雪旺细胞源营养神经活性物质对大鼠视网膜节细胞损伤的作用

目的 观察雪旺细胞 (Schwann cells,SC)源营养神经活性物质 (SC derived neurotrophic ac-tivity,SCNA)对 Sprague- Dawly(SD)大鼠视神经损伤后视网膜节细胞 (retinal ganglion cells,RGC)存活的影响。 方法 体外培养日龄 3～ 5 d SD乳鼠 SC,收集无血清条件培养液 ,经超滤浓缩后制成冻干粉。SD大鼠分为正常对照组 ,视神经夹伤对照组 ,视神经夹伤溶剂对照组 ,视神经夹伤 SCNA治疗组 ,每组 2 0只眼。荧光金逆行标记 RGC后 7d,除正常对照组外均行球后视神经夹伤 ,SCNA治疗组将 10 0 ng SCNA注入大鼠玻璃体腔内。分别于视神经夹伤后第 5、7、14、2 1、2 8d将动物灌注固定 ,做全视网膜铺片 ,行 RGC计数。 结果 视神经夹伤后第 7d RGC开始减少 ,14d时降至正常对照的 70 .2 % ,2 8d时降至 40 .5 %。 SCNA治疗组 7d时 RGC数开始减少 ,但 14、2 1、2 8d RGC数均明显多于视神经夹伤对照组及视神经夹伤溶剂对照组 (P<0 .0 1)。 结论 在视神经夹伤后眼内注射 SCNA能减少 RGC的死亡对 RGC损伤有保护作用。

Effects of Qingguang’an Granules on mitochondrial autophagy of retinal ganglion cells in rats with chronic ocular hypertension

Objective To investigate the effect and underlying mechanism of Qingguang’an Granules(青光安颗粒剂,QGAG)on mitochondrial autophagy(mitophagy)of retinal ganglion cells(RGCs)in rats with chronic ocular hypertension(COH).Methods Sixty Sprague Dawley(SD)rats,half males and half females,were randomly assigned to three groups:the control,model,and QGAG(2.5 g/kg)groups,with 20 rats in each group.Rats’model of COH was established by cauterizing episcleral veins in the model group and QGAG group.Three weeks after successful modeling,rats in the QGAG group were intra-gastrically administered with QGAG,while rats in the control group and the model group received an equal dose of normal saline.After three months of intragastric administration,intraocular pressure(IOP)of all rats was measured.The mitophagy was monitored by the immunofluorescence method,the mitochondrial membrane potential was measured using the JC-1 method,and the morphological changes of mitophagy in RGCs were observed by transmission electron microscopy.Meanwhile,rat RGCs were labeled using the fluorescent gold method,and RGCs density in each group was calculated.Moreover,RGCs apoptosis was observed by TdT-mediated dUTP Nick-End Labeling(TUNEL)assay.Finally,the expression levels of Parkin,optineurin,microtubule-associated protein 1 light chain 3-Ⅱ/microtubule-associated protein 1 light chain 3-Ⅰ(LC3-Ⅱ/LC3-Ⅰ),recombinant lysosomal associated membrane protein 1(LAMP1),and B-cell lymphoma-2(Bcl-2)in RGCs were determined by Western blot assay.The corresponding mRNAs were detected through quantitative real-time polymerase chain reaction(qRT-PCR).Results The QGAG reduced IOP in COH rats,and inhibited mitophagy and apoptosis of RGCs(P<0.05).Besides,the QGAG significantly increased the expression levels of Parkin and Bcl-2(P<0.05),and inhibited the expression levels of optineurin,LAMP1,and LC3-Ⅱ/LC3-Ⅰ(P<0.05)in RGCs of COH rats.Conclusion The QGAG can inhibit mitophagy in RGCs of COH rats and show a protective effect against optic nerve damage caused by glaucoma,which may be mediated through the mitophagy ubiquitination via the Parkin/PINK1-related pathway.

Retinal ganglion cells of high cytochrome oxidase activity in the rat

Retinal ganglion cells in the rat were studied using the heavy metal intensified oytochrome oxidase and horseradish peroxidase histochemieal methods. The results show that a population of large retinal ganglion cells was consistently observed with the eyto3hrome oxidase staining method in retinas of normal rats or rats which received unilateral thalamotomy at birrth. These oytochrome oxidase rich ganglion cells appeared to have large somata, 3-6 primary dendrites and extensive dendritic arbors, and are comparable to ganglion cells labeled by the wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). However, the morphological details of some of the cells revealed by the cytoahrome oxidase staining method are frequently better than those shown by the HRP histochemieal method. These results suggest that the mit03hondrial enzyme oytoohrome oxidase can be used as a simple but reliable marker for identifying and studying a population of retinal ganglion cells with high metabolie rate in the rat.

