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Despite the rapid increase of greenhouse gases (GHGs) in the atmosphere during the past 50 years, observed global mean surface temperature (GMST) showed a pause in the warming trend during the first decade of the twenty-first century. This is referred to as the global warming “hiatus”. A dominant hypothesis emphasizes that the superimposition of the cold phase of the Pacific decadal variability and the global warming trend can lead to the hiatus. Using simply energy balance models, we explore two potential mechanisms that may supress the GMST warming trend: enhanced negative climate feedback and downward heat mixing. Forced by linearly increasing heating, a stronger negative climate feedback can reduce the GMST warming rate, but cannot result in a warming hiatus. Downward mixing of heat can cause a short-lived hiatus of surface warming rate due to enhanced nonlinear ocean heat uptake by the lower ocean, but the surface warming would be accelerated in the long run due to the decline of downward heat mixing rate. This study provides further evidence, both theoretically and numerically, that in the long run, the only route to contain the global warming effectively is to reduce GHG emissions.

General anesthesia induces a profound but reversible unconscious state, which is accompanied by changes in various neurotransmitters in the cortex. Unlike the "brain silencing" effect of γ-aminobutyric acid (GABA) receptor potentiator anesthesia, ketamine anesthesia leads the brain to a paradoxical active state with higher cortical activity, which is manifested as dissociative anesthesia. However, how the overall neurotransmitter network evolves across conscious states after ketamine administration remains unclear. Using in vivo microdialysis, high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis, and electroencephalogram (EEG) recording technique, we continuously measured the concentrations of six neurotransmitters and the EEG signals during anesthesia with esketamine, an

Acute lung injury (ALI) is a life-threatening acute inflammatory disease with high morbidity and mortality rates. N6-methylation of adenosine (m6A) is an abundant mRNA nucleotide modification, which is critical for mRNA metabolism and function. However, the role of m6A modifications in ALI progression remains unclear. To address this problem, we modeled ALI in vivo by intraperitoneally injecting lipopolysaccharide (LPS) to C57BL/6 mice. In addition, we isolated alveolar type II cells (AECII) from the lungs of C57BL/6 mice and treated them in vitro with various concentrations of LPS. An m6A RNA methylation quantification kit was used to detect methylation levels. An m6A microarray and mRNA transcriptomic sequencing were used to screen the downstream targets of methyltransferase-like 3 (METTL3)-mediated m6A modifications. Gene and protein expression levels were determined by qRT-PCR and western blotting. The biological effects of modulating METTL3 expression level were evaluated both in vitro and in vivo. The binding between proteins and mRNA was verified by the RNA immunoprecipitation assay. The CCK-8 and EdU assays were used to examine changes in AECII viability and proliferation. The global number of mRNA m6A-modified loci and the “writer” methyltransferase METTL3 expression were significantly increased in the lung tissue following ALI and in LPS-treated AECII. METTL3 knockdown reduced the viability and proliferation of LPS-treated AECII. Furthermore, METTL3-mediated m6A modification of phosphatase and tensin homolog (Pten) mRNA enhanced its stability. Collectively, our results showed that METTL3 contributes to ALI progression by increasing stability and translation efficiency of Pten mRNA by means of m6A modification, indicating that METTL3 might become a new and innovative therapeutic target for the treatment of ALI.

Aims Sevoflurane preconditioning (SPC) results in cerebral ischemic tolerance; however, the mechanism remains unclear. Promoting microglia/macrophages polarization from pro‐inflammatory state to anti‐inflammatory phenotype has been indicated as a potential treatment target against ischemic stroke. In this study, we aimed to assess the effect of SPC on microglia polarization after stroke and which signaling pathway was involved in this transition. Methods Mouse primary microglia with SPC were challenged by oxygen‐glucose deprivation (OGD) or lipopolysaccharide (LPS), and mice with SPC were subjected to middle cerebral artery occlusion (MCAO). Then, the mRNA and protein levels of pro‐inflammatory/anti‐inflammatory factors were analyzed. GSK‐3β phosphorylation and Nrf2 nuclear translocation were measured. The mRNA and protein expression of pro‐inflammatory/anti‐inflammatory factors, neurological scores, infarct volume, cellular apoptosis, the proportion of pro‐inflammatory/anti‐inflammatory microglia/macrophages, and the generation of super‐oxidants were examined after SPC or GSK‐3β inhibitor TDZD treatment with or without Nrf2 deficiency. Results Sevoflurane preconditioning promoted anti‐inflammatory and inhibited pro‐inflammatory microglia/macrophages phenotype both in vitro and in vivo. GSK‐3β phosphorylation at Ser9 was increased after SPC. Both SPC and TDZD administration enhanced Nrf2 nuclear translocation, reduced pro‐inflammatory microglia/macrophages markers expression, promoted anti‐inflammatory markers level, and elicited a neuroprotective effect. Nrf2 deficiency abolished the promoted anti‐inflammatory microglia/macrophages polarization and ischemic tolerance induced by TDZD treatment. The reduced percentage of pro‐inflammatory positive cells and super‐oxidants generation induced by SFC or TDZD was also reversed by Nrf2 knockdown. Conclusions Our results indicated that SPC exerts brain ischemic tolerance and promotes anti‐inflammatory microglia/macrophages polarization by GSK‐3β‐dependent Nrf2 activation, which provides a novel mechanism for SPC‐induced neuroprotection., SPC promoted microglia/macrophages polarization toward to anti‐inflammatory phenotype in vitro and in vivo. Both SPC and TDZD administration enhanced Nrf2 nuclear translocation.SPC promoted microglia/macrophages toward to anti‐inflammatory phenotype shifting via GSK‐3β‐dependent Nrf2 activation.

