Anatomical Substrates of Rapid Eye Movement Sleep Rebound in a Rodent Model of Post-sevoflurane Sleep Disruption.

BACKGROUND: Previous research suggests that sevoflurane anesthesia may prevent the brain from accessing rapid eye movement (REM) sleep. If true, then patterns of neural activity observed in REM-on and REM-off neuronal populations during recovery from sevoflurane should resemble those seen after REM sleep deprivation. In this study, the authors hypothesized that, relative to controls, animals exposed to sevoflurane present with a distinct expression pattern of c-Fos, a marker of neuronal activation, in a cluster of nuclei classically associated with REM sleep, and that such expression in sevoflurane-exposed and REM sleep-deprived animals is largely similar. METHODS: Adult rats and Targeted Recombination in Active Populations mice were implanted with electroencephalographic electrodes for sleep-wake recording and randomized to sevoflurane, REM deprivation, or control conditions. Conventional c-Fos immunohistochemistry and genetically tagged c-Fos labeling were used to quantify activated neurons in a group of REM-associated nuclei in the midbrain and basal forebrain. RESULTS: REM sleep duration increased during recovery from sevoflurane anesthesia relative to controls (157.0 ± 24.8 min vs. 124.2 ± 27.8 min; P = 0.003) and temporally correlated with increased c-Fos expression in the sublaterodorsal nucleus, a region active during REM sleep (176.0 ± 36.6 cells vs. 58.8 ± 8.7; P = 0.014), and decreased c-Fos expression in the ventrolateral periaqueductal gray, a region that is inactive during REM sleep (34.8 ± 5.3 cells vs. 136.2 ± 19.6; P = 0.001). Fos changes similar to those seen in sevoflurane-exposed mice were observed in REM-deprived animals relative to controls (sublaterodorsal nucleus: 85.0 ± 15.5 cells vs. 23.0 ± 1.2, P = 0.004; ventrolateral periaqueductal gray: 652.8 ± 71.7 cells vs. 889.3 ± 66.8, P = 0.042). CONCLUSIONS: In rodents recovering from sevoflurane, REM-on and REM-off neuronal activity maps closely resemble those of REM sleep-deprived animals. These findings provide new evidence in support of the idea that sevoflurane does not substitute for endogenous REM sleep.

Sleep Fragmentation, Electroencephalographic Slowing, and Circadian Disarray in a Mouse Model for Intensive Care Unit Delirium.

BACKGROUND: We aimed to further validate our previously published animal model for delirium by testing the hypothesis that in aged mice, Anesthesia, Surgery and simulated ICU conditions (ASI) induce sleep fragmentation, electroencephalographic (EEG) slowing, and circadian disarray consistent with intensive care unit (ICU) patients with delirium. METHODS: A total of 41 mice were used. Mice were implanted with EEG electrodes and randomized to ASI or control groups. ASI mice received laparotomy, anesthesia, and simulated ICU conditions. Controls did not receive ASI. Sleep was recorded at the end of ICU conditions, and hippocampal tissue was collected on EEG recording. Arousals, EEG dynamics, and circadian gene expression were compared with t tests. Two-way repeated measures analysis of variance (RM ANOVA) was used to assess sleep according to light. RESULTS: ASI mice experienced frequent arousals (36.6 ± 3.2 vs 26.5 ± 3.4; P = .044; 95% confidence interval [CI], 0.29-19.79; difference in mean ± SEM, 10.04 ± 4.62) and EEG slowing (frontal theta ratio, 0.223 ± 0.010 vs 0.272 ± 0.019; P = .026; 95% CI, -0.091 to -0.007; difference in mean ± SEM, -0.05 ± 0.02) relative to controls. In ASI mice with low theta ratio, EEG slowing was associated with a higher percentage of quiet wakefulness (38.2 ± 3.6 vs 13.4 ± 3.8; P = .0002; 95% CI, -35.87 to -13.84; difference in mean ± SEM, -24.86 ± 5.19). ASI mice slept longer during the dark phases of the circadian cycle (nonrapid eye movement [NREM], dark phase 1 [D1]: 138.9 ± 8.1 minutes vs 79.6 ± 9.6 minutes, P = .0003, 95% CI, -95.87 to -22.69, predicted mean difference ± SE: -59.28 ± 13.89; NREM, dark phase 2 (D2): 159.3 ± 7.3 minutes vs 112.6 ± 15.5 minutes, P = .006, 95% CI, -83.25 to -10.07, mean difference ± SE, -46.66 ± 13.89; rapid eye movement (REM), D1: 20.5 ± 2.1 minutes vs 5.8 ± 0.8 minutes, P = .001, 95% CI, -24.60 to -4.71, mean difference ± SE, -14. 65 ± 3.77; REM, D2: 21.0 ± 2.2 minutes vs 10.3 ± 1.4 minutes, P = .029, 95% CI, -20.64 to -0.76, mean difference ± SE, -10.70 ± 3.77). The expression of essential circadian genes was also lower in ASI mice (basic helix-loop-helix ARNT like [BMAL1] : -1.3 fold change; circadian locomotor output cycles protein kaput [CLOCK] : -1.2). CONCLUSIONS: ASI mice experienced EEG and circadian changes mimicking those of delirious ICU patients. These findings support further exploration of this mouse approach to characterize the neurobiology of delirium.

