Laminin is a basement membrane glycoprotein, widely distributed in body tissues. It has been found on capillary basement membranes in both developing and adult retinal vessels. Oxygen-induced retinopathy was produced in kittens and mice by exposing them to a high oxygen environment during the newborn period. When the animals developed a proliferative retinopathy after a period of survival in room air, they were sacrificed and the eyes enucleated and embedded in paraffin wax. Cross-sections were cut and de-waxed. After preliminary digestion with pepsin, we used a fluorescein isothiocyanate (FlTC)-labelled double antibody technique to identifv laminin-like immunoreactivity (LLlRj. LLlR was found on both intravitreal and intraretinal new vessels in the kitten and mouse, indicating that it is probably secreted by endothelial cells during the formation of pathological new vessels.Key words: Laminin, retinal vessels, angiogenesis, neovascularization, basement membrane, immunohistochemistry. There is an increasing awareness that components of the extracellular matrix may affect endothelial cell behaviour.1.2 Laminin is a noncollagenous glycoprotein (MW = 1 x lo6) found in the extracellular m a t r i~,~.~ and is thought to be involved in the attachment of endothelial, epithelial, and tumour cells to basement membrane^.^-' Endothelial cells in culture secrete l a m i~~i n .~,~ Using immunohistochemical methods on cross-sections, laminin-like immunoreactivity (LLIR) has been reported on the retinal vessels of adult bovine and human Using a whole mount technique, we have previously reported that LLIR is present on the developing retinal vessels of the newborn mouse, on the retinal vessels of the adult rat and mouse, on retracted capillaries, and that LLIR encases pericytes. 12. 13 There is little known concerning the extracellular matrix of pathologically induced new vessel^.'^ Using an immunohistochemical technique on cross-sections of the retina, we have demonstrated the presence of LLIR on induced new vessels in the kitten and mouse models of oxygen-induced retinopathy.
METHODS
Induction of Oxygen-induced RetinopathyA litter of three-day-old kittens was placed in a ChambeF and exposed to an 80% oxygen environment for 96 hours. On day 14 or day 21 of life, when the kittens exhibited a florid proliferative retinopathy,16 they were sacrificed by an overdose of intraperitoneal pentobarbitone. The eyes were enucleated and fixed in 10% buffered formalin. Routine embedding in paraffin wax was performed and 5 pm crosssections were cut. Newborn mice were placed in a perspex chamber measuring 16 x 16 x 22 cm and exposed to a 98% oxygen environment for five days, according to the protocol described by Ashton." On day ten of life, they were sacrificed by halothane overdosage and the eyes were enucleated, fixed in 10% buffered formalin, and embedded in paraffin wax. Cross-sections were cut and processed as for the kitten material.
Immunohistochemical MethodImmunohistochemical localization o f laminin was achieved usin...