Effects of Graded Hypothermia on Hypoxic-ischemic Brain Damage in the Neonatal Rat

Objective To investigate the effect of graded hypothermia on neuropathologic alterations of neonatal rat brain after exposed to hypoxic-ischemic insult at 37℃,33℃,31℃,and 28℃,respectively,and to observe the effect of hypothermia on 72-kDa heat shock protein (HSP72) expression after hypoxic-ischemic insult.Methods Seven days old Wistar rats were subjected to unilateral common carotid artery ligation followed by exposure to hypoxia in 8% oxygen for 2 hours at 37℃,33°C,31℃,and 28℃,respectively.The brain temperature was monitored indirectly by inserting a mini-thermocouple probe into the temporal muscle during hypoxia.After hypoxia-ischemia their mortality was assessed.Neuronal damage was assessed with HE staining 72 hours after hypoxia.HSP72 expression at 0.5,24,and 72 hours of recovery was immunohis-tochemically assessed using a monoclonal antibody to HSP72.Results Hypoxia-ischemia caused 10.5% (2/19) of mortality in rat of 37°C group,but no death occurred in 33℃,31℃ or 28℃ groups.HE staining showed neuropathologic damage was extensive in rats exposed to hypoxia-ischemia at 37℃ (more than 80.0%).The incidence of severe brain damage was significantly decreased in 33℃ (53.3%) and 31℃ groups (44.4%),and no histologic injury was seen in the 28℃ group of rats.Expression of HSP72 was manifest and persistent in the rat brain of 37°C group,but minimum in the rat brain of 28℃ group.Conclusion Mild and moderate hypothermia might prevent cerebral visible neuropathologic damage associated with hypoxic-ischemic injury by decreasing stress response.     

Anoxic and hypoxic immature rat model for measurement of monoamine using in vivo microdialysis

The immature brain is considered relatively resistant to anoxia and ischemia. Although hypoxia without ischemia has not been considered to produce brain damage in immature rats as well as in adult rats (S. Levine, Anoxic-ischemic encephalopathy in rats, Am. J. Pathol., 36 (1960) 1-17 [8]; D.E. Levy, J.B. Brieley, D.G. Silverman, F. Plum, Brief hypoxia-ischemia initially damages cerebral neurons, Arch. Neurol., 32 (1975) 450-456 [9]; J.E. Rice, R.C. Vannucci, J.B., Brieriey, The influence of immaturity on hypoxic-ischemic brain damage in rat, Ann. Neurol., 9 (1981) 131-141 [14]), hypoxia in postnatal period is possible to cause a functional brain damage (T. Hender, P. Lundborg, Regional changes in monoamine synthesis in the developing rat brain during hypoxia, Acta. Physiol. Scand., 106 (1979) 139-143 [3]; W. Ihle, J. Gross, R. Moller, Effect on chronic postnatal hypoxia on dopamine uptake by synaptosomes from striatum of adult rats, Biomed. Biochem. Acta., 44 (1985) 433-437 [7]; A. Lun, J. Gross, M. Beyer, H.D. Fischer, C. Wustmann, J. Schmidt, K. Hecht, The vulnerable period of perinatal hypoxia with regard to dopamine release and behavior in adult rats, Biomed. Biochem. Acta., 45 (1986) 619-627 [10]). Using microdialysis, we studied the anoxic or hypoxic effect on catecholamine metabolism in immature rat brain by measuring extracellular concentrations of norepinephrine (NE), dopamine (DA), and its metabolites and also 5-hydroxyindole-3-acetic acid (5-HIAA), the serotonin metabolite. DA is a well established excitatory neurotransmitter (R.C. Vannucci, Experimental biology of cerebral hypoxia-ischemia: relation to perinatal brain damage, Pediatr. Res., 27 (1990) 317-326 [16]), and in the previous report using hypoxic 7-day-old rat pups increase of DA was not detected without additional stimulations (K. Gordon, D. Johnston, M.V. Robinson, T.E. Statman, J.B. Becker, F. Silverstein, Transient hypoxia alters striatal catecholamine metabolism in immature brain: An in vivo microdialysis study, J. Neurochem., 54 (1990) 605-611 [2]). Whereas recently in newborn piglets, hypoxic hypoxia produced increase of extracellular DA (C.-C. Huang, N.S. Lajevardi, O. Tammela, A. Pastuszko, Relationship of extracellular dopamine in striatum of newborn piglets to cortical oxygen pressure, Neurochem. Res., 19 (1994) 649-655 [6]; Olano, M., Song, D., Murphy, S., Wilson, D. F. and Pastuszko, A., Relationships of dopamine, cortical oxygen pressure, and hydroxyl radicals in brain of newborn piglets during hypoxia and posthypoxic recovery, J. Neurochem., 65 (1995) 1205-1212 [13]). We consider that hypoxic ischemic brain damage of human newborns that we can treat is a damage, which does not show overt neuropathological changes. We therefore tried to show that transient anoxia and hypoxia caused biochemical alteration if the exposure did not produce marked morphological changes. This rodent model is adequate to study perinatal asphyxia and alteration of monoamine level could be useful for evaluation of brain damage, even if it is not detected histologically.     

