Hydroxyl radical generation after the third hour following ischemia contributes to brain damage

The effects of 10 antiallergic drugs (astemizole, azelastine, ebastine, emedastine, epinastine, ketotifen, oxatomide, terfenadine, pemirolast and tranilast) on neuronal dopamine uptake were examined. Some drugs examined showed a concentration-dependent inhibition of [3H]dopamine uptake into synaptosomal preparations of the rat striatum. The inhibition constant (Ki) values were 231-876 nM for ebastine, terfenadine, oxatomide and astemizole. The specific binding of [3H] (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine) (GBR12935) to the rat striatal membranes was also inhibited by these antiallergic drugs. There was a good correlation between the degrees of inhibition of [3H]dopamine uptake and [3H]GBR12935 binding. Then, the behavioral excitement induced by L-DOPA (100 mg/kg, s.c.) plus pargyline hydrochloride (80 mg/kg, i.p.) in mice was significantly enhanced by i.p. treatment with ebastine (10 mg/kg) and astemizole (5 mg/kg). These results suggest that the neuronal dopamine uptake is inhibited by some antiallergic drugs, especially ebastine.     

Hydroxyl radical formation in diabetic rats induced by streptozotocin

Production of hydroxyl radicals was examined in the diabetic rats induced by streptozotocin to prove its involvement to the pathogenesis of diabetes. Hydroxyl radicals generated in plasma, heart muscle, liver and brain of the hyperglycemic rats were quantitatively assayed by trapping hydroxyl radicals with salicylic acid as 2,3- and 2,5-dihydroxybenzoic acid. The concentrations of 2,3- and 2,5-dihydroxybenzoic acid were significantly increased in all the tissues of the diabetic rats. In the brain and heart muscle of the diabetic rats, the increase of 2,3-dihydroxybenzoic acid was more manifest than that of 2,5-dihydroxybenzoic acid, while in liver 2,5-dihydroxybenzoic acid increased markedly. All the values of 2,3-dihydroxybenzoic acid detected in the tissues of the diabetic rats were quite higher than those in control. Hydroxyl radical production and blood glucose concentration were depended almost linearly on the amount of streptozotocin injected to rats up to 60 mg/kg body weight. It was suggested that 2,3-dihydroxybenzoic acid was produced from hydroxyl radicals themselves, while 2,5-dihydroxybenzoic acid was produced by hydroxylation of salicylic acid not only with hydroxyl radicals, but also by enzymatic reaction of microsomal cytochrome-P450. Hydroxyl radical formation may account for some pathological process especially in the heart muscle and brain.     

Hydroxyl radical scavengers inhibit TNF-alpha production in mononuclear cells but not in polymorphonuclear leukocytes

The hydroxyl radical (HO*) scavengers dimethylthiourea (DMTU), tetramethylthiourea (TMTU), dimethylsulfoxide (DMSO) and deferoxamine (DFX), the latter being an iron chelator which prevents HO* formation by blocking the Fenton reaction, were found to inhibit TNF-alpha production in LPS-stimulated human PBMC but not in PMN. Furthermore, this effect was not LPS-specific, as TNF-alpha production was reduced by HO* radical scavengers to a similar extent upon stimulation of PBMC with immune complexes (IC), concanavalin A (Con A) and phorbol myristate acetate (PMA). Other scavengers such as glutathione (GSH), N-acetylcysteine (NAC), ascorbic acid (ASC) and mannitol (MAN) do not have effect on the production of TNF-alpha either in PBMC or PMN. These results provide evidence that the participation of ROI in the regulation of TNF-alpha production differ in different cell types. Particularly, the data presented in this work indicate that HO* radicals have a central role in the production of this inflammatory cytokine by human PBMC.     

