Diazoxide Attenuates Postresuscitation Brain Injury in a Rat Model of Asphyxial Cardiac Arrest by Opening Mitochondrial ATP-Sensitive Potassium Channels.

Objective. We investigated whether and how diazoxide can attenuate brain injury after cardiopulmonary resuscitation (CPR) by selective opening of mitochondrial ATP-sensitive potassium (mitoKATP) channels. Methods. Adult male Sprague-Dawley rats with induced cerebral ischemia (n = 10 per group) received an intraperitoneal injection of 0.1% dimethyl sulfoxide (1 mL; vehicle group), diazoxide (10 mg/kg; DZ group), or diazoxide (10 mg/kg) plus 5-hydroxydecanoate (5 mg/kg; DZ + 5-HD group) 30 min after CPR. The control group (sham group, n = 5) underwent sham operation, without cardiac arrest. Mitochondrial respiratory control rate (RCR) was determined. Brain cell apoptosis was assessed using TUNEL staining. Expression of Bcl-2, Bax, and protein kinase C epsilon (PKCε) in the cerebral cortex was determined by Western blotting and immunohistochemistry. Results. The neurological deficit scores (NDS) in the vehicle group decreased significantly at 24 h and 48 h after CPR. Diazoxide significantly improved NDS and mitochondrial RCR after CPR at both time points; 5-HD cotreatment abolished these effects. Diazoxide decreased TUNEL-positive cells following CPR, upregulated Bcl-2 and PKCε, downregulated Bax, and increased the Bcl-2/Bax ratio; 5-HD cotreatment reversed these effects. Conclusions. Diazoxide attenuates postresuscitation brain injury, protects mitochondrial function, inhibits brain cell apoptosis, and activates the PKC pathway by opening mitoKATP channels.

Perinatal asphyxia leads to PARP-1 overactivity, p65 translocation, IL-1ß and TNF-α overexpression, and apoptotic-like cell death in mesencephalon of neonatal rats: prevention by systemic neonatal nicotinamide administration.

Perinatal asphyxia (PA) is a leading cause of neuronal damage in newborns, resulting in long-term neurological and cognitive deficits, in part due to impairment of mesostriatal and mesolimbic neurocircuitries. The insult can be as severe as to menace the integrity of the genome, triggering the overactivation of sentinel proteins, including poly (ADP-ribose) polymerase-1 (PARP-1). PARP-1 overactivation implies increased energy demands, worsening the metabolic failure and depleting further NAD(+) availability. Using a global PA rat model, we report here evidence that hypoxia increases PARP-1 activity, triggering a signalling cascade leading to nuclear translocation of the NF-κB subunit p65, modulating the expression of IL-1ß and TNF-α, pro-inflammatory molecules, increasing apoptotic-like cell death in mesencephalon of neonate rats, monitored with Western blots, qPCR, TUNEL and ELISA. PARP-1 activity increased immediately after PA, reaching a maximum 1-8 h after the insult, while activation of the NF-κB signalling pathway was observed 8 h after the insult, with a >twofold increase of p65 nuclear translocation. IL-1ß and TNF-α mRNA levels were increased 24 h after the insult, together with a >twofold increase in apoptotic-like cell death. A single dose of the PARP-1 inhibitor nicotinamide (0.8 mmol/kg, i.p.), 1 h post delivery, prevented the effect of PA on PARP-1 activity, p65 translocation, pro-inflammatory cytokine expression and apoptotic-like cell death. The present study demonstrates that PA leads to PARP-1 overactivation, increasing the expression of pro-inflammatory cytokines and cell death in mesencephalon, effects prevented by systemic neonatal nicotinamide administration, supporting the idea that PARP-1 inhibition represents a therapeutic target against the effects of PA.

Proteomic analysis of cardiac metabolic enzymes in asphyxiated newborn piglets.

