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Melatonin pretreatment prevents isoflurane-induced cognitive dysfunction by modulating sleep–wake rhythm in mice
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Research Report
Melatonin pretreatment prevents isoflurane-induced cognitive dysfunction by modulating sleep–wake rhythm in mice Q1
Tianjiao Xia1, Yin Cui1, Shuaishuai Chu, Jia Song, Yue Qian, Zhengliang Man, Xiaoping Gunn Department of Anesthesiology, Affiliated Drum Tower Hospital of Medical Department of Nanjing University, 321 Zhong Shan Road, Nanjing, Jiangsu 210008, PR China
art i cle i nfo
ab st rac t
Article history:
Background: Sleep plays an important role in memory processing. However, its role in
Accepted 22 October 2015
anesthesia-induced cognitive dysfunction was not revealed. Our study sought to investigate the connection between the cognition decline and sleep–wake rhythm disorders after
Keywords:
long-term isoflurane anesthesia in mice. Also, we examined the effect of exogenous
Sleep–wake rhythm
melatonin pretreatment on both cognitive function and circadian rhythm. Furthermore,
Isoflurane
we discussed whether NR2B (N-methyl–D-aspartate receptor 2B subunit)–CREB (cAMP-
Cognitive
response element binding protein) signaling pathway was involved in this course.
Melatonin
Methods: 2-month-old male C57/BL-6J mice were submitted to long-term anesthesia using
NR2B
1% isoflurane from CT (Circadian Time) 14 to CT20. Melatonin pretreatment were
CREB
conducted before anesthesia for 7 Days. Intellicage for mice and Mini-Mitter were applied to monitor spatial memory and gross motor activity which can reflect cognition and sleep– wake rhythm. Messenger RNA and protein expression of right hippocampus NR2B and CREB were examined by RT-PCR and Western blot. Results: 6 h isoflurane anesthesia led to impaired spatial memory from Day 3 to Day 10 in mice accompanied by the disruption of sleep–wake rhythm. Meanwhile, the hippocampus CREB and NR2B expression declined in step. Melatonin pretreatment ameliorated disturbed sleep–wake cycle, improved isoflurane-induced cognitive dysfunction, and reversed the down-regulation of CREB and NR2B expression. Conclusions: Our data demonstrate that sleep–wake rhythm is involved in the isofluraneinduced cognition impairment and pretreatment of melatonin has a positive effect on circadian normalization and cognition reversal. Also, NR2B–CREB signaling pathway has a critical role in this process. This study provides us a new strategy for anesthesia-induced cognitive dysfunction therapy. & 2015 Published by Elsevier B.V.
n
Corresponding author. Fax: þ86 25 8310 5502. Corresponding author. Fax: þ86 25 8310 5502. E-mail addresses: [email protected] (T. Xia), [email protected] (Y. Cui), [email protected] (S. Chu), [email protected] (J. Song), [email protected] (Y. Qian), [email protected] (Z. Ma), [email protected] (X. Gu). 1 Tianjiao Xia and Yin Cui contributed equally to this work and should be considered as co-first authors. nn
http://dx.doi.org/10.1016/j.brainres.2015.10.036 0006-8993/& 2015 Published by Elsevier B.V.
Please cite this article as: Xia, T., et al., Melatonin pretreatment prevents isoflurane-induced cognitive dysfunction by modulating sleep–wake rhythm in mice. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.10.036
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1.
Introduction
Postoperative cognitive dysfunction (POCD) is a common and well-known complication after surgery (Steinmetz et al.,
2.
Results
2.1 Cognitive performance of animals after anesthesia and melatonin treatment
2009). It features disturbance of memory, attention, consciousness, information processing and sleep–wake cycle, leading to postoperative morbidity and mortality (Bekker and Weeks, 2003). The etiology of POCD is likely multifactorial. Different preoperative and operative factors are associated with the development of cognitive dysfunction. Factors such as increasing age, presence of diabetes, hypotension during surgery and anesthesia were believed to be involved (Shaw et al., 1989; Grocott et al., 2005). Sleep, which shares some common neuronal mechanisms with general anesthesia (Allada, 2008; Kelz et al., 2008) is important to memory processing (Turner et al., 2007). It is known to be poor in the hospital (Vico-Romero et al., 2014), and the prevalence of sleep disorders showed a marked increase in patients admitted to the Intensive Care Unit (Gomez, 2013). The disturbance of sleep–wake rhythm is considered to be one of the most important causes of sleep disorders. This rhythm is influenced by various factors, including light (Friedman et al., 2012), melatonin (Koch et al., 2009) and orexin (Diniz et al., 2010). It has also been reported that general anesthesia could impact the sleep–
Following anesthesia, animals were housed in the intellicage. Their continuous locomotive activity was tracked in terms of the number of visits and their correct rate of visits were also recorded In Place learning phase, the results showed a reducement in locomotive activity from the first day after isoflurane anesthesia based on an decreased numbers of visits in anesthesia group (Po0.05). Significantly increased on correct rate of visit two days after isoflurane anesthesia were observed and later significantly decreased from Day 3 to Day 7 when compared with control group (Po0.05) were observed, suggesting that isoflurane anesthesia could lead to cognitive dysfunction in mice. The restoration of normal locomotive activity and correct corner visits were occurred in anesthesia group 10 days and 11 days respectively after anesthesia. Compared with anesthesia group, pretreated with melatonin mice when underwent a 6 h isoflurane anesthesia showed a significant increased on the numbers of visits on Day 1, Day 4, day 5, Day 6 and Day 7. Meanwhile, higher correct visits were also detected from Day 3 to Day 7 (Po0.05). During place reversal learning phase, melatonin pretreated mice showed that the locomotive activity and correct rate of visit recovered to control level on Day 13 and Day 14. (P40.05) (Figs. 1A and E).
