Dispase金属蛋白酶诱导的兔增殖性玻璃体视网膜病变模型

目的建立并评价dispase诱导的增殖性玻璃体视网膜病变(proliferativevitreoretinopathy,PVR)动物模型。方法选择健康成年的青紫蓝兔40只,右眼分别行玻璃体腔注射dispase0.05ml,按注射浓度不同,随机分成4组,A组(0.05U)、B组(0.075U)、C组(0.1U)、D组(0.2U)。左眼分别注射PBS0.05ml作为对照。术后第3小时、第1天、第7天至第70天每周进行眼部检查。检查项目包括裂隙灯、间接眼底镜和眼底照相。在处死动物后,典型的PVR眼进行组织病理学检查及增殖细胞核抗原(proliferatingcellmuclearantigen,PCNA)免疫组织化学染色。结果术后3h所有dispase眼均见视网膜前或玻璃体出血。大约在3周后,dispase眼依次出现视网膜前膜、髓线和血管抬高、固定视网膜皱折以及牵拉性视网膜脱离。组织病理学见视网膜前增殖膜对视网膜的牵拉以及视网膜结构的紊乱。在dispase注药后2周,视网膜节细胞层抗增殖细胞核抗原(PCNA)呈阳性表达,4周时内核层细胞表达阳性,至发生视网膜脱离后视网膜色素上皮开始表达PCNA。对照组未见PVR发生,也未见PCNA的阳性表达。结论Dispase可以在不使用外源性细胞、生长因子和细胞因子的情况下诱导视网膜细胞增殖从而建立PVR模型,操作简单且成功率高。     

The Classification of Retinal Detachment with Proliferative Vitreoretinopathy

The term proliferative vitreoretinopathy (PVR) is proposed as a designation for the clinical condition previously known variously as massive vitreous retraction, massive preretinal retraction, or massive periretinal proliferation. This is an abnormality in which rhegmatogenous retinal detachment is complicated by proliferation of membranes on both surfaces of the detached retina and on the posterior surface of the detached vitreous gel. The new classification subdivides PVR into four stages: A, minimal; B, moderate; C, marked; and D, massive. The extent of the fixed retinal folds is subdivided to indicate the number of involved quadrants. The degree of retinal shrinkage into a funnel-like configuration is subdivided into three grades.     

Population and proportion of component cells in preretinal membranes

Cell population and proportion of each component cell type in preretinal membranes formed after retinal detachment were studied by light microscopy (LM) and transmission electron microscopy (TEM). The preretinal membranes were surgically obtained from 20 proliferative vitreoretinopathy (PVR) and 7 macular pucker patients. A thick section from the middle of Epok-embedded specimens stained by toluidine blue was photographed by LM and the area was determined. The cell nuclei were counted on the light micrographs, and the membrane cell population was estimated. All the cells in an ultrathin section of each membrane were photographed by TEM and classified into six types: pigment epithelial cells, astrocytes, macrophages, fibroblasts, cells of other types and unclassified cells. The proportion of these cell types in each membrane was analyzed. There was no significant correlation between the cell populations and duration of retinal detachment or grades of PVR. The proportions of macrophages and pigment epithelial cells were large in early stages of PVR and decreased in late stages, whereas those of astrocytes were the same as those of macrophages or pigment epithelial cells in the early stage and then increased as the duration of retinal detachment became prolonged. These results suggest that macrophages and pigment epithelial cells play important roles in the initiation of preretinal membrane formation in PVR, and that astrocytes participate in the development of rigid membranes. The main component cells in the preretinal membranes obtained from macular pucker patients were astrocytes. The population and the proportion of the astrocytes were almost similar among different stages. These facts suggest that astrocytes play an important role in preretinal membrane formation in macular pucker.     

