组织病理学切片与频域OCT对大鼠视网膜组织形态的对比研究

目的 研究组织病理学切片（石蜡切片和冰冻切片）及频域OCT在评价大鼠视网膜各亚层结构中的特点及其异同,并分析其原因。设计 实验研究。研究对象 健康Brown Norway （BN）大鼠12只。方法 选择正常BN大鼠12只（24眼）,频域OCT（Cirrus HD-OCT）对大鼠视网膜距视盘2 PD处进行扫描并测量各亚层厚度;处死大鼠后左眼行视网膜石蜡切片,右眼行冰冻切片,并分别测量距视盘2 PD处视网膜各亚层厚度,分析其形态学特点,并对所得实验数据进行统计学分析。主要指标 大鼠视网膜各个亚层的厚度及形态学特点。结果 石蜡切片所测大鼠距视盘2PD处视网膜厚度（166.98±9.07）μm,冰冻切片所测大鼠距视盘2 PD处视网膜厚度（209.06±10.41）μm,频域OCT测量值为（208.75±11.19）μm。频域OCT图像与冰冻切片测量结果接近,两者之间差异无统计学意义（P=0.425）;而石蜡切片与其他两者之间差异均有统计学意义（P均<0.001）。除内核层外,频域OCT、石蜡切片以及冰冻切片测量的大鼠视网膜各亚层厚度间差异均有统计学意义（P均<0.05）,石蜡切片较冰冻切片和频域OCT所测量的视网膜各亚层均有不同程度的收缩,但组织层次更加完整清晰,可以更清楚地显示至细胞水平。通过频域OCT可以得到大鼠视网膜图像,但内界膜、神经纤维层和神经节细胞层之间分界不明显;冰冻切片方便快捷,相对石蜡切片更接近OCT活体测量的结果,但容易造成玻璃体后脱离致视网膜神经纤维层不完整。结论 组织病理学切片（石蜡切片和冰冻切片）及频域OCT在评价大鼠视网膜各亚层结构中具有各自独特的特点,又具有一定的一致性。在进行视网膜的定量研究时,冰冻切片和频域OCT更加准确,而在定性研究特别是细胞水平的研究中,石蜡切片的组织形态更加完整清晰。（眼科, 2013, 22：344-348）     

不同分期糖尿病视网膜病变患者黄斑区结构和微血管改变定量分析

目的：通过光相干断层扫描(OCT)和光相干断层扫描血管造影(OCTA)检查,观察不同分期糖尿病视网膜病变(DR)患者黄斑区神经节细胞-内丛状层(GCIPL)厚度及各象限视网膜浅层毛细血管密度特征并作定量分析。

方法：回顾性病例对照研究。选取2019-12/2020-05我院确诊DR患者33例54眼,并根据眼底情况分为无糖尿病视网膜病变(NDR)组6例8眼、非增殖型糖尿病视网膜病变(NPDR)组18例28眼和增殖型糖尿病视网膜病变(PDR)组9例18眼。选取同期与患者年龄相匹配的健康志愿者18例26眼作为对照组。观察并定量分析不同分期DR患者黄斑区GCIPL厚度及各象限视网膜浅层毛细血管线性密度(VD)和血管灌注密度(PD)。

结果：DR组患者黄斑区各象限视网膜浅层毛细血管VD、PD及GCIPL厚度最小值均小于对照组(P<0.05)。不同分期DR患者黄斑区GCIPL厚度最小值及各象限视网膜浅层毛细血管VD组间比较均有差异(P<0.05),下方视网膜浅层毛细血管VD对DR的诊断价值最高(AUC=0.807、最佳诊断界限值为18.60mm^-1、灵敏度为0.923、特异度为0.648)。DR患者黄斑区GCIPL厚度最小值与各象限视网膜浅层毛细血管VD均呈正相关(r=0.342、0.480、0.384、0.342,均P<0.05)。

