糖尿病大鼠视网膜和肾小球微血管病变的对比研究

寻找糖尿病性视网膜病变和糖尿病性肾病两者间组织学改变的关系。方法采用普通电镜和免疫组化电镜方法,并经电镜下定量分析视网膜和肾小球毛细血管基底膜的厚度及基底膜上负电荷位点的计数。     

高血压大鼠视网膜微血管病变的病理基础

目的 探讨实验性高血压性视网膜病变的病理基础。 方法 16只大鼠手术切除其一侧肾脏,部分结扎另一侧肾动脉建立高血压动物模型,对照组16只大鼠模拟手术。采用常规光镜组织化学染色、免疫组织化学染色、电镜和组织化学电镜等技术,观察两组大鼠术后2周,1,2,4个月4个不同时间点视网膜微血管的改变。 结果 ①高血压大鼠视网膜毛细血管基底膜(retinal capillary basement membrane,RBM)从术后2个月起即普遍增厚,至病程4个月时,其厚度约为对照组的2～2.5倍;②高血压大鼠RBM的增厚伴有其构成成分Ⅳ型胶原和层粘连蛋白的增多,并伴有RBM上负电荷位点数目的减少;③高血压大鼠术后2周出现视网膜毛细血管内皮细胞下水肿,与基底膜剥离的现象,病程4个月时周细胞可有水肿变性。 结论 高血压大鼠视网膜毛细血管壁的三层结构均有改变,这些改变是高血压性视网膜病变的病理基础。〖ＨＴＨ〗 (中华眼底病杂志, 1999, 15: 163-166)     

糖尿病视网膜病变的药物治疗及思路

糖尿病视网膜病变(diabetic retinopathy，DR)是糖尿病最常见的和最严重的并发症之一，并已成为四大主要致盲疾病之一。DR发生的关键在于视网膜组织缺氧，早期病理改变为毛细血管周细胞丧失、微血管瘤形成、毛细血管基底膜增厚、血-视网膜屏障破坏、出血、渗血和视网膜水肿，晚期可见新生血管、     

链脲佐菌素-糖尿病大鼠视网膜微血管病理改变

目的：观察SIZ-糖尿病大鼠视网膜微血管的早期病理变化。方法：45只SD大鼠被随机分为糖尿病组(DM)和对照组(CON)，用链脲佐菌素(streptozotocin，STZ)诱导SD大鼠建立糖尿病动物模型，分别饲养3个月和6个月。各组大鼠到期后，取眼球，采用视网膜消化铺片PAS染色、细胞图像分析及透射电镜技术进行早期糖尿病视网膜微血管形态学检查，记数微血管细胞数和无细胞血管数目，观察微血管及细胞超微结构。结果：糖尿病视网膜早期微血管周细胞数目逐渐减少，无细胞血管增多。超微结构显示微血管细胞结构异常，基底膜变性、增厚。结论：糖尿病视网膜病变早期存在微血管细胞病变、基底膜变性增厚及无细胞血管形成的改变。临床应重视糖尿病视网膜病变(DR)的早期防治，改善微血管循环。     

非肥胖性糖尿病小鼠的早期视网膜病变

目的:研究早期糖尿病视网膜病变的小鼠模型。方法:NOD小鼠随机分为正常对照组(4,12wk组,n=8)和糖尿病组(4,12wk组,n=12),每组取4wk和12wk处死小鼠,光镜和电镜观察视网膜结构的改变。结果:糖尿病组4wk时毛细血管基底膜增厚,可见内皮细胞、周细胞和神经节细胞超微结构改变。12wk时基底膜明显增厚,内皮细胞、周细胞和神经节细胞凋亡增加。结论:在糖尿病早期发生了视网膜神经元细胞超微结构病变和微血管病变。     

实验性糖尿病大鼠视网膜病理学研究（电镜观察）

报告了对四氧嘧啶(50mg/kg体重)诱导的糖尿病大鼠视网膜组织电镜观察结果,高血糖(400～600mg/dl)持续6个月,结果显示毛细血管周细胞退行性改变、基底膜电子密度不均和结节增厚、感光细胞外节以及色素上皮细胞微绒毛结构紊乱。认为毛细血管周细胞和基底膜病变是糖尿病视网膜病变的最初改变,色素上皮功能减退可能是感光细胞外节结构紊乱的原因。     

