Saito Yuta, Geisen Pete, Uppal Abhineet, Hartnett M Elizabeth
Department of Ophthalmology, University of North Carolina, Chapel Hill, NC 27599-7041, USA.
Mol Vis. 2007 Jun 12;13:840-53.
To study the mechanisms of action of the antioxidants, n-acetylcysteine (NAC), and the nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase oxidase inhibitor, apocynin, on intravitreous neovascularization (IVNV), and retinal avascularity in a rat model of retinopathy of prematurity (ROP).
Newborn rats exposed to oxygen-induced retinopathy underwent intraperitoneal (IP) injections of NAC (150 mg/kg) at post-natal day (p)2, p6, p10 (early NAC-treated), or p12 through p17 (late NAC-treated), apocynin (10 mg/kg) from p12 through p17, or phosphate buffered saline (PBS; controls). Lipid hydroperoxide (LHP) was measured in early NAC-treated oxygen-induced retinopathy (OIR) at p7, p14 and p18. Pups were placed in room air at p14. At p18, retinal flat mounts were scored for IVNV and avascular/total retinal area, or retinas were assayed for cleaved caspase-3 and vascular endothelial growth factor (VEGF) protein. In non-injected OIR pups, retinas were assayed for gp91(phox). Cryosections were stained with isolectin B4, cleaved caspase-3, CD68, CD31, gp91(phox), neuron-glial antigen 2 (NG-2), or anti-glial fibrillary acidic protein (GFAP) and visualized with confocal microscopy.
LHP increased over time in retinas from OIR exposed pups in association with IVNV. Early NAC-treated retinas had significantly reduced LHP compared to PBS-control at p18 (p<0.012). However, neither early nor late treatment with NAC had an effect on IVNV or retinal avascularity. Although apocynin had no effect on IVNV, it reduced both avascular retina (p=0.017) and retinal cleaved caspase-3 determined by western blot (p=0.021). In cryosections from OIR eyes, cleaved caspase-3 positive cells co-labeled with some lectin-stained vessels, NG2 labeled cells, and with GFAP positive cells in the inner nuclear layer. We found that the intravascular expression of gp91(phox) co-localized mostly with CD31 and some CD68 positive cells.
Our results do not support the antioxidant properties of NAC as effective in reducing IVNV or avascular retina in the 50/10 OIR rat model. Apocynin reduced avascularity and apoptosis in the OIR model perhaps through pathways triggered by ROS generation but upstream from LHP production. Further study and consideration may be given to apocynin or NAD(P)H oxidase inhibitors as adjunctive therapy for ROP to reduce the avascular retina.
研究抗氧化剂N - 乙酰半胱氨酸（NAC）和烟酰胺腺嘌呤二核苷酸磷酸（NAPDH）氧化酶抑制剂阿朴吗啡对早产儿视网膜病变（ROP）大鼠模型玻璃体内新生血管形成（IVNV）和视网膜无血管化的作用机制。
暴露于氧诱导性视网膜病变的新生大鼠在出生后第（p）2天、p6天、p10天（早期NAC治疗组）或p12至p17天（晚期NAC治疗组）腹腔注射NAC（150 mg/kg），从p12至p17天注射阿朴吗啡（10 mg/kg），或注射磷酸盐缓冲盐水（PBS；对照组）。在早期NAC治疗的氧诱导性视网膜病变（OIR）模型中，于p7、p14和p18测量脂质过氧化氢（LHP）。幼崽在p14时置于空气中。在p18时，对视网膜平铺片进行IVNV和无血管/总视网膜面积评分，或检测视网膜中裂解的半胱天冬酶 - 3和血管内皮生长因子（VEGF）蛋白。在未注射的OIR幼崽中，检测视网膜中的gp91(phox)。冰冻切片用异凝集素B4、裂解的半胱天冬酶 - 3、CD68、CD31、gp91(phox)、神经胶质抗原2（NG - 2）或抗胶质纤维酸性蛋白（GFAP）染色，并用共聚焦显微镜观察。
与IVNV相关，OIR暴露幼崽视网膜中的LHP随时间增加。与p18时的PBS对照组相比，早期NAC治疗的视网膜LHP显著降低（p<０.０１２）。然而，NAC的早期和晚期治疗均对IVNV或视网膜无血管化无影响。尽管阿朴吗啡对IVNV无影响，但它减少了无血管视网膜（p = 0.017）以及通过蛋白质印迹法测定的视网膜裂解半胱天冬酶 - 3（p = 0.021）。在OIR眼的冰冻切片中，裂解的半胱天冬酶 - 3阳性细胞与一些凝集素染色的血管、NG2标记的细胞以及内核层中的GFAP阳性细胞共标记。我们发现gp91(phox)的血管内表达主要与CD31和一些CD68阳性细胞共定位。
我们的结果不支持NAC的抗氧化特性在50/10 OIR大鼠模型中有效减少IVNV或无血管视网膜。阿朴吗啡可能通过ROS产生触发但在LHP产生上游的途径减少了OIR模型中的无血管化和细胞凋亡。对于阿朴吗啡或NAD(P)H氧化酶抑制剂作为ROP辅助治疗以减少无血管视网膜，可能需要进一步研究和考虑。
