Immunohistologic study of proliferative vitreoretinopathy

An immunohistologic study was performed on pars plana specimens obtained by biopsy in ten patients with rhegmatogenous retinal detachment, with or without proliferative vitreoretinopathy. Using immunofluorescence or immunoperoxidase procedures, linear deposits of IgG, IgA, and complement components were found in the eight cases of retinal detachment with proliferative vitreoretinopathy at the basal pole of the pigment epithelial cells and within the stroma. In contrast, these deposits were absent from the normal pars plana and from the retinal detachment without proliferative vitreoretinopathy. Moreover, pigment and nonpigment epithelial cells were found to express HLA-DR and HLA-DQ determinants in six of the eight patients with proliferative vitreoretinopathy. Our results are similar to those obtained in a previous study on proliferative diabetic retinopathy, which suggests the involvement of autoimmune phenomena in proliferative diseases and eventual interactions between the immune system and peptide growth factors. However, whether or not this immune reaction functions in the initiation or extension of intraocular proliferative syndromes remains to be determined.     

Origin of fibronectin in epiretinal membranes of proliferative vitreoretinopathy and proliferative diabetic retinopathy.

Fibronectins, high molecular multifunctional glycoproteins of the extracellular matrix and plasma, have been a popular area of research in the pathogenesis of proliferative disorders of the retina. Several immunohistochemical studies have revealed that fibronectin is a major constituent of epiretinal membranes and that the cell types involved in proliferative intraocular disorders may synthesise it. However, owing to the fact that plasma and cellular fibronectin are similar in their overall structure, the origin of fibronectin in epiretinal membranes has not yet been clearly defined. In this study, we used two monoclonal antibodies: FN-3, which recognises an extra domain present in the cellular but not plasma form of fibronectin; and FN-4, which reacts with an antigenic site on both plasma and cellular fibronectin. In 37 epiretinal membranes obtained from eyes with proliferative vitreoretinopathy and proliferative diabetic retinopathy, we demonstrated the presence of cellular fibronectin, thus indicating local production. The significantly stronger and positive immunostain with FN-4 in the same specimens suggests the colocalisation of plasma fibronectin, that may be derived from the breakdown of the blood-retinal barrier and trapped in membranes during their formation. In pathological vitreous we demonstrated both types of fibronectin by western blot analysis.     

Expression of myofibroblast activation molecules in proliferative vitreoretinopathy epiretinal membranes

Purpose: Fibrotic disorders are associated with activation of fibroblasts into extracellular matrix‐secreting myofibroblasts expressing α‐smooth muscle actin (α‐SMA). Myofibroblasts are the predominant cellular component of proliferative vitreoretinopathy (PVR) epiretinal membranes. We investigated the expression of molecules involved in myofibroblast activation, migration and proliferation in PVR epiretinal membranes. Methods: Fifteen membranes were studied by immunohistochemical techniques using monoclonal and polyclonal antibodies directed against snail, fibroblast activation protein (FAP), CD44, hydrogen peroxide‐inducible clone‐5 (Hic‐5), galectin‐3, interleukin‐13 receptor α2 (IL‐13Rα2) and receptor for advanced glycation end products (RAGE). Results: Myofibroblasts expressing α‐SMA were present in all membranes. Myofibroblasts expressed nuclear immunoreactivity for Snail and Hic‐5, cytoplasmic immunoreactivity for FAP, IL‐13Rα2 and RAGE and membranous immunoreactivity for CD44. There was no immunoreactivity for galectin‐3. The number of cells expressing α‐SMA correlated significantly with the number of cells expressing Snail (r = 0.56; p = 0.03), Hic‐5 (r = 0.526; p = 0.044), IL‐13Rα2 (r = 0.773; p = 0.001) and RAGE (r = 0.734; p = 0.002). Conclusions: Snail, FAP, CD44, Hic‐5, IL13Rα2 and RAGE may be involved in proliferative events occurring in PVR.     

增生性玻璃体视网膜病变视网膜前膜CD44v6的表达

目的：观察增生性玻璃体视网膜病变视网膜前膜中CD44v6的表达情况。方法：用免疫组化方法对13例PVR患行玻璃体切割术的剥离的视网膜前膜进行观察。结果：CD44v6阳性表达10例，表达率77％。结论：提示CD44v6在PVR的形成和发展中有一定的作用。     

Proliferative vitreoretinopathy. Lymphocytes in epiretinal membranes.

