Chloral Hydrate Preconditioning Protects Against Ischemic Stroke via Upregulating Annexin A1.
ABSTRACT: AIMS:Preconditioning is promising for treating cerebral ischemic stroke. Annexin A1 (ANXA1) is a homeostatic antiinflammatory mediator that participates in countering against ischemic injuries. We investigated whether chloral hydrate preconditioning (CH) exerts neuroprotection via regulation of ANXA1 in stroke. METHODS:Adult male C57BL/6J mice or ANXA1 knockout (ANXA1(-/-) ) mice were randomly allocated to control (NCH) and CH groups [2%, 6%, and 10% chloral hydrate (i.p.) 1 h before the middle cerebral artery occlusion (MCAO)]. Neurological performances were evaluated by modified 7-point neurological scales and rotarod test. Cerebral infarction was analyzed by triphenyltetrazolium chloride (TTC) staining and MRI. The expression of ANXA1, pro-inflammatory (TNF-α, IL-1β, IL-6), and antiinflammatory (IL-4, IL-10, TGF-β) cytokines was investigated by RT-PCR, western blot, and immunofluorescence. RESULTS:Chloral hydrate preconditioning significantly improved the neurological outcomes and reduced the infarction and brain edema after ischemia. In addition, CH increased the expression of ANXA1 in the microglia, decreased the levels of TNF-α, IL-1β, and IL-6, while elevated the levels of IL-4, IL-10, and TGF-β in the MCAO mice. Furthermore, both ANXA1 blocker Boc1 (5 mg/kg, i.c.v.) or ANXA1 gene deficiency restrained the protective effects of CH against stroke. CONCLUSIONS:Chloral hydrate preconditioning protects against ischemic injuries through upregulating the expression of ANXA1, and the followed antiinflammatory mechanisms.
Project description:OBJECTIVE:To study the short-term effects of a single-dose chloral hydrate on neonatal auditory perception by measuring auditory event-related potentials (aERPs). METHODS:Thirty-nine full-term neonates, aged 2-28 days and weighing 2980-4350 g, were divided into two groups including a chloral hydrate group (CH group, n = 17) and a non-chloral hydrate control group (non-CH group, n = 22). The CH group was given single-dose chloral hydrate (30 mg/kg) orally before aERPs measurement. An auditory oddball paradigm was used to elicit aERPs. P2 and N2 components of the ERP were recorded from electrodes at the Fz and Cz locations, and the areas under their curves (P2 and N2 areas) were calculated for the comparison between two groups. RESULTS:Significant differences was found in the P2 area between the two groups at Fz and Cz (Fz: F (1,37) = 487.75, P < 0.05; Cz: F (1,37) = 1465.94, P < 0.05). Similarly, significant difference was also in the N2 area between the two groups at both locations (Fz: F(1,37) = 153.38, P < 0.05; Cz: F(1,37) = 798.42, P < 0.05). CONCLUSION:A single-dose of chloral hydrate impacts neonatal auditory perception in the short-term. Long-term effects will also be studied in future.
Project description:Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) have been shown to ameliorate cerebral ischemia in animal models. In this study we investigated the effects of hUCB-MSCs on inflammatory responses and neuronal apoptosis during the early stage of focal cerebral ischemia in rabbits.Focal cerebral ischemia was induced in male New Zealand rabbits by occlusion of MCA for 2 h. The blood samples were collected at different time points prior and during MCAO-reperfusion. The animals were euthanized 3 d after MCAO, and the protein levels of IL-1β, IL-6, IL-10 and TNF-α in the serum and peri-ischemic brain tissues were detected using Western blot and ELISA, respectively. Inflammatory cell infiltration, neuronal apoptosis and neuronal density were measured morphologically. hUCB-MSCs (5 × 10(6)) were iv injected a few minutes after MCAO.The serum levels of IL-1β, IL-6 and TNF-α were rapidly increased, and peaked at 2 h after the start of MCAO. hUCB-MSC transplantation markedly and progressively suppressed the ischemia-induced increases of serum IL-1β, IL-6 and TNF-α levels within 6 h MCAO-reperfusion. Focal cerebral ischemia decreased the serum level of IL-10, which was prevented by hUCB-MSC transplantation. The expression of IL-1β, IL-6, IL-10 and TNF-α in the peri-ischemic brain tissues showed similar changes as in the serum. hUCB-MSC transplantation markedly suppressed the infiltration of inflammatory cells, and increased the neuronal density around the ischemic region. Furthermore, hUCB-MSC transplantation significantly decreased the percentage of apoptosis around the ischemic region.hUCB-MSCs transplantation suppresses inflammatory responses and neuronal apoptosis during the early stage focal cerebral ischemia in rabbits.
