测b波部分恢复;而缺血60min或更长时间组则b波未恢复。结论：缺血60min或更长时间导致内层视网膜严重损伤,而不同的眼高压维持时间和再灌时间都是影响视网膜损伤程度的重要因素。     

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。     

急性高眼压后大鼠视网膜葡萄糖转运因子-1的表达

目的:观察急性高眼压后大鼠视网膜葡萄糖转运因子-1(GLUT-1)的表达变化及其对视网膜损伤的影响.方法:利用急性高眼压大鼠模型,通过免疫组织化学和免疫印迹检测急性高眼压后大鼠视网膜GLUT-1蛋白的表达变化;Nissl染色观察视网膜神经细胞层次改变及节细胞数的变化.结果:正常视网膜血管内皮细胞有GLUT-1表达,急性高眼压后3h开始下调,6h达最低值,之后1d、3d、7d逐渐恢复至正常水平.Nissl染色结果显示急性高眼压后3h、6h和12 h视网膜节细胞数减少不明显,到1、3d,视网膜层次紊乱、变薄,节细胞数明显减少.结论:急性高眼压后早期大鼠视网膜GLUT-1表达下调,影响了视网膜葡萄糖的转运,这可能是视网膜结构紊乱,节细胞丢失的重要原因.     

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。     

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。     

Immunohistochemical localization of phospholipase D1 in the retina of pigs

The expression of phospholipase D1 (PLD1) was examined in the retinas of pigs. Western blot analysis detected the expression of PLD1 in the retinas of 1-day-old piglets and showed that it was enhanced in the retinas of 2 years old adult pigs. Immunohistochemically, PLD1 was mainly immunostained in ganglion cell bodies in the ganglion cell layer, in some radial processes of Muller cells in the retinal layer and in the inner and outer segments of the rod and cone layer in newborn and adult pigs, but not in astrocytic bundles in nerve fiber layers. The immunoreactivity of PLD1 in the radial processes of Muller cells across the retinal layers was enhanced in adult pig retinas compared to those of newborn piglets. This was the first demonstration to show that PLD1 is constitutively expressed in the retina of pigs, implying that retinal PLD1 expression is enhanced in radial fibers of Muller cells with age. This finding suggests that PLD1 plays an important role in signal transduction of glial cells and neuronal cells in the retina.     

Phospholipase D1 is up-regulated in the retina of Lewis rats with experimental autoimmune uveoretinitis

To investigate whether phospholipase D1 (PLD1) is involved in autoimmune damage to the eyes, we used Western blotting and immunohistochemistry to examine the expression and distribution of PLD1 in the retinas of Lewis rats with experimental autoimmune uveoretinitis (EAU). Western blot analysis showed that the level of expression of PLD1 was significantly increased in EAU-affected retinas compared to that of control. Immunohistochemical analysis showed that ganglion cells and Muller cells, which express PLD1 weakly in the normal retina, showed increased expression of PLD1 in EAU-affected retinas; some ED1-immunopositive cells were also immunopositive for PLD1 in lesions at post-immunization days 14 and 21. These results suggest that the increased expression of PLD1 in inflammatory cells exacerbates an autoimmune response in EAU, while its expression in ganglion cells limits cell loss by activation of survival factors in eyes with autoimmune injury.     

信号转导子与转录激活子3蛋白在金黄地鼠视网膜生后早期发育过程中的表达

目的 观察信号转导子与转录激活子3(STA13)蛋白在金黄地鼠视网膜生后早期发育过程中的表达。方法 STA13蛋白免疫细胞化学染色和Westem免疫印迹方法结果生后发育早期整个视网膜神经层均有STA13阳性产物分布,以视网膜节细胞层(GcL)、内网层(IPL)和成神经细胞层(NBL)的内层更明显,随着发育进程,其阳性产物逐渐局限于视网膜节细胞的胞浆和胞核;蛋白半定量结果显示,生后1周内STA13蛋白表达水平较高,之后逐渐降低,成年后最低。结论 STA13蛋白的表达及变化可能与视网膜生后早期发育密切相关。     

Enhanced protein expressions of sortilin and p75NTR in retina of rat following elevated intraocular pressure-induced retinal ischemia

Elevated introcular pressure (IOP)-induced retinal neuron ischemic death includes an early phase of necrosis and prolonged phase of apoptosis. We used this ischemic model to observe the changes of sortilin and p75(NTR) protein expressions in rat retina. The results of Western blot analysis showed the expression of p75(NTR) at the band of 75 (mature form), 60 (non-glycosylated pieces) and 50 kDa (ectodomain shedding pieces), and the expression of sortilin at the 95 and 90 kDa (the mature form). The protein expressions of p75(NTR) (60 and 50 kDa pieces) and sortilin (90 kDa) increased significantly (p < 0.05) at days 3, 5 and 7 after retinal ischemia. This effect was also confirmed by immunofluorescence staining. Sortilin was primarily present in cell membrane of the ganglion cells layer (GCL) and large ganglion cell bodies by immunofluorescence labeling. There was little expression of p75(NTR) in the normal retina, while expression increased extensively in GCL, inner plexiform layer (IPL) and inner nuclear layer (INL) after retinal ischemia. p75(NTR) was shown to co-localize with neurofilament in the axons of neuronal cells by double-labeling. These results suggested that the protein expressions of 60 and 50 kDa forms of p75(NTR), and the 90 kDa mature form of sortilin increased in ischemia-induced retinal neuron of rats.     

