© 1990 S. Karger AG. Basel 0030-3755/90/2014-0187 S 2.75/0
Ophthalmologica 1990;201:187-195
Characterization of Glial Involvement in Proliferative Diabetic Retinopathy1 Akihiro Ohira, Eugene cle Juan, Jr. Duke Eye Center, Duke University Medical Center, Durham, N.C., USA
Key Words. Proliferative diabetic retinopathy • Glial cells • Neovascularization
Introduction Diabetic retinopathy is a major cause of visual loss in the United States [1|. The most severe visual impairments result from the pro liferative stages of the disease. Due to the striking appearance of developing blood ves sels, clinically histologic descriptions of the proliferative stages in diabetes have empha' This work was supported in part by grant EYO5903 from the National Eye Institute.
sized the vessels and their growth f2-4|. Spe cial techniques such as trypsin digest and India ink preparations have been used to demon strate and emphasize the early vascular chang es in diabetes [5, 6]. Similar emphasis on vas cular damages has been placed in the ultrastructural studies of membranes removed during vitreous surgery for proliferative dia betic retinopathy [7, 8]. The purpose of this study is to emphasize and characterize the glia and other supporting tissues associated with newly formed blood
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:45:48 AM
Abstract. We studied the relationship of glial cells and other supporting tissues associated with newly formed blood vessels in proliferative diabetic retinopathy. Seventeen postmortem, freshly enucleated eyes from diabetic patients and 34 epiretinal and preretinal membranes removed during vitreous surgery for proliferative diabetic retinopathy were analyzed using the peroxidaseantiperoxidase method for light microscopy and protein A/gold labeling of ultrathin cryosections for transmission electron microscopy in addition to routine transmission and scanning electron microscopy. We found that glial cells are commonly and characteristically found in elevated diabetic proliferations and present at the vitreous surface. The newly formed blood vessels, however, were not seen at the edge of elevated epiretinal and preretinal membranes in early and intermediate stages. These results suggest that glial cells may extend beyond the vascularized areas of the proliferative tissue. It is possible that glial cells and their extracellular matrix contribute to the framework leading to the development of new blood vessels.
Ohira/de Juan
188
Materials and Methods Post mortem and freshly enucleated eyes from dia betic patients were obtained from the Duke University Medical Center pathology laboratory files. Eyes with various stages in the development of proliferative mem branes were selected for further studies (16 eyes). The formalin-fixed specimens were prepared for histologic examination after staining with hematoxylin-eosin (HE) and periodic acid-Schiff (PAS). Phosphotungstic acidhematoxylin stain was used to demonstrate the presence of glia. Immunoperoxidase staining for glial fibrillary acidic protein (GFAP) was performed on sections from formalin-fixed Paraplast-embedded eyes (7 eyes). Com mercially obtained rabbit antibody to human GFAP (Dako) was used with the peroxidase-antiperoxidase technique [9], Briefly, 10-mm-thick sections mounted on Histostick-coated slides were deparaffinized in xylene and hydrated in graded alcohol solutions. The slides were treated with 3% hydrogen peroxide for 5 min to eliminate endogenous peroxidase activity. The speci mens were rinsed in Tris buffer and then incubated with rabbit antibody to human GFAP for 20 min and rinsed in Tris buffer for 20 min. Swine antirabbit IgG antibody was reacted with the specimens for 20 min. After rins ing, the specimens were reacted with the peroxidaseantiperoxidase immunocomplex for 20 min and rinsed. Freshly prepared diaminobenzidine staining solution was reacted with the specimens under visualization, fol lowed by another rinse in distilled water. The slides were then counterstained with Mayer's hematoxylin. Nonspe cific staining was judged by repeating the procedure with nonimmune rabbit serum. Positive controls for GFAP were performed by using formalin-fixed human cerebral tissue. In addition, 34 epiretinal and preretinal membranes removed during vitreous surgery for proliferative dia betic retinopathy were studied by light electron micros copy. The membranes were rinsed in 0.1 M cacodylate buffer with 5% sucrose (pH 7.4) immediately after re moval. They were then placed in 2% glutaraldehyde in 0.1 M cacodylate buffer for at least 1 h and postfixed in 2% osmium tetroxide in 0.1 M cacodylate buffer for 1 h rinsed in buffer and dehydrated in graded ethanol. The
mebranes were embedded in Spurr low-viscosity medi um. and 1-um sections were stained with toluidine blue and basic fuchsin for light microscopy. Sixty-nanomctcr sections were cut and stained with uranyl acetate and lead citrate for transmission electron microscopy (JEM-1200EX. Jeol). When a specimen was large enough, a portion of the specimen was critical-point dried, sputter coated with gold palladium and examined with a scanning electron microscope (JSM-35C, Jeol). The immunocytochemical staining to demonstrate GFAP was carried out using protein A/gold labeling on ultrathin cryosections according to published methods [ 10].