Hypoxia/reoxygenation (H/R) creates an energetic deficiency in the heart, which may contribute to myocardial dysfunction. We hypothesized that H/R-induced impairment of cardioenergetic enzymes occurs in asphyxiated newborn animals. After hypoxia for 2 h (10-15% oxygen), newborn piglets were resuscitated with 100% oxygen for 1 h, followed by 21% oxygen for 3 h. Sham-operated control piglets had no H/R. Hemodynamic parameters in the piglets were continuously measured. At the end of experiment, hearts were isolated for proteomic analysis. In asphyxiated hearts, the level of isocitrate dehydrogenase and malate dehydrogenase was reduced compared to controls. Inverse correlations between the level of myocardial malate dehydrogenase and cardiac function were observed in the control, but not the H/R hearts. We conclude that reoxygenation of asphyxiated newborn piglets reduces the level of myocardial isocitrate dehydrogenase and malate dehydrogenase. While the cause is not clear, it may be related to the impaired tricarboxylic acid cycle pathway and energy production in the heart.

Altered expression of cyclooxygenase-2, presenilins and oxygen radical scavenging enzymes in a rat model of global perinatal asphyxia.

Cyclooxygenase-2 (COX-2) and presenilin (PSEN) 1 and 2 genes are regulated during development as well as in pathological conditions associated to hypoxia. In this study, we investigated their patterns of expression during the first 2 weeks of postnatal life in a rat model of moderate global perinatal asphyxia that we have previously reported to be characterized by early oxidative stress and delayed behavioral alterations. In the hippocampus, global perinatal asphyxia induced an early up-regulation COX-2 mRNA (postnatal day, pnd, 1), which preceded those of PSEN 1 and 2 genes, observed at pnd 4. At pnd 11, the expression of all three genes was decreased compared to control animals. In addition, we analyzed the expression of the scavenging enzymes catalase, copper/zinc- and manganese-superoxide dismutase. As for COX-2, the three enzymes were up-regulated in the hippocampus at pnd 1 and returned at or below baseline by pnd 4. In the cortex, only PSEN 1 and 2 showed a moderate up-regulation at pnd 1 followed by a down-regulation at pnd 11. These findings suggest that moderate perinatal hypoxic episodes are associated with a dysregulation of the several genes involved in brain development and anti-oxidant defenses, which follows a rapid and transient oxidative stress. Such alterations are particularly evident in the hippocampus and could represent an adaptive response to the hypoxic condition. The delayed down-regulation of the scavenging enzymes could set the ground for brain damage and delayed behavioral alterations and further support the potential benefit of early anti-oxidant treatments in a short therapeutic window soon after birth.

Hypothermia and ERK activation after cardiac arrest.

Mild hypothermia improves survival and neurological outcome after cardiac arrest, as well as increasing activation of the extracellular-signal-regulated kinase (ERK) in hippocampus. ERK signaling is involved in neuronal growth and survival. We tested the hypothesis that the beneficial effects of hypothermia required ERK activation. ERK activation was measured by immunoblotting with phosphorylation-specific antibodies. Rats (n = 8 per group) underwent 8 min of asphyxial cardiac arrest and were resuscitated with chest compressions, ventilation, epinephrine and bicarbonate. At 30 min after resuscitation, vehicle (50% saline:50% DMSO) or the ERK kinase inhibitor U0126 (100 microg) was infused into the lateral ventricle. Cranial temperature was kept at either 33 degrees C (hypothermia) or 37 degrees C (normothermia) between 1 and 24 h. Neurological function was assessed daily for 14 days. Surviving neurons were counted in the hippocampus. A dose of 100 mug U0126 inhibited ERK bilaterally for 12 to 24 h and decreased phosphorylation of the ERK substrates ATF-2 and CREB. As in previous studies, hypothermia improved survival, neurological and histological outcome after cardiac arrest. However, survival, neurological score and histology did not differ between U0126 and vehicle-treated rats after cardiac arrest. Therefore, a dose of U0126 sufficient to inhibit biochemical markers of ERK signaling in hippocampus does not alter the beneficial effects of hypothermia induced after resuscitation in rats and did not affect recovery of normothermia-treated rats. These results suggest that hypothermia-induced improvement in outcomes does not require ERK activation.