wake rhythm. Propofol anesthesia, for instance, induced phase advances when administered at the rest/activity transition point(Challet et al., 2007).Moreover, inhalational anesthetics sevoflurane and isoflurane caused phase delays when administered during the subjective Day (Ohe et al., 2011; Cheeseman et al., 2012). However, whether the disruption of sleep–wake rhythm resulted from anesthesia plays an important role in cognitive dysfunction remains unknown. Melatonin, a hormone secreted by the pineal gland during the dark period of the Day, mediates a diverse array of biological and physiological actions. In addition to its effects on antioxidant, immunomodulatory, and oncostatic activities (Pandi‐Perumal et al., 2006), melatonin has an important role in sleep and circadian regulations. Small dosages of melatonin were found to regulate sleep–wake rhythm (Hughes et al., 1998) and ameliorate the sleep quality by normalizing sleep–wake cycle (Finati et al., 2013). Despite these advances, little is known about the effect of melatonin on cognitive function after anesthesia. In this study, we asked whether the sleep–wake disorders induced by anesthesia contributed to the development of cognitive dysfunction, and whether this effect of anesthesia
2. 2 Gross motor activity of animals after anesthesia and melatonin treatment A 6 h anesthetic during the subjective night caused a persistent and marked shift of motor activity rhythm. On Day 1 after anesthesia, compared with control group, anesthesia mice demonstrated greater spontaneous activity levels on CT0, 2 and 4(1771.207270.97 vs. 1242.507329.38, P¼0.022; 1737.507 399.40 vs. 1203.307123.43, P¼ 0.008; 1578.707297.58 vs. 691.67107.49, Po0.001). This phenomenon lasted for at least 4 Days after anesthesia: CT4 (1498.807 310.66 vs. 1062.20796.64, P¼0.008); CT6 (1173.507 221.53 vs. 672.337141.96, Po0.001). On Day 1 after anesthesia, when compared with anesthesia group, mice which pretreated with melatonin showed lower activity levels during sleeping period on CT0 (1305.007432.50, P ¼0.048), CT6(685.177180.27, Po0.001), and this effect lasted for at least 4 Days after anesthesia on CT0, 2, 4 and 6 (828.507180.09, Po0.001; 837.83771.63, Po0.001; 1152.707199.54, P¼ 0.048; 668.177191.17, Po0.001). These marked difference indicate that pretreatment with melatonin could normalize the disrupted sleep–wake rhythm caused by isoflurane anesthesia (Fig. 2A and C).
could be blocked by exogenous melatonin treatment. By using an international advanced cognitive behavioral instrumentautomatic mouse intellicage, we found that 6 h isoflurane anesthesia induced cognitive dysfunction, accompanied by the disruption of sleep–wake rhythm. Pretreatment of melatonin had a positive effect on sleep–wake cycle normalization and cognition reversal.NR2B–CREB signaling pathway was thought to be among the molecular mechanisms.
2.3 The expression of CREB and NR2B at the hippocampus level after anesthesia and melatonin treatment Compared with control group, the expression of NR2B mRNA had decreased on Day 1 (0.5170.09 vs. 1.1270.15, Po0.001), Day 3 (0.6270.15 vs. 1.1770.16, Po0.001), and Day 7 (0.6970.12 vs. 1.1370.14, P¼ 0.002) after anesthesia in the hippocampus. Compared with anesthesia group, melatoninþanesthesia group
Please cite this article as: Xia, T., et al., Melatonin pretreatment prevents isoflurane-induced cognitive dysfunction by modulating sleep–wake rhythm in mice. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.10.036
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Fig. 1 – Number and correct rate of visits during Day 1–14. Schematic presentation of experimental design. (A). The number of visits (C,E) and correct rate of visits (B,D)in control group (open square), Iso group (closed square), melatonin group (open circle), Iso þMel group (closed circle) are all compared in pair. Data are showing with Mean7S.D. ( aPo0.05, compared with control group, bPo0.05, compared with Isoflance group, cPo0.05, compared with Melatonin group).
showed increased NR2B mRNA level on Day 1 (1.0470.15, Po0.001), Day 3 (1.0570.15, P¼0.002), and Day 7 (1.0670.18, P¼0.010). On Day 14 after anesthesia, the NR2B mRNA level was similar in all groups. Compared with control group, the expression of anesthesia group CREB mRNA was significantly decreased on Day 1 (0.6470.07 vs.1.2870.10, Po0.001), Day 3 (0.6670.12 vs. 1.1770.17, Po0.001), Day 7(0.7270.10 vs. 1.1570.14, P¼0.005). When compared with anesthesia group, Pre-administration melatonin increased the declining CREB mRNA expression after anesthesia on Day 1 (0.9770.15, P¼0.002), Day 3 (1.1670.14, Po0.001), Day 7 (1.1270.16, P¼0.008). On Day 14, the CREB mRNA expression was similar in each group (Fig. 3A and B). We next explored the expression of p-NR2B (Tyr 1472) and p-CREB (Ser 133) in the hippocampus in all groups. The pCREB expression was significantly increased on Day 1 after anesthesia when compared with control group (1.8870.17 vs. 170.14, Po0.001). On Day 3, the levels of NR2B phosphorylation (0.1170.02) and CREB phosphorylation (0.2970.04) in
anesthesia group were significantly decreased compared with the control group (pNR2B, 1.0070.10, Po0.001; pCREB, 170.07, Po0.001). While when compared with anesthesia group, the expression of pNR2B (0.9670.12, Po0.001) and pCREB (0.5770.05, Po0.001) in melatoninþanesthesia group were upregulated. On Day 14, the pNR2B 1.1270.11) and pCREB (1.0870.10) expression in anesthesia group retured to control level (pNR2B, 1.0070.04, P ¼ 0.250; pCREB, 170.05, P¼ 0.794), and no differences were observed in all groups (Fig. 4A and D).
3.
Discussion
Exposed to general anesthesia has been linked to a persistent deficit of cognitive function in both humans and rodents, and this effect is independent of underlying disease or tissue injury (Stratmann et al., 2014). In our study, 6 h anesthesia was conducted mainly because that operations lasting for a long time are increasingly found clinically and the negative effect
Please cite this article as: Xia, T., et al., Melatonin pretreatment prevents isoflurane-induced cognitive dysfunction by modulating sleep–wake rhythm in mice. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.10.036
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Fig. 2 – Gross motor activity amounts averaged (Mean7S.D) in 2-h intervals during Day 1–14.Schematic presentation of experimental design.(A). The amounts of activity (B,C)in control group (black line), anesthesia group (red line), melatonin group (green line), anesthesiaþmelatonin group (blue line) are all compared in pair. (aPo0.05, compared with control group, b Po0.05, compared with Isoflane group, cPo0.05, compared with Melatonin group). Horizontal gray bar: light phase of the day; horizontal black bar: dark phase of the day.
cannot be ignored. In this study, we found impaired spatial memory from Day 3 to Day10 of the study and memory phase in 2-month-old mice after long-term isoflurane anesthesia. As with our study, Zhu also found that isoflurane could induce persistent and progressive memory impairment in young rodents (Zhu et al., 2010). These findings suggest that anesthesia can be a cause of cognitive deficits. At the meanwhile, striking changes were also discovered in gross motor activity in mice after 6 h isoflurane anesthesia monitored by Mini-Miter. Despite the fact that fewer activities do not mean sleep, the activity pattern measured by MiniQ2 Miter may indicate sleep-related mechanism explaining our research (Fernandez et al., 2014). Researchers have found that
a deeper level of isoflurane anesthesia could disturb or delay the restoration of normal sleep–wake architecture (Takahashi et al., 2001; Jang et al., 2010). Cheeseman's research and our previous study also saw thatclock gene expression, which is important in regulating the sleep–wake rhythm, changed significantly after isoflurane anesthesia (Cheeseman et al., 2012; Xia et al., 2015). Other biological factors like hormonal desynchronization, liver dynamics, blood chemistry also cannot be ignored (Celic-Spuzic, 2011; Kajimoto et al., 2014; Schwarzkopf et al., 2013). Nevertheless, all these data indicate that sleep–wake rhythm changed markedly after isoflurane anesthesia in mice.