Results Overall glial cell involvement was docu mented in many types of elevated diabetic pro liferations in eyes obtained postmortem. De tails of the glial relationships to the developing vessels and overall membrane architecture were demonstrated by electron microscopy of tissue removed during vitreous surgery. The glial changes ranged in complexity from intraretinal cellular changes to complex elevated membranes. One mild change that was observed in all eyes studied (fig. 1) was increased GFAP stain ing of the Müller cell foot plates (fig. 2). This was often associated with some hypercellularity (presumably glial) of the nerve fiber and ganglion cell layers. The cells were small and had densely stained nuclei. The same eyes had a few areas with GFAP staining in an overlying epiretinal membrane but not in the underlying inner retina. Other eyes showed areas of thin cellular epiretinal membranes (fig. 3a) in the presence of either an attached or detached posterior vit reous face. No vessels were observed in these membranes. Positive immunoperoxidase staining of GFAP indicated their glial nature in 3 eyes (fig. 3b). When the vitreous was at-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:45:48 AM
vessels in proliferative diabetic retinopathy as well as to outline possible mechanisms leading to their development.
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Glial Involvement in Proliferative Diabetic Retinopathy
Fig. 1. a Photomicrograph showing vascularized preretinal membrane with thin cellular layer on vit reous surface. HE. x 450. b Immunoperoxidase-positive GFAP epiretinal gliovascular membrane showing the glial nature of cells on the innermost portion of the membrane on the vitreous side, x 450.
r
Fig. 2. Photomicrograph of immunoperoxidasestained (top) GFAP in Müller end-feet in the eye of a patient with proliferative diabetic retinopathy. Note small spindle-like cells within the retina causing a hypercellularity. x 450.
increased collagen production toward the reti nal side. Immunoperoxidase staining and ul trastructural examinations demonstrated that the glia were usually limited to the vitreal bor der of the mebrane with collagen and vascular tissue adjacent to the retinal side (fig. la, b). In
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:45:48 AM
tached, the membrane was usually thin with a smooth retinal surface. When the vitreous was detached, the membranes were often associ ated with surface wrinkling (fig. 4). Anti-GFAP as marked by the gold particles was seen at the end-feet and processes of Müller cells in background diabetic retinas (fig. 5a, b). Transmission electron microscopy of thin epiretinal membranes (6 membranes) obtained during vitreous surgery, demonstrat ed cells with large intracellular filaments (10 nm), a relatively organelle-free cytoplasm and a basal lamina (fig. 6). These ultrastructu ral features are consistent with reactive glia. The extraretinal glial cells rested on a collage nous layer of variable thickness. Scanning elec tron microscopy of the mebranes (4 mem branes) revealed that the cells on the vitreous surface had a mosaic-like appearance with mi crovilli on their surfaces (fig. 7). Glial cells ad jacent to the vitreous were polarized with their microvilli directed the vitreous and the basal lamina toward the retinal side. Epiretinal glial membrames that contained vessels (16 membranes) were associated with
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Ohira/de Juan
Fig. 3. a Photomicrograph showing thin cellular epiretinal membrane on the surface of the retina of a patient with proliferative diabetic retinopathy. HE. x 450. b Positive GFAP immunoperoxidase staining indicating glial nature of cells in epiretinal membrane, x 450.
such as polarity were more evident at this stage. Elevated membranes showed glia on the vitreous side and collagen and vessels on the retinal side despite being separated from the retinal surface (fig. 8).
