Effects of fasting and insulin-induced hypoglycemia on brain cell membrane function and energy metabolism during hypoxia-ischemia in newborn piglets.

This study was done to determine the effects of 12 h fasting-induced mild hypoglycemia (blood glucose 60 mg/dl) and insulin-induced moderate hypoglycemia (blood glucose 35 mg/dl) on brain cell membrane function and energy metabolism during hypoxia-ischemia in newborn piglets. Sixty-three ventilated piglets were divided into six groups; normoglycemic control (NC, n=8), fasting-induced mildly hypoglycemic control (FC, n=10), insulin-induced moderately hypoglycemic control (IC, n=10), normoglycemic/hypoxic-ischemic (NH, n=11), fasting-induced mildly hypoglycemic/hypoxic-ischemic (FH, n=12) and insulin-induced moderately hypoglycemic/hypoxic-ischemic (IH, n=12) group. Cerebral hypoxia-ischemia was induced by occlusion of bilateral common carotid arteries and simultaneous breathing with 8% oxygen for 30 min. The brain lactate level was elevated in NH group and this change was attenuated in FH and IH groups. The extent of cerebral lactic acidosis during hypoxic-ischemic insult showed significant positive correlation with blood glucose level (r=0.55, p<0.001). Cerebral Na+, K+-ATPase activity and concentrations of high-energy phosphate compounds were reduced in NH group and these changes were not ameliorated in FH or IH group. Cortical levels of conjugated dienes, measured as an index of lipid peroxidation of brain cell membrane, were significantly elevated in NH, FH and IH groups compared with NC, FC and IC groups and these increases were more profound in FH and IH with respect to NH. Blood glucose concentration showed significant inverse correlation with levels of conjugated dienes (r=-0.35, p<0.05). These findings suggest that, unlike in adults, mild or moderate hypoglycemia, regardless of methods of induction such as fasting or insulin-induced, during cerebral hypoxia-ischemia is not beneficial and may even be harmful in neonates.     

Effect of hyperglycemia on brain cell membrane function and energy metabolism during hypoxia–ischemia in newborn piglets

The purpose of this study was to test the hypothesis that hyperglycemia ameliorates changes in brain cell membrane function and preserves cerebral high energy phosphates during hypoxia–ischemia in newborn piglets. A total of 42 ventilated piglets were divided into 4 groups, normoglycemic/normoxic(group 1, n=9), hyperglycemic/normoxic(group 2, n=8), normoglycemic/hypoxic–ischemic(group 3, n=13) and hyperglycemic/hypoxic–ischemic(group 4, n=12) group. Cerebral hypoxia–ischemia was induced by occlusion of bilateral common carotid arteries and simultaneous breathing with 8% oxygen for 30 min. Hyperglycemia (blood glucose 350–400 mg/dl) was maintained for 90 min before and throughout hypoxia–ischemia using modified glucose clamp technique. Changes in cytochrome aa3 were continuously monitored using near infrared spectroscopy. Blood and CSF glucose and lactate were monitored. Na⁺, K⁺-ATPase activity, lipid peroxidation products (conjugated dienes), tissue high energy phosphates (ATP and phosphocreatine) levels and brain glucose and lactate levels were determined biochemically in the cerebral cortex. During hypoxia–ischemia, glucose levels in blood and CSF were significantly elevated in hyperglycemic/hypoxic–ischemic group compared with normoglycemic/hypoxic–ischemic group, but lactate levels in blood and CSF were not different between two groups. At the end of hypoxia–ischemia of group 3 and 4, Cyt aa3, Na⁺, K⁺-ATPase activity, ATP and phosphocreatine values in brain were significantly decreased compared with normoxic groups 1 and 2, but were not different between groups 3 and 4. Levels of conjugated dienes and brain lactate were significantly increased in groups 3 and 4 compared with groups 1 and 2, and were significantly elevated in group 4 than in group 3 (0.30±0.11 vs. 0.09±0.02 μmol g⁻¹ protein, 26.4±7.6 vs. 13.1±2.6 mmol kg⁻¹, p<0.05). These findings suggest that hyperglycemia does not reduce the changes in brain cell membrane function and does not preserve cerebral high energy phosphates during hypoxia–ischemia in newborn piglets. We speculate that hyperglycemia may be harmful during hypoxia–ischemia due to increased levels of lipid peroxidation in newborn piglet.     

