Study On The Mechanism Of Brain Injury Induced By Hypoglycemia In Neonatal Rats And The Neuroprotectective Effect Of Pyruvate

Background and objectivesContinuous blood glucose availability is the basic requirement to maintain the function of the brain. The neonate suffers the most risk of hypoglycemia because of the deviations or perturbations in the adaptive responses of energy metabolism in the postnatal period. Hypoglycemia is a common disease of new born infants. Repititive and profound neonatal hypoglycemia can induce irreversible brain injury and result in severe neurologic sequelae, of which the mechanism was not elucidaled by hitherto. Studies on isolated neonatal hypoglycemia are limited in clinical practice, because hypoglycemia is often accompanied by other conditions, making it difficult to separate the effects of hypoglycemia from those of concurrent pathophysiologic states. Moreover no reliable animal model of brain injury induced by neonatal hypoglycemia is available to carry out more researches. So it is very necessary to establish a reliable animal model of brain injury induced by neonatal hypoglycemia. Studies on mature brain injury resulted from severe hypoglycemia indicated that decreased availability of glucose induces energy crisis in the brain that alters electrical activity, results in dysbolism of free fatty acid and amino acid, elevates the glutamate concentrations in brain extracellutar. Hypoglycemic brain injury is the end-result of excitotoicity in central nervous system induced by sustained glutamate receptor activation.Neuronal death resulting from hypoglycemia is the result of a series of events triggered by reduced glucose availability, and the normalization of blood glucose levels does not necessarily block or reverse this cell death process once it has begun. Because of the abnormality of metabolism and the decline of ATP level, hypoglycemia causes several-fold elevations in brain extracellular glutamate concentrations, resulted from the increased generation and the decreased uptake of glutamate, inducing sustained glutamate receptor activation. Sustained action of glutamate on neuronal NMDA (N-methyl-D-asparate) receptors, which concerned with channels of potassium, sodium and calcium, induces a massive influx of calcium ions into brain cells and the loss of ion homeostasis. Furthermore, hypoglycemia also can increase the sensibility of NMDA receptor to the activation of glutamate. The threshold of glutamate activation concentreation declines. The energy dependent revert mechanism of normal ion concentreation gradient across membrane lose efficacy because of the deficiency of ATP and phosphagen. It results intracellular calcium increases, mitochondrial dysfunction, production of reactive oxygen species (ROS), DNA damage, PARP-1(poly (ADP-ribose) polymerase-1) activation. The abnormality of Ca⁺ distribution and the extensive activation of PARP-1 can induce mitochondrial permeability transition (MPT), mitochondrial membrane potential (ΔΨm) disaggregate and the protein which promote apoptosis released, including cytochrome c (cyt c). With the participation of ATP/dATP, cyt c, released into cytoplasm, combines apoptotic protease activating factor-1 (Apaf-1) to form cyt c/Apaf-1oligomer in the ratio of 2:1. The N-terminal of Apaf-1 interacts with the domain of procaspase-9 and form cyt c/Apaf-1/caspase-9 complex. Then the complex activates caspase-3 to switch on apoptosis. And all the inducing factors have been demonstrated that they work through caspase. Caspase-8 and caspase-9 are the initiators that trigger the apotosis pathway, and caspase-3 is the executor. Active caspase-3 has been used as apoptosis marker now.Activated PARP-1 consumes cytosolic oxidized form of nicotinamide-adenine dinucleotide (NAD⁺), and because NAD⁺ is required for glycolysis, hypoglycemia-induced PARP-1 activation may render cells unable to use glucose even when glucose availability is restored. Administration of PARP inhibitors, NAD⁺, or pyruvate,α-ketoglutarate and other substrates, which can be metabolized in the absence of cytosolic NAD⁺, at the termination of hypoglycemia substantially reduced neuronal death in vulnerable brain regions and prevented cognitive impairment. In recent years researchers paid more attention on pyruvate because it is inexpensive, readily available, and innocuous. Many studies were carried out in the models of mature brain injury induced by hypoglycemia and hypoxia. Whether pyruvate can offer a neuroprotectective