Fig. 4. Epiretinal membrane which has spontane ously separated from the retinal surface causing a fold in the internal limiting membrane and retina. PAS. x 450.
some membranes, the gliovascular architec ture was well defined; however, in the newer (developing) membranes the relationships were not as clear and the cellular character istics not well defined. Additionally, cells which lacked distinguishing characteristics
Detailed clinical descriptions of prolifera tive diabetic retinopathy are given in the litera ture. These descriptions emphasize vascular development and pay less attention to the sup porting tissues except in the fibrotic late stages. The retinal vasculature has been ele gantly studied using trypsin digest and other methods [2, 5], but these studies also deemphasize the surrounding tissues. Since very few eyes with early or intermediate stages of dia betic retinopathy are available for ultrastructu ral studies, most electron-microscopic studies have concentrated on surgically removed membranes [7,8]. Again much of the emphasis was placed on the developing vessels and not the supporting tissues. Hamilton et al. [7] de-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:45:48 AM
Discussion
Glial Involvement in Proliferative Diabetic Retinopathy
191
scribed glia in the membranes that they stud ied, but no effort was made to differentiate vascularized from nonvascularized tissue. We have shown that the glia, when present, are often at the surface of the membrane, so a systematic effort must be made to demonstrate glia. Although extraretinal glia were not found in all surgical specimens, they were seen in all whole eyes with proliferative diabetic retinop athy that were studied with immunoperoxidase methods as well as the vast majority of wellpreserved surgical specimens. It is our opinion that retinal glia are commonly and character istically found in elevated diabetic prolifera-
tions and are present at the vitreous surface. In addition, they may extend beyond the vascu larized areas of the proliferation. It is not possible with techniques applied in this study to establish the exact origin of the glia found in the membranes, i.e. either Müller cells or retinal astrocytes - both of which are normally present in the human retina. How ever, a recent study of 4 diabetic eyes [11] in dicates that Müller glia are present in the preretinal membranes. Additionally, we do not know the role of glial tissues in proliferative diabetic retinopathy. Glial migration also pre cedes extraretinal neovascularization or at
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:45:48 AM
Fig. 5a, b. Electron micrographs of ultrathin cryosection of diabetic retina incubated with anti-GFAP and stained with protein A/gold. Gold particles can be seen distributed in the Müller end-feet and the processes of Müller cells indicating GFAP presence. Bar = 1 pm.
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192 Ohira/de Juan
Glial Involvement in Proliferative Diabetic Retinopathy
193
Fig. 8. Electron micrograph showing gliovascular collagenous membrane. Note the thin layer of overlapping glial cells towards the vitreous side of the membrane overlying the vessel and dense collagenous extracellular matrix. Bar = 5 pm.
The proposed structure of elevated diabetic membranes (i.e. glia vascular-collagenous membrane), although not invariable, appears to be typical, particularly in the later stages of development. Additionally, glial membranes can develop without the incorporation of ves sels and may be important in the development of elevated diabetic membranes.
Fig. 6. Electron micrograph demonstrating glial cells in the elevated thin preretinal membrane in a patient with proliferative diabetic retinopathy. Note polarity of cells with microvilli (top right) directed toward the vitreous side and basement membrane and collagen matrix on the retinal side of the membrane (upper left). Bar = 1pm. Fig. 7. Scanning electron micrograph showing the epithelial nature of glial cells viewed from the vitreous side of the membrane. Note microvillous projections into the vitreous cavity and well-delineated cell borders. Bar = 10 pm.
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least develops in areas where vessels are not present. Glial cells may also provide a cellu lar scaffold with an extracellular matrix along which vessels can grow (fig. 9a, b). This may play a role in the production of posterior vit reous separation, which is common in pa tients which proliferative diabetic retinopa thy [12],
194
Ohira/de Juan
References 1 Kini MM, Heibowitz HM. Colton T. et al: Preva lence of senile cataract diabetic retinopathy, senile macular degeneration and open-angle glaucoma in the Framingham Eye Study. Am J Ophthalmol 1976; 85:28-34.
2 de Venecia G, Davis M, Engerman R: Clinicopathologic correlations in diabetic retinopathy. I. Histol ogy and fluorescein angiography of microaneu rysms. Arch Ophthalmol 1976:94:1766. 3 Bresnick CH, Davis MD, Myer FL. et al: Clinicopathologic correlation in diabetic retinopathy. II. Clinical and histologic appearances of retinal capil
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:45:48 AM
Fig. 9. a Drawing demonstrating typical features of the glial epiretinal membrane in a patient with diabetes. Note that the vitreous may be at tached to the glial membrane which in turn rests on the internal limiting lamina of the retina, b Elevated gliovascular collagenous membrane giv ing the appearance of vessels growing on the posterior surface of the vit reous. Note that the glial cellular lay er may be transparent, until excess collagenous tissue causes the mem brane to become opaque.
