Isoflurane Anesthesia In Rats Research Articles

Repetitive Anodal Transcranial Direct Current Stimulation Hastens Isoflurane-Induced Emergence and Recovery and Enhances Memory in Healthy Rats.

Attaining a rapid and smooth return to consciousness after general anesthesia is a goal for clinical anesthesiologists. This study aimed to investigate the effects of repeated anodal transcranial direct current stimulation (atDCS) on emergence and recovery from isoflurane anesthesia in rats. Four days after surgery for atDCS socket implantation, rats received either sham stimulation or repetitive anodal direct electrical current of 0.2 mA intensity applied to the right motor cortex for 20 minutes/d for 10 consecutive days. Isoflurane potency and emergence and recovery from a 2-hour isoflurane challenge were evaluated 24 hours after the last atDCS session. Cognitive performance on novel object recognition and spontaneous alternation Y-maze tests were measured 48 hours after the last atDCS session. Locomotor activity was assessed via automated counting of electric infrared beam crossings. Data are expressed as mean ± standard error of mean (SEM). Isoflurane potency was not affected by atDCS (sham: 1.69% ± 0.06%, transcranial direct current stimulation [tDCS]: 1.73% ± 0.11%, mean difference [MD]: 0.045, 95% confidence interval [CI]: -0.22 to 0.30; P = .72). However, the time to appearance of emergence behavioral marker (eg, return of righting reflex) was hastened in rats receiving atDCS (sham: 486 ± 31 seconds, tDCS: 330 ± 45 seconds, MD: 157, 95% CI: 30-284; P = .008). Similarly, time to acknowledgment of adhesive tape ("sticky dot" applied while anesthetized) was also decreased in atDCS-treated rats as compared to sham (sham: 1374 ± 179 seconds, tDCS: 908 ± 151 seconds, MD: 466, 95% CI: 73-858; P = .015), indicating a faster recovery of isoflurane anesthesia. Rats treated with atDCS spent more time exploring the novel object and environment when compared to sham without affecting activity cycles, indicating visual and working memory can be enhanced by atDCS. Taken together, our findings suggest that atDCS over cortical areas might hasten recovery from isoflurane anesthesia and could potentially be used as a preventative strategy for disruptions in higher order functions related to sedation/anesthesia.

Selective optogenetic activation of orexinergic terminals in the basal forebrain and locus coeruleus promotes emergence from isoflurane anaesthesia in rats

BackgroundThe 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. MethodsHcrtcre 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. ResultsIn 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. ConclusionsActivation of orexinergic terminals in the FB or LC mediates facilitation of emergence from anaesthesia by orexinergic neurones during isoflurane anaesthesia.

GABAA Receptor/STEP61 Signaling Pathway May Be Involved in Emulsified Isoflurane Anesthesia in Rats.

(1) Background: Emulsified isoflurane (EISO) is a type of intravenous anesthetic. How emulsified isoflurane works in the brain is still unclear. The aim of this study was to explore whether epigenetic mechanisms affect anesthesia and to evaluate the anesthetic effects of emulsified isoflurane in rats. (2) Methods: Rats were randomly divided into four groups (n = 8/group): The tail vein was injected with normal saline 0.1 mL·kg−1·min−1 for the control (Con) group, with intralipid for the fat emulsion (FE) group, with EISO at 60 mg·kg−1·min−1 for the high-concentration (HD) group, and 45 mg·kg−1·min−1 for the low-concentration (LD) group. The consciousness state, motor function of limbs, and response to nociceptive stimulus were observed after drug administration. (3) Results: Using real-time polymerase chain reaction (PCR) to assess the promoter methylation of ion channel proteins in the cerebral cortex of rats anesthetized by EISO, we demonstrated that the change in the promoters’ methylation of the coding genes for gamma-aminobutyric acid A receptor α1 subunit (GABAAα1), N-methyl-D-aspartate receptor subunit 1 (NMDAR1), and mu opioid receptor 1 (OPRM1) was accompanied by the change in messenger ribonucleic acid (mRNA) and protein expression by these genes. (4) Conclusion: These data suggest that the epigenetic factors’ modulation might offer a novel approach to explore the anesthetic mechanism of EISO.

Electrical Stimulation in the Claustrum Area Induces a Deepening of Isoflurane Anesthesia in Rat.

