Role of the Toll‑like receptor 3 signaling pathway in the neuroprotective effect of sevoflurane pre‑conditioning during cardiopulmonary bypass in rats

Abstract

The aim of the present study was to explore the roles and possible molecular mechanism of the alleviating effect of sevoflurane pre‑treatment on the extracorporeal circulation and to investigate the possible involvement of the Toll‑like receptor (TLR3) signaling pathway. A total of 64 male Sprague Dawley rats were randomly divided into three groups: The sham operation group (H group; n=8), cardiopulmonary bypass (CPB) group (C group; n=24) and sevoflurane pre‑conditioning group (S group; n=32). The C group was subjected to tracheal intubation and mechanical ventilation, vessel puncture and catheter placement in the right femoral artery and right internal jugular vein, while no CPB was performed in the H group. The S group was pre‑treated with 2.4% sevoflurane for 1 h prior to establishing the CPB model. The CPB in the C and S groups was performed for 1 h. Blood of the rats was analyzed and clinical parameters were detected prior to, during and at various time‑points after CPB. In addition, eight rats from the C and S groups each were sacrificed at these time‑points and brain tissue samples were analyzed. The levels of the brain damage‑specific protein S100‑β as well as IL‑6 and IFN‑β in the serum were detected by ELISA; furthermore, the expression levels of TLR3 and TIR‑domain‑containing adapter‑inducing interferon‑β (TRIF) in the left hippocampus were assessed by ELISA and/or western blot analysis. The right hippocampus was assessed for neuronal apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The mean arterial pressure, heart rate and hematocrit were significantly decreased following CPB (P<0.05), while there was no significant changes in any other clinical parameters. The serum levels of S100‑β and IL‑6 in the C group were significantly increased compared with those in the H group (P<0.05), which was attenuated by sevoflurane‑pre‑treatment. Compared with the H group, the serum levels of IFN‑β as well as hippocampal protein levels of TLR3 and TRIF were significantly increased in the C group during and after CPB (P<0.05), which was markedly aggravated in the S group (P<0.05). The number of apoptotic hippocampal neurons, although being generally low, was significantly increased in the C group compared with that in the H group (P<0.05), while apoptosis was significantly attenuated by sevoflurane‑pre‑treatment (P<0.05). The present study therefore concluded that 2.4% sevoflurane pre‑treatment has a protective effect on the rat brain against CPB‑induced injury, which may be mediated via the TLR3 signaling pathway through upregulating the expression levels of anti‑inflammatory and downregulating pro‑inflammatory proteins.

Figure 1

Concentration of S100-β in the experimental groups at various time-points. Values are expressed as the mean ± standard deviation. ^#P<0.05 compared with T₀; ^*P<0.05 compared to H group; ^Δ0P<0.05 compared with S group. H, sham group; C, CPB group; S, sevoflurane-pre-conditioned group; T₀, prior to CPB; T₁, CPB for 30 min; T₂, CPB for 1 h; T₃, 1 h after 1-h CPB; T₄, 2 h after 1-h CPB; T₅, 3 h after 1-h CPB; CPB, cardiopulmonary bypass.

Figure 2

Serum levels of IL-6 in the experimental groups at various time-points. Values are expressed as the mean ± standard deviation. ^#P<0.05 compared with T₀; ^*P<0.05 compared to H group; ^ΔP<0.05 compared with H group. H, sham group; C, CPB group; S, sevoflurane-pre-conditioned group; T₀, prior to CPB; T₁, CPB for 30 min; T₂, CPB for 1 h; T₃, 1 h after 1-h CPB; T₄, 2 h after 1-h CPB; T₅, 3 h after 1-h CPB; CPB, cardiopulmonary bypass; IL, interleukin.

Figure 3

Concentration of IFN-β in the experimental groups at various time-points. Values are expressed as the mean ± standard deviation. ^#P<0.05 compared with T₀; ^*P<0.05 compared to H group; ^ΔP<0.05 compared with C group. H, sham group; C, CPB group; S, sevoflurane-pre-conditioned group; T₀, prior to CPB; T₁, CPB for 30 min; T₂, CPB for 1 h; T₃, 1 h after 1-h CPB; T₄, 2 h after 1-h CPB; T₅, 3 h after 1-h CPB; CPB, cardiopulmonary bypass; IFN, interferon.

Figure 4

TLR3 protein levels in rat hippocampi in the experimental groups at various time-points. Values are expressed as the mean ± standard deviation. #P<0.05 compared with T₀; ^*P<0.05 compared to H group; ^ΔP<0.05 compared with C group. H, sham group; C, CPB group; S, sevoflurane-pre-conditioned group; T₀, prior to CPB; T₁, CPB for 30 min; T₂, CPB for 1 h; T₃, 1 h after 1-h CPB; T₄, 2 h after 1-h CPB; T₅, 3 h after 1-h CPB; CPB, cardiopulmonary bypass; TLR, Toll-like receptor.

Figure 5

TRIF protein levels in rat hippocampi in the experimental groups at various time-points. Values are expressed as the mean ± standard deviation. #P<0.05 compared with T₀; ^*P<0.05 compared to H group; ^ΔP<0.05 compared with H group. H, sham group; C, CPB group; S, sevoflurane-pre-conditioned group; T₀, prior to CPB; T₁, CPB for 30 min; T₂, CPB for 1 h; T₃, 1 h after 1-h CPB; T₄, 2 h after 1-h CPB; T₅, 3 h after 1-h CPB; CPB, cardiopulmonary bypass; TRIF, TIR-domain-containing adapter-inducing interferon-β.

Figure 6

Western blot analysis of TLR3 expression levels in rat hippocampi. H, sham group; C, CPB group; S, sevoflurane-pre-conditioned group at T5.

Figure 7

Relative expression levels of TLR3 in rat hippocampi determined by western blot analysis. Values are expressed as the mean ± standard deviation. ^*P<0.05 compared with H group; ^ΔP<0.05 compared with C group. H, sham group; C, CPB group; S, sevoflurane-pre-conditioned group.

Figure 8

Apoptosis of hippocampal neurons as indicated by terminal deoxynucleotidyl transferase dUTP nick end labeling in (A) H group, (B) C group and (C) S group (magnification, ×400). H, sham group; C, CPB group; S, sevoflurane-pre-conditioned group.

Figure 9

Integrated Optical Density Average value of positive hippocampal neuronal cells in each group. Values are expressed as the mean ± standard deviation. ^*P<0.05 compared with H group; ^ΔP<0.05 compared with C group. H, sham group; C, CPB group; S, sevoflurane-pre-conditioned group.