Glial Involvement in Proliferative Diabetic Retinopathy
195
lary microaneurysms. Arch Ophthalmol 1977:95: 1215-1220. Asthon W: Studies of the retinal capillaries in rela tion to diabetic and other retinopathies. Br J O ph thalmol 1963:47:521. Kohner EM, Henkind P: Correlation of fluorescein angiogram and retinal digest in diabetic retinopathy. Am J Ophthalmol 1970:69:403. Cogan DG, Kuwabara I: Capillary shunts in the pathogenesis of diabetic retinopathy. Diabetes 1963: 12:292. Hamilton CW, Chandler D. Klintworth GK, Machemer R: A transmission and scanning electron microscopic study of surgically excised preretinal membrane proliferations in diabetes mellitus. Am J Ophthalmol 1982:94:473-488. Miller S, Miller B, Zonis S, Nir I: Diabetic neo vascularization: permeability and ultrastructure. In vest Ophthalmol Vis Sci 1984:25:1338-1342. Ohira A, Oshima K. Kikuchi M: Immunohisto chemical study of preretinal proliferating membrane in human MPP. Acta Soc Ophthalmol Jpn 1982: 86:803-809.
10 Chang LY, Slott JW, Geuze HJ, Crapo JD: Molec ular immunocytochemistry of the CuZn Superoxide dismutase in rat hépatocytes. J Cell Biol 1988: 107:2169-2179. 11 Nork TM, Wallow IH. Sramek S. Anderson G: Müller's cell involvement in proliferative diabetic retinopathy. Arch Ophthalmol 1987:105:1424-1429. 12 de Juan E: Mechanism of elevated membrane for mation and posterior vitreous detachment in dia betes mellitus: in BenEzra D. Ryan SJ (eds): Ocular Circulation and Neovascularization. Dordrecht, Nijhoff, 1987.
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6
7
8
9
Received: May 18,1990 Accepted: May 29.1990 Eugene de Juan. Jr.. MD Duke Eye Center Box 3802 Durham, NC 27710 (USA)
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:45:48 AM
4
Ophthalmologica 1990;201:187-195
Characterization of Glial Involvement in Proliferative Diabetic Retinopathy1 Akihiro Ohira, Eugene cle Juan, Jr. Duke Eye Center, Duke University Medical Center, Durham, N.C., USA
Key Words. Proliferative diabetic retinopathy • Glial cells • Neovascularization
Introduction Diabetic retinopathy is a major cause of visual loss in the United States [1|. The most severe visual impairments result from the pro liferative stages of the disease. Due to the striking appearance of developing blood ves sels, clinically histologic descriptions of the proliferative stages in diabetes have empha' This work was supported in part by grant EYO5903 from the National Eye Institute.
sized the vessels and their growth f2-4|. Spe cial techniques such as trypsin digest and India ink preparations have been used to demon strate and emphasize the early vascular chang es in diabetes [5, 6]. Similar emphasis on vas cular damages has been placed in the ultrastructural studies of membranes removed during vitreous surgery for proliferative dia betic retinopathy [7, 8]. The purpose of this study is to emphasize and characterize the glia and other supporting tissues associated with newly formed blood
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:45:48 AM
Abstract. We studied the relationship of glial cells and other supporting tissues associated with newly formed blood vessels in proliferative diabetic retinopathy. Seventeen postmortem, freshly enucleated eyes from diabetic patients and 34 epiretinal and preretinal membranes removed during vitreous surgery for proliferative diabetic retinopathy were analyzed using the peroxidaseantiperoxidase method for light microscopy and protein A/gold labeling of ultrathin cryosections for transmission electron microscopy in addition to routine transmission and scanning electron microscopy. We found that glial cells are commonly and characteristically found in elevated diabetic proliferations and present at the vitreous surface. The newly formed blood vessels, however, were not seen at the edge of elevated epiretinal and preretinal membranes in early and intermediate stages. These results suggest that glial cells may extend beyond the vascularized areas of the proliferative tissue. It is possible that glial cells and their extracellular matrix contribute to the framework leading to the development of new blood vessels.