The role of the claustrum in consciousness and vigilance states was proposed more than two decades ago; however, its role in anesthesia is not yet understood, and this requires more investigation. The aim of our study was to assess the impact of claustrum electrical stimulation during isoflurane anesthesia in adult rats. The claustrum in the left hemisphere was electrically stimulated using a bipolar tungsten electrode inserted stereotaxically. In order to monitor the anesthetic depth, the electrocorticogram (ECoG) was recorded before, during, and after claustrum stimulation using frontal and parietal epidural electrodes placed over the left hemisphere. After reaching stabilized slow-wave isoflurane anesthesia, twenty stimuli, each of one second duration with ten seconds interstimulus duration, were applied. ECoG analysis has shown that, after a delay from the beginning of stimulation, the slow-wave ECoG signal changed to a transient burst suppression (BS) pattern. Our results show that electrical stimulation of the claustrum area during slow-wave isoflurane anesthesia induces a transitory increase in anesthetic depth, documented by the appearance of a BS ECoG pattern, and suggests a potential role of claustrum in anesthesia.

Serotonergic neurons in the dorsal raphe nucleus mediate the arousal-promoting effect of orexin during isoflurane anesthesia in male rats

Previous studies have demonstrated that the activation of orexinergic neurons facilitates the recovery of animals from general anesthesia. Moreover, serotonergic neurons that receive projections from orexin neurons have also been shown to participate in sleep-wakefulness regulation. In the present study, we aimed to explore whether orexinergic neurons facilitate emergence from isoflurane anesthesia in rats by activating serotonergic neurons. Orexin A (30 or 100 pmol), orexin B (30 or 100 pmol), and their respective antagonists SB-334867 and TCS-OX2–29 (5 or 20 μg) were microinjected into the dorsal raphe nucleus (DRN) of rats, and their effects on induction and emergence times were analyzed. Electroencephalogram (EEG) changes were also recorded and analyzed to illuminate the effect of orexin injection into the DRN on cortical excitability under isoflurane anesthesia. Activation of serotonergic neurons was detected via immunohistochemical analysis of c-Fos expression following orexin administration. Our results indicated that injection of neither orexins nor orexin antagonists into the rat DRN exerted an impact on induction time, whereas orexin-A injection (100 pmol) enhanced arousal when compared with the saline group. In contrast, administration of orexin receptor type 1 antagonist SB-334867 (20 μg) prolonged emergence time from isoflurane anesthesia. Microinjection of orexin-A induced an arousal pattern on EEG, and decreased the burst suppression ratio under isoflurane anesthesia. Isoflurane anesthesia inhibited the activity of serotonergic neurons, as shown by decrease in the number of c-Fos-immunoreactive serotonergic neurons when compared with the sham group. This inhibitory effect was partially reversed by administration of orexin-A. Taken together, our findings suggest that orexinergic signals facilitate emergence from isoflurane anesthesia, at least partially, by reversing the effects of isoflurane on serotonergic neurons of the DRN.

Sex effects on behavioral markers of emergence from propofol and isoflurane anesthesia in rats

Clinical studies have demonstrated sex-related differences in recovery from surgical anesthesia. This study aimed to characterize the emergence pattern following two anesthesia regimens in both sexes of rats.We considered six different markers of emergence from anesthesia: sigh, eye blinking, forelimb movement, mastication, neck extension, and recovery of the righting reflex (RORR). Spontaneous motor activity 24 h after the anesthesia induction was also examined.Our results showed that the rank order of the emergence latency after intraperitoneal propofol, PRO, exposure was forelimb movement < sigh < blink < mastication < neck extension < RORR, while after inhaled isoflurane, ISO, anesthesia the sequence was changed as sigh < blink < mastication < forelimb movement < neck extension < RORR in both male and female rats. Moreover, the latency to emergence after PRO in female rats was significantly higher than male rats, although following ISO there was no difference between the sexes (P < 0.001; P > 0.05, respectively). Open-field testing revealed no difference in PRO and ISO spontaneous locomotor activity due to drug administration (P > 0.05).These two anesthetics presented different emergence sequences. Although clinical data suggests that females arouse faster than males from anesthesia with propofol, our intraperitoneal technique in a rodent model had the opposite effect. Pharmacokinetic analysis demonstrated increased absorption of injected propofol for the female rats in our study, emphasizing the role of sexual dimorphism in drug distribution in rodents. Despite these pharmacokinetic differences, the pharmacodynamic effects of the drugs were remarkably consistent among both sexes through emergence.

Neuroprotective effect of ginsenoside Rg1 prevents cognitive impairment induced by isoflurane anesthesia in aged rats via antioxidant, anti-inflammatory and anti-apoptotic effects mediated by the PI3K/AKT/GSK-3β pathway.