Ontogenetic changes and developmental adjustments in lactate dehydrogenase isozymes of an obligate air-breathing fish Channa punctatus during deprivation of air access.

In air-breathing snakehead Channa punctatus, Ldh-B is expressed at all ontogenetic and developmental stages, while Ldh-A is expressed temporally in pre-hatchlings 12-13 days ahead of bimodal respiration marked by air-breathing. Remarkable differences are observed in the LDH isozyme expression among various ontogenetic and developmental stages upon denying air access. When denied air access, water-breathing larvae show two distinct characteristics: (i) they survive longer than transitory air-breathers due to independence from air-breathing and (ii) there is more transient induction of Ldh-B than Ldh-A. Transition to bimodal breathing, which occurred post-hatching in 15-day old larvae, is coincidental with inducibility of Ldh-A and concomitant down-regulation of Ldh-B. Heart tissue from air-breathing adults denied air access shows a preferential expression of LDH-A subunit and slight down-regulation of LDH-B. Heterotetramers of A and B subunits participate in adjusting LDH levels among those stages which either precede air-breathing switchover, or are subsequent to this transition. The contribution of heterotetramers depends on the stage-specific levels of LDH homotetramers A(4) or B(4). Scaling of muscle mass during growth, tolerance to extended deprivation of air access and induction of Ldh-A are correlated. Response to restoring air contact indicated that advanced air-breathing stages of C. punctatus possess an inherent capacity to sense surface air. In kinetic properties, LDH isozymes of C. punctatus are teleost-like but species specificity is displayed in oxidative potential by cardiac muscle and in L-lactate reduction by skeletal muscle.

Up-regulation of cerebral carbonic anhydrase by anoxic stress in piglets.

The resuscitation of asphyxiated babies is associated with changes in cerebral protein synthesis that can influence the neurological outcome. Insufficient gas exchange results in rapid shifts in extracellular and intracellular pH. Carbonic anhydrase (CA) plays an important role in buffering acute changes in pH in the brain. We investigated whether asphyxia/re-ventilation influences the expression of cerebral CA isoforms (CA-II, CA-III and CA-IV) in anaesthetized newborn pigs. The cerebral cortex, hippocampus, cerebellum and retina were sampled, and prepared for either CA immunohistochemistry or CA immunoblotting from piglets subjected to asphyxia (10 min) followed by 2-4 h of re-ventilation, and also from normoxic controls. The CA immunoreactivity (IR) of all the isoforms studied was weak in the controls, apart from staining of a few oligodendrocytes in the subcortical white matter, some astrocytes in the superficial layer of the cerebral cortex, the cerebellar Purkinje cells and the retinal Müller cells that possessed moderate CA-II IR. However, asphyxia induced a marked increase in the CA IR of all isoforms in all the cerebral regions investigated and the retina after 4 h of survival. The pyramidal cells of the frontal cortex and hippocampus displayed the most conspicuous increase in CA IR. Immunoblotting confirmed increased levels of all the CA isoenzymes. We conclude that raised CA levels after asphyxia may contribute to the compensatory mechanisms that protect against the pathological changes in the neonatal CNS.

Deficient brain RNA polymerase and altered nucleolar structure persists until day 8 after perinatal asphyxia of the rat.