U1 snRNP inhibits pre-mRNA polyadenylation through a direct interaction between U1 70K and poly(A) polymerase

The brain damage produced by unilateral cerebral hypoxia-ischemia in the immature rat results from major alterations in cerebral energy metabolism and glucose utilization which begin during the course of the insult and proceed into the recovery period. Consistent with a lack of pathology, the alterations in the hemisphere contralateral to the carotid artery ligation are transient and return to normal within 24 h of recovery, whereas the hemisphere ipsilateral to the ligation exhibits both early and late responses, and infarction. The facilitative glucose transporter proteins mediate glucose transport across the blood-brain barrier (55 kDa GLUT1), and into neurons and glia (GLUT3 and 45 kDa GLUT1), and demonstrate both early and late responses to perinatal hypoxia-ischemia. This study employed in situ hybridization histochemistry to investigate the temporal and regional patterns of GLUT1 and GLUT3 gene expression following a severe (2.5 h) hypoxic-ischemic insult in the 7-day old rat brain. Enhanced GLUT1 mRNA expression was apparent in cerebral microvessels of both hemispheres and remained elevated in the ipsilateral hemisphere through 24 h of recovery, consistent with our previous observation of increased microvascular 55 kDa GLUT1 protein. The expression of the neuronal isoform, GLUT3, was enhanced in penumbral regions, such as piriform cortex and amygdala, but was rapidly reduced in the affected areas of cortex, hippocampus and thalamus, reflecting necrosis. The late response, observed at 72 h of recovery, was characterized by extensive necrosis in the ipsilateral hemisphere, loss of GLUT3 expression, and a gliotic reaction including increased GLUT1 in GFAP-positive astrocytes. This study demonstrates that cerebral hypoxia-ischemia in the immature rat produces both immediate-early and long-term effects on the glucose transporter proteins at the level of gene expression.     

Reduction in cerebral ischemic injury in the newborn rat by potentiation of endogenous adenosine

Because of ontogenic influences on the pathophysiologic mechanisms of brain injury in the perinatal brain, and in particular, the incomplete development of adenosine receptor systems, we investigated the potential for adenosine to provide cerebro-protection in a well established newborn rat model of hypoxia-ischemia. Fifteen litters of postnatal d 7 animals were subjected to unilateral carotid ligation and exposure to hypoxia (8% oxygen) for 3 h. Immediately after hypoxia-ischemia, animals received either the adenosine deaminase inhibitor deoxycoformycin (DCF; 2.5 mg/kg intraperitoneally) or the adenosine uptake inhibitor propentofylline (PPF; 10 mg/kg intraperitoneally); paired littermates received an equivalent volume of normal saline. On postnatal d 14, injury or protection was assessed by differences in hemispheric weights, morphometric determinations of infarct area, and histopathologic analyses. DCF resulted in a 34% (p = 0.02) and 31% (p = 0.03) reduction in hemispheric weight disparities and infarct area, respectively; for PPF, these reductions were 46% (p = 0.03) and 32% (p = 0.04), respectively. Light microscopic examinations of striatum, thalamus, hippocampus, and cortex revealed that both drugs significantly improved histologic scores as well. Measurements in six separate litters indicated that neither drug significantly reduced core body temperature for at least 6 h postadministration. These findings indicate that potentiation of endogenous adenosine levels in the perinatal brain can significantly ameliorate brain injury. Each of these treatment strategies was effective even when administered after the hypoxic-ischemic insult. Thus, further investigations of adenosinergic therapies are warranted in this and other perinatal models of cerebral ischemia to elucidate in detail their potential for clinical application.     

