Sevoflurane Exacerbates Cognitive Impairment Induced by A β 1-40 in Rats through Initiating Neurotoxicity, Neuroinflammation, and Neuronal Apoptosis in Rat Hippocampus

Abstract

Objective: This study was aimed at investigating whether sevoflurane inhalation induced cognitive impairment in rats with a possible mechanism involved in the event.

Methods: Thirty-two rats were randomly divided into four groups of normal saline (NS) + O₂, NS + sevoflurane (sevo), amyloid-β peptide (Aβ) + O₂, and Aβ + sevo. The rats in the four groups received bilateral intrahippocampus injections of NS or Aβ. The treated hippocampus was harvested after inhaling 30% O₂ or 2.5% sevoflurane. Evaluation of cognitive function was performed by Morris water maze (MWZ) and an Aβ _1-42 level was determined by ELISA. Protein and mRNA expressions were executed by immunohistochemical (IHC) staining, Western blotting, and qRT-PCR.

Results: Compared with the NS-treated group, sevoflurane only caused cognitive impairment and increased the level of Aβ _1-42 of the brain in the Aβ-treated group. Sevoflurane inhalation but not O₂ significantly increased glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule (IBA)1 expression in Aβ-treated hippocampus of rats. Expression levels for Bcl-xL, caspase-9, receptor for advanced glycation end products (RAGE) and brain-derived neurotrophic factor (BDNF) were significantly different in quantification of band intensity between the rats that inhaled O₂ and sevoflurane in Aβ-treated groups (all P < 0.05). Interleukin- (IL-) 1β, nuclear factor-κB (NF-κB), and inducible nitric oxide synthase (iNOS) mRNA expression increased after the rats inhaled sevoflurane in the Aβ-treated group (both P < 0.01). There were no significant differences in the change of GFAP, IBA1, Bcl-xL, caspase-9, RAGE, BDNF, IL-1β, NF-κB, and iNOS in the NS + O₂ and NS + sevo group (all P > 0.05).

Conclusion: Sevoflurane exacerbates cognitive impairment induced by Aβ _1-40 in rats through initiating neurotoxicity, neuroinflammation, and neuronal apoptosis in rat hippocampus.

Figure 1

Schematic representation of study designs. Animal model of Alzheimer disease was established with injection of NS or Aβ on day 7. Those animals started to inhale 30% O₂ or 2.5% sevoflurane after 14 days of the above injection.

Figure 2

Effects of sevoflurane on animal health. To verify whether inhalation anesthesia with sevoflurane was toxic to animals, the NS- and Aβ-treated rats inhaled 2.5% sevoflurane for 1–4 hours, and then MAP (a), heart beats (b), pH (c), PaCO₂ (d), PaO₂ (e), and HCO₃⁻ (f) were continuously monitored in the animals. There were no statistical significance in the above-stated parameters among 4 different time points in the NS + sevo group or Aβ + sevo group (all P > 0.05 and n = 4 in each group).

Figure 3

Sevoflurane influenced cognitive function and Aβ_1–42 level in rats. Rats received bilateral intrahippocampus injections of either NS or Aβ_1–40 after training. The spatial probe test was examined in the time course after inhalation of 30% O₂ or 2.5% sevoflurane, and the time spent in exploration of the original platform was recorded (a). The results were expressed as a ratio (%) as compared to the NS + O₂ or Aβ + O₂ group on day 7. Navigation test was performed after inhalation of 30% O₂ or 2.5% sevoflurane on day 7, and the escape latency was recorded (b). The results were expressed as seconds as compared to the NS + O₂ or Aβ + O₂ group at the given times. After the rats inhaled 30% O₂ or 2.5% sevoflurane in the presence and absence of Aβ, hippocampus tissues of the treated rats were harvested. And then, the level of Aβ_1–42 in the supernatant from the tissue was determined by ELISA (c). ^∗P < 0.05 vs. Aβ + O₂ (n = 8).

Figure 4

Sevoflurane inhalation increased protein expression of GFAP and IBA-1 in the hippocampus determined by immunochemistry and Western blot. Immunochemistry was used to determine the GFAP (a) and IBA1 (b) expression levels in the NS- or Aβ-treated hippocampus of rats after inhaling 30% O₂ and 2.5% sevoflurane at the indicated times. Western blot was also used to determine the expression of GFAP and IBA1 in the four groups (c). The results in (d) and (e) were calculated as % of the intensity of the β-actin protein band and expressed as a fold change in reference to their controls. ^∗∗P < 0.01 vs. Aβ + O₂ (n = 8).

Figure 5

Sevoflurane inhalation influenced protein expression of Bcl-xL, caspase-9, BDNF, RAGE, and mRNA level of IL-1β, NF-κB, and iNOS in the hippocampus. Caspase-9 (a, b), Bcl-xL (a, c), BDNF (d, e), and RAGE (d, f) protein levels in the NS- or Aβ-treated hippocampus of rats were determined using Western blot after inhaling 30% O₂ and 2.5% sevoflurane at the indicated times. Relative band intensity of each protein expression was calculated as % of the intensity of the β-actin protein band and expressed as a fold change in reference to their controls. IL-1β (g), NF-κB (h), and iNOS (i) mRNA levels in the NS- and Aβ-treated hippocampus were determined after inhaling 30% O₂ or 2.5% sevoflurane on day 7. The results were expressed as a fold change in reference to mRNA levels of their own controls. ^∗P < 0.05 vs. A β + O₂ (n = 8); ^∗∗P < 0.01 vs. A β + O₂ (n = 8).