Myocardial injury after hypoxia in immature, adult and aged rats

We evaluated the abilities of isolated perfused hearts from immature (IM) (2.5-3 months), ADULT (11-13 months) and OLD (24-26 months) Fischer 344 rats to tolerate and recover from oxygen deprivation. Hearts were perfused at 60 mmHg for a 30-minute prehypoxic period with oxygenated buffer supplemented with 10 mM glucose (+insulin) and 2 mM acetate, then 30 minutes with substrate-free, hypoxic buffer gassed with 95% N2:5% CO2, and finally reoxygenated for an additional 45 minutes with the same buffer used during the prehypoxic period. During prehypoxia, all groups were similar in ventricular mechanical function, glycogen content, high-energy phosphates (HEP), reduced glutathione (GSH), Ca+2 content, and mitochondrial state 3 rates. At the end of the hypoxic period, glycogen levels were similar and almost completely depleted in all groups, HEP were lower (p < 0.05) in ADULT vs other groups, mitochondrial state 3 rates were decreased (24%, p < 0.05) only in ADULT, and GSH was depleted by 34% in IM vs only 13% in OLD (p < 0.05). After 45 minutes of reoxygenation, IM and OLD had recovered 48% and 45% of their respective prehypoxic function which was two-fold greater than the 23% recovery by ADULT. Loss of cytosolic enzymes, an indicator of sarcolemmal damage, was estimated by measuring lactate dehydrogenase (LDH) release. LDH release and Ca+2 content during reoxygenation in IM were only about half of that observed in ADULT or OLD. We conclude that immature and aged hearts tolerate and recover from hypoxia better than hearts from adults, and that the sarcolemmal membranes of immature rat hearts are less susceptible to damage from hypoxic stress than those of either older group.     

Synaptic remodeling and free radical formation after brain contusion injury in the rat

The purpose of this study was to explore whether bilateral frontal cortex contusion in rats would demonstrate changes relevant for understanding the pathology of frontal lobe injury in humans. Rats were allowed to survive for 3, 7, or 18 days postinjury (dpi). In the contused rats, albumin was trapped in frontal cortices, as well as in other brain areas, showing that neurons were exposed to plasma components. In the sham-operated rats, which had only craniotomy but no penetration of dura, the level of trapped albumin was also increased compared to intact controls, suggesting a partial lesion-like condition. Choline acetyltransferase activity was severely decreased in the frontal cortices of contused rats, compared to the sham-operated controls. The decrease was most pronounced at 3 dpi and less pronounced 18 dpi, suggesting that after the initial damage, regeneration of the cholinergic terminals occurred. The concentration of the mature presynaptic membrane protein D3(SNAP-25) was also decreased in the frontal cortices of contused rats at 3 and 7 dpi, whereas it was normalized at 18 dpi. Previously, we have evaluated changes in the rate of synaptic remodeling in brain injury by calculating the ratio of the neural cell adhesion molecule (NCAM) to D3(SNAP-25). The NCAM/D3(SNAP-25) ratio at 3 dpi was elevated by more than 60% in the frontal cortices of contused rats, suggesting a high initial rate of synaptic remodeling. The ratios were smaller at 7 and 18 dpi, suggesting that after the initial burst, the rate of remodeling leveled off. In contrast, astrocyte activation was less pronounced at 3 dpi than at 7 and 18 dpi, as measured by the levels of glial fibrillary acidic protein and glutamine synthetase immunoactivities. The immunoreactivity of glutamine synthetase more than doubled in the contused brains but its enzymatic activity increased less than 50%, suggesting that many enzymatic centers had been inactivated by free radicals. Calculated as the difference between the relative immunoreactivity and the relative enzymatic activity the "lost glutamine synthetase activity" increased continuously in frontal cortex and striatum from 3 to 18 dpi, indicating the production of free radicals long after the initial contusion event. In conclusion, following frontal cortical contusions the early synaptic damage was partly compensated by synaptic remodeling. We suggest that the continuous production of free radicals may have contributed to the declining remodeling rate and impair functional recovery.     

Lack of major hypoxia and significant brain damage in rats despite dramatic hyponatremic encephalopathy

The small but growing number of men in the nursing profession does not herald a progressive integration of masculine and feminine sex roles. The evidence presented in this paper suggests that even in female-dominated occupations such as nursing, patriarchal gender relations which reflect a high valuation of all that is male and masculine, play a significant role in situating a disproportionate number of men in administrative and elite specialty positions. At the heart of this gender dynamic is the need to separate the masculine from the lesser valued feminine. Male nurses do this by employing strategies that allow them to distance themselves from female colleagues and the quintessential feminine image of nursing itself, as a prerequisite to elevating their own prestige and power. They are aided in this task by patriarchal cultural institutions that create and perpetuate male advantage, as well as by women nurses themselves who, consciously or unconsciously, nurture the careers of men colleagues.     

