ascorbic-acid and Hypoxia-Ischemia--Brain

This article explains the mechanisms underlying choices of pharmacotherapy for hypoxic-ischemic insults of both preterm and term babies. Some preclinical data are strong enough that clinical trials are now underway. Challenges remain in deciding the best combination therapies for each age and insult.

Topics: Acetylcysteine; Allopurinol; Antioxidants; Ascorbic Acid; Biopterins; Erythropoietin; Excitatory Amino Acid Antagonists; Free Radical Scavengers; Fructose; Humans; Hypoxia-Ischemia, Brain; Infant, Extremely Premature; Infant, Newborn; Infant, Premature; Infant, Premature, Diseases; Melatonin; Memantine; Neuroprotective Agents; Nitric Oxide Synthase Type III; Resveratrol; Stilbenes; Topiramate; Vitamin E; Xenon

Free oxygen radicals and proinflammatory cytokines are important causes for brain injury in neonates with hypoxic ischemic encephalopathy (HIE). Our objectives were to test the hypothesis that a combination of antioxidants (ascorbic acid) and anti-inflammatory agents (ibuprofen) can ameliorate the brain injury in HIE and improve neurodevelopmental outcomes when given to term infants immediately after birth.. In a prospective, randomized, double-blinded controlled trial, 60 asphyxiated term infants were assigned to one of two groups, intervention and control. The intervention group (n=30) received intravenous ascorbic acid and oral ibuprofen for 3 days; and the control group (n=30) received similar volumes of a placebo. We measured a panel of cytokines at enrollment and administered the treatment drugs within 2 h after birth. Neurological evaluations and developmental screenings were performed for all survivors at 6 months of age.. The Intervention and Control groups did not differ in the severity of HIE at enrollment, the concentrations of IL-1 beta and IL-6, the incidence of mortality (37 vs 33%), the incidence of neurological abnormalities at hospital discharge (47 vs 55%) and the incidence of developmental delay at 6 months of age (32 vs 40%), respectively. None of the observed complications were related to intervention. Serum interleukin (IL)-1 beta and IL-6 concentrations correlated positively with the severity of HIE at birth (P<0.01), whereas only serum IL-6 correlated with neurodevelopmental outcome at 6 months (P<0.001).. Early administration of ascorbic acid and ibuprofen did not affect outcomes in infants with perinatal asphyxia. This study does not explain whether our intervention was not effective in blocking free radicals and inflammatory cytokines, if the dosing and route of administration were inadequate, or if other mediators existed that could have a more powerful role in brain injury during hypoxia-ischemia.

Topics: Anti-Inflammatory Agents; Antioxidants; Ascorbic Acid; Asphyxia Neonatorum; Developmental Disabilities; Double-Blind Method; Drug Therapy, Combination; Female; Humans; Hypoxia-Ischemia, Brain; Ibuprofen; Infant, Newborn; Male

The intracerebral antioxidant ability of mature rats after neonatal hypoxic-ischemic (HI) brain injury was estimated using the microdialysis-electron spin resonance method.. Seven-day-old Wistar rats were subjected to a modified Levine's procedure for producing HI brain injury. After HI insult, pups were returned and reared with their dams. Seven weeks after HI insult, their intracerebral antioxidant abilities were measured using the microdialysis-electron spin resonance method after the intraperitoneal injection of 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl. Ascorbic acid, L-cysteine, and glutathione (GSH) were also determined. The rats without HI insult were used as a control.. The decay rate of 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl in the non-ligated side of the cerebral hemisphere of the HI group was significantly larger than that of the control group. The amounts of ascorbic acid in the perfusate from the non-ligated side of the HI group were about four times larger than those of the control group. The amounts of L-cysteine and GSH of the HI group were about 10 times larger than those of the control group.. The antioxidant ability in the non-ligated sides of the cerebral hemispheres of the mature rats 7 weeks after neonatal HI insult was higher than that of the control group. Higher amounts of ascorbic acid and GSH supported the higher antioxidant ability. The increase of the intracerebral antioxidant ability of the non-ligated side indicates the compensation of motor function for the lost side. The present results should offer important insights into the prognosis for hypoxic-ischemic encephalopathy.

