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Interphotoreceptor matrix in the frog retina
INTERPHOTORECEPTOR MATRIX IN THE FROG RETINA
N. J. Philp and M. H. Bernstein Department of Anatomy, Wayne State University School of Medicine, Detroit, Michigan.
Glycosaminoglycans (e.g., hyaluronate, chondroitin sulfates, heparan sulfate) are high molecular weight anionic polysaccharides. They are common constituents of extracellular matrices although their composition varies depending on the tissue (i). Basic dyes such as Ruthenium Red (2) or Alcian Blue (3) can be used to visualized extracellular glycosaminoglycans both at light and electron microscopic levels. In the present study the nature and distribution of glycosaminoglycans in the interphotoreceptor space of the retina are examined The interphotoreceptor space is a complex extracellular domain extending from the ora serrata to the optic disc and from Verhoeff's membrane of the retinal pigment epithelium to the outer limiting membrane and is invaded by the cellular processes of the MHller cells, the photoreceptors and the apical processes of the pigment epithelium (Fig. i). It was previously demonstrated histochemically (4) that the interphotoreceptor space contains anionic polysaccharides (interphotoreceptor matrix). Biochemical analysis of the matrix isolated from the bovine retinae indicated that the matrix is composed of both sulfated and nonsulfated polysaccharides. Frog (Rana Pipiens) retinae were fixed in 2% glutaraldehyde in 0.i M cacodylate buffer containing 0.5% Ruthenium Red or Aleian Blue and post-fixed in osmium tetroxide. Scanning and transmission electron microscopy of the retinae demonstrated that both Ruthenium Red and Alcian Blue allowed for the retention and visualization of interphotoreceptor matrix as a thick coat covering the surface of the photoreceptors (Figs. 2 and 3). A less extensive retention of matrix was seen in retinae that were fixed in glutaraldehyde alone without the addition of a stain (Fig. i). The susceptibility of the interphotoreceptor matrix to enzymatic digestion was examined with the trypsin, hyaluronidase and neuraminidase. Trypsin (Fig. 4) and similarly hyalurondase (Fig. 5) removed most of the matrix material; neuraminidase (Fig. 6) removed much less material and left a considerable amount of matrix residue. Trypsin is a protease, lacking substrate specificity, which removes cell surface glycoproteins and extracellular glycosaminoglycans. Hyaluronidase is a carbohydrase which cleaves chondroitin sulfates in addition to hyaluronate and is similarly effective in removing extracellular materials. Neuraminidase is a more specific enzyme which cleaves only sialic acid residues. The observations in this study suggest that the IMP found in the frog retinae is composed of anionic polysaccharides that are sensitive to hyluronidase and have protein backbones that are cleaved by trypsin. The partial removal of the IMP by neuraminidase suggests that some of the glycosaminoglycans present in the IMP may be bound to the surface coat glycoproteins through sialic acid residues. References (i) R. U. Margolis and R. K. Margolis, Metabolism and function of glycoproteins and glycosaminoglycans in nervous tissue, Int. J. Biochem. 8, 85 (1977). (2) J. H. Luft, Ruthenium Red and Violet, Anatomical Rec. 171, 347 (1971). (3) O. Behnke and T. Zelander, Preservation of intercellular substances by the cationic dye Alcian Blue in preparative procedures for electron microscopy, J. Ultrastruct. Res. 31, 424 (1970). (4) P. Rohlich, The interphotoreceptor matrix: electron microscopic and histochemical observations on the vertebrate retina, Exptl. Eye Res. i0, 80 (1970). 215
216
N . J . Philp and M. H. Bernstein
i ¸ ] Transmission electron micrograph of frog retina fixed in glutara]dehyde containing Alcian B]ue. Note surface coat (SC) on the photoreceptors (PR) and the interphotoreceptor matrix (IPM). bar = lO0 um
Fig. 1 Control, frog retina fixed in glutaraldehyde without stain. Demonstrates pigment epithelium (PE) and outer limiting membrane (OLH) and lack of matrix covering the photoreceptors (PR). bar = l~m
Fig. 2 Frog retina fixed in glutaraldehyde containing Ruthenium Red. Extensive surface coat (SC) of matrix is seen covering the photoreceptors (PR). bar = l~Jm
Fig.
Fig. 4 Frog retina incubated in trypsin prior to fixation in glutaraldehyde containing Ruthenium Red. The interphotoreceptor matrix has been degraded, bar = l~m
Fig. 5 Frog retina incubated in hyaluronidase prior to fixation in glutaraldehyde containing Ruthenium Red. Interphotoreceptor matrix is degraded. (cf. fig. 4). bar = l~m
Fig. 6 Frog retina incubated in neuraminidase prior to fixation in glutaraldehyde containing Ruthenium Red, Only a partial removal of the matrix is seen. bar = i~
N. J. Philp and M. H. Bernstein Department of Anatomy, Wayne State University School of Medicine, Detroit, Michigan.
Glycosaminoglycans (e.g., hyaluronate, chondroitin sulfates, heparan sulfate) are high molecular weight anionic polysaccharides. They are common constituents of extracellular matrices although their composition varies depending on the tissue (i). Basic dyes such as Ruthenium Red (2) or Alcian Blue (3) can be used to visualized extracellular glycosaminoglycans both at light and electron microscopic levels. In the present study the nature and distribution of glycosaminoglycans in the interphotoreceptor space of the retina are examined The interphotoreceptor space is a complex extracellular domain extending from the ora serrata to the optic disc and from Verhoeff's membrane of the retinal pigment epithelium to the outer limiting membrane and is invaded by the cellular processes of the MHller cells, the photoreceptors and the apical processes of the pigment epithelium (Fig. i). It was previously demonstrated histochemically (4) that the interphotoreceptor space contains anionic polysaccharides (interphotoreceptor matrix). Biochemical analysis of the matrix isolated from the bovine retinae indicated that the matrix is composed of both sulfated and nonsulfated polysaccharides. Frog (Rana Pipiens) retinae were fixed in 2% glutaraldehyde in 0.i M cacodylate buffer containing 0.5% Ruthenium Red or Aleian Blue and post-fixed in osmium tetroxide. Scanning and transmission electron microscopy of the retinae demonstrated that both Ruthenium Red and Alcian Blue allowed for the retention and visualization of interphotoreceptor matrix as a thick coat covering the surface of the photoreceptors (Figs. 2 and 3). A less extensive retention of matrix was seen in retinae that were fixed in glutaraldehyde alone without the addition of a stain (Fig. i). The susceptibility of the interphotoreceptor matrix to enzymatic digestion was examined with the trypsin, hyaluronidase and neuraminidase. Trypsin (Fig. 4) and similarly hyalurondase (Fig. 5) removed most of the matrix material; neuraminidase (Fig. 6) removed much less material and left a considerable amount of matrix residue. Trypsin is a protease, lacking substrate specificity, which removes cell surface glycoproteins and extracellular glycosaminoglycans. Hyaluronidase is a carbohydrase which cleaves chondroitin sulfates in addition to hyaluronate and is similarly effective in removing extracellular materials. Neuraminidase is a more specific enzyme which cleaves only sialic acid residues. The observations in this study suggest that the IMP found in the frog retinae is composed of anionic polysaccharides that are sensitive to hyluronidase and have protein backbones that are cleaved by trypsin. The partial removal of the IMP by neuraminidase suggests that some of the glycosaminoglycans present in the IMP may be bound to the surface coat glycoproteins through sialic acid residues. References (i) R. U. Margolis and R. K. Margolis, Metabolism and function of glycoproteins and glycosaminoglycans in nervous tissue, Int. J. Biochem. 8, 85 (1977). (2) J. H. Luft, Ruthenium Red and Violet, Anatomical Rec. 171, 347 (1971). (3) O. Behnke and T. Zelander, Preservation of intercellular substances by the cationic dye Alcian Blue in preparative procedures for electron microscopy, J. Ultrastruct. Res. 31, 424 (1970). (4) P. Rohlich, The interphotoreceptor matrix: electron microscopic and histochemical observations on the vertebrate retina, Exptl. Eye Res. i0, 80 (1970). 215
216
N . J . Philp and M. H. Bernstein
i ¸ ] Transmission electron micrograph of frog retina fixed in glutara]dehyde containing Alcian B]ue. Note surface coat (SC) on the photoreceptors (PR) and the interphotoreceptor matrix (IPM). bar = lO0 um
Fig. 1 Control, frog retina fixed in glutaraldehyde without stain. Demonstrates pigment epithelium (PE) and outer limiting membrane (OLH) and lack of matrix covering the photoreceptors (PR). bar = l~m
Fig. 2 Frog retina fixed in glutaraldehyde containing Ruthenium Red. Extensive surface coat (SC) of matrix is seen covering the photoreceptors (PR). bar = l~Jm
Fig.
Fig. 4 Frog retina incubated in trypsin prior to fixation in glutaraldehyde containing Ruthenium Red. The interphotoreceptor matrix has been degraded, bar = l~m
Fig. 5 Frog retina incubated in hyaluronidase prior to fixation in glutaraldehyde containing Ruthenium Red. Interphotoreceptor matrix is degraded. (cf. fig. 4). bar = l~m
Fig. 6 Frog retina incubated in neuraminidase prior to fixation in glutaraldehyde containing Ruthenium Red, Only a partial removal of the matrix is seen. bar = i~