Effects of hyperglycemia or hypoglycemia on brain cell membrane function and energy metabolism during the immediate reoxygenation–reperfusion period after acute transient global hypoxia–ischemia in the newborn piglet

This study was done to determine the effects of hyperglycemia or hypoglycemia on brain cell membrane function and energy metabolism during the immediate reoxygenation–reperfusion period after hypoxia–ischemia (HI). Forty-five newborn piglets were divided randomly into four experimental groups: normoxia control (NC, n=9); HI/reoxygenation–reperfusion (RR) control (HC, n=11); HI/RR hyperglycemia (HE, n=12); and HI/RR hypoglycemia (HO, n=13) group. Animals were subjected to transient HI for 30 min followed by 2 h of RR. Cerebral HI was induced by temporary but complete occlusion of bilateral common carotid arteries with surgical clips and simultaneous breathing with 8% oxygen. Glucose was unregulated in HC group, and controlled by modified glucose clamp technique immediately after HI in HE (350 mg/dl) and HO (50 mg/dl) groups. During HI, heart rate, base deficit, glucose and lactate level in the blood and cerebrospinal fluid increased, and arterial pH, oxygen saturation and blood pressure decreased significantly in HC, HE and HO groups. During RR, these abnormalities returned to normal values, but lactic acidosis persisted especially in HO group. Cerebral Na⁺,K⁺-ATPase activity decreased, and lipid peroxidation products increased significantly in HC group than in NC group, and these abnormalities were significantly aggravated in HE, but not in HO, group. Brain ATP and phosphocreatine levels in HE group were significantly reduced compared to the corresponding values in NC, HC and HO groups. In summary, hyperglycemia, but not hypoglycemia immediately after HI interfered with the recovery of brain cell membrane function and energy metabolism. These findings suggest that post-hypoxic–ischemic hyperglycemia is not beneficial and might even be harmful in neonatal hypoxic–ischemic encephalopathy.     

Mild hyperglycemia and insulin treatment in experimental cerebral ischemia in rats

We investigated the effects of mild hyperglycemia and insulin treatment on the metabolism of the ischemic brain in spontaneously hypertensive rats with acute hyperglycemia (n = 9), acute hyperglycemia treated with insulin during ischemia (n = 10), and normoglycemia (n = 10). Cerebral blood flow was measured by the H₂ clearance method. Cerebral ischemia induced occlusion of the bilateral carotid arteries. Cerebral glycolytic metabolites were measured enzymatically. Blood glucose levels were significantly higher in hyperglycemic animals (11.8 to 13.7 mM/I) than in normoglycemic animals (6.0 mM/I). At 60 min of ischemia, the blood flow to the parietal cortex was decreased to 3% of the resting value in all groups. Blood glucose levels at 60 min of ischemia in the hyperglycemic rats were 1.9–3 times higher than the treated hyperglycemic rats and normoglycemic rats. Glucose concentrations were significantly and positively correlated with the ATP level (p < .0001) but not with the lactate levels in the ischemic brain. Our results suggest that mild hyperglycemia may preserve glucose metabolism in the presence of ischemic insult.     

Interactions among glucose, lactate and adenosine regulate energy substrate utilization in hippocampal cultures

Glucose is the major energy source during normal adult brain activity. However, it appears that glial-derived lactate is preferred as an energy substrate by neurons following hypoxia–ischemia. We examined factors influencing this switch in energetic bias from glucose to lactate in cultured hippocampal neurons, focusing on the effects of the physiological changes in lactate, glucose and adenosine concentrations seen during hypoxia–ischemia. We show that with typical basal concentrations of lactate and glucose, lactate had no effect on glucose uptake. However, at the concentrations of these metabolites found after hypoxia–ischemia, lactate inhibited glucose uptake. Reciprocally, glucose had no effect on lactate utilization regardless of glucose and lactate concentrations. Furthermore, we find that under hypoglycemic conditions adenosine had a small, but significant, inhibitory effect on glucose uptake. Additionally, adenosine increased lactate utilization. Thus, the relative concentrations of glucose, lactate and adenosine, which are indicative of the energy status of the hippocampus, influence which energy substrates are used. These results support the idea that after hypoxia–ischemia, neurons are biased in the direction of lactate rather than glucose utilization and this is accomplished through a number of regulatory steps.     