结论：OCT结合OCTA检查为早期评估及定期随访DR的进展提供了可重复、可定量的检测方法和监测指标。     

急性心肌缺血对视网膜的影响及其机制

目的 探讨SD大鼠心肌缺血急性期视网膜组织形态学改变及发生机制。方法 将34只大鼠68眼分2组：(1)对照组：冠状动脉左对角支下穿线，不结扎；(2)结扎组：结扎冠状动脉左对角支。2组大鼠于结扎冠脉或冠脉下穿线后30min和3h取视网膜，进行组织病理学检查、5-羟色胺和去甲肾上素免疫组织化学实验。结果与对照组比较，光镜及电镜下可见结扎组大鼠视网膜组织损伤明显。结扎组结扎冠状动脉30min时，光镜下见视网膜组织水肿增厚，细胞空泡变性。结扎冠状动脉3h，视网膜组织萎缩、变薄。电镜下见视网膜组织细胞线粒体嵴断裂明显，呈空泡变性；视细胞膜盘排列紊乱；外核层细胞变小、变形．细胞核固缩。免疫组织化学实验结果显示，结扎冠状动脉30min，视网膜5-羟色胺和去甲肾上腺素阳性染色显著增多。结论 心肌缺血急性期可引起视网膜组织病理学改变。视网膜组织内5-羟色胺、去甲肾上腺素于缺血后增多，可能与心肌缺血时机体内伤害性刺激的作用以及机体的反应有关。     

快速减压后大鼠视网膜形态和胶质反应变化

目的观察快速减压对大鼠视网膜形态学结构、胶质反应和细胞凋亡的影响。方法24只大鼠随机分为6组,即正常对照组、安全减压组、快速减压处理后0h、6h、24h、48h组。安全减压组、快速减压处理各组实验动物暴露于加压舱内,舱内气压在30s内升至1．0MPa,维持5．5min,快速减压组打开放气阀55s减至常压,安全减压组采用动物安全减压方案减到常压。采用HE染色观察视网膜组织结构和神经节细胞密度变化情况,采用TUNEL染色观察视网膜内细胞凋亡的分布和比例,采用免疫组化方法观察视网膜胶质细胞胶质纤维酸性蛋白（GFAP）和波形蛋白（vimentin）表达变化。结果大体观察发现快速减压后视网膜水肿明显,其中部分血管闭塞,视网膜内点片状出血。HE染色发现快速减压后24h内随时间延长,视网膜水肿逐渐加重,其中神经节细胞层、神经纤维层、内丛状层和外丛状层水肿明显。快速减压后48h视网膜水肿较24h组减轻,神经节细胞数量减少,视锥视杆细胞减少,内核层、外核层变薄、核溶解、坏死,结构较紊乱。减压后24h神经节细胞密度为（6．41±1．39）个／100um,开始明显降低,减压后48h细胞密度为（5．31±1．94）个／100um,与正常对照组（8．62±1．75）个／100um和安全减压组（9．03±2．66）个／100um相比显著降低（P〈0．05）。GFAP和vimentin在快速减压后24h和48h表达最强,正常对照组表达最弱,安全减压组介于两者之间。TUNEL染色显示快速减压后24h和48h时视网膜节细胞层及内、外核层均有大量浓染的阳性细胞。结论快速减压可造成视网膜缺氧,导致多种病理改变,其中主要病变特点是神经元细胞凋亡和胶质细胞反应增强。     

缺氧缺血对新生大鼠视网膜线粒体Ca2+含量的影响

目的 :观察缺氧缺血不同时期新生大鼠视网膜线粒体Ca2 含量的变化规律 ,探讨缺氧缺血对视网膜的损伤机制。方法 :将新生SD(sprague dawley)大鼠结扎右颈总动脉并吸入 96%N2 4%O2 制成缺氧缺血模型 ,以原子吸收火焰法测定线粒体Ca2 含量。结果 :假手术对照组 ,缺氧缺血组 (缺氧 1 5,30 ,60和 1 2 0min)和缺氧后供氧组Ca2 含量 ( μg/gpro)分别为 32± 0 0 3,4 7 5± 5 98,97 5± 3 54,54±6 0 2 ,4 5 3± 3 66和 1 51 5± 9 2 0。结论 :缺氧缺血使视网膜线粒体Ca2 含量增高     

