MORPHOLOGIC EVIDENCE FOR A PREFERENTIAL STORAGE OF TISSUE-PLASMINOGENACTIVATOR (T-PA) IN PERIVASCULAR AXONS OF THE RAT UVEA

The uveal layer is thought to hold the largest stores of tissue plasmi nogen activator (t-PA) within the eye. However, the uveal cell types t hat contain and could release t-PA to contiguous tissues and fluids ha ve not been clearly identified, In the present study the general distr ibution pattern of t-PA antigen in fresh rat iris and choroid tissue w as determined by immunofluorescence in preliminary light microscopic ( LM) cryosections. Transmission electron microscopic (TEM) immunogold l ocalization was then used to detect specific cellular and subcellular sites of t-PA antigen, The primary antibody was rabbit anti-mouse t-PA IgG. The immunofluorescence in preliminary LM cryosections of both ti ssues was most intense over discrete linear and cross-sectioned struct ures that resembled the contours of axon bundles. This impression was strengthened when silver impregnation highlighted similar structures i n separate sections of the same tissue samples. TEM immunogold labelin g of thin sections then confirmed that the t-PA antigen was confined t o the axoplasm of both myelinated and unmyelinated perivascular nerve fibers in both the iris and choroid, Gold particles were not observed over axonal membranes, myelin sheaths, Schwann cells, retinal pigment epithelium or vascular endothelial cells. Ultrathin TEM cryosections o f the iris showed a localization of some particles over structures tha t resembled tubules and vesicles within the axoplasm, but not over mit ochondria. The axonal location of t-PA was shown by the co-localizatio n of t-PA with an antibody against rat neurofilaments. The typical axo n morphology that enclosed the t-PA particle markers in all TEM sectio ns also indicated an axonal location. Separate TEM sections were proce ssed with conventional fixatives and stains to highlight the typical u veal axon morphology, which also confirmed the identity of perivascula r axons as the sites of t-PA localization. Affinity of the primary ant ibody for rat t-PA was shown by an inhibition ELISA against rat uveal tissue extracts and by the inhibition of t-PA activity in aqueous humo r. An amidolytic assay was used to quantify t-PA activity. Possible ex planations for the preferential immunolocalization of t-PA antigen to the axoplasm of uveal nerve terminals and the need for additional func tional studies to confirm a putative neural t-PA synthesis are discuss ed. (C) 1997 Academic Press Limited.