Diffusion- andT2-Weighted Increases in Magnetic Resonance Images of Immature Brain during Hypoxia–Ischemia: Transient Reversal Posthypoxia

Hypoxic–ischemic changes in brain are detected earlier with diffusion-weighted (DW) than withT₂-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 orT₂-weighted images. By the second hour of hypoxia–ischemia there were marked areas of increased intensity inboththeT₂and the DW images, with cortex and striatum being affected prior to thalamus and hippocampus. The area of DW exceeded that ofT₂hyperintensities. In the first hour after hypoxia–ischemia there was a transient recovery of hyperintensities on bothT₂and DW images. Between 24 and 72 h the hyperintense area on DW images decreased, whereas that onT₂-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.     

The effects of systemic glucose concentration on brain metabolism following repeated brain ischemia

Since systemic glucose concentration is an important determinant of ischemic brain metabolism in neonates, we sought to determine if the systemic glucose concentration influences brain metabolic alterations following repeated partial ischemia. A group of hyperglycemic pigles (n = 12) were compared to a group of modestly hypoglycemic piglets (n = 12) using in vivo²H and ³¹P magnetic resonance spectroscopy to simultaneously measure cerebral blood flow and phosphorylated metabolites before, during and 30 min after two 10-min episodes of ischemia (i.e. Recovery 1 and 2). For both groups, β-ATP levels at Recovery 1 and 2 were lower than Control (91 ± 11and83 ± 15% of Control, respectively for both groups combined, P = 0.002 vs Control). Inorganic phosphorus was elevated in hyperglycemic piglets at Recovery 1 and 2 (117 ± 15and118 ± 10% of Control). In contrast, in modestly hypoglycemic piglets inorganic phosphorus progressively rose from Recovery 1 (131 ± 24% of Control) to Recovery 2 (149 ± 37% of Control), and differed from the hyperglycemic group (P = 0.02). These changes did not correlate with post-ischemic cerebral blood flow, cerebral O₂ delivery or cerebral glucose delivery. In both groups phosphocreatine and intracellular pH returned to Control values during Recovery 1 and 2. The progressive increase in inorganic phosphorus post-ischemia in hypoglycemic piglets suggests that modest hypoglycemia during and following repeated partial ischemia adversely affects immediate brain metabolic recovery.     

Clinical restitution following cerebral ischemia in hypo-, normo- and hyperglycemic rats

Rats with different levels of blood glucose concentration were exposed to 10 min of complete brain ischemia achieved by compression of neck vessels by a pneumatic cuff. All normoglycemic rats survived the ischemic period and made the best clinical recovery. Hyperglycemic rats died within 12 h. Seizure activity was observed in all animals in this group. Three of eight hypoglycemic rats died between 3 and 16 days. The clinical recovery was less complete than in the control group. Thus, recovery from cerebral ischemia depends upon preischemic blood glucose concentration. Hyper- and hypoglycemia hamper the clinical recovery after transient cerebral ischemia.     

Effect of ketamine on hypoxic–ischemic brain damage in newborn rats

The present study tests the hypothesis that ketamine, a dissociative anesthetic known to be a non-competitive antagonist of the NMDA receptor, will attenuate hypoxic–ischemic damage in neonatal rat brain. Studies were performed in 7-day-old rat pups which were divided into four groups. Animals of the first group, neither ligated nor exposed to hypoxia, served as controls. The second group was exposed to hypoxic–ischemic conditions and sacrificed immediately afterwards. Animals of the third and fourth groups were treated either with saline or ketamine (20 mg/kg, i.p.) in four doses following hypoxia. Hypoxic–ischemic injury to the left cerebral hemisphere was induced by ligation of the left common carotid artery followed by 1 h of hypoxia with 8% oxygen. Measurements of high energy phosphates (ATP and phosphocreatine) and amino acids (glutamate and glutamine) and neuropathological evaluation of the hippocampal formation were used to assess the effects of hypoxia–ischemia. The combination of common carotid artery ligation and exposure to an hypoxic environment caused major alterations in the ipsilateral hemisphere. In contrast, minor alterations in amino acid concentrations were observed after the end of hypoxia in the contralateral hemisphere. These alterations were restored during the early recovery period. Post-treatment with ketamine was associated with partial restoration of energy stores and amino acid content of the left cerebral hemisphere. Limited attenuation of the damage to the hippocampal formation as demonstrated by a reduction in the number of damaged neurons was also observed. These findings demonstrate that systemically administered ketamine after hypoxia offers partial protection to the newborn rat brain against hypoxic–ischemic injury.     

