Protective effects of hypercapnic acidosis on Ischemia-reperfusion-induced retinal injury

Abstract

Ischemia-reperfusion (I/R) injury is associated with numerous retinal diseases, such as diabetic retinopathy, acute glaucoma, and other vascular retinopathies. Hypercapnic acidosis (HCA) has a protective effect on lung, myocardial, and central nervous system ischemic injury models. However, no study has evaluated its protective effects in an experimental retinal I/R injury model. In this study, retinal I/R injury was induced in Sprague Dawley rats by elevating the intraocular pressure to 110 mmHg for 60 minutes. HCA was induced before and after the injury. After 24 hours, the terminal dUTP nick end labeling assay was performed. Moreover, the ratios of cleaved caspase-3/total caspase-3, phosphorylated IκB/IκB, and phosphorylated p38 were measured through Western blotting. After 7 days, the rats' aqueous humor was analyzed. In addition, electroretinography and retinal thickness measurement were performed in the rats. Moreover, the retinal neural cell line RGC-5 was exposed to 500 μM H2O2 for 24 hours to induce a sustained oxidative stress in vitro. The effects of HCA were evaluated by comparing oxidative stress, MAPK signals, NF-κB signals, survival rates, and apoptosis rates in the RGC-5 cells before and after H2O2 exposure. We further investigated whether the potent I/R-protective heat shock protein (HSP) 32 contribute to protective effects of HCA. Our results indicated that HCA has protective effects against retinal I/R injury both in vivo and in vitro, at multiple levels, including antiapoptotic, anti-inflammatory, antioxidative, and functional retinal cell protection. Further research clarifying the role of HCA in retinal I/R injury prevention and treatment is warranted.

Fig 1

Numbers of cells (A) and protein concentrations (B) in the aqueous humor. The results are the mean ± SEM of three independent experiments. ***P < 0.001 versus control group, **P < 0.01 versus control group; ###P < 0.001, ##P < 0.01, #P < 0.05 versus IR group.

Fig 2

(A) Hematoxylin and eosin staining of the retinas. Representative photomicrographs showing the histologic appearance of retinas in the five groups. Significant decrease in the mean retinal thickness is visible in the IR group. (B) Representative ERG waveforms from the five groups. I/R injury significantly reduced the the α and β wave amplitudes. (C)Whole retinas were significantly thicker in the IR-HCA group than in the IR group. (D) ERG revealed significant recovery in the difference of cursor of α and β waves in the IR-HCA group. The results are mean ± SEM of three independent experiments. ***P < 0.001, *P < 0.05 versus control group; ###P < 0.001, #P < 0.05 versus IR group.

Fig 3

(A) Colocalization in TUNEL (apoptosis, green) and 4′,6-diamidino-2-phenylindole (DAPI; nuclei, blue).(B)Number of TUNEL-positive cells significantly decreased in both IR-HCA and HCA-IR groups. (C and D) Effects of HCA on the expression of the caspase-3 in the five groups. The results are mean ± SEM of three independent experiments. ***P < 0.001, **P < 0.01 versus control group; ###P < 0.001, ##P < 0.01, #P < 0.05 versus IR group.

Fig 4

(A) Annexin V/PI flow cytometry of RGC-5cells in the five groups. The upper right quadrants (Annexin V+/PI+) represent apoptotic/necrotic cells, and the lower right quadrants (Annexin V+/PI−) represent early apoptotic cells.(B and C) Ratio of cleaved caspase-3/total caspase-3 was markedly increased by H₂O₂ compared with the control and post groups. The results are mean ± SEM of three independent experiments. ***P < 0.001 versus control group; ###P < 0.001 versus H₂O₂ group.

Fig 5

(A) Representative Western blots for the effects of HCA induction on the in vitro IκB expression in the five groups. (B) Effects of HCA induction on the IκB (pIκB/IκB ratio) in the five groups in vitro. (C and D) Activation of P38MAPK pathways in the five groups in vitro. The results are mean ± SEM of three independent experiments. ***P < 0.001 versus control group; ###P < 0.001, ## P<0.01 versus H₂O₂ group. (E) Representative Western blots for the effects of HCA induction on the expression of IκB in the five groups in vivo. (F) Effects of HCA on the IκB (pIκB/IκB ratio) in the five groups in vivo. (G and H) Activation of p38 MAPK in the five groups in vivo. The results are mean ± SEM of three independent experiments. ***P < 0.001,**P < 0.01,*P < 0.05 versus control group; ##P < 0.01, #P < 0.05 versus IR group.

Fig 6. HSP32 expression was detected by measure protein activity.

The results are mean ± SEM of three independent experiments. **P < 0.01 versus control group; ##P < 0.01 versus IR group.