Fibrovascular proliferation and retinal detachment after intravitreal injection of activated macrophages in the rabbit eye

Injection of activated macrophages into the posterior vitreous of the rabbit induced vigorous fibrovascular proliferation over the optic disk and medullary rays, as demonstrated by 3H-thymidine autoradiography. One week after injection, endothelial cells and pericytes of the capillaries near the inner surface of the optic disk and rays were labeled; fibroblast-like cells, which were also labeled, migrated and formed vitreous strands. By the second week after injection, the fibrovascular tissue proliferated most actively, and traction medullary ray detachment and peripapillary retinal fold formation were observed. The cellular proliferation was accompanied by inflammatory cell infiltration. Glial cells within the optic disk, as well as retinal pigment epithelial cells beneath the detached retina, were labeled by 3H-thymidine. These results demonstrate that the fibrovascular proliferation originates from the vessel complex of the optic disk and medullary rays in this experimental model of retinal detachment.     

Acoustic studies in proliferative diabetic retinopathy

Based on analysis of published reports and his own experience of acoustic studies (B-method) in more than 500 patients with proliferative diabetic retinopathy, the author concludes that preretinal fibrovascular membranes (posterior detachment of the hyaloid membrane with neovascularization) are often mistaken for retinal detachment. Acoustically detected solitary or multiple adhesions between fibrovascular and eye membranes are an important differential diagnostic sign indicating the absence of retinal detachment. These adhesions result from gradual staged "creeping" posterior vitreal detachment and indicate the sites of previous contact of the hyaloid membrane with the retina. In subtotal detachment of the retina there are no adhesions with ocular membranes and the retina acquires the configuration of a rigid fibrovascular membrane.     

Perisilicone proliferation after vitrectomy for proliferative vitreoretinopathy

From 1983 to 1986, silicone oil injections were used to treat 31 patients with retinal detachment (RD) and advanced proliferative vitreoretinopathy (PVR). In 19 eyes (61%), perisilicone proliferation (PSP) developed causing recurrent RDs in 15 eyes (49%). At an average of 5 weeks after surgery, PSP occurred and was characterized by extensive transparent preretinal membranes with denser focal areas. Microscopic examination of five preretinal membranes showed droplets of silicone oil and necrotic cells on the silicone side and glial or retinal pigment epithelial cells, or both, on the retinal side, often in layers separated by extracellular matrix. Silicone oil was present in periretinal membranes removed several months after the intraocular silicone had been evacuated indicating that silicone within cells may persist despite the removal of silicone. The use of silicone oil to provide tamponade in eyes with recurrent PVR is associated with a high incidence of periretinal proliferation that frequently leads to recurrent RD and visual failure.     

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。     

Proliferative vitreoretinopathy. Histopathology of retroretinal membranes

Light- and electron-microscopic studies of 43 eyes with retroretinal membranes are presented. Associated features were severe blunt or penetrating injury, total retinal detachment, surbretinal proteinaceous exudate, and concomitant presence of preretinal fibrocellular or fibrovascular proliferations. The retinal pigment epithelium was found to be the major cellular component in early (as soon as 1 week) and chronic retinal detachment cases. Isolated glial-derived membranes were less common and were found in cases with chronic retinal detachment. Macrophages and fibrocytes, along with the retinal pigment epithelium and glia, were detected in mixed membranes. Subretinal proteins are thought to influence migration and attachment of the retinal pigment epithelium, which in turn provides a template for proliferation of the various cell types described.     

Immunocytochemical study of an eye with proliferative vitreoretinopathy and retinal tacks

After an eye-wall resection for a choroidal melanoma, a 32-year-old woman had subsequent retinal detachment with proliferative vitreoretinopathy (PVR), and an unsuccessful attempt at repair with retinal tacks. Gross and light-microscopic examination of the globe revealed a total retinal detachment with extensive preretinal and subretinal membranes. The membranes surrounded the tack heads and extended in taut bands to form a tractional detachment of the pars plana. The membranes contained glial and nonglial cells. The glial cells immunolabeled for glial fibrillary acidic protein (GFAP), carbonic anhydrase-C (CA-C), vimentin, and glutamine synthetase (GS), thus suggesting that they were Müller's cells. While the tacks did not seem to cause PVR, in this case they may have provided an anchoring point from which membranes were able to exert traction on the retina and pars plana.     

