Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Correspondence: Zhihou Liang, MD, PhD, Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022, China. Email: email@example.com
Anesthesia is related to cognitive impairment and the risk for Alzheimer's disease. Hypothermia during anesthesia can lead to abnormal hyperphosphorylation of tau, which has been speculated to be involved in anesthesia-induced cognitive impairment. The aim of this study was to investigate whether maintenance of the tau phosphorylation level by body temperature control during anesthesia could reverse the cognitive dysfunction in C57BL/6 mice.
Eighteen-month-old mice were repeatedly anesthetized during a 2-week period with or without maintenance of body temperature, control mice were treated with normal saline instead of anesthetics. Tau phosphorylation level in mice brain was detected on western blot, and cognitive performance was measured using the Morris water maze (MWM).
After anesthesia-induced hypothermia in old mice, tau was hyperphosphorylated and the cognitive performance, measured on MWM, was impaired. When body temperature was controlled during anesthesia, however, the tau hyperphosphorylation was completely avoided, and there was partial recovery in cognitive impairment measured on the MWM.
Hyperphosphorylation of tau in the brain after anesthesia is an important event, and it might be, although not solely, responsible for postoperative cognitive decline.
ALZHEIMER'S DISEASE (AD) is a neurodegenerative disease that has affected a growing elderly population throughout the world. AD is characterized by neuronal loss, the presence of extracellular aggregates of the β-amyloid peptide (Aβ), and intracellular neurofibrillary tangles, which are composed of hyperphosphorylated tau protein assembled in paired helical filaments.[1, 2] A growing body of evidence has suggested that abnormal hyperphosphorylation and aggregation of tau are crucial to neurodegeneration in AD.[3-6] The causes of tau phosphorylation and the nature of its postulated toxicity in AD, however, remain to be determined.
General anesthesia has provided immeasurable health and societal benefits for almost two centuries, and anesthesia is still irreplaceable in medical activities, but there is growing interest in the potential relationship between anesthesia and the onset and progression of neurodegenerative disorders such as AD.[7-9] It has been reported that anesthesia may be associated with cognitive impairment[10-13] and increased risk for AD, especially in individuals undergoing cardiac surgery who need deeper and longer anesthesia.[10, 15-17] Symptoms of cognitive impairment after surgery or anesthesia are described as ‘postoperative cognitive decline’ (POCD). Most researchers agree that the etiology of POCD following surgery/anesthesia is most likely multifactorial. The probable risk factors include anesthetics, other drugs given during surgery, age, changes in hemodynamics, and the hospital environment. Investigation is still needed, however, into the link between anesthetics and POCD as well as the link between POCD and the onset of AD.
Recently, our team, along with others, showed that anesthesia induced rapid and massive tau hyperphosphorylation at several epitopes.[18, 19] Based on these results, we suspected that tau hyperphosphorylation after anesthesia might explain the observed POCD. Planel et al. suggested that hyperphosphorylation of tau during anesthesia resulted not from anesthesia per se, but from the hypothermia consequent to anesthesia. Thus, if POCD was mediated by tau hyperphosphorylation, we hypothesized that POCD might be avoided by controlling the temperature during anesthesia. To investigate this hypothesis in the present study, we anesthetized C57BL/6 mice with or without body temperature maintenance and analyzed the relationship between the level of tau phosphorylation and the learning/memory performance using the Morris water maze (MWM).
Animals and group assignment
This study used old (18 months old) female C57BL/6 mice purchased from Hunan Slac Jynda Laboratory Animal Co. Ltd. (SJL Animal, Changsha, China). In the preliminary study, we had used young mice (approx. 14 weeks old) for the test, but the MWM results showed that there was no cognitive impairment in the young mice after single or repeated anesthesia. Negative MWM results in young mice prompted us to switch to old mice (18 months old) for the current study. These animals were allowed free access to food and water and randomly divided into two teams. For one team, the mice were killed, and their brains immediately removed, frozen in dry ice, and stored at −80°C until they were homogenized for western blot. The other team of mice underwent the MWM procedure (n = 60). In both teams, mice were further divided into three groups: the control group (group C), which received an equal volume of saline; the anesthesia group (group A), which received anesthetics; and the anesthesia plus normothermia group (group A + N), which received anesthetics and body temperature control. Animal experiments were performed in accordance with a protocol approved by the Ethics Committee on Laboratory Animals of Huazhong University of Science and Technology. Anesthesia was induced by i.p. injection of sodium pentobarbital (100 mg/kg bodyweight) or propofol (50 mg/kg bodyweight). Some mice were designed to be anesthetized by repeated i.p. injections of sodium pentobarbital (4 × 100 mg/kg in a 2-week period). Both anesthetics were obtained from Sigma (St Louis, MO, USA).