Aims General anesthesia has been widely applied in surgical or nonsurgical medical procedures, but the mechanism behind remains elusive. Because of shared neural circuits of sleep and anesthesia, whether serotonergic system, which is highly implicated in modulation of sleep and wakefulness, regulates general anesthesia as well is worth investigating. Methods Immunostaining and fiber photometry were used to assess the neuronal activities. Electroencephalography spectra and burst‐suppression ratio (BSR) were used to measure anesthetic depth and loss or recovery of righting reflex to indicate the induction or emergence time of general anesthesia. Regulation of serotonergic system was achieved through optogenetic, chemogenetic, or pharmacological methods. Results We found that both Fos expression and calcium activity were significantly decreased during general anesthesia. Activation of 5‐HT neurons in the dorsal raphe nucleus (DRN) decreased the depth of anesthesia and facilitated the emergence from anesthesia, and inhibition deepened the anesthesia and prolonged the emergence time. Furthermore, agonism or antagonism of 5‐HT 1A or 2C receptors mimicked the effect of manipulating DRN serotonergic neurons. Conclusion Our results demonstrate that 5‐HT neurons in the DRN play a regulative role of general anesthesia, and activation of serotonergic neurons could facilitate emergence from general anesthesia partly through 5‐HT 1A and 2C receptors., General anesthesia decreases the activities of serotonergic neurons in the dorsal raphe. Activation of DRN 5‐HT neurons reduced the depth of anesthesia and accelerated the emergence from general anesthesia, partially through the 5‐HT 1A and 2C receptors.

The lateral hypothalamic area (LHA) plays a pivotal role in regulating consciousness transition, in which orexinergic neurons, GABAergic neurons, and melanin-concentrating hormone neurons are involved. Glutamatergic neurons have a large population in the LHA, but their anesthesia-related effect has not been explored. Here, we found that genetic ablation of LHA glutamatergic neurons shortened the induction time and prolonged the recovery time of isoflurane anesthesia in mice. In contrast, chemogenetic activation of LHA glutamatergic neurons increased the time to anesthesia and decreased the time to recovery. Optogenetic activation of LHA glutamatergic neurons during the maintenance of anesthesia reduced the burst suppression pattern of the electroencephalogram (EEG) and shifted EEG features to an arousal pattern. Photostimulation of LHA glutamatergic projections to the lateral habenula (LHb) also facilitated the emergence from anesthesia and the transition of anesthesia depth to a lighter level. Collectively, LHA glutamatergic neurons and their projections to the LHb regulate anesthetic potency and EEG features.

Background The neuropeptide orexin promotes arousal from general anaesthesia, however the neuronal circuits that mediate this effect have not been defined. We investigated whether orexinergic neurones modulate the basal forebrain (BF) and locus coeruleus (LC) in emergence from anaesthesia. Methods Hcrtcre rats were generated using a CRISPR/Cas9-based approach. Viruses encoding optogenetic probes were injected into the perifornical lateral hypothalamic (PeFLH) area, optogenetic fibres were embedded in the PeFLH, BF, or LC, and changes in anaesthesia state under 1.4 vol% or 0.8 vol% isoflurane were determined. Results In the PeFLH, 98.8% (0.4%) of orexin-A-positive cells expressed tdTomato, and 91.9% (2.2%) of tdTomato cells were orexin-A-positive. Under 1.4 vol% isoflurane anaesthesia, compared with control groups, burst suppression ratio was less, and emergence time was shorter in groups with optogenetic activation of orexinergic cell bodies in the PeFLH (923 [162] vs 493 [68] s, P=0.0003) or orexinergic terminals in the BF (937 (122) vs 674 (108) s, P=0.0049) or LC (913 [128] vs 742 [76] s, P=0.022). Optical stimulation of orexinergic terminals in the BF and LC also improved the movement scores of rats under 0.8 vol% isoflurane anaesthesia. Conclusions Activation of orexinergic terminals in the FB or LC mediates facilitation of emergence from anaesthesia by orexinergic neurones during isoflurane anaesthesia.

A growing number of studies have identified sex differences in response to general anesthesia; however, the underlying neural mechanisms are unclear. The medial preoptic area (MPA), an important sexually dimorphic structure and a critical hub for regulating consciousness transition, is enriched with estrogen receptor alpha (ERα), particularly in neuronal clusters that participate in regulating sleep. We found that male mice were more sensitive to sevoflurane. Pharmacological inhibition of ERα in the MPA abolished the sex differences in sevoflurane anesthesia, in particular by extending the induction time and facilitating emergence in males but not in females. Suppression of ERα in vitro inhibited GABAergic and glutamatergic neurons of the MPA in males but not in females. Furthermore, ERα knockdown in GABAergic neurons of the male MPA was sufficient to eliminate sex differences during sevoflurane anesthesia. Collectively, MPA ERα positively regulates the activity of MPA GABAergic neurons in males but not in females, which contributes to the sex difference of mice in sevoflurane anesthesia.