诺帝抑制糖尿病大鼠视网膜毛细血管基底膜增厚的体视学观测

目的检测诺帝对糖尿病大鼠视网膜毛细血管基底膜增厚的影响及意义。方法采用链脲佐菌素诱导大鼠糖尿病模型,将糖尿病大鼠随机分为糖尿病组(10只)、生理盐水注射组(10只)和诺帝注射组(10只),另取10只正常大鼠作为正常对照组。其中,诺帝注射组在出现糖尿病表现后按27mg·kg-1腹腔注射诺帝溶液(隔日1次),生理盐水注射组只在同期注射等量生理盐水,糖尿病组和正常对照组不予任何处理。注射后1个月、3个月分别取视网膜制片,通过PAS染色、透射电镜观察视网膜毛细血管形态的改变,对视网膜毛细血管基底膜行超微结构体视学定量。结果糖尿病组和生理盐水注射组大鼠3个月视网膜制片PAS染色可见毛细血管壁着色区增厚,其余各组大鼠PAS染色均未发现明显异常改变。糖尿病组、生理盐水注射组、诺帝注射组和正常对照组视网膜毛细血管基底膜平均体积(×106nm3)在1个月时分别为:1.22±0.11、1.20±0.09、0.92±0.14、0.62±0.08;在3个月时分别为:2·15±0.48、2.14±0.44、1.33±0.34、0.88±0.17。糖尿病组和生理盐水注射组大鼠在1个月和3个月时,毛细血管基底膜平均体积均较同期正常对照组增加(P<0.01)。诺帝腹腔注射明显减轻糖尿病大鼠视网膜毛细血管基底膜增厚(与无诺帝处理的糖尿病组大鼠和生理盐水注射组大鼠比较差异有显著性,P<0.01)。结论诺帝能抑制糖尿病大鼠视网膜毛细血管基底膜的增厚,提示诺帝可能对糖尿病视网膜病变具有一定的抑制作用。     

基于OCTA观察眼底不同分期的2型糖尿病患者黄斑区脉络膜及视网膜血流密度变化

目的 应用光学相干断层扫描血管成像(OCTA)观察眼底不同分期的2型糖尿病患者黄斑区脉络膜和视网膜血流密度变化。方法 收集蚌埠医学院第一附属医院2型糖尿病患者150例150眼纳入研究,依据国际临床糖尿病视网膜病变(DR)严重程度分级标准将患眼分为5组,无DR组和轻、中、重度非增生型DR(NPDR)组及增生型DR(PDR)组,每组各30例30眼。OCTA对所有受试者行黄斑部6 mm×6 mm区域的扫描,测量视网膜浅层、深层和脉络膜毛细血管层的血流密度,观察不同分期DR患者脉络膜及视网膜血流密度的变化,分析血流密度与病变程度的相关性。结果 随着DR患者的严重程度加深,视网膜浅层、深层和脉络膜毛细血管层血流密度均呈显著减少趋势(均为P<0.001);但不同分层血流密度中,中度NPDR组与重度NPDR组比较差异均无统计学意义(P=0.216、0.896、0.350)。视网膜浅层、深层和脉络膜毛细血管层血流密度与病变程度均呈正相关(均为P<0.001)。视网膜深层血流密度识别DR严重程度的能力(敏感性和特异性分别为92.5%和93.1%)优于视网膜浅层和脉络膜毛细血管层。结论 OCT...     