Topics: Animals; Antioxidants; Ascorbic Acid; Birth Injuries; Cerebral Cortex; Cyclic N-Oxides; Cysteine; Disease Models, Animal; Disease Progression; Electron Spin Resonance Spectroscopy; Glutathione; Humans; Hypoxia-Ischemia, Brain; Infant, Newborn; Microdialysis; Neurons; Oxidative Stress; Pyrrolidines; Rats, Wistar; Spin Labels

The early antecedents of cerebral palsy (CP) are unknown but are suspected to be due to hypoxia-ischemia (H-I). In our rabbit model of CP, the MRI biomarker, apparent diffusion coefficient (ADC) on diffusion-weighted imaging, predicted which fetuses will develop postnatal hypertonia. Surviving H-I fetuses experience reperfusion-reoxygenation but a subpopulation manifested a continued decline of ADC during early reperfusion-reoxygenation, which possibly represented greater brain injury (RepReOx). We hypothesized that oxidative stress in reperfusion-reoxygenation is a critical trigger for postnatal hypertonia. We investigated whether RepReOx predicted postnatal neurobehavior, indicated oxidative stress, and whether targeting antioxidants at RepReOx ameliorated motor deficits, which included testing of a new superoxide dismutase mimic (MnTnHex-2-PyP). Rabbit dams, 79% gestation (E25), were subjected to 40 min uterine ischemia. Fetal brain ADC was followed during H-I, immediate reperfusion-reoxygenation, and 4-72 h after H-I. Endpoints were postnatal neurological outcome at E32, ADC at end of H-I, ADC nadir during H-I and reperfusion-reoxygenation, and area under ADC curve during the first 20 min of reperfusion-reoxygenation. Antioxidants targeting RepReOx were administered before and/or after uterine ischemia. The new MRI-ADC biomarker for RepReOx improved prediction of postnatal hypertonia. Greater superoxide production, mitochondrial injury, and oligodendroglial loss occurred in fetal brains exhibiting RepReOx than in those without. The antioxidants, MnTnHex-2-PyP and Ascorbate and Trolox combination, significantly decreased postnatal motor deficits and extent of RepReOx. The etiological link between early injury and later motor deficits can thus be investigated by MRI, and allows us to distinguish between critical oxidative stress that causes motor deficits and noncritical oxidative stress that does not.

Topics: Age Factors; Animals; Animals, Newborn; Antioxidants; Ascorbic Acid; Benzimidazoles; Blood Flow Velocity; Brain; Brain Mapping; Carbocyanines; Chromans; Diffusion Magnetic Resonance Imaging; Disease Models, Animal; Embryo, Mammalian; Female; Flow Cytometry; Hypoxia-Ischemia, Brain; Ionophores; Laser-Doppler Flowmetry; Membrane Potential, Mitochondrial; Metalloporphyrins; Microvessels; Mitochondria; Movement Disorders; Muscle Hypertonia; O Antigens; Pregnancy; Rabbits; Reperfusion Injury; Superoxides; Time Factors; Valinomycin