General anesthesia has been successfully used in the clinic for over 170 years, but its mechanisms of effect remain unclear. Behaviorally, general anesthesia is similar to sleep in that it produces a reversible transition between wakefulness and the state of being unaware of one’s surroundings. A growing discussion has been imposed regarding the common circuits of sleep and general anesthesia, as an increasing number of sleep-arousal regulatory nuclei are reported to participate in the consciousness shift occurring during general anesthesia. Recently, with progress in research technology, both positive and negative evidence for overlapping neural circuits between sleep and general anesthesia have emerged. This article provides a review of the latest evidence on the neural substrates for sleep and general anesthesia regulation by comparing the roles of pivotal nuclei in sleep and anesthesia.

Background Orexin, a neuropeptide derived from the perifornical area of the hypothalamus (PeFLH), promotes the recovery of propofol, isoflurane, and sevoflurane anesthesias, without influencing the induction time. However, whether the orexinergic system also plays a similar role in desflurane anesthesia, which is widely applied in clinical practice owing to its most rapid onset and offset time among all volatile anesthetics, has not yet been studied. In the present study, we explored the effect of the orexinergic system on the consciousness state induced by desflurane anesthesia. Methods The c-Fos staining was used to observe the activity changes of orexinergic neurons in the PeFLH and their efferent projection regions under desflurane anesthesia. Chemogenetic and optogenetic techniques were applied to compare the effect of PeFLH orexinergic neurons on the induction, emergence, and maintenance states between desflurane and isoflurane anesthesias. Orexinergic terminals in the paraventricular thalamic nucleus (PVT) were manipulated with pharmacologic, chemogenetic, and optogenetic techniques to assess the effect of orexinergic circuitry on desflurane anesthesia. Results Desflurane anesthesia inhibited the activity of orexinergic neurons in the PeFLH, as well as the neuronal activity in PVT, basal forebrain, dorsal raphe nucleus, and ventral tegmental area, as demonstrated by c-Fos staining. Activation of PeFLH orexinergic neurons prolonged the induction time and accelerated emergence from desflurane anesthesia but only influenced the emergence in isoflurane anesthesia, as demonstrated by chemogenetic and pharmacologic techniques. Meanwhile, optical activation of orexinergic neurons exhibited a long-lasting inhibitory effect on burst-suppression ratio (BSR) under desflurane anesthesia, and the effect may be contributed by the orexinergic PeFLH-PVT circuitry. The orexin-2 receptor (OX2R), but not orexin-1 receptor (OX1R), in the PVT, which had been inhibited most significantly by desflurane, mediated the proemergence effect of desflurane anesthesia. Conclusions We discovered, for the first time, that orexinergic neurons in the PeFLH could not only influence the maintenance and emergence from isoflurane and desflurane anesthesias but also affect the induction under desflurane anesthesia. Furthermore, this specific effect is probably mediated by orexinergic PeFLH-PVT circuitry, especially OX2Rs in the PVT.

Background Orexin can facilitate emergence after general anaesthesia via multiple neural pathways. Dopaminergic neurones in the ventral tegmental area (VTA) participate in behavioural arousal from anaesthesia. We investigated the regulation of dopaminergic VTA neurones by orexinergic neurones during emergence from general anaesthesia. Methods Orexins were microinjected into the VTA to determine the effects on isoflurane anaesthesia induction, emergence, and maintenance. Orexin receptors and dopaminergic neurones in the VTA were identified using immunofluorescence. Orexinergic terminals in the VTA were optogenetically regulated to detect the endogenous orexin-mediated regulation of dopaminergic neurones during anaesthesia in Hcrtcre rats. Results Injection of orexin-A (100 pmol) into the VTA reduced emergence time [from 949 (118) to 727 (101) s; P=0.0058] and reduced the electroencephalographic burst–suppression ratio (BSR) (26.6 [10.2]% vs 44.3 [6.8]%; P=0.0027) during isoflurane anaesthesia. The percentage of dopaminergic neurones that expressed either orexin-1 receptor or orexin-2 receptor was 73.4 (5.0)% and 74.4 (62.4)%, respectively. Optogenetic activation of orexinergic projections to the VTA reduced the BSR (from 40.5 [2.7]% to 22.4 [11.8]%; P=0.0019) and facilitated emergence (915 [89] vs 685 [68] s; P=0.0026), whereas optical inhibition prolonged the time to wakefulness (from 941 [92] to 1279 [250] s; P=0.011). Dopaminergic neurones in the VTA showed increased firing frequency (387 [78]% of control, P=0.005) after bath application of orexin-A. Conclusions Orexin promotes emergence from isoflurane anaesthesia through activation of dopaminergic neurones in the VTA.

