Inhibition of ROCK2 kinase activity improved behavioral deficits and reduced neuron damage in a DEACMP rat model.

The main pathological changes that occur in delayed encephalopathy after acute carbon monoxide poisoning (DEACMP) are extensive demyelination of brain white matter and neuron damage. Previous studies suggested that demyelination and neuron injury are related to activating the Rho/ROCK signaling pathway. Inhibition of the Rho/ROCK signaling pathway can alleviate neuron injury and promote myelin repair. This study utilized a DEACMP model in which rats were prepared by space injection of CO gas intraperitoneally (CO group), and the association between the Rho/ROCK signaling pathway and DEACMP was investigated. The ROCK2 kinase inhibitor Y-27632 was used to prevent the effects of the DEACMP model to elucidate its protective mechanism. The results demonstrated that the cognitive and motor functions were significantly impaired, and the GFAP, NSE, RhoA, and ROCK2 protein levels were significantly increased in the CO group within three weeks after the model was established. After Y-27632 intervention, the cognitive and motor functions of the CO+Y-27632 group were significantly improved within three weeks after the model was established. In the CO+Y-27632 group, the RhoA, ROCK2, GFAP, and NSE (indicating neuron injury) protein levels decreased significantly, and the MBP protein levels (indicating myelin repair) increased significantly within three weeks after the model was established. These results suggested that the pathogenesis of DEACMP was associated with activation of the Rho/ROCK pathway and that Y-27632 inhibited ROCK2 kinase activity in the CO exposed rats, resulting in improved behavioral deficits, reduced neuron damage, and promotion of myelin repair. Therefore, Y-27632 might be a potentially effective drug for the treatment of DEACMP-induced brain damage.

Serum neuron-specific enolase as an early predictor of delayed neuropsychiatric sequelae in patients with acute carbon monoxide poisoning.

Delayed onset of neuropsychiatric symptoms after apparent recovery from acute carbon monoxide (CO) poisoning has been described as delayed neuropsychiatric sequelae (DNS). To date, there have been no studies on the utility of serum neuron-specific enolase (NSE), a marker of neuronal cell damage, as a predictive marker of DNS in acute CO poisoning. This retrospective observational study was performed on adult patients with acute CO poisoning consecutively treated over a 9-month period. Serum NSE was measured after emergency department arrival, and patients were divided into two groups. The DNS group comprised patients with delayed sequelae, while the non-DNS group included patients with none of these sequelae. A total of 98 patients with acute CO poisoning were enrolled in this study. DNS developed in eight patients. The median NSE value was significantly higher in the DNS group than in the non-DNS group. There was a statistical difference between the non-DNS group and the DNS group in terms of CO exposure time, Glasgow Coma Scale (GCS), loss of consciousness, creatinine kinase, and troponin I. GCS and NSE were the early predictors of development of DNS. The area under the curve according to the receiver operating characteristic curves of GCS, serum NSE, and GCS combined with serum NSE were 0.922, 0.836, and 0.969, respectively. In conclusion, initial GCS and NSE served as early predictors of development of DNS. Also, NSE might be a useful additional parameter that could improve the prediction accuracy of initial GCS.

Hydroxyl radical production via NADPH oxidase in rat striatum due to carbon monoxide poisoning.