Impact of inflammation on brain subcellular energetics in anesthetized rats.

BACKGROUND: Emerging data suggests that volatile anesthetic agents may have organ protection properties in the setting of critical illness. The purpose of this study was to better understand the effect of inflammation on cerebral subcellular energetics in animals exposed to two different anesthetic agents-a GABA agonist (propofol) and a volatile agent (isoflurane). RESULTS: Forty-eight Sprague-Dawley rats were anesthetized with isoflurane or propofol. In each group, rats were randomized to celiotomy and closure (sham) or cecal ligation and puncture (inflammation [sepsis model]) for 8 h. Brain tissue oxygen saturation and the oxidation state of cytochrome aa3 were measured. Brain tissue was extracted using the freeze-blow technique. All rats experienced progressive increases in tissue oxygenation and cytochrome aa3 reduction over time. Inflammation had no impact on cytochrome aa3, but isoflurane caused significant cytochrome aa3 reduction. During isoflurane (not propofol) anesthesia, inflammation led to an increase in lactate (+ 0.64 vs. - 0.80 mEq/L, p = 0.0061). There were no differences in ADP:ATP ratios between groups. In the isoflurane (not propofol) group, inflammation increased the expression of hypoxia-inducible factor-1α (62%, p = 0.0012), heme oxygenase-1 (67%, p = 0.0011), and inducible nitric oxide synthase (31%, p = 0.023) in the brain. Animals exposed to inflammation and isoflurane (but not propofol) exhibited increased expression of protein carbonyls (9.2 vs. 7.0 nM/mg protein, p = 0.0050) and S-nitrosylation (49%, p = 0.045) in the brain. RNA sequencing identified an increase in heat shock protein 90 and NF-κß inhibitor mRNA in the inflammation/isoflurane group. CONCLUSIONS: In the setting of inflammation, rats exposed to isoflurane show increased hypoxia-inducible factor-1α expression despite a lack of hypoxia, increased oxidative stress in the brain, and increased serum lactate, all of which suggest a relative increase in anaerobic metabolism compared to propofol. Differences in oxidative stress as well as heat shock protein 90 and NF-κß inhibitor may account for the differential expression of cerebral hypoxia-inducible factor-1α during inflammation.

Comparison of Broadband and Discrete Wavelength Near-Infrared Spectroscopy Algorithms for the Detection of Cytochrome aa3 Reduction.