BACKGROUND: To investigate the potential contribution of inflammatory and immune-mediated processes contributing to the pathogenesis of proliferative vitreoretinopathy (PVR), an immunohistochemical study was undertaken to characterize the infiltrating inflammatory cells in epiretinal membranes surgically removed from the eyes of patients with PVR. METHODS: Twenty-one epiretinal membranes obtained surgically from eyes with PVR complicating rhegmatogenous retinal detachment were studied immunohistochemically using the ABC technique and a panel of monoclonal and polyclonal antibodies. RESULTS: T lymphocytes were found in 18 of the 21 specimens and generally constituted a small percentage of the total cell number. CD4+ T cells were found in 14 of the 18 membranes containing T cells. Three of six frozen membranes contained T cells that were positive for the interleukin-2 receptor. In 5 of 16 membranes studied, cells positive for the macrophage/monocyte marker were found. No B lymphocytes or neutrophils were identified, and there were no deposits of complement or immunoglobulins. Positive staining for the class II MHC antigen HLA-DR was found in 7 of the 21 membranes, a result that was more consistent in frozen than in fixed tissues. CONCLUSION: The study suggests that T lymphocytes are present in PVR epiretinal membranes and may be activated. These cells have the potential to play a role in the pathobiology of PVR.     

肾上腺髓质素及其受体在增生性玻璃体视网膜病变前膜中的表达

目的 探讨肾上腺髓质素(ADM)及其受体(ADMR)在增生性玻璃体视网膜病变(PVR)前膜中的表达。方法 标本取自2001年7-11月在我院眼科研究所确诊为孔源性视网膜脱离合并PVR行玻璃体手术的10例连续病例,PVR均为C级以上,采用原位杂交法对术中所取标本的ADM和ADMR mRNA表达情况进行观察。结果10例增生性玻璃体视网膜病变前膜中有7例ADM和ADMR mRNA阳性。阳性细胞内分布不均匀,部分细胞以胞质阳性为主,部分细胞的阳性主要出现在胞核。结论 ADM和ADMR在PVR前膜中表达,PVR的发病过程中有ADM和ADMR的参与。(中华眼科杂志,2004,4D：317-320)     

Subretinal membranes in proliferative vitreoretinopathy

Subretinal membranes (SRMs) are an important but rarely identified component of proliferative vitreoretinopathy (PRV). In 153 consecutive cases that had vitreoretinal surgery for this condition and were followed for at least 6 months, SRMs were encountered in 72 eyes (47%). In 20 (28%) of the 72 eyes, the SRMs prevented complete retinal reattachment and needed to be removed or excised through one or multiple retinotomies. Intraoperative complications related to the SRMs or their removal included choroidal or retinal hemorrhage in three eyes (15%), subretinal air in three eyes (15%), and unplanned extension of the retinotomies in two eyes (10%). The 20 eyes requiring SRMs removal were followed for a median of 11 months. Retinas were reattached in 13 eyes (65%), although only 4 eyes (20%) had a visual acuity of 5/200 or better. Recognizing SRMs as a component of PVR is important in helping to maximize the anatomic success rate although the effects on visual function are not fully known.     

Indocyanine green-assisted peeling of the epiretinal membrane in proliferative vitreoretinopathy

BACKGROUND: We stained the internal limiting membrane of patients suffering from proliferative vitreoretinopathy with indocyanine green solution during proliferative vitreoretinopathy surgery to improve the visibility of the membranes, and thereby histopathologically confirmed the excised epiretinal membranes. METHODS: Three patients underwent a standard three-port pars plana vitrectomy with indocyanine green staining. After performing a subtotal vitrectomy we spread 0.5% indocyanine green solution, approximately 1 ml, on the retinal surface and peeled off the epiretinal membranes. RESULTS: The epiretinal membranes did not stain clearly, while the internal limiting membranes did stain clearly. We could therefore distinguish the epiretinal membranes from the retina. We cut the internal limiting membrane, grasped it, and peeled off the internal limiting membrane underlying the epiretinal membranes using vitreoretinal forceps. A histopathologic examination confirmed the presence of proliferative cells and an extracellular matrix underlying the internal limiting membranes. CONCLUSION: The technique for staining the epiretinal membranes in proliferative vitreoretinopathy using indocyanine green gives better visualization and allows surgeons to remove the epiretinal membranes more safely and effectively, as well as with less risk of retinal damage.     