The platelet-activating factor antagonist BN 52021 attenuates hypoxic-ischemic brain injury in the immature rat

Platelet-activating factor (PAF) is overproduced in ischemic brain. Although postischemic PAF antagonist administration protects the mature brain in some models, little is known about the effects of PAF antagonists in the immature brain. We hypothesized that the PAF antagonist BN 52021 would attenuate perinatal cerebral hypoxic-ischemic injury. To elicit focal hypoxic-ischemic brain injury, 7-d-old (P7) rats (n = 111) underwent right carotid ligation, followed by 2.5-3.25 h of hypoxia (fractional concentration of inspired O2 = 0.08). BN 52021 neuroprotection was evaluated in three groups of experiments: 1) 25 mg/kg/dose, 0 and 2 h posthypoxia; 2), 25 mg/kg/dose immediately before and 1 h after hypoxia; and 3) posthypoxia-ischemia treatment with BN 52021 12.5, 25, or 50 mg/kg/dose in 2 doses 0 and 2 h after hypoxia. All experiments included concurrent vehicle-injected controls. To quantitate severity of injury, bilateral regional cross-sectional areas (groups 1 and 2) or hemisphere weights (group 3) were evaluated on P12. Both pre- and posthypoxic treatment with BN 52021 (25 mg/kg/dose, two serial doses) decreased the incidence of cerebral infarction from 90% to about 30% (p < 0.02, Fisher's exact test). Measurement of cross-sectional areas confirmed neuroprotection and indicated some benefit of pre- over posthypoxic-ischemic treatment in hippocampus and cortex. Over the dose range tested, the neuroprotective effect of BN 52021 administration was not dose-dependent. In contrast, BN 52021 did not attenuate N-methyl-D-aspartate-induced hippocampal excitotoxic injury in P7 rats. Either prophylactic or "rescue" administration of PAF antagonists decreases the incidence and severity of brain injury associated with an episode of perinatal cerebral hypoxia-ischemia.     

Advanced laboratory techniques for diagnosing Toxoplasma gondii encephalitis in AIDS patients: significance of intrathecal production and comparison with PCR and ECL-western blotting

The aim of this study was to determine the influence of neonatal focal cerebral hypoxia-ischemia (HI) on sleep-waking pattern, electrocorticogram (ECoG) power spectra and locomotor activity (LA) in adult Wistar rats. Seven-day old pups were subjected to permanent unilateral ligation of the common carotid artery and transient hypoxia (8% O2). At 10 weeks of age, the extent of brain damages was evaluated by magnetic resonance imaging (MRI) and homogenous injured animals were selected before chronic implantation of radiotelemetry device. Using a single ECoG recording channel method, waking (W), paradoxical sleep (PS) and slow wave sleep (SWS) were continuously recorded for 72 h and they were semi-automatically analyzed off-line. We observed that neonatal HI triggers a cascade of events leading, in adult rats, to brain dysfunction characterized by an increase in SWS (55.0 vs. 40.2% in sham-operated rats, p<0.05) and a marked decrease in W phases duration (43.4 vs. 51.5%, p<0.05) while PS was almost suppressed in HI rats (1.6 vs. 8.3%, p<0.05). In addition, power spectral analysis of ECoG revealed significant (p<0.05) alteration in PS power density with a shift of the dominant frequency peak (5.0 to 7.5 Hz for HI and sham-operated rats, respectively). During the light period, we found that HI induced a pronounced reduction of LA (-30%, p<0.05). These results indicate that Wistar rats exposed to a neonatal unilateral cerebral HI present significant ECoG activity, sleep-waking pattern and behavioral disturbances when adults. However, it remains to establish whether such alterations can be prevented by neuroprotective agents.     