RNA polymerases (POL) are integral constituents of the protein synthesis machinery, with POL I and POL III coding for ribosomal RNA and POL II coding for protein. POL I is located in the nucleolus and transcribes class I genes, those that code for large ribosomal RNA. It has been reported that the POL system is seriously affected in perinatal asphyxia (PA) immediately after birth. Because POL I is necessary for protein synthesis and brain protein synthesis was shown to be deranged after hypoxic-ischemic conditions, we aimed to study whether POL derangement persists in a simple, well-documented animal model of graded global PA at the activity, mRNA, protein, and morphologic level until 8 d after the asphyctic insult. Nuclear POL I activity was determined according to a radiochemical method; mRNA steady state and protein levels of RPA4O-an essential subunit of POL I and III-were evaluated by blotting methods; and the POL I subunit polymerase activating factor-53 was evaluated using immunohistochemistry. Silver staining and transmission electron microscopy were used to examine the nucleolus. At the eighth day after PA, nuclear POL I decreased with the length of the asphyctic period, whereas mRNA and protein levels for RPA4O were unchanged. The subunit polymerase activating factor-53, however, was unambiguously reduced in several brain regions. Dramatic changes of nucleolar morphology were observed, the main finding being nucleolar disintegration at the electron microscopy level. We suggest that severe acidosis and/or deficient protein kinase C in the brain during the asphyctic period may be responsible for disintegration of the nucleolus as well as for decreased POL activity persisting until the eighth day after PA. The biologic effect may be that PA causes impaired RNA and protein synthesis, which has been already observed in hypoxic-ischemic states.

[Detection of metalloproteinases in pulmonary tissue after sudden and violent death]. / Prukaz metaloproteináz v plicní tkáni u náhlé a násilné smrti.

During early stages of chronic hypoxia under experimental conditions in the lungs alveolar macrophages are activated and in the latter an increased amount of matrix-metalloproteinase-collagenolytic enzymes is produced. Their presence was assessed in the present study in the lungs of infants who died suddenly (SIDS) and also in subjects who died from a violent death incl. acute and protracted external suffocation. The positive findings of these matrixins, in particular MMP-9 (gelatinase) were assessed in alveolar macrophages not only after protracted suffocation but also in the majority of sudden infant deaths. These findings supplement the mosaic of changes which indicate that sudden infant deaths are preceded by a period when the infant was exposed to hypoxia.

A decade of pediatric homicide: a retrospective study at the Medical University of South Carolina.

More than 3 million children are abused and/or neglected each year in the United States. Unfortunately, a significant percentage of these cases result in homicide by child abuse or child neglect. Causes of death range from blunt force trauma and shaking to asphyxia to immolation. We retrospectively reviewed all pediatric forensic cases referred to the Medical University of South Carolina Forensic Pathology Section over the past 10 years, from January 1986 to December 1995. Of these, we looked only at children < or =5 years of age. The majority (342 cases, 69%) of these deaths were classified as natural, 96 (19%) as accident, and 60 (12%) as homicide. Of the homicides, we examined the cause of death; age, gender, and race of the victim; relationship to the perpetrator; time interval between injury and death; and the initial history given as to the cause of the injury. The cause of death fell into nine categories, the number one category being head trauma. Forty-five percent of the homicides were by head trauma, 12% by abdominal or body trauma, 25% by asphyxia (with half of these due to drowning), 10% by carbon monoxide poisoning or thermal injury, and the remaining 8% involving cases of neglect, stabbing, and poisoning. The majority of the homicide victims were male (67%) and black (67%). Forty-six percent were < or =1 year of age. Approximately 25% of the homicide cases were designated as shaken baby syndrome (SBS). In 97% of the cases, the assailant was known to the victim and was a family relative in 77%. Sixty-three percent of the assailants were female and 45% of the assailants were male; in 12%, the assailants were both parents, and in 1 case, the assailant remains unknown. Of the asphyxia deaths, 87% of the assailants were female. The time interval between injury and death ranged from minutes to hours in most cases to months in cases of repeated abuse and chronic injury and sequelae. The time interval between injury and the onset of symptoms remains unknown in most cases due to inconsistencies in the history and lack of credibility of the caretaker. The most common initial history given was "a fall" (20%). We report our findings of a decade of pediatric homicides to increase awareness of the common scenarios and case histories, demographics of the victims, causes of death, and perpetrators of pediatric homicide.

Upregulation of the aromatic amino acid decarboxylase under neonatal asphyxia.