Immunomorphological study of the vitreous body and subretinal fluid in vitreoretinal proliferation

Proliferative vitreoretinopathy (PVR) is a major complication of rhegmatogenous retinal detachment. Its pathogenesis remains poorly understood and the accurate nature of the growing cells on both surfaces of the detached retina has not been yet determined. We undertook an immunocytological study on 28 specimens of vitreous or subretinal fluid removed from patients with PVR. Five main types of cells could be identified: heavily pigmented cells, poorly pigmented ones, large totally unpigmented macrophage-like ones, smaller unpigmented cells and lymphocytes. Analysis of intravitreal pigment granules showed two different types of pigmented cells, those with lipofuscin and melanin and those with melanin without any granules of lipofuscin, which could originate from ciliary or iris pigment epithelia. Immunostaining procedures confirmed the epithelial non macrophagic lineage of the intravitreal and subretinal cells. Lymphocytes were only B cells. These results confirm the importance of proliferative process during the course of PVR and shows the involvement of other ocular structures other than the retinal pigment epithelium.     

Acidic FGF and other growth factors in preretinal membranes from patients with diabetic retinopathy and proliferative vitreoretinopathy.

The development and extension of fibrovascular or fibroglial membranes onto the retinal surface are a major cause of visual loss in diabetic patients with proliferative retinopathy and in patients suffering from retinal detachment with proliferative vitreoretinopathy. The pathogenesis of these proliferative diseases, however, remain poorly understood and the nature of growth-promoting mediators implicated in these phenomena has not been determined yet. Using indirect immunofluorescence procedures, three different growth factors known to be mitogenic for various cell components of preretinal membranes, acidic fibroblast growth factor, epidermal growth factor and insulin-like growth factor type I, were sought in 14 specimens of preretinal proliferative tissues. Similar results were obtained in diabetic preretinal membranes and tissues from patients with proliferative vitreoretinopathy. The three different growth factors were found diffusely in the connective stroma and around new blood vessels within the vascular walls. Some fibroblast-like and pigment epithelial-derived cells more markedly reacted with anti-growth factor antibodies. These results provide indications on the eventual involvement of three potent growth factors in intraocular proliferative diseases, but whether or not these mediators play an active role in the development of preretinal membranes remains to be determined.     

Pathogenesis and classification of massive periretinal proliferation.

Massive periretinal proliferation (MPP), a serious complication of retinal detachment, is caused by proliferation and fibrous metaplasia of cells mostly deriving from retinal pigment epithelium and retinal glial cells. Contracting fibrous membranes in the vitreous, and on and also under the retina, cause the intraocular changes of MPP. Early signs such as increased 'tobacco dust', pigmented and unpigmented clumps in the vitreous, and subtle preretinal and even retroretinal membranes are usually overlooked. The late signs such as starfolds, irregular retinal folds, circumferential folds, and funnel-shaped detachments are well known. The pathogenesis of the clinically visible signs is described, and a 4-stage classification of the disease is given.     

Intraocular osseous metaplasia. A clinico-pathological study

PURPOSE: To evaluate the clinico-pathologic features of intraocular osseous metaplasia. METHODS: Pathology specimens of enucleated eyes submitted to the ophthalmic pathology service at a tertiary eye-care referral center between January 1995 and June 1999 were studied for intraocular osseous metaplasia. Specific histopathologic features noted in specimens with osseous metaplasia were the presence of retinal detachment, gliosis, retinal pigment epithelial hyperplasia, drusen, epiretinal membrane, fibrovascular proliferation and inflammation. Immunohistochemistry using monoclonal antibody against glial fibrillary acidic protein was performed to assess the glial component within the membranes and the proliferative vitreoretinal mass. Clinical records were reviewed and correlated with histopathologic findings. RESULTS: Osseous metaplasiaS was noted in 8 of 151 (5.2%) eyes examined. Clinical diagnosis in these was phthisis bulbi, staphyloma, absolute glaucoma and microphthalmos. Enucleation was performed for relief of symptoms (in painful blind eyes) or for cosmesis, and in an eye inciting sympathetic ophthalmia. Retinal detachment, gliosis and retinal pigment epithelial hyperplasia were noted in all the cases. Drusen with calcification or ossification (5 of 8), fibrovascular proliferation in the vitreous (5 of 8) and active inflammation (4 of 8) were the other associated histologic features. Location of ossification was subretinal in 3 cases, preretinal (ora serrata) in 1 case and in both locations in 4 cases. The eyes with subretinal osseous metaplasia had associated calcified drusen, while preretinal ossification was seen within the fibrovascular membranes. CONCLUSION: Chronic retinal detachment, hyperplasia and transdifferentiation of retinal pigment epithelium appear to be a few of the prerequisites for intraocular osseous metaplasia. Ossification can occur at isolated subretinal and preretinal locations or can involve both. Though a larger study is required to postulate the chronology of events, in this small series, isolated subretinal ossification appears to be initiated by calcification and ossification of drusen, while in the pre-retinal region it is associated with vitreoretinal proliferation.     

