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Xyloside-Lnduced Disruption of Inferphororeceptor Matrix Proteoglycans Results in Retinal Detachment

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Xyloside-Lnduced Disruption of Inferphororeceptor Matrix Proteoglycans Results in Retinal Detachment Investigative Ophthalmology & Visual Science, Vol. 33, No. 2, February 1992 Copyright © Association for Research in Vision and Ophthalmology Xyloside-lnduced Disruption of Inferphororeceptor Matrix Proteoglycans Results in Retinal Detachment Howard 5. Lazarus and Gregory S. Hageman Unique domains of the retinal interphotoreceptor matrix (IPM), termed cone matrix sheaths, are composed largely of chondroitin 6-sulfate proteoglycan in most higher mammalian species. Recent investigations suggest that cone matrix sheaths participate in the maintenance of normal retinal attach- ment. To investigate the potential functional roles of IPM proteoglycans further, the synthesis of cone matrix sheath chondroitin 6-sulfate proteoglycan was perturbed in vivo. Intravitreal injections of p-ni- trophenyl-0-D-xylopyranoside (xyloside), a sugar that inhibits chondroitin sulfate proteoglycan synthe- sis, were administered to Yucatan micropigs. Their eyes were examined funduscopically and electrore- tinographically. At selected times, the eyes were enucleated and examined histochemically and immuno- histochemically with various probes directed against cone photoreceptor cells and cone matrix sheaths. The IPM was affected selectively after xyloside administration; no inner retinal pathology or dysfunc- tion was detected morphologically or electroretinographically. The degree of xyloside-induced pertur- bation was dependent on the duration of xyloside exposure and dose. It was classified into three stages, based on morphologic and histochemical criteria. Although all three stages could be observed in a given retina, a single stage typically predominated, depending on the particular dosage regimen. The early stage was characterized by IPM disruption, as evidenced by disorganization of chondroitin 6-sulfate- and peanut agglutinin (PNA)-binding glycoconjugates. Cone photoreceptor cell outer segment degener- ation and markedly decreased chondroitin 6-sulfate immunoreactivity distinguished the middle stage. During the late stage, there was a near complete absence of both immunoreactive chondroitin 6-sulfate and PNA-binding glycoconjugates in the IPM. Shallow retinal detachments that appeared funduscopi- cally as patches of retinal whitening frequently were observed after moderate durations of xyloside exposure; these progressed peripherally with continued xyloside exposure. Histologically, the areas of retinal whitening corresponded to regions in which cone matrix sheaths were split transversely (ie, in a plane perpendicular to the longitudinal axes of the photoreceptor cell outer segments) or were sepa- rated completely from cone outer segments. Similar effects were not observed in control eyes. These results suggest that adhesion between the neural retina and retinal pigmented epithelium may be dependent, in part, on continuous synthesis of cone matrix sheath-associated proteoglycans and, poten- tially, other IPM proteoglycans. In addition, these proteoglycans appear to be necessary for the mainte- nance of cone photoreceptor cell outer segment integrity. Invest Ophthalmol Vis Sci 33:364-376,1992 The molecules that occupy the retinal interphoto- lia, the neural retina, and the retinal pigment epithe- receptor space, collectively termed the interphoto- lium (RPE). There is a consensus that these molecules receptor matrix (IPM), are situated uniquely between mediate interactions between (1) photoreceptor, the apices of two neuroectodermally derived epithe- RPE, and Muller cells and (2) the choroidal vascula- ture.1"3 However, the specific functional roles of most of these constituents, especially those of recently de- 1 3 From the Department of Ophthalmology, Bethesda Eye Insti- scribed proteoglycans, are not understood fully. " tute, St. Louis, University School of Medicine, St. Louis, Missouri. Evidence for compartmentalization of some IPM- Supported by National Institutes of Health (Bethesda, Maryland) associated molecules has been provided by lectin his- grant EY06463 (GSH), by Research to Prevent Blindness, Inc. tochemical and antibody immunohistochemical stud- ([New York, New York] an Olga Keith Wiess Scholar Award to GSH and an unrestricted grant to the BEI), and by the Bethesda Eye ies. Photoreceptor cell-specific domains of IPM glyco- Institute Resident Research Fund (HSL). conjugates have been identified based on selective 4 6 Presented in part at the annual meeting of the Association for binding of peanut agglutinin (PNA) " and wheat Research in Vision and Ophthalmology, Sarasota, Florida, and the germ agglutinin.7"9 The PNA-binding molecules are Ninth International Congress of Eye Research, Helsinki, Finland. located