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The mRNA and protein expression levels of CXCL10 and CXCR3 were significantly increased after IOP-induced retinal ischemia

The mRNA and protein expression levels of CXCL10 and CXCR3 were significantly increased after IOP-induced retinal ischemia. deleting CXCR3 gene significantly attenuated ischemic injury-induced upregulation of inflammatory molecules (interleukin-1and E-selectin), inhibited the recruitment of microglia/monocyte to the superficial retina, reduced peroxynitrite formation, and prevented the loss of neurons within the ganglion cell layer. In contrast, intravitreal delivery of CXCL10 increased leukocyte recruitment and retinal cell apoptosis. Inhibition of endoplasmic reticulum (ER) stress with chemical chaperones partially blocked ischemic injury-induced CXCL10 upregulation, whereas induction of ER stress with tunicamycin enhanced CXCL10 expression in ICAM4 retina and primary retinal ganglion cells. Interestingly, deleting CXCR3 attenuated ER stress-induced retinal cell death. In conclusion, these results indicate that ER stress-medicated activation of CXCL10/CXCR3 pathway has an important role in retinal inflammation and neuronal injury after high IOP-induced ischemia. Acute glaucoma is the major form of glaucoma in East Asia where it is a leading cause of irreversible blindness.1 In Western countries, it is less common, but it still has much higher rate to induce vision impairment and blindness than open-angle glaucoma.2 Acute glaucoma is a medical emergent condition when intraocular pressure (IOP) is suddenly increased because of blocked drainage canals.1, 2 Prompt treatment is needed to avoid irreversible glaucomatous optic nerve damage.1 Nevertheless, in a substantial portion of patients, acute glaucoma continues progressing to blindness in spite of intensive medical treatment.3 A rapid rise in IOP that exceeds retinal perfusion pressure is known to cause retinal ischemia and induce retinal neuronal cell death.2, 4 However, the mechanisms by which elevated IOP induces retinal neuronal injury in acute glaucoma are largely unknown. Inflammation is the body’s defense system against pathogens,5 whereas excessive or uncontrolled inflammation induces tissue injury and results in diseases. In the central nervous system (CNS), inflammation has been recognized as a key player in many ZM 449829 neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease.6 Inflammation is involved in the development of glaucoma given that the levels of inflammatory cytokines (e.g., TNF-hybridization of CXCL10 mRNA. Retinal frozen sections from control and IR-performed mice at 6?h after IR were hybridized with a probe against mouse CXCL10 and detected with RNAscope Fluorescent Multiplex Kit. Green fluorescent signal reflects CXCL10 mRNA expression and DAPI (blue) stains nuclei. Arrows indicate CXCL10-expressed retinal ganglion cells. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer. (d and e) The mRNA levels of CXCL4 and CXCR3 were determined by qPCR. (f) Retinal frozen sections from control and IR-performed mice at 6?h after IR were incubated with an antibody against CXCR3. ZM 449829 Fluorescent signal (red) reflects CXCR3 staining. *and E-selectin mRNA expression in retina at 24?h after IR. (f) Nitrotyrosine level in retina was analyzed by western blot at 24?h after IR. Actin was used as an internal ZM 449829 loading control. *and E-selectin in WT retinas after ischemic injury, which were markedly blocked by deleting CXCR3 (Figures 2d and e). Together, these data indicate that this activation of CXCR3 pathway mediates microglia/monocyte recruitment and activation and retinal inflammatory reactions after IOP-induced retinal ischemia. CXCR3 is usually critically involved in oxidative and nitrosative stress after ischemic injury During inflammation, local retinal cells and/or recruited leukocytes produce superoxide and nitric oxide, which can not only kill pathogens but also induce tissue injury. To determine whether the activation of CXCR3 is usually involved in oxidative and nitrosative stress after retinal ischemia, we examined the formation of peroxynitrite in retinal lysates. Peroxynitrite is usually rapidly formed through the reaction of superoxide and nitric oxide and is an indicator for oxidative and nitrosative stress. Western blot analysis of nitrotyrosine, a marker of peroxynitrite, revealed a prominent increase of peroxynitrite formation in WT retina after ischemic injury. However, this increase was blocked by CXCR3 deletion (Physique 2f). This result suggests that CXCR3 pathway is usually involved in retinal oxidative and nitrosative stress after IOP-induced retinal ischemia. CXCR3 pathway has a predominant role in IOP-induced retinal neuronal cell damage The loss of retinal neurons in the GCL is usually a hallmark of glaucoma,2, 4, 27 and both inflammation and oxidative stress can cause neuronal cell death; therefore, we investigated whether blocking CXCR3 pathway would safeguard retinal neuronal cells from IOP-induced cell death. At 24?h after retinal.