Purpose Chemokine CX3CL1 and its receptor CX3CR1 in the lumbar spinal cord play crucial roles in pain processing. Electroacupuncture (EA) is recognized as an alternative therapy in pain treatment due to its efficacy and safety. However, the analgesic mechanism of EA remains unclear. The aim of this study was to investigate whether EA suppressed complete Freund's adjuvant (CFA)-induced pain via modulating CX3CL1-CX3CR1 pathway. Materials and methods Inflammatory pain was induced by intraplantar injection of CFA to the left hind paw of Sprague-Dawley rats. EA with 2 Hz for 30 mins was given to bilateral Zusanli acupoints (ST36) on the first and third day after CFA injection. Mechanical allodynia and thermal hyperalgesia were tested with von Frey tests and Hargreaves tests, respectively. The expressions of CX3CL1, CX3CR1 and p38 mitogen-activated protein kinase (MAPK) were quantified with Western blots. The release of IL-1β, IL-6 and TNF-α were evaluated with ELISA. Recombinant CX3CL1 or control IgG were then injected through intrathecal catheters in the EA-treated CFA model rats. The behavioral tests, p38 MAPK activation and cytokine release were then evaluated. Results EA significantly inhibited inflammatory pain induced by CFA for 3 days. Meanwhile, EA downregulated the expression of CX3CL1 but not CX3CR1 in the lumbar spinal cord of the CFA rats. Besides, activation of p38 MAPK and the release of pain-related cytokines (IL-1β, IL-6 and TNF-α) were inhibited by EA. Intrathecal injection of CX3CL1 largely reversed the analgesic effect of EA treatment and re-activated p38 MAPK signaling, and resulted in pro-inflammatory cytokines increase in acupuncture-treated rats. Conclusion Our findings indicate that EA alleviates inflammatory pain via modulating CX3CL1 signaling in lumbar spinal cord, revealing a potential mechanism of anti-nociception of EA in inflammatory pain.

Postmenopausal women have a higher incidence of stroke compared to the age-matched males, and the estrogen was thought to be the main cause of such difference. However, estrogen replacement therapy for the prevention of postmenopausal stroke shows controversial results and is widely disputed because of its serious side effects after chronic administration. Genistein (Gen), a natural phytestrogen with fewer side effects, has a protective effect against cerebral ischemia damage. However, whether Gen could effectively prevent postmenopausal stroke has not been elucidated. In the current study, reproductively senescent mice were treated with Gen (10 mg/kg) for 2 weeks before having transient cerebral ischemia insults. Neurological scores, infarct volumes, and cell apoptosis were evaluated 24 h after reperfusion. The levels of inflammatory factors and nod-like receptor protein 3 (NLRP3) inflammasome-related proteins were also examined. The results showed that Gen treatment reduced infarct volumes, improved neurological scores, attenuated apoptosis, and decreased inflammatory factor release. The expression of NLRP3 inflammasome-related proteins in microglia was downregulated by Gen. However, the overexpression of NLRP3 in microglia abrogated the Gen-induced inhibition of inflammatory factor release and reversed the neuroprotective effect of Gen. Taken together, the results suggest that Gen treatment could attenuate the acute injury induced by cerebral ischemia in reproductively senescent mice via the inhibition of the NLRP3 inflammasome in microglia, indicating that Gen could be a candidate drug for the treatment of stroke in postmenopausal women.

Background Previous clinical studies have shown that emergence from isoflurane anaesthesia takes longer in elderly patients compared with middle-aged patients. The current study investigated whether delayed emergence from anaesthesia in older age is associated with the age-related decrease in orexin receptors by using a rat model. Methods Adult and aged Sprague–Dawley rats were used to assess the time to emergence after 30 min isoflurane anaesthesia (1.4 vol%), and differences in the orexinergic systems, including the number of orexinergic neurones, plasma orexin concentrations, and expression of orexin-1 (OX1R) and orexin-2 receptors, compared using immunofluorescence, radioimmunoassay, western blot, and real-time polymerase chain reaction. The effects of OX1R expression on emergence time were determined by virus-mediated overexpression of OX1R using intra-cerebroventricular injection. Results The median (range) emergence time of aged rats was longer than that in adult rats [1082 (1010–1130) compared with 848 (829–938) s; P=0.0009]. Plasma orexin concentrations were higher in the aged group than the adult group [34 (33–37) and 25 (22–31) pg ml−1, respectively; P=0.04], but the number of orexinergic neurones was similar in both groups. Protein expression of OX1R was lower in the aged group compared with the adult group [0.47 (0.35–0.58) compared with 0.97 (0.86–1.32), respectively; P=0.002]. Overexpression of OX1R significantly shortened the emergence time in aged rats from [1120 (1040–1190) s] to [769 (576–928) s; P=0.03]. Conclusions Age-related decrease in OX1R expression is associated with delayed emergence from isoflurane anaesthesia in aged rats.