Ginsenoside Rg1 is the primary active substance in ginseng, and it has multiple pharmacological actions. Investigations on the pharmacologic action of ginsenoside Rg1 have developed, with a particular focus on the regulation of metabolism. The present study hypothesized that the neuroprotective effects of ginsenoside Rg1 prevent cognitive impairment induced by isoflurane anesthesia via antioxidant, anti‑inflammatory and anti‑apoptotic effects, mediated by the phosphoinositide 3‑kinase (PI3K)/AKT/glycogen synthase kinase‑3β (GSK‑3β) pathway in aged rats. Sprague‑Dawley rats were divided into isoflurane and ginsenoside Rg1 groups and were treated with 20mg/kg ginsenoside Rg1 for 7days. Morris water maze was performed to analyze the cognitive function of the rats. Enzyme‑linked immunosorbent assays were used to analyze the levels of malondialdehyde, glutathione, interleukin (IL)‑1β, IL‑6 and caspase3. The protein expression levels of AKT, GSK3β, p21WAF1/CIP1 and p53 were measured using western blot analysis. Ginsenoside Rg1 significantly improved cognitive function, and exhibited antioxidant and anti‑inflammatory effects, demonstrating the neuroprotective effects of ginsenoside Rg1 against the effect of isoflurane anesthesia in the rats. In addition, ginsenoside Rg1 significantly reduced caspase‑3 activity, upregulated the expression of PI3K/AKT/GSK‑3β and downregulated the mRNA expression levels of p21WAF1/CIP1 and p53 in the aged rats exposed to isoflurane anesthesia. The data obtained in the present study provided evidence that the neuroprotective effects of ginsenoside Rg1 prevented the cognitive impairment induced by isoflurane anesthesia via antioxidant, anti‑inflammatory and anti‑apoptotic effects, mediated by the PI3K/AKT/GSK‑3β pathway.

Role of orexin in sleep disorder after isoflurane anesthesia in rats

Objective To evaluate the role of orexin in the sleep disorder after isoflurane anesthesia in rats.Methods Sixty-four male Sprague-Dawley rats,aged 10-12 weeks,weighing 280-320 g,were randomly divided into 2 groups (n =32 each) using a random number table:control group (C) and isoflurane group (Ⅰ).Group Ⅰ inhaled 1.2％ isoflurane from 8:00 to 13:30 to induce anesthesia,followed by 0.5 h of recovery.Group C received no anesthesia and the other procedures were similar to those previously described in group Ⅰ.The induction time and awakening time were recorded.Eight rats were randomly chosen to record the movement condition (locomotor time and activity) from 14:00 to 8:00 the next morning.Before beginning of anesthesia,at 4 h after beginning of anesthesia,and at 4 and 10 h after the end of anesthesia,6 rats were randomly chosen in each group to count the orexin/c-fos double-labeled neurons in hypothalamus.The ratio of activated orexin neurons (orexin/c-fos double-labeled neurons to orexin positive neurons) was calculated and plasma orexin-A concentration was detected.Results The induction time was (2.14 ± 0.17) min,awakening time was (8.7 ± 0.5) min,and EEG showed that there was no typical burst and suppression patterns in group Ⅰ.There was no significant difference in the number of orexin positive neurons between the two groups (P ＞ 0.05).Compared with group C,the time for locomotor activity was significantly prolonged,and the activity was increased during the night (P ＜ 0.01),the number of activated neurons,ratio of activated orexin neurons and plasma orexin-A concentration were decreased at 4 h after beginning of anesthesia in group Ⅰ (P ＜ 0.01).The plasma orexin-A concentration was lower at 4 h after beginning of anesthesia,while higher at 10 h after the end of anesthesia than before beginning of anesthesia in group Ⅰ (P ＜ 0.05).The number of activated neurons was significantly larger and ratio of activated orexin neurons was higher before beginning of anesthesia and at 10 h after the end of anesthesia and the plasma orexin-A concentration was higher at 4 and 10 h after the end of anesthesia than at 4 h after beginning of anesthesia in group Ⅰ (P ＜ 0.05 or 0.01).Conclusion The development of sleep disorder after isoflurane anesthesia during the day time in rats is related to the regulatory role of orexin in it. Key words: Sleep initiation and maintenance disorders; Isoflurane; Orexin