Please cite this article as: Xia, T., et al., Melatonin pretreatment prevents isoflurane-induced cognitive dysfunction by modulating sleep–wake rhythm in mice. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.10.036
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in CA1 neurons which may produce hippocampal-dependent cognitive improvement after anesthesia. In our experiment, melatonin intervention was used to retrieve sleep disturbance. It is primarily synthesized and secreted at night and that the circadian rhythm of its production is determined by the prevailing light–dark cycle (Axelrod et al., 1965). In our experiment, CT2 was chosen to be exogenous melatonin supplement as the period of low level melatonin secretion. We found that pretreated with melatonin could reverse isoflurane anesthesia induced cognitive dysfunction. Dispersyn demonstrated that general anesthesia had disturbing effects on the circadian rhythm of plasma melatonin in rats under normal light conditions (Dispersyn et al., 2010). Melatonin has the capacity to change the timing of circadian rhythms and functions, synchronize them with prevailing LD cycles and thereby ameliorate the sleep quality in patients with sleep– wake rhythm disturbance (Wyatt et al., 2006; Srinivasan et al., 2010; Finati et al., 2013). The role of melatonin in regulating the sleep/wake cycle is mediated through MT1 and MT2 melatonin receptors present in SCN. These two melatonin receptors modulate gamma‑aminobutyric acid (GABA) receptors in the SCN differentially; these GABA receptors, reportedly, both phase shift and synchronize SCN clock cells (Kumar and Singh, 2009). Furthermore, we explored the possible molecular mechanism of cognitive dysfunction and sleep–wake rhythm disorders induced by isoflurane. We found that the downregulation of cognitive function is relatively synchronous Fig. 3 – Quantitative real-time reverse transcription-polymerase chain reaction analysis of changes of hippocampus NR2B and CREB messenger RNA(mRNA) expression after treatment on mice. Hippocampus NR2B(A) and CREB(B) mRNA expression progressively decreased on Day 1, 3, 7 after anesthesia in mice. Each group used five mice. Data were presented as the means7SD. aPo0.05 compared with the control group,bPo0.05 compared with the anesthesia group,cPo0.05 compared with the melatonin group.
with suppression of NR2B/CREB and pNR2B/pCREB expression in the hippocampus. NR2B is involved in pain, learning and memory (Qiu et al., 2011). Synaptic NR2B in the medial prefrontal cortex is also involved in surgical incisioninduced nociception in POCD (Zhang et al., 2013). P-NR2B (Tyr 1472) is important for spatial memory because it is implicated in long-term potentiation and hippocampal synaptic plasticity (Rostas et al., 1996). Moreover; p-NR2B (Tyr 1472) affects the downstream transduction (Nakazawa et al., 2006). CREB, originally identified as a 43 KDa nuclear protein, was believed to play an important role in cognition
In recent years, increasing studies have discovered that disordered sleep–wake rhythm could be a main reason of deficits in cognitive performance (Rouch et al., 2005). Clinical studies have found that declined in circadian founction with age resulted to dementia or mild cognitive impairment. (Tranah et al., 2011; Schlosser et al., 2012). Animal studies also affirmed that dysrhythmia of Siberian hamsters in the SCN severely impairs spatial and recognition memory processing (Fernandez et al., 2014). In our study we found sleep rhythm notably changed after 6 h isoflurane anesthesia. Moreover, spatial memory also impaired significantly. All these data suggest that sleep–wake rhythm is closely related to learning and memory. Interestingly, the isoflurane anesthesia caused learning enhancement at 2 Days after anesthesia, and this phenomenon is consistent with Rammes and Culley's researches (Rammes et al., 2009; Culley et al., 2003). The underlying mechanisms of the observations remain unknown. It is possible that hippocampusspecific elevation of NR2B subunit composition and enhances LTP
(Sakamoto et al., 2011). Cognitive enhancements elicited by exposure to young blood are mediated partly by activation of CREB in the aged hippocampus (Villeda et al., 2014). P-CREB (Ser 133) is a protein that depends on circadian time, it plays a valuable role in circadian rhythm (Ginty et al., 1993). Meanwhile, it also affects learning and memory by regulating gene transcription. Consistently, our current data suggested that isoflurane anesthesia can decrease NR2B and CREB in hippocampus of mice, and melatonin pretreatment can reverse the down-regulation of NR2B and CREB. In conclusion, our work demonstrates that sleep–wake rhythm is involved in the isoflurane-induced cognition impairment and pretreatment of melatonin has a positive effect on circadian normalization and cognition reversal. Also, NR2B–CREB signaling pathway has a critical role in this process. Our findings provide a new possible strategy for the treatment of anesthesia-induced cognitive dysfunction.
Please cite this article as: Xia, T., et al., Melatonin pretreatment prevents isoflurane-induced cognitive dysfunction by modulating sleep–wake rhythm in mice. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.10.036
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Fig. 4 – Changes of hippocampus NR2B phosphorylation and CREB phosphorylation protein expression on Day 1 (A), Day 3(B), Day 7(C) and Day 14(D) after anesthesia in all groups. Representative blot of each protein and statistical analysis of the relative protein expression were shown. (n ¼ 5 in each group). The data are expressed as the means7SD. aPo0.05 compared with the control group, bPo0.05 compared with the anesthesia group, cPo0.05 compared with the melatonin group.
4.1 Experimental animals
were housed under a 12 h light/12 h dark cycle (light, Circadian time 0–12; dark, Circadian time 12–24) for at least 3 weeks, with a constant room temperature (2071 1C) and food and water available ad libitum.
All animal experiments were conducted in accordance with a
4.2 General anesthesia
4.
Experimental procedures
protocol, approved by the Policy on the Use of Animals in Nanjing University. All processing methods are in accordance to Directive 2010/63/EU. Male 2-month-old C57/BL-6J mice weighting 20–25 g were used in the experiments. Animals
Animals were placed in a chamber and exposed to a mixture of 1% (Abbott, 176715 U) in 100% oxygen at a flow rate of 4 L/min for 6 h from CT14 to CT20. The behavioral marker of anesthesia was the
Please cite this article as: Xia, T., et al., Melatonin pretreatment prevents isoflurane-induced cognitive dysfunction by modulating sleep–wake rhythm in mice. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.10.036
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lack of tail-clip responses. In the whole process, respiratory rate was observed to keep normal vital sign. To maintain normal body temperature, the chambers were placed on a heated sheet. Body temperature was kept at 36.871 1C throughout the anesthesia.
