Western blot analysis showed that isoflurane exposure time dependently altered phosphorylation of hippocampal tau protein at the Ser262 site (tau[pS262]) in both WT and transgenic APP695 mice (Fig. 3, A and B). However, isoflurane exposure had no significant effect on the expression of total tau protein in the hippocampi of all mice (Fig. 3C, P ≥ 0.54). The widths of the 99% confidence intervals are all <46% of the corresponding means and an associated SE is 2% (Table S2, Supplemental Digital Content 3, http://links.lww.com/AA/A926). In WT mice, the levels of hippocampal tau[pS262] were significantly increased on days 1 and 3 after isoflurane exposure (Fig. 3A, P < 0.0001 for day 1 and P = 0.0008 for day 3), and the tau phosphorylation reached peak level on day 1 and returned to baseline (levels in control group) on day 7 after isoflurane (Fig. 3A, P = 0.8 for day 7). In the transgenic APP695 mice, the levels of hippocampal tau[pS262] were markedly increased on days 1, 3, and 7 after isoflurane exposure (Fig. 3B, P < 0.0001 for all 3 time points), and the tau phosphorylation also reached peak level on day 1 but did not return to baseline (levels in control group) on day 7 after isoflurane (Fig. 3B).
Compared with WT mice, transgenic APP695 mice displayed increased levels of hippocampal tau[pS262] before and after isoflurane exposure (Fig. 4). In normal conditions, the level of hippocampal tau[pS262] in the APP695 mice was significantly higher than in WT mice (Fig. 4A, P < 0.0001). On days 1, 3, and 7 after isoflurane exposure, the levels of hippocampal tau[pS262] in the APP695 mice were also significantly higher than those in WT mice (Fig. 4, B−D, P < 0.0001).
In the present study, we investigated the role of hippocampal tau protein phosphorylation in isoflurane-induced cognitive dysfunction in a transgenic mouse model of AD. We found that isoflurane exposure markedly impairs spatial learning and memory in transgenic APP695 mice. Isoflurane-induced cognitive dysfunction may be correlated with phosphorylation of hippocampal tau protein at the Ser262 site. Our results demonstrate that isoflurane may induce cognitive impairment by enhancing phosphorylation of hippocampal tau protein at the Ser262 site, and this effect is more significant in APP695 mice, suggesting isoflurane might have a detrimental effect on the development of AD by enhancing hippocampal tau protein phosphorylation and cognitive dysfunction.
Our data showed that isoflurane MAC was higher in transgenic APP695 mice than in WT mice (Table 1), which is consistent with 2 studies from other laboratories that used other AD mouse models.23,24 Previous studies have shown that Aβ oligomers directly activate N-methyl-D-aspartate (NMDA) receptors25 and that Aβ also induces astrocytic glutamate release, which in turn activates extrasynaptic NMDA receptors on neurons.26 An increase in the cytosolic concentration of Ca2+ induced by Aβ oligomers in cortical neurons is prevented by AP5, a broad spectrum NMDA receptor antagonist.25 In our study, we observed that Aβ(1–42) expression was markedly increased in APP695 mice (Fig. 1). Therefore, the excitatory effect of Aβ may contribute to increased isoflurane MAC in APP695 mice. To investigate the effect of isoflurane exposure on cognitive function in our AD mouse model, we treated both APP695 and WT mice with 1 MAC isoflurane to reach the same anesthesia depth. MAC is defined as the concentration of inhaled anesthetic that is needed to prevent movement in 50% of subjects in response to surgical (pain) stimulus. MAC value is accepted as a valid measure of potency for inhaled anesthetics, which remains fairly constant for a given species. Thus, by treating animals with 1 MAC isoflurane, we can avoid overdose of isoflurane-produced off-target effects in the 2 types of mice. However, APP695 mice may have been overdosed with isoflurane relative to WT mice for tau phosphorylation, because it is well known that not all anesthetic end points have the same sensitivity.
Inhaled anesthesia has a variety of effects, both short term and long term, on central nervous system function. Isoflurane exposure has been shown to influence cognitive function in rodent animals, but previous studies have reported controversial results regarding the effect of isoflurane anesthesia on cognitive function.4,22,27–30 For instance, Rammes et al.28 showed that isoflurane reversibly improves cognitive function and long-term enhancement, and Su et al.29 also observed that repeated isoflurane exposure improves spatial memory. However, several other studies have illustrated that isoflurane exposure leads to cognitive dysfunction.4,22,27,30 These discrepancies may have been due to different ages of animals used in different studies. It has been reported that isoflurane anesthesia impairs acquisition of hippocampus-dependent spatial memory 2 weeks after cessation of anesthesia in aged rats,31 but in adult rats, previous isoflurane exposure produces improved spatial memory performance 2 weeks later.32 Isoflurane treatment with different concentrations and exposure times might also produce differential effects. In our study, 12-month-old male mice received 1.0 MAC of isoflurane treatment for 4 hours, and our data in the Morris Water Maze test showed that isoflurane exposure significantly impaired spatial learning and memory. Compared with WT mice, transgenic AD mice (APP695 mice) displayed enhanced impairment of spatial learning and memory after isoflurane exposure, suggesting that isoflurane may promote AD development by aggravating cognitive dysfunction.
