Original Paper
Ophthalmic Res 1992:24:260-264
Department of Ophthalmology, Kyoto University, Faculty of Medicine. Sakyo-ku, Kyoto, Japan; Department of Ophthalmology. Aichi Medical University, Aichi, Japan; Department of Tissue Physiology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
Keywords Proliferative diabetic retinopathy Intravitreal neovascular tissue Collagen Fibronectin Glial fibrillary acidic protein
Intravitreal IMeovascular Tissue of Proliferative Diabetic Retinopathy: An Immunohistochemical Study
Abstract Intravitreal neovascular tissue in 8 cases of proliferative dia betic retinopathy was investigated using immunohistochemical techniques. All 8 cases yielded positive immunoreactivity for type II collagen (vitreous collagen). The intravitreal neo vascular tissue was classified into two groups (A or B), depend ing upon the distribution of type II collagen. In group A (3 cases), blood vessels were entirely surrounded by vitreous collagen, and in group B (5 cases), the vessels proliferated on one side of a mass of vitreous collagen. Type I and III colla gens were distributed diffusely within the extracellular space of the tissue, whereas type IV collagen and fibronectin (FN) formed a basement membrane-like foundation for the newly formed vessels. Glial fibrillary acidic protein (GFAP)-immunoreactive cells were not clearly detected in any of the cases. Neovascular tissue typically proliferated on the posterior vitreous surface (as found in group B), but was also found to penetrate the vitreous gel (as found in group A). As ncovascular tissue proliferation proceeded, types I, III and IV collagens and FN were produced. Glial cells (GFAP-positive cells) were not essential for neovascular tissue formation.
Introduction Fibrovascular membranes removed during vitreous surgery and autopsy from patients with proliferative diabetic retinopathy
Supported by grant-in-aid for Scientific Research C-03670828 from the Ministry of Education. Science and Culture of the Japanese government.
Received: June 25, 1990 Accepted: January 14. 1992
(PDR), have been described morphologically [1-7], Some of these studies have shown that diabetic proliferative membranes contain cel lular and extracellular collagenous compo nents in addition to vascular elements. How-
Yasuko Hosoda. MD Department of Ophthalmology Faculty of Medicine Kyoto University Sakyo-ku. Kyoto 606 (Japan)
©1992 S. Karger AG. Basel 0030-3747/92/ 024 5-0260S2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 11:21:14 PM
Yasuko Hosodaa Mario Okadaa Miyo Matsum urna Nobuchika Oginob Yoshihito Hondaa Yutaka Nagaic
Materials and Methods Amisera Sprague-Dawlcy rats were immunized with human type I, III, or IV collagen. The antisera against each type of collagen were obtained from 1 week to 10 days after the last booster injection. Type-specific anticol lagen antibodies were purified from crude antisera by cross-adsorption on affinity columns consisting of each of the other types of collagen coupled with acti vated CH-Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden), followed by immunoadsorption on affinity columns prepared from the same type of colla gen immunized and by elution. The specificity of each antibody was analyzed by the inhibition test, using ELISA. Antiserum against human type II collagen was raised in rabbits. Specific antibody to human type IIcollagen was obtained from the antisera by immunoad sorption and affinity chromatography on antigen colla gen. The antibody was tested for type-specificity by passive hemagglutination. Details of these procedures
and the results have been described elsewhere [11. 12]. Rabbit antihuman fibronectin antibody was pur chased from Dakopatts (Glostrup. Denmark). The specificity of this antibody was determined by immunoabsorbance using affinity-purified human fibronec tin (Pierce Chemical Company. 111., USA). Rabbit anti-GFAP antibody was purchased from DAKOPATTS. The antibody specificity was tested using an immunoblotting technique, and its reactivity to the human retinal astrocytes was also tested as described elsewhere [ 13].
