左卡尼汀对糖尿病大鼠视网膜神经节细胞保护作用的实验研究

目的研究左卡尼汀对糖尿病大鼠视网膜神经节细胞的保护作用。方法通过STZ诱导建立I型糖尿病大鼠模型,成模后采用灌胃方式给予不同剂量的左卡尼汀口服液,6周后行电镜观察,比较疾病模型组与治疗组之间视网膜节细胞的超微结构变化,并进行DNA末端转移酶介导缺口末端标记(TUNEL)染色,观察细胞的凋亡情况。结果电镜显示药物治疗组神经节细胞核异染色质及线粒体空泡化较对照组减少,200 mg·kg-1的治疗剂量效果优于100 mg·kg-1组(P<0.05)。药物治疗组减少了由糖尿病导致的视网膜神经节细胞的凋亡数目(P<0.05)。结论左卡尼汀对糖尿病大鼠模型视网膜节细胞具有保护作用,抗凋亡可能是作用机制之一。

2型糖尿病患者视网膜神经纤维层厚度水平及临床意义分析

目的分析2型糖尿病患者视网膜神经纤维层厚度水平及临床意义。方法选取2018年5月-2019年4月在我院复查的40例2型糖尿病患者设为观察组,共80眼;选取同期我院体检中心正常体检者42例设为对照组,共84眼。对比2组下象限视网膜神经纤维层(RNFL)厚度及黄斑区视网膜厚度。结果对照组的下象限RNFL厚度大于观察组(P<0.05),2组视盘周围平均值、颞象限、上象限、鼻象限RNFL厚度对比无统计学差异(P>0.05);2组中心凹厚度无统计学差异(P>0.05),观察组平均视网膜厚度大于对照组(P<0.05)。结论糖尿病患者存在早期视网膜神经纤维层变薄现象,且黄斑部视网膜厚度增加,应给予关注及干预。

青光眼睫状体炎综合征反复发作对视网膜神经纤维层厚度及视野影响的研究

目的观察青光眼睫状体炎综合征反复发作是否会对视网膜神经纤维层厚度及视野产生影响。方法收集2014年7月—2016年7月在我院诊断为青光眼睫状体炎综合征且发作2次的患者16例,将其发病眼作为发作眼组,对侧眼列为对侧眼组,选取在我院健康体检者为正常对照组。在第1次及第2次发作眼压控制后3 d内及满3个月时均记录一般资料、眼部常规检查,并运用自动视野计行标准全视野检查,眼科相干光断层扫描仪行以视乳头为中心1.73 m m半径内不同象限视网膜神经纤维层厚度检查。结果各组不同时间点视网膜神经纤维层厚度的比较差异无统计学意义(P>0.05);16例患者在反复发作后出现视野缺损和出现鼻侧阶梯或者旁中心暗点的患者均为0例(0眼)。结论青光眼睫状体炎综合征反复发作对视网膜神经纤维层厚度的改变不明显,但可能出现视野损害。

MGB probe assay for rapid detection of mtDNA11778 mutation in the Chinese LHON patients by real-time PCR

Objective： Leber＇s hereditary optic neuropathy （LHON） is a maternally inherited degeneration of the optic nerve caused by point mutations of mitochondrial DNA （mtDNA）. Many unsolved questions regarding the penetrance and pathophysiological mechanism of LHON demand efficient and reliable mutation testing. This study aims to develop a minor groove binder （MGB） probe assay for rapid detection of mtDNA11778 mutation and heteroplasmy in Chinese LHON patients by real-time polymerase chain reaction （PCR）. Methods： Forty-eight patients suspected of having LHON and their maternal relatives underwent a molecular genetic evaluation, with 20 normal individuals as a control group at the same time. A real-time PCR involving two MGB probes was used to detect the mtDNA 1 1778 mutation and heteroplasmy. A linear standard curve was obtained by pUCmLHONG and pUCmLHONA clones. Results： All 48 LHON patients and their maternal relatives were positive for rntDNA11778 mutation in our assay, 27 heteroplasmic and 21 homoplasmic. Eighteen cases did not show an occurrence of the disease, while 9 developed the disease among the 27 heteroplasmic mutation cases. Eleven did not show an occurrence of the disease, while 10 cases developed the disease among 21 homoplasmic mutation cases. There was a significant difference in the incidence between the heteroplasmic and the homoplasmic mutation types. The time needed for running a real-time PCR assay was only 80 min. Conclusion： This real-time PCR assay is a rapid, reliable method for mtDNA mutation detection as well as heteroplasmy quantification. Detecting this ratio is very important for predicting phenotypic expression of unaffected carriers.