Severe poisoning induced by carbon monoxide (CO) at 3000 ppm, but not 1000 ppm, enhances hydroxyl radical (OH) production in rat striatum, which is greatly susceptible to inhibitors of NADPH oxidase (NOX), including diphenyleneiodonium (DPI), but not xanthine oxidase. The quantitative real-time PCR confirmed the previous microarray finding that CO at 3000 ppm, but not 1000 ppm, enhanced mRNA expression of dual oxidase 2 (DUOX2), but not DUOX1, in rat striatum, both of which are NOX family members producing reactive oxygen species. However, the protein levels of DUOX2 and DUOX1 were decreased by 3000 ppm CO. The CO-induced OH production was resistant to chelerythrine and SB230580, inhibitors of protein kinase C and p38MAPK, respectively, which are reported to mediate activation of DUOX1 and DUOX2, respectively. Deprivation of Ca2+, which is required for activation of both DUOXs, failed to suppress the CO-induced OH production. The CO-induced OH production was strongly suppressed by EHT1864, an inhibitor of Rac (Ras-related C3 botulinum toxin substrate), which is a factor for activation of NOX1, NOX2 and NOX3 (the role of Rac on Nox3 activation is controversial) as much as that was suppressed by DPI. In addition, EHT1864 in combination with DPI further suppressed the CO-induced OH production. There were no significant changes in the protein levels of NOX1 through NOX4 and Rac1. It is likely that the CO-induced OH production is mediated through the activation of Rac-dependent NOX enzymes, such as Nox1, Nox2, and Nox3.

Modulation of JAK2, STAT3 and Akt1 proteins by granulocyte colony stimulating factor following carbon monoxide poisoning in male rat.

Carbon monoxide (CO) is an odorless, colorless, tasteless and non-irritating by-product of inefficient combustion of hydrocarbon fuels such as motor vehicle exhausted gases. It is the leading cause of mortality in the USA among all unintentional toxicants. Male rats exposed to CO poisoning in the heart has many cardiovascular effects such as, cardiomyopathy, tachycardia, arrhythmias, and ischemia and in severe cases, myocardial infarction (MI) and cardiac arrest. Cardiomyocyte apoptosis is one of the most frequent consequences in the heart. Granulocyte colony stimulating factor (G-CSF) is a cytokine that mobilizes and differentiates granulocytes from stem cells. It can stimulate many anti-apoptotic pathways such as JAK2-STAT3 and PI3-Akt kinases following cardiac ischemia. G-CSF exerts its anti-apoptotic effects through binding to its specific cell surface receptor. The purpose of this study was to elucidate the mechanism of anti-apoptotic effect of G-CSF following CO poisoning. Rats were exposed to CO 1500 or 3000 ppm for 60 min. Animals received G-CSF 100 µg/kg subcutaneously for five consecutive days after CO intoxication. Western blot analysis was used to evaluate the expression of six proteins namely JAK2, p-JAK2, STAT3, p-STAT3, Akt1 and p-Akt1 following G-CSF 100 µg/kg consecutive dose administration after CO poisoning. There was a significant difference between phosphorylated proteins including p-JAK2, p-STAT3 and p-Akt1 in the G-CSF groups and those in control groups and there were not any significant differences in total protein among the groups.

Elevated serum ubiquitin C-terminal hydrolase-L1 levels in patients with carbon monoxide poisoning.

OBJECTIVE: Ubiquitin C-terminal hydrolase-L1 (UCH-L1) has been established as a reliable and potential biomarker of neuronal damage after acute neurologic insults, such as ischemic stroke, subarachnoid hemorrhage, and traumatic brain injury. However, the effect of serum UCH-L1 levels has not been investigated in carbon monoxide (CO)-poisoned patients. The aim of the present study was to evaluate whether serum UCH-L1 levels are a reliable marker of brain damage and the association of UCH-L1 with outcome. DESIGN AND METHODS: This case-control study enrolled 46 CO-poisoned subjects and 30 controls. Using an enzyme-linked immunosorbent assay (ELISA) kit, we studied the temporal profile of serum UCH-L1 levels at 6, 12, 24 and 48 h after acute CO poisoning. Poisoning severity was assessed using the Glasgow Coma Scale (GCS) score. Long-term outcome was assessed using the Glasgow Outcome Scale (GOS) at 6 months after poisoning. RESULTS: Compared with controls, CO-poisoned patients had significantly elevated serum levels of UCH-L1 at each time point after poisoning. There were significantly higher levels of UCH-L1 in CO-poisoned patients with a lower GCS score as well as in those with a poor 6-month outcome dichotomized GOS. CONCLUSIONS: Serum levels of UCH-L1 appear to have potential clinical utility in providing valuable information about poisoning severity and outcome after CO poisoning.