改良法制作的视网膜切片内核层神经元的形态和电学特性

目的:探讨低温琼脂包埋振动切片机切片法制作的大鼠视网膜切片内核层神经元的形态和基本电生理学特性。方法:采用低温琼脂包埋振动切片机切片的方法制作大鼠视网膜切片,对内核层的神经元进行膜片钳全细胞记录,同时在胞内液中加入荧光黄观察记录细胞的形态。结果:该方法制作的视网膜切片切面平整、细胞活性好、保留了细胞之间的突起联系,能够根据细胞胞体的大小、位置初步辨别细胞的种类。在视网膜切片上荧光黄显示的细胞形态表明,双极细胞胞体呈梭形,突起主要沿纵向延伸;而水平细胞和无长突细胞胞体圆形或椭圆形、胞体较大,分别位于内核层的最外层和最内层。水平细胞和无长突细胞的静息膜电位(RMP)和膜电容(Cm)明显高于双极细胞。给予时程40ms,步阶10mV从-60mV至+40mV的电压刺激,41.7%的视锥双极细胞和64.7%的无长突细胞表现出内向钠电流和外向钾电流,其他细胞则只表现出外向钾电流。结论:采用低温琼脂包埋振动切片机切片的方法操作简单,制作的切片质量稳定可靠,使得在视网膜切片上对包括水平细胞在内的不同内核层神经元进行膜片钳记录成为可能。进一步研究视网膜内核层神经元的电生理学特性,有助于揭示视觉信号的发生、传导和调控机制。     

Proteomic analysis of rat retina in a steroid-induced ocular hypertension model: Potential vulnerability to oxidative stress

Purpose To investigate global protein expression profiles in the retinas of normal and glucocorticoid-induced ocular hypertensive rats by proteomic analysis. Methods Ocular hypertension was induced by topical application of dexamethasone (DEX) for 4 weeks. Age-matched untreated rats served as controls. Intraocular pressure (IOP) was monitored by an electronic tonometer. Retinal protein expression profiling was carried out by two-dimensional fluorescence difference gel electrophoresis (2-D DIGE). Proteins were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Results In DEX-treated rats, average IOP was elevated significantly compared with controls. With DEX treatment, levels of four proteins were altered, as revealed by 2-D DIGE and MALDI-TOF mass spectrometry: apolipoprotein A1 (apoA1), a lipid-binding protein, upregulated 1.9-fold, P < 0.05; alpha A crystallin (CRYAA), a molecular chaperone, downregulated 2.7-fold, P < 0.01; superoxide dismutase 1 (SOD1), an antioxidant enzyme, downregulated 2.3-fold, P < 0.05; and triosephosphate isomerase 1 (TPI1), a glycolytic enzyme, downregulated 2.3-fold, P < 0.01. Conclusions Downregulation of CRYAA, SOD1, and TPI1, observed here after a short period of DEX-induced ocular hypertension, may be involved in the onset of neural damage in steroid-induced glaucoma.     

黄芪对大鼠视网膜缺血再灌注损伤中Bcl-2和Bax表达的影响

目的：探讨大鼠视网膜缺血再灌注损伤(retinal ischemia-reperfusion injury,RIRI)过程中黄芪对Bcl-2和Bax蛋白表达的影响及作用机制。 方法：前房加压法制作实验性RIRI的大鼠模型。将66只SD大鼠随机分为正常组、缺血再灌注模型组、黄芪注射液治疗组。后两组各分为 6,12,24,48h和3d五个时间段,HE染色后光镜下观察视网膜组织变化, 采用免疫组织化学法与Western-blot法测定大鼠RIRI后视网膜中Bcl-2和Bax蛋白表达的变化。 结果：Bcl-2和Bax在正常视网膜组织中几乎不表达,在缺血再灌注6h开始表达,24h表达显著,48h开始下降,3d后表达已经明显减弱。黄芪注射液治疗组各观察指标变化趋势基本与单纯缺血再灌注组相似。黄芪注射液治疗组与模型组比较,Bcl-2表达均明显增强,Bax表达均明显减弱,两组间比较差异均有显著统计学意义(P<0.01)。 结论：黄芪注射液预处理可使神经节细胞Bcl-2表达增强,Bax表达减弱,减少神经节细胞凋亡,对RIRI神经节细胞有明显的保护作用。     