Intravitreal and subretinal proliferation induced by platelet-rich plasma injection in rabbits.

We developed an experimental model of proliferative vitreoretinopathy (PVR) in albino rabbits by combining some factors suspected of causing the disease. Sixty nine eyes divided into six groups served as controls (Groups C 1-6). Forty nine eyes were divided into four experimental groups (Groups E 1-4). Group E1 (n = 12) was injected with 0.15 ml of platelet-rich plasma. In addition, Groups E2 (n = 12) and E3 (n = 12) underwent cryotherapy or vitrectomy. Group E4 (n = 13) underwent both procedures. Seven of the 13 Group 4 experimental eyes developed total retinal detachment and giant holes. None of the other groups developed more than two total retinal detachments or giant holes (P < 0.05). Light and electron microscopy showed intravitreal or preretinal proliferation composed of fibroblast-like cells. Retroretinal membranes appeared only in Group E4 eyes, composed of elongated cells with oval nuclei and abundant organelles in the cytoplasm. We believe these lesions mimic human PVR more closely than other models previously developed.     

Proliferative vitreoretinopathy and chemotherapeutic agents

Proliferative vitreoretinopathy (PVR) is a disease process that occurs in eyes with rhegmatogenous retinal detachments and accounts for the majority of failures following retinal detachment surgery. PVR involves the uncontrolled proliferation of non-neoplastic cells capable of forming membranes, which may occur on either surface of the retina or along the detached surface of the vitreous gel. Contraction of these membranes creates tractional forces that can distort or detach the retina. Various surgical procedures have been used to repair retinal detachments associated with PVR. The results have not been encouraging in many instances. Recent efforts have been directed toward the chemical inhibition of cellular proliferation in PVR. The majority of drugs used in these studies have been antineoplastic agents that affect various phases in the cycle of cell growth.     

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.     

Class II histocompatibility antigen expression by cellular components of vitreous and subretinal fluid in proliferative vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is the major cause of failure in retinal detachment surgery. It is characterized by the formation of membranes extending along both surfaces of the detached retina and within the vitreous, but the nature of the growing cells has not yet been determined. Using cytologic and immunocytologic procedures with 13 different monoclonal antibodies directed against Class II histocompatibility antigens and various markers of epithelial and immunocompetent cells, 30 specimens were studied of vitreous or subretinal fluid removed from patients with PVR. Five main types of cells could be identified: heavily pigmented cells, poorly pigmented ones, large totally unpigmented macrophage-resembling ones, smaller unpigmented cells, and lymphocytes. Analysis of intravitreal pigment granules, using autofluorescence by epiillumination and cytologic procedures, showed two different populations of pigmented cells: one with autofluorescent lipofuscin granules and the other with exclusively melanin pigment. Immunostaining procedures confirmed the epithelial nonmacrophage lineage of the intravitreal and subretinal cells because most of these cells were positive for cytokeratin but negative for macrophage markers. In addition, 40-100% of these epithelial-derived cells strongly expressed Class II histocompatibility antigens HLA-DR and -DQ. Lymphocytes were found in 13 specimens; B-cells were seen, but no T-lymphocytes could be identified. These results confirm the involvement of retinal pigment epithelial cells and the strong morphologic changes they undergo during the course of PVR. Moreover, the expression of Class II histocompatibility antigens by the growing cells may be related to inflammatory phenomena, but their eventual role in the development and the extension of periretinal proliferation has not been determined.     