Project description:Ischemic tolerance can be induced by numerous preconditioning stimuli, including various Toll-like receptor (TLR) ligands. We have shown previously that systemic administration of the TLR4 ligand, lipopolysaccharide (LPS) or the TLR9 ligand, unmethylated CpG ODNs prior to transient brain ischemia in mice confers substantial protection against ischemic damage. To elucidate the molecular mechanisms of preconditioning, we compared brain and blood genomic profiles in response to preconditioning with these TLR ligands and to preconditioning via exposure to brief ischemia. The experiment is a comparison of multiple treatment groups with sampling at multiple time points. The objective is to identify differentially regulated genes associated with preconditioning. Time points are examined both following preconditioning alone and following subsequent ischemic challenge (middle cerebral artery occlusion (MCAO)). Brain ipsilateral cortex tissue and blood were collected and processed from each animal. 6 experimental conditions: (n=3-4 mice/condition) LPS treated (i.p. 0.2mg/kg) + ischemic challenge (45min MCAO) CpG treated (i.p. 0.8mg/kg) + ischemic challenge (45min MCAO) Saline treated (i.p.) + ischemic challenge (45min MCAO) brief ischemia (12 min MCAO) + ischemic challenge (45min MCAO) Sham of brief ischemia (12 min) + ischemic challenge (45min MCAO) Non-treated + ischemic challenge (45min MCAO) Time points: Pre-ischemic challenge 3hr 24hr 72hr Post-ischemic challenge 3hr 24hr Unhandled (6 mice)-BASELINE
Project description:LapA is the largest surface adhesion protein of Pseudomonas putida that initiates biofilm formation. Here, by using transposon insertion mutagenesis and a conditional lapA mutant, we demonstrate for the first time that LapA influences chloral hydrate (CH) dechlorination in P. putida LF54.
Project description:Inflammatory responses play a critical role in ischemic brain injury. MicroRNA-155 (miR-155) induces the expression of inflammatory cytokines, and acetylbritannilactone (ABL) exerts potent antiinflammatory actions by inhibiting expression of inflammation-related genes. However, the functions of miR-155 and the actual relationship between ABL and miR-155 in ischemia-induced cerebral inflammation remain unclear. In this study, cerebral ischemia of wild-type (WT) and miR-155(-/-) mice was induced by permanent middle cerebral artery occlusion (MCAO). pAd-miR-155 was injected into the lateral cerebral ventricle 24 h before MCAO to induce miR-155 overexpression. MCAO mice and oxygen-glucose deprivation (OGD)-treated BV2 cells were used to examine the effects of ABL and miR-155 overexpression or deletion on the expression of proinflammatory cytokines. We demonstrated that ABL treatment significantly reduced neurological deficits and cerebral infarct volume by inhibiting tumor necrosis factor-? (TNF-?) and interleukin-1? (IL-1?) expression in ischemic cerebral tissue and OGD-treated BV2 cells. Mechanistic studies suggested that the observed decrease in TNF-? and IL-1? expression was attributable to the ABL-induced suppression of the expression of nuclear factor-kappa B (NF-?B) and Toll-like receptor 4 (TLR4). We further found that miR-155 promoted TNF-? and IL-1? expression by upregulating TLR4 and downregulating the expression of suppressor of cytokine signaling 1 (SOCS1) and myeloid differentiation primary response gene 88 (MyD88), while ABL exerted an inhibitory effect on miR-155-mediated gene expression. In conclusion, miR-155 mediates inflammatory responses in ischemic cerebral tissue by modulating TLR4/MyD88 and SOCS1 expression, and ABL exerts its antiinflammatory action by suppressing miR-155 expression, suggesting a novel miR-155-based therapy for ischemic stroke.