Effects of intraocular rifampicin on retinal ganglion cell structure:a stereological and histopathological study

AIM: To determine the histopathological changes of rifampicin applied intravitreally on retinal ganglion cells by means of stereological and histopathological methods.METHODS: For this study twenty-four New Zealand adult rabbits were divided into four groups (n=6 for each group). 50μg/0.1mL (group 1), 100μg/0.1mL (group 2), 150μg/0.1mL (group 3) and 200μg/0.1mL (group 4), rifampicin were injected into the vitreous of the right eyes of animals, their left eyes were used as control (group 5). After the 28^th day of application, animals were anesthetised with xylazine (8mg/kg, IM) and then their eyes were enucleated immediately. Patterns were taken away and eyes were prepared for both stereological and electromicroscopic observation.RESULTS: Depending on the high dose of rifampicin, some histopathological changes such as cytoplasmic dilatation and damaged membrane were observed on the electromicroscopic level. Using quantitative examination, which was done at the light microscopic level, it was shown that the number of neurons decreased linearly as rifampicin dose increased when compared with the control group.CONCLUSION: Based on these findings, low-dose rifampicin (50μg/0.1mL) may be useful for treatment of the ocular diseases.     

Lipid hydroperoxide formation in the retina: correlation with retinal degeneration and light damage in a rat model of Smith-Lemli-Opitz syndrome

The Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disease presenting with multiple congenital anomalies, caused by a defect in cholesterol biosynthesis that results in abnormally elevated levels of 7-dehydrocholesterol (7DHC). Progressive retinal degeneration has been demonstrated in a rat model of SLOS, which is markedly exacerbated by intense light, far more so than occurs in normal albino rats under the same conditions. Herein, we demonstrate that, by six postnatal weeks, retinas in the SLOS rat model contain levels of lipid hydroperoxides (LPOs) comparable to those found in light-damaged albino rats (twice the normal steady-state levels), and that intense light exposure results in a three-fold elevation of LPOs with concomitant severe retinal degeneration. These results suggest a correlation between retinal degeneration and LPO levels. We propose that the presence of 7DHC in the SLOS rat retina potentiates LPO formation, and promotes the observed hypersensitivity to light-induced retinal degeneration.     

Effects of antioxidant nutrient deficiency on the retina and retinal pigment epithelium of albino rats: a light and electron microscopic study

Male Sprague-Dawley rats were maintained, from weaning, on diets deficient either in vitamin E, selenium, chromium and sulfur amino acids (?E?Se?S?Cr), or only in vitamin E and selenium (?E?Se+S+Cr). Control animals (+E+Se+S+Cr) received all four nutrients. After 24–26 weeks on their respective diets, both deficient groups showed a dramatic accumulation of an autofluorescent pigment, similar to lipofuscin, in the retinal pigment epithelium. This increased autofluorescence was correlated with a large increase in the number of electron-dense inclusion bodies observed in the pigment epithelium by transmission electron microscopy. Accompanying the build-up of autofluorescent pigment was the development of an irregularity and an overall increase in retinal pigment epithelium cell height. There was also an increase in the number of lipid droplets in the retinal pigment epithelium, particularly in the periphery of the eye. In the eyes of deficient animals, cells were occasionally seen which appeared to have detached from Bruch's membrane and migrated into the region of the photoreceptor outer segments. Photoreceptor outer segment phagocytosis by the retinal pigment epithelium appeared to be decreased as a consequence of dietary antioxidant deficiency, since a reduction of over 75% in the number of phagosomes per unit retinal pigment epithelium cell length was seen in some areas of the eye in both deficient groups.These changes in the retinal pigment epithelium were accompanied by a pronounced loss of photoreceptor cells, particularly from the central retina (20–34% fewer cells than supplemented controls). The disk membranes of the photoreceptor outer segments of deficient animals were often swollen, disoriented and vesiculated, and areas were frequently seen where outer segment debris had accumulated at the interface between the photoreceptors and retinal pigment epithelium.Many of the changes in the retina and retinal pigment epithelium were more severe in the fully deficient (?E?Se?S?Cr) group, than in the group deficient only in vitamin E and selenium.     