Ohira/de Juan
188
Materials and Methods Post mortem and freshly enucleated eyes from dia betic patients were obtained from the Duke University Medical Center pathology laboratory files. Eyes with various stages in the development of proliferative mem branes were selected for further studies (16 eyes). The formalin-fixed specimens were prepared for histologic examination after staining with hematoxylin-eosin (HE) and periodic acid-Schiff (PAS). Phosphotungstic acidhematoxylin stain was used to demonstrate the presence of glia. Immunoperoxidase staining for glial fibrillary acidic protein (GFAP) was performed on sections from formalin-fixed Paraplast-embedded eyes (7 eyes). Com mercially obtained rabbit antibody to human GFAP (Dako) was used with the peroxidase-antiperoxidase technique [9], Briefly, 10-mm-thick sections mounted on Histostick-coated slides were deparaffinized in xylene and hydrated in graded alcohol solutions. The slides were treated with 3% hydrogen peroxide for 5 min to eliminate endogenous peroxidase activity. The speci mens were rinsed in Tris buffer and then incubated with rabbit antibody to human GFAP for 20 min and rinsed in Tris buffer for 20 min. Swine antirabbit IgG antibody was reacted with the specimens for 20 min. After rins ing, the specimens were reacted with the peroxidaseantiperoxidase immunocomplex for 20 min and rinsed. Freshly prepared diaminobenzidine staining solution was reacted with the specimens under visualization, fol lowed by another rinse in distilled water. The slides were then counterstained with Mayer's hematoxylin. Nonspe cific staining was judged by repeating the procedure with nonimmune rabbit serum. Positive controls for GFAP were performed by using formalin-fixed human cerebral tissue. In addition, 34 epiretinal and preretinal membranes removed during vitreous surgery for proliferative dia betic retinopathy were studied by light electron micros copy. The membranes were rinsed in 0.1 M cacodylate buffer with 5% sucrose (pH 7.4) immediately after re moval. They were then placed in 2% glutaraldehyde in 0.1 M cacodylate buffer for at least 1 h and postfixed in 2% osmium tetroxide in 0.1 M cacodylate buffer for 1 h rinsed in buffer and dehydrated in graded ethanol. The
mebranes were embedded in Spurr low-viscosity medi um. and 1-um sections were stained with toluidine blue and basic fuchsin for light microscopy. Sixty-nanomctcr sections were cut and stained with uranyl acetate and lead citrate for transmission electron microscopy (JEM-1200EX. Jeol). When a specimen was large enough, a portion of the specimen was critical-point dried, sputter coated with gold palladium and examined with a scanning electron microscope (JSM-35C, Jeol). The immunocytochemical staining to demonstrate GFAP was carried out using protein A/gold labeling on ultrathin cryosections according to published methods [ 10].
Results Overall glial cell involvement was docu mented in many types of elevated diabetic pro liferations in eyes obtained postmortem. De tails of the glial relationships to the developing vessels and overall membrane architecture were demonstrated by electron microscopy of tissue removed during vitreous surgery. The glial changes ranged in complexity from intraretinal cellular changes to complex elevated membranes. One mild change that was observed in all eyes studied (fig. 1) was increased GFAP stain ing of the Müller cell foot plates (fig. 2). This was often associated with some hypercellularity (presumably glial) of the nerve fiber and ganglion cell layers. The cells were small and had densely stained nuclei. The same eyes had a few areas with GFAP staining in an overlying epiretinal membrane but not in the underlying inner retina. Other eyes showed areas of thin cellular epiretinal membranes (fig. 3a) in the presence of either an attached or detached posterior vit reous face. No vessels were observed in these membranes. Positive immunoperoxidase staining of GFAP indicated their glial nature in 3 eyes (fig. 3b). When the vitreous was at-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:45:48 AM
vessels in proliferative diabetic retinopathy as well as to outline possible mechanisms leading to their development.