4.3 Spatial memory tests Spatial memory tests were performed in the Intellicage, an automated and computer controlled system, which can be used for monitoring long-term behavior of group-housed mice (NewBehavior AG; Zürich, Switzerland). The plastic cage (size 55 37.5 20.5 cm3) was equipped with four operant learning chambers, each of which can be accessed into via a tube, with a built-in transponder codes reader. A single mouse is restricted access to the learning chamber only for at the time. The chamber contained two openings permitting access to drinking bottles. Poking a nose into the openings automatically operated door controlled access to liquid. Four triangular red shelters were placed in the middle of the Intellicage to permit mice to reach food ad libitum. Several cohorts consisting of 10–12 mice were subjected to the 28-day Intellicage protocol; it is an established protocol which was divided into two phases: Adaption Phase (14 days) comprised of 2 stages. At stage 1, all doors were opened continuously, animals could enter any corner and drink for an unlimited time; at stage 2, all doors were closed and opened only when an animal poked a nose into openings placed inside each corner. The door closed automatically when removed from the opening. Study and memory phase (14 days), during the first 7 days of this phase (Place learning phase), nose-pokes in only the selected corner would trigger the doors to open and the water bottle to be reached for each mouse (“correct” corner).Thus, the ability of each mouse to learn to find the “correct” corner provided a simple measure of place learning. During the following 7 days of this phase (Place reversal learning phase), each mouse again had free access to another “correct” corner and had access to the water bottles. All other conditions of the experiment were the same as the first 7 days and visits were recorded for each individual animal.
4.4 Measurements of gross motor activity A radio transmitter device (G2 E-Mitter; Mini Mitter Co. Inc., a Respironics Company, Bend, Ore, USA) was used to measure core body temperature and gross motor activity of mice. Under sodium pentobarbital anesthesia (50 mg/kg body i.p.), G2 EMitters were implanted into the abdominal cavity and sutured to the inside of the abdominal wall by sterile techniques. Radio signals for all physiological parameters were recorded by a receiver board (ER-4000 receiver) underneath the cage housing each animal and stored via VitalView Data Acquisition System (version 4.2; Respironics, Inc.) on a personal computer in 6min bins. The mice were allowed to recover from surgery for at least 2 weeks before onset of the experiments. The ambient temperature was 21–22 1C within the cabin that stored 24 cages that were not equipped with running wheels.
4.5 RT-PCR Total RNA from the right hippocampus was extracted using a PureLink RNA Mini Kit (Invitrogen, 12183020) according to the
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manufacturer's protocol. The RNA concentrations were determined using UV spectrophotometry (Biotek). Total RNA was reverse transcribed with a cDNA Reverse Transcriptase Kit (TakaRa, RR036A). Real Time Q-PCR was performed using an ABI StepOne Plus Real-Time PCR system (Applied Biosystems) and SYBR Premix Ex Taq (Takara, RR420A) with the following genespecific primers:CREB1 forward primer50 -TTCTACAGTATGCACAGACCACTG-30 , reverse primer50 -GGTATGTTTGTACATCGCCTGA3; NR2B forward primer 50 -GGATCTACCAGTCTAACATG-30 ,reverse primer 50 -GATAGTTAGTGATCCCACTG-30 ; β-actin forward primer 50 - CTGTCCCTGTATGCCTCTG-30 , reverse primer 50 -ATGTCACGCACGATTTCC-30 . β-actin was used as a housekeeping gene. To ensure specificity of the PCR amplification, a temperature controlled melting curve analysis was performed as the last step of the PCR reaction. As expected, each melting curve revealed a single peak, corresponding to the desired specific amplification product.
4.6 Western blot The mice were rapidly sacrificed by cervical dislocation. Right hippocampus was quickly removed and rapidly frozen in liquid nitrogen and stored at 80 1C for further processing. Samples were homogenized in SDS buffer containing a mixture of proteinase inhibitors (Sigma, USA). The quantification of the protein contents was performed using the BSA method. The protein samples (40 μg) were separated on a SDS-PAGE gel and transferred to polyvinylidenedifluoride filters (Millipore, USA). The filters were blocked with 5% milk and immunoblotted using anti-phosphor-Ser133 CREB (abcam, UK, 1:1000 dilution) and anti-phosphor-Tyr1472 NR2B (abcam, UK, 1:1000 dilution).After washed with TBST, membranes (pCREB and pNR2B) were incubated with a goat polyclonal secondary antibody to rabbit IgG (abcam, UK, 1:5000 dilution).The blots were visualized in ECL solution (DuPont-NEN, USA) for 1 min and exposed to hyperfilms (Amersham Biosciences) for 1– 10 min. The density of specific bands was measured using a computer-assisted imaging analysis system and normalized against the corresponding loading control bands. β-actin (abcam, UK, 1:1000 dilution) was used as the loading control.
4.7 Experimental design 4.7.1.
Experiment 1
In this experiment, we examined the question of whether isoflurane anesthesia could lead to prolonged cognitive impairment and whether melatonin had effect on this process.32mice were divided randomly into four groups: control group, anesthesia group, melatonin group, melatoninþanesthesia group. In melatonin group and melatoninþanesthesia group, melatonin 10 mg/kg was administered intragastrically once a day for 7 consecutive days, while equal volume of normal saline was given in control group and anesthesia group. Anesthesia group and melatoninþanesthesia group were anesthetized for 6 h from CT14 to CT20 on the first day of Study and memory phase. All mice were put into intellicage for monitoring long-term behavior after anesthesia.
Please cite this article as: Xia, T., et al., Melatonin pretreatment prevents isoflurane-induced cognitive dysfunction by modulating sleep–wake rhythm in mice. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.10.036
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4.7.2.
Experiment 2
To examine the effect of sleep disorders on the isoflurane anesthesia induced prolonged cognitive impairment, and whether melatonin had effect on this process, 24 mice were also divided randomly into four groups and each group was treated as experiment 1. All mice were put into cage for monitoring gross motor activity by Mini-Mitter.
4.7.3.
Experiment 3
In this experiment, to explore the molecular mechanism of isoflurane anesthesia induced cognitive impairment, we investigated the expression of CREB and NR2B mRNA and pCREB (CREB phosphorylation) and pNR2B (NR2B phosphorylation) in the right hippocampus. The tissue samples were obtained on Days 1, 3, 7 and 14 for all groups after sodium pentobarbital anesthesia (n¼ 5).
4.8 Statistical analysis All of the data were expressed as the mean7SD (standard deviation). The data from the behavioral tests were analyzed using repeated measures ANOVA across testing time points to detect overall differences among the treatment groups. The data from quantitative real-time reverse transcription-polymerase chain reaction, western blot data were analyzed by One-way ANOVA to determine the differences among all of the experimental groups. When significant main effects were observed, the Bonferroni post hoc tests were performed to determine the sources of the differences. Statistical analysis was performed using SPSS 16.0 software (IBM Corporation, Armonk, NY). The differences were considered statistically significant at the level of Po0.05.
Conflict of interest All authors have declared that no conflict of interest exists.
Acknowledgments This research was supported by National Natural Science Foundation of China (81371207, 81171047, 81070892 and 81171048), Natural Science Foundation of Jiangsu Province (BK2010105), and the Grant from the Department of Health of Jiangsu Province of China (XK201140 and RC2011006).