Chemiluminescent study of free radical lipid peroxidation in the rabbit brain after cranio-cerebral trauma

The method of chemiluminescence was applied for the determination of free radicals content in rabbit brain after the experimental craniocerebral trauma. The level of malondialdehyde was measured spectrophotometrically. The quantity of free radical products in hemispheres, brain stem and cerebellum of rabbits with traumatic injury was shown to be significantly increased and correlated with MDA level. Data obtained indicate that the chemiluminescent technique can be useful for the investigation of lipid peroxidation in brain pathology.     

Human ventilatory response to CO2 after 8 h of isocapnic or poikilocapnic hypoxia

During ventilatory acclimatization to hypoxia (VAH), the relationship between ventilation (VE) and end-tidal PCO2 (PETCO2) changes. This study was designed to determine 1) whether these changes can be seen early in VAH and 2) if these changes are present, whether the responses differ between isocapnic and poikilocapnic exposures. Ten healthy volunteers were studied by using three 8-h exposures: 1) isocapnic hypoxia (IH), end-tidal PO2 (PETO2) = 55 Torr and PETCO2 held at the subject's normal prehypoxic value; 2) poikilocapnic hypoxia (PH), PETO2 = 55 Torr; and 3) control (C), air breathing. The VE-PETCO2 relationship was determined in hyperoxia (PETO2 = 200 Torr) before and after the exposures. We found a significant increase in the slopes of VE-PETCO2 relationship after both hypoxic exposures compared with control (IH vs. C, P < 0.01; PH vs. C, P < 0.001; analysis of covariance with pairwise comparisons). This increase was not significantly different between protocols IH and PH. No significant changes in the intercept were detected. We conclude that 8 h of hypoxia, whether isocapnic or poikilocapnic, increases the sensitivity of the hyperoxic chemoreflex response to CO2.     

Lipid free radical generation and brain cell membrane alteration following nitric oxide synthase inhibition during cerebral hypoxia in the newborn piglet

Nitric oxide (NO) is reported to cause neuronal damage through various mechanisms. The present study tests the hypothesis that NO synthase inhibition by N(omega)-nitro-L-arginine (NNLA) will result in decreased oxygen-derived free radical production leading to the preservation of cell membrane structure and function during cerebral hypoxia. Ten newborn piglets were pretreated with NNLA (40 mg/kg); five were subjected to hypoxia, whereas the other five were maintained with normoxia. An additional 10 piglets without NNLA treatment underwent the same conditions. Hypoxia was induced with a lowered FiO2 and documented biochemically by decreased cerebral ATP and phosphocreatine levels. Free radicals were detected by using electron spin resonance spectroscopy with a spin trapping technique. Results demonstrated that free radicals, corresponding to alkoxyl radicals, were induced by hypoxia but were inhibited by pretreatment with NNLA before inducing hypoxia. NNLA also inhibited hypoxia-induced generation of conjugated dienes, products of lipid peroxidation. Na+,K+-ATPase activity, an index of cellular membrane function, decreased following hypoxia but was preserved by pretreatment with NNLA. These data demonstrate that during hypoxia NO generates free radicals via peroxynitrite production, presumably causing lipid peroxidation and membrane dysfunction. These results suggest that NO is a potentially limiting factor in the peroxynitrite-mediated lipid peroxidation resulting in membrane injury.     