Immunohistochemical Studies Intravitreal tissue removed during pars plana vitrec tomy from 8 patients with severe PDR was dropped into phosphatc-bulTcrcd saline (PBS) and immediately observed using a phase-contrast microscope. Soon after the observation, the specimens were fixed in a mixture containing 3% paraformaldehyde and 0.1 %glutaraldehyde in PBS for 2 h at room temperature. Following fixation, the tissues were dehydrated in a serial ethanol gradient and embedded in Lowicryl K4M (Palaron Equipment Ltd., Watford, UK). Semithin sections for light microscopy were treated as follows: sections were incubated with PBS containing 1% bovine serum albu min to block nonspecific binding. The sections were then incubated at 4 °C overnight with the primary anti bodies: types I, II. Ill, and IV antihuman collagen anti bodies (1:200). and for 1 h at room temperature with antihuman FN antibody (1:2,000) and antirabbit GFAP antibody (1:1.000). Normal rat serum (1:200) was used as a negative control for anticollagcn anti bodies (types I, III and IV), and normal rabbit serum (1:200) was used instead of anticollagen type II anti body, anti-FN antibody and anti-GFAP antibody. Nor mal human retinas obtained from eye-bank eyes within 24 h postmortem were embedded in the same way and incubated with anti-GFAP antibody (1:1,000) as a posi tive control. After washing 3 times with PBS. the sec tions were incubated for 1 h with a secondary antibodycoated colloidal gold (1:80) (Janssen Life Science Prod ucts, Olen. Belgium) and rinsed again with PBS. The sections were subsequently fixed in 2.5% glutaraldehyde to prevent dissociation of the antibody-antigen complex during the subsequent procedure. Fixation was followed by washing with distilled water and treatment with a silver enhancer (77 mM hydroquinone and 5.5 mM silver lactate in 200 mM citrate buffer, pH 3.85). The reactions were stopped with a fixing solu tion (Janssen Life Science Products). Finally, the sec tions were counterstained with 0.5% toluidine blue and viewed using a light microscope.
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ever, morphologic techniques alone cannot determine the origin of the cellular and extra cellular components of this proliferative tis sue. In PDR. two types of proliferative neovascular tissue have been recognized: epiretinal and intravitreal. Biomicroscopically, diabetic epiretinal tissue is observed as a dense fibrovascular membrane on the retinal surface, whereas intravitreal tissue appears as a simple extension of vascular tissue into the vitreous cavity. Although these two types of tissue are considered to have a number of different characteristics, the two types have not been distinguished from each other in previous studies of PDR [8. 9], Furthermore, most pre vious studies of PDR have focused primarily on epiretinal, rather than intravitreal, fibrovascular tissue. In this study, the components of surgically removed intravitreal neovascular tissue were examined using immunogoldsilver staining (IGSS) [10] to determine the distribution of types I, II, III, and IV collagen, fibronectin (FN) and glial fibrillary acidic protein (GFAP).
Phase-contrast microscopic observation revealed that the intravitreal neovascular tis sue was comprised of a highly complicated vascular network in addition to vitreous gel (fig- 1). The distribution of type II (vitreous) colla gen, which was detectable in all specimens, was used to separate the two categories of tis sue. In group A, vascular components were surrounded entirely by a mass of type II colla gen (fig. 2a). In group B. the vascular compo nents were found on one side of a mass of type II collagen (fig. 2b). Types I and III colla gens were distributed diffusely throughout the extracellular space of the tissue, but in all cases, the highest concentrations were de tected at a short distance from the vessels (fig. 2c, d). Type IV collagen was also present in all specimens and was exclusively localized in close proximity to the vessels (fig. 2e). FN reactivity was weakly detected throughout the tissue, but similarly concentrated around the vessels in most cases (fig. 2f). Type IV colla gen and FN were colocalized in a layer sur rounding the vessels. In normal human reti nas, GFAP immunolabelling was observed (fig. 3a). GFAP, however, was not detected in any of the specimens (fig. 3b). No significant immunoreactivity was observed in any of the negative control sections (fig. 4a-4c).
Discussion It is well known that the neovascular tis sues in PDR proliferate into the vitreous cav ity along the posterior vitreous surface [7], Scheiffarth et al. [9] demonstrated that type II collagen was present in 12 of 13 diabetic membranes, but the origin of this tissue (epiretinal vs. intravitreal) was not elucidated. In our study, type II collagen was found in all
262
intravitreal proliferative tissue, and these tis sues were classified into two groups on the basis of vitreous collagen distribution (ta ble 1). In group B, the vascular components were present on one side of a mass of type II collagen (vitreous collagen), suggesting that the neovascular tissue developed on the pos terior vitreous surface. In group A, however, the neovascular tissue was entirely sur rounded by type II collagen, suggesting that tissue proliferation had penetrated the vi treous gel as described previously by Eisner [ 1 ].