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.     

Localization of p27kip1 in the developing avian retina: Sustained expression in the mature tissue

p27kip1 is a cyclin/CDK inhibitor that is expressed in cells that exit cell cycle and turn post-mitotic. Here, we characterized the expression and localization of p27kip1 during the development of the chick embryo retina. Expression of p27kip1 in this tissue begins at embryonic day 5 (E5), increasing as development proceeds. In contrast to the expression in the developing rat retina that markedly decreases after postnatal day 6, expression of p27kip1 in the chick retina decreases only slightly (30%) after E12. Thereafter, it remains highly expressed in the tissue. p27kip1 expression increases in an orderly succession. By E5, immunoreactivity was observed over β-tubulin III (TUJ-1) positive cell bodies located in the prospective Ganglion Cell Layer. By E7, p27kip1 was also detected over elongated cell nuclei located in the inner and outer portions of the Neuroblastic Layer and over cell bodies in the middle of the Inner Plexiform Layer. By E12, besides labeled cell bodies, labeled processes from amacrine cells and from cells at the GCL in the IPL were identified. In retinas from post-hatched chicken, immunoreactivity was observed over cell bodies located at all nuclear layers. Several differentiated ChAT positive cholinergic cells were labeled for p27kip1. Our data suggest that, as in the retina of other species, p27kip1 is expressed in cells that are exiting cell cycle and differentiating in the early developing chick embryo retina. However, as opposed to rodents and amphibians, neuronal expression of p27kip1 is sustained in the adult chick retina, indicating that its expression is differently regulated during development in this specie.     

Distribution of GABA immunoreactivity in kainic acid-treated rabbit retina

Ischaemic retinal cell degeneration seems to involve both NMDA and non-NMDA receptor over stimulation. However, different retinal cell types differ largely in their susceptibility to excitatory amino acid induced neurotoxicity. We have investigated the vulnerability of GABAergic cells in the rabbit retina to the non-NMDA receptor agonist kainic acid (KA). The distribution of GABA immunoreactivity (GABA-IR) was examined in the central inferior retina at different survival times (5 h–6 days) following an intra-ocular injection of 140 nmol KA and compared to that of control and untreated retinas. In the normal retina, the majority of GABA-positive cells (79%) were located in the inner nuclear layer (INL), in one to four cell rows next to the inner plexiform layer (IPL), and in one cell row next to the outer plexiform layer (OPL). The remainder (21%) were found in the ganglion cell layer (GCL). Dense immunoreactivity was seen throughout the IPL. In the OPL, stained dots and occasional immunoreactive large processes could be seen. KA-exposed retinas processed for GABA immunocytochemistry 5 and 24 h after the injection showed an 85% reduction in the number of GABA immunoreactive cells. About the same degree of depletion was seen among GABA-IR cells located at different retinal levels. However, at these survival times, immunostaining was observed in three distinct bands in the IPL, indicating that the vulnerability to KA is not uniformly distributed among all GABAergic cells. At 48 h, an additional decrease in the number of labelled cells was noted, but immunoreactive cells were still found both in the INL and GCL. Even 6 days after KA treatment, a few stained cell bodies were seen in the INL next to the IPL, as well as a few processes in the IPL. The study shows that KA receptor overstimulation induces a marked depletion of the endogenous cellular GABA pools of the central rabbit retina, most likely as a result of GABAergic cell loss. However, a small population of GABAergic cells located in the INL appears to be less vulnerable to the toxic effects of 140 nmol KA.     

游离锌离子在小鼠视网膜的定位研究

目的研究游离锌离子在小鼠视网膜的定位分布。方法应用ZnSe金属自显影技术(AMG)检测硒酸钠注射40 m in后小鼠视网膜内的锌离子。结果注射硒酸钠40 m in后发现游离锌离子主要分布于小鼠视网膜的色素上皮细胞层、光感受器的内节、外核层、外网层、内核层、内网层和神经节细胞层。在色素上皮细胞层、光感受器的内节和内核层与内网层交界处AMG阳性反应最为明显,在光感受器外节和神经纤维层几乎没有AMG阳性反应产物。结论小鼠视网膜内锌离子,在视网膜神经元视觉信息的传导和形成过程中可能起着重要作用。     

Expression of voltage-dependent calcium channel subunits in the rat retina

The expression patterns of different Ca(2+) channel alpha(1) subunits (alpha(1A-E)) were immunohistochemically studied in the rat retina. Intense immunoreactivity (IR) for alpha(1A) (P/Q-type) and alpha(1B) (N-type) Ca(2+) channels was observed in both the outer and inner plexiform layers (OPL and IPL). In addition, alpha(1B)-IR was found in the outer and inner nuclear layers. Staining for alpha(1E) (R-type) was diffusely distributed in all three nuclear layers and in the IPL. The alpha(1C) and alpha(1D), two L-type Ca(2+) channel subunits, exhibited distinct expression patterns, with alpha(1C) being almost exclusively expressed on bipolar cells, and alpha(1D) mainly on photoreceptor cell bodies and in the OPL. Staining for alpha(1D) was also observed on Müller cells. The differential expression pattern of the alpha(1) subunits suggests that these Ca(2+) channel subtypes may be associated with different retinal functions.