Fibronectin synthesis in subretinal membranes of proliferative vitreoretinopathy.

In situ hybridisation and immunohistochemical studies were conducted on six surgically excised subretinal membranes of proliferative vitreoretinopathy to investigate whether displacement of retinal pigment epithelial and glial cells to subretinal membranes was associated with fibronectin production by the subretinal membrane cells. Fibronectin messenger RNA (mRNA) and fibronectin immunoreactivity were observed in some cells in all of the subretinal membranes studied and up to 30% of the cells in individual specimens showed intense labelling for fibronectin mRNA. The results support the concept that the cells in subretinal membranes produce fibronectin. Locally produced fibronectin may play a role in subretinal membrane cohesion, and displacement of retinal pigment epithelial and glial cells from their normal location may induce the cells to manufacture fibronectin. Fibronectin production may be more prominent in migrating subretinal cells.     

Immunochemical studies of epiretinal membranes using APAAP complexes: Evidence for macrophage involvement in traumatic proliferative vitreoretinopathy

Proliferative vitreoretinopathy is characterized by cellular proliferations in the periretinal space resulting in traction retinal detachment. Numerous cellular elements and connective tissue components have been identified by morphologic criteria as well as immunochemical techniques. In this study, we used the recently introduced APAAP (alkaline phosphatase - anti-alkaline phosphatase) immunostaining procedure to identify macrophages, T-lymphocytes, the structural proteins fibronectin, vimentin, and cytokeratin, and a proliferating cell antigen, in eleven human epiretinal membranes obtained during vitreoretinal surgery. Our results confirm that the pathologic processes in PVR are not immunologically mediated, but reveal the features of physiologic wound healing and scar formation.Posttraumatic PVR seems to be characterized by a severe initial inflammatory reaction as evidenced by the presence of numerous macrophages, whereas idiopathic PVR, as a complication of retinal detachment, may be caused by different mechanisms in the early pathogenesis.This study was supported by the Retinovit-Foundation.     

Localization of acidic fibroblast growth factor in proliferative vitreoretinopathy membranes.

The pathogenesis of proliferative vitreoretinopathy (PVR) membranes remains poorly understood. We have studied the presence of acidic fibroblast growth factor (aFGF), a potent mitogen for many cells, within these membranes. We have used affinity purified monospecific anti-aFGF polyclonal antibodies, in conjunction with highly sensitive immunofluorescence techniques. The labelling was exclusively localized to cell bodies and was absent from the extracellular matrix. Double labelling techniques revealed that all cytokeratin positive cells (probably pigmented epithelial cells) and macrophages contained aFGF-like immunoreactivity, whilst glial cells were unlabelled. Appropriate controls indicated the specificity of the antibodies. Hence, the presence of this mitogenic molecule within certain cell types constituting PVR membranes may contribute to the pathogenesis.     

三种视网膜病视网膜前膜中基质金属蛋白酶及其天然抑制分子的表达

目的 研究增殖性糖尿病视网膜病变(proliferative diabetic retinopathy,PDR)、增殖性玻璃体视网膜疾病(proliferative vitreoretinopathy,PVR)和急性视网膜坏死(acute retinalnecrosis,ARN)患者视网膜前膜中基质金属蛋白酶(matrixmetalloproteinases:MMPs)及其天然抑制物(tissueinhibitorsofmetalloproteinages,TIMPs)的表达情况.方法 玻璃体手术中剥取PVR、PDR和ARN患者的视网膜前膜,同供体眼视网膜作为正常对照,冰冻切片后进行免疫组织化学染色,包括:MMP-1,MMP-2,MMP-3,MMP-7,MMP-9,TIMP-1和TIMP-2.结果 正常视网膜中能够观察到MMP-1,MMP-3,TIMP-1和TIMP-2的表达,在PVR、PDR和ARN患者标本中各种分子的表达都增强,尤以MMP-2,MMP-3和MMP-7明显.结论 正常视网膜中存在MMPs和TIMPs分子维持着细胞外基质动态的平衡,在PVR,PDR和ARN患者中MMP-2,MMP-3和MMP-7等MMPs分子表达增强,在其病变过程中可能起重要作用.     