Perinatal hypoxic-ischemic cerebral injury is a major determinant of neurologic morbidity and mortality in the neonatal period and later in childhood. There is evidence that the dopaminergic system is sensitive to asphyxia. However, the respective enzyme activities have not yet been measured in the living neonatal brain. In this study, we have used 18F-labeled 6-fluoro-L-3,4-dihydroxyphenylalanine (FDOPA) together with positron-emission tomography (PET) to estimate the activity of the aromatic amino acid decarboxylase (AADC), the ultimate enzyme in the synthesis of dopamine (DA), in the brain of newborn piglets. Simultaneously, the cerebral blood flow (CBF) was measured with colored microspheres. Asphyxia elicited an up to threefold increase of the CBF. Despite this, the blood-brain transfer of FDOPA as well as the clearance rate constants from brain were unchanged. However, the synthesis rate of FDA from FDOPA was significantly increased in frontal cortex, striatum, and midbrain. This increase of the AADC activity and the decrease of monoamine oxidase activity may contribute to the increase of extracellular DA during asphyxia which is expected to be involved in severe disturbances of neuronal metabolism, e.g., by generating free radicals.

Value of myocardial hypoxia markers (creatine kinase and its MB-fraction, troponin-T, QT-intervals) and serum creatinine for the retrospective diagnosis of perinatal asphyxia.

Neonatal asphyxia is a major topic of neonatal research. However, no clear-cut physiologic parameters exist which enable an early identification of neonatal infants who are either at risk to develop brain damage or posthypoxic heart failure. Parameters indicating dysfunction of the heart and kidneys as creatinine and creatinine kinase have been evaluated. In our study, 47 asphyxiated infants (umbilical artery pH < 7.18 and either a 1-min Apgar score < 4 or a 5-min Apgar score < 7) were compared to 27 nonasphyxiated controls regarding significant differences in creatinine, creatinine kinase, its MB fraction, and a newly introduced myocardial hypoxia indicator -- troponin T -- to establish the value of these parameters in the retrospective diagnosis of asphyxia. Further we evaluated two subsets of these 47 asphyxiated infants with either subsequent signs of encephalopathy (seizures) or heart failure. Creatinine, creatinine kinase and troponin T were significantly elevated in asphyxiated infants compared with controls; no differences were found in creatinine kinase and its MB fraction. In asphyxiated infants with heart failure, troponin T was significantly higher than in the other asphyxiated infants. However, none of the parameters studied was significantly different in patients with brain damage compared with asphyxiated infants without neurological sequelae. Troponin T has a high positive predictive value in the postnatal diagnosis of asphyxia. The diagnostic power of troponin T equals that of creatinine. However, troponin T is more sensitive in the identification of infants with asphyxia and cardiocirculatory failure than creatinine. Creatinine kinase and its MB fraction have no diagnostic value.

Effects of angiotensin II on brain monoamine oxidase activity in non-hypoxic and hypoxic mice.

The effects of angiotensin II (ATII) administered intracerebroventricularly (i.c.v.) at a dose of 0.5 microgram per mouse on the activity of monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) in the forebrain of normoxic and hypoxic mice were studied. The influence of hypoxia (asphyctic and haemic) on MAO-A and MAO-B activity was also investigated. MAO-A activity was increased in haemic hypoxia; MAO-B activity increased in both asphyctic and haemic hypoxia. ATII increased MAO-A activity without affecting MAO-B activity under normoxic conditions. ATII increased MAO-A activity but decreased MAO-B in hypoxic (asphyctic) mice as compared to normoxic controls. The results suggest the role of MAO-A and MAO-B in the ATII-induced increase of susceptibility to acute hypoxia.

The effect of post-asphyxial reoxygenation with 21% vs. 100% oxygen on Na+,K(+)-ATPase activity in striatum of newborn piglets.