In vivo free radical production after cross-clamping and reperfusion of the renal artery in the rabbit

Postischaemic reperfusion injury is often attributed to the generation of oxygenated free radicals which may subsequently promote lipid peroxidation in cell membranes. Electron paramagnetic resonance spectroscopy in association with the spin trap molecule alpha-phenyl-N-tert-butyl-nitrone allowed direct confirmation of lipid free radical production after renal ischaemia-reperfusion in an in vivo rabbit model. A 60-min period of ischaemia followed by reperfusion caused free radical production twofold greater than after 15 min of ischaemia. Glutathione and alpha-tocopherol have been measured in renal tissue, as indirect markers of lipid peroxidation. After 15 min of ischaemia followed by 10 min of reperfusion, the mean(s.e.m.) glutathione content of the ischaemic kidney was slightly but significantly reduced by 11.9(2.5)% (P < 0.003). The content of alpha-tocopherol was unchanged. However, 10 min of reperfusion following 60 min of ischaemia led to significant decrease in mean(s.e.m.) content of both glutathione (30.4(3.7)%) (2.23(0.2) versus 3.14(0.18) mumol/g wet tissue, P < 0.001) and alpha-tocopherol (46.1(7.8)%) (0.57(0.10) versus 1.09(0.14) micrograms/g wet tissue, P < 0.001) when compared to the control kidney. Under these experimental conditions, desferrioxamine (15 mg/kg administered intravenously before inducing ischaemia), a drug known to limit free radical production, significantly limited the decrease of alpha-tocopherol to 20.8(6.4)% (0.83(0.08) versus 1.05(0.04) micrograms/g wet tissue, P < 0.05), but did not prevent glutathione consumption in the reperfused kidney.     

Oxygen free radical generation during in-utero hypoxia in the fetal guinea pig brain: the effects of maturity and of magnesium sulfate administration

Previous studies have shown, employing direct measurements with electron spin resonance (ESR) spectroscopy, that hypoxia induces an increased production of oxygen free radicals (OFR) in the brain of the guinea pig fetus. The present study using the same approach, investigated the effects of maturity and Mg2+-pretreatment on hypoxia-induced OFR formation in the guinea pig fetal brain. The normoxic and the hypoxic groups were exposed for 60 min to 21% or 7% oxygen, respectively. The control group consisted of term fetuses exposed to normoxia (n=7) and hypoxia (n=7). The experimental groups consisted of the following: (a) for the investigation on maturity effect, preterm fetuses (40 days) exposed to normoxia (n=6) or hypoxia (n=6); and (b) for the Mg2+-pretreatment investigation, term fetuses (60 days) exposed to normoxia (n=6) or hypoxia (n=6) following maternal pretreatment with Mg2+ which consisted of an initial bolus of MgSO4 (600 mg/kg, i.p.) 1 h prior to hypoxia followed by a second dose (300 mg/kg, i.p.). Oxygen free radicals were measured by ESR spectroscopy in the fetal cerebral cortical tissue utilizing phenyl-N-tert-butylnitrone (PBN) spin trapping. Fetal brain tissue hypoxia was documented biochemically by decreased tissue levels of ATP and phosphocreatine. In the control group of term fetuses, the cortical tissue from hypoxic fetuses showed a significant increase in spin adducts (71% increase, p<0.01). In the preterm group, the cortical tissue from hypoxic fetuses showed a 33% increase in spin adducts (p<0.001). The baseline free radical generation during normoxia was 22.5% higher at preterm than at term (41.4+/-3.5 units/g issue vs. 33.8+/-9.3 units/g tissue, p<0.05). In Mg2+-treated groups, spin adduct levels in cortical tissue from hypoxic fetuses did not significantly differ from those of the normoxic group (30.2+/-9.9 units/g tissue, normoxic-Mg2+ vs. 30. 6+/-8.1 units/g tissue, hypoxic-Mg2+). The results indicate that the fetal brain at term may be more susceptible to hypoxia-induced free radical damage than at preterm and that Mg2+ administration significantly decreased the hypoxia-induced increase in oxygen free radical generation in the term fetal guinea pig brain in comparison with non-treated hypoxic group.     

The effect of metal ions on free radical formation in lung and brain of rats

The influence of 14 metal ions on free radical (FR) formation in subcellular systems of lung mitochondria and brain synaptosomes (cortex and cerebellum) of rats was investigated. Iron and manganese showed a highly significant increase in FR formation in lung, whereas other investigated metal ions showed relatively slight, insignificant decreases in FR. The significant enhancement effects of iron and manganese increased with their content in the media. Scandium exhibited an insignificant enhancement effect at a low concentration of 0.01 microM, but also an insignificant decrease in FR formation in lung at higher concentrations. In brain cerebellum only iron significantly increased FR formation but manganese, as well as trivalent metals (Al, La, Sc), showed insignificant changes in FR formation. The possible causes of differences induced by metal ions at various concentrations, as well as the biological functions of the investigated tissues, were considered in the explanation of obtained results.     

