Dexmedetomidine alleviates early brain injury following traumatic brain injury by inhibiting autophagy and neuroinflammation through the ROS/Nrf2 signaling pathway

Abstract

Traumatic brain injury (TBI) is a major public health problem and a major cause of mortality and disability that imposes a substantial economic burden worldwide. Dexmedetomidine (DEX), a highly selective α‑2‑adrenergic receptor agonist that functions as a sedative and analgesic with minimal respiratory depression, has been reported to alleviate early brain injury (EBI) following traumatic brain injury by reducing reactive oxygen species (ROS) production, apoptosis and autophagy. Autophagy is a programmed cell death mechanism that serves a vital role in neuronal cell death following TBI. However, the precise role of autophagy in DEX‑mediated neuroprotection following TBI has not been confirmed. The present study aimed to investigate the neuroprotective effects and potential molecular mechanisms of DEX in TBI‑induced EBI by regulating neural autophagy in a C57BL/6 mouse model. Mortality, the neurological score, brain water content, neuroinflammatory cytokine levels, ROS production, malondialdehyde levels and neuronal death were evaluated by TUNEL staining, Evans blue extravasation, ELISA, analysis of ROS/lipid peroxidation and western blotting. The results showed that DEX treatment markedly increased the survival rate and neurological score, increased neuron survival, decreased the expression of the LC3, Beclin‑1 and NF‑κB proteins, as well as the cytokines IL‑1β, IL‑6 and TNF‑α, which indicated that DEX‑mediated inhibition of autophagy and neuroinflammation ameliorated neuronal death following TBI. The neuroprotective capacity of DEX is partly dependent on the ROS/nuclear factor erythroid 2‑related factor 2 signaling pathway. Taken together, the results of the present study indicated that DEX improves neurological outcomes in mice and reduces neuronal death by protecting against neural autophagy and neuroinflammation.

Keywords: autophagy; dexmedetomidine; early brain injury; neuroinflammation; traumatic brain injury.

Figure 1.

DEX alleviates neurological deficits and brain edema following TBI. (A) Schematic of the experimental paradigm for establishing the TBI model in mice. (B) Neurological scores of mice in the sham group, TBI group and TBI group treated with DEX at 72 h after TBI (n=10, P<0.05). (C) Comparison of the brain water content between the three groups (n=5; P<0.01). DEX, dexmedetomidine; TBI, traumatic brain injury; mNSS, modified neurological severity score.

Figure 2.

DEX alleviates neuronal apoptosis and increased BBB permeability following TBI. (A) TUNEL staining showed that DEX alleviated neuronal apoptosis in the hippocampus at 72 h after TBI and representative images of apoptotic neurons are shown. Scale bar=50 µm. (B) Levels of caspase-3 and cleaved caspase-3 in the brain cortex of mice following TBI were determined using western blotting. Quantification of (C) caspase-3 and (D) cleaved caspase-3 in the brain cortex relative to β-actin, the loading control. DEX reduced caspase-3 and cleaved caspase-3 levels in mice with TBI. (E) DEX reduces BBB permeability (n=5, P<0.05; ANOVA; means ± standard error of mean). DEX, dexmedetomidine; BBB, blood-brain barrier; TBI, traumatic brain injury; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI, 4′6-diamidino-2-phenylindole.

Figure 3.

DEX alleviates neuroinflammation following TBI. DEX significantly reduced hippocampal (A) TNF-α, (B) IL-1β, (C) IL-6 and (D) NF-κB levels at 72 h after TBI (n=5, P<0.05, ANOVA; means ± standard error of mean). DEX, dexmedetomidine; TBI, traumatic brain injury.

Figure 4.

DEX inhibits TBI-induced autophagy activation in the hippocampus. (A) The expression of LC3 and Beclin-1 in the brain cortex of mice with TBI was determined using western blotting. (B) Quantification of LC3 and Beclin-1 levels in the brain cortex relative to β-actin, the loading control. DEX reduced LC3 and Beclin-1 expression in mice with TBI (n=5, P<0.01; ANOVA; means ± standard error of mean). (C) Representative pictures of double staining for LC3 and NeuN in the injured hippocampus following TBI. LC3-positive neurons were rarely observed in the hippocampus of the sham group and DEX decreased the number of LC3-positive neurons. Scale bar=20 µm (magnification, ×400). DEX, dexmedetomidine; TBI, traumatic brain injury.

Figure 5.

Rap stimulates autophagy and reverses the neuroprotective effect of DEX. Pretreatment with rapamycin significantly increased the (A) mNSS, (B) The brain water content and (C) BBB permeability compared with the TBI + DEX group (n=10, P<0.05). (D) Rapamycin increased neuronal apoptosis in the injured hippocampus compared with the TBI + DEX group (magnification, ×400). (E) Levels of LC3, Beclin-1 and caspase-3 in the brain cortex of mice with TBI were determined using western blotting. (F) Quantification of LC3, Beclin-1 and caspase-3 levels in the brain cortex relative to β-actin, the loading control. Rap increases the LC3, Beclin-1 and caspase-3 levels in DEX-treated mice following TBI (n=5, ANOVA; means ± standard error of mean). Rap, rapamycin; DEX, dexmedetomidine; mNSS, modified neurological severity score; TBI, traumatic brain injury; BBB, blood-brain barrier; DAPI, 4′6-diamidino-2-phenylindole.

Figure 6.

DEX regulates autophagy following TBI by modulating the ROS/Nrf2 signaling pathway. A-B: The levels of (A) ROS and (B) MDA were increased following TBI and decreased significantly following DEX treatment. (C) The expression of Nrf2 and HO-1 in the brain cortex of mice with TBI was determined using western blotting. Quantification of (D) Nrf2 and (E) HO-1 levels in the brain cortex relative to the β-actin loading control. DEX increased Nrf2 and HO-1 expression in mice with TBI (n=5, P<0.01; ANOVA; mean ± SEM). DEX, dexmedetomidine; TBI, traumatic brain injury; ROS, reactive oxygen species; Nrf2, nuclear factor erythroid 2-related factor 2; MDA, malondialdehyde; HO, heme oxygenase.

Figure 7.

A diagram of the proposed model explaining the observations of ROS/Nrf2-mediated regulation of autophagy following TBI and potential mechanisms underlying the effect of the DEX intervention. ROS, reactive oxygen species; Nrf2, nuclear factor erythroid 2-related factor 2; TBI, traumatic brain injury; HO, heme oxygenase; DEX, dexmedetomidine.