增生性玻璃体视网膜病变增殖膜的免疫组织化学及细胞凋亡研究

目的　检测增生性玻璃体视网膜病变 (PVR)增殖膜标本中细胞Fas、Fas配体 (Fasligand ,FasL)的表达和凋亡 ,并探讨Fas/FasL与凋亡的关系。方法　通过免疫组织化学的方法检测 12例增殖膜标本Fas和FasL的表达 ,TUNEL技术检测凋亡细胞。结果　PVR增殖膜标本中Fas和FasL阳性细胞百分率分别为 60 6%和 43 75 % ,偶见两者同时表达的细胞。 5例前膜和 2例下膜标本可见TUNEL染色阳性细胞核。凋亡与Fas、FasL相关系数均为 0 77(t =3 82 ,P <0 0 5 ) ,PVR病程与凋亡的r值为 0 74(t =3 11,P <0 0 1)。结论　Fas、FasL高表达与凋亡的正相关性提示 ,通过Fas/FasL系统诱导增殖细胞及炎症细胞凋亡 ,可能成为临床防治PVR的新策略     

增生性玻璃体视网膜病变膜剥离标本的免疫组化研究

目的 探讨不同增生性玻璃体视网膜病变(PVR)的增殖特征。方法采用5种特异性抗体对12例PVR膜样本进行免疫组织化学研究。结果成纤维细胞、神经胶质细胞为参与PVR膜的主要细胞成分,视网膜色素上皮细胞(RPE)、巨噬细胞、纤维连接蛋白和新生血管也参与了PVR的病理过程。结论新生血管主要参与了增生性视网膜血管病变的病理过程。增殖膜中增殖的细胞、细胞外基质和血管成分参与了PVR的病理过程并起着不同的作用。     

大鼠外伤性PVR视网膜前膜形成中TERT、PCNA和Bcl-2的变化

目的：探讨与细胞增殖相关的端粒酶逆转录酶TERT、增殖细胞核抗原(PCNA)和凋亡抑制蛋白Bcl-2在外伤性PVR视网膜前膜形成中的动态变化及其相关性。方法：S-P法对外伤性PVR大鼠模型视网膜前膜做TERT、PCNA、Bcl-2免疫组织化学染色和HE染色,染色结果行图像分析。结果：伤后PCNA蛋白表达阳性细胞率和平均光密度值出现先升高后降低的动态变化,14d组阳性率最高,与7d组和28d组比较有显性差异。伤后TERT蛋白和Bcl-2表达阳性细胞率和平均光密度值出现先升高后稍降低的动态变化,14d组和21d组阳性率最高,与7d组比较有显性差异,TERT、PCNA和Bcl-2蛋白表达之间有显相关性(P≤0．01)。结论：TERT、Bcl-2参与外伤性PVR视网膜前膜增殖细胞的生长调控,与细胞增殖的动态变化呈高度相关性。     

Proliferative vitreoretinopathy membranes. An immunohistochemical study

Proliferative vitreoretinopathy (PVR) is the leading cause of failure after retinal detachment surgery. Therefore, both the extracellular matrix and cellular components of preretinal membranes from 23 eyes with PVR were characterized immunohistochemically. The membrane stroma was composed primarily of types I, II, and III collagen. Laminin and both heparan sulfate proteoglycans and collagens types IV and V were co-distributed in discrete regions within the stroma. Glial and retinal pigment epithelial (RPE) cell populations were identified in these membranes using specific immunohistochemical markers as was a small but significant macrophage population. Double-labeling experiments indicated that RPE cells in these membranes expressed the class II histocompatibility antigen HLA-DR, although neither the RPE monolayer in situ nor cultured RPE cells was HLA-DR positive unless induced by gamma interferon. Only rare isolated vascular endothelial cells were detected in 5 of the 23 membranes.