Esketamine at a Clinical Dose Attenuates Cerebral Ischemia/Reperfusion Injury by Inhibiting AKT Signaling Pathway to Facilitate Microglia M2 Polarization and Autophagy

Abstract

Purpose: This study aimed to assess the protective effect of a clinical dose esketamine on cerebral ischemia/reperfusion (I/R) injury and to reveal the potential mechanisms associated with microglial polarization and autophagy.

Methods: Experimental cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in adult rats and simulated by oxygen-glucose deprivation (OGD) in BV-2 microglial cells. Neurological and sensorimotor function, cerebral infarct volume, histopathological changes, mitochondrial morphological changes, and apoptosis of ischemic brain tissues were assessed in the presence or absence of esketamine and the autophagy inducer rapamycin. The expression of biomarkers related to microglial M1 and M2 phenotypes in the ischemic brain tissues was determined by immunofluorescence staining and RT-qPCR, and the expression of proteins associated with autophagy and the AKT signaling pathway in the ischemic brain tissues was assayed by Western blotting.

Results: Esketamine alone and esketamine combined with rapamycin alleviated neurological impairment, improved sensorimotor function, decreased cerebral infarct volume, and mitigated tissue injury in the MCAO rats. Importantly, esketamine promoted microglial phenotypic transition from M1 to M2 in both the MCAO rats and the OGD-treated BV-2 microglia, induced autophagy, and inactivated AKT signaling. Furthermore, the effects of esketamine were enhanced by addition of autophagy inducer rapamycin.

Conclusion: Esketamine at a clinical dose attenuates cerebral I/R injury by inhibiting AKT signaling pathway to facilitate microglial M2 polarization and autophagy. Furthermore, esketamine combined autophagy inducer can provide an improved protection against cerebral I/R injury. Thus, this study provides new insights into the neuroprotective mechanisms of esketamine and the potential therapeutic strategies of cerebral I/R injury.

Keywords: autophagy; cerebral ischemia/reperfusion injury; esketamine; ischemic stroke; microglia polarization.

Figure 1

Esketamine alleviated neurological impairments and sensorimotor dysfunction by MCAO surgery. (A) Longa score (***P<0.001). (B and C) The times required to contact and to remove the sticker during the double-sided sticker removal experiment (***P<0.001). (D) Navigational path map of the MWM test in each group; (E and F) Mean daily escape latencies and swimming distance of rats in the MWM test (*P<0.05 vs control group; #P<0.05 vs MCAO group; *P < 0.05 vs Esketamine group). (G and H) Numbers of platform crossing and time spent in the target quadrant of rats during the probe trial (*P<0.05, **P<0.01, ***P< 0.001). n=10.

Figure 2

(A) Representative TTC-stained cerebral slices and the between-group comparisons of infarct volume; (B) The histopathological changes of hippocampus in the ischemic side with H&E staining. Scale bar = 50 μm; (C) Mitochondrial morphological changes using transmission electron microscope. Scale bar = 5 μm; (D) Representative pictures of apoptosis in the ischemic brain tissues using TUNEL staining and the between-group comparisons for the percents of TUNEL positive cells. *P<0.01; ***P<0.001. Scale bar = 50 μm. n=5.

Figure 3

Esketamine promoted the phenotype transition of microglia from M1 to M2 in the MCAO rats. Representative images of double immunofluorescence staining for Iba-1⁺CD16⁺ (A) and Iba-1⁺CD206⁺ (B) in the peri-infarct regions and the between-group comparisons of their quantitative data. **P < 0.01, ***P<0.001. Scale bar = 50 μm. n=5.

Figure 4

Esketamine promoted the expression of microglia M2 phenotype biomarkers and inhibited the expression of microglia M1 phenotype markers in the MCAO rats. (A) The between-group comparisons for mRNA expression of microglia M1 (CD16 and iNOS) and M2 (CD206 and Arg-1) phenotype biomarkers in the ischemic brain tissues by RT-qPCR; (B) The between-group comparisons for mRNA expression of TNF-α, IL-1β and IL-10 in the ischemic brain tissues by RT-qPCR. **P < 0.01, ***P < 0.001. n=5.

Figure 5

Esketamine enhanced autophagy and inhibited AKT signaling activation in the MCAO rats. (A) Representative Western blotting images for expression of autophagy-related markers in the ischemic brain tissues and the between-group comparisons of their quantitative data; (B) Representative Western blotting images for expression of p-AKT and AKT in the ischemic brain tissues and the between-group comparisons of their quantitative data. **P<0.01, ***P<0.001. Scale bar = 50 μm. n=5.

Figure 6

The OGD treatment suppressed the phenotype transition of microglia from M1 to M2. Representative images of double immunofluorescence staining for Iba-1⁺CD16⁺ (A) and Iba-1⁺CD206⁺ (B) in the BV-2 microglia exposed to the OGD treatment for 6 h, 12 h and 24 h and the between-group comparisons of their quantitative data. ***P<0.001.

Figure 7

Esketamine increased the viability of OGD-treated BV-2 microglia and facilitated the phenotype transition of microglia from M1 to M2. (A) The viability of OGD-treated BV-2 microglia; (B and C) Representative images of double immunofluorescence staining for Iba-1⁺CD16⁺ and Iba-1⁺CD206⁺ in the OGD-treated BV-2 microglia and the between-group comparisons of their quantitative data. *P < 0.05, ***P<0.001. Scale bar = 50 μm.

Figure 8

Esketamine promoted the expression of M2 phenotype biomarkers and inhibited the expression of M1 phenotype biomarkers in the OGD-treated BV-2 microglia. (A) The between-group comparisons of quantitative data for mRNA expression of microglia M1 phenotype biomarkers (CD16 and iNOS) and M2 phenotype biomarkers (CD206 and Arg-1); (B) The between-group comparisons of quantitative data for mRNA expression of TNF-α, IL-1β and IL-10. ***P <0.001.

Figure 9

Esketamine inhibited AKT signaling activation in the OGD-treated BV-2 microglia. (A) Representative Western blotting images for expression of p-AKT and AKT; (B) The between-group comparisons of their quantitative data. ***P<0.001.