Role of intermittent hypoxic training combined with methazolamide in the prevention of high-altitude cerebral edema in rats

Abstract

Although intermittent hypoxia training (IHT) and methazolamide (MTZ) alone can prevent high-altitude cerebral edema (HACE) to varying degrees, their efficacy and dispersion remain limited. However, only a handful of trials have explored the effectiveness of the IHT and MTZ combination in preventing HACE. Rats were first exposed to hypobaric hypoxia (5000 m, 54.02 kPa, 10.8% fraction of inspired oxygen (FiO₂)) with simultaneous exhaustive exercise (EE) for different durations to determine the ideal condition for establishing a rat model of HACE. Rats receiving various courses of IHT were subjected to this condition, and changes in behaviour, brain water content (BWC), pathology and brain protein expression were evaluated. Meanwhile, rats received different doses of MTZ before and during hypoxia exposure with simultaneous EE. Finally, rats receiving the IHT and MTZ combination were then exposed to hypoxia with simultaneous EE. Systemic inflammation and mild cerebral edema developed in rats after 6 h of hypobaric hypoxia with simultaneous EE. Rats showed severe impairment of spatial and memory functions after 2 days of hypobaric hypoxia with simultaneous EE, and the pathology of their brain showed significant dilated perivascular spaces, cell swelling, vacuolar degeneration and reduced neuron count. BWC, serum inflammatory factors and expression of vascular endothelial growth factor (VEGF) and aquaporin 4 (AQP4) proteins in the hippocampus increased significantly. Both IHT and MTZ differentially counteracted hypobaric hypoxia-induced spatial and memory function impairments and increased BWC, pathological changes and expression of AQP4 and VEGF proteins in the hippocampus. Among these, the long-course IHT (BID, 14 d) combined with MTZ (200 mg/kg/d) showed the most significant improvement, restoring the rats' indices to normal levels. Continuous hypobaric hypoxia with simultaneous EE for 2 days resulted in significant HACE in rats, which may be used to establish a rat model of HACE. Both IHT and MTZ alleviated HACE in rats to varying degrees, among which long-course IHT (BID, 14 d) combined with MTZ (200 mg/kg/d) effectively prevented HACE in rats.

Keywords: Acute mountain sickness; High-altitude cerebral edema; Hypobaric hypoxia; Intermittent hypoxia training; Methazolamide.

Fig. 1

Graphic schema of experimental design.

Fig. 2

Establishment of the experimental rat model of HACE. (A) MWM, the platform quadrant dwell time and platform crossing number of rats in each group; (B) BWC was significantly increased in all hypoxia groups compared to the NC group, among which the mean BWC was highest in the HH 2d group; (C) The histopathological features in the cortex and hippocampus regions were examined by HE staining. The cortex and hippocampus regions of the NC group showed normal histological features (a and f). Only dilated perivascular spaces and a little pyknotic neurons were observed in the HH 6h group (b and g). However, pyramidal cells in the cortex and hippocampus regions of rats in the HH 1d group presented massive derangement, karyopyknosis, hyperchromatic nuclei and irregular shape (c and h). The HH 2d group (d and i) and HH 5d group (e and j) showed the most severe lesions than the other hypoxia groups with the feature of cell swelling, vacuolar degeneration, dilated perivascular spaces and reduced number of neurons in the cortex and hippocampus regions. (D) Inflammatory and anti-inflammatory cytokines in serum; (E) The expression of AQP4 and VEGF proteins. The expression of target proteins was normalized to that of the reference protein β-actin; (F) The relative level of VEGF and AQP4 mRNA expression. The expression of AQP4 and VEGF mRNA was normalized to that of actin as the internal reference, calculated using the 2^-ΔΔCt; Asterisks are used to indicate statistical significance as follows: *, **, ***, and **** indicate p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively.

Fig. 3

IHT intervention. (A) MWM, the platform quadrant dwell time and platform crossing number of rats in each group; (B) Compared to the HH group, both S-IHT and L-IHT groups showed a significant improvement in BWC, with L-IHT group closer to the NC group, but there was a statistically significant difference between the two groups; (C) In HE staining, cell swelling, vacuolar degeneration, and dilated perivascular spaces in the cortex and hippocampus regions of the S-IHT group (c and g) were significantly relieved, compare with the HH group (b and f). Only dilated perivascular spaces and a little pyknotic neurons were observed in the L-IHT groups (d and h), which are very close to the normal histological features (a and e). (D) Inflammatory and anti-inflammatory cytokines in serum; (E) The expression of AQP4 and VEGF proteins. The expression of target proteins was normalized to that of the reference protein β-actin; (F) The relative level of VEGF and AQP4 mRNA expression. The expression of AQP4 and VEGF mRNA was normalized to that of actin as the internal reference, calculated using the 2^-ΔΔCt; Asterisks are used to indicate statistical significance as follows: *, **, ***, and **** indicate p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively.

Fig. 4

MTZ intervention. (A) MWM, the platform quadrant dwell time and platform crossing number of rats in each group; (B) Compared to the HH + NS group, both HH + MTZ1 and HH + MTZ2 showed a significant improvement in BWC, with the HH + MTZ2 group closer to the NC group, but there was statistical significance between the two groups; (C) In HE staining, cell swelling, vacuolar degeneration, and dilated perivascular spaces in the cortex and hippocampus regions of the HH + MTZ1 group (c and g) were significantly relieved, compare with the HH group (b and f). Only dilated perivascular spaces and a little pyknotic neurons were observed in the HH + MTZ2 group (d and h), which are very close to the normal histological features (a and e); (D) Inflammatory and anti-inflammatory cytokines in serum; (E) The expression of AQP4 and VEGF proteins. The expression of target proteins was normalized to that of the reference protein β-actin; (F) The relative level of VEGF and AQP4 mRNA expression. The expression of AQP4 and VEGF mRNA was normalized to that of actin as the internal reference, calculated using the 2^-ΔΔCt; Asterisks are used to indicate statistical significance as follows: *, **, ***, and **** indicate p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively.

Fig. 5

Long-course IHT combined with MTZ intervention. (A) MWM, the platform quadrant dwell time and platform crossing number of rats in each group; (B) Compared to the L-IHT and HH + MTZ2 groups, the IHT&MTZ group showed a significant further improvement in BWC, with no statistical significance between the IHT&MTZ and NC groups; (C) In HE staining, there was no cell swelling, vacuolar degeneration and dilated perivascular spaces in the cortex and hippocampus regions of the IHT&MTZ1 group (d and h), which is basically consistent with the NC group (a and e); (D) Inflammatory and anti-inflammatory cytokines in serum; (E) The expression of AQP4 and VEGF proteins. The expression of target proteins was normalized to that of the reference protein β-actin; (F) The relative level of VEGF and AQP4 mRNA expression. The expression of AQP4 and VEGF mRNA was normalized to that of actin as the internal reference, calculated using the 2^-ΔΔCt; Asterisks are used to indicate statistical significance as follows: *, **, ***, and **** indicate p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively.