Objective:To evaluate the role of microglial hypoxia-inducible factor-3α (HIF-3α) in cognitive impairment after hemorrhagic shock and resuscitation(HSR) and the relationship with neuronal ferroptosis in mice.Methods:Twenty-four specific pathogen-free healthy male C57BL/6 mice, aged 10 weeks, weighing 25-30 g, were divided into 3 groups ( n=8 each) using a random number table method: sham operation group (Sham group), HSR group, and HSR+ ferroptosis inhibitor (ferrostatin-1) group (HSR+ Fer-1 group). Sixteen C57BL/6 mice and 16 HIF-3α flox/flox: Cx3crl Cre (HIF-3α CKO) mice were selected and assigned to 2 groups ( n=8 each) using a random number table method: sham operation group (WT-Sham group, HIF-3α CKO-Sham group) and HSR group (WT-HSR group, HIF-3α CKO-HSR group). To establish the HSR model, 40% of the total blood volume was withdrawn at a steady rate via the right carotid artery within 30 min and 1 h later reinfused through the jugular vein over a period of 30 min. Ferrostatin-1 10 mg/kg was nasally administered once mice recovered after HSR in HSR+ Fer-1 group. The cognitive function was evaluated by the novel object recognition test at 72 h after developing the model. The hippocampal tissues were collected under deep anesthesia after evaluation for determination of the expression of glutathione peroxidase 4 (GPX4) and ferritin heavy chain 1 (FTH1) in the ipsilateral hippocampi (by Western blot) and expression of microglial HIF-3α and GPX4 and FTH1 in neurons in the hippocampal CA3 region (by immunofluorescence staining) and for examination of the ultrastructure of mitochondria in hippocampal neurons (with a transmission electron microscope). Results:Compared to Sham group, the cognitive and discrimination indexes were significantly decreased, and the expression of GPX4 and FTH1 was down-regulated in HSR group ( P<0.05). Compared to HSR group, the cognitive and discrimination indexes were significantly increased, and the expression of GPX4 and FTH1 in the hippocampi was up-regulated in HSR+ Fer-1 group ( P<0.05). Compared to WT-Sham group, the cognitive and discrimination indexes were significantly decreased, and the expression of microglial HIF-3α in the hippocampal CA3 region was up-regulated, and the expression of neuronal GPX4 and FTH1 was down-regulated in WT-HSR group ( P<0.05), and no statistically significant change was found in the aforementioned parameters in HIF-3α CKO-Sham group ( P>0.05). Compared to WT-HSR group, the cognitive and discrimination indexes were significantly increased, and the expression of microglial HIF-3α in the hippocampal CA3 region was down-regulated, the expression of GPX4 and FTH1 was up-regulated ( P<0.05), and mitochondrial damage in the neurons was significantly attenuated in HIF-3α CKO-HSR group. Conclusions:Microglial HIF-3α-mediated ferroptosis in hippocampal neurons is involved in cognitive impairment following HSR in mice.

Objective:To evaluate the role of microglial hypoxia-inducible factor-3α (HIF-3α) in cognitive impairment after hemorrhagic shock and resuscitation(HSR) and the relationship with neuronal ferroptosis in mice.Methods:Twenty-four specific pathogen-free healthy male C57BL/6 mice, aged 10 weeks, weighing 25-30 g, were divided into 3 groups ( n=8 each) using a random number table method: sham operation group (Sham group), HSR group, and HSR+ ferroptosis inhibitor (ferrostatin-1) group (HSR+ Fer-1 group). Sixteen C57BL/6 mice and 16 HIF-3α flox/flox: Cx3crl Cre (HIF-3α CKO) mice were selected and assigned to 2 groups ( n=8 each) using a random number table method: sham operation group (WT-Sham group, HIF-3α CKO-Sham group) and HSR group (WT-HSR group, HIF-3α CKO-HSR group). To establish the HSR model, 40% of the total blood volume was withdrawn at a steady rate via the right carotid artery within 30 min and 1 h later reinfused through the jugular vein over a period of 30 min. Ferrostatin-1 10 mg/kg was nasally administered once mice recovered after HSR in HSR+ Fer-1 group. The cognitive function was evaluated by the novel object recognition test at 72 h after developing the model. The hippocampal tissues were collected under deep anesthesia after evaluation for determination of the expression of glutathione peroxidase 4 (GPX4) and ferritin heavy chain 1 (FTH1) in the ipsilateral hippocampi (by Western blot) and expression of microglial HIF-3α and GPX4 and FTH1 in neurons in the hippocampal CA3 region (by immunofluorescence staining) and for examination of the ultrastructure of mitochondria in hippocampal neurons (with a transmission electron microscope). Results:Compared to Sham group, the cognitive and discrimination indexes were significantly decreased, and the expression of GPX4 and FTH1 was down-regulated in HSR group ( P<0.05). Compared to HSR group, the cognitive and discrimination indexes were significantly increased, and the expression of GPX4 and FTH1 in the hippocampi was up-regulated in HSR+ Fer-1 group ( P<0.05). Compared to WT-Sham group, the cognitive and discrimination indexes were significantly decreased, and the expression of microglial HIF-3α in the hippocampal CA3 region was up-regulated, and the expression of neuronal GPX4 and FTH1 was down-regulated in WT-HSR group ( P<0.05), and no statistically significant change was found in the aforementioned parameters in HIF-3α CKO-Sham group ( P>0.05). Compared to WT-HSR group, the cognitive and discrimination indexes were significantly increased, and the expression of microglial HIF-3α in the hippocampal CA3 region was down-regulated, the expression of GPX4 and FTH1 was up-regulated ( P<0.05), and mitochondrial damage in the neurons was significantly attenuated in HIF-3α CKO-HSR group. Conclusions:Microglial HIF-3α-mediated ferroptosis in hippocampal neurons is involved in cognitive impairment following HSR in mice.