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Available online at www.sciencedirect.com
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Research Report
Melatonin pretreatment prevents isoflurane-induced cognitive dysfunction by modulating sleep–wake rhythm in mice Q1
Tianjiao Xia1, Yin Cui1, Shuaishuai Chu, Jia Song, Yue Qian, Zhengliang Man, Xiaoping Gunn Department of Anesthesiology, Affiliated Drum Tower Hospital of Medical Department of Nanjing University, 321 Zhong Shan Road, Nanjing, Jiangsu 210008, PR China
art i cle i nfo
ab st rac t
Article history:
Background: Sleep plays an important role in memory processing. However, its role in
Accepted 22 October 2015
anesthesia-induced cognitive dysfunction was not revealed. Our study sought to investigate the connection between the cognition decline and sleep–wake rhythm disorders after
Keywords:
long-term isoflurane anesthesia in mice. Also, we examined the effect of exogenous
Sleep–wake rhythm
melatonin pretreatment on both cognitive function and circadian rhythm. Furthermore,
Isoflurane
we discussed whether NR2B (N-methyl–D-aspartate receptor 2B subunit)–CREB (cAMP-
Cognitive
response element binding protein) signaling pathway was involved in this course.
Melatonin
Methods: 2-month-old male C57/BL-6J mice were submitted to long-term anesthesia using
NR2B
1% isoflurane from CT (Circadian Time) 14 to CT20. Melatonin pretreatment were
CREB
conducted before anesthesia for 7 Days. Intellicage for mice and Mini-Mitter were applied to monitor spatial memory and gross motor activity which can reflect cognition and sleep– wake rhythm. Messenger RNA and protein expression of right hippocampus NR2B and CREB were examined by RT-PCR and Western blot. Results: 6 h isoflurane anesthesia led to impaired spatial memory from Day 3 to Day 10 in mice accompanied by the disruption of sleep–wake rhythm. Meanwhile, the hippocampus CREB and NR2B expression declined in step. Melatonin pretreatment ameliorated disturbed sleep–wake cycle, improved isoflurane-induced cognitive dysfunction, and reversed the down-regulation of CREB and NR2B expression. Conclusions: Our data demonstrate that sleep–wake rhythm is involved in the isofluraneinduced cognition impairment and pretreatment of melatonin has a positive effect on circadian normalization and cognition reversal. Also, NR2B–CREB signaling pathway has a critical role in this process. This study provides us a new strategy for anesthesia-induced cognitive dysfunction therapy. & 2015 Published by Elsevier B.V.
n
Corresponding author. Fax: þ86 25 8310 5502. Corresponding author. Fax: þ86 25 8310 5502. E-mail addresses: [email protected] (T. Xia), [email protected] (Y. Cui), [email protected] (S. Chu), [email protected] (J. Song), [email protected] (Y. Qian), [email protected] (Z. Ma), [email protected] (X. Gu). 1 Tianjiao Xia and Yin Cui contributed equally to this work and should be considered as co-first authors. nn
http://dx.doi.org/10.1016/j.brainres.2015.10.036 0006-8993/& 2015 Published by Elsevier B.V.
Please cite this article as: Xia, T., et al., Melatonin pretreatment prevents isoflurane-induced cognitive dysfunction by modulating sleep–wake rhythm in mice. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.10.036
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1.
Introduction
Postoperative cognitive dysfunction (POCD) is a common and well-known complication after surgery (Steinmetz et al.,
2.
Results
2.1 Cognitive performance of animals after anesthesia and melatonin treatment
2009). It features disturbance of memory, attention, consciousness, information processing and sleep–wake cycle, leading to postoperative morbidity and mortality (Bekker and Weeks, 2003). The etiology of POCD is likely multifactorial. Different preoperative and operative factors are associated with the development of cognitive dysfunction. Factors such as increasing age, presence of diabetes, hypotension during surgery and anesthesia were believed to be involved (Shaw et al., 1989; Grocott et al., 2005). Sleep, which shares some common neuronal mechanisms with general anesthesia (Allada, 2008; Kelz et al., 2008) is important to memory processing (Turner et al., 2007). It is known to be poor in the hospital (Vico-Romero et al., 2014), and the prevalence of sleep disorders showed a marked increase in patients admitted to the Intensive Care Unit (Gomez, 2013). The disturbance of sleep–wake rhythm is considered to be one of the most important causes of sleep disorders. This rhythm is influenced by various factors, including light (Friedman et al., 2012), melatonin (Koch et al., 2009) and orexin (Diniz et al., 2010). It has also been reported that general anesthesia could impact the sleep–
Following anesthesia, animals were housed in the intellicage. Their continuous locomotive activity was tracked in terms of the number of visits and their correct rate of visits were also recorded In Place learning phase, the results showed a reducement in locomotive activity from the first day after isoflurane anesthesia based on an decreased numbers of visits in anesthesia group (Po0.05). Significantly increased on correct rate of visit two days after isoflurane anesthesia were observed and later significantly decreased from Day 3 to Day 7 when compared with control group (Po0.05) were observed, suggesting that isoflurane anesthesia could lead to cognitive dysfunction in mice. The restoration of normal locomotive activity and correct corner visits were occurred in anesthesia group 10 days and 11 days respectively after anesthesia. Compared with anesthesia group, pretreated with melatonin mice when underwent a 6 h isoflurane anesthesia showed a significant increased on the numbers of visits on Day 1, Day 4, day 5, Day 6 and Day 7. Meanwhile, higher correct visits were also detected from Day 3 to Day 7 (Po0.05). During place reversal learning phase, melatonin pretreated mice showed that the locomotive activity and correct rate of visit recovered to control level on Day 13 and Day 14. (P40.05) (Figs. 1A and E).