Interaction of carbon monoxide with transition metals: evolutionary insights into drug target discovery.

The perception that carbon monoxide (CO) is poisonous and life-threatening for mammalian organisms stems from its intrinsic propensity to bind iron in hemoglobin, a reaction that ultimately leads to impaired oxygen delivery to tissues. From evolutionary and chemical perspectives, however, CO is one of the most essential molecules in the formation of biological components and its interaction with transition metals is at the origin of primordial cell signaling. Not surprisingly, mammals have gradually evolved systems to finely control the synthesis and the sensing of this gaseous molecule. Cells are indeed continuously exposed to small quantities of CO produced endogenously during the degradation of heme by constitutive and inducible heme oxygenase enzymes. We have gradually learnt that heme oxygenase-derived carbon monoxide (CO) serves as a ubiquitous signaling mediator which could be exploited for therapeutic purposes. The development of transition metal carbonyls as prototypic carbon monoxide-releasing molecules (CO-RMs) represents a novel stratagem for a safer delivery of CO-based pharmaceuticals in the treatment of various pathological disorders. This review will look back at evolution to analyze and argue that a dynamic interaction of CO with specific intracellular metal centers is the common denominator for the diversified beneficial effects mediated by this gaseous molecule.

[Expression of tryptase and chymase in human lung tissue of anaphylactic shock].

OBJECTIVE: To explore the expression of tryptase and chymase in human lung tissue of anaphylactic shock and its value for forensic medicine. METHODS: With ten carbon monoxide poisoning cases as control group, the levels of tryptase and chymase were observed by immunofluorescence and analyzed using the Image Analyze and the Image-pro plus 5.0.2. The positive mast cells were counted and the levels of the tryptase and chymase were calculated respectively. RESULTS: There was a statistically significant difference (P < 0.05) for the tryptase and chymase concentrations in the lung tissue between the anaphylactic shock group and the control group. CONCLUSION: The levels of the tryptase and the chymase expression are greatly increased in human lung tissue of anaphylactic shock, which may provide the morphological evidence and reference for the diagnosis of anaphylactic shock in forensic practice.

Induction of heme oxygenase-1 with hemin attenuates hippocampal injury in rats after acute carbon monoxide poisoning.

Carbon monoxide (CO) poisoning is a major cause of brain injury and mortality; delayed neurological syndrome (DNS) is encountered in survivors of acute CO exposure. The toxic effects of CO have been attributed to oxidative stress induced by hypoxia. Heme oxygenase-1 (HO-1) is the inducible heme oxygenase isoform, and its induction acts as an important cellular defense mechanism against oxidative stress, cellular injury and disease. In this study, we examined the functional roles of HO-1 induction in a rat model of CO-exposured hippocampal injury. We report that acute CO exposure produces severe hippocampal injury in rats. However, hemin pretreatment reduced both the CO-induced rise in hippocampal water content and levels of neuronal damage in the hippocampus; survival rates at 24 h were significantly improved. Upregulation of HO-1 by hemin pretreatment resulted in a significant decrease in hippocampal levels of malondialdehyde (MDA), a marker of oxidative stress; levels of pro-apoptotic caspase-3 were also reduced. In contrast, inhibition of HO activity by administration of tin protoporphyrin IX (SnPP, a specific inhibitor of HO) abolished the neuroprotective effects of HO-1 induction. These data suggested that the upregulation of endogenous HO-1 expression therefore plays a pivotal protective role in CO neurotoxicity. Though the precise mechanisms underlying hemin-mediated HO-1 induction and neuroprotection are not known, these may involve the anti-oxidant and anti-apoptotic effects of HO-1 enzyme activity.