Neuroprotective effect of human placental extract on hypoxic–ischemic brain injury in neonatal rats

We investigated the neuroprotective effects of human placental extracts (HPE) and the effects of HPE on recovery of cognitive and behavioral function on hypoxic–ischemic brain injury in the newborn rat. The right common carotid arteries of 7-day-old rats were coagulated, and rats were then exposed to 8% oxygen. Immediately before and again at three times after the hypoxia–ischemia (pre-treatment group), and immediately after and three times again after hypoxia–ischemia (post-treatment group), the rats were intraperitoneally injected with HPE (0.1, 0.25, or 0.5 mL/10 g/dose). No-treatment rats received saline only. On postnatal day 12, brains were removed and gross morphological damage was evaluated. To quantify the severity of brain injury, bilateral cross-sectional areas of the anterior commissural and posterior hippocampal levels were analyzed with NIH Image. Assessments of the open field activity levels at 2, 4, 6 and 8 week and, the Morris water maze test at 8 weeks after hypoxia–ischemia were carried out according to standard methods. HPE pre-treatment decreased the incidence of liquefactive cerebral infarction, at an optimally neuroprotective dose of 0.5 mL/10 g/dose (P < 0.05). In the Morris water maze test, the group injected with HPE at 0.5 mL/10 g/dose concentration showed shorter escape latencies than the no-treatment group (P < 0.05). These findings support a protective effect of the HPE treatment on neuronal integrity and cognitive function following hypoxic–ischemic brain injury. Injected at an appropriate dose prior to exposure, HPE may significantly reduce or prevent hypoxic–ischemic injury in the immature brain.     

Cerebral mitochondrial respiration in diabetic and chronically hypoglycemic rats

The respiratory function of cerebral mitochondria harvested from genetically diabetic (BB/W) and streptozotocin-diabetic rats deprived of insulin for 3-4 weeks was found to be unchanged from control values. Furthermore, insulin-deprived BB/W rats subjected to 30 min of insulin-induced hypoglycemic coma demonstrated a normal mitochondrial respiration following a 60 min period of glucose restitution, a finding consistent with earlier results in non-diabetic rats. However, in rats exposed to 1 week of moderate hypoglycemia (plasma glucose = 3.0 mumol.ml-1), both state 3 respiration and the respiratory control ratio (RCR) were reduced from control. In fact, when the chronic hypoglycemia was imposed following a 3-4 week period of diabetic hyperglycemia, the state 3 rate and RCR were found to be reduced to a greater degree than in chronically hypoglycemic, non-diabetic, previously normoglycemic rats. Finally, when 1 week of moderate hypoglycemia preceded a 30 min period of insulin-induced hypoglycemic coma, a disturbed pattern of mitochondrial respiration (i.e. increased state 4, decreased RCR) was found at 60 min of recovery following coma. These results indicate that chronic increases in glucose (and insulin deprivation) have no effect on cerebral mitochondrial respiratory function, whereas prolonged, albeit moderate, reductions in cerebral glucose supply result in perturbations in mitochondrial respiration. These results demonstrate the importance of an adequate glucose supply for normal mitochondrial activity.     

Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in perinatal asphyxia

Matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) play important roles in the function of the blood–brain-barrier (BBB). We investigated the roles of MMP-9 and TIMP-1 in the pathogenesis of hypoxic–ischemic encephalopathy following perinatal asphyxia. Serum concentrations of MMP-9 and TIMP-1 were determined by ELISA in 12 neonates with perinatal asphyxia and 15 controls on the birth day and the next day. Serum MMP-9 concentrations in asphyxiated neonates with neurological sequelae (n = 5) were significantly higher than concentration in asphyxiated neonates without sequelae (n = 7) and controls on birth day (p = 0.003 and p < 0.001, respectively). The ratios of serum MMP-9/TIMP-1 on birth day in asphyxiated neonates with neurological sequelae were significantly higher than those in asphyxiated neonates without sequelae (p = 0.048). There were no significant differences in the serum MMP-9 concentrations or the ratios of MMP-9/TIMP-1 between asphyxiated neonates with and without neurological sequelae on the day after birth. Our preliminary study suggests that serum MMP-9 levels on birth day are important for predicting neurological prognosis of neonates with asphyxia.     

