Inhibition of Tissue Plasminogen Activator Attenuates Non–NMDA Receptor–Mediated Excitotoxic Retinal Degeneration

Abstract

Abstract: : Purpose: Increased levels of extracellular L–glutamate have been suggested to play a role in retinal damage in a number of blinding retinal diseases including glaucoma and diabetic retinopathy. Although glutamate can over–stimulate its receptors and cause retinal damage due to excitotoxicity, the down–stream events that lead to retinal damage are poorly understood. The objective of this study is to determine the role of tissue plasminogen activator (tPA) in excitotoxic retinal damage. Methods: Excitotoxicity was induced by injection of kainic acid (KA), a non–NMDA type receptor agonist, into the vitreous humor in CD–1 mice. tPA activity in retinal protein extracts was determined by zymography and protein levels were determined by western blot analysis. Localization of tPA in the retina was determined by immunohistochemistry. Activation of microglial cells was determined by immunolocalization. Retinal degeneration was assessed by immunostaining of retinal cross sections using antibodies against neurofilament–light (NF–L) and calretinin and retrograde labeling studies. Apoptotic cell death in both tPA knockout mice and their normal littermate was determined by TUNEL staining Results: Injection of KA into the vitreous humor in CD–1 mice led to an up–regulation in tPA activity in the retina and this was associated with activation of zymogen plasminogen to active plasmin. Immunocytochemical evidence indicated that retinal ganglion cells (RGCs), constitutively, synthesize tPA in the retina. Intravitreal injection of KA led to the release of tPA from RGCs into the extracellular space and this was correlated with activation of microglial cells. In addition, release of tPA was associated with significant degeneration of the retina as evidenced by the loss of RGCs and amacrine cells. These phenomena were inhibited when a tPA inhibitor, recombinant plasminogen activator inhibitor (rPAI–1), was injected into the vitreous humor along with KA. Furthermore, compared to normal littermate mice, tPA knockout mice show attenuated retinal degeneration after intravitreal injection of KA. Conclusions:Taken together, the above results suggest that inhibition of tPA activity might protect retina in blinding retinal diseases in which over–stimulation of glutamate receptors has been implicated as a causative factor in retinal damage.

Keywords: excitatory amino acid receptors • ganglion cells • neuroprotection