The cholinergic amacrine cells of rabbit retina receive on and off input: An analysis of [H]-ACh release using 2-amino-4-phosphonobutyric acid (APB) and chloride free medium

Using thein vitro rabbit eye-cup we have examined the light-evoked release of ACh from cholinergic amacrine cells under conditions known to eliminate On responses in the retina. APB (100 μM), which blocks the photoreceptor/depolarizing bipolar cell synapse, reduced the light-evoked release of ACh by 80% but a small light-evoked response remained, which was potentiated by bicuculline. Depolarizing bipolar cells are also Cl⁻ dependent. Cl⁻ free medium caused a tenfold Ca²⁺-dependent increase in the release of ACh but some small light evoked release remained. These results indicate that the cholinergic amacrine cells receive On and Off input. Our findings are consistent with anatomical and electrophysiological evidence which suggests that the displaced cholinergic amacrine cells are On cells and the conventionally placed cholinergic amacrine cells are Off cells.     

The cholinergic amacrine cells of rabbit retina receive on and off input: An analysis of [3H]-ACh release using 2-amino-4-phosphonobutyric acid (APB) and chloride free medium

Using thein vitro rabbit eye-cup we have examined the light-evoked release of ACh from cholinergic amacrine cells under conditions known to eliminate On responses in the retina. APB (100 μM), which blocks the photoreceptor/depolarizing bipolar cell synapse, reduced the light-evoked release of ACh by 80% but a small light-evoked response remained, which was potentiated by bicuculline. Depolarizing bipolar cells are also Cl^? dependent. Cl^? free medium caused a tenfold Ca²⁺-dependent increase in the release of ACh but some small light evoked release remained. These results indicate that the cholinergic amacrine cells receive On and Off input. Our findings are consistent with anatomical and electrophysiological evidence which suggests that the displaced cholinergic amacrine cells are On cells and the conventionally placed cholinergic amacrine cells are Off cells.     

The Precorneal Tear Film as a Fluid Shell: The Effect of Blinking and Saccades on Tear Film Distribution and Dynamics

We conducted a series of experiments to elucidate the behavior of the human precorneal tear film (PCTF) during blinking and horizontal and vertical saccades. Methodology included video-interferometry with subsequent image cross-correlation (tear film lipid layer [TFLL]) and video-microscopy (mucoaqueous subphase [MAS]). We observed that the TFLL interference pattern deteriorates rapidly with successive blinks and degrades slowly with repeated horizontal saccades during blink suppression when dark arcs of thinning appear in the fluorescein-stained PCTF. Furthermore, after full downgaze and a return to the primary position, a transient horizontal bright band appears, deep to the spreading TFLL. It may be followed by local disturbances in the interference pattern. Two horizontal dark bands form in the stained PCTF after the return saccade. PCTF disruption may occur below the lower band during blink suppression. We concluded that shearing during horizontal saccades is insufficient to disturb the tear film structure greatly. The MAS and TFLL move together as a fluid shell. The dark arcs/bands are caused by meniscus-induced thinning, imprinted onto the PCTF at the lid margin. Their stability during blink suppression suggests that the MAS has gel-like properties. The horizontal bright bands are probably due to transient corneal indentation in downgaze. In downgaze, the disturbance of the TFLL and MAS below the dark bands is possibly due to shearing across the MAS in the return phase. This could cause desiccating stress in everyday activities, such as working at a computer.     

Best vitelliform macular dystrophy in a Swedish family: genetic analysis and a seven‐year follow‐up of photodynamic treatment of a young boy with choroidal neovascularization

