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. 2022 Oct;28(10):1509-1518.
doi: 10.1111/cns.13875. Epub 2022 Jun 14.

Normobaric hyperoxia plays a neuroprotective role after cerebral ischemia by maintaining the redox homeostasis and the level of connexin43 in astrocytes

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Normobaric hyperoxia plays a neuroprotective role after cerebral ischemia by maintaining the redox homeostasis and the level of connexin43 in astrocytes

Zhifeng Qi et al. CNS Neurosci Ther. 2022 Oct.

Abstract

Introduction: Acute cerebral ischemia is caused by an insufficient blood supply to brain tissue. Oxygen therapy, which is able to aid diffusion to reach the ischemic region, has been regarded as a possible treatment for cerebral ischemia. Recent animal and pilot clinical studies have reported that normobaric hyperoxia (NBO) showed neuroprotective effects if started soon after the onset of stroke. However, little is known about the role and mechanism of NBO treatment in astrocytes. Connexin43, one of the main gap junction proteins in astrocytes, is extremely sensitive to hypoxia and oxidative stress after cerebral ischemia.

Aims: In the present study, we used sutures to develop an ischemia/reperfusion model in rats to mimic clinical recanalization and investigated the role of connexin43 in NBO-treated stroke rats, as well as the underlying mechanism of NBO therapy.

Results: Normobaric hyperoxia treatment maintained the homeostasis of oxidoreductases: glutathione peroxidase 4 (GPX4) and NADPH oxidase 4 (two important oxidoreductases) and rescued the ischemia/reperfusion-induced downregulation of connexin43 protein in astrocytes. Furthermore, NBO treatment attenuated cerebral ischemia-induced cytochrome c release from mitochondria and was involved in neuroprotective effects by regulating the GPX4 and connexin43 pathway, using Ferrostatin-1 (an activator of GPX4) or Gap27 (an inhibitor of connexin43).

Conclusions: This study showed the neuroprotective effects of NBO treatment by reducing oxidative stress and maintaining the level of connexin43 in astrocytes, which could be used for the clinical treatment of ischemic stroke.

Keywords: acute ischemic stroke; astrocyte; gap junction connexin43; glutathione peroxidase 4 (GPX4); normobaric hyperoxia; oxidative stress.

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Conflict of interest statement

None.

Figures

FIGURE 1
FIGURE 1
Experimental design. (A) The chart shows the experimental design including drug administration, MCAO surgery, NBO treatment, tissue collection, and neurobehavioral assessments. MCAO, middle cerebral artery occlusion; NBO, normobaric hyperoxia. (B) The schematic diagram of the focusing area (context in peri‐ischemic region, red‐dashed box) collected for western blotting or observed for immunohistochemistry. The ischemic region is outlined with the black‐dashed line
FIGURE 2
FIGURE 2
Normobaric hyperoxia maintained the homeostasis of oxidoreductases (GPX4/NOX4) in astrocytes at the early phase of ischemia. The level of GPX4, NOX4, and downstream HO‐1 was measured after 90 min ischemia plus 24 h reperfusion, using western blotting, with or without NBO treatment. (A) The representative bands of western blotting. (B–D) Quantitative analysis of expression of GPX4, NOX4, and HO‐1. Data are presented as the mean ± SEM. ** p < 0.01, versus the sham group; # p < 0.05, versus the NBO group. (E) The co‐staining images of HO‐1 (green) and GFAP (red) showed the expression of HO‐1 in astrocytes with or without NBO treatment. The nucleus was stained with DAPI (blue). The images in white‐dashed boxes are enlarged. Scale bar: 50 μm (merge); 20 μm (enlarged). All data represent at least three independent experiments (three animals in each group)
FIGURE 3
FIGURE 3
Normobaric hyperoxia treatment improved the expression of connexin43 in astrocytes after 90‐min ischemia/24‐h reperfusion. The level of connexin43 was measured using western blotting and immunohistochemistry staining after 90 min ischemia/24 h reperfusion. (A) Representative bands of western blotting. (B) Statistical analysis of connexin43 expression. Data are presented as the mean ± SEM. ** p < 0.01, versus the sham group; # p < 0.05, versus the air group. (C) The co‐staining images of connexin43 (green) and anti‐glial fibrillary acidic protein (red) showed the expression of connexin43 in astrocytes with or without NBO treatment. The nucleus was stained with DAPI (blue). The images in white‐dashed boxes are enlarged. Scale bar: 50 μm (merge); 20 μm (enlarged). All data represent at least three independent experiments (three animals in each group)
FIGURE 4
FIGURE 4
Normobaric hyperoxia treatment attenuated ischemia‐induced cytochrome c release via the GPX4‐connexin43 pathway. To investigate the role of the GPX4 or connexin43 pathway in NBO‐induced protection, rats were pretreated with connexin43 inhibitor (Gap27) or GPX4 activator (Ferrostatin‐1) by tail vein injections. The mitochondrial and cytosolic fractions were isolated from ischemic tissues to measure the levels of CytC in mitochondrial and cytosol fractions. (A) The level of CytC in isolated mitochondrial and cytosolic fractions. The level of Cox‐IV was shown as the loading control in the mitochondria fraction. The levels of NOX4, GPX4, HO‐1, and connexin43 in each group are also illustrated, showing the successful regulation of Gap27 and Ferrostatin‐1. (B) Statistical analysis of CytC release. Data are presented as the mean ± SEM. ** p < 0.01, versus the saline + air group; ## p < 0.01, versus the saline + NBO group. All data represent at least three independent experiments (three animals in each group)
FIGURE 5
FIGURE 5
The GPX4‐connexin43 pathway participated in the normobaric hyperoxia (NBO)‐induced infarction reduction after cerebral ischemia. (A) TUNEL co‐staining (specific marker for apoptotic cells, colored green) and NeuN (specific marker for neurons, colored red). The nucleus was stained with DAPI (blue). The images in white‐dashed boxes were enlarged. Arrows refer to the TUNEL+/NeuN+ cells (neuronal cell death). Scale bar: 50 μm (merge); 20 μm (enlarged). (B) Typical TTC staining after 90‐min ischemia/24‐h reperfusion. (C) Statistical analysis. Data are presented as the mean ± SEM. ** p < 0.01, *** p < 0.001 versus the saline + air group; # p < 0.05, versus the saline + NBO group. All data represent at least five animals in each group
FIGURE 6
FIGURE 6
The GPX4‐connexin43 pathway participated in normobaric hyperoxia (NBO)‐induced neuroprotection after cerebral ischemia. The neurological functions and body weight were evaluated at 1, 3, 5, 7 and 14 days post ischemia/reperfusion. (A) The mNSS scores. (B) The foot‐fault test. (C) The contact time of the adhesive removal test. (D) The removal time of the adhesive removal test. (E) Body weight. Data are presented as median (interquartile range, A–D) or as the mean ± SEM (E). ** p < 0.01, *** p < 0.001 versus the saline + air group; # p < 0.05, ## p < 0.01, ### p < 0.001 versus the saline + NBO group. All data represent at least eight animals in each group

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