Total tau was detected using the polyclonal antibody 134d.[20, 21] Phosphorylated tau was detected with the polyclonal tau antibody pT205 (Biosource, Carlsbad, CA, USA) because Thr 205 was the site that changed most prominently after anesthesia in our previous study. The secondary antibody was peroxidase-conjugated goat anti-rabbit IgG from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).
Mouse forebrains were homogenized in radio immunoprecipitation assay (RIPA) lysis buffer (Cat.P0013B; Beyotime Biotechnology, Shanghai, China). Aliquots of the homogenates were immediately mixed with a triple volume of four-fold concentrated Laemmeli buffer and heated in a 95°C water bath for 5 min. The levels of tau and tau phosphorylation were determined on western blot. Phosphorylation-dependent and site-specific tau antibody pT205 was used to determine the extent of tau phosphorylation at the corresponding individual phosphorylation site. The blots were developed with an enhanced chemiluminescence kit (Cat.KC131037; Pierce, Loughborough, Leics, UK). Immunoreactivity of the blots was quantified densitometrically. For quantification, the phosphorylation levels of tau were normalized to the levels of total tau.
Measurement and control of body temperature
Using an indwelling rectal thermograph, the body temperature of the mice in group A was manually recorded every 5 min throughout the whole course of anesthesia. In group A + N, the temperature of the mice was controlled using an automated infant incubator (Cat.ZD16BB100; Midwest, Beijing, China) according to the manufacturer's instructions. Because the mice in the group C were conscious, the body temperature was not detected to avoid a stress reaction.
Morris water maze
The experimental apparatus was a circular pool (120 cm diameter and 50 cm depth) filled with water (25 ± 0.5°C), which was made opaque with Nestle full cream powdered milk. The pool surface was 30 cm below the edge of the metallic wall. The apparatus was located in a separate room containing several extra-maze cues and illuminated by two white lights (60 W). Four marks (north, south, east, and west) were equally spaced around the perimeter of the tank, which divided the pool into four equal quadrants. For the 5-day learning performance test (LPT), a circular goal platform (9 cm diameter and 19 cm height) was painted white and placed approximately 1 cm below the surface of the water. This platform was kept in the same position in the third quadrant throughout the LPT. The platform had a rough surface providing sufficient grip for the animals to climb on top of it (Fig. 1).
The procedure consisted of two different phases: a 5-day LPT and the subsequent 1-day spatial memory test (SMT).[22, 23]
In the LPT, each mouse received three training/learning sessions per day. The starting point of each session was the midpoint of the circumference of quadrants 1, 2, and 4 (Fig. 1). Mice were introduced into the maze facing the wall at one of the three designated starting points and allowed to escape onto the hidden platform. Each training/learning session lasted for 90 s or until the mouse located the platform. Mice that did not find the platform within 90 s were manually guided to the platform by the experimenter. After mounting the platform, mice remained on the platform for 20 s before they were taken back to the cage. Each mouse had been given at least 20 min of rest between the training/learning sessions. Each mouse was tested for learning performance on five consecutive days.
Data were collected using a video camera (Olympus, Tokyo, Japan) suspended above the pool that was interfaced with a video tracking system (Ethovision 3.0; Noldus Information Technology, Wageningen, Netherlands). The time to find the platform, calculated by averaging the three training/learning values, was used to represent learning performance each day. If the mouse failed to find the platform within 90 s, the maximum value of 90 s was automatically recorded.
After 5 days of the LPT, animals were subjected to the SMT on the sixth day. For this test, the platform was removed from the arena, and the animals were introduced into the maze and allowed to swim freely for 90 s. Mice who remembered the platform would spend more time searching for it in quadrant 3. Thus, percentages of time spent in quadrant 3 (target quadrant) were calculated as the index for spatial memory.