Compliance considerations with hormone replacement therapy

Lactate accumulation, amino acid aspartate and glutamate levels, and hypoxanthine, xanthine and malondialdehyde (MDA) concentrations were compared in neonate rat brain after transient global hypoxia induced alone or in association with unilateral ligation of a carotid artery. Lactate production in both hemispheres was higher in cerebral hypoxia-ischemia (CHI) than in cerebral hypoxia (CH), and was lower in CHI after 2 h than at 15 min of recovery. Aspartate and glutamate levels were reduced 15 min after CHI in both hemispheres, but aspartate alone was decreased 2 h after CHI in the ipsilateral (left) hemisphere and 15 min after CH in both hemispheres. Hypoxanthine was increased 15 min after CHI in the ipsilateral hemisphere but decreased at 2 h, whereas xanthine was increased. MDA production was not modified after CH or CHI. These data, compared to those obtained in adult animals suggest that glutamate release and the capacity to generate oxygen-derived radicals are lower in neonates after ischemia. These differences might explain why the brain of the mammalian neonate is much more resistant to CH and CHI than that of the adult.     

The effect of focal cerebral cooling on perinatal hypoxic-ischemic brain damage

We describe a method of focal cooling of the head and its effects on hypoxic-ischemic cerebral damage in neonatal rat. Focal cooling of the head was obtained by positioning a catheter under the scalp ipsilateral to the ligated common carotid artery and by running cold water through the catheter during 2 h of systemic hypoxia. Hypoxia was produced in neonatal rats by breathing 8% oxygen for 2 h in a 37 degrees C chamber. Animals underwent focal cooling with ipsilateral scalp temperatures ranging from 22 degrees C to 35 degrees C. Temperature recordings from the ipsilateral scalp, cerebral hemisphere (dorsal hippocampus) and core (rectal) were obtained. The results suggest that the method is effective in cooling of brain and also to a lesser extent in lowering of the core temperature. At a mean scalp temperature of 28 degrees C, mean hippocampal temperature in hypoxic rat was 29.5 degrees C and mean core temperature in hypoxic rat was 32.8 degrees C. At a lower scalp temperature of 22 degrees C, mean hippocampal temperature in hypoxic rat was 24.7 degrees C and mean core temperature was 31.3 degrees C. Neuropathologic examination 3-4 days following hypoxia-ischemia showed that focal cooling with a scalp temperature of lower than 28 degrees C completely protected from brain damage, and that there was a trend towards greater damage with higher scalp temperatures.     

Studies on the eosinophilic granule cells in the gills of the rainbow trout, Oncorhynchus mykiss

Hypoxic-ischemic changes in brain are detected earlier with diffusion-weighted (DW) than with T2-weighted magnetic resonance (MR) imaging techniques in adults, whereas the response in immature brain is not known. We investigated MR imaging changes prior to, during, and/or after 2 h of hypoxia-ischemia (right carotid artery occlusion + 2 h of hypoxia) in 7-day-old rats anesthetized with isoflurane. In general, within the first 45 min of hypoxia-ischemia there were no changes in the DW or T2-weighted images. By the second hour of hypoxia-ischemia there were marked areas of increased intensity in both the T2 and the DW images, with cortex and striatum being affected prior to thalamus and hippocampus. The area of DW exceeded that of T2 hyperintensities. In the first hour after hypoxia-ischemia there was a transient recovery of hyperintensities on both T2 and DW images. Between 24 and 72 h the hyperintense area on DW images decreased, whereas that on T2-weighted images increased. The distribution of pathological damage assessed histologically correlated with the areas of hyperintensity on the MR images. In contrast to adult brain, early hypoxic-ischemic injury in immature brain is detected as an increase in intensity in both diffusion- and T2-weighted images, indicating a unique alteration in brain water dynamics in this neonatal model of hypoxia-ischemia. These imaging changes and alterations in brain water can rapidly but transiently reverse upon the start of normoxia and reperfusion, suggestive of secondary energy failure or delayed neuronal death.     