The Protective Effect of Ischemic Preconditioning Against Hepatic Ischemic-Reperfusion Injury Under Isoflurane Anesthesia in Rats

PurposeApoptosis is a central mechanism of ischemic-reperfusion injury (IRI) to the liver. Among the methods to reduce IRI, ischemic preconditioning (IP) has been shown to confer protection. Therefore, the aim of this study was to determine if IP conferred protection against hepatic IRI under isoflurane anesthesia in rats and to investigate underlying protective mechanisms. Materials and MethodsTwenty-three rats weighing 270 to 300 grams were randomly divided into three groups: (1) the sham operated group (n = 5); (2) the non-IP group (n = 9; 45 minutes of hepatic ischemia followed by 2 hours of reperfusion); and (3) the IP group (n = 9); IP induced by 10 minutes of hepatic ischemia followed by 15 minutes of reperfusion before 45 minutes of prolonged hepatic ischemia). Anesthesia was maintained with isoflurane (1.5%). We compared the degrees of hepatic injury and expressions of B cell lymphoma 2 (Bcl-2) and caspase 3 and 8 mRNAs. ResultsThe IP group showed significantly lower levels of aspartate transaminase and alanine transaminase as well as reduced histological grades of hepatocyte injury compared with the non-IP group at 2 hours after reperfusion. At the corresponding time, the Bcl-2 mRNA level was 2-fold higher in the IP group. Caspase 3 mRNA levels were highest in the non-IP group significantly compared with the sham cohort. Similarly, caspase 8 mRNA levels were highest in the Non_IP group albeit not significancely. ConclusionIP protected against hepatic IRI under isoflurane anesthesia in rats. The mechanism of protection appeared to involve upregulation of Bcl-2 expression resulting in inhibited apoptosis.

Dopamine-enhancing Medications to Accelerate Emergence from General Anesthesia

“The anatomical and neurochemical signatures of the dopamine system for arousal under conditions of general anesthesia are clinically important to understand.” Dopaminergic neurotransmission is prominently implicated in emergence from the minimal conscious state,1,2general anesthesia,3and in sleep–wake regulation.4The molecular mechanisms for these effects are still incompletely understood. The anatomical and neurochemical signatures of the dopamine (DA) system for arousal under conditions of general anesthesia are clinically important to understand. First, from a clinical point of view, accurate knowledge of the molecular basis for emergence from general anesthetics with agents for which no specific antagonists exists (i.e ., inhalational agents) and for other anesthetic regimens could lead to the development of a range of new drugs specifically designed for rapid emergence. Such therapeutics could perhaps be useful in the future for routine anesthesia practice. For example, rapid arousal of elderly patients undergoing surgical procedures requiring general anesthesia may prove valuable for reducing cognitive dysfunction and/or early delirium postoperatively. Second, a better understanding of the neuronal mechanisms by which DA increases the emergence from anesthesia will also help in the management of patients with DA abnormalities as may be the case for patients with Parkinson disease, schizophrenic patients treated with depot neuroleptics (long-acting dopamine-2 receptor [D2R] antagonists) and drug abusers.In this issue of ANESTHESIOLOGY, Taylor et al .5report that the dopamine-1 receptor (D1R) agonist 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide but not the D2R agonist quinpirole reduces the time to emergence from isoflurane anesthesia in rats by 85% compared with placebo. The authors conclude that selective activation of D1Rs is sufficient to induce emergence from isoflurane general anesthesia and that D2Rs are not needed.5These new data in the setting of isoflurane anesthesia are intriguing and supplement older data reporting similar findings with phenobarbital anesthesia.6,7Specifically, Horita et al .7investigated the effects of a D1R agonist (1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol) and a D2R antagonist (raclopride) on duration of phenobarbital anesthesia in rats (administered only 20 min after induction with 40 mg/kg intraperitoneal phenobarbital to reduce pharmacokinetic effects on emergence). They documented that the D1R agonist reduced the time to emergence by approximately 30% and found that this effect could be abolished by a D1R antagonist.7However, in contrast to the current study,5Horita et al .7reported that the D2R antagonist raclopride and atropine also reversed the effect of the D1R agonist. Thus, the findings of Horita et al . suggest that the arousing effects of D1R agonists require the coactivation of these two receptors (D1R and D2R). Interestingly, although nonsignificant, the study by Taylor et al .5showed decreases in the time from emergence of anesthesia in the rats treated with the D2R agonist quinpirole (from 330 to 189 s) and had their sample (n = 6) been larger, this effect may have been significant. Regardless, it is clear that the effects of the D1R agonist was stronger than that of the D2R agonist (quinpirole), which could reflect in part its effects on D2R autoreceptors that would lead to a decrease in DA release. In the previous study, Horita et al .7also provide evidence that the dopaminergic effects (driven by D1R but presumably requiring background D2R tone) are mediated by downstream cholinergic effects (because these effects were blocked by atropine). In the context of the importance of D2Rs, Solt et al .3reported that droperidol (a potent D2 antagonist) abolished the ability of the DA-enhancing drug methylphenidate to accelerate emergence in rats anesthetized with isoflurane, which also strongly suggests that D2Rs are important for anesthesia emergence.Another intriguing question is which brain pathways mediate the effects of DA in emergence from anesthesia. In general, studies on DA networks have focused mostly on pathways involved with movement (mesostriatal), reward (mesoaccumbens), and cognition (mesocortical), and much less is known about the DA pathways implicated in arousal. Although it is plausible that some or all of these DA pathways participate in arousal, it is also likely that DA mediates its arousing effects through additional brain-wide systems. For example, the orexin/hypocretin lateral hypothalamic nucleus, which mediates arousal and is implicated in narcolepsy, is modulated by both D1R and D2R.8,9Similarly, the tuberomammillary nucleus, which is involved in the control of wakefulness, signals not only through histamine receptors but also through D1R and D2R.10In addition, as discussed by Taylor et al. ,5DA neurons in the ventral periaqueductal gray could mediate the effects of DA on the emergence from anesthesia.11Finally, it is also possible that DA mediates its effects on arousal via thalamic activation.12Over the past decades, scientists have studied the DA system’s role in cognition, motor behavior, and reward and its disruption in neuropsychiatric diseases. The data of Taylor et al .5and that of others now highlight the importance of DA in arousal. These findings have direct clinical implications for the practice of anesthesia for they may help direct the development of new medications to accelerate the emergence from anesthesia.