To compare the effect of 21% vs. 100% oxygen during post-asphyxial reoxygenation on brain cell membrane function in the striatum, 20 anesthetized, ventilated newborn piglets were studied: group 1 (normoxia, n = 5), group 2 (asphyxia, no reoxygenation, n = 5), group 3 (asphyxia followed by reoxygenation with 21% O2, n = 5), and group 4 (asphyxia followed by reoxygenation with 100% O2, n = 5). Asphyxia was induced by a stepwise reduction in FiO2 at 20 min intervals from 21% to 14%, 11%, and 8%. Following a total 60 min of asphyxia, piglets in groups 3 and 4 were recovered for 2 h with either 21% or 100% O2. Na+,K(+)-ATPase activity (mumol Pi/mg protein/h) in striatal cell membranes was 31 +/- 1, 22 +/- 2, 32 +/- 2 and 26 +/- 1 in groups 1, 2, 3 and 4, respectively. Na+,K(+)-ATPase activities in groups 2 and 4 were significantly lower than in groups 1 and 3 (p < 0.01). Piglets recovered post-asphyxia for 2 h with 21% O2 had restoration of Na+,K(+)-ATPase activity to baseline levels, while those treated with 100% O2 during recovery had persistent Na+,K(+)-ATPase inhibition of 16%. This could result from increased free radical production during reoxygenation with 100% O2 which could contribute to post-asphyxial cellular injury in the striatum.

Brain cell membrane dysfunction following acute asphyxia in newborn piglets.

Brain cell membrane function during and following single and repeated episodes of asphyxia was investigated. Asphyxia in 24 anesthetized, paralyzed, mechanically-ventilated newborn piglets was produced by stopping ventilation for 2-3 min followed by recovery with reventilation. Measurements of cerebral Na+,K(+)-ATPase activity and of lipid peroxidation products, conjugated dienes and fluorescent compounds, were made during control (n = 12), asphyxia (n = 5), recovery after a single asphyxia event (n = 4), and recovery following 7 repeated asphyxia episodes (n = 3). Cerebral Na+,K(+)-ATPase activity remained unchanged from control during asphyxia (14.57 +/- 2.43 compared to 15.33 +/- 4.27 mumol Pi/mg protein/h, mean +/- SD), but was significantly reduced both during recovery after single (3.87 +/- 1.66) and after repeated (2.59 +/- 1.58) asphyxias, representing a 73 and 82% reduction in enzyme activity, respectively. Conjugated dienes and fluorescent compounds were similarly unchanged during asphyxia compared to control, but increased during recovery from single and from repeated episodes. Decreased cerebral Na+,K(+)-ATPase activity, simultaneous with an increase in lipid peroxidation products, reflects significant cellular membrane damage consistent with oxygen free radical formation during the recovery from acute asphyxia in the newborn piglet.

The postmortem determination of CK isozymes in the pericardial fluid in various causes of death.

The authors have studied the CK isozyme pattern in the pericardial fluid of 100 cadavers autopsied in the Anatomic Forensic Institute of Granada. The samples were classified into several groups according to the cause of death: --Multiple trauma with thoracic contusion --Pneumonia and pulmonary embolism --Mechanical asphyxia --Cranio-cerebral trauma --Acute haemorrhage --Myocardial infarction --Others. The results showed that the CK isozyme pattern of pericardial fluid provides useful postmortem information of cardiac "status", adding to the diagnostic potential of CK isozymes.

[Possible mechanisms of rat liver peroxidation of lipids in the recovery period after mechanical asphyxia]. / O vomozhnykh mekhanizmakh perekisnogo okisleniia lipidov pecheni krys v vosstanovitel'nom periode posle mekhanicheskoi asfiksii.

Lipid peroxidation was stimulated in rat liver tissue after mechanical asphyxia due to inhibition of the antiradical and antiperoxidase systems simultaneously with an apparent increase in production of free radicals. Lipid peroxidation in liver membranes was maintained at the high level after reanimation despite of normalization (and occasional increase) of the activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase.