Sulfated proteoglycans in Bruch's membrane of the human eye: localization and characterization using cupromeronic...

The distribution of sulfated proteoglycans in Bruch's membrane of the human eye was evaluated histochemically using Cupromeronic Blue in combination with specific enzyme digestions and nitrous acid treatment. Five distinct categories of filament-shaped profiles were present following staining with this dye. Type 1 (90 +/- 13 nm long and 7 +/- 1 nm in diameter) (mean +/- S.D.) and type 2 (43 +/- 7 nm long and 5 +/- 1 nm in diameter) filaments were associated with collagen fibrils in the inner and outer collagenous zones. Type 3 profiles (70 +/- 18 nm long and 8 +/- 1 nm in diameter) were present in two locations--along the cortical border of the central elastic zone and within the basal infoldings of the pigment epithelium. Type 4 (60 +/- 11 nm long and 6 +/- 1 nm in diameter) and type 5 (200 +/- 100 nm long and 100 +/- 50 nm in diameter) filaments were associated with the basal laminae of the retinal pigment epithelium and choriocapillaris. Chondroitinase AC treatment eliminated the staining of type 1 filaments. Chondroitinase ABC treatment eliminated the staining of both type 1 and type 2 filaments. Nitrous acid eliminated the staining of type 4 and type 5 filaments. Incubations with keratanase or hyaluronidase did not alter the staining of any filament type. Type 3 filaments were resistant to all enzyme digestions and nitrous acid treatment. These results are consistent with an interpretation that Bruch's membrane contains chondroitin sulfate, dermatan sulfate and heparan sulfate-type proteoglycans. Proteoglycans containing chondroitin sulfate (type 1) and dermatan sulfate (type 2) are associated uniquely with collagen fibrils. Heparan sulfate type proteoglycans (types 4 and 5) are associated with the basal lamina of the pigment epithelium and choriocapillaris. The identity of type 3 profiles, which were resistant to all enzyme and nitrous acid digestions employed, could not be established at this time.