Increased electroencephalographic gamma activity reveals awakening from isoflurane anaesthesia in rats

Animal studies often require reliable measures for anaesthetic drug effects. Lately, EEG-based depth of anaesthesia estimation has been widely applied to rat models. This study investigated the reliability of different EEG spectral properties in revealing awakening from isoflurane anaesthesia in rats. Adult Wistar rats with previously implanted frontal epidural electrodes were anaesthetized using isoflurane. The anaesthesia was slowly lightened until awakening, as observed by the first spontaneous movement, after which anaesthesia was induced again by increasing the isoflurane concentration. EEG was recorded during the recovery and induction periods, and the spectrograms and 23 quantitative spectral parameters used in the depth of anaesthesia estimation were calculated from the signals. The awakening was accompanied by a decrease in EEG activity at frequencies below 25 Hz, while the activity at higher frequencies (25-150 Hz) was increased. Whereas the behaviour of parameters used to measure activity in the lower frequencies was subject to variability between animals, the increase in higher frequency activity was more consistent, resulting in a statistically significant change in the relative gamma power parameters at the moment of awakening. The increase in frontal relative gamma activity, especially in the 50-150 Hz frequency band, seems to be the most reliable EEG indicator for the awakening of a rat from isoflurane anaesthesia. A number of other spectral measures can also be used to detect this event. However, the role of gamma frequencies in the performance of these parameters is crucial.

The Protective Effect of Ischemic Preconditioning Against Hepatic Ischemic/Reperfusion Injury Under Isoflurane Anesthesia in Rats