Pretreatment with a competitive NMDA antagonist D-CPPene attenuates focal cerebral infarction and brain swelling in awake rats

The purpose of the study was to assess effects of the competitive N-methyl-D-aspartate (NMDA) receptor antagonist D-(E)-4-(3-phosphonoprop-2-enyl)piperazine-2-carboxylic acid (D-CPPene) upon focal cerebral infarction and brain oedema in the rat. Focal cerebral ischaemia was produced by permanent occlusion of the middle cerebral artery under halothane anaesthesia. The anaesthetic gas was discontinued immediately after the occlusion and the rats were killed 24 hours later. Cerebral infarction and brain swelling were each assessed on the frozen brain sections at 8 predetermined coronal planes. Pretreatment with D-CPPene (4.5 mg/kg i.v. followed by continuous infusion at 3 mg/kg/h until sacrifice) 15 minutes prior to MCA occlusion, significantly reduced the volume of infarction in the cerebral hemisphere by 29% (p < 0.05). Brain swelling, obtained by subtracting the nonischaemic hemispheric volume from the ischaemic hemispheric volume, was significantly reduced with D-CPPene treatment and the mean reduction in swelling (34% less than the controls: p < 0.001) proportionately similar to the decrease in infarct volume in the same animals. These data indicate that systemic administration of the competitive NMDA receptor antagonist D-CPPene has neuroprotective effects against ischaemic brain damage, and the reduction in brain swelling occurs in parallel with the reduction in ischaemic damage.     

Hypoxia-ischemia induces a rapid elevation of ubiquitin conjugate levels and ubiquitin immunoreactivity in the immature rat brain

Postnatal rats at 7 and 21 days of age were subjected to unilateral hypoxia-ischemia (H/I) by right carotid artery ligation followed by 1.5 to 2 hours of hypoxia (8% oxygen). Brains were frozen at specific intervals of recovery from 0 to 24 hours. Western blots of samples of right and left forebrain were immunodeveloped with a monoclonal antibody specific for ubiquitin, RHUb1. An elevation of ubiquitin conjugate levels in the right compared with the left forebrain of 7-day-old animals was detectable immediately following H/I and increased by close to 60% of control level within 1 hour of recovery. The conjugate immunoreactivity remained at this level for 6 hours but had declined to control levels by 24 hours of recovery. No such increase was observed in response to hypoxia alone. Similar changes were observed in samples from the 21-day-old rat brain. However, the elevation of ubiquitin conjugate levels was of slower onset and persisted longer than observed for the 7-day-old animals. Immunocytochemical studies of brain fixed by immersion in formaldehyde/acetone/methanol showed that ubiquitin-like immunoreactivity was increased in the right, but not left, cerebral cortex and hippocampus of animals subjected to H/I. The data suggest that elevated ubiquitination may represent a neuroprotective response to H/I.     

Apoptosis in a neonatal rat model of cerebral hypoxia-ischemia

BACKGROUND and PURPOSE: The mechanisms of excitotoxic cell death in cerebral ischemia are poorly understood. In addition to necrosis, apoptotic cell death may occur. The purpose of this study was to determine whether an established model of cerebral hypoxia-ischemia in the neonatal rat demonstrates any features of apoptosis. METHODS: Seven-day-old neonatal rats underwent bilateral, permanent carotid ligation followed by 1 hour of hypoxia, and their brains were examined 1, 3, and 4 days after hypoxia-ischemia. The severity of ischemic damage was assessed in the dentate gyrus and frontotemporal cortex by light microscopy. Immunocytochemistry was performed to detect the cleavage of actin by caspases, a family of enzymes activated in apoptosis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) reactivity was examined in the cortical infarction bed and dentate gyrus. Neonatal rat brain DNA was run on agarose gel electrophoresis to detect DNA fragmentation. Ethidium bromide-staining and electron microscopy were used to determine whether apoptotic bodies, 1 of the hallmarks of apoptosis, were present. RESULTS: The frontotemporal cortex displayed evidence of infarction, and in most rats the dentate gyrus showed selective, delayed neuronal death. Immunocytochemistry demonstrated caspase-related cleavage of actin. TUNEL and DNA electrophoresis provided evidence of DNA fragmentation. Ethidium bromide-staining and electron microscopy confirmed the presence of chromatin condensation and apoptotic bodies. CONCLUSIONS: Features of apoptosis are present in the described model of cerebral hypoxia-ischemia. Apoptosis may represent a mode of ischemic cell death that could be the target of novel treatments that could potentially expand the therapeutic window for stroke.     