specifically in IPM domains termed "cone Submitted for publication: July 5, 1990; accepted September 5, matrix sheaths," that surround cone, but not rod, 1991. photoreceptor outer segments and ellipsoids in Reprint requests: Dr. Gregory S. Hageman, PhD, Bethesda Eye 3 10 Institute, 3655 Vista Avenue, St. Louis, MO 63110. various species, including humans. " Immunohisto- 364 Downloaded from iovs.arvojournals.org on 09/30/2021 No. 2 XYLOSIDE-INDUCED PERTURBATION OF IPM / Lozorus and Hageman 065 chemical and biochemical investigations have shown Materials and Methods that chondroitin 6-sulfate glycosaminoglycan is also a specific structural component of cone matrix Animals sheaths34" and that it is part of a larger, membrane- We used 11 female Yucatan micropigs (age range, associated proteoglycan or proteoglycan aggregate 4-6 months old; weight range, 9.6-14.2 kg) obtained that cannot be extracted by aqueous buffers.3'4'12'13 from Charles River (Wilmington, MA) in these stud- Based on our understanding of the roles of proteo- ies. The micropigs were maintained on Purina (St. glycans in other tissues and organ systems,14"16 several Louis, MO) Mini-Pig Grower or Mini-Pig Breeder putative roles, including retinal adhesion, can be pos- chows. Yucatan micropigs were used in these studies tulated for the proteoglycans associated with cone because (1) the porcine retina contains numerous matrix sheaths. Recent observations from a number cone photoreceptor cells23 with well-defined cone ma- of laboratories suggest a potential role for cone matrix trix sheaths;6 (2) their retinal vasculature24 and vitre- sheaths in retina-RPE adhesion.317"20 For example, ous body25 are similar to those of humans; and (3) a in experiments in which porcine, monkey, or human library of probes specific for various porcine retinal retinas were peeled away gently from the RPE, cone cell types and both cone matrix sheath- and noncone matrix sheaths remain attached to the RPE and be- matrix sheath-specific IPM components is available. come elongated up to four to six times their normal Although these animals do not have a true macula or length before (1) splitting along their transverse fovea, there is an area centralis with decreased vascu- axes,313 (2) detaching the RPE from Bruch's mem- larity that is populated densely by cone photorecep- brane,3' '3 or (3) tearing apical RPE microvilli.'9 Collec- tors.23 The animals were treated in conformity with tively, these studies suggest that the strength of attach- the ARVO Resolution on the Use of Animals in Re- ment of cone matrix sheaths to the RPE and cone search, the National Institutes of Health (Bethesda, photoreceptor cell outer segments is greater than that MD) "Guide for the Care and Use of Laboratory Ani- of the cone matrix sheaths themselves or than that mals" (NIH publication 86-23), and the guidelines set between the RPE and Bruch's membrane. Other stud- forth by the St. Louis University Department of Com- ies show that retinal adhesion is weakened signifi- parative Medicine. cantly without concomitant loss of photoreceptor function after intravitreal or subretinal injections of Reagents protease-free chondroitinase ABC.20 Taken together, these studies suggest that cone matrix sheaths may We obtained p-nitrophenyl-/3-D-xylopyranoside provide a molecular bond of considerable strength from Research Products International (Mt. Prospect, that participates in the adhesion between the RPE and IL) and Koch-Light (Suffolk, England). Antibodies di- neural retina. rected against chondroitin-6-sulfate (ADi-6S) were obtained from ICN (Lisle, IL). A cone photoreceptor To investigate further the potential functional role cell-specific monoclonal antibody, designated CSA-1, of cone matrix sheath-associated chondroitin 6-sul- has been generated and characterized.26 Fluorescein- fate proteoglycan in retinal adhesion, studies directed conjugated goat anti-mouse immunoglobulins G and toward perturbing the synthesis of this molecule by M antibodies were purchased from Sigma (St. Louis, administering intravitreal injections of p-nitro- MO) and Cappel (Durham, NC). Protease-free chon- phenol-/3-D-xylopyranoside (xyloside) to Yucatan droitinase ABC (Proteus vulgaris) was obtained from micropigs were conducted. Xyloside inhibits chon- Seikagaku America (St. Petersburg, FL). The fluores- droitin sulfate proteoglycan (also dermatan sulfate cein-conjugated PNA lectin, Arachis hypogaea agglu- and, to some extent, heparan sulfate proteoglycan, nei- tinin, was obtained from Vector (Burlingame, CA) ther of which is present in the IPM) synthesis by com- and EY (San Mateo, CA). Dimethyl sulfoxide peting with the xylosylated proteoglycan core protein (DMSO), globulin-free bovine serum albumin (BSA), as an acceptor for the first galactosyltranferase reac- MgCl , and CaCl were purchased from Sigma. Acryl- tion.21-22 As such, the proteoglycans
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