Project description:We have shown that aq. 100% (w/v) chloral hydrate (2,2,2-trichloroethane-1,1-diol) dissociates bovine heart cytochrome c oxidase. We have developed new procedures of polyacrylamide-gel electrophoresis in the presence of chloral hydrate that permit variation in the pH of the separation, and, by using these procedures, we have observed 15 components in preparations of the enzyme. This number contrasts with the eight bands that were seen on electrophoresis in the presence of SDS (sodium dodecyl sulphate) and urea. We have isolated material from these eight bands and have characterized each by electrophoresis in the presence of chloral hydrate. Twelve of the fifteen components that were seen by electrophoresis in chloral hydrate were identified as constituents of the eight bands seen by electrophoresis in the presence of SDS and urea. Two-dimensional electrophoretic separations confirmed these identifications ans showed that the other three components which were resolved as discrete bands by electrophoresis in the presence of chloral hydrate appeared to be diffusely present in the electrophoretic separations performed in the presence of SDS and urea, which suggested anomalous behaviour in that detergent. Trypsin treatment of cytochrome c oxidase caused total loss, as observed by electrophoretic separations in the presence of chloral hydrate, of a number of components. The trypsin-sensitive components included all of those that behaved anomalously in the presence of SDS and urea. Chloral hydrate is a potent non-ionic dissociating agent for cytochrome c oxidase and its use in polyacrylamide-gel electrophoresis, with variation in the pH of the gel, permits charge-dependent separations that should have general application in the analysis of membrane proteins.
Project description:This study evaluates the safety and efficacy of chloral hydrate administration for the conscious sedation of infants in the pediatric cardiovascular intensive care unit (PCICU).We conducted a retrospective review of the charts of 165 infants with congenital heart disease who received chloral hydrate in our PCICU between January 2014 and December 2014. Chloral hydrate was administered orally or rectally to infants using doses of 50?mg/kg. We collected and analyzed relevant clinical parameters.The overall length of time to achieve sedation was ranged from 5 to 35 min (10.8?±?6.2?min); the overall mean duration of sedation was ranged from 15 to 60 min (33.5?±?11.3?min); and the overall mean length of time to return to normal activity was 10 min to 6?h (34.3?±?16.2?min). The length of the PCICU stay was ranged from 3 to 30 days (8.2?±?7.1 days). Physiologically, there were no clinically significant changes in heart rate, mean arterial pressure, respiratory rate, or peripheral oxygen saturation before, during, or after use of the chloral hydrate. There were no significant differences regarding sedative effects in the subgroups (cyanotic vs acyanotic group, with pulmonary infection vs without pulmonary infection group, and with pulmonary hypertension vs without pulmonary hypertension group).Our experience suggests that chloral hydrate is a safe and efficacious agent for conscious sedation of infants in the PCICU.
Project description:We documented previously the entity of trichloroethylene (TCE) hypersensitivity syndrome (THS) in occupational workers.To identify the culprit causative compound, determine the type of hypersensitivity of THS, and establish a screening test for subjects at risk of THS.TCE and its main metabolites chloral hydrate (CH), trichloroethanol (TCOH) and trichloroacetic acid (TCA) were used as allergens at different concentrations in skin patch tests. The study included 19 case subjects diagnosed with occupational THS, 22 control healthy workers exposed to TCE (exposure >12 weeks), and 20 validation new workers exposed to TCE for <12 weeks free of THS. All subjects were followed-up for 12 weeks after the patch test.The highest patch test positive rate in subjects with THS was for CH, followed by TCOH, TCA and TCE. The CH patch test positive rate was 100% irrespective of CH concentrations (15%, 10% and 5%). The TCOH patch test positive rate was concentration-dependent (89.5%, 73.7% and 52.6% for 5%, 0.5% and 0.05%, respectively). Lower patch test positive rates were noted for TCA and TCE. All patch tests (including four allergens) were all negative in each of the 22 control subjects. None of the subjects of the validation group had a positive 15% CH patch test.Chloral hydrate seems to be the culprit causative compound of THS and type IV seems to be the major type of hypersensitivity of THS. The CH patch test could be potentially useful for screening workers at risk of THS.