The influence of nimodipine and MK-801 on the brain free arachidonic acid level and the learning ability in hypoxia-exposed rats

1. The influence of voltage dependent calcium channel blocker (VDCC), nimodipine and N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 on the brain free arachidonic acid (FAA) level and on the learning ability in hypoxia-exposed rats was examined. 2. Some animals were decapitated after cerebral hypoxia had been obtained and the brain FAA level was determined by gas chromatography. The other animals were trained in a passive avoidance procedure and were exposed to hypoxic conditions immediately after the learning trial response had been acquired. A passive avoidance retention test was performed 24 hours later. 3. Various doses of nimodipine (0.03; 0.1; 0.3 and 1.0 mg/kg) and MK-801 (0.03; 0.1 and 0.3 mg/kg) had been injected 30 minutes before biochemical or behavioral procedures started. 4. It was found that hypoxia strongly increased the brain FAA level and impaired the retention of the passive avoidance response. 5. Pretreatment with 0.3 mg/kg and 1.0 mg/kg of nimodipine prevented the brain FAA accumulation. It has also been shown that all tested doses of nimodipine significantly improved the retention deficit in the animals exposed to hypoxia. 6. Neither the one of tested doses of MK-801 influenced significantly the increase of the brain FAA level and/or passive avoidance behavior in hypoxic animals. 7. These results confirm the hypothesis that the brain FAA accumulation and cognitive impairment, caused by hypoxia, are maybe associated with disturbances in calcium homeostasis and that nimodipine may be useful in ameliorating the hypoxia-induced brain tissue damage. Blocade of NMDA receptor-channel complex by MK-801 was not sufficient to prevent hypoxia-induced neuronal damage.     

An animal model of hypoxia-induced perinatal seizures

Clinically, neonatal hypoxic encephalopathy is commonly associated with seizure activity. Here we describe a rodent model of cerebral hypoxia in which there is are age dependent effects of hypoxia, with hypoxia inducing seizure activity in the immature rat, but not in the adult. Global hypoxia (3-4% O2) induced acute seizure activity during a window of development between postnatal day (P5-17), peaking at P10-12. Animals which had been rendered hypoxic between P10-12 had long term decreases in seizure threshold, while animals exposed at younger (P5) or older (P60) ages did not. Antagonists of excitatory amino acid (EAA) transmission appear to be superior to benzodiazepines in suppressing the acute and long term effects of perinatal hypoxia, suggesting involvement of the EAA system in these phenomena. No significant histologic damage occurs in this model, suggesting that functional alterations take place in neurons when exposed to an hypoxic insult at a critical developmental stage. Future work is directed at evaluating molecular and cellular events underlying the permanent increase in seizure susceptibility produced by this model.     

Norepinephrine transmitter metabolite generates free radicals and activates mitochondrial permeability transition: a mechanism for DOPEGAL-induced apoptosis

The aim of this study was to investigate the role of cysteine in development of brain damage after hypoxia-ischemia (HI) in neonatal rats. Rat pups were subjected to unilateral carotid ligation and exposure to hypoxia (7.7% oxygen) for 60 or 90 min. A subtoxic dose of cysteine were administered before or after HI and the unilateral brain injury was evaluated 14 days after the insult and expressed as ipsilateral weight deficit as % of the contralateral hemisphere. In some experiments the changes of extracellular (e.c.) cysteine in the cerebral cortex were sampled with microdialysis and analyzed with HPLC. Cysteine in a dose of 0.2 mg/g s.c. given before 60 min of HI increased the extent of brain injury by 59%. The effect of posttreatment was limited and dependent on the duration of HI: 0.2 mg/g of cysteine given after 90 min of HI increased the degree of brain injury by 25%, whereas the same dose administered after 60 min of HI was ineffective in spite of that this combination of cysteine and HI resulted in e.c. cysteine concentrations 3-4 times higher than those observed in non-treated HI controls. These data show that subtoxic doses of cysteine administered before or after HI enhances brain injury. However, e.c. cysteine levels exceeding those induced by HI are required which makes a substantial contribution of cysteine in the pathophysiology of HI brain injury in the neonatal rat unlikely.     