Previous studies have shown that diabetic epiretinal membranes contain type I collagen
Table 1. Positive immunoreactivity for collagen, FN, and GFAP
Collagen
FN GFAP
Type Type Type Type
I II III IV
Group A
Group B
3/3 3/3 3/3 3/3 2/3 0/3
5/5 5/5 5/5 5/5 4/5 0/5
F ig .1. Phase-contrast micrograph of intravitreal neovascular tissue. The tissue consists of highly com plicated vascular networks (arrows) and vitreous gel (asterisks). Bar = 500 pm. Fig. 3. 1GSS were performed using antiglial fibril lary acidic protein (GFAP) antibody, a In normal reti nas, glial cells showed GFAP immunoreactivity (ar rows). b GFAP-immunoreactive cells were not ob served in the intravitreal neovascular tissue. IL = Inter nal limiting membrane; OL = outer limiting mem brane. Bar= 10 pm. Fig. 4. For the negative control, nonimmune rabbit serum (a) and nonimmune rat serum (b)were used instead of the primary antibodies. The primary antibody-omitted experiment was performed (c). No sig nificant immunoreaction was observed. Bar = 10 pm.
Hosoda/Okada/Matsumura/Ogino/ Honda/Nagai
Intravitreal Neovascular Tissue of PDR
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 11:21:14 PM
Results
also diffusely distributed within the neovas cular tissue. FN is an important component of the basement membrane and is known to be required by a variety of cells for attachment. These observations suggest that type IV colla gen and FN form a basement membrane-like framework for neovascular tissue formation. GFAP-immunoreactive cells have been re ported in the epiretinal membranes in dia betic retinopathy [8, 14] and other diseases [15, 16], In this study, however, GFAP-immunoreactive cells were not detected in the intravitreal neovascular tissue. Glial cells are not considered to play an active role during intravitreal neovascular tissue formation in diabetic retinopathy, although they may be important during epiretinal membrane for mation. In conclusion, intravitreal neovascular tis sue consists of vitreous gel, extracellular ma trix components and new vessels. These new vessels can penetrate into the vitreous cavity, producing types I, III and IV collagens and FN in the process. Glial cells do not appear to play an important role during intravitreal neovascular tissue formation in PDR.
Fig. 2. Light micrograph of the intravitreal neovas cular tissue. Immunogold-silver staining (IGSS) was performed using anticollagen antibodies and an antifibronectin antibody. Positive reactions are represented as black-colored silver precipitates, a Group A: The vascular components were surrounded by type II colla gen (vitreous collagen) (arrows), b Group B: The vas cular components were found on one side of a mass of type II collagen (asterisks), c Type I collagen was dis tributed diffusely in the extracellular space but concen trated in close proximity to the vessels (arrows), d Type III collagen was distributed similarly to type I collagen (arrows), e Type IV collagen was concen trated exclusively around the vessles (arrows), f Fibronectin was weakly distributed in the extracellular space of the tissue (asterisks) and was concentrated around the vessels (arrows), b, c, d, e are serial sections of the same specimen. Bar = 10 pm.
263
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[8, 9]. In this study, type I collagen was simi larly detected in intravitreal neovascular tis sue. Jerdan et al. [8] reported negative stain ing for type III collagen in diabetic epiretinal membranes. In contrast, Scheiffarth et al. [9] who observed type III collagen in all cases of PDR, did not differentiate between epiretinal or intravitreal specimens. In our study, both type I and III collagens were distributed dif fusely in the extracellular space of the tissue and were concentrated at a short distance from the vessels. These findings indicated that proliferating cells produced both type I and type III collagens, as these collagens are not normally found in the vitreous cavity. Previous studies have suggested the pres ence of type IV collagen in the collagenous meshwork of PDR [8, 9], Scheiffarth et al. [9] reported histochemical staining of type IV collagen in basement membranes, and noted more variability in both concentration and distribution than was observed for both type I and type III collagens and FN. In our study, type IV collagen staining was localized around the vessels in all cases. FN was simi larly concentrated around the vessels, but was
1 Eisner G: Biomicroscopy of the Pe ripheral Fundus. Berlin. Springer. 1973. p 17. 2 Hamilton CW, Chandler D. Klintworth GK. Machemer R: A trans mission and scanning electron mi croscopic study of surgically excised preretinal membrane proliferation in diabetes mellitus. Am J Ophthal mol 1982:94:473-488. 3 Miller H. Miller B. Zois S. Nir 1: Diabetic neovascularization: Per meability and ultrastructure. Invest Ophthalmol Vis Sci 1984:25:1338— 1342. 4 Wallow IHL. Stevens TS, Greaser ML. Bindley C, Wilson R: Actin filaments in contracting preretinal membranes. Arch Ophthalmol 1984:102:1370-1375. 5 Williams JM. de Juan E. Machemer R: Ultrastructural characteristics of new vessels in proliferative diabetic retinopathy. Am J Ophthalmol 1988:105:491-499. 6 Taniguchi Y: Ultrastructure of newly formed blood vessels in dia betic retinopathy. Jpn J Ophthalmol 1976:20:19-31.