189
Glial Involvement in Proliferative Diabetic Retinopathy
Fig. 1. a Photomicrograph showing vascularized preretinal membrane with thin cellular layer on vit reous surface. HE. x 450. b Immunoperoxidase-positive GFAP epiretinal gliovascular membrane showing the glial nature of cells on the innermost portion of the membrane on the vitreous side, x 450.
r
Fig. 2. Photomicrograph of immunoperoxidasestained (top) GFAP in Müller end-feet in the eye of a patient with proliferative diabetic retinopathy. Note small spindle-like cells within the retina causing a hypercellularity. x 450.
increased collagen production toward the reti nal side. Immunoperoxidase staining and ul trastructural examinations demonstrated that the glia were usually limited to the vitreal bor der of the mebrane with collagen and vascular tissue adjacent to the retinal side (fig. la, b). In
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:45:48 AM
tached, the membrane was usually thin with a smooth retinal surface. When the vitreous was detached, the membranes were often associ ated with surface wrinkling (fig. 4). Anti-GFAP as marked by the gold particles was seen at the end-feet and processes of Müller cells in background diabetic retinas (fig. 5a, b). Transmission electron microscopy of thin epiretinal membranes (6 membranes) obtained during vitreous surgery, demonstrat ed cells with large intracellular filaments (10 nm), a relatively organelle-free cytoplasm and a basal lamina (fig. 6). These ultrastructu ral features are consistent with reactive glia. The extraretinal glial cells rested on a collage nous layer of variable thickness. Scanning elec tron microscopy of the mebranes (4 mem branes) revealed that the cells on the vitreous surface had a mosaic-like appearance with mi crovilli on their surfaces (fig. 7). Glial cells ad jacent to the vitreous were polarized with their microvilli directed the vitreous and the basal lamina toward the retinal side. Epiretinal glial membrames that contained vessels (16 membranes) were associated with
190
Ohira/de Juan
Fig. 3. a Photomicrograph showing thin cellular epiretinal membrane on the surface of the retina of a patient with proliferative diabetic retinopathy. HE. x 450. b Positive GFAP immunoperoxidase staining indicating glial nature of cells in epiretinal membrane, x 450.
such as polarity were more evident at this stage. Elevated membranes showed glia on the vitreous side and collagen and vessels on the retinal side despite being separated from the retinal surface (fig. 8).
Fig. 4. Epiretinal membrane which has spontane ously separated from the retinal surface causing a fold in the internal limiting membrane and retina. PAS. x 450.
some membranes, the gliovascular architec ture was well defined; however, in the newer (developing) membranes the relationships were not as clear and the cellular character istics not well defined. Additionally, cells which lacked distinguishing characteristics
Detailed clinical descriptions of prolifera tive diabetic retinopathy are given in the litera ture. These descriptions emphasize vascular development and pay less attention to the sup porting tissues except in the fibrotic late stages. The retinal vasculature has been ele gantly studied using trypsin digest and other methods [2, 5], but these studies also deemphasize the surrounding tissues. Since very few eyes with early or intermediate stages of dia betic retinopathy are available for ultrastructu ral studies, most electron-microscopic studies have concentrated on surgically removed membranes [7,8]. Again much of the emphasis was placed on the developing vessels and not the supporting tissues. Hamilton et al. [7] de-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:45:48 AM
Discussion
Glial Involvement in Proliferative Diabetic Retinopathy
191
scribed glia in the membranes that they stud ied, but no effort was made to differentiate vascularized from nonvascularized tissue. We have shown that the glia, when present, are often at the surface of the membrane, so a systematic effort must be made to demonstrate glia. Although extraretinal glia were not found in all surgical specimens, they were seen in all whole eyes with proliferative diabetic retinop athy that were studied with immunoperoxidase methods as well as the vast majority of wellpreserved surgical specimens. It is our opinion that retinal glia are commonly and character istically found in elevated diabetic prolifera-
tions and are present at the vitreous surface. In addition, they may extend beyond the vascu larized areas of the proliferation. It is not possible with techniques applied in this study to establish the exact origin of the glia found in the membranes, i.e. either Müller cells or retinal astrocytes - both of which are normally present in the human retina. How ever, a recent study of 4 diabetic eyes [11] in dicates that Müller glia are present in the preretinal membranes. Additionally, we do not know the role of glial tissues in proliferative diabetic retinopathy. Glial migration also pre cedes extraretinal neovascularization or at
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:45:48 AM
Fig. 5a, b. Electron micrographs of ultrathin cryosection of diabetic retina incubated with anti-GFAP and stained with protein A/gold. Gold particles can be seen distributed in the Müller end-feet and the processes of Müller cells indicating GFAP presence. Bar = 1 pm.