Cyclooxygenase-2 (COX-2) activity has been implicated in the pathogenesis of ischemic injury, but the exact mechanisms responsible for its toxicity remain unclear. Infection of primary neurons with an adenovirus expressing wild type (WT) COX-2 increased the susceptibility of neurons to hypoxia. Infection with an adenoviral vector expressing COX-2 with a mutation at the cyclooxygenase site did not increase susceptibility to hypoxia, whereas over-expression of COX-2 with a mutation in the peroxidase site produced similar susceptibility to hypoxia as WT COX-2. Primary neuronal cultures obtained from transgenic mice bearing a mutation in the COX-2 cylooxygenase site were protected from hypoxia. Mice with a mutation in the cyclooxygenase site had smaller infarctions 24 h after 70 min of middle cerebral artery occlusion than WT control mice. COX-2 activity had no effect on the formation of protein carbonyls. Ascorbate radicals were detected by electron paramagnetic resonance as a product of recombinant COX-2 activity and were blocked by COX-2 inhibitors. Similarly, formation of ascorbate radicals was inhibited in the presence of COX-2 inhibitors and in homogenates obtained from COX-2 null mice. Taken together, these results indicate that the cyclooxygenase activity of COX-2 is necessary to exacerbate neuronal hypoxia/ischemia injury rather than the peroxidase activity of the enzyme.

Topics: Animals; Arachidonic Acid; Ascorbic Acid; Brain Infarction; Catalytic Domain; Cell Line; Cells, Cultured; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Free Radicals; Humans; Hypoxia-Ischemia, Brain; Mice; Mice, Transgenic; Nerve Degeneration; Oxidative Stress; Peroxidase; Prostaglandin H2; Rats

Recent clinical trials have demonstrated the efficacy and safety of therapeutic hypothermia for neonatal hypoxic ischemic encephalopathy (HIE). We previously reported that the levels of non-protein-bound iron and ascorbic acid (AA) are increased in the CSF of infants with HIE. In this study, we investigated the effect of hypothermia on the combined cytotoxicity of Fe and AA for differentiated PC12 cells. The optimal settings for hypothermic treatment were a temperature of 30-32 degrees C, rescue time window of less than 6 h, and minimum duration of at least 24 h. Hypothermia effectively prevented the loss of the mitochondrial transmembrane potential from 6 h to 72 h (end of the study period) and attenuated the release of apoptotic proteins (cytochrome c and apoptosis-inducing factor) at 6 h of exposure to Fe-AA. Activation of caspase-3 was also delayed until 24 h. Akt was transiently activated, although no influence of temperature was observed. Elevation of oxidative stress markers, including ortho-, meta-, and di-tyrosine (markers of protein oxidation) and 4-hydroxynonenal (lipid peroxidation) was significantly attenuated when the temperature was reduced by 5 degrees C. The half-cell reduction potential (Ehc) of GSSG/2GSH redox couple ranged from -220 to -180 mV in unstressed differentiated PC12 cells, and apoptosis was triggered when Ehc exceeded -180 mV. Hypothermia prevented Ehc from rising above -180 mV within 24 h of exposure to Fe-AA. In conclusion, hypothermia prevented cell death due to Fe-AA toxicity by inhibiting apoptotic pathways through maintenance of a reduced cellular environment, as well as by alleviating oxidative stress.

Topics: Aldehydes; Animals; Apoptosis; Apoptosis Regulatory Proteins; Ascorbic Acid; Biomarkers; Body Temperature; Drug Synergism; Glutathione; Hypothermia, Induced; Hypoxia-Ischemia, Brain; Iron; Membrane Potentials; Neurons; Neurotoxins; Oxidation-Reduction; Oxidative Stress; PC12 Cells; Proto-Oncogene Proteins c-akt; Rats; Temperature; Tyrosine