Project description:BACKGROUND AND AIMS: The colons of patients with pneumatosis cystoides coli produce excessive H2. Exposure to alkyl halides could explain this. Six consecutive patients who had pneumatosis cystoides coli while taking chloral hydrate (1-5+ g/day) are reported. Patients 2 and 3 were investigated after they had ceased chloral hydrate treatment. One produced methane, the other did not. (Pneumatosis cystoides coli patients are non-methanogenic according to the literature.) Both had overnight fasting breath H2 of less than 10 ppm. A literature review disclosed just one patient who was using chloral at the time of diagnosed pneumatosis cystoides coli, but an epidemic of the disease in workers exposed to trichloroethylene. METHODS: (i) In vitro experiments with human faeces: chloral or closely related alkyl halides were added to anaerobic faecal cultures derived from four methane-producing and three non-methanogenic human subjects. H2 and CH4 gases were measured. (ii) In vivo animal experiment: chloral hydrate was added to drinking water of four Wistar rats, and faecal H2 compared with control rats. RESULTS: Alkyl halides increased H2 up to 900 times in methanogenic and 10 times in non-methanogenic faecal cultures. The Ki of chloral was 0.2 mM. Methanogenesis was inhibited in concert with the increase in net H2. In the rat experiment, chloral hydrate increased H2 10 times, but did not cause pneumatosis. CONCLUSIONS: Chloral and trichloroethylene are alkyl halides chemically similar to chloroform, a potent inhibitor of H2 consumption by methanogens and acetogens. These bacteria are the most important H2-consuming species in the colon. It is postulated that exposure to these alkyl halides increases net H2 production, which sets the scene for "counterperfusion supersaturation" and the formation of gas cysts. In recent times, very low prescribing rates for chloral have caused primary pneumatosis cystoides to become extremely rare. As with primary pneumatosis, secondary pneumatosis cystoides, which occurs if there is small bowel bacterial overgrowth distal to a proximally located gut obstruction, is predicted by counterperfusion supersaturation. "Inherent unsaturation" due to metabolism of O2 is a safety factor, which could explain why gas bubbles do not form more often in tissue with high H2 tension.
Project description:Tissue sampling for gene expression analysis is usually performed under general anesthesia. Anesthetics are known to modulate hemodynamics, receptor-mediated signaling cascades, and outcome parameters. The present study determined the influence of anesthetic paradigms typically used for euthanization and tissue sampling on cerebral mRNA expression in mice. Naïve mice and animals with acute traumatic brain injury induced by controlled cortical impact (CCI) were randomized to the following euthanasia protocols (n=10-11/group): no anesthesia (NA), 1?min of 4?vol% isoflurane in room air (ISO), 3?min of a combination of 5?mg/kg midazolam, 0.05?mg/kg fentanyl, and 0.5?mg/kg medetomidine intraperitoneally (COMB), or 3?min of 360?mg/kg chloral hydrate intraperitoneally (CH). mRNA expression of actin-1-related gene (Act1), FBJ murine osteosarcoma viral oncogene homolog B (FosB), tumor necrosis factor alpha (TNF?), heat shock protein beta-1 (HspB1), interleukin (IL)-6, tight junction protein 1 (ZO-1), IL-1ß, cyclophilin A, micro RNA 497 (miR497), and small cajal body-specific RNA 17 were determined by real-time polymerase chain reaction (PCR) in hippocampus samples. In naïve animals, Act1 expression was downregulated in the CH group compared with NA. FosB expression was downregulated in COMB and CH groups compared with NA. CCI reduced Act1 and FosB expression, whereas HspB1 and TNF? expression increased. After CCI, HspB1 expression was significantly higher in ISO, COMB, and CH groups, and TNF? expression was elevated in ISO and COMB groups. MiR497, IL-6, and IL-1ß were upregulated after CCI but not affected by anesthetics. Effects were independent of absolute mRNA copy numbers. The data demonstrate that a few minutes of anesthesia before tissue sampling are sufficient to induce immediate mRNA changes, which seem to predominate in the early-regulated gene cluster. Anesthesia-related effects on gene expression might explain limited reproduciblity of real-time PCR data between studies or research groups and should therefore be considered for quantitative PCR data.