wake rhythm. Propofol anesthesia, for instance, induced phase advances when administered at the rest/activity transition point(Challet et al., 2007).Moreover, inhalational anesthetics sevoflurane and isoflurane caused phase delays when administered during the subjective Day (Ohe et al., 2011; Cheeseman et al., 2012). However, whether the disruption of sleep–wake rhythm resulted from anesthesia plays an important role in cognitive dysfunction remains unknown. Melatonin, a hormone secreted by the pineal gland during the dark period of the Day, mediates a diverse array of biological and physiological actions. In addition to its effects on antioxidant, immunomodulatory, and oncostatic activities (Pandi‐Perumal et al., 2006), melatonin has an important role in sleep and circadian regulations. Small dosages of melatonin were found to regulate sleep–wake rhythm (Hughes et al., 1998) and ameliorate the sleep quality by normalizing sleep–wake cycle (Finati et al., 2013). Despite these advances, little is known about the effect of melatonin on cognitive function after anesthesia. In this study, we asked whether the sleep–wake disorders induced by anesthesia contributed to the development of cognitive dysfunction, and whether this effect of anesthesia
2. 2 Gross motor activity of animals after anesthesia and melatonin treatment A 6 h anesthetic during the subjective night caused a persistent and marked shift of motor activity rhythm. On Day 1 after anesthesia, compared with control group, anesthesia mice demonstrated greater spontaneous activity levels on CT0, 2 and 4(1771.207270.97 vs. 1242.507329.38, P¼0.022; 1737.507 399.40 vs. 1203.307123.43, P¼ 0.008; 1578.707297.58 vs. 691.67107.49, Po0.001). This phenomenon lasted for at least 4 Days after anesthesia: CT4 (1498.807 310.66 vs. 1062.20796.64, P¼0.008); CT6 (1173.507 221.53 vs. 672.337141.96, Po0.001). On Day 1 after anesthesia, when compared with anesthesia group, mice which pretreated with melatonin showed lower activity levels during sleeping period on CT0 (1305.007432.50, P ¼0.048), CT6(685.177180.27, Po0.001), and this effect lasted for at least 4 Days after anesthesia on CT0, 2, 4 and 6 (828.507180.09, Po0.001; 837.83771.63, Po0.001; 1152.707199.54, P¼ 0.048; 668.177191.17, Po0.001). These marked difference indicate that pretreatment with melatonin could normalize the disrupted sleep–wake rhythm caused by isoflurane anesthesia (Fig. 2A and C).
could be blocked by exogenous melatonin treatment. By using an international advanced cognitive behavioral instrumentautomatic mouse intellicage, we found that 6 h isoflurane anesthesia induced cognitive dysfunction, accompanied by the disruption of sleep–wake rhythm. Pretreatment of melatonin had a positive effect on sleep–wake cycle normalization and cognition reversal.NR2B–CREB signaling pathway was thought to be among the molecular mechanisms.
2.3 The expression of CREB and NR2B at the hippocampus level after anesthesia and melatonin treatment Compared with control group, the expression of NR2B mRNA had decreased on Day 1 (0.5170.09 vs. 1.1270.15, Po0.001), Day 3 (0.6270.15 vs. 1.1770.16, Po0.001), and Day 7 (0.6970.12 vs. 1.1370.14, P¼ 0.002) after anesthesia in the hippocampus. Compared with anesthesia group, melatoninþanesthesia group
Please cite this article as: Xia, T., et al., Melatonin pretreatment prevents isoflurane-induced cognitive dysfunction by modulating sleep–wake rhythm in mice. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.10.036
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Fig. 1 – Number and correct rate of visits during Day 1–14. Schematic presentation of experimental design. (A). The number of visits (C,E) and correct rate of visits (B,D)in control group (open square), Iso group (closed square), melatonin group (open circle), Iso þMel group (closed circle) are all compared in pair. Data are showing with Mean7S.D. ( aPo0.05, compared with control group, bPo0.05, compared with Isoflance group, cPo0.05, compared with Melatonin group).
showed increased NR2B mRNA level on Day 1 (1.0470.15, Po0.001), Day 3 (1.0570.15, P¼0.002), and Day 7 (1.0670.18, P¼0.010). On Day 14 after anesthesia, the NR2B mRNA level was similar in all groups. Compared with control group, the expression of anesthesia group CREB mRNA was significantly decreased on Day 1 (0.6470.07 vs.1.2870.10, Po0.001), Day 3 (0.6670.12 vs. 1.1770.17, Po0.001), Day 7(0.7270.10 vs. 1.1570.14, P¼0.005). When compared with anesthesia group, Pre-administration melatonin increased the declining CREB mRNA expression after anesthesia on Day 1 (0.9770.15, P¼0.002), Day 3 (1.1670.14, Po0.001), Day 7 (1.1270.16, P¼0.008). On Day 14, the CREB mRNA expression was similar in each group (Fig. 3A and B). We next explored the expression of p-NR2B (Tyr 1472) and p-CREB (Ser 133) in the hippocampus in all groups. The pCREB expression was significantly increased on Day 1 after anesthesia when compared with control group (1.8870.17 vs. 170.14, Po0.001). On Day 3, the levels of NR2B phosphorylation (0.1170.02) and CREB phosphorylation (0.2970.04) in
anesthesia group were significantly decreased compared with the control group (pNR2B, 1.0070.10, Po0.001; pCREB, 170.07, Po0.001). While when compared with anesthesia group, the expression of pNR2B (0.9670.12, Po0.001) and pCREB (0.5770.05, Po0.001) in melatoninþanesthesia group were upregulated. On Day 14, the pNR2B 1.1270.11) and pCREB (1.0870.10) expression in anesthesia group retured to control level (pNR2B, 1.0070.04, P ¼ 0.250; pCREB, 170.05, P¼ 0.794), and no differences were observed in all groups (Fig. 4A and D).
3.
Discussion
Exposed to general anesthesia has been linked to a persistent deficit of cognitive function in both humans and rodents, and this effect is independent of underlying disease or tissue injury (Stratmann et al., 2014). In our study, 6 h anesthesia was conducted mainly because that operations lasting for a long time are increasingly found clinically and the negative effect
Please cite this article as: Xia, T., et al., Melatonin pretreatment prevents isoflurane-induced cognitive dysfunction by modulating sleep–wake rhythm in mice. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.10.036
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Fig. 2 – Gross motor activity amounts averaged (Mean7S.D) in 2-h intervals during Day 1–14.Schematic presentation of experimental design.(A). The amounts of activity (B,C)in control group (black line), anesthesia group (red line), melatonin group (green line), anesthesiaþmelatonin group (blue line) are all compared in pair. (aPo0.05, compared with control group, b Po0.05, compared with Isoflane group, cPo0.05, compared with Melatonin group). Horizontal gray bar: light phase of the day; horizontal black bar: dark phase of the day.
cannot be ignored. In this study, we found impaired spatial memory from Day 3 to Day10 of the study and memory phase in 2-month-old mice after long-term isoflurane anesthesia. As with our study, Zhu also found that isoflurane could induce persistent and progressive memory impairment in young rodents (Zhu et al., 2010). These findings suggest that anesthesia can be a cause of cognitive deficits. At the meanwhile, striking changes were also discovered in gross motor activity in mice after 6 h isoflurane anesthesia monitored by Mini-Miter. Despite the fact that fewer activities do not mean sleep, the activity pattern measured by MiniQ2 Miter may indicate sleep-related mechanism explaining our research (Fernandez et al., 2014). Researchers have found that
a deeper level of isoflurane anesthesia could disturb or delay the restoration of normal sleep–wake architecture (Takahashi et al., 2001; Jang et al., 2010). Cheeseman's research and our previous study also saw thatclock gene expression, which is important in regulating the sleep–wake rhythm, changed significantly after isoflurane anesthesia (Cheeseman et al., 2012; Xia et al., 2015). Other biological factors like hormonal desynchronization, liver dynamics, blood chemistry also cannot be ignored (Celic-Spuzic, 2011; Kajimoto et al., 2014; Schwarzkopf et al., 2013). Nevertheless, all these data indicate that sleep–wake rhythm changed markedly after isoflurane anesthesia in mice.