264
7 Yanoff M: Ocular pathology of dia betes mellitus. Am J Ophthalmol 1969:67:21-38. 8 Jerdan JA. Michels RG. Glaser BM: Diabetic preretinal membranes. An immunohistochemical study. Arch Ophthalmol 1986:104:286-290. 9 Scheiffarth OF. Kampik A. Gunter H. von der Mark K: Proteins of the extracellular matrix in vitreoretinal membranes. Graefes Arch Clin Exp Ophthalmol 1988:226:357-361. 10 Danascher G: Localization of gold in biological tissues. A photochemi cal method for light and electron mi croscopy. Histochemistry' 1981:71: 81-88. ’ 11 Yasui N. Ono K. Yamaura I. Konomi H, Nagai Y: Immunohisto chemical localization of type I. II. and III collagens in the ossified pos terior longitudinal ligament of hu man cervical spine. CalcifTissue Int 1983;35:159-163.
12 Minamoto T. Ooi A. Okada Y. Mai M. Nagai Y. Nakanishi I: Desmo plastic reaction of gastric carcino ma: A light- and electron-micro scopic immunohistochemical analy sis using collagen type-specific anti bodies. Hum Pathol 1988:19:815821. 13 Okada M. Matsumura M. Ogino N, Honda Y: Muller cells in detached human retina express glial fibrillary acidic protein and vimentin. Graefes Arch Clin Exp Ophthalmol 1990:228:467-474. 14 Michael TN. Wallow IHL. Sramek SJ. Anderson G: Muller’s cell in volvement in proliferative diabetic retinopathy. Arch Ophthalmol 1987:105:1424-1429. 15 Hiscott PG. Grierson I. Trombetta CJ. Rahi AHS. Marshall J. Mcleod D: Retinal and epiretinal glia: An immunohistochemical study. Br J Ophthalmol 1984;68:698-707. 16 Yamashita H. Hori S, Kitano S. Ishii Y. Masuda K: Glial cells in cul ture of preretinal membrane of pro liferative vitreoretinopathy. Jpn J Ophthalmol 1985:29:42-53.
Hosoda/Okada/Matsumura/Ogino/ Honda/Nagai
Intravitrcal Neovascular Tissue of PDR
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 11:21:14 PM
References
Ophthalmic Res 1992:24:260-264
Department of Ophthalmology, Kyoto University, Faculty of Medicine. Sakyo-ku, Kyoto, Japan; Department of Ophthalmology. Aichi Medical University, Aichi, Japan; Department of Tissue Physiology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
Keywords Proliferative diabetic retinopathy Intravitreal neovascular tissue Collagen Fibronectin Glial fibrillary acidic protein
Intravitreal IMeovascular Tissue of Proliferative Diabetic Retinopathy: An Immunohistochemical Study
Abstract Intravitreal neovascular tissue in 8 cases of proliferative dia betic retinopathy was investigated using immunohistochemical techniques. All 8 cases yielded positive immunoreactivity for type II collagen (vitreous collagen). The intravitreal neo vascular tissue was classified into two groups (A or B), depend ing upon the distribution of type II collagen. In group A (3 cases), blood vessels were entirely surrounded by vitreous collagen, and in group B (5 cases), the vessels proliferated on one side of a mass of vitreous collagen. Type I and III colla gens were distributed diffusely within the extracellular space of the tissue, whereas type IV collagen and fibronectin (FN) formed a basement membrane-like foundation for the newly formed vessels. Glial fibrillary acidic protein (GFAP)-immunoreactive cells were not clearly detected in any of the cases. Neovascular tissue typically proliferated on the posterior vitreous surface (as found in group B), but was also found to penetrate the vitreous gel (as found in group A). As ncovascular tissue proliferation proceeded, types I, III and IV collagens and FN were produced. Glial cells (GFAP-positive cells) were not essential for neovascular tissue formation.