The Bjerknes compensation (BJC) under global warming is studied using a simple box model and a coupled Earth system model. The BJC states the out-of-phase changes in the meridional atmosphere and ocean heat transports. Results suggest that the BJC can occur during the transient period of global warming. During the transient period, the sea ice melting in the high latitudes can cause a significant weakening of the Atlantic meridional overturning circulation (AMOC), resulting in a cooling in the North Atlantic. The meridional contrast of sea surface temperature would be enhanced, and this can eventually enhance the Hadley cell and storm-track activities in the Northern Hemisphere. Accompanied by changes in both ocean and atmosphere circulations, the northward ocean heat transport in the Atlantic is decreased while the northward atmosphere heat transport is increased, and the BJC occurs in the Northern Hemisphere. Once the freshwater influx into the North Atlantic Ocean stops, or the ocean even loses freshwater because of strong heating in the high latitudes, the AMOC would recover. Both the atmosphere and ocean heat transports would be enhanced, and they can eventually recover to the state of the control run, leading to the BJC to become invalid. The above processes are clearly demonstrated in the coupled model CO2 experiment. Since it is difficult to separate the freshwater effect from the heating effect in the coupled model, a simple box model is used to understand the BJC mechanism and freshwater’s role under global warming. In a warming climate, the freshwater flux into the ocean can cool the global surface temperature, mitigating the temperature rise. Box model experiments indicate clearly that it is the freshwater flux into the North Atlantic that causes out-of-phase changes in the atmosphere and ocean heat transports, which eventually plays a stabilizing role in global climate change.

We screened for novel circuits in the mouse brain that promote wakefulness. Chemogenetic activation experiments and electroencephalogram recordings pointed to glutamatergic/nitrergic (NOS1) and GABAergic neurons in the ventral tegmental area (VTA). Activating glutamatergic/NOS1 neurons, which were wake- and rapid eye movement (REM) sleep-active, produced wakefulness through projections to the nucleus accumbens and the lateral hypothalamus. Lesioning the glutamate cells impaired the consolidation of wakefulness. By contrast, activation of GABAergic VTA neurons elicited long-lasting non-rapid-eye-movement-like sleep resembling sedation. Lesioning these neurons produced an increase in wakefulness that persisted for at least 4 months. Surprisingly, these VTA GABAergic neurons were wake- and REM sleep-active. We suggest that GABAergic VTA neurons may limit wakefulness by inhibiting the arousal-promoting VTA glutamatergic and/or dopaminergic neurons and through projections to the lateral hypothalamus. Thus, in addition to its contribution to goal- and reward-directed behaviors, the VTA has a role in regulating sleep and wakefulness.

Background Isoflurane preconditioning (IPC) induces cerebral ischaemic tolerance, but the mechanism remains poorly understood. The aim of this study was to determine changes in mitochondrial function in the brain after IPC, and whether the cannabinoid receptor 1 (CB1R) could be involved in the mechanism of mitochondrial protection mediated by IPC. Methods Adult male Sprague–Dawley rats were pretreated with isoflurane 2% for 1 h day−1, for 5 days consecutively, and then subjected to 120 min right middle cerebral artery occlusion. Cannabinoid receptor 1 expression in the cellular and mitochondrial membrane was measured. The CB1R agonist HU-210 was administered alone, or the antagonists AM251 and SR141716A were given to the animals before each preconditioning. Neurological scores, infarct volume, apoptosis, and mitochondrial function were examined after middle cerebral artery occlusion. Results Expression of CB1R on cellular and mitochondrial membranes was increased 6 h after preconditioning. Both IPC and HU-210 administration before middle cerebral artery occlusion improved neurological outcomes and reduced infarct volume. Isoflurane preconditioning increased the expression of the anti-apoptotic proteins Bcl-2 and Bcl-XL and reduced apoptosis in neurones. Isoflurane preconditioning and HU-210 also markedly preserved the activity of respiratory chain complexes, reduced mitochondrial radical generation, preserved mitochondrial membrane potential, and inhibited mitochondrial permeability transition pore opening. Cannabinoid receptor 1 antagonists abolished the improvement in mitochondrial function and the neuroprotective effects induced by IPC. Conclusions Our results indicate that IPC elicits brain ischaemic tolerance and mitochondrial protection by activating the CB1R, which provides a new mechanism for IPC-induced neuroprotection against cerebral ischaemia.

Our previous studies have shown that hyperbaric oxygen preconditioning (HBO-PC) induces tolerance to cerebral ischemia/reperfusion (I/R). This study aimed to investigate whether SirT1, a class III histone deacetylase, is involved in neuroprotection elicited by HBO-PC in animal and cell culture models of ischemia. Rats were subjected to middle cerebral artery occlusion for 120 minutes after HBO-PC (once a day for 5 days). Primary cultured cortical neurons were exposed to 2 hours of HBO-PC after 2 hours of oxygen–glucose deprivation (OGD). We showed that HBO-PC increased SirT1 protein and mRNA expression, promoted neurobehavioral score, reduced infarct volume, and improved morphology at 24 hours and 7 days after cerebral I/R. Neuroprotection of HBO-PC was attenuated by SirT1 inhibitor EX527 and SirT1 knockdown by short interfering RNA (siRNA), whereas it was mimicked by SirT1 activator resveratrol. Furthermore, HBO-PC enhanced SirT1 expression and cell viability and reduced lactate dehydrogenase release 24 hours after OGD/re-oxygenation. The neuroprotective effect of HBO-PC was emulated through upregulating SirT1 and, reversely, attenuated through downregulating SirT1. The modulation of SirT1 was made by adenovirus infection carrying SirT1 or SirT1 siRNA. Besides, SirT1 increased B-cell lymphoma 2 (Bcl-2) expression and decrease cleaved caspase 3. These results indicate that SirT1 mediates HBO-PC-induced tolerance to cerebral I/R through inhibition of apoptosis.