All numerical data are presented as mean ± SD and were analyzed with SPSS 11.5. Statistical significance was determined using one-way analysis of variance (ANOVA) followed by the Tukey test when appropriate. Differences with P < 0.05 were considered statistically significant.
Temperature control reversed tau hyperphosphorylation induced by 1-h anesthesia in mice
We have previously shown marked increases in tau phosphorylation in several sites in young mouse brain homogenates 1 h after the induction of anesthesia by pentobarbital. These phosphorylation sites included Thr 181, Ser 199, Thr 205, Thr 212, Ser 262, and Ser 404. We found that Thr 205 was the site that changed most prominently during anesthesia, therefore phosphorylation at Thr 205 was chosen to represent the phosphorylation of tau in the present study.
In the current study, we monitored the body temperature of anesthetized mice. After anesthesia, the body temperature of the mice in group A rapidly dropped from 37.9 ± 0.4°C to 30.2 ± 0.6°C. The temperature of mice in group A + N remained steady after anesthesia (38.1 ± 0.5°C before anesthesia to 37.8 ± 0.3°C after anesthesia). We then performed western blots on C57BL/6 mice (18 months old) 1 h after exposure to normal saline, pentobarbital, or propofol. Compared with group C (control group), tau in the group A mice was significantly hyperphosphorylated at Thr 205, but there was no significant change in the phosphorylation level at the same site of tau in the group A + N mice. There was no change in the quantity of total tau in both groups compared with the control (Fig. 2).
Single 1-h anesthesia did not induce cognitive impairment in mice during the MWM test
Twenty-four hours after the exposure of the single 1-h anesthesia, the mice were introduced to the MWM test. Compared with group C, there was no learning impairment detected in group A at the 5-day LPT or the SMT on the sixth day. There was also no statistical difference between group A + N and group C in the LPT or SMT (data not shown).
Temperature control reversed tau hyperphosphorylation induced by repeated anesthesia in mice
To enhance the sensitivity of the subsequent cognition measurement, we exposed old C57BL/6 mice to anesthetics (100 mg/kg pentobarbital or 50 mg/kg propofol) twice a week for 2 weeks (four repeated anesthesia exposures in a 2-week period). Then we analyzed the phosphorylation level of tau on western blot in cerebrum samples 2 h after the last exposure. We found that four exposures to anesthesia led to an increase in tau phosphorylation, which was indicated by an enhanced relative immunoreactivity and mobility shift detected with a Thr 205 phosphorylation-dependent antibody (Fig. 3), as well as that after a single 1-h exposure. There was also a mobility shift in total tau without a significant change in immunoreactivity, which was observed using the phosphorylation-independent total tau antibody 134d (Fig. 3).
Temperature control had a mild protective effect on anesthesia-induced MWM performance impairment in old mice
Twenty-four hours after the last exposure of repeated anesthesia, the mice were introduced to the MWM test (n = 20 in each group). Compared with group C, a significant increase in time to find the platform was observed in group A mice on days 2 and 3 (P < 0.01) of the 5-day LPT (Fig. 4a). Although there was still a significant difference between the escape latency of group A + N and group C (P < 0.05), the group A + N mice showed a mild decrease in the time to find the hidden platform compared with the group A mice (P < 0.05) on the third day of training. This indicates that repeated anesthesia induced learning impairments in old mice, and maintenance of the normal body temperature eased this complication to some extent.
When the platform was removed from the pool for the SMT on day 6, a significant effect was observed across the three groups. Group A mice spent less time in the target quadrant (quadrant 3, Fig. 1) compared with the control mice (P < 0.01; Fig. 3b), and the group A + N mice showed a slight increase in the time spent in the target quadrant compared with group A (P < 0.05; Fig. 4b). Similar to the LPT results, a partly protective effect of anesthesia-induced spatial memory impairment in group A + N mice was observed when compared with group A (Fig. 4b).
As is well known, aging is considered to be the most important risk factor for AD. In addition, other factors are also suspected to be associated with AD, such as common procedures in surgery, anesthesia and the cold environment in the operating room.[7, 25-27] The aim of the present study was to investigate whether maintenance of the tau phosphorylation level by body temperature control during anesthesia could reverse the cognitive dysfunction in C57BL/6 mice. It should be noted that the goal of this study was to investigate ways to reduce the incidence or extent of neurodegenerative complications from anesthesia but not to oppose anesthesia per se. Then we found that the anesthesia-induced tau hyperphosphorylation almost disappeared after temperature control, and the cognitive impairment in anesthetized mice was partially improved.