Temporal characteristics of the protective effect of aminoguanidine on cerebral ischemic damage

We investigated the temporal profile of the reduction in focal cerebral ischemic damage exerted by aminoguanidine (AG), an inhibitor of inducible nitric oxide synthase (iNOS). In anesthetized spontaneously hypertensive rats, the middle cerebral artery (MCA) was occluded distal to the origin of the lenticulostriate arteries. Rats were treated with vehicle (saline) or AG (100 mg kg⁻¹, i.p.) immediately after MCA occlusion and, thereafter, two times per day. Rats were sacrificed 1 (n=7), 2 (n=8), 3 (n=6) or 4 days (n=5) after MCA occlusion. Injury volume (mm³) was determined in thionin-stained sections using an image analyzer. Volumes were corrected for ischemic swelling. Administration of AG up to 2 days after MCA occlusion did not reduce cerebral ischemic damage (p>0.05 from vehicle; t-test). Treatment for a longer period decreased injury volume, the reduction averaging 21±5% at 3 days (p<0.05) and 30±9% at 4 days (p<0.05). Aminoguanidine did not affect ischemic brain swelling (p>0.05). Administration of AG did not substantially modify arterial pressure, arterial blood gases, pH, hematocrit, plasma glucose and rectal temperature. We conclude that the protective effect of AG is time-dependent and occurs only when the drug is administered for longer than 2 days, starting after induction of ischemia. Because iNOS enzymatic activity develops more than 24 h after MCA occlusion [C. Iadecola, X. Xu, F. Zhang, E.E. El-Fakahany, M.E. Ross, Marked induction of calcium-independent nitric oxide synthase activity after focal cerebral ischemia, J. Cereb. Blood Flow. Metab. 14 (1995) 52–59; C. Iadecola, F. Zhang, X. Xu, R. Casey, M.E. Ross, Inducible nitric oxide synthase gene expression in brain following cerebral ischemia, J. Cereb. Blood Flow Metab. 15 (1995) 378–384.], the data support the hypothesis that the protective effect of AG is mediated by inhibition of iNOS in the post-ischemic brain.     

Focal ischemia of the rat brain,with special reference to the influence of plasma glucose concentration

Summary Focal cerebral ischemia was induced by occlusion of the right middle cerebral artery in hypoglycemic, normoglycemic, as well as in acute and chronic diabetic rats. The brain damage was studied after 4 days. The volume of infarction was decreased in hypoglycemia (29±19 mm³ (mean±SD) versus 58±35 mm³,P<0.0046), unaltered in acute diabetes (61±45 mm³), and increased in chronic diabetes (91±22 mm³,P<0.0463). The cortex adjacent to the infarct showed selective neuronal injury affecting the cortical layers 2 and 3. The damage was enhanced by hypoglycemia and prevented in most of the diabetic animals. The findings indicate that different mechanisms cause infarction and selective neuronal injury outside infarcts, but that both are influenced by the plasma glucose concentration.     

The lumped constant of the deoxyglucose method in hypoglycemia: effects of moderate hypoglycemia on local cerebral glucose utilization in the rat

The applicability of the [14C]deoxyglucose method for measuring local cerebral glucose utilization (lCMRglc) has been extended for use in hypoglycemia by determination of the values of the lumped constant to be used in rats with plasma glucose concentrations ranging from approximately 2 to 6 mM. Lumped constant values were higher in hypoglycemia and declined from a value of 1.2 at the lowest arterial plasma glucose level (1.9 mM) to about 0.48 in normoglycemia. The distribution of glucose, and therefore also of the lumped constant, was found to remain relatively uniform throughout the brain at the lowest plasma glucose levels studied. lCMRglc in moderate, insulin-induced hypoglycemia (mean arterial plasma glucose concentration +/- SD of 2.4 +/- 0.3 mM) was determined with the appropriate lumped constant corresponding to the animal's plasma glucose concentration and compared with the results obtained in six normoglycemic rats. The weighted average rate of glucose utilization for the brain as a whole was significantly depressed by 14% in the hypoglycemic animals, i.e., 61 mumols/100 g/min in hypoglycemia compared to 71 mumols/100 g/min in the normoglycemic controls (p less than 0.05). lCMRglc was lower in 47 of 49 structures examined but statistically significantly below the rate in normoglycemic rats in only six structures (p less than 0.05) by multiple comparison statistics. Regions within the brainstem were most prominently affected. The greatest reductions, statistically significant or not, occurred in structures in which glucose utilization is normally high, suggesting that glucose delivery and transport to the tissue became rate-limiting first in those structures with the greatest metabolic demands for glucose.     