黏附分子E选择素在大鼠糖尿病视网膜 病变中的表达

目的研究黏附分子E选择素在早、中、晚期糖尿病视网膜病变(DR) 大鼠微血管中的表达。方法雄性SD大鼠90只，随机分为6、9、12个月正常对照组，每组10只大鼠；及链脲佐菌素（STZ）DR大鼠6、9、12个月模型组，每组20只大鼠。STZ DR组用STZ按60 mg/kg的剂量腹腔一次性注射，制备DR模型。分别按期处死大鼠，取眼球进行视网膜微血管消化铺片，免疫组织化学EnVision方法显示微血管黏附分子E选择素，采用Leica Q550W图像分析仪定量分析E选择素在大鼠视网膜微血管中含量表达，并同时观察其形态学改变，了解DR病损程度与E选择素表达有无相关性。结果形态改变： DR 6个月组内皮细胞增生，周细胞减少。DR 9、12个月组毛细血管网排列紊乱，基底膜明显增厚，毛细血管腔内有较多白细胞黏附、聚集，大面积无细胞毛细血管形成和周细胞内皮细胞凋亡。定量分析结果：正常组无E选择素表达， DR各组均有不同程度E选择素表达，DR各组之间差异有统计学意义（P<0.01），其中以DR 9、12个月组表达量最高。结论随DR病程延长，视网膜毛细血管E 选择素表达逐渐增高，并与DR微血管的病损程度呈正相关。(中华眼底病杂志,2005,21:318-321)     

白细胞淤滞对糖尿病视网膜病变作用的研究

目的:观察糖尿病小鼠视网膜血管内白细胞淤滞和细胞间黏附因子-1(ICAM-1)在视网膜的表达.方法:选取C57型小鼠45只,随机分为2组:糖尿病组22只、正常组23只.10wk后取视网膜,荧光显微镜计数小鼠全视网膜微血管内淤滞的白细胞数目及其含有ICAM-1表达的荧光小球数目,免疫蛋白印迹法检测视网膜血管内皮生长因子(VEGF)和ICAM-1的表达.结果:10wk后,糖尿病小鼠视网膜血管内白细胞数目及含有ICAM-1表达的荧光小球数目明显高于正常对照组(P<0.01),其视网膜内VEGF和ICAM-1明显增加,有统计学意义(P<0.05).结论:视网膜血管内白细胞淤滞与糖尿病视网膜病变(DR)早期的视网膜毛细血管无灌注有关.     

Changes in extracellular matrix components after excimer laser photoablation in rat cornea

PURPOSE: To learn more about corneal wound healing after excimer laser photoablation. METHODS: Observations were made on the chronological changes in type I collagen, fibronectin, laminin, and type IV collagen after excimer ablation of rat cornea. Immunohistochemical techniques were used. RESULTS: There was no obvious change in the localization of type I collagen in the ablated area, but the localization of fibronectin, laminin, and type IV collagen changed remarkably. One day after ablation, immunofluorescent staining for fibronectin increased on the ablated surface. Subsequently, the fluorescence of fibronectin, laminin, and type IV collagen increased remarkably; in particular, the localization of these extracellular matrix proteins was sustained in the shallow layer of the stroma until about 24 weeks after ablation. Hematoxylin-eosin staining indicated that keratocytes temporarily disappeared 1 day after ablation, and activated keratocytes then migrated to the ablated areas. CONCLUSIONS: These results suggest that activated keratocytes might be synthesizing the extracellular matrix components. Therefore sustained responses of keratocytes may be induced by excimer laser photoablation.     

Immuno-electron labelling of matrix components in congenital hereditary endothelial dystrophy

Two corneal buttons were obtained from a patient with congenital hereditary endothelial dystrophy (CHED) at the ages of 2.5 years (right eye) and 14 years (left eye) and were studied by light and electron microscopy including immunogold labelling for collagen types I -V and laminin. The posterior collagenous layer (PCL) of Descemet's membrane contained collagen types I, III-V, and laminin: the latter was also localised to finebanded and granular material in the posterior non-banded zone (PNBZ). Comparison of the endothelium at 2.5 years and 14 years revealed occasional dystrophic changes in the former and extensive dystrophic changes in the latter. The distribution of collagen types I, III and V within the PCL supports previous morphological observations of fibroblast-like change of the endothelium in CHED. Persisting endothelial properties were manifest as positive labelling of type IV collagen and laminin. An excessive amount of laminin found in PNBZ and PCL is another stress-related endothelial reaction.Parts of this publication were presented at the First Annual Meeting of the European Community Ophthalmic Research Association (ECORA), 1993     