The hydroxyl radical scavenger Nicaraven inhibits glutamate release after spinal injury in rats

Neuronal degeneration after trauma is mediated in part by release of excitatory amino acids (EAAs) and oxygen free radicals (OFR). We evaluated the effect of i.v. treatment with a hydroxyl radical scavenger ((+/-)-N,N'-propylenedinicotinamide; AVS) and spinal hypothermia (33 degrees C) on spinal CSF glutamate release after spinal trauma. In a control group, spinal compression evoked at 10 min a significant increase (5-fold) in glutamate which declined over 4 h (2.1-fold). AVS treatment attenuated glutamate release but had no additive effect. These data suggest that this compound can be effective in modulating spinal excitotoxicity resulting from increased OFR synthesis and corresponding potentiation of EAA release.     

Increase in the chronically monitored cerebrospinal fluid pressure after experimental brain injury in rats

An enzyme-linked immunosorbent assay to detect thyroglobulin autoantibodies (TGAB) in canine serum was developed and validated. The test result for each sample was derived from the optical density readings (OD) and expressed as an Ab-score(%) calculated from three in-house calibrators. The assay specifically detected TGAB as judged from lack of response in the assay after samples had been incubated with specific antigen. Intra- and interassay coefficients of variation ranged from 2.0-4.9% and 4.6-9.9%, respectively. The detection limit, an Ab-score of 5.6%, was close to the median Ab-score of 10% observed in healthy dogs (n = 132). The median Ab-score of dogs with primary hypothyroidism and lymphocytic thyroiditis (n = 11), skin diseases (n = 35), and non-thyroidal diseases (n = 63) was 340%, 12%, and 8%, respectively. The prevalence of TGAB in hypothyroid dogs with lymphocytic thyroiditis (sensitivity) was 91% (95% confidence limits: 59%-99%). In dogs with dermatological diseases without lymphocytic thyroiditis the prevalence of TGAB was 3% corresponding to a specificity of 97% (95% confidence limit: 85%-100%). In dogs with non-thyroidal diseases and healthy dogs the prevalence of TGAB was 5% and 6%, respectively. The diagnostic accuracy of serum TGAB was evaluated by subjecting the data from 11 dogs with lymphocytic thyroiditis and 35 control dogs without lymphocytic thyroiditis to receiver-operating characteristic curve analysis. The area under the receiver-operating characteristic curve (W = 0.966; 95% confidence limit 87%-100%) was significantly higher than that of a worthless test (0.5) (P < 0.0001), thereby indicating that serum TGAB measurements distinguished between dogs with and without lymphocytic thyroiditis.     

Copper/zinc superoxide dismutase transgenic brain accumulates hydrogen peroxide after perinatal hypoxia ischemia

OBJECTIVE: This study aims to evaluate the risk of esophagectomy in the elderly compared with younger patients and to determine whether results of esophagectomy in the elderly have improved in recent years. SUMMARY BACKGROUND DATA: An increased life expectancy has led to more elderly patients presenting with carcinoma of the esophagus in recent years. Esophagectomy for carcinoma of the esophagus is associated with significant morbidity and mortality, and advanced age is often considered a relative contraindication to esophagectomy despite advances in modern surgical practice. METHODS: The perioperative outcome and long-term survival of 167 elderly patients (70 years or more) with esophagectomy for carcinoma of the esophagus were compared with findings in 570 younger patients with esophagectomy in the period 1982 to 1996. Changes in perioperative outcome and survival between 1982 to 1989 and 1990 to 1996 were separately analyzed. RESULTS: The resection rate in the elderly was 48% (167/345), lower than the 65% (570/874) resection rate in younger patients (p < 0.001). There were significantly more preoperative risk factors and postoperative medical complications in the elderly, but no significant differences were observed in surgical complications. The 30-day mortality rate was higher in the elderly (7.2%) than in younger patients (3.0%) (p = 0.02), but the hospital mortality rate was not significantly different in the elderly (18.0%) and younger age groups (14.4%) (p = 0.27). The long-term survival after curative resection in elderly patients was worse than younger patients (p = 0.01). However, when deaths from unrelated medical conditions were excluded from analysis, survival was similar between the two age groups (p = 0.23). A comparison of data for the periods 1982 to 1989 and 1990 to 1996 revealed that the resection rate had increased from 44% to 54% in the elderly, with significantly fewer postoperative complications and lower 30-day and hospital mortality rates. Long-term survival has also improved, although this has not reached a statistically significant level. CONCLUSIONS: With current surgical management, esophagectomy for carcinoma of the esophagus can be carried out with acceptable risk in the elderly, but intensive perioperative support is required. The improved results of esophagectomy in the elderly in recent years are attributed to increased experience and better perioperative management. Long-term survival was similar to that of younger patients, excluding deaths caused by unrelated medical conditions.     