Background A wealth of evidence has demonstrated that sevoflurane preconditioning induces brain ischemic tolerance, but the mechanism remains poorly understood. This study was designed to investigate the role of canonical Notch signaling in the neuroprotection induced by sevoflurane preconditioning in a mouse model. Methods C57BL/6 mice were pretreated with 1-h sevoflurane exposure at a dose of 2.5% for 5 consecutive days. Twenty-four hours after the last exposure, all mice were subjected to focal cerebral ischemia by right middle cerebral artery occlusion for 60 min. Neurobehavioral scores, brain infarct volumes, and cellular apoptosis were determined at 72 h after reperfusion (n = 10 per group). The activation of Notch signaling was evaluated (n = 5 per group), and its role in ischemic tolerance was assessed by intraperitoneal administration of γ-secretase inhibitor DAPT (100 mg/kg, n = 10 per group) and conditional Notch-RBP-J knockout technique (n = 8 per group). Results Sevoflurane preconditioning reduced brain infarct volumes (42.5%), improved neurologic outcomes (P < 0.01 vs. control), and attenuated neuronal cell apoptosis (cells positive for terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling reduced to 21.2%). The expression of Notch1 intracellular domain (1.35 folds) and the transcriptions of Hes1 (1.95 times) and Hes5 (1.48 times) were up-regulated. DAPT augmented the brain infarcts (1.64-fold) and decreased neurologic scores (P = 0.43 vs. sevoflurane) in sevoflurane-preconditioned mice. Brain infarct volumes, neurobehavioral scores, and apoptotic cell numbers showed no significance between Notch knockout mice with sevoflurane preconditioning and wild-type mice without preconditioning. Conclusions Sevoflurane preconditioning-induced protective effects against transient cerebral ischemic injuries are mediated by the activation of canonical Notch signaling pathway in mice.

Circadian clocks allow anticipation of daily environmental changes. The suprachiasmatic nucleus (SCN) houses the master clock, but clocks are also widely expressed elsewhere in the body. Although some peripheral clocks have established roles, it is unclear what local brain clocks do. We tested the contribution of one putative local clock in mouse histaminergic neurons in the tuberomamillary nucleus to the regulation of the sleep-wake cycle. Histaminergic neurons are silent during sleep, and start firing after wake onset; the released histamine, made by the enzyme histidine decarboxylase (HDC), enhances wakefulness. We found that hdc gene expression varies with time of day. Selectively deleting the Bmal1 (also known as Arntl or Mop3) clock gene from histaminergic cells removes this variation, producing higher HDC expression and brain histamine levels during the day. The consequences include more fragmented sleep, prolonged wake at night, shallower sleep depth (lower nonrapid eye movement [NREM] δ power), increased NREM-to-REM transitions, hindered recovery sleep after sleep deprivation, and impaired memory. Removing BMAL1 from histaminergic neurons does not, however, affect circadian rhythms. We propose that for mammals with polyphasic/nonwake consolidating sleep, the local BMAL1-dependent clock directs appropriately timed declines and increases in histamine biosynthesis to produce an appropriate balance of wake and sleep within the overall daily cycle of rest and activity specified by the SCN.

Background:Mechanism of sevoflurane preconditioning–induced cerebral ischemic tolerance is unclear. This study investigates the role of N-myc downstream–regulated gene-2 (NDRG2) in the neuroprotection of sevoflurane preconditioning in ischemic model both in vivo and in vitro.Methods:At 2 h after sevoflurane (2%) preconditioning for 1 h, rats were subjected to middle cerebral artery occlusion for 120 min. Neurobehavioral scores (n = 10), infarct volumes (n = 10), cellular apoptosis (n = 6), and NDRG2 expression (n = 6) were determined at 24 h after reperfusion. In vitro, cultural astrocytes were exposed to oxygen–glucose deprivation for 4 h. Cellular viability, cytotoxicity, apoptosis, and NDRG2 expression (n = 6) were evaluated in the presence or absence of NDRG2-specific small interfering RNA or NDRG2 overexpression plasmid.Results:Sevoflurane preconditioning decreased apoptosis (terminal deoxynucleotidyl transferase–mediated 2’-deoxyuridine 5’-triphosphate nick-end labeling–positive cells reduced to 31.2 ± 5.3% and cleaved Caspase-3 reduced to 1.42 ± 0.21 fold) and inhibited NDRG2 expression (1.28 ± 0.15 fold) and nuclear translocation (2.21 ± 0.29 fold) in ischemic penumbra. Similar effects were observed in cultural astrocytes exposed to oxygen–glucose deprivation. NDRG2 knockdown by small interfering RNA attenuated oxygen–glucose deprivation–induced injury (cell viability increased to 80.5 ± 4.1%; lactate dehydrogenase release reduced to 30.5 ± 4.0%) and cellular apoptosis (cleaved Caspase-3 reduced to 1.55 ± 0.21 fold; terminal deoxynucleotidyl transferase–mediated 2’-deoxyuridine 5’-triphosphate nick-end labeling–positive cells reduced to 18.2 ± 4.3%), whereas NDRG2 overexpression reversed the protective effects of sevoflurane preconditioning. All the data are presented as mean ± SD.Conclusion:Sevoflurane preconditioning inhibits NDRG2 up-regulation and nuclear translocation in astrocytes to induce cerebral ischemic tolerance via antiapoptosis, which represents one new mechanism of sevoflurane preconditioning and provides a novel target for neuroprotection.