Histogenesis of the intravitreal membrane and secondary vitreous in the mouse

PURPOSE: The intravitreal membrane (IVM) is a membranous structure between the primary and secondary vitreous bodies in developing mammalian eyes. In this study, for the first time the histogenesis of the IVM and the relationship between the hyaloid vasculature and the IVM was characterized in newborn mice. METHODS: Eyes of mice less than 12 days old were fixed and embedded. From these, serial paraffin-embedded sections were made for lectin histochemistry, immunohistochemistry, and picrosirius red (PSR) staining, and ultrathin sections were made for transmission electron microscopy (TEM). Eight biotinylated lectins and antibodies for laminin and type IV collagen were used. RESULTS: Among the eight lectins tested, concanavalin A (Con A) agglutinin, Ricinus communis agglutinin I, and wheat germ agglutinin demonstrated strong positive staining in the IVM and vitreous fibrils of the primary and secondary vitreous bodies. They also bound to the internal limiting membrane (ILM) of the retina. At postgestational day 4, the secondary vitreous first appeared between the ILM and the vasa hyaloidea propria (VHP). Immunohistochemical staining revealed that the IVM consists of extracellular matrix components including laminin and type IV collagen, whereas PSR staining and TEM showed that collagen fibrils in the IVM are bundled and continuous with the basement membrane of hyaloid capillaries or the VHP. CONCLUSIONS: Lectin histochemistry and immunohistochemistry provided good methods for visualizing the structures of the IVM and vitreous fibrils. These results suggest that the IVM is separated from the basement membrane of the retinal ILM along with the vascular network of the VHP when the secondary vitreous begins to form.     

Extracellular matrix changes of the optic nerve lamina cribrosa in monkey eyes with experimentally chronic glaucoma

Using light microscopic immunohistochemistry, we studied the immunolocalization and immunoreactivity of the extracellular matrix, including collagen types III, IV, VI, laminin, and alpha elastin in the lamina cribrosa of monkey eyes with normal and experimentally chronic glaucoma. Our results showed: (1) abnormal linearlike immunodeposits of both collagen type IV and laminin in the margin of the lamina cribrosa with significant density in the glaucomatous eyes; (2) the immunoreactivity of collagen type III resembled that of the normal eye, but was slightly stronger at the laminar surface; (3) findings with collagen type VI resembled those of type III with an enhanced linearlike staining surrounding the nerve-fiber bundles. Furthermore, staining of alpha elastin demonstrated dramatic changes in both reactivity and localization.The lamina cribrosa of glaucomatous eyes showed a markedly reduced immunoreactivity as well as an irregular, interrupted pattern. These observations suggest that the changes might be a secondary to the long-standing elevation of intraocular pressure. The alteration of these macromolecules may modify the course of glaucomatous optic damage.Correspondence to: T. Fukuchi     

Laser in situ keratomileusis in human corneas: new organ culture model

PURPOSE: To establish an in vitro model of laser in situ keratomileusis (LASIK) in human donor eyes and to test its validity in comparison with animal models. SETTING: Department of Anatomy, Friedrich-Alexander Unviersity, Erlangen, Germany. METHODS: Laser in situ keratomileusis was performed on 20 organ-cultured human corneal buttons. The excimer laser ablations ranged from 0 to 12.0 diopters. The corneas were maintained in culture for up to 6 months and then evaluated with light microscopy and transmission electron microscopy. In addition, corneal sections were immunohistochemically stained for collagen type III, laminin, and fibronectin. The main outcome measures were the ultrastructural and immunohistochemical features of the stromal incision interface. RESULTS: Ultrastructural investigations in the peripheral cornea revealed a disarrangement of collagen fibers, indicating scar formation. These findings were not observed in the central area. Immunohistochemical staining for fibronectin and collagen type III was detected over the entire stromal incision interface, whereas laminin staining was related to the ingrowth of epithelial cells. CONCLUSIONS: The morphological changes after LASIK in an organ culture model can simulate the in vivo situation. Therefore, this model appears appropriate to use in further study of corneal wound-healing changes after LASIK.     