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.

Identification of connexin 50 and 57 mRNA in A-type horizontal cells of the rabbit retina

Horizontal cells (HCs) mediate negative feedback to photoreceptors. In the mammalian retina, there are two types of HCs, which are extensively coupled to neighboring cells through homologous gap junctions. The permeability and therefore the strength of feedback can be regulated by light intensity, dopamine and many other factors. However, the component(s) of the most prominent gap junctions, those between A-type HCs in the rabbit retina, is still unknown. In this study, we compared the sequences of many types of mammalian connexins, obtained partial sequences of rabbit connexin 50 and 57. Using specific primers designed against the rabbit sequences, we identified mRNAs of connexin 50 and/or 57 in visually selected single A-type HC using multiplex RT-PCR.

Retinal Prosthesis:A Potential Benefit for the Blind

Retinal degenerative diseases may induce the degeneration of outer retina and in turn,blindness.Nevertheless,due to the maintenance of inner retina,the coding and processing of visual neurons responses are still able to be executed naturally.Therefore,an effective retinal prosthesis device may be developed by mimicking the function of outer retina:transferring the visual light into artificial stimulus and delivering the stimulus to the retina aiming to evoke the neural responses.As two main developing directions for current retinal prosthesis,epiretinal(ER)and subretinal(SR)prosthesis are both undergoing experimental stage and possessing advantages and limitations.Further investigations in power supply,biocompatibility,etc.are still required.Additionally,suprachoroidal transretinal stimulation(STS)and neurotransmitter-induced stimulation as some other alternatives in retinal prosthesis are also considered as promising research directions,although they are not mature enough to be applied commercially,either.

Optical coherence tomography in measuring retinal nerve fiber layer thickness in normal subjects and patients with open-angle glaucoma

Objectives To investigate image characteristics and thickness of the retinal nerve fiber layer (RNFL) in normal and glaucomatous eyes using optical coherence tomography (OCT), and analyze the relationship between RNFL thickness and visual field index.Methods Eighty-three normal persons (150 eyes) and 83 patients with primary open angle glaucoma (POAG, 149 eyes) underwent OCT examinations with 3.4 mm diameter circle scan to calculate the RNFL thickness. Statistical analysis was used to compare differences in RNFL thickness in quadrants and means between the normal and glaucomatous groups and the different stages of POAG. Linear correlation and regression analysis were used to show the correlation between RNFL thickness and visual field index of 115 eyes in glaucomatous patients. Reproducibility, sensitivity and specificity of RNFL measurements using OCT were evaluated.Results RNFL thickness measured by OCT in normal subjects was thicker in superior and inferior, less in temporal, and thinnest in nasal quadrants. The curve showed double peaks. RNFL of glaucomatous patients showed local thinning or defect, diffuse thinning, or both. The mean RNFL thicknesses of the normal group in the temporal, superior, nasal and infeior quadrants were 90.1 ± 10.8 lμm, 140.4 ± 10.5μm, 85.2 ± 14.0 μm, and 140.4 + 9.7 μm, respectively with a mean of 114.2 ± 6.0 μm. The numbers for the glaucomatous group were respectively 56.0 ± 31.0 μm, 81.0 ± 36.3 μm, 47.1 ± 27.5 μm, and 73.4 ±38.4 μm for the four quadrants, with a mean of 64.6 ± 28.8 μm. There was a significant difference in RNFL thickness between the normal and glaucomatous groups (P ＜ 0.000), and the three stages (early,developing and late) of glaucornatous groups (P ＜ 0.000). There was a close negative relationship between RNFL thickness and visual field index ( r = - 0.796, P ＜ 0.0001 ). The sensitivity and specificity of RNFL thickness in POAG measured using OCT were 93.3％ and 92.0％, respectively.Conclusions OCT can quantitatively measure RNFL thickness differences between normal persons and glaucomatous patients. RNFL thickness gradually decreases while visual field defect increases with the development of POAG.