Information on the change in extracellular ascorbic acid (AA) during the acute period of cerebral ischemia is of great importance in the early therapeutic intervention of the cerebral ischemic injury since AA is known to be involved into most kinds of neurochemical changes in the cerebral ischemia. This study describes a fast and efficient method through integration of in vivo microdialysis with on-line electrochemical detection for continuous monitoring cerebral AA, allowing comparative study of the change in the extracellular AA level in different brain ischemia/reperfusion models. The method exhibits a high specificity for AA measurements, bearing a good tolerance against the fluctuation in the brain anoxia and acidity induced by cerebral ischemia/reperfusion. In the global two-vessel occlusion (2-VO) ischemia model, the striatum AA did not change with statistic significance until 60 min after occlusion and was decreased to be 91+/-3% (n=5, P<0.05) of the basal level (8.05+/-0.23 microM) at the time point of 60 min after occlusion. In the 2-VO ischemia/reperfusion model, AA remained unchanged during the 10 min of ischemia, and was sharply increased to be 267+/-74% (n=5, P<0.05) of the basal level after the initial 15 min of reperfusion, and then decreased to be 122+/-33% (n=5, P<0.05) of the basal level after 50 min of reperfusion. Extracellular AA was largely increased after 5 min of left middle cerebral artery occlusion (LMCAO) and was then gradually increased to be 257+/-49% (n=5, P<0.05) of the basal level after 60 min of LMCAO ischemia. In the LMCAO ischemia/reperfusion model, AA was greatly increased during 10 min of ischemia and then gradually increased to be 309+/-69% (n=5, P<0.05) of the basal level after the consecutive 50 min of reperfusion. The results demonstrated here may be useful for understanding the neurochemical processes in the acute period of cerebral ischemia and could thus be important for neuroprotective therapeutics for cerebral ischemic injury.

Topics: Animals; Ascorbic Acid; Brain; Corpus Striatum; Disease Models, Animal; Electrochemistry; Extracellular Fluid; Hydrogen-Ion Concentration; Hypoxia-Ischemia, Brain; Infarction, Middle Cerebral Artery; Male; Microdialysis; Neurochemistry; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Processing, Computer-Assisted; Time Factors

Neuronal cell damage following hypoxic-ischemic (HI) brain injury is partly caused by production of free radicals and reactive oxygen species (ROS). Ascorbic acid (AA) is a potent antioxidant, which scavenges various types of ROS. Some studies have shown that it is neuroprotective, however, the issue is still controversial. In this study, we examined the effect of intraventricular AA administration on immature HI brain using the Rice-Vannucci model. After unilateral carotid artery ligation under isoflurane anesthesia, 7-day-old rat pups received varying concentrations of AA (0.04, 0.2, 1 and 5 mg/kg) by intraventricular injection and were exposed to 8% oxygen for 90 min. Vehicle controls received an equal volume of phosphate saline buffer. We assessed the neuroprotective effect of AA at 7 days post-HI. The percent brain damage measured by comparing the wet weight of the ligated side of hemisphere with that of contralateral one was reduced in both 1 and 5 mg/kg groups but not in either 0.04 or 0.2 mg/kg groups compared to vehicle controls (5 mg/kg 16.0 +/- 4.3%, 1 mg/kg 10.9 +/- 5.0%, vs. controls 36.7 +/- 3.6%, P < 0.05). Macroscopic evaluation of brain injury revealed the neuroprotective effect of AA in both 1 and 5 mg/kg groups (5 mg/kg 1.1 +/- 0.4, 1 mg/kg 0.4 +/- 0.3, vs. controls 2.9 +/- 0.3, P < 0.05). Western blots of fodrin on the ligated side also showed that AA significantly suppressed 150/145-kDa bands of fodrin breakdown products, which suggested that AA suppressed activation of calpain. Neuropathological quantitative analysis of cell death revealed that 1 mg/kg of AA injection significantly reduced the number of necrotic cells in cortex, caudate putamen, thalamus and hippocampus CA1, whereas that of apoptotic cells was only reduced in cortex. These findings show that intraventricular AA injection is neuroprotective after HI in immature rats.