Please cite this article as: Xia, T., et al., Melatonin pretreatment prevents isoflurane-induced cognitive dysfunction by modulating sleep–wake rhythm in mice. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.10.036
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in CA1 neurons which may produce hippocampal-dependent cognitive improvement after anesthesia. In our experiment, melatonin intervention was used to retrieve sleep disturbance. It is primarily synthesized and secreted at night and that the circadian rhythm of its production is determined by the prevailing light–dark cycle (Axelrod et al., 1965). In our experiment, CT2 was chosen to be exogenous melatonin supplement as the period of low level melatonin secretion. We found that pretreated with melatonin could reverse isoflurane anesthesia induced cognitive dysfunction. Dispersyn demonstrated that general anesthesia had disturbing effects on the circadian rhythm of plasma melatonin in rats under normal light conditions (Dispersyn et al., 2010). Melatonin has the capacity to change the timing of circadian rhythms and functions, synchronize them with prevailing LD cycles and thereby ameliorate the sleep quality in patients with sleep– wake rhythm disturbance (Wyatt et al., 2006; Srinivasan et al., 2010; Finati et al., 2013). The role of melatonin in regulating the sleep/wake cycle is mediated through MT1 and MT2 melatonin receptors present in SCN. These two melatonin receptors modulate gamma‑aminobutyric acid (GABA) receptors in the SCN differentially; these GABA receptors, reportedly, both phase shift and synchronize SCN clock cells (Kumar and Singh, 2009). Furthermore, we explored the possible molecular mechanism of cognitive dysfunction and sleep–wake rhythm disorders induced by isoflurane. We found that the downregulation of cognitive function is relatively synchronous Fig. 3 – Quantitative real-time reverse transcription-polymerase chain reaction analysis of changes of hippocampus NR2B and CREB messenger RNA(mRNA) expression after treatment on mice. Hippocampus NR2B(A) and CREB(B) mRNA expression progressively decreased on Day 1, 3, 7 after anesthesia in mice. Each group used five mice. Data were presented as the means7SD. aPo0.05 compared with the control group,bPo0.05 compared with the anesthesia group,cPo0.05 compared with the melatonin group.
with suppression of NR2B/CREB and pNR2B/pCREB expression in the hippocampus. NR2B is involved in pain, learning and memory (Qiu et al., 2011). Synaptic NR2B in the medial prefrontal cortex is also involved in surgical incisioninduced nociception in POCD (Zhang et al., 2013). P-NR2B (Tyr 1472) is important for spatial memory because it is implicated in long-term potentiation and hippocampal synaptic plasticity (Rostas et al., 1996). Moreover; p-NR2B (Tyr 1472) affects the downstream transduction (Nakazawa et al., 2006). CREB, originally identified as a 43 KDa nuclear protein, was believed to play an important role in cognition
In recent years, increasing studies have discovered that disordered sleep–wake rhythm could be a main reason of deficits in cognitive performance (Rouch et al., 2005). Clinical studies have found that declined in circadian founction with age resulted to dementia or mild cognitive impairment. (Tranah et al., 2011; Schlosser et al., 2012). Animal studies also affirmed that dysrhythmia of Siberian hamsters in the SCN severely impairs spatial and recognition memory processing (Fernandez et al., 2014). In our study we found sleep rhythm notably changed after 6 h isoflurane anesthesia. Moreover, spatial memory also impaired significantly. All these data suggest that sleep–wake rhythm is closely related to learning and memory. Interestingly, the isoflurane anesthesia caused learning enhancement at 2 Days after anesthesia, and this phenomenon is consistent with Rammes and Culley's researches (Rammes et al., 2009; Culley et al., 2003). The underlying mechanisms of the observations remain unknown. It is possible that hippocampusspecific elevation of NR2B subunit composition and enhances LTP
(Sakamoto et al., 2011). Cognitive enhancements elicited by exposure to young blood are mediated partly by activation of CREB in the aged hippocampus (Villeda et al., 2014). P-CREB (Ser 133) is a protein that depends on circadian time, it plays a valuable role in circadian rhythm (Ginty et al., 1993). Meanwhile, it also affects learning and memory by regulating gene transcription. Consistently, our current data suggested that isoflurane anesthesia can decrease NR2B and CREB in hippocampus of mice, and melatonin pretreatment can reverse the down-regulation of NR2B and CREB. In conclusion, our work demonstrates that sleep–wake rhythm is involved in the isoflurane-induced cognition impairment and pretreatment of melatonin has a positive effect on circadian normalization and cognition reversal. Also, NR2B–CREB signaling pathway has a critical role in this process. Our findings provide a new possible strategy for the treatment of anesthesia-induced cognitive dysfunction.
Please cite this article as: Xia, T., et al., Melatonin pretreatment prevents isoflurane-induced cognitive dysfunction by modulating sleep–wake rhythm in mice. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.10.036
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Fig. 4 – Changes of hippocampus NR2B phosphorylation and CREB phosphorylation protein expression on Day 1 (A), Day 3(B), Day 7(C) and Day 14(D) after anesthesia in all groups. Representative blot of each protein and statistical analysis of the relative protein expression were shown. (n ¼ 5 in each group). The data are expressed as the means7SD. aPo0.05 compared with the control group, bPo0.05 compared with the anesthesia group, cPo0.05 compared with the melatonin group.
4.1 Experimental animals
were housed under a 12 h light/12 h dark cycle (light, Circadian time 0–12; dark, Circadian time 12–24) for at least 3 weeks, with a constant room temperature (2071 1C) and food and water available ad libitum.
All animal experiments were conducted in accordance with a
4.2 General anesthesia
4.
Experimental procedures
protocol, approved by the Policy on the Use of Animals in Nanjing University. All processing methods are in accordance to Directive 2010/63/EU. Male 2-month-old C57/BL-6J mice weighting 20–25 g were used in the experiments. Animals
Animals were placed in a chamber and exposed to a mixture of 1% (Abbott, 176715 U) in 100% oxygen at a flow rate of 4 L/min for 6 h from CT14 to CT20. The behavioral marker of anesthesia was the
Please cite this article as: Xia, T., et al., Melatonin pretreatment prevents isoflurane-induced cognitive dysfunction by modulating sleep–wake rhythm in mice. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.10.036
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lack of tail-clip responses. In the whole process, respiratory rate was observed to keep normal vital sign. To maintain normal body temperature, the chambers were placed on a heated sheet. Body temperature was kept at 36.871 1C throughout the anesthesia.