Immunohistochemistry and electron microscopy of retrocorneal scrolls in syphilitic interstitial keratitis

PURPOSE: To describe the immunohistochemical and electron microscopic characteristics of retrocorneal scrolls in syphilitic interstitial keratitis. METHODS: Five eyes of five patients with congenital syphilitic interstitial keratitis who underwent keratoplasty for corneal opacities and corneal edema were studied. The corneal buttons were processed for histologic examination with hematoxylin and eosin staining and underwent immunohistochemistry stainings for collagen types I, III, IV, V, VI, VIII, fibronectin, laminin, and decorin. The corneal buttons were also processed for transmission electron microscopy and immunoelectron microscopy. RESULTS: Light microscopy revealed that the retrocorneal scrolls had a multilayered, amorphous, acellular matrix. All scrolls were lined with attenuated corneal endothelial cells. The Descemet membranes in all specimens had areas of irregular thickening with attenuated endothelium. Immunohistochemical assessment of the scrolls showed positive staining for collagens I, III, IV, VI, VIII, fibronectin, laminin, and decorin but not for alpha -SMA. Immunoelectron microscopy confirmed these findings. Transmission electron microscopy showed multilaminar disorganized structures in scrolls composed of long- and short-fiber collagens. CONCLUSIONS: We confirmed the presence of collagens I, III, IV, VI, VIII and proteoglycans in the retrocorneal scrolls lined with attenuated endothelium. Our findings may provide further insight into the pathogenesis of keratopathy in syphilitic interstitial keratitis.     

Abnormal deposition of laminin and type IV collagen at corneal epithelial basement membrane during wound healing in diabetic rats

PURPOSE: To understand the pathophysiology of the corneal basement membrane in diabetes, we compared the localization of laminin and type IV collagen in the epithelial basement membrane during corneal epithelial wound healing in diabetic and nondiabetic rats. METHODS: Streptozotocin was used to induce diabetes in half the rats. Two weeks later, the whole corneal epithelium was debrided. Diabetic and healthy rats (3-5 per group) were sacrificed before debridement and 1, 3, and 7 days and 1 month afterwards. The localization of laminin and type IV collagen was observed in cryosections by epifluorescence microscopy. RESULTS: In unwounded corneas of both diabetic and normal rats, laminin and type IV collagen were localized in the corneal epithelial basement. The intensity of fluorescence, however, was clearly stronger in the diabetic rats. In normal rats, wounding initially removed laminin and type IV collagen, but during healing these two proteins reappeared beneath the resurfacing corneal epithelium. Although similar results were observed in diabetic rats, the expression of laminin and type IV collagen was delayed, and their deposition was fragmented and irregular. CONCLUSIONS: These results suggest that delayed corneal epithelial wound healing in diabetes might involve delayed reappearance and abnormal reformation of epithelial basement membrane proteins.     

Localization of extracellular matrix proteins after holmium YAG laser radiation in rat cornea

Purpose: To understand corneal responses to holmium YAG (Ho:YAG) laser radiation, we used immunofluorescent microscopy to examine changes in the localization of extracellular matrix proteins. Methods: Rats were radiated with an Ho:YAG laser. On days 1, 3, and 7 after radiation, the eyes were enucleated and frozen. The cryosections were stained by immunofluorescent microscopy using antibodies against type I collagen, fibronectin, type IV collagen, and laminin.Results: One day after Ho:YAG laser radiation, contraction of the stromal collagen fibrils was observed. Keratocytes could not be observed at the radiated stromal region on day 1 after radiation. One week after radiation, keratocytes returned to the radiated area. Although the stromal collagen fibrils were contracted, they were stained by an antibody against type I collagen. Dense fluorescence for fibronectin was observed at the margin of the stromal acellular zone. Both laminin and type IV collagen were observed at the basement membrane under the corneal epithelium regardless of whether the corneas were radiated or not. Conclusions: These results suggest that Ho:YAG laser radiation might be useful for collagen contraction of the stroma, without causing serious damage to the corneal epithelium or the basement membrane.