Vulnerability to cerebral hypoxic-ischemic insult in neonatal but not in adult rats is in parallel with disruption of the blood-brain barrier

BACKGROUND AND PURPOSE: Vulnerability to cerebral hypoxic-ischemic (H-I) insult and its relation to disruption of the blood-brain barrier were investigated in postnatal rats. METHODS: Pups of postnatal day (P) 7, P14, and P21 underwent ligation of a unilateral carotid artery and were exposed to hypoxic conditions. For the detection of early-phase deterioration, brains were perfusion-fixed 24 hours after H-I insult and examined by argyrophil III method. For the detection of later infarction, animals were fixed at 72 hours after the H-I insult. RESULTS: In either case, tissue damage was detected in the striatum, parietal cortex, and hippocampus. The vulnerability of P7 and P21 rats was remarkable, as compared with P14 rats. Although the developmental status of the vasculature was not significantly different at each age, the permeability of IgG after H-I injury was prominent in P7 rats and to a lesser extent in P14 rats. In P21 rats, however, there was little IgG leakage even 24 hours after the insult. Dexamethasone pretreatment blocked the extravasation of IgG and reduced the damaged tissue in P7 and P14 rats but not in P21 rats. Percentages of reduction in infarcted areas by the dexamethasone became smaller in proportion to ages. CONCLUSIONS: The results suggest that in younger rats vulnerability to H-I insult was in parallel with permeability of the blood-brain barrier, whereas in adults in might be more dependent on cellular vulnerability.     

The effect of age on susceptibility to brain damage in a model of global hemispheric hypoxia-ischemia

Stroke occurs in all age groups, ranging from the newborn to the elderly. The immature brain is generally believed to be more resistant to the damaging effects of cerebrovascular compromise compared to the more mature brain. However, recent experiments suggest that the correlation between brain damage and age is not linear. To determine the effects of age and development on hypoxic-ischemic brain damage, we developed a model whereby rats of increasing age received identical cerebrovascular insults, and assessed neuropathologic outcome. Male Wistar rats of 1, 3, 6, and 9 weeks and 6 months underwent unilateral common carotid artery ligation and exposure to 12% oxygen for 35 min. Animals were all spontaneously breathing under light halothane anesthesia (0.5%). Core temperatures were maintained at 37 degrees C. Blood pressures were monitored via indwelling carotid artery catheters on the side ipsilateral to the carotid artery ligation. Cerebral blood flow was assessed in separate groups utilizing Laser Doppler flowmetry. Physiologic monitoring revealed that under these experimental conditions, mean arterial blood pressure and cerebral blood flow decreased to the same extent in each of the age groups, verifying that all animals experienced an identical insult. Neuropathologic assessment at 7 days of recovery showed that brain damage was most severe in the 1 and 3 week old animals followed by those that were 6 months. The 6 and 9 week old groups had significantly less injury than the other 3 age groups. Hippocampal damage was most severe in the 3 week and 6 month old rats compared to all other age groups. Our findings contrast previously held beliefs regarding the enhanced tolerance of the immature brain to hypoxic-ischemic damage and demonstrates that, in a physiologically controlled in vivo model of hemispheric global ischemia, (1) the immature brain is, in fact, less resistant to hypoxic-ischemic brain damage than its adult counterpart, (2) the brain damaging effects of hypoxic-ischemia are age dependent, but do not increase linearly with advancing age and development, and (3) the intermediate age groups are more tolerant to hypoxic-ischemic brain injury than either very young or more mature ages.     

Does concurrent or prior nicotine exposure interact with neonatal hypoxia to produce cardiac cell damage?