Increased brain damage after stroke or excitotoxic seizures in melatonin-deficient rats

The pineal hormone melatonin is neuroprotective in vitro, and in vivo it is neuroprotective when given in pharmacological doses. Consequently, it has been hypothesized that with aging, as circulating levels of melatonin in mammals normally decrease, the brain might be at increased risk of neurodegeneration. However, direct evidence that melatonin deficiency leads to increased brain vulnerability is still lacking. We created melatonin deficiency in rats by pinealectomy and induced neurodegeneration by two models of focal brain ischemia/stroke and by glutamate receptor-mediated, epilepsy-like seizures. We observed greater neurodegeneration in melatonin-deficient animals than in controls. Our results suggest that endogenous melatonin may play a neuroprotective role, and that melatonin deficiency might be a pathophysiological mechanism in neurodegenerative diseases.     

Sustained decrease in brain regional blood flow after microsphere embolism in rats

BACKGROUND AND PURPOSE: An experimental model that induces sustained ischemia and infarction may provide useful information relevant to prevention of the development of ischemic brain disease. The purpose of the present study was to elucidate the pathophysiological consequences of cerebral blood flow under sustained cerebral ischemia or oligemia and infarction in rats after microsphere embolism. METHODS: We injected 900 microspheres (48 microns in diameter) into the right internal carotid artery of 146 rats and determined the time course of changes in blood flow of the cerebral cortex, striatum, and hippocampus of both hemispheres by the hydrogen clearance method for a period of 28 days after the operation. Infarct area was determined by triphenyltetrazolium chloride staining and hematoxylin and eosin staining methods. RESULTS: Cortical and striatal blood flow of the right hemisphere of microsphere-injected rats was significantly decreased after the embolism, and this was sustained throughout the experiment. Hippocampal blood flow of the microsphere-injected hemisphere was also decreased on days 1 and 3 but tended to return toward control levels thereafter. In the left hemisphere, reduction in regional blood flow was detected in the cortex and hippocampus on day 1 and the striatum on day 3. A triphenyltetrazolium chloride-unstained area had developed by day 3 after the embolism. The extent of the area was similar to that on days 7 and 28. Microscopic examination revealed degenerative areas scattered mainly in the parietotemporal cortex, corpus callosum, hippocampus, thalamus, and lenticular nucleus of the embolized hemisphere, demonstrating the induction of widespread necrosis after embolism. CONCLUSIONS: Microsphere embolism resulted in a sustained decrease in regional blood flow and production of cerebral infarction in the brain regions of the microsphere-injected hemisphere.     

The effect of hypoxia on the pentose phosphate pathway in brain

Sodium, potassium, and chloride levels are artifactually depressed in hyperlipemic sera. Accurate electrolyte levels are needed for management of patients with hyperlipemia, but present methods for correcting the values (serum water and/or osmolality determinations) either are technically cumbersome or fail to provide accurate data to correct the falsely low levels. Alternatively, to determine true sodium, potassium, and chloride concentrations in hyperlipemic sera, only the required electrolyte values and the triglycerides are measured. The percentage by which the measured electrolyte levels in the hyperlipemic sample must be increased to approximate the true values is given by the following equation: per cent increase = 2.1 X triglycerides (Gm./dl.) - 0.6.