Stroke has severe consequences in postmenopausal women. As replacement therapy of estrogen have various adverse effects and the undermined outcomes. Genistein, a natural phytoestrogen, has been suggested to be a potential neuroprotective agent for such stroke patients. However, the role of genistein and its underlying mechanism in ovariectomized mice has not yet been evaluated. In the present study, ovariectomized mice were treated with genistein (10 mg/kg) or vehicle daily for two weeks before developing transient cerebral ischemia (middle cerebral artery occlusion). The neurological manifestation was evaluated, and infarct volumes were demonstrated by 2,3,5-triphenyltetrazolium chloride staining at 24 h after reperfusion. In addition, phosphorylation of extracellular signal-regulated kinase (ERK) was detected by Western blotting and immunofluorescence staining, and cellular apoptosis was evaluated in the ischemic penumbra. We found that treatment with genistein reduced infarct volumes, improved neurological outcomes and attenuated cellular apoptosis at 24 h after reperfusion. ERK1/2 showed increased phosphorylation by genistein treatment after reperfusion, and an ERK1/2 inhibitor U0126 abolished this protective effect of genistein in terms of infarct volumes, neurological scores and cellular apoptosis. Our findings indicate that treatment with genistein can reduce the severity of subsequent stroke episodes, and that this beneficial function is associated with ERK activation.

Ginsenoside Rd (Rd), one of the main active ingredients in Panax ginseng, has been demonstrated to protect against ischemic cerebral damage in vitro and in vivo. In this study, we aimed to further define the preclinical characteristics of Rd. We show that Rd passes the intact blood-brain barrier and exerts protection in both transient and permanent middle cerebral artery occlusion (MCAO) in rats. In the dose-response study, Rd (10–50 mg/Kg) significantly reduced the infarct volume on postoperative days (PODs) 1, 3, and 7. This protection was associated with an improved neurological outcome for as many as 6 weeks after transient MCAO, as assessed by modified neurological severity score, modified sticky-tape test, and corner test. For comparison, Rd was significantly more effective than edaravone and slightly more effective than N-tert-butyl-alpha-phenylnitrone (PBN). In the therapeutic window study, Rd exhibited remarkable neuroprotection, even when administered for as many as 4 h after the recirculation of transient MCAO or after the onset of permanent MCAO. Furthermore, in female rats or 16-month-old male rats, the salutary effects of Rd were also observed. These findings suggest Rd is a promising neuroprotectant and provide support for future clinical studies to confirm whether Rd is beneficial in ischemic stroke.

Background Signal transducer and activator of transcription 3 (STAT3) activation in ischemic brain has been verified. However, the mechanism and the role of STAT3 activation after cerebral ischemia-reperfusion are poorly elucidated. In the present study, we sought to test the hypothesis that STAT3 activation after cerebral ischemia-reperfusion was related to reactive oxygen species (ROS) production. Methods Adult male Sprague-Dawley rats were subjected to focal cerebral ischemia induced by middle cerebral artery occlusion. STAT3 activation was evaluated by immunohistochemistry and Western blotting. Rats were subjected to permanent ischemia or ischemia-reperfusion to clarify the temporal profile of STAT3 activation. The role of ROS in inducing STAT3 activation was assessed by administration of the ROS scavenger dimethylthiourea (DMTU). The effects of DMTU and the STAT3 activation inhibitor AG490 administration on brain ischemic injuries were evaluated by neurologic behavior scores and brain infarct volumes. Results The activation of STAT3 after middle cerebral artery occlusion was significantly increased within peri-ischemia neurons and astrocytes. STAT3 activation mainly occurred in the reperfusion phase rather than in the ischemia phase. In addition, DMTU suppressed STAT3 activation in a dose-dependent manner, indicating that STAT3 activation may be a subsequent event after ROS production. DMTU and AG490 significantly reduced infarct sizes and improved neurologic outcomes. Conclusion In comparison with ischemia, reperfusion is a more powerful stimulus for STAT3 activation. ROS scavenging is closely correlated with an inhibition of STAT3 activation. Neuroprotective effects are achieved through ROS scavenging and down-regulation of STAT3 activation.