Introduction Fibrovascular membranes removed during vitreous surgery and autopsy from patients with proliferative diabetic retinopathy
Supported by grant-in-aid for Scientific Research C-03670828 from the Ministry of Education. Science and Culture of the Japanese government.
Received: June 25, 1990 Accepted: January 14. 1992
(PDR), have been described morphologically [1-7], Some of these studies have shown that diabetic proliferative membranes contain cel lular and extracellular collagenous compo nents in addition to vascular elements. How-
Yasuko Hosoda. MD Department of Ophthalmology Faculty of Medicine Kyoto University Sakyo-ku. Kyoto 606 (Japan)
©1992 S. Karger AG. Basel 0030-3747/92/ 024 5-0260S2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 11:21:14 PM
Yasuko Hosodaa Mario Okadaa Miyo Matsum urna Nobuchika Oginob Yoshihito Hondaa Yutaka Nagaic
Materials and Methods Amisera Sprague-Dawlcy rats were immunized with human type I, III, or IV collagen. The antisera against each type of collagen were obtained from 1 week to 10 days after the last booster injection. Type-specific anticol lagen antibodies were purified from crude antisera by cross-adsorption on affinity columns consisting of each of the other types of collagen coupled with acti vated CH-Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden), followed by immunoadsorption on affinity columns prepared from the same type of colla gen immunized and by elution. The specificity of each antibody was analyzed by the inhibition test, using ELISA. Antiserum against human type II collagen was raised in rabbits. Specific antibody to human type IIcollagen was obtained from the antisera by immunoad sorption and affinity chromatography on antigen colla gen. The antibody was tested for type-specificity by passive hemagglutination. Details of these procedures
and the results have been described elsewhere [11. 12]. Rabbit antihuman fibronectin antibody was pur chased from Dakopatts (Glostrup. Denmark). The specificity of this antibody was determined by immunoabsorbance using affinity-purified human fibronec tin (Pierce Chemical Company. 111., USA). Rabbit anti-GFAP antibody was purchased from DAKOPATTS. The antibody specificity was tested using an immunoblotting technique, and its reactivity to the human retinal astrocytes was also tested as described elsewhere [ 13].
Immunohistochemical Studies Intravitreal tissue removed during pars plana vitrec tomy from 8 patients with severe PDR was dropped into phosphatc-bulTcrcd saline (PBS) and immediately observed using a phase-contrast microscope. Soon after the observation, the specimens were fixed in a mixture containing 3% paraformaldehyde and 0.1 %glutaraldehyde in PBS for 2 h at room temperature. Following fixation, the tissues were dehydrated in a serial ethanol gradient and embedded in Lowicryl K4M (Palaron Equipment Ltd., Watford, UK). Semithin sections for light microscopy were treated as follows: sections were incubated with PBS containing 1% bovine serum albu min to block nonspecific binding. The sections were then incubated at 4 °C overnight with the primary anti bodies: types I, II. Ill, and IV antihuman collagen anti bodies (1:200). and for 1 h at room temperature with antihuman FN antibody (1:2,000) and antirabbit GFAP antibody (1:1.000). Normal rat serum (1:200) was used as a negative control for anticollagcn anti bodies (types I, III and IV), and normal rabbit serum (1:200) was used instead of anticollagen type II anti body, anti-FN antibody and anti-GFAP antibody. Nor mal human retinas obtained from eye-bank eyes within 24 h postmortem were embedded in the same way and incubated with anti-GFAP antibody (1:1,000) as a posi tive control. After washing 3 times with PBS. the sec tions were incubated for 1 h with a secondary antibodycoated colloidal gold (1:80) (Janssen Life Science Prod ucts, Olen. Belgium) and rinsed again with PBS. The sections were subsequently fixed in 2.5% glutaraldehyde to prevent dissociation of the antibody-antigen complex during the subsequent procedure. Fixation was followed by washing with distilled water and treatment with a silver enhancer (77 mM hydroquinone and 5.5 mM silver lactate in 200 mM citrate buffer, pH 3.85). The reactions were stopped with a fixing solu tion (Janssen Life Science Products). Finally, the sec tions were counterstained with 0.5% toluidine blue and viewed using a light microscope.