Topics: Analysis of Variance; Animals; Animals, Newborn; Antioxidants; Apoptosis; Ascorbic Acid; Blotting, Western; Body Weight; Carrier Proteins; Cell Count; Functional Laterality; Hypoxia-Ischemia, Brain; Hypoxia, Brain; Injections, Intraventricular; Microfilament Proteins; Molecular Weight; Necrosis; Organ Size; Rats; Rats, Wistar

Among various hypothetical mechanisms for the in vivo production of reactive oxygen species, transition metal-catalyzed reactions in cooperation with a biologic reducing agent like ascorbic acid or superoxide may be some of the most important. In the present study, we retrospectively examined the existence of non-protein-bound metal ions, an essentially hazardous pro-oxidant form of various transition metals, and the occurrence of metal-catalyzed reactive oxygen species production in cerebrospinal fluid (CSF) of 10 infants with hypoxic ischemic encephalopathy (HIE) subsequent to perinatal asphyxia and 12 control infants within 72 h of birth. Non-protein-bound iron was detected in eight out of 10 CSF samples from the HIE infants and its level was significantly correlated with Sarnat's clinical stage, whereas none of the control infants had detectable non-protein-bound iron levels. Non-protein-bound copper was below the detection limit in all CSF samples from both groups. Ascorbic acid was significantly increased in the CSF of HIE infants when compared with that of controls (means, 664.9 versus 449.4 microM, p = 0.008). ortho-Tyrosine and meta-tyrosine, which are highly specific and sensitive markers of protein oxidation induced by hydroxyl radicals, were significantly higher in HIE infants than in controls when evaluated by the ratio relative to their source amino acid, phenylalanine [means, 110.5 versus 75.4, p = 0.018 for ortho-tyrosine/phenylalanine; 104.6 versus 67.7 (nM/microM x 10(2)), p = 0.048 for meta-tyrosine/phenylalanine]. Both ratios were significantly correlated with non-protein-bound iron, but not with ascorbic acid. Our preliminary observations provide direct evidence that hydroxyl radicals are generated in the CNS during asphyxiation. Iron chelation therapy could be worth developing as a neuroprotective strategy for perinatal asphyxia.

Topics: Allantoin; Ascorbic Acid; Biomarkers; Copper; Dehydroascorbic Acid; Female; Humans; Hydroxyl Radical; Hypoxia-Ischemia, Brain; Infant, Newborn; Iron; Male; Oxidative Stress; Protein Binding; Reactive Oxygen Species

Of particular physiological interest, ascorbate, the ionized form of ascorbic acid, possesses strong reducing properties. However, it has been shown to induce oxidative stress and lead to apoptosis under certain experimental conditions. Ascorbate in the brain is released during hypoxia, including stroke, and is subsequently oxidized in plasma. The oxidized product (dehydroascorbate) is transported into neurons via a glucose transporter (GLUT) during a reperfusion period. The dehydroascorbate taken up by cells is reduced to ascorbate by both enzymatic and non-enzymatic processes, and the ascorbate is stored in cells. This reduction process causes an oxidative stress, due to coupling of redox reactions, which can induce cellular damage and trigger apoptosis. Ascorbate treatment decreased cellular glutathione (GSH) content, and increased the rates of lipid peroxide production in rat cortical slices. Wortmannin, a specific inhibitor of phosphatidylinositol (PI)-3-kinase (a key enzyme in GLUT translocation), prevented the ascorbate induced-decrease of GSH content, and suppressed ascorbate-induced lipid peroxide production. However, wortmannin was ineffective in reducing hydrogen peroxide (H(2)O(2))-induced oxidative stress. The oxidative stress caused ceramide accumulation, which was proportionally changed with lipid peroxides when the cortical slices were treated with ascorbate. These differential effects support the hypothesis that GLUT efficiently transports the dehydroascorbate into neurons, causing oxidative stress.

Topics: Androstadienes; Animals; Apoptosis; Ascorbic Acid; Brain; Ceramides; Dehydroascorbic Acid; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glutathione; Hypoxia-Ischemia, Brain; Lipid Peroxides; Male; Monosaccharide Transport Proteins; Nerve Degeneration; Organ Culture Techniques; Oxidative Stress; Rats; Rats, Sprague-Dawley; Rotenone; Signal Transduction; Sphingomyelins; Stroke; Uncoupling Agents; Wortmannin