Xanthine oxidoreductase and neurological sequelae of carbon monoxide poisoning.

Neurological sequelae (NS) is a common complication of carbon monoxide (CO) poisoning and structural alterations of myelin basic protein have been proven to initiate immunological reactions leading to NS. To determine whether xanthine oxidoreductase (XOR) participates in the pathophysiology of CO-mediated NS, we examined myelin basic protein in CO poisoned XOR-depleted rats and performed radial maze studies to evaluate the alteration of cognitive function. Carbon monoxide poisoned XOR-depleted rats did not exhibit myelin basic protein alterations or impaired cognitive function, both found in CO poisoned control rats. These results indicate that XOR is essential to the pathological cascade of CO-mediated NS.

Blockade of haem oxygenase and nitric oxide synthetase causes cortical dysfunction in sheep exposed to carbon monoxide.

Twenty adult ewes underwent common surgery and following recovery were exposed to 1% carbon monoxide (CO) for 2 h. Ten of these sheep were randomly selected for treatment with haem oxygenase (HO) and nitric oxide synthetase (NOS) blockers. All sheep were killed 5 days later. The CO exposure was narcotic and EEG frequency was suppressed. The EEG recovery was rapid in the control sheep and both slow and incomplete in the treated sheep. This difference was statistically significant (P<0.05). For the first time in our CO studies in sheep, one showed multiple cortical infarcts. This sheep was blocked for HO and NOS function. No significant differences were seen in peri-ventricular white matter infarction distribution and frequency. We have previously shown brain protection against CO- and inert diluent-hypoxemia by way of an increase in brain blood flow (BBF) that maintains adequate brain O2 uptake, and by an increase in circulating red blood cells. From this study, we propose that the induction of neuronal and glial HO and NOS in sheep exposed to CO is protective, especially for the cortex. We intend to study this further by both selective and collective enzyme blockade and by measuring regional BBF changes.

Carbon monoxide specifically inhibits cytochrome c oxidase of human mitochondrial respiratory chain.

Carbon monoxide (CO) toxicity is the result of a combination of tissue hypoxia and direct CO-mediated damage at a cellular level, since not all the signs and symptoms presented can be explained only by the formation of carboxyhaemoglobin. Mitochondria, specially the electron transport chain, seem to be the target for CO at a subcellular level. However, the direct effect of CO in individual complexes of the human mitochondrial respiratory chain has not been completely elucidated. We here studied the in vitro effect of CO on individual complexes of the mitochondrial respiratory chain of human mitochondria. We obtained muscle tissue from 10 healthy people who underwent orthopaedic surgery for hip replacement. Isolated mitochondria were incubated for 5 min. under CO concentrations of 50, 100 and 500 ppm. Afterwards, enzymatic activities of individual complexes of the mitochondrial respiratory chain were assessed in vitro and compared with those obtained in basal (synthetic air without CO) conditions. Cytochrome c oxidase (complex IV of the mitochondrial respiratory chain) activity showed a decrease from 836+/-439 nmol/min./mg of mitochondrial protein after air incubation to 670+/-401, 483+/-182, and 379+/-131 nmol/min./mg after 50, 100 and 500 ppm of CO incubation, respectively (20%, 42% and 55% decrease in cytochrome c oxidase activity). This gradual decrease in cytochrome c oxidase was found to be statistically significant (P<0.001). Other complex activities showed no any significant variation. Carbon monoxide is toxic for mitochondria in man, altering the mitochondrial respiratory chain at the cytochrome c oxidase level. This inhibition in cytochrome c oxidase may play a role in the development of the symptoms observed in acute CO poisoning, and in some diseases related to smoking.

[Changes of myocardial enzymes in patients with acute carbon monoxide poisoning].

