青光眼 2879 PO0541
Cellular mechanisms associated with acute IOP elevation induced apoptosis of adult rat retinal ganglion cells
Yao Huang1,2, Ning Li Wang2, Dennis S.C. Lam1, Chi Pui Pang1, Qi Cui1
Department of Ophthalmology & Visual Sciences, The Chinese University of HongKong, HongKong, China (DOVS, CUHK, 3/F, HongKong Eye Hospital, 147K, Argyle street, Kowloon, HongKong)
Purpose: High intraocular pressure(IOP) is the most important factor to cause apoptosis of the retinal ganglion cells(RGCs) in glaucoma. JAK/STAT3, PI3K/Akt and CK2 signal transduction pathways can facilitate cell survival in vitro. Out purpose is to investigate the involvement of the above three pathways in acute IOP elevation induced RGC apoptosis in vivo.
Methods: Sprague-Dawley Rats were subjected to acute IOP elevation by anterior chamber infusion for 2h. JAK inhibitor I (an inhibitor of JAK, 2mM, 3μL) , Wortmannin (an inhibitor of PI3K, 2mM, 3μL) and DMAT(an inhibitor of CK2, 1mM, 3μL), or DMSO(control solution) was injected into the vitreous cavity 3d later and repeated on 9 and 15d. Assayed 3w after infusion. To assess the RGC death quantitatively, we labeled RGCs retrogradely by putting a piece of gel foam soaked in fluorogold behind the proximal stump of the transected optic nerve and subsequently counting the fluorescently labeled RGCs in retina wholemounts. Thereafter, ED-1 immunostaining was performed to show the activated macrophages. Phosphorylation of downstream product of the three pathways STAT3, Akt and CK2 was assessed by immunoblot of whole retinal lysates. Data were analyzed using ANOVA and student’s t-test, with P <0.05 considered significant.
Results: Anterior chamber infusion can induce significant RGC death. The mean RGC density of SD rat was 2148±180 cells/mm2. Within 3w after anterior chamber infusion, it decreased to 1277±231cell/mm2. JAK inhibitor I, and Wortmannin decreased the number of surviving RGCs, while injection of DMAT increased the surviving RGCs. Whereas injection of DMSO did not significantly influence the survival of RGCs, injection of JAK inhibitor I led the mean RGC density decrease to 681±314 cell/mm2(P<0.05), injection of Wortmannin led the RGC density decrease to 132±34 cell/mm2 (P<0.01), while injection of DMAT led the RGC density increase to 2074±106 cell/mm2 (P<0.01). The results of ED-1 immunostaining showed that with the decreasing of RGCs, the activated macrophages increased accordingly. Injection of JAK-1 inhibitor led the activated macrophages increased from 80±19 cell/mm2 to 222±60 cell/mm2 (P<0.01), and the injection of Wortmannin increased the macrophages to 861±82 cell/mm2 (P<0.01), while after the injection of DMAT the activated macrophages decreased to 12±2 cell/mm2 (P<0.01). Results of Westernblot showed that the above three inhibitors can inhibit the phosphorylation of the three kinases effectively.
Conclusions: JAK/STAT3, PI3K/Akt and CK2 pathways are involved in signal transduction in retina after anterior chamber infusion. These inhibitor studies suggest that the previous two signal transduction pathways play anti-apoptotic roles in RGC death induced by acute IOP elevation, while the third pathway play pro-apoptotic role. These three pathways could be potentially useful for the treatment of optic nerve damage and neurodegenerative diseases that affect RGCs, such as glaucoma.
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