Introduction: Apoptosis is a central mechanism of ischemic/reperfusion (I/R) injury in the liver. Ischemic preconditioning (IP) has been shown to confer protection against I/R injury in various organs. Therefore, the aim of this study was to determine if application of IP would confer protection against hepatic I/R injury under isoflurane anesthesia in rats and investigate underlying protective mechanism. Materials and methods: Twenty-three rats weighing 270-300 g were enrolled in this study. The rats were randomly divided into three groups: (1) Sham group (n=5): sham operated group; (2) Non-IP group (n=9): 45 minutes of hepatic ischemia followed by 2 hours of reperfusion; (3) IP group (n=9): ischemic preconditioning induced by 10 minutes of hepatic ischemia followed by 15 minutes of reperfusion prior to 45 minutes of prolonged hepatic ischemia. Isoflurane (1.5%) was maintained throughout the study period (from induction to 2 h after reperfusion when animals were sacrificed). The degree of hepatic injury and the expressions of Bcl-2 and caspase 3 and 8 mRNA were compared. Results: IP group showed significantly lower levels of AST (227 ± 188 vs. 562 ± 244 U/L, P < 0.05) and ALT (136 ± 145 vs. 344 ± 178 U/L, P < 0.05) and reduction in histological grade of hepatocyte injury (Table 1) compared with Non_IP group at 2 h after reperfusion.Table: [Histologic Liver Changes at 2 h after Reperfusion]At the corresponding time, the Bcl-2 mRNA level was significantly higher (two-fold, P < 0.05) in the IP group compared to the Non_IP group (Figure 1).[Figure 1. Bcl-2 mRNA expressions]Caspase 3 mRNA level was highest in Non_IP group with statistical significance between Non_IP group and sham group. Similarly, caspase 8 mRNA level was highest in Non_IP group but no statistical significance was observed among the groups (Figure 2).[Figure 2. Caspases 3 & 8 mRNA]Conclusions: Ischemic preconditioning protected against hepatic ischemic/reperfusion injury under isoflurane anesthesia in rats. The mechanism of protection appears to occur through up-regulation of Bcl-2 expression resulting in inhibition of apoptosis.

Sleep and Anesthesia

Sleep and Anesthesia

Tau hyperphosphorylation is associated with memory impairment after exposure to 1.5% isoflurane without temperature maintenance in rats

There is increasing interest in studying the role of tau hyperphosphorylation associated with memory impairment. We examined the involvement of tau hyperphosphorylation in memory impairment after hypothermia following isoflurane anaesthesia in rats. Adult rats were randomly divided into three groups: the control group received no treatment and others were subjected to 1.5% isoflurane anaesthesia with or without temperature control for 2 h. On the day before anaesthesia and on postanaesthetic days 1, 3 and 7, cognitive functions were assessed in a Y-maze test paradigm. To find the relationship between memory results and tau, we measured the site-specific phosphorylation of tau at Thr-205 and Ser-396 and the activity of protein phosphatase 2A within the hippocampus. The spatial learning and memory of animals with hypothermia were impaired at day 1 after anaesthesia, compared with nonanaesthetized rats. Anaesthesia and hypothermia led to tau hyperphosphorylation at the Thr-205 and Ser-396 epitopes in the hippocampus. There was no significant difference in the protein phosphatase 2A activity between the control and the postanaesthetic rat hippocampal samples, whereas nearly 45% protein phosphatase 2A inhibition was detected in the anaesthetized without temperature maintenance rat samples. Our results indicate that tau phosphorylation is not a direct result of anaesthesia per se, but it is due to anaesthesia-induced hypothermia and this leads to the inhibition of phosphatase activity as well as tau hyperphosphorylation. Tau hyperphosphorylation is associated with the observed deficits in spatial learning and memory following anaesthesia in hypothermic rats.

Isoflurane Anesthesia Elicits Protein Pattern Changes in Rat Hippocampus

Postoperative cognitive dysfunction (POCD) is a known phenomenon occurring after anesthesia with volatile anesthetics (VA), such as isoflurane. Recent reports suggest that VA interact with neurodegenerative disease-associated proteins including compounds with pathogenic relevance in Alzheimer disease (AD) and induce processes that may be linked to AD neuropathology. Unfortunately, our present understanding of the exact anesthetics' molecular mechanisms of action, their side effects on the brain, and their catenation with AD pathology is still limited. The present study analyzes the differential proteome of the hippocampus immediately after and 3 days after a 3-hour 1 minimal alveolar concentration isoflurane anesthesia in rats. Differential 2-dimensional electrophoresis, mass spectrometry, and functional network mapping were used to identify and functionally classify 12 different hippocampal proteins, which were significantly regulated after isoflurane anesthesia (6 up-regulated, 11 down-regulated with P<0.01). Induction of differential expression ranged from 0.05 (25-fold down-regulation) to 4.4 (4.4-fold up-regulation). Ten proteins were regulated immediately after and 7 proteins 3 days after isoflurane exposure. The proteome displays isoflurane-responsive protein candidates, which have also been shown to play a role in AD. They were grouped according to their key biologic activities, which showed that isoflurane affects selected biologic processes including synaptic plasticity, stress response, detoxification, and cytoskeleton in early and late recovery phases after anesthesia. These processes are also affected in AD. Results are discussed in view of AD, the toxicity mechanisms of isoflurane as well as the implications for our present understanding and conduction of clinical anesthesia.