As reported in 2007, Planel et al. observed an effect of anesthesia on tau phosphorylation in vivo, and soon we confirmed this phenomenon. In their research, Planel et al. suggested that hyperphosphorylation of tau after anesthesia was elicited by anesthesia-induced hypothermia, which destroyed the balance of phosphorylation and dephosphorylation by down-regulation of protein phosphatase 2A (PP2A) activity. Other studies insisted that tau hyperphosphorylation after anesthesia was induced by ether stress as well as stress in cold-water swimming.[29, 30] Nevertheless, because tau hyperphosphorylation is one of the neuropathologic markers in AD brains, it was speculated that hyperphosphorylated tau might be the mechanism of POCD or the increasing risk of AD. The present results showed that repeated anesthesia caused a significant impairment in learning performance and spatial memory in old C57BL/6 mice. Fortunately, we found that the cognitive impairment could be partially avoided by reversing the anesthesia-induced tau hyperphosphorylation with body temperature control. When body temperature was controlled during anesthesia, however, the tau hyperphosphorylation was completely avoided while there was partial recovery in cognitive impairment. From only these experiments, we cannot conclude that there is a clearly causal relationship between tau hyperphosphorylation and impairment of cognitive performance in MWM. Nevertheless, at least, these results indicate that anesthesia-induced tau hyperphosphorylation in the brain is probably associated with POCD in old mice. If prevention or a reversal of both tau hyperphosphorylation and concomitant cognitive decline is observed in other animal models or by using other methods than temperature control used in the present study, causal connections between tau hyperphosphorylation and cognitive impairment would be identified.
Interestingly, body temperature control during anesthesia could completely reverse tau hyperphosphorylation, and it could offer only partial protection against cognitive impairment. There are two possible explanations for this phenomenon. One is that anesthesia-induced tau hyperphosphorylation is a key factor for POCD, although not the only one. The other possibility is that, if body temperature is maintained at a normal level during anesthesia, it provides a potential protective effect by reversing tau hyperphosphorylation, but gives up the hypothermia-induced protection in some other aspects that have been previously identified.[31-35] Although more and more researchers are questioning the adverse effects of hypothermia induced by anesthesia or surgery, studies have shown that hypothermia has neuronal protective effects.[33, 35, 36]
Additionally, it should perhaps be noted that the sensitivity of behavioral tests including MWM can be influenced by a ceiling effect and a floor effect,[37-39] which may explain the inconsistent MWM results between different test days in this study. The differences in learning or memory between the groups could be identified only when the difficulty of the task is appropriate to the animals. During the 5-day LPT, the mission on the first day may be too difficult or complicated, and that on the fourth and fifth days may be too easy or simple for all the mice, so the differences in learning ability among the groups could not be identified. The mission of learning on the second and third days, and the mission of memory on the sixth day may be at an appropriate level of difficulty, enabling the differences among the three groups to be successfully detected.
The present results show that body temperature control may be able to relieve the cognitive impairment to some extent, but we should not rush to use this approach to reduce cognitive complications during operations involving anesthesia. In other words, we should distinguish different surgical treatment and provide different temperature management. For cardiac surgery, in which cerebral blood flow might be temporarily cut off, reducing body temperature has been shown to be a rigid requirement for providing brain protection from ischemia or reperfusion injury. Thus, in some types of surgery, increasing the risk of some loss of cognitive function is necessary to prevent brain damage. For common surgery involving anesthesia, maintaining body temperature at or close to the normal level is important to avoid the increased risk and extent of POCD. Thus, temperature control is a double-edged sword. Other ways to reduce the risk of POCD would be more valuable, especially in cardiac surgery, and it should be extensively explored.
In short, we found that anesthesia-induced tau hyperphosphorylation almost disappeared after temperature control in the current study, and the cognitive impairment was partially improved. This indicates that tau may play an important role in POCD. Exploring the mechanism of POCD may provide important information for the prevention and treatment of AD.
This work was supported by Grants from the National Natural Science Foundation of China (Grant number: 30901386), the Wuhan Science and Technology Bureau (Grant number: 200960323132), and the Research Fund for the Doctoral Program of Higher Education of China (Grant number: 200804871026). There is no conflict of interest.