BACKGROUND: Cytochrome aa3, the terminal component of the electron transport chain, absorbs near-infrared radiation (NIR) differentially depending on its oxidation state (Cytox), which can in theory be measured using near-infrared spectroscopy (NIRS) by relating light absorption at specific wavelengths to chromophore concentrations. Some NIRS algorithms use discrete wavelengths, while others analyze a band of NIR (broadband NIRS). The purpose of this study was to test the ability of discrete wavelength and broadband algorithms to measure changes in Cytox (primary outcome), and to determine whether or not a discreet wavelength NIRS algorithm could perform similarly to a broadband NIRS algorithm for the measurement of Cytox in a staged hypoxia-cyanide model (hypoxia and cyanide have oppositional effects on tissue saturation, but both cause cytochrome reduction). METHODS: Twenty Sprague-Dawley rats were anesthetized with isoflurane, intubated, and instrumented. Blood pressure, end-tidal carbon dioxide, and arterial oxygen saturation were measured. A halogen light source transmitted NIR transcranially. NIR from the light source and the skull was transmitted to 2 cooled charge-coupled device spectrometers. Rats were subjected to anoxia (fraction of inspired oxygen, 0.0) until arterial oxygen saturation decreased to 70%. After recovery, 5 mg/kg sodium cyanide was injected intravenously. The cycle was repeated until cardiac arrest occurred. Relative concentrations of hemoglobin and cytochrome aa3 were calculated using discreet wavelength and broadband NIRS algorithms. RESULTS: Hypoxia led to an increase in calculated deoxyhemoglobin (0.20 arbitrary units [AUs]; 95% confidence interval [CI], 0.17-0.22; P < .0001), a decrease in calculated oxyhemoglobin (-0.16 AUs; 95% CI, -0.19 to -0.14; P < .0001), and a decrease in calculated Cytox (-0.057 AUs; 95% CI, -0.073 to 0.0040; P < .001). Cyanide led to a decrease in calculated deoxyhemoglobin (-0.037 AUs; 95% CI, 0.046 to -0.029; P < .001), an increase in calculated oxyhemoglobin (0.053 AUs; 95% CI, 0.040-0.065; P < .001), and a decrease in calculated Cytox (-0.056 AUs; 95% CI, -0.064 to -0.048; P < .001). The correlations between "discreet" wavelength algorithms (using 4, 6, 8, and 10 wavelengths) and the broadband algorithm for the measurement of calculated Cytox were 0.54 (95% CI, 0.52-0.56), 0.87 (0.87-0.88), 0.88 (0.88-0.89), and 0.95 (0.95-0.95), respectively. CONCLUSIONS: The broadband and 10 wavelength algorithm were able to accurately track changes in Cytox for all experiments.

Silencing the α2 subunit of γ-aminobutyric acid type A receptors in rat dorsal root ganglia reveals its major role in antinociception posttraumatic nerve injury.

BACKGROUND: Neuropathic pain (NPP) is likely the result of repetitive high-frequency bursts of peripheral afferent activity leading to long-lasting changes in synaptic plasticity in the spinal dorsal horn. Drugs that promote γ-aminobutyric acid (GABA) activity in the dorsal horn provide partial relief of neuropathic symptoms. The authors examined how in vivo silencing of the GABA receptor type A (GABAA) α2 gene in dorsal root ganglia (DRG) controls NPP. METHODS: After crush injury to the right sciatic nerve of female rats, the α2 GABAA antisense and mismatch oligodeoxynucleotides or NO-711 (a GABA uptake inhibitor) were applied to the L5 DRG. In vivo behavioral assessment of nociception was conducted before the injury and ensuing 10 days (n = 4 to 10). In vitro quantification of α2 GABAA protein and electrophysiological studies of GABAA currents were performed on acutely dissociated L5 DRG neurons at relevant time points (n = 6 to 14). RESULTS: NPP postcrush injury of a sciatic nerve in adult female rats coincides with significant down-regulation of the α2 subunit expression in the ipsilateral DRG (approximately 30%). Selective down-regulation of α2 expression in DRGs significantly worsens mechanical (2.55 ± 0.75 to 5.16 ± 1.16) and thermal (7.97 ± 0.96 to 5.51 ± 0.75) hypersensitivity in crush-injured animals and causes development of significant mechanical (2.33 ± 0.40 to 5.00 ± 0.33) and thermal (10.80 ± 0.29 to 7.34 ± 0.81) hypersensitivity in sham animals (data shown as mean ± SD). Conversely, up-regulation of endogenous GABA via blockade of its uptake in DRG alleviates NPP. CONCLUSION: The GABAA receptor in the DRG plays an important role in pathophysiology of NPP caused by sciatic nerve injury and represents promising target for novel pain therapies.

Intraoperative administration of isoflurane improves survival in rats exposed to caecal ligation and puncture.

