Sevoflurane postconditioning improves spatial learning and memory ability involving mitochondrial permeability transition pore in hemorrhagic shock and resuscitation rats

Abstract

Background: Hemorrhagic shock induces the cognitive deficiency. Sevoflurane postconditioning has been documented to provide neuroprotection against ischemic-reperfusion injury by suppressing apoptosis. Mitochondrial permeability transition pore (mPTP) plays an important role in apoptosis, but it is unknown if the protective effect of sevoflurane postconditioning on hemorrhagic shock and resuscitation is associated with the change of mPTP opening. Hence, the aim of the study was to find out the precise mechanism of the sevoflurane postconditioning.

Methods: Sprague Dawley rats were subjected to hemorrhage shock for 60 min and then exposed to 2.4% sevoflurane for 30 min at the instant of reperfusion. Additionally, an opener (atractyloside) or an inhibitor (cyclosporine A) of mPTP was used in the animal model before sevoflurane postconditioning. Rats were randomly assigned into groups of Sham, Shock, Shock+Sevoflurane, Shock+Atractyloside, Shock+Sevoflurane+Atractyloside, Shock+Cyclosporin A, and Shock+Sevoflurane+Cyclosporin A treatment. Rat behavior was assessed for 5 days by Morris water maze 72 hr after surgery, and then hippocampus CA1 region was immunohistochemically stained. Brains were harvested 24 hr after surgery to detect the protein expression levels of Bcl-2, Bax, and cytochrome C by Western blot, the changes of mPTP opening, and mitochondrial membrane potential (MMP).

Results: We found that sevoflurane postconditioning significantly improved rats' spatial learning and memory ability, down-regulated the expression of Bax, cytochrome C, and caspase-3, up-regulated the expression of Bcl-2, decreased the mPTP opening, and increased the MMP. The neuroprotective effect of sevoflurane postconditioning was abolished by atractyloside, but cyclosporin A played the similar protective role as sevoflurane postconditioning.

Conclusion: These findings proved that sevoflurane postconditioning improved spatial learning and memory ability in hemorrhage shock and resuscitation rats, the mechanism of which may be related to block mPTP opening, increase MMP, and reduce neuron apoptosis in the hippocampus.

Keywords: apoptosis; hemorrhagic shock and resuscitation; mitochondrial membrane potential; mitochondrial permeability transition pore; sevoflurane postconditioning; spatial learning and memory.

Figure 1

Procedures of hemorrhagic shock and resuscitation rat model and animal grouping. Atr, atractyloside; CsA, cyclosporin A; Sev, sevoflurane postconditioning

Figure 2

Mean arterial pressure during hemorrhage shock and resuscitation in seven groups. Compared with the baseline period, the mean arterial pressure of six groups was <50 mmHg except for Sham group. Then, the mean arterial pressure returned to baseline level after resuscitation. N = 6 in each group, values are presented as mean ± standard error of mean, *p < .01 and **p < .001 versus baseline level

Figure 3

Effects of sevoflurane postconditioning on spatial learning and memory in the Morris water maze. Escape latency (expressed in s; a); swimming distances in target quadrant (expressed in cm; b); numbers of platform crossings (expressed in times; c); proportion of time spent in the target quadrant (expressed in %; d); swimming speed (expressed in cm/s; e). N = 6 in each group, data are presented as mean ± standard error of mean, **p < .01 and ***p < .001 versus Sham group, ^# p < .05, ^## p < .01, and ^### p < .001 versus Shock group, ^&& p < .01 and ^&&& p < .001 versus Shock+Sev group

Figure 4

Effect of sevoflurane postconditioning on the expression of protein following hemorrhagic shock and resuscitation. Representative immunoblots of Bcl‐2, Bax, Cyt C, and β‐Actin proteins (a) in hippocampus in all groups. The proteins were determined 24 hr after the intervention. Quantification of the relative expression of Bcl‐2 (b), Bax (c), and Cyt C (d) were detected, and β‐Actin was respectively served as the loading control. N = 6 in each group, values are presented as mean ± standard error, ***p < .001 versus Sham group, ^### p < .001 versus Shock group, and ^&&& p < .001 versus Shock+Sev group

Figure 5

Effect of sevoflurane postconditioning on the staining of caspase‐3 immunoreactive positive cells following hemorrhagic shock and resuscitation. Immunochemical staining of caspase‐3 in the hippocampus CA1 region after the water maze test were shown in the Sham (a), Shock (b), Shock+Sev (c), Shock+Atr (d), Shock+Sev+Atr (e), Shock+CsA (f), and Shock+Sev+CsA (g) groups. Quantitative analysis of caspase‐3 expression in the hippocampal CA1 region was shown in (h). N = 6 in each group, data are presented as mean ± standard error, ***p < .001 versus Sham group, ^### p < .001 versus Shock group, and ^&&& p < .001 versus Shock+Sev group

Figure 6

Effect of sevoflurane postconditioning on the opening of mitochondrial permeability transition pore following hemorrhagic shock and resuscitation. The opening of mitochondrial permeability transition pore in isolated mitochondria from hippocampus was measured 24 hr after resuscitation. The changes in mitochondrial permeability transition pore opening were detected with calcein‐AM as a fluorescence indicator by confocal microscopy in the Sham (a), Shock (b), Shock+Sev (c), Shock+Atr (d), Shock+Sev+Atr (e), Shock+CsA (f), and Shock+Sev+CsA (g) groups. Quantitative analysis for the relative changes in calcein fluorescence of the opening of mPTP in the hippocampal CA1 region was shown in (h). N = 6 in each group, data are presented as mean ± standard error, ***p < .001 versus Sham group, ^### p < .001 versus Shock group, and ^&&& p < .001 versus Shock+Sev group

Figure 7

Effect of sevoflurane postconditioning on mitochondrial membrane potential (MMP) following hemorrhagic shock and resuscitation. The mitochondrial membrane potential in isolated mitochondria from hippocampus was measured 24 hr after resuscitation in the Sham (a), Shock (b), Shock+Sev (c), Shock+Atr (d), Shock+Sev+Atr (e), Shock+CsA (f), and Shock+Sev+CsA (g) groups. Summarized data for the relative changes in JC‐1 fluorescence were analyzed in (h). N = 6 in each group, data are presented as mean ± standard error, ***p < .001 versus Sham group, ^### p < .001 versus Shock group, and ^&&& p < .001 versus Shock+Sev group