Weight measurements of experimental and control eyes showed that the reduction in eye weight in the experimental eyes was paralleled by an even greater reduction in the weight of their VB gels; the weight of the retina was not affected
(Fig. 7A) . The reduced vitreous gel indicated an interference of Hep with one of the VB ECM components. To investigate this further, the binding of VB proteins to heparin was examined. Incubation of VB with heparin Sepharose showed that collagen II and collagen IX were retained, and both proteins were released from the heparin Sepharose with only 1 M salt
(Figs. 7B 7C) . The binding of Hep to collagen II and IX suggests that intravitreally injected Hep could interfere with collagen II fibrillogenesis or (and) binding of collagen IX to collagen II fibrils. Ultrastructural analysis showed that the collagen fibrils from the heparin-injected eyes
(Fig. 7E)had less of the electron-dense ECM material associated with it than collagen II fibrils from the control eyes (
Fig. 7D , arrows). Immunostaining confirmed that the collagen II fibrils from experimental eyes
(Fig. 7G)had less collagen IX immunoreactivity associated with it than collagen II fibrils from the control eyes (
Fig. 7F , arrows). Finally, Western blots showed that the concentration and banding pattern of collagen II and IX in total VB samples from Hep-treated eyes (
Fig. 7H , lanes 2 for coll2 and coll9) and control eyes (
Fig. 7H , lanes 1 for coll2 and coll9) were similar. The insoluble vitreous gel from heparin-treated eyes (
Fig. 7H , lane 4 for col9), however, contained less collagen IX than the VB gel from the control eyes (
Fig. 7H , lane 3 for coll9;
n = 4). In contrast, there was more of the fully processed, low molecular-weight collagen II band
34 35 (
Fig. 7H , star, lane 4 of coll2) in the heparin-treated eyes (
Fig. 7H , lane 4 for col2;
n = 4) compared with VB gels from the control eyes (
Fig. 7H , lane 3 for col2). Thus, intraocular Hep affected VB gel formation in two ways, by decreasing the binding of collagen IX to collagen II fibrils and by promoting the processing of collagen II from the proform to the fully processed trimer.