261
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ever, morphologic techniques alone cannot determine the origin of the cellular and extra cellular components of this proliferative tis sue. In PDR. two types of proliferative neovascular tissue have been recognized: epiretinal and intravitreal. Biomicroscopically, diabetic epiretinal tissue is observed as a dense fibrovascular membrane on the retinal surface, whereas intravitreal tissue appears as a simple extension of vascular tissue into the vitreous cavity. Although these two types of tissue are considered to have a number of different characteristics, the two types have not been distinguished from each other in previous studies of PDR [8. 9], Furthermore, most pre vious studies of PDR have focused primarily on epiretinal, rather than intravitreal, fibrovascular tissue. In this study, the components of surgically removed intravitreal neovascular tissue were examined using immunogoldsilver staining (IGSS) [10] to determine the distribution of types I, II, III, and IV collagen, fibronectin (FN) and glial fibrillary acidic protein (GFAP).
Phase-contrast microscopic observation revealed that the intravitreal neovascular tis sue was comprised of a highly complicated vascular network in addition to vitreous gel (fig- 1). The distribution of type II (vitreous) colla gen, which was detectable in all specimens, was used to separate the two categories of tis sue. In group A, vascular components were surrounded entirely by a mass of type II colla gen (fig. 2a). In group B. the vascular compo nents were found on one side of a mass of type II collagen (fig. 2b). Types I and III colla gens were distributed diffusely throughout the extracellular space of the tissue, but in all cases, the highest concentrations were de tected at a short distance from the vessels (fig. 2c, d). Type IV collagen was also present in all specimens and was exclusively localized in close proximity to the vessels (fig. 2e). FN reactivity was weakly detected throughout the tissue, but similarly concentrated around the vessels in most cases (fig. 2f). Type IV colla gen and FN were colocalized in a layer sur rounding the vessels. In normal human reti nas, GFAP immunolabelling was observed (fig. 3a). GFAP, however, was not detected in any of the specimens (fig. 3b). No significant immunoreactivity was observed in any of the negative control sections (fig. 4a-4c).
Discussion It is well known that the neovascular tis sues in PDR proliferate into the vitreous cav ity along the posterior vitreous surface [7], Scheiffarth et al. [9] demonstrated that type II collagen was present in 12 of 13 diabetic membranes, but the origin of this tissue (epiretinal vs. intravitreal) was not elucidated. In our study, type II collagen was found in all
262
intravitreal proliferative tissue, and these tis sues were classified into two groups on the basis of vitreous collagen distribution (ta ble 1). In group B, the vascular components were present on one side of a mass of type II collagen (vitreous collagen), suggesting that the neovascular tissue developed on the pos terior vitreous surface. In group A, however, the neovascular tissue was entirely sur rounded by type II collagen, suggesting that tissue proliferation had penetrated the vi treous gel as described previously by Eisner [ 1 ].
Previous studies have shown that diabetic epiretinal membranes contain type I collagen
Table 1. Positive immunoreactivity for collagen, FN, and GFAP
Collagen
FN GFAP
Type Type Type Type
I II III IV
Group A
Group B
3/3 3/3 3/3 3/3 2/3 0/3
5/5 5/5 5/5 5/5 4/5 0/5
F ig .1. Phase-contrast micrograph of intravitreal neovascular tissue. The tissue consists of highly com plicated vascular networks (arrows) and vitreous gel (asterisks). Bar = 500 pm. Fig. 3. 1GSS were performed using antiglial fibril lary acidic protein (GFAP) antibody, a In normal reti nas, glial cells showed GFAP immunoreactivity (ar rows). b GFAP-immunoreactive cells were not ob served in the intravitreal neovascular tissue. IL = Inter nal limiting membrane; OL = outer limiting mem brane. Bar= 10 pm. Fig. 4. For the negative control, nonimmune rabbit serum (a) and nonimmune rat serum (b)were used instead of the primary antibodies. The primary antibody-omitted experiment was performed (c). No sig nificant immunoreaction was observed. Bar = 10 pm.
Hosoda/Okada/Matsumura/Ogino/ Honda/Nagai
Intravitreal Neovascular Tissue of PDR
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 11:21:14 PM
Results
also diffusely distributed within the neovas cular tissue. FN is an important component of the basement membrane and is known to be required by a variety of cells for attachment. These observations suggest that type IV colla gen and FN form a basement membrane-like framework for neovascular tissue formation. GFAP-immunoreactive cells have been re ported in the epiretinal membranes in dia betic retinopathy [8, 14] and other diseases [15, 16], In this study, however, GFAP-immunoreactive cells were not detected in the intravitreal neovascular tissue. Glial cells are not considered to play an active role during intravitreal neovascular tissue formation in diabetic retinopathy, although they may be important during epiretinal membrane for mation. In conclusion, intravitreal neovascular tis sue consists of vitreous gel, extracellular ma trix components and new vessels. These new vessels can penetrate into the vitreous cavity, producing types I, III and IV collagens and FN in the process. Glial cells do not appear to play an important role during intravitreal neovascular tissue formation in PDR.
