Maternal sevoflurane exposure induces neurotoxicity in offspring rats via the CB1R/CDK5/p-tau pathway

Abstract

Sevoflurane is widely used for maternal anesthesia during pregnancy. Sevoflurane exposure of rats at mid-gestation can cause abnormal development of the central nervous system in their offspring. Sevoflurane is known to increase the expression of cannabinoid 1 receptor (CB1R) in the hippocampus. However, the effect of cannabinoid 1 receptor on fetal and offspring rats after maternal anesthesia is still unclear. At gestational day 14, pregnant rats were subjected to 2-h exposure to 3.5% sevoflurane or air. Rats underwent intraperitoneal injection with saline or rimonabant (1 mg/kg) 30 min prior to sevoflurane or air exposure. cannabinoid 1 receptor, cyclin-dependent kinase 5 (CDK5), p35, p25, tau, and p-tau expression in fetal brains was measured at 6, 12, and 24 h post-sevoflurane/air exposure. Neurobehavioral and Morris water maze tests were performed postnatal days 3-33. The expression of cannabinoid 1 receptor/cyclin-dependent kinase 5/p-tau and histopathological staining of brain tissues in offspring rats was observed. We found that a single exposure to sevoflurane upregulated the activity of cyclin-dependent kinase 5 and the level of p-tau via cannabinoid 1 receptor. This was accompanied by the diminished number of neurons and dendritic spines in hippocampal CA1 regions. Finally, these effects induced lower scores and platform crossing times in behavioral tests. The present study suggests that a single exposure to 3.5% sevoflurane of rats at mid-gestation impairs neurobehavioral abilities and cognitive memory in offspring. cannabinoid 1 receptor is a possible target for the amelioration of postnatal neurobehavioral ability and cognitive memory impairments induced by maternal anesthesia.

Keywords: CB1R; hippocampus; learning and memory; rimonabant; sevoflurane; tau.

FIGURE 1

Schematic timeline of this research. Pregnant rats received 2-h exposure of 3.5% sevoflurane or air in the day 0 of gestation (G0). Thirty min prior to sevoflurane or air exposure, rats were subjected to intraperitoneal injection with Rimonabant (RIM, 1 mg/kg) or vehicle (Veh). Brain tissues were harvested from fetal rats at 6, 12, or 24 h subsequent to sevoflurane exposure. The offspring rats in each group were employed for neurobehavioral tests and Morris water maze (MWM) tests during P3–33. Next, the hippocampus was harvested 1 h after MWM test.

FIGURE 2

Maternal sevoflurane exposure caused dysfunction in neurobehaviors and cognitive memory in neonatal rats. Neurobehavioral tests (n = 12 per group) were performed during P3–13 and included surface righting reflex (A), negative geotaxis reflex (B), forelimb grip test (C), cliff avoidance test (D), and sensory reflexes (E). The MWM tests (n = 12 per group) were carried out during P28–33. Escape latency (F), the times of crossing the platform (G), and time spent in each quadrant (H) were recorded. Data are depicted as mean ± S.E.M. *p < .05, **p < .01, vs. the Con + Veh group; #p < .05, vs. the Sevo + Veh group.

FIGURE 3

Functions of maternal sevoflurane exposure in neuronal density and dendritic spine density in the hippocampal CA1 area of neonatal rats. Nissl staining under ×400 (A) and neuronal density ratio changes (B) in each group (n = 3). Golgi staining under ×1000 (D) and histograms of spine density/10 µm (C) in each group (n = 3). Data are displayed as mean ± S.E.M. **p < .01, vs. the Con + Veh group; #p < .05, ###p < .001, vs. the Sevo + Veh group.

FIGURE 4

Sevoflurane upregulated p-tau in the fetal brains via the CB1R/CDK5/p25 pathway. Assays of western blotting (n = 3) demonstrating the expression of CB1R, CDK5, p35, p25, tau, and p-tau (A,B) and the value of p25/p35 (C) and p-tau/tau (D). Immunofluorescence double staining of CB1R and Nestin (E, n = 3) and CDK5 and Nestin (F, n = 3) in the fetal rat brains. Quantification of CB1R/Nestin-positive cells (G) and CDK5/Nestin-positive cells (H). Data are manifested as mean ± S.E.M. *p < .05, **p < .01, vs. the Con + Veh group; #p < .05, ##p < .01, vs. the Sevo + Veh group.

FIGURE 5

Sevoflurane augmented p-tau expression in the hippocampus of offspring rats. Representative western blots (A) and quantitation (B–I) of western blotting assays (n = 3 rats/group) showing CB1R, p35, CDK5, p25, tau, and p-tau expression in the hippocampus. Data are summarized as mean ± S.E.M. *p < .05, **p < .01, vs. the Con + Veh group; #p < .05, vs. the Sevo + Veh group.