4.3 Spatial memory tests Spatial memory tests were performed in the Intellicage, an automated and computer controlled system, which can be used for monitoring long-term behavior of group-housed mice (NewBehavior AG; Zürich, Switzerland). The plastic cage (size 55 37.5 20.5 cm3) was equipped with four operant learning chambers, each of which can be accessed into via a tube, with a built-in transponder codes reader. A single mouse is restricted access to the learning chamber only for at the time. The chamber contained two openings permitting access to drinking bottles. Poking a nose into the openings automatically operated door controlled access to liquid. Four triangular red shelters were placed in the middle of the Intellicage to permit mice to reach food ad libitum. Several cohorts consisting of 10–12 mice were subjected to the 28-day Intellicage protocol; it is an established protocol which was divided into two phases: Adaption Phase (14 days) comprised of 2 stages. At stage 1, all doors were opened continuously, animals could enter any corner and drink for an unlimited time; at stage 2, all doors were closed and opened only when an animal poked a nose into openings placed inside each corner. The door closed automatically when removed from the opening. Study and memory phase (14 days), during the first 7 days of this phase (Place learning phase), nose-pokes in only the selected corner would trigger the doors to open and the water bottle to be reached for each mouse (“correct” corner).Thus, the ability of each mouse to learn to find the “correct” corner provided a simple measure of place learning. During the following 7 days of this phase (Place reversal learning phase), each mouse again had free access to another “correct” corner and had access to the water bottles. All other conditions of the experiment were the same as the first 7 days and visits were recorded for each individual animal.
4.4 Measurements of gross motor activity A radio transmitter device (G2 E-Mitter; Mini Mitter Co. Inc., a Respironics Company, Bend, Ore, USA) was used to measure core body temperature and gross motor activity of mice. Under sodium pentobarbital anesthesia (50 mg/kg body i.p.), G2 EMitters were implanted into the abdominal cavity and sutured to the inside of the abdominal wall by sterile techniques. Radio signals for all physiological parameters were recorded by a receiver board (ER-4000 receiver) underneath the cage housing each animal and stored via VitalView Data Acquisition System (version 4.2; Respironics, Inc.) on a personal computer in 6min bins. The mice were allowed to recover from surgery for at least 2 weeks before onset of the experiments. The ambient temperature was 21–22 1C within the cabin that stored 24 cages that were not equipped with running wheels.
4.5 RT-PCR Total RNA from the right hippocampus was extracted using a PureLink RNA Mini Kit (Invitrogen, 12183020) according to the
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manufacturer's protocol. The RNA concentrations were determined using UV spectrophotometry (Biotek). Total RNA was reverse transcribed with a cDNA Reverse Transcriptase Kit (TakaRa, RR036A). Real Time Q-PCR was performed using an ABI StepOne Plus Real-Time PCR system (Applied Biosystems) and SYBR Premix Ex Taq (Takara, RR420A) with the following genespecific primers:CREB1 forward primer50 -TTCTACAGTATGCACAGACCACTG-30 , reverse primer50 -GGTATGTTTGTACATCGCCTGA3; NR2B forward primer 50 -GGATCTACCAGTCTAACATG-30 ,reverse primer 50 -GATAGTTAGTGATCCCACTG-30 ; β-actin forward primer 50 - CTGTCCCTGTATGCCTCTG-30 , reverse primer 50 -ATGTCACGCACGATTTCC-30 . β-actin was used as a housekeeping gene. To ensure specificity of the PCR amplification, a temperature controlled melting curve analysis was performed as the last step of the PCR reaction. As expected, each melting curve revealed a single peak, corresponding to the desired specific amplification product.
4.6 Western blot The mice were rapidly sacrificed by cervical dislocation. Right hippocampus was quickly removed and rapidly frozen in liquid nitrogen and stored at 80 1C for further processing. Samples were homogenized in SDS buffer containing a mixture of proteinase inhibitors (Sigma, USA). The quantification of the protein contents was performed using the BSA method. The protein samples (40 μg) were separated on a SDS-PAGE gel and transferred to polyvinylidenedifluoride filters (Millipore, USA). The filters were blocked with 5% milk and immunoblotted using anti-phosphor-Ser133 CREB (abcam, UK, 1:1000 dilution) and anti-phosphor-Tyr1472 NR2B (abcam, UK, 1:1000 dilution).After washed with TBST, membranes (pCREB and pNR2B) were incubated with a goat polyclonal secondary antibody to rabbit IgG (abcam, UK, 1:5000 dilution).The blots were visualized in ECL solution (DuPont-NEN, USA) for 1 min and exposed to hyperfilms (Amersham Biosciences) for 1– 10 min. The density of specific bands was measured using a computer-assisted imaging analysis system and normalized against the corresponding loading control bands. β-actin (abcam, UK, 1:1000 dilution) was used as the loading control.
4.7 Experimental design 4.7.1.
Experiment 1
In this experiment, we examined the question of whether isoflurane anesthesia could lead to prolonged cognitive impairment and whether melatonin had effect on this process.32mice were divided randomly into four groups: control group, anesthesia group, melatonin group, melatoninþanesthesia group. In melatonin group and melatoninþanesthesia group, melatonin 10 mg/kg was administered intragastrically once a day for 7 consecutive days, while equal volume of normal saline was given in control group and anesthesia group. Anesthesia group and melatoninþanesthesia group were anesthetized for 6 h from CT14 to CT20 on the first day of Study and memory phase. All mice were put into intellicage for monitoring long-term behavior after anesthesia.
Please cite this article as: Xia, T., et al., Melatonin pretreatment prevents isoflurane-induced cognitive dysfunction by modulating sleep–wake rhythm in mice. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.10.036
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4.7.2.
Experiment 2
To examine the effect of sleep disorders on the isoflurane anesthesia induced prolonged cognitive impairment, and whether melatonin had effect on this process, 24 mice were also divided randomly into four groups and each group was treated as experiment 1. All mice were put into cage for monitoring gross motor activity by Mini-Mitter.
4.7.3.
Experiment 3
In this experiment, to explore the molecular mechanism of isoflurane anesthesia induced cognitive impairment, we investigated the expression of CREB and NR2B mRNA and pCREB (CREB phosphorylation) and pNR2B (NR2B phosphorylation) in the right hippocampus. The tissue samples were obtained on Days 1, 3, 7 and 14 for all groups after sodium pentobarbital anesthesia (n¼ 5).
4.8 Statistical analysis All of the data were expressed as the mean7SD (standard deviation). The data from the behavioral tests were analyzed using repeated measures ANOVA across testing time points to detect overall differences among the treatment groups. The data from quantitative real-time reverse transcription-polymerase chain reaction, western blot data were analyzed by One-way ANOVA to determine the differences among all of the experimental groups. When significant main effects were observed, the Bonferroni post hoc tests were performed to determine the sources of the differences. Statistical analysis was performed using SPSS 16.0 software (IBM Corporation, Armonk, NY). The differences were considered statistically significant at the level of Po0.05.
Conflict of interest All authors have declared that no conflict of interest exists.
Acknowledgments This research was supported by National Natural Science Foundation of China (81371207, 81171047, 81070892 and 81171048), Natural Science Foundation of Jiangsu Province (BK2010105), and the Grant from the Department of Health of Jiangsu Province of China (XK201140 and RC2011006).
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