Cigarette smoking during pregnancy exposes the fetus to both nicotine and hypoxia/ischemia; postnatal exposure to second-hand smoke also involves substances that cause hypoxia (CO, HCN). Although developing cardiac cells are more resistant to hypoxia-induced damage than are mature cells, we examined whether nicotine affects this resistance, either when exposure is concurrent with hypoxia, or when animals are exposed to nicotine prenatally and receive subsequent hypoxic exposure. One, 8-, or 15-day-old rats exposed to 7% O2 for 2 hr all showed inhibition of cardiac DNA synthesis. By contrast, administration of nicotine at either low (0.3 mg/kg) or high (3 mg/kg) doses failed to alter DNA synthesis. To examine effects on cells that were not undergoing mitosis, we examined ornithine decarboxylase (ODC), an enzymatic marker for cell damage. One day old rats showed inhibition of ODC by hypoxia, a response that represents preservation of cell integrity; by 8 days of age, ODC was increased by hypoxia, evidence of cell damage. The high dose of nicotine evoked an increase in ODC at all ages and the low dose exacerbated the effects of hypoxia at 8 days of age. Prenatal nicotine exposure caused a transient inhibition of cardiac DNA synthesis but did not produce evidence of cell damage (ODC, protein synthesis markers) by itself, nor did it alter the effect of a subsequent postnatal exposure to hypoxia. These results suggest that cardiac cell damage could emerge as a consequence of concurrent, repeated exposures to nicotine and hypoxia. Such effects could contribute to the elevated incidence of perinatal morbidity/mortality and Sudden Infant Death Syndrome associated with smoking.     

A positive crossmatch in liver transplantation--no effect or inappropriate analysis? A prospective study

Secondary brain damage after transient cerebral hypoxia-ischemia (HI) is caused by a cascade of cellular events. In this study, complementary methods of magnetic resonance imaging and histochemistry were used to investigate the formation of cytotoxic and vasogenic edema during secondary brain damage induced by transient HI in 7-d-old rats. To elicit injury, 21 rats underwent right common carotid artery ligation followed by 1.5 h of 8% O2 exposure. Sequential apparent diffusion coefficient (ADC) and transversal relaxation time (T2) weighted magnetic resonance imaging were recorded for up to 3 d in 13 7-d-old rats. Eight animals were killed at various intervals between the end of HI and 21 h of recovery to perform histochemical assays using neuronal and astrocytic markers. Changes of the ADC revealed a biphasic function for the evolution of cytotoxic edema during the recovery period. At the end of HI, the ADC in the ipsilateral cortex was significantly decreased. Upon reoxygenation, it returned transiently to normal followed by a secondary, although less pronounced, decline after 8-48 h. After this, the ADC rose steadily. From 8 h of recovery, the proportion of vasogenic edema steadily increased as indicated by the T2 prolongation. At 21 h, the majority of glial cells showed immunoreactivity for glial fibrillary acidic protein and were of larger size, whereas the neurons were apoptotic. These results indicate that the delayed cerebral injury is accompanied by late glial swelling in conjunction with an enlarged interstitial space due to cell damage.     

Effects of neonatal exposure to anti-nerve growth factor on the number and size distribution of trigeminal neurones projecting to the molar dental pulp in rats

The primary cause of neurologic impairment in newborn infants is hypoxic-ischemic injury. Studies of the mechanisms involved in the damaging effects of hypoxia-ischemia and reperfusion in brain tissue indicate significant contributions from reactive oxygen species, with the loss of homeostatic control of intracellular iron an important determinant of oxidant-mediated damage. We investigated the effects of cerebral hypoxia-ischemia and reperfusion on the redistribution of nonheme iron in newborn piglets. Anesthetized newborn piglets were subjected to reductions in cerebral blood flow by phlebotomy and cervical cuff compression. Control animals were sham-operated. Subcellular fractions were isolated from brain tissue homogenates by differential centrifugation, and nonheme iron contents of these fractions were measured with ferene-S. Iron contents in the homogenates were not altered. However, iron contents of the microsomal fractions of animals subjected to 30 minutes of hypoxia-ischemia increased from 0.517 +/- 0.053 to 0.930 +/- 0.061 nmol/mg protein (p < 0.01); 120 minutes of reperfusion caused no further changes. This translocation of iron may be linked to oxidative alterations of brain proteins, which we investigated by detection of dinitrophenylhydrazine-derivitized protein carbonyls, which are characteristic of iron-catalyzed oxidation reactions.