BACKGROUND: It has been reported that sevoflurane preconditioning can induce neuroprotection, the mechanisms of which, however, are poorly elucidated. We designed the present study to examine the hypothesis that sevoflurane preconditioning could reduce cerebral ischemia– reperfusion injury through up-regulating antioxidant enzyme activities before ischemic injury by generating reactive oxygen species (ROS). METHODS: In preconditioning groups, adult male Sprague–Dawley rats were pretreated with 1 hour sevoflurane exposure at a dose of 1%, 2%, or 4% for 5 consecutive days. At 24 hours after the last exposure, all rats were subjected to focal brain ischemia induced by middle cerebral artery occlusion for 120 minutes followed by 72-hour reperfusion. The role of ROS in ischemic tolerance was assessed by administration of the free radical scavenger dimethylthiourea and antioxidant N-acetylcysteine before each preconditioning. Brain ischemic injury was evaluated by neurologic behavior scores and brain infarct volume calculation. Antioxidant enzyme activities (superoxide dismutase, catalase, and glutathione peroxidase [GSH-px]) of brain tissue and blood serum were tested at 24 hours after the last sevoflurane preconditioning. RESULTS: Sevoflurane preconditioning reduced infarct size and improved neurobehavioral outcome in a dose-dependent manner. The neuroprotective effects of sevoflurane preconditioning were abolished by dimethylthiourea and N-acetylcysteine. The activities of catalase and glutathione peroxidase (GSH-px) in the brain tissue were elevated by sevoflurane preconditioning before ischemic injury. The up-regulated activity of GSH-px in serum negatively correlated with brain infarct volume percentage. CONCLUSION: Sevoflurane preconditioning induces cerebral ischemic tolerance in a dose– response manner through ROS release and consequent up-regulation of antioxidant enzyme activity before ischemic injury in rats. Serum GSH-px activity could be developed as a marker to assess the effectiveness of sevoflurane preconditioning before ischemia.

Background and Purpose— Our previous study has demonstrated that the rapid tolerance to cerebral ischemia by electroacupuncture (EA) pretreatment was possibly mediated through an endocannabinoid system-related mechanism. The purpose of this study was to investigate whether activation of epsilon protein kinase C (εPKC) was involved in EA pretreatment-induced neuroprotection via cannabinoid receptor type 1 in a rat model of transient focal cerebral ischemia. Methods— The activation of εPKC in the ipsilateral brain tissues after EA pretreatment was investigated in the presence or absence of cannabinoid receptor antagonists. At 2 hours after the end of EA pretreatment, focal cerebral ischemia was induced by middle cerebral artery occlusion for 120 minutes in rats. The neurobehavioral scores, infarction volumes, neuronal apoptosis, and the expression of Bcl-2 and Bax were evaluated after reperfusion in the presence or absence of εPKC-selective peptide inhibitor (TAT-εV1–2) or activator (TAT–ψεRACK). Results— EA pretreatment enhanced εPKC activation. Systemic delivery of TAT–ψεRACK conferred neuroprotection against a subsequent cerebral ischemic event when delivered 2 hours before ischemia. Pretreatment with EA reduced infarct volumes, improved neurological outcome, inhibited neuronal apoptosis, and increased the Bcl-2 -to- Bax ratio after reperfusion, and the beneficial effects were attenuated by TAT-εV1–2. In addition, the blockade of cannabinoid receptor type 1, but not cannabinoid receptor type 2 receptor, reversed the increase in εPKC activation and neuroprotection induced by EA pretreatment. Conclusion— EA pretreatment may activate endogenous εPKC-mediated anti-apoptosis to protect against ischemic damage after focal cerebral ischemia via cannabinoid receptor type 1, which represents a new mechanism of EA pretreatment-induced rapid tolerance to focal cerebral ischemia in rats.

Background We have previously reported that electroacupuncture (EA) pretreatment induced tolerance against cerebral ischemic injury, but the mechanisms underlying this effect of EA are unknown. In this study, we assessed the effect of EA pretreatment on the expression of α7 nicotinic acetylcholine receptors (α7nAChR), using the ischemia-reperfusion model of focal cerebral ischemia in rats. Further, we investigated the role of high mobility group box 1 (HMGB1) in neuroprotection mediated by the α7nAChR and EA. Methods Rats were treated with EA at the acupoint "Baihui (GV 20)" 24 h before focal cerebral ischemia which was induced for 120 min by middle cerebral artery occlusion. Neurobehavioral scores, infarction volumes, neuronal apoptosis, and HMGB1 levels were evaluated after reperfusion. The α7nAChR agonist PHA-543613 and the antagonist α-bungarotoxin (α-BGT) were used to investigate the role of the α7nAChR in mediating neuroprotective effects. The roles of the α7nAChR and HMGB1 release in neuroprotection were further tested in neuronal cultures exposed to oxygen and glucose deprivation (OGD). Results Our results showed that the expression of α7nAChR was significantly decreased after reperfusion. EA pretreatment prevented the reduction in neuronal expression of α7nAChR after reperfusion in the ischemic penumbra. Pretreatment with PHA-543613 afforded neuroprotective effects against ischemic damage. Moreover, EA pretreatment reduced infarct volume, improved neurological outcome, inhibited neuronal apoptosis and HMGB1 release following reperfusion, and the beneficial effects were attenuated by α-BGT. The HMGB1 levels in plasma and the penumbral brain tissue were correlated with the number of apoptotic neurons in the ischemic penumbra. Furthermore, OGD in cultured neurons triggered HMGB1 release into the culture medium, and this effect was efficiently suppressed by PHA-543,613. Pretreatment with α-BGT reversed the inhibitory effect of PHA-543,613 on HMGB1 release. Conclusion These data demonstrate that EA pretreatment strongly protects the brain against transient cerebral ischemic injury, and inhibits HMGB1 release through α7nAChR activation in rats. These findings suggest the novel potential for stroke interventions harnessing the anti-inflammatory effects of α7nAChR activation, through acupuncture or pharmacological strategies.