Late measures of microstructural alterations in severe neonatal hypoxic–ischemic encephalopathy by MR diffusion tensor imaging

Neonatal hypoxic–ischemic encephalopathy is a major cause of brain damage in infants, and is associated with periventricular white matter injury and chronic neurological dysfunctions. However, the mechanisms of the chronic white matter injury and reorganization are still unclear. In this study, in vivo diffusion tensor imaging (DTI) was employed to evaluate the late changes of white matter microstructural integrity in the rat brains at 10 weeks after severe neonatal hypoxic–ischemic insults at postnatal day 7. In the fractional anisotropy directionality map, qualitative evaluation showed that a dorsoventrally oriented fiber bundle extended from the corpus callosum into the cyst in the anterior brain, whilst the posterior peri-infarct areas had similar fiber orientations as the contralateral internal capsule, optic tract and fimbria of hippocampus. Compared to the contralateral hemisphere, significantly higher fractional anisotropy, axial diffusivity and diffusion trace value were observed quantitatively in the distal end of the extended fiber bundle connecting the anterior and posterior white matters rostrocaudally. A significantly lower fractional anisotropy but higher axial and radial diffusivities and trace were also found in the ipsilateral corpus callosum, proximal external capsule and anterior commissure, while slightly lower fractional anisotropy and axial diffusivity were noticed in the ipsilateral internal capsule and optic nerve. It was suggested that increased fractional anisotropy, axial diffusivity and trace characterize white matter reorganization in chronic neonatal hypoxic–ischemic insults, whereas reduction in fractional anisotropy appears to characterize two types of white matter lesions, with significantly higher axial and radial diffusivities and trace being primary and slightly lower axial diffusivity being secondary. Combined with fractional anisotropy directionality map, in vivo DTI provides important indices to differentiate the chronic effects of severe neonatal hypoxic–ischemic injury and recovery globally, quantitatively and non-invasively.     

Microtubule-associated protein 2 (MAP2)—a promising approach to diagnosis of forensic types of hypoxia-ischemia

The loss of neuronal immunoreactivity of the cytoskeletal microtubule-associated protein 2 (MAP2) is known to be a marker of—at least—transient functional failure of neurons following ischemia. Because there are no specific neuropathological findings in forensic types of acute hypoxia-ischemia, detection of this relevant cause of death is often complicated and a reliable ischemic biomarker would be of great importance. We therefore investigated the neuronal immunoreactivity of MAP2 in human cases of forensic significance. A control group (n=27) was compared to a group of cases of hypoxia-ischemia (n=45), comprising death due to hanging (n=19), drowning (n=14) and carbon monoxide (CO) poisoning (n=12). Using immunohistochemical staining, the percentage of MAP2-positive neurons in the hippocampus (areas CA1–CA4) and frontal cortex (layers II–VI) was evaluated and compared. The hypoxia-ischemia group showed decreased MAP2 immunostaining in the hippocampal areas CA2–CA4 (P<0.05) and in cortical layers II–VI (P<0.001) compared to controls. Most vulnerable regions seem to be the hippocampal CA4 area and cortical layers III–V. Within the hypoxia-ischemia group, death due to CO poisoning was characterized by the lowest MAP2 immunoreactivity. The hypoxic-ischemic groups differ from controls by a distinct decrease of MAP2 immunostaining. Thus, the loss of MAP2 immunoreactivity may support the diagnosis of neuronal injury in forensic types of hypoxia-ischemia, although investigations on postmortem tissue must be interpreted cautiously.     

The influence of preischemic hyperglycemia on acute changes in the apparent diffusion coefficient of brain water following global ischemia in rats

We report the effect of increased plasma glucose levels on changes in the apparent diffusion coefficient of brain water (ADCw) during the first few minutes of global ischemia in rats. Brain ADCw values were acquired every 15 s using a diffusion-weighted line-scan MR pulse sequence. Preischemic hyperglycemia was achieved by infusion of 50% dextrose (i.v.) prior to KCl-induced cardiac arrest global ischemia. Analysis based on single voxels (3.4 μl) in brain demonstrated significant differences in the time course of ADCw decline between normoglycemic (n=8) and hyperglycemic (n=6) groups. Mean data from the hyperglycemic group indicated a biphasic decline of ADCw that was characterized by an initial rapid drop followed by a plateau of approximately 1 min before gradually declining and leveling off to its minimum value. In the normoglycemic group, ADCw declined to the same value as in the hyperglycemic group, but without a notable plateau. In the cerebral cortex, the times to maximal and half maximal ADCw drop following global ischemia in the hyperglycemic group were 3.96 and 2.26 min respectively. Corresponding time intervals for the normoglycemic group were 1.86 and 1.14 min, respectively. The time course for changes in ADCw demonstrated here is significantly different than that for anoxic depolarization reported under similar experimental conditions and suggests that events other than the complete loss of membrane ionic homeostasis and subsequent cell swelling may be involved in the initial decline of ADCw in global cerebral ischemia.     