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.

Diabetic retinopathy: recent advances towards understanding neurodegeneration and vision loss

Diabetic retinopathy （DR） is one of the most common retinal diseases world-wide. It has a complex pathology that involves the vasculature of the inner retina and breakdown of the blood-retinal barrier. Extensive research has determined that DR is not only a vascular disease but also has a neurodegenerative component and that essentially all types of ceils in the retina are affected, leading to chronic loss of visual function. A great deal of work using animal models of DR has established the loss of neurons and pathology of other cell types, including supporting glial cells. There has also been an increased emphasis on measuring retinal function in the models, as well as further validation and extension of the animal studies by clinical and translational research. This article will attempt to summarize the more recent developments in research towards understanding the complexities of retinal neurodegeneration and functional vision loss in DR.

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.

Diagnostic value of optic disc retinal nerve fiber layer thickness for diabetic peripheral neuropathy

Objective:To investigate the value of optic disc retinal nerve fiber layer(RNFL)thickness in the diagnosis of diabetic peripheral neuropathy(DPN).Methods:Ninety patients with type 2 diabetes,including 60 patients without DPN(NDPN group)and 30 patients with DPN(DPN group),and 30 healthy participants(normal group)were enrolled.Optical coherence tomography(OCT)was used to measure the four quadrants and the overall average RNFL thickness of the optic disc.The receiver operator characteristic curve was drawn and the area under the curve(AUC)was calculated to evaluate the diagnostic value of RNFL thickness in the optic disc area for DPN.Results:The RNFL thickness of the DPN group was thinner than those of the normal and NDPN groups in the overall average((101.07±12.40)μm vs.(111.07±6.99)μm and(109.25±6.90)μm),superior quadrant((123.00±19.04)μm vs.(138.93±14.16)μm and(134.47±14.34)μm),and inferior quadrant((129.37±17.50)μm vs.(143.60±12.22)μm and(144.48±14.10)μm),and the differences were statistically significant.The diagnostic efficiencies of the overall average,superior quadrant,and inferior quadrant RNFL thicknesses,and a combined index of superior and inferior quadrant RNFL thicknesses were similar,and the AUCs were 0.739(95%confidence interval(CI)0.635–0.826),0.683(95%CI 0.576–0.778),0.755(95%CI 0.652–0.840),and 0.773(95%CI 0.672–0.854),respectively.The diagnostic sensitivity of RNFL thickness in the superior quadrant reached 93.33%.Conclusions:The thickness of the RNFL in the optic disc can be used as a diagnostic method for DPN.

Association between retinal neuronal degeneration and visual function impairment in type 2 diabetic patients without diabetic retinopathy

The changes in retinal thickness and visual function in type 2 diabetic patients without clinical evidence of diabetic retinopathy were evaluated. A total of 141 diabetic subjects without retinopathy and 158 healthy subjects were enrolled in this study. Superior macular ganglion cell complex thicknesses were significantly decreased in diabetic cases, and no significant peripapillary retinal nerve fiber layer thickness changes were observed. The contrast sensitivities at all space frequencies were significantly different between diabetic patients and controls. The mean P50 amplitude from pattern electroretinogram results was reduced significantly in the diabetic group. In the diabetic group, average superior ganglion cell complex thicknesses positively correlated with both contrast sensitivities at high spatial frequencies and P50 amplitudes. The results indicated that ganglion cell complex thickness and visual function changes could be observed in diabetic subjects before the onset of any significant diabetic retinopathy. Macular ganglion cell complex reduction occurred much earlier than peripapillary retinal nerve fiber layer thinning in diabetic patients without retinopathy.