Tau protein can functionally bind microtubules, which is crucial for the processes of neuronal outgrowth and axonal integrity.33 Tau protein plays an important role in synaptic plasticity and memory formation, and tau hyperphosphorylation may contribute to cognitive dysfunction in tauopathies.34 The hyperphosphorylated form of tau protein is present in neurofibrillary tangles.35 The formation of neurofibrillary tangles is considered to be a common mechanism underlying neurodegeneration in the pathogenesis of AD.36,37 In addition, the progression of tau phosphorylation has been shown to correlate with the evolution of tau pathology.38 The development of intraneuronal lesions as a result of the progressive deposition of hyperphosphorylated tau at specific brain regions (such as hippocampus and cortex) plays a key role in the pathological process of AD. It has been reported that tau phosphorylation at Ser262 is relevant to the pathogenesis of AD.39,40 It is noteworthy that phosphorylation at this residue is modified in the brain of patients with AD.39 In double transgenic mice (APPSwe/tauVLW) that reproduce Aβ and tau pathologies, tau phosphorylation at Ser262 correlates with an increase in the formation of filamentous tau aggregates showing a diameter similar to that of tau filaments observed in AD.40 Moreover, tau hyperphosphorylation has been associated with memory impairment after isoflurane exposure.22 Tan et al.22 observed that isoflurane anesthesia combined with hypothermia, but not isoflurane anesthesia alone, increases tau phosphorylation at the Thr205 and Ser396 sites in the hippocampus.22 They also found that approximately 45% of protein phosphatase 2A are inhibited in isoflurane-anesthetized rats without temperature control.22 Their results indicate that tau hyperphosphorylation is not induced by isoflurane anesthesia directly, but may be a consequence of isoflurane anesthesia-produced hypothermia, because the inhibition of phosphatase activity and subsequent tau hyperphosphorylation could occur in hypothermic rats. Thus, tau hyperphosphorylation may be associated with isoflurane-induced spatial learning and memory impairment through isoflurane anesthesia-produced hypothermia. Recently, Dong et al.12 observed that isoflurane exposure increases tau phosphorylation at the Ser262 site both in vitro and in vivo. In their in vitro study, primary neurons were exposed to 2% isoflurane for 6 hours and were harvested at the end of isoflurane exposure12; in their in vivo study, 5- to 8-month-old mice were treated with 1.4% isoflurane for 2 hours, and whole brain tissues were harvested at 6, 12, and 24 hours after isoflurane anesthesia.12 By conducting Western blot analysis, they showed that isoflurane exposure increases the level of phospho-tau at the Ser262 site in both primary neurons and whole brain tissues.12 They also found that isoflurane-induced tau phosphorylation is mediated by caspase activation and Aβ generation.12 To further investigate the relationship between hippocampus-dependent cognitive deficits and hippocampal tau phosphorylation after isoflurane anesthesia in aged animals, we determined whether isoflurane exposure alters tau phosphorylation in the hippocampus of 12-month-old mice. We found that isoflurane exposure time dependently increases hippocampal tau phosphorylation at the Ser262 site. Compared with WT mice, transgenic AD mice (APP695 mice) displayed greater enhancement of hippocampal tau phosphorylation after isoflurane exposure. Our results suggest that isoflurane-induced cognitive dysfunction may be correlated with phosphorylation of hippocampal tau protein at the Ser262 site. Because of its role in tau phosphorylation at the Ser262 site, the effect of isoflurane exposure on the pathology of AD should be studied in the future.
In conclusion, our study demonstrates that isoflurane may induce cognitive dysfunction by enhancing phosphorylation of hippocampal tau protein at the Ser262 site and this effect is more significant in transgenic APP695 mice. Tau phosphorylation at the Ser262 site has been illustrated to prevent tau from binding and stabilizing microtubules, which is associated with increasing severity of neuronal cytopathology in AD.41–43 Therefore, hippocampal tau phosphorylation might be involved in the molecular mechanisms by which isoflurane anesthesia impairs cognitive function and promotes AD development, and isoflurane might be a deleterious factor contributing to the neuropathogenesis of AD.
The authors are grateful to the invaluable assistance and insight from Professor Haiyan Yang (Department of Epidemiology and Biostatistics, Zhengzhou University College of Public Health, Zhengzhou, Henan, China) with regard to statistical analysis.
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