OBJECTIVE: To study the clinical significance of changes of serum myocardial enzymes in patients with acute carbon monoxide poisoning. METHODS: To determine the dynamic changes of the activity of myocardial enzymes and ECG in 62 patients with acute CO poisoning. RESULTS: In patients with acute CO poisoning 5 kinds of myocardial enzymes begin to increase within 24 hours, the activities of aspartate aminotransferase (AST), creatine phosphokinase (CPK), lactic dehydrogenase (LDH), alpha-hydroxybutyrate dehydrogenase (alpha-HBDH), CPK isoenzyme (CK-MB) were (20.2 +/- 12.3), (151.6 +/- 91.8), (146.8 +/- 50.4), (154.8 +/- 47.7), (13.8 +/- 8.1) U/L respectively, while those in control group were (12.1 +/- 6.7), (90.6 +/- 17.3), (118.7 +/- 13.5), (89.9 +/- 27.9), (5.9 +/- 3.3) U/L respectively. There was significant difference between two groups (P < 0.01); 3 d later, the activities of 5 enzymes were still increased [(21.3 +/- 12.3), (105.8 +/- 51.4), (144.8 +/- 51.4), (159.8 +/- 35.4), (16.2 +/- 9.1) U/L respectively]. 7 and 12 d later, the activities of alpha-HBDH and CK-MB were still higher than those of control (P < 0.01). LDH(1) and LDH(2) increased to peak value in 24 h after poisoning (35.3 +/- 5.8), (43.8 +/- 5.7) U/L vs (24.8 +/- 3.9), (36.9 +/- 4.3) U/L, P < 0.01. The abnormal rate of serum LDH(1) was 78.7%, LDH(2) 58.3%, LDH 45.2%, CK-MB 37.1%, alpha-HBDH 33.6% and the abnormal rate of ECG was less than 10%. CONCLUSION: Acute carbon monoxide poisoning may cause myocardial injury. Determination of serum myocardial enzymes may contribute to showing myocardial injury, early diagnosis and treatment, results of treatment and prognosis.

Mitochondrial cytochrome c oxidase inhibition during acute carbon monoxide poisoning.

Clinical symptoms of acute carbon monoxide (CO) poisoning are mainly related to the capability of haemoglobin to bind CO. However, the persistence of some clinical alterations after carboxyhaemoglobin normalization suggests that other heme containing proteins, like cytochrome c oxidase, could play a role in its pathogenesis. We studied mitochondrial enzyme activities of lymphocytes from three patients suffering from acute CO poisoning. HbCO levels were 11.6%. 19.6% and 22.3% in the acute phase, 2.3%, 2.4% and 1.5% on day 3 after admission, and 1.2%, 3.3% and 1.1% on day 12. Complex II, III and glycerol-3-phosphate dehydrogenase activities remained normal along the study, while cytochrome c oxidase (complex IV) activity showed a 76% inhibition compared to controls during acute poisoning (P < 0.01) and 48% at day 3 (P < 0.05). The activity was normal already on day 12 after the complete disappearance of symptomatology. Our results suggest that mitochondrial cytochrome c oxidase is also a target site in human acute CO poisoning, and its extended and generalized inhibition could explain the persistence of different symptoms after the normalization of HbCO levels.

Dehydrogenase conversion to oxidase and lipid peroxidation in brain after carbon monoxide poisoning.

The conversion of xanthine dehydrogenase to xanthine oxidase and lipid peroxidation were measured in brain from carbon monoxide- (CO) poisoned rats. Sulfhydryl-irreversible xanthine oxidase increased from a control level of 15% to a peak of 36% over the 90 min after CO poisoning, while the conjugated diene level doubled. Reversible xanthine oxidase was 3-6% of the total enzyme activity over this span of time but increased to 31% between 90 and 120 min after poisoning. Overall, reversible and irreversible xanthine oxidase represented 66% of total enzyme activity at 120 min after poisoning. Rats depleted of this enzyme by a tungsten diet and those treated with allopurinol before CO poisoning to inhibit enzyme activity exhibited no lipid peroxidation. Treatment immediately after poisoning with superoxide dismutase or deferoxamine inhibited lipid peroxidation but had no effect on irreversible oxidase formation. Biochemical changes only occurred after removal from CO, and changes could be delayed for hours by continuous exposure to 1,000 ppm CO. These results are consistent with the view that CO-mediated brain injury is a type of postischemic reperfusion phenomenon and indicate that xanthine oxidase-derived reactive oxygen species are responsible for lipid peroxidation.