Burst Activation of the Cerebral Cortex by Flash Stimuli during Isoflurane Anesthesia in Rats

The degree of suppression of sensory functions during general anesthesia is controversial. Here, the authors investigated whether discrete flash stimuli induced cortical field potential responses at an isoflurane concentration producing burst suppression and compared the spatiotemporal properties and frequency spectra of flash-induced burst responses with those occurring spontaneously. Rats were equipped with multiple epidural and intracortical electrodes to record cortical field potentials in the right hemisphere at several locations along the anterior-posterior axis. At isoflurane concentrations of 1.1, 1.4, and 1.8%, discrete light flashes were delivered to the left eye while cortical field potentials were continuously recorded. Isoflurane at 1.4-1.8% produced burst suppression. Each flash produced a visual evoked potential in the primary visual cortex followed by secondary bursting activity in more anterior regions. The average latency and duration of these bursts were 220 and 810 ms, respectively. The spontaneous and flash-induced bursts were similar in frequency, duration, and spatial distribution. They had maximum power in the frontal (primary motor) cortex with a dominant frequency of 10 Hz. The results suggest that discrete flash stimuli activate the motor regions of the cerebral cortex during isoflurane anesthesia and that these activations are analogous with those that occur spontaneously during burst suppression. Electrocortical suppression of the cortex during anesthesia does not prevent its response to visual stimuli.

Cholinergic reversal of isoflurane anesthesia in rats as measured by cross-approximate entropy of the electroencephalogram.

Pharmacologic modulation of the state of consciousness is of interest for clinical practice and for a better understanding of anesthetic mechanisms. The cholinergic activating system is an important regulator of the state of consciousness during general anesthesia. Entropy of the electroencephalogram has been proposed as a promising measure of anesthetic depth. The authors have shown that volatile anesthetics decrease cross-approximate entropy (C-ApEn) of the bihemispheric frontal electroencephalogram in rats. The effect of cholinergic agents on C-ApEn has not been examined. Here, the authors test the hypothesis that cholinergic activation reverses the effect of isoflurane anesthesia on C-ApEn. An electroencephalogram in the 1- to 100-Hz range was recorded bipolarly, with epidural leads from the frontal cortex of both hemispheres, and used to calculate C-ApEn, which reflects statistical independence of bihemispheric electroencephalographic activity. Cholinesterase inhibitor, neostigmine (25 mug), or the muscarinic agonist oxotremorine (25 mug) were infused intracerebroventricularly while the rats were inhaling 1.0% (0.7 minimum alveolar concentration) isoflurane. In other animals, isoflurane was lowered to 0.4% (0.3 minimum alveolar concentration) to assess the electroencephalogram in a sedated, waking state. At 1.0% isoflurane, C-ApEn decreased by 54% compared with that at 0.4%, but the motor reflex response to tail pinch was still present. Cholinergic agents reversed the electroencephalogram-depressant effect of isoflurane, i.e., C-ApEn rose to the level measured at 0.4% isoflurane. The rise in C-ApEn was paralleled by the appearance of spontaneous limb and orofacial explorative movements, suggesting a return of consciousness. In contrast, cholinergic agents fully blocked the motor reflex to tail pinch. C-ApEn of the bihemispheric electroencephalogram correlates with the return of spontaneous motor signs but not with the nociceptive reflex. Cerebral cholinergic activation dissociates central and peripheral anesthetic effects. C-ApEn, a novel measure of interhemispheric electroencephalogram independence, is a promising correlate of depth of sedation and state of consciousness.

Hyperglycolysis is exacerbated after traumatic brain injury with fentanyl vs. isoflurane anesthesia in rats