Pro-protein convertase-2/carboxypeptidase-E mediated neuropeptide processing of RGC-5 cell after in vitro ischemia

Objective To observe the change of the neuropeptide pro-protein processing system in the ischemic retina ganglion cell-5(RGC-5) cells,pro-protein convertase-2(PC2),carboxypeptidase-E(CPE) and preproneuropeptide Y(preproNPY) protein levels in the ischemic RGC-5 cells and conditioned medium were analyzed. Methods The RGC-5 cell was differentiated in 0.1 μmol/L staurosporine for 24 h and then stressed by different doses of oxygen and glucose deprivation(OGD). The acute or chronic OGD-induced cell death rates w...

Epac2-deficiency leads to more severe retinal swelling, glial reactivity and oxidative stress in transient middle cerebral artery occlusion induced ischemic retinopathy

Ischemia occurs in diabetic retinopathy with neuronal loss, edema, glial cell reactivity and oxidative stress. Epacs, consisting of Epac 1 and Epac2, are cAMP mediators playing important roles in maintenance of endothelial barrier and neuronal functions To investigate the roles of Epacs in the pathogenesis of ischemic retinopathy, transient middle cerebral artery occlusion （tMCAO） was performed on Epacl-deficient （Epacl-/- ） mice, Epac2-deficient （Epac2-/-） mice, and their wild type counter-parts （Epacl＋/＋ and Epac2＋/＋）. Two-hour occlusion and 22-hour reperfusion were conducted to induce ischemia/reperfusion injury to the retina. After tMCAO, the contralateral retinae displayed similar morphology between different genotypes. Neu-ronal loss, retinal edema and increase in immunoreactivity for aquaporin 4 （AQP4）, glial fibrillary acidic protein （GFAP）, peroxiredoxin 6 （Prx6） were observed in ipsilateral retinae. Epac2 / ipsilateral retinae showed more neuronal loss in retinal ganglion cell layer, increased retinal thickness and stronger immunostaining of AQP4, GFAP, and Prx6 than those of Epac2＋/＋. However, Epacl-/- ipsilateral retinae displayed similar pathology as those in Epacl＋/＋ mice. Our observations suggest that Epac2-deficiency led to more severe ischemic retinopathy after retinal ischemia/reperfusion injury.

microRNA-21a-5p/PDCD4 axis regulates mesenchymal stem cell-induced neuroprotection in acute glaucoma

Mesenchymal stem ceils （MSCs） have been demonstrated to have promising therapeutic benefits for a variety of neurological dis- eases; however, the underlying mechanisms are poorly understood. Here, we showed that intravitreal infusion of MSCs promoted retinal ganglion cell （RGC） survival in a mouse model of acute glaucoma, with significant inhibition of microglial activation, production of TNF-α, IL-1β, and reactive oxygen species, as well as caspase-8 and caspase-3 activation. In vitro, MSCs inhibited both caspase-8-mediated RGC apoptosis and microgUal activation, partly via the action of stanniocalcin 1 （STCl）. Furthermore, we found that microRNA-21a-Sp （miR-21） and its target, PDCD4, were essential for STC1 production and the neuroprotective property of MSCs in vitro and in vivo. Importantly, miR-21 overexpression or PDCD4 knockdown augmented MSC-mediated neuroprotective effects on acute glaucoma. These data highlight a previously unrecognized neuroprotective mechanism by which the miR-21/ PDCD4 axis induces MSCs to secrete STC1 and other factors that exert neuroprotective effects. Therefore, modulating the miR- 21/PDCD4 axis might be a promising strategy for clinical treatment of acute glaucoma and other neurological diseases.

Melanopsin-expressing retinal ganglion cell loss and behavioral analysis in the Thy1-CFP-DBA/2J mouse model of glaucoma

In this study, the role of melanopsin-expressing retinal ganglion cells （mRGCs） in the glaucoma-induced depressive behavioral response pattern was investigated. The CFP-D2 transgenic glaucoma animal model from five age groups was used in this study. Immunohistochemical labeling, quantitative analysis of mRGC morphology, open field test （OFT）, and statistical analysis were used. In comparison with C57 BL/6 mice, the age-matched CFP-D2 mice had significantly elevated intraocular pressure （lOP）. We observed parallel morphological changes in the retina, including a reduction in the density of cyan fluorescent protein- （CFP） expressing cells （cells mm^-2 at 2 months of age, 1309±26; 14 months, 878±30, P〈0.001）, mRGCs （2 months, 48_＋3; 14 months, 19±4, P〈0.001）, Brn3b-expressing RGCs （2 months, 1283±80; 14 months, 950±31, P〈0.001）, Brn-3b expressing mRGCs （5 months, 50.17%±5.5%; 14 months, 12.61%±3.8%, P〈0.001）, and reduction in the dendritic field size of mRGCs （mm^2 at 2 months, 0.077±0.015; 14 months, 0.065±0.015, P〈0.05）. CFP-D2 mice had hyperactive locomotor activity patterns based on OFT findings of the total distance traveled, number of entries into the center, and time spent in the center of the testing apparatus. The glaucoma induced hyperactive response pattern could be associated with dysfunctional mRGCs, most likely Brn-3b-positive mRGCs in CFP-D2 mice.