Rhabdomyolysis and acute renal failure following carbon monoxide poisoning: two case reports with muscle histopathology and enzyme activities.

Two patients with carbon monoxide poisoning are presented, both of whom suffered rhabdomyolysis complicated by acute renal failure. One patient, an attempted suicide, developed a compartment syndrome of the right thigh that required fasciotomy and recovered after a period of hemofiltration and hemodialysis. Muscle biopsy appearances were consistent with partial muscle infarction. The other patient, rescued from a smoke filled room, exhibited raised creatine kinase but no evidence of muscle swelling. He developed anuric renal failure and adult respiratory distress syndrome and died despite maximum intensive care. Muscle biopsy showed early evidence of muscle necrosis. In both cases there was a marked reduction of enzyme activities in the muscle biopsy consistent with metabolic derangement. Although there was a clinical compartment syndrome in the first case, there was no muscle swelling at the time of biopsy or subsequently in the second case. A direct toxic effect of carbon monoxide may thus have been an important mechanism contributing to the muscle necrosis in the second case, although local ischemia may have been an exacerbating factor in the first case.

Cerebral regional capillary perfusion and blood flow after carbon monoxide exposure.

Alterations in regional cerebral blood flow (rCBF) and percent perfused capillaries (indicative of functional intercapillary distance) were determined in conscious male Long-Evans rats after reducing their blood O2-carrying capacity by exposing them to 1% CO for 12 min. rCBF was determined by the iodoantipyrine method. rCBF increased from a mean of 106 +/- 8 (SE) ml.min-1.100 g-1 before CO exposure to 173 +/- 14 ml.min-1.100 g-1 after CO exposure. There was a greater flow increase (126%) in the cerebral cortex than in the lower brain stem [pons (45%), medulla (39%)]. Presence of fluorescein isothiocyanate-labeled dextran identified the perfused capillaries before and after CO exposure. The volume fraction (Vv) and number/mm2 (Na) of all capillaries (perfused and nonperfused) in a given area of brain were determined after staining for alkaline phosphatase. The percent Vv and percent Na of perfused capillaries increased uniformly (from approximately 50% to approximately 80%) in all parts of the brain after CO exposure. In the presence of tissue hypoxia with undiminished plasma PO2, the brain vasculature allowed greater flow of blood while the microvasculature adjusted to reduce the diffusion distance for O2.

Effect of carbon monoxide inhalation exposure in mice on drug metabolism in vivo.

Experiments were undertaken to evaluate the action of carbon monoxide (CO) on the mixed-function oxidase (MFO) system in vivo. Mice were exposed to 500 ppm CO for 8 h per day in an inhalation chamber under dynamic airflow conditions. Hexobarbital (150 mg/kg, i.p.), zoxazolamine (150 mg/kg, i.p.) or ethanol (2 mg/g, i.p.) was given to each group of mice during CO exposure and disappearance of the drug from blood or brain was determined while CO exposure continued. The experiments were repeated with different groups of animals which were exposed to CO for 3 or 5 days. Hexobarbital and ethanol metabolism were not affected by CO following either one day exposure or repeated exposure. There was no statistically significant difference in the brain level of zoxazolamine in animals exposed to CO when compared to control. These studies indicate that in vivo metabolism of hexobarbital, zoxazolamine and ethanol in mice is not affected by exposure to 500 ppm CO under the conditions employed in the present study.