Despite common use of narcotics in the clinical management of severe traumatic brain injury (TBI), in experimental models rats treated with fentanyl have exhibited worse functional outcome and more CA1 hippocampal death than rats treated with standard isoflurane anesthesia. We hypothesized that greater post-traumatic excitotoxicity, reflected by cerebral glucose utilization (CMRglu), may account for detrimental effects of fentanyl vs. isoflurane. Rats were anesthetized with either isoflurane (1% by inhalation) or fentanyl (10 mcg/kg iv bolus then 50 mcg/kg/h infusion). 14C-deoxyglucose autoradiography was performed 45 min after controlled cortical impact (CCI) to left parietal cortex ( n=4 per anesthetic group) or in uninjured rats after 45 min of anesthesia ( n=3 per anesthetic group). Uninjured rats treated with fentanyl vs. isoflurane showed 35–45% higher CMRglu in all brain structures ( p<0.05) except CA3. After TBI in rats treated with isoflurane, CMRglu increased significantly only in ipsilateral CA1 and ipsilateral parietal cortex ( p<0.05 vs. isoflurane uninjured). Conversely, after TBI in rats treated with fentanyl, CMRglu increased markedly and bilaterally in CA1 and CA3 ( p<0.05 vs. fentanyl uninjured), but not ipsilateral parietal cortex. In contralateral CA1, CMRglu was nearly two times greater after TBI in fentanyl vs. isoflurane treated rats ( p<0.05). Hyperglycolysis was exacerbated in CA1 and CA3 hippocampus after TBI in rats treated with fentanyl vs. isoflurane anesthesia. This post-traumatic hyperglycolysis suggests greater excitotoxicity and concurs with reports of worse functional outcome and more CA1 hippocampal death after TBI with fentanyl vs. isoflurane anesthesia.

Comparison of electroencephalogram activity and auditory evoked responses during isoflurane and halothane anaesthesia in the rat

ObjectivesTo compare the second differential index (SDI) calculated from the auditory evoked potential (AEP) and electroencephalogram (EEG) parameters: median frequency (MF), spectral edge frequency (SEF) and burst suppression rate (BSR) determined at four equivalent minimum alveolar concentrations (MAC) of isoflurane or halothane. AnimalsTwelve male Wistar rats weighing 418 g (SD ± 18.4 g). MethodsAuditory evoked potentials and EEG responses were recorded in animals implanted with electrodes at established anaesthetic concentrations. Depth of anaesthesia was assessed using the strength of the pedal withdrawal reflex (PWR), and data were analysed using repeated measures anova and paired t-tests. ResultsThe SEF tended to decrease with increasing depth of halothane anaesthesia (F = 4.198, p = 0.05), but not with isoflurane. The MF and SDI were significantly higher during halothane than with isoflurane (F = 5.82, p = 0.036 and F = 5.263, p = 0.045, respectively) at equivalent depths of anaesthesia, and EEG burst suppression occurred at deeper planes of isoflurane but not halothane anaesthesia. ConclusionsThe study demonstrated that EEG and AEP characteristics recorded at MAC equivalent concentrations were suppressed to a greater degree by isoflurane than by halothane. These findings have strong implications for research projects where EEG recordings are collected, and also cast more general doubts upon the value of such parameters for evaluating depth of isoflurane anaesthesia in rats.

Tolerance against ischemic neuronal injury can be induced by volatile anesthetics and is inducible NO synthase dependent.

We tested whether volatile anesthetics induce neuroprotection that is maintained for a prolonged time. Rats were pretreated for 3 hours with 1 minimal anesthetic concentration of isoflurane or halothane in normal air (anesthetic preconditioning [AP]). The animals were subjected to permanent middle cerebral artery occlusion (MCAO) at 0, 12, 24, or 48 hours after AP. Halothane-pretreated animals were subjected to MCAO 24 hours after AP. Histological evaluation of infarct volumes was performed 4 days after MCAO. Cerebral glucose utilization was measured 24 hours after AP with isoflurane. Primary cortical neuronal cultures were exposed to 1.4% isoflurane for 3 hours. Oxygen-glucose deprivation (OGD) was performed 24 hours after AP. Injury was assessed 24 hours later by measuring the release of lactate dehydrogenase into the medium 24 hours after OGD. Isoflurane anesthesia at 0, 12, and 24 hours before MCAO or halothane anesthesia 24 hours before MCAO significantly reduced infarct volumes (125+/-42 mm3, P=0.024; 118+/-51 mm3, P=0.008; 120+/-49 mm3, P=0.009; and 121+/-48 mm3, P=0.018, respectively) compared with control volumes (180+/-51 mm3). Three hours of isoflurane anesthesia in rats did not have any effect on local or mean cerebral glucose utilization measured 24 hours later. Western blot analysis from cortical extracts of AP-treated animals revealed an increase of the inducible NO synthase (iNOS) protein beginning 6 hours after AP. The iNOS inhibitor aminoguanidine (200 mg/kg IP) eliminated the infarct-sparing effect of AP. In cultured cortical neurons, isoflurane exposure 24 hours before OGD decreased the OGD-induced release of lactate dehydrogenase by 49% (P=0.002). Pretreatment with volatile anesthetics induces prolonged neuroprotection in vitro and in vivo, a process in which iNOS seems to be critically involved.