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 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 induced hyperglycemia on brain cell membrane function and energy metabolism during the early phase of experimental meningitis in newborn piglets

This study was done to elucidate the mechanism of hypoglycorrhachia and elevated lactate concentrations leading to neuronal dysfunction in neonatal meningitis, and to determine the effects of induced hyperglycemia on these disturbances. Thirty-eight newborn piglets were divided into three groups: 12 in the control group (CG), 12 in the normoglycemic meningitis group (NG), and 14 in the hyperglycemic meningitis group (HG). Meningitis was induced by intracisternal injection of 108 cfu of Escherichia coli. Hyperglycemia (blood glucose 300–400 mg dl⁻¹) was induced and maintained for 60 min before induction of meningitis and throughout the experiment using modified glucose clamp technique. CSF-to-blood glucose ratio decreased significantly in NG. In HG, baseline CSF-to-blood glucose ratio was lower than two other groups, but increased at 1 h after induction of meningitis. CSF lactate concentration was increased progressively in both meningitis groups, and positively correlated with CSF leukocyte numbers (r=0.41, p<0.001) and TNF-α level (r=0.43, p<0.001). Brain glucose concentration was significantly increased in HG and showed inverse correlation with CSF leukocyte numbers (r=−0.59, p<0.01). Brain lactate concentration was not significantly different among three groups and positively correlated with the CSF TNF-α level (r=0.51, p<0.05). Lipid peroxidation products were increased in NG. Na⁺,K⁺-ATPase activity, ATP/PCr concentrations were not different among three groups. Increased intracranial pressure, CSF pleocytosis (214±59 vs. 437±214/mm³, p<0.02) and increased lipid peroxidation products observed in NG were reduced in HG. These results suggest that hypoglycorrhachia and elevated lactate concentration in the CSF during meningitis originates primarily from the increased anaerobic glycolysis in the subarachnoid space, induced by TNF-α and leukocytes. Induced hyperglycemia attenuates the inflammatory responses of meningitis and might be beneficial by providing an increased glucose delivery to meet its increased demand in meningitis.     

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.     

Effect of hypothermia on bilirubin-induced alterations in brain cell membrane function and energy metabolism in newborn piglets

The aim of this study was to evaluate the effects of hypothermia on bilirubin-induced alterations in brain cell membrane function and energy metabolism in the developing brain. Thirty-seven newborn piglets were divided randomly into four groups: normothermic control (NC, n=9); hypothermic control (HC, n=7); normothermic bilirubin infusion (NB, n=11); and hypothermic bilirubin infusion (HB, n=10) groups. In bilirubin infusion groups (NB and HB), a loading dose of bilirubin (35 mg/kg) was given over 5 min, followed by a continuous infusion (25 mg/kg/h) for 4 h. The control groups (NC, HC) received a bilirubin-free buffer solution. Sulfadimethoxine was administered to animals in all experimental groups. Rectal temperature was maintained between 38.0 and 39.0 degrees C in normothermic groups, and between 34.0 and 35.0 degrees C in hypothermic groups for 4 h after the start of bilirubin infusion. The final blood and brain bilirubin concentrations in the bilirubin infusion groups (NB and HB) were not significantly different. Decreased cerebral cortical cell membrane Na(+),K(+)-ATPase activity and increased lipid peroxidation products observed in the NB group, indicative of bilirubin-induced brain damage, were significantly attenuated in the HB group. Hypothermia also significantly improved the bilirubin-induced reduction in brain ATP and phosphocreatine levels and increase in blood and brain lactate levels. In summary, hypothermia significantly attenuated the bilirubin-induced alterations in brain cell membrane function and energy metabolism in the newborn piglet. These findings suggest the possibility that hypothermia could be a good neuroprotective therapeutic modality in neonatal bilirubin encephalopathy.     

