Conflicting Actions of Inhalational Anesthetics, Neurotoxicity and Neuroprotection, Mediated by the Unfolded Protein Response

Abstract

Preclinical studies have shown that exposure of the developing brain to inhalational anesthetics can cause neurotoxicity. However, other studies have claimed that anesthetics can exert neuroprotective effects. We investigated the mechanisms associated with the neurotoxic and neuroprotective effects exerted by inhalational anesthetics. Neuroblastoma cells were exposed to sevoflurane and then cultured in 1% oxygen. We evaluated the expression of proteins related to the unfolded protein response (UPR). Next, we exposed adult mice in which binding immunoglobulin protein (BiP) had been mutated, and wild-type mice, to sevoflurane, and evaluated their cognitive function. We compared our results to those from our previous study in which mice were exposed to sevoflurane at the fetal stage. Pre-exposure to sevoflurane reduced the expression of CHOP in neuroblastoma cells exposed to hypoxia. Anesthetic pre-exposure also significantly improved the cognitive function of adult wild-type mice, but not the mutant mice. In contrast, mice exposed to anesthetics during the fetal stage showed cognitive impairment. Our data indicate that exposure to inhalational anesthetics causes endoplasmic reticulum (ER) stress, and subsequently leads to an adaptive response, the UPR. This response may enhance the capacity of cells to adapt to injuries and improve neuronal function in adult mice, but not in developing mice.

Keywords: ER stress; KDEL receptor; anesthetics; chaperone; endoplasmic reticulum; neuroprotection; neurotoxicity; unfolded protein response.

Figure 1

Exposure to sevoflurane resulted in the initiation of the unfolded protein response (UPR). Neuro2a cells were cultured and exposed to 3% sevoflurane for 2.5, 5, or 7.5 h. The cells were then collected and analyzed by western blotting. Sevoflurane exposure activated ATF6 and induced the expression of XBP-1, a transcription factor that enhanced the expression of BiP, a major endoplasmic reticulum (ER) chaperone. GRP94, another ER chaperone is recognized by the anti-KDEL antibody. CHOP, a transcription factor responsible for initiating apoptotic cell death during ER stress, was expressed at significantly higher levels after 7.5 h of exposure.

Figure 2

Anesthetic exposure during the perinatal period had negative effects on spatial working memory in young mice. Eight-arm radial maze testing was performed in wild-type mice (+/+; circles) and Bip mutant mice (Bm; triangles) at 10 weeks after sevoflurane exposure (+/+ sevo, n = 12, closed red circles; Bm/+ sevo, n = 16, closed red triangles), and without exposure (+/+, n = 6, open blue circles; Bm/+, n = 8; open blue triangles). Tests were performed for 5 days. Statistical significance was determined by one-way repeated measures analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison testing. (a) Correct answer rates are shown as the mean value of each group + standard error of the mean (SEM). The correct answer rate was defined as the ratio of successful attempts to overall attempts (0.0–1.0). (b) Mean completion times for each group + SEM (data extracted from DATASET S1 and DATASET S2, Front. Neurosci. 12:753. doi:10.3389/fnins. 2018.00753). Fetal anesthetic exposure resulted in a lower correct answer rate (p < 0.05) (a), and a longer completion time (p < 0.01) (b), compared with unexposed mice; this was the case for both Bip mutant mice and wild-type mice.

Figure 3

Pre-exposure to sevoflurane may induce the UPR and reduce the damage caused by hypoxia. (a) Cells were exposed to 3% sevoflurane for 5 h. After 12 h of recovery time, cells were exposed to hypoxic conditions (1% O₂) for 17 h before collection. Hypoxia alone induced the expression of CHOP. Pre-exposure to sevoflurane reduced the expression of CHOP while siBiP enhanced the expression of CHOP. (b) Densitometry was performed for three experiments. The density of CHOP relative to that of tubulin was measured by Image J and analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. Values indicate mean + standard error of the mean (SEM). Control vs. siBip; p < 0.05.

Figure 4

Anesthetic exposure had positive effects on spatial working memory in adult wild-type mice, but not mutant BiP mice. Eight-arm radial maze testing was performed in adult wild-type mice (+/+; circles) and mutant-Bip mice (Bm; triangles) 7 days after sevoflurane (sevo) exposure (+/+ sevo, n = 4, closed red circles; Bm/+ sevo, n = 5, closed red triangles) and without exposure (+/+, n = 7, open circles; Bm/+, n = 10; open triangles). Tests were performed over 5 days. Significance was determined by one-way repeated measures ANOVA followed by Bonferroni’s multiple comparison testing. (a) Correct answer rates are shown as the mean value for each group + standard error of the mean (SEM). The correct answer rate was defined as the ratio of successful attempts to the number of overall attempts (0.0–1.0). (b) Completion times represent the mean for each group + SEM.