Hyperglycemic but not normoglycemic global ischemia induces marked early intraneuronal expression of β-amyloid precursor protein

Preischemic hyperglycemia is known to accentuate acute ischemic injury to neurons, microglia, and endothelia. In the present study, we used a monoclonal antibody to the N-terminal portion of β-APP to examine how the immunoreactivity of this normal membrane glycoprotein is differentially influenced by transient cerebral ischemia when carried out under normoglycemic vs. hyperglycemic conditions. Anesthetized, physiologically regulated rats received 12.5 min of global forebrain ischemia by bilateral carotid artery occlusions plus systemic hypotension. Hyperglycemia was induced by intraperitoneal dextrose administration prior to ischemia. One or three days later, brains were examined by β-APP immunohistochemistry. Ischemia under hyperglycemic conditions led to the robust, widespread intraneuronal expression of β-APP immunoreactivity in neocortex, hippocampus, thalamus, and striatum of all 11 rats; this was most prominent at 24 h postischemia. Compared to rats with normoglycemic ischemia, numbers of β-APP-immunopositive neurons in the parietal cortex of hyperglycemic rats were increased by 5.9 fold at 24 h, and by 10.6 fold at 3 days postischemia. β-APP-immunopositive neurons in hyperglycemic rats often exhibited striking morphological alterations typical of ischemic necrosis; however, no β-APP immunoreaction was observed in zones of frank infarction. Brains of normoglycemic rats (n=11), by contrast, showed only weak β-APP immunostaining in occasional non-necrotic pyramidal neurons of parietal neocortex; no necrosis was present in thalamus. In sham-operated hyperglycemic rats, β-APP immunostaining of thalamic neurons was somewhat increased at 24 h. Western analysis revealed that the hyperglycemia-induced intraneuronal overexpression of β-APP was not associated with an overall increase in tissue levels. The results of this study demonstrate that transient forebrain ischemia under hyperglycemic conditions leads to the early intraneuronal expression of β-APP within neuronal populations showing a heightened susceptibility to hyperglycemia-induced accentuation of ischemic injury. Our data suggest that β-APP or its metabolites may be involved in the injury process.     

Comorbidity and metabolic context are crucial factors determining neurological sequelae of hypoglycaemia

Aim To determine risk factors for neurological sequelae following hypoglycemia. Method We analysed the neurological outcome in 164 patients (mean age 10y 10mo, SD 5.9) following hypoglycemia due to three diseases with various metabolic contexts, different ages at onset, and combinations with comorbidity (fever/infection, hypoxia/ischemia): glycogen storage disease type I (GSDI) (21 patients, mean age at first hypoglycemic episode 3.8mo, SD 3.5); fatty acid β‐oxidation defects (FAOD) (29 patients, mean age at first hypoglycemic episode 14.8mo, SD 12.6); and hyperinsulinism (HIns) (114 patients, mean age at first hypoglycemic episode 2.3mo, SD 4.7). Results Risk factors of poor neurological outcome were aetiology (p<0.006), comorbidity (p<0.001), and prolonged convulsions (p<0.001). Ordinal logistic regression showed that comorbidity (p<0.001) and status epilepticus (p=0.002) were the main determinants of sequelae. Asymptomatic hypoglycemia did not lead to sequelae, whatever the aetiology. Age was not correlated to sequelae, whatever the aetiology. The highest prevalence of hypoglycemic sequelae was found in FAOD and HIns combined with comorbidity, the lowest in GSDI (p<0.001) in which hypoglycemia is often asymptomatic, associated with increased plasma lactate, and rarely combined with comorbidity. Interpretation Hypoglycemia is severely deleterious for the brain in the context of fever/infection and/or hypoxia/ischemia, and status epilepticus. The metabolic context providing alternative fuels may improve neurological outcome.