The Roles Of Altered Expression Of NIDD/nNOS And Retinal NNOS Inhibition By L-NAME In Glaucoma Model Of DBA/2J Mice

Background Glaucoma is a serious irreversible blinding eye disease featured in progressive optic nerve degeneration. The disease is the second leading cause of blindness in the world with a variety of pathological processes. Intraocular pressure (IOP) is not always the cause of glaucoma. Even if the IOP is at normal levels, some patients still develop glaucomatous visual impairment. Retinal ganglion cells (RGCs) death is the final common pathway of optic nerve injury, among which the apoptosis of RGCs plays an important role in the pathogenesis of glaucoma. The NIDD gene, nNOS-interacting DHHC domain-containing protein with dendritic mRNA, codes a protein that up-regulates nNOS enzyme activity by the interaction with the PDZ (postsynaptic density protein95/discsslarge/zon occlusens-1) domain of nNOS. The correlation of nNOS and glia activation has been reported. Glial cell activation, especially Miiller cells, may be an important factor contributing to RGCs death in glaucoma.Objective The present study is designed to establish the clinical pathology diagnostic criteria of DBA/2J mice based on a series of eye phenotypic changes and to investigate the NIDD/nNOS expression changes and their correlation with glaucomatous phenotypes.Methods The female DBA/2J mice aged from3to15-month old (n=100) were examined with slit-lamp biomicroscopy, fundus photography, IOP measurement, hematoxylin-eosin staining of the optic nerve head and optic nerve axon counts. According to these examinations, DBA/2J mice were classified into normal group (Nor), pre-glaucoma group (pre-G), glaucoma group (G), late-glaucoma group (late-G) and terminal-glaucoma group (ter-G). Quantitative RT-PCR and Western blot analysis were used to analyze mRNA and protein expression of nNOS and NIDD in the ocular tissue. The spatial distribution of them was evaluated by immunohistochemistry. Immunofluorescence was performed to observe the colocalization of nNOS and NIDD and the association of NIDD with Muller cells.Results First, with the progression of glaucoma, pigment dispersion, iris strorna atrophy, transillumination defects and pupil deformation were found in DBA/2J mice. IOP gradually increased and reached the highest in G-group, which then decreased to normal base line. The abnormality of optic nerve cupping and optic nerve axons were found at stage G. The optic nerve glaucomatous damage was developed with the progress of glaucoma. Second, the mRNA and protein expression of nNOS reached the peak at G stage. The protein of NIDD underwent the similar change while the mRNA of NIDD significantly increased at pre-G stage. Third, the expression of NIDD was physically co-existed with nNOS in Muller cells. Forth, administration of NOS inhibitor L-NAME by i.p. prevented RGCs from apoptosis as shown the increase of Brn-3a (RGC marker) expression, which accompanied with decreased expression of NIDD.Conclusions First, we combined with clinical and pathological examinations to establish the clinical diagnostic criteria of glaucoma in DBA/2J mice. Second, NIDD possible activated Muller cells through nNOS involved in the pathological process of glaucomatous RGCs damage. Third, mice injected with nitric oxide synthase inhibitor L-NAME in vivo can effectively protect RGCs survival. Forth, NIDD/nNOS may be involved in the pathological process of glaucomatous damage.