Purpose: To determine the mutation in a Swedish family with Best disease (vitelliform macular dystrophy; VMD) and to investigate the short‐ and long‐term effects of photodynamic treatment (PDT) on subretinal neovascularization in a young boy. Methods: The five members of three generations of a family with VMD underwent a thorough ophthalmological examination, including best‐corrected visual acuity (VA), visual field, colour vision, biomicroscopy of the posterior segment (dilated), fundus photography and electro‐oculography (EOG). For the proband, an eleven‐year‐old boy, his father and grandfather, dark adaptation test, angiography and electroretinography (ERG) were also performed. After PCR amplification, the genotype was determined by cleavage with restriction enzyme, specific for the W93C allele. Results: Four family members had an abnormal EOG response. All showed the W93C mutation in the VMD2 gene. Visual acuity ranged from 20/20 to 20/250. The fundus manifestations varied from minor pigmentary changes over egg yolk‐like lesions to chorioretinal atrophy, and fluorescein angiography showed corresponding pathology. In the proband, VA decreased during follow‐up from 0.5 (20/40) to 0.08 (20/250) due to a subfoveal neovascularization with haemorrhage, and PDT with visudyne was begun. The haemorrhage resolved within 2 months, and after three treatments, VA had increased to 0.25 (20/80). One year later, acuity had improved to 0.5 (20/40), and this result was stable throughout the 7 years of the follow‐up. Conclusion: The mutation was determined to be W93C, the most common mutation in VMD in Sweden. In an eleven‐year‐old boy with subretinal neovascularization, PDT seemed to be beneficial also in a long‐term follow‐up.     

A bibliometric analysis of academic publication on diabetic retinopathy disease trends during 1980-2014: a global and medical view

AIM: To investigate diabetic retinopathy (DR) literature using the Institute for Scientific Information (ISI) Web of Science (WoS) database and to analyse the correlation results between socio-economic development datas and number of DR publications. METHODS: The statistical analysis of the documents published during 1980-2014 was analysed. The data of this study were based on the database of WoS. “Diabetic retinopathy” was used as the keywords to search the WoS database. RESULTS: The United States ranked first in the DR research with 1840 publications and 24.38% of the world production followed by England and Japan. Besides, the most productive country was Iceland. A high correlation was found between number of publications and 2014 gross domestic product (GDP) values of 81 countries (r=0.800, P<0.001). We found a significant correlation between number of publications and Human Development Index (HDI) (r=0.645, P=0.001). There is a moderate correlation between people with diabetes and number of DR publications for 81 countries (r=0.514, P<0.01). It could be analysed that estimated publication number with DR title will be 445 according to the regression curve constituted with cubic model in 2015 (R2=1.000). CONCLUSION: More DR studies have been published in developed countries, DR and other complications of diabetes have gradually increased in developing countries over recent decades. It can be expected that the number of DR studies will gradually increase in developing countries.     

Effect of intravitreal bevacizumab (Avastin®) in neovascular age‐related macular degeneration using a treatment regimen based on optical coherence tomography: 6‐ and 12‐month results

Purpose: To study the effect of intravitreal bevacizumab therapy on visual and anatomical outcomes in patients with neovascular age‐related macular degeneration (AMD) within a follow‐up period of 6 and 12 months. Methods: A retrospective analysis of 102 eyes of 102 consecutive patients with neovascular AMD evaluated repeated intravitreal bevacizumab (1 or 2.5 mg) injections. Retreatment was performed following an optical coherence tomography (OCT)‐based regimen. Ophthalmic examination included best‐corrected visual acuity (BCVA), dilated fundus examination and OCT imaging. Data were analysed at baseline, 6 months (24 weeks) and 12 months (48 weeks) after treatment initiation. Results: BCVA remained stable at 6 months (mean: 0.00 ± 0.41 logMAR; p = 0.95) and 12 months (mean: +0.02 ± 0.43 logMAR; loss of ～ 1 letter; p = 0.70) after the first treatment. OCT retinal thickness decreased by a mean of ?37.8 ± 101.6 μm (p < 0.05) compared to baseline at month 6 and ?38.6 ± 93.3 μm (p < 0.05) at month 12. A mean of 2.6 ± 1.2 injections were needed to obtain absence of fluid by OCT, and the time to recurrence was 23 ± 11 weeks thereafter. There was no difference in BCVA and OCT outcomes between treatment‐naive eyes and eyes that had undergone prior treatment. Conclusion: The 6‐ and 12‐month follow‐up of repeated intravitreal bevacizumab therapy in eyes with neovascular AMD demonstrated stabilization of vision and no safety concerns. An OCT‐based retreatment strategy appears appropriate in the management of patients treated with intravitreal bevacizumab.