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192 Ohira/de Juan
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193
Fig. 8. Electron micrograph showing gliovascular collagenous membrane. Note the thin layer of overlapping glial cells towards the vitreous side of the membrane overlying the vessel and dense collagenous extracellular matrix. Bar = 5 pm.
The proposed structure of elevated diabetic membranes (i.e. glia vascular-collagenous membrane), although not invariable, appears to be typical, particularly in the later stages of development. Additionally, glial membranes can develop without the incorporation of ves sels and may be important in the development of elevated diabetic membranes.
Fig. 6. Electron micrograph demonstrating glial cells in the elevated thin preretinal membrane in a patient with proliferative diabetic retinopathy. Note polarity of cells with microvilli (top right) directed toward the vitreous side and basement membrane and collagen matrix on the retinal side of the membrane (upper left). Bar = 1pm. Fig. 7. Scanning electron micrograph showing the epithelial nature of glial cells viewed from the vitreous side of the membrane. Note microvillous projections into the vitreous cavity and well-delineated cell borders. Bar = 10 pm.
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least develops in areas where vessels are not present. Glial cells may also provide a cellu lar scaffold with an extracellular matrix along which vessels can grow (fig. 9a, b). This may play a role in the production of posterior vit reous separation, which is common in pa tients which proliferative diabetic retinopa thy [12],
194
Ohira/de Juan
References 1 Kini MM, Heibowitz HM. Colton T. et al: Preva lence of senile cataract diabetic retinopathy, senile macular degeneration and open-angle glaucoma in the Framingham Eye Study. Am J Ophthalmol 1976; 85:28-34.
2 de Venecia G, Davis M, Engerman R: Clinicopathologic correlations in diabetic retinopathy. I. Histol ogy and fluorescein angiography of microaneu rysms. Arch Ophthalmol 1976:94:1766. 3 Bresnick CH, Davis MD, Myer FL. et al: Clinicopathologic correlation in diabetic retinopathy. II. Clinical and histologic appearances of retinal capil
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:45:48 AM
Fig. 9. a Drawing demonstrating typical features of the glial epiretinal membrane in a patient with diabetes. Note that the vitreous may be at tached to the glial membrane which in turn rests on the internal limiting lamina of the retina, b Elevated gliovascular collagenous membrane giv ing the appearance of vessels growing on the posterior surface of the vit reous. Note that the glial cellular lay er may be transparent, until excess collagenous tissue causes the mem brane to become opaque.
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lary microaneurysms. Arch Ophthalmol 1977:95: 1215-1220. Asthon W: Studies of the retinal capillaries in rela tion to diabetic and other retinopathies. Br J O ph thalmol 1963:47:521. Kohner EM, Henkind P: Correlation of fluorescein angiogram and retinal digest in diabetic retinopathy. Am J Ophthalmol 1970:69:403. Cogan DG, Kuwabara I: Capillary shunts in the pathogenesis of diabetic retinopathy. Diabetes 1963: 12:292. Hamilton CW, Chandler D. Klintworth GK, Machemer R: A transmission and scanning electron microscopic study of surgically excised preretinal membrane proliferations in diabetes mellitus. Am J Ophthalmol 1982:94:473-488. Miller S, Miller B, Zonis S, Nir I: Diabetic neo vascularization: permeability and ultrastructure. In vest Ophthalmol Vis Sci 1984:25:1338-1342. Ohira A, Oshima K. Kikuchi M: Immunohisto chemical study of preretinal proliferating membrane in human MPP. Acta Soc Ophthalmol Jpn 1982: 86:803-809.
10 Chang LY, Slott JW, Geuze HJ, Crapo JD: Molec ular immunocytochemistry of the CuZn Superoxide dismutase in rat hépatocytes. J Cell Biol 1988: 107:2169-2179. 11 Nork TM, Wallow IH. Sramek S. Anderson G: Müller's cell involvement in proliferative diabetic retinopathy. Arch Ophthalmol 1987:105:1424-1429. 12 de Juan E: Mechanism of elevated membrane for mation and posterior vitreous detachment in dia betes mellitus: in BenEzra D. Ryan SJ (eds): Ocular Circulation and Neovascularization. Dordrecht, Nijhoff, 1987.
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Received: May 18,1990 Accepted: May 29.1990 Eugene de Juan. Jr.. MD Duke Eye Center Box 3802 Durham, NC 27710 (USA)
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:45:48 AM
4