Mild cerebral hypoxia–ischemia produces a sub-acute transient inflammatory response that is less selective and prolonged after a substantial insult

Cerebral ischemia initiates various injurious processes including neuroinflammatory responses such as activation of microglia and increases in cytokine and nitric oxide release. Evidence primarily from in vitro studies, indicates that neuroinflammatory effects can be either beneficial or harmful, possibly related to stimulus strength. We investigated using in vivo models, the effect of a mild or substantial cerebral hypoxia–ischemia on: cerebral microglial/macrophage activation (ED1), pro-inflammatory cytokines (tumor necrosis factor-alpha), nitrosative stress (nitrotyrosine) and permanent brain damage. A mild insult produced a transient (1–2 days post) increase in activated microglia/macrophages within subcortical white and not gray matter but transiently increased cytokine or nitrotyrosine expression in cortex and not white matter. There was also prolonged scattered cell death in cortex and white matter over weeks along with loss of myelin/axons and cortical atrophy at 4 weeks post-insult. In contrast, a substantial insult produced white and gray matter necrosis, cyst formation and atrophy, along with increases in tumor necrosis factor and nitrotyrosine staining within both white and gray matter starting at 1–2 days post-insult. Microglial/macrophage staining was increased starting at 1-week post a substantial insult and remained elevated for weeks thereafter.Thus, a transient neuroinflammatory response occurs following a mild insult whereas prolonged scattered cell death occurs for weeks, particularly in white matter. Insult severity also affects the progression of the neuroinflammatory response, which is prolonged after a substantial insult. Effective therapy will need to be customized for insult severity and timing; and, monitoring the injury processes with imaging or biomarkers may help guide treatment.     

Early appearance of activated matrix metalloproteinase-9 and blood–brain barrier disruption in mice after focal cerebral ischemia and reperfusion

Blood–brain barrier (BBB) disruption is thought to play a critical role in the pathophysiology of ischemia/reperfusion. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that can degrade all the components of the extracellular matrix when they are activated. Gelatinase A (MMP-2) and gelatinase B (MMP-9) are able to digest the endothelial basal lamina, which plays a major role in maintaining BBB impermeability. The present study examined the expression and activation of gelatinases before and after transient focal cerebral ischemia (FCI) in mice. Adult male CD1 mice were subjected to 60 min FCI and reperfusion. Zymography was performed from 1 to 23 h after reperfusion using the protein extraction method with detergent extraction and affinity-support purification. MMP-9 expression was also examined by both immunohistochemistry and Western blot analysis, and tissue inhibitors to metalloproteinase-1 was measured by reverse zymography. The BBB opening was evaluated by the Evans blue extravasation method. The 88-kDa activated MMP-9 was absent from the control specimens, while it appeared 3 h after transient ischemia by zymography. At this time point, the BBB permeability alteration was detected in the ischemic brain. Both pro-MMP-9 (96 kDa) and pro-MMP-2 (72 kDa) were seen in the control specimens, and were markedly increased after FCI. A significant induction of MMP-9 was confirmed by both immunohistochemistry and Western blot analysis. The early appearance of activated MMP-9, associated with evidence of BBB permeability alteration, suggests that activation of MMP-9 contributes to the early formation of vasogenic edema after transient FCI.     

Inhibition of transient receptor potential vanilloid 4 decreases the expressions of caveolin‐1 and caveolin‐2 after focal cerebral ischemia and reperfusion in rats

This study aimed to investigate the effects of transient receptor potential vanilloid 4 (TRPV4) inhibition on blood–brain barrier (BBB) integrity and the expressions of caveolae structural proteins caveolin‐1 and caveolin‐2 in rats with focal cerebral ischemia and reperfusion. BBB permeability was assessed by Evans blue extravasation. The mRNA and protein expressions of caveolin‐1 and caveolin‐2 were determined by RT‐PCR, Western blot and immunohistochemistry assays. We found that BBB permeability significantly increased and reaches its peak at 72 h of reperfusion in cerebral ischemia‐reperfusion rats and is able to be ameliorated by administration of HC‐067047, an antagonist of TRPV4. Additionally, it shows a significant upregulation of caveolin‐1 and caveolin‐2 expression in cerebral microvessels of ischemic tissue. However, treatment with HC‐067047 was shown to downregulate caveolin‐1 and caveolin‐2 expression during cerebral ischemia‐reperfusion. This study demonstrates that inhibition of TRPV4 ameliorates BBB leakage induced by ischemia‐reperfusion injury through the downregulation of caveolin‐1 and caveolin‐2.