Glial Reactivity In Early Diabetic Retinopathy And Its Effect On Blood-Retinal Barrier

1. GFAP and Occludin changes in STZ-induced diabetic ratsPurpose To observe the animal model of diabetic retinopathy induced by STZ in rat and to investigate how diabetes alters vascular endothelial cell tight junction protein and glial cell morphology at the blood-retinal barrier( BRB ).Methods Diabetes was induced in rats with a single intraperitoneal injection of streptozotocin( STZ ). 3 days later, rats with blood sugar over 11.0mmol/l are induced successfully. The distribution of the glial marker, glial fibrillary acidic protein (GFAP), and the endothelial cell tight junction protein occludin were explored by immunofluorescence histochemistry and Westernt blot in flatmounted retinas of STZ-diabetic and age-matched control rats.Results GFAP immunoreactivity was limited to astrocytes in control retinas. Two weeks of STZ-diabetes reduced GFAP immunoreactivity in astrocytes and increased GFAP immunoreactivity in small groups of Müller cells. After 3 months of STZ-induced diabetes, all Müller cells had intense GFAP immunoreactivity, whereas thera was virtually none in the astrocytes.Occludin immunoreactivity in normal rats was greatest in the deeper capillary bed of retina and arterioles of the inner retina but much less initense in the postcapillary venules. Diabetes reduced occludin immunoreactivity in the capillaries and induced redistribution from continuous cell border to interrupted punctate immunoreactivity in the arterioles.Conclusion Diabetes alters GFAP expression in retinal glial cells, accompanied by reduction and redistribution fo occludin in endothelial cells. These changes are consistent with the concept that altered glial-endothelial cell interactions at the BRB contribute to diabetic retinopathy.2.Microvascular Endothelial Cell and Muller Cell Culture and Morphological Observation.Purpose To obtain purer Muller cells and retinal vascular endothelial cells( BREC) for the physiological and pathological study of BBB. Method We used the filtration and enzyme digestion techniques to isolate BREC from bo vein retina and Muller cell from rat retina. The isolated cells were cultured and observed morphologically. The cells were seen under electronmicroscope, Immunocytochemistry demonstrated that more than 95% of the cultured cells were positive to Von Willebrand factor and GFAP respectively. Results The cultured cells attached completely after 24-48h in vitro, reached confluence in 8-10 days with the tiypical appearance and were passed on time. Immuunohistochemically these cells were stained positively by GFAP and Von Willebrand factor antibody respectively.Conclusion Conditioned primary culture of retinal Miiller cells and BREC is a reliable and quick method.3. Advanced glycosylation end products ( AGEs) promoted the growth of retinalMüller cells in vitro.Purpose To obsrve the effect of AGEs on the growth of retinal glial cells invitro.Methods The Miiller cells were exposted to different concentrations of AGEs,the cells growth characteristics were measured with MTT assay and VEGF inmedium concentration was assayde by ELISA.Results It was demonstrated that AGEs with high concentration promoted thegrowth of Miiller cells and this effect was dosage dependent.Conclusin Interaction of Miiller cells and AGEs is related to the formationand development of DR.4.Retinal Miiller Cells Contioned Medium affected Occludin expression in BRECPurose To observe the effect of retinal retinal Miiller cells conditioned medium (MCM )on the Occludin expression of BREC.Methods BREC were exposed to MCM of normal Miiller cells andAGEs-Müller cells respectively.The expression of Occludin in BREC wasmeasured with ELISA.Results Normal MCM can accerlarate the Occlludin expression in BRECwhile abnormal MCM affected by AGEs restrain the expression.Conclusion Miiller cells can affect the function of BRB by modulating theexpression of Occludin in BREC.