Fig. 2. Light micrograph of the intravitreal neovas cular tissue. Immunogold-silver staining (IGSS) was performed using anticollagen antibodies and an antifibronectin antibody. Positive reactions are represented as black-colored silver precipitates, a Group A: The vascular components were surrounded by type II colla gen (vitreous collagen) (arrows), b Group B: The vas cular components were found on one side of a mass of type II collagen (asterisks), c Type I collagen was dis tributed diffusely in the extracellular space but concen trated in close proximity to the vessels (arrows), d Type III collagen was distributed similarly to type I collagen (arrows), e Type IV collagen was concen trated exclusively around the vessles (arrows), f Fibronectin was weakly distributed in the extracellular space of the tissue (asterisks) and was concentrated around the vessels (arrows), b, c, d, e are serial sections of the same specimen. Bar = 10 pm.
263
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[8, 9]. In this study, type I collagen was simi larly detected in intravitreal neovascular tis sue. Jerdan et al. [8] reported negative stain ing for type III collagen in diabetic epiretinal membranes. In contrast, Scheiffarth et al. [9] who observed type III collagen in all cases of PDR, did not differentiate between epiretinal or intravitreal specimens. In our study, both type I and III collagens were distributed dif fusely in the extracellular space of the tissue and were concentrated at a short distance from the vessels. These findings indicated that proliferating cells produced both type I and type III collagens, as these collagens are not normally found in the vitreous cavity. Previous studies have suggested the pres ence of type IV collagen in the collagenous meshwork of PDR [8, 9], Scheiffarth et al. [9] reported histochemical staining of type IV collagen in basement membranes, and noted more variability in both concentration and distribution than was observed for both type I and type III collagens and FN. In our study, type IV collagen staining was localized around the vessels in all cases. FN was simi larly concentrated around the vessels, but was
1 Eisner G: Biomicroscopy of the Pe ripheral Fundus. Berlin. Springer. 1973. p 17. 2 Hamilton CW, Chandler D. Klintworth GK. Machemer R: A trans mission and scanning electron mi croscopic study of surgically excised preretinal membrane proliferation in diabetes mellitus. Am J Ophthal mol 1982:94:473-488. 3 Miller H. Miller B. Zois S. Nir 1: Diabetic neovascularization: Per meability and ultrastructure. Invest Ophthalmol Vis Sci 1984:25:1338— 1342. 4 Wallow IHL. Stevens TS, Greaser ML. Bindley C, Wilson R: Actin filaments in contracting preretinal membranes. Arch Ophthalmol 1984:102:1370-1375. 5 Williams JM. de Juan E. Machemer R: Ultrastructural characteristics of new vessels in proliferative diabetic retinopathy. Am J Ophthalmol 1988:105:491-499. 6 Taniguchi Y: Ultrastructure of newly formed blood vessels in dia betic retinopathy. Jpn J Ophthalmol 1976:20:19-31.
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7 Yanoff M: Ocular pathology of dia betes mellitus. Am J Ophthalmol 1969:67:21-38. 8 Jerdan JA. Michels RG. Glaser BM: Diabetic preretinal membranes. An immunohistochemical study. Arch Ophthalmol 1986:104:286-290. 9 Scheiffarth OF. Kampik A. Gunter H. von der Mark K: Proteins of the extracellular matrix in vitreoretinal membranes. Graefes Arch Clin Exp Ophthalmol 1988:226:357-361. 10 Danascher G: Localization of gold in biological tissues. A photochemi cal method for light and electron mi croscopy. Histochemistry' 1981:71: 81-88. ’ 11 Yasui N. Ono K. Yamaura I. Konomi H, Nagai Y: Immunohisto chemical localization of type I. II. and III collagens in the ossified pos terior longitudinal ligament of hu man cervical spine. CalcifTissue Int 1983;35:159-163.
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Hosoda/Okada/Matsumura/Ogino/ Honda/Nagai
Intravitrcal Neovascular Tissue of PDR
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