Background: Emerging data suggest that volatile anaesthetic agents may be protective during critical illness. Methods: Three-month-old Sprague Dawley rats were randomly allocated to one of four groups: isoflurane during surgery followed by 3 days of isoflurane 0.8% (and intralipid i.v.), propofol during surgery and 314 µg kg-1 h-1 propofol for 3 days, isoflurane during surgery and intralipid for 3 days, and propofol during surgery and intralipid for 3 days. After induction with propofol or isoflurane, rats breathed oxygen 100% spontaneously via a nose cone. Propofol or intralipid was administered through a 22-gauge jugular vein i.v. catheter. Caecal ligation and puncture was performed through a paramedian incision. The surgical concentration of isoflurane was kept at 2%, and propofol was maintained at 800 µg kg-1 h-1. After recovery and 3 days of exposure to intralipid or anaesthetic agents, the rats were allowed to roam free in an adequately vented, temperature- and humidity-controlled cage with food and water ad libitum. Results: Rats that received isoflurane for 3 days survived longer than the postoperative propofol group (P=0.0002, log-rank test). Among rats receiving no postoperative anaesthetic, those receiving isoflurane during surgery survived longer than those that received propofol during surgery group (P=0.0081). Conclusions: Exposure to isoflurane, as opposed to propofol, may improve survival in rats exposed to caecal ligation and puncture.

Neonatal anesthesia impairs synapsin 1 and synaptotagmin 1, two key regulators of synaptic vesicle docking and fusion.

Early exposure to anesthetics may interfere with synaptic development and lead to cognitive deficits. We previously demonstrated a decrease in vesicles docked at and within 100 nm from the presynaptic membrane in hippocampal nerve terminals of neonatal rats after anesthesia. Hence, we designed this study to assess the effects of neonatal anesthesia on synapsin 1 (Syn1) and synaptotagmin 1 (Syt1), two key regulators of vesicle docking and fusion. To test the link between changes in Syn1 and Syt1 and behavioral deficits observed after neonatal anesthesia, we also assessed retention memory and fear conditioning in adolescent rats after neonatal anesthesia. Pups received a combination of clinical anesthetics, then Syn1 and Syt1 mRNA and protein expression were determined at the peak (postnatal day 8, P8), part-way through (P12) and end of synaptogenesis (P24) in the CA1-subiculum by qPCR and western blotting. Anesthesia decreased Syn1 and Syt1 mRNA expression at P8 (P<0.01 and <0.001) and P12 (P=0.001 and 0.017), but not P24 (P=0.538 and 0.671), and impaired Syn1, p-Syn1, and Syt1 protein levels at P8 (P=0.038, 0.041, and 0.004, respectively), P12 (P<0.001, P=0.001, and P<0.0001), and P24 (P=0.025, 0.031, and 0.001). Anesthetic-challenged rats displayed deficient long-term retention memory (P=0.019) and hippocampus-dependent fear conditioning (P<0.001). These results suggest that anesthetics alter Syn1 and Syt1 during synapse assembly and maturation, raising the possibility that anesthetic interference with Syn1 and Syt1 could initiate changes in synaptic function that contribute to the cognitive deficits observed after neonatal anesthesia.

Early Exposure to Ketamine Impairs Axonal Pruning in Developing Mouse Hippocampus.

Mounting evidence suggests that prolonged exposure to general anesthesia (GA) during brain synaptogenesis damages the immature neurons and results in long-term neurocognitive impairments. Importantly, synaptogenesis relies on timely axon pruning to select axons that participate in active neural circuit formation. This process is in part dependent on proper homeostasis of neurotrophic factors, in particular brain-derived neurotrophic factor (BDNF). We set out to examine how GA may modulate axon maintenance and pruning and focused on the role of BDNF. We exposed post-natal day (PND)7 mice to ketamine using a well-established dosing regimen known to induce significant developmental neurotoxicity. We performed morphometric analyses of the infrapyramidal bundle (IPB) since IPB is known to undergo intense developmental modeling and as such is commonly used as a well-established model of in vivo pruning in rodents. When IPB remodeling was followed from PND10 until PND65, we noted a delay in axonal pruning in ketamine-treated animals when compared to controls; this impairment coincided with ketamine-induced downregulation in BDNF protein expression and maturation suggesting two conclusions: a surge in BDNF protein expression "signals" intense IPB pruning in control animals and ketamine-induced downregulation of BDNF synthesis and maturation could contribute to impaired IPB pruning. We conclude that the combined effects on BDNF homeostasis and impaired axon pruning may in part explain ketamine-induced impairment of neuronal circuitry formation.