RPE-mediated collagen gel contraction. Inhibition by colchicine and stimulation by TGF-beta

Retinal detachment secondary to proliferative vitreoretinopathy (PVR) is often precipitated by traction on the retina from preretinal membranes and traction on the vitreous base. Contraction and proliferation of retinal pigment epithelial (RPE) cells appear to play an important role in the pathogenesis of PVR. Contraction of collagen gels by human RPE cells was evaluated as a model of tractional forces on the vitreous gel. The area of collagen gels with admixed RPE cells in 15-mm wells was measured daily. The collagen gels contracted to less than 50% of original area in 89% of wells. Colchicine (0.01-1 microM) inhibited RPE-induced collagen gel contraction, whereas 1 ng/ml of transforming growth factor-beta enhanced it. Potential stimulators or inhibitors of RPE-mediated gel contraction can be tested using this model.     

Anterior proliferative vitreoretinopathy. Clinicopathologic, light microscopic, and ultrastructural findings

Proliferative vitreoretinopathy (PVR) involving the posterior and equatorial retina is an established clinicopathologic entity. Clinically, a similar process, anterior PVR (APVR), results in anterior dragging of the peripheral retina by membranes which connect to the ciliary body or iris and cause circumferentially and radially fixed retinal folds. The pathology of APVR, however, has not been reported. The authors describe pathologic findings in 28 cases of APVR and ultrastructural pathologic findings in 6 surgical APVR specimens. Anterior PVR was frequently associated with retinal detachment (RD) repair (96%) and trauma (38%). Residual vitreous at the vitreous base virtually always provided a scaffold for membranes containing proliferating cells and deposited extracellular matrix. Major components of APVR membranes were fibrovascular tissue (71%), pigment epithelial cells (43%), fibrous and corneal stromal ingrowth (32%), and glial proliferation (18%). Because of its anterior location, APVR membranes also incorporated ciliary epithelium and corneal endothelium. Contraction of APVR membranes caused anterior retinal displacement and detachment in anatomic configurations corresponding to narrow and wide peripheral troughs. The authors' findings indicate that APVR is a distinctive clinicopathologic entity which may complicate rhegmatogenous RD and its repair.     

Matrix metalloproteinases and their natural inhibitors in fibrovascular membranes of proliferative diabetic retinopathy

AIM: To examine epiretinal membranes of proliferative diabetic retinopathy (PDR) for the presence of selective matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs), in order to determine whether neovascularisation and fibrosis, characteristic of this complication of diabetes mellitus, are associated with specific anomalies of MMP or TIMP expression. METHODS: The presence of selected MMPs and TIMPs was investigated in 24 fibrovascular epiretinal membranes of PDR, and the findings compared with that observed in 21 avascular epiretinal membranes of proliferative vitreoretinopathy (PVR) and five normal retinas. Specimens were examined for deposition of interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), gelatinase A (MMP-2), gelatinase B (MMP-9), and three tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2, and TIMP-3). RESULTS: The results showed that unlike normal retina, which constitutively expresses MMP-1 and TIMP-2, a large proportion of PDR membranes (> 62%) stained for MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2, and TIMP-3. There were no differences in the expression of these molecules when compared with PVR membranes. A characteristic staining for MMP-9 was observed within the perivascular matrix of PDR membranes, and there was a significant increase in TIMP-2 expression by PDR membranes (p= 0.036) when compared with PVR membranes. CONCLUSIONS: The findings that MMPs involved in degradation of fibrovascular tissue matrix, as well as TIMP-1 and TIMP-2, are found in a large proportion of PDR membranes, and that their expression does not differ from that of PVR membranes, suggest the existence of common pathways of extracellular matrix degradation in pathological processes leading to retinal neovascularisation and fibrosis.