effect in repetitive and profound neonatal hypoglycemic brain injury still keep unknown.In the present study, we established a reliable animal model of brain injury induced by neonatal hypoglycemia first, then to study the mechanism of hypoglycemic brain injury induced by mitochondrial dysfunction. Fanily, the neuroprotectection of pyruvate to repetitive and profound neonatal hypoglycemic brain injury was investigated by administration of pyruvate at the termination of hypoglycemia and also provide new ideas and theories for treating repetitive and profound neonatal hypoglycemia.Methods:1. To establish a repetitive and profound neonatal hypoglycemic brain injury rat model. Neonatal hypoglycemia was induced by insulin (15 U/kg) hypodermic injection and fasting for 12h on postnatal day 2 (P 2), P 4 and P 6. One hundred and sixty experimental animals were randomly divided into four groups: insulin-treated rats with short hypoglycemia (INS-S, n=40), insulin-treated rats with prolonged hypoglycemia (INS-P, n=40), fasted rats (FAS, n=40) and control rats (CON, n=40). The period of hypoglycemia in INS-S was 1.5h and in INS-P was 2.5h. New born rats in FAS were fasting for 12h. The blood glucose was measured with a One Touch II blood glucose meter. FJB staining was used to quantify cell death and determine cellular localization of neuronal degeneration 2h, 6h, 1d, 3d, 7d and 14d after the last challenge of hypoglycemia. The dead neurons were counted in parasagittal cortex, piriform cortex, cornu ammonis sector 1 (CA1), CA3 and dentate gyrus (DG) of hippocampus, thalamus and hypothalamus within each section, and compared among groups. At 6h after the last challenge of hypoglycemia, CA3 of hippocampus of each group was separated for electron microscopic examination.2. To study the mechanism of brain injury induced by repetitive and profound neonatal hypoglycemia. One hundred and fifty-eight experimental animals were randomly divided into two groups: insulin-treated rats with prolonged hypoglycemia (INS-P, n=79) and control rats (CON, n=79).â‘ To detect the mitochondrialΔΨm. Suspensions of neuron of each group were prepared 6h after the last challenge of hypoglycemia. The suspensions were detected by flow cytometry after Rho 123 stainning.â‘¡To detect the level of cyt c in neuronal endochylema. Tissue of cerebral cortex of each group was removed at 2h, 6h, 1d, 3d, 7d and 14d after the last challenge of hypoglycemia. Neuronal plasmosin was extracted by differential centrifugation in subambient temperature and detected by western bloting.â‘¢To detect activated caspase-3. Immunohistochemistry was performanced to detect activated caspase-3 P20 2h, 6h, 1d, 3d, 7d and 14d after the last challenge of hypoglycemia. Cell phenotype was determined using double-label immunofluorescent staining with caspase-3 P20 and neuronal nuclear protein (NeuN), and the morphous of caspase-3 P20 positive neurons were observed.3. To investigate the neuroprotectection of pyruvate to repetitive and profound neonatal hypoglycemic brain injury. Thirty-six experimental animals were randomly divided into three groups: insulin-treated rats with prolonged hypoglycemia (INS-P, n=12), insulin-treated rats with prolonged hypoglycemia + pyruvate (INS-PP, n=12) and control rats (CON, n=12). Pyruvate was administrated to INS-PP new born rats at the termination of hypoglycemia. FJB staining was used to quantify cell death of each brain region, and compared among groups. Morris water maze test was performed to evaluate rat's ability of spatial learning and memory.Results:1. The establishment of the repetitive and profound neonatal hypoglycemic brain injury rat model. Insulin injection and fasting both could induce consistent hypoglycemia in newborn rats. But on P 2, fasting could induce considerable hypoglycemic events, but on P 4 and P 6, the blood glucose levels were not very low. Newborn Wistar rats are very responsive to insulin. Only single dose insulin given by hypodermic injection can induce consistent hypoglycemia. The blood glucose of rats in INS-S and INS-P was <2.5mmol/l at 0.5h after injection and <1.5 mmol/l at 1.5h, and <1.2 mmol/1 at 2.5 h of rats in INS-P. FJB staining of brains of CON, FAS and INS-S rats did not result in any detectable fluorescence, while FJB+ cells were seen in all brains of INS-P 6h after injection of insulin. FJB+ cells were numerous in all sections of brains of INS-P. The regions with the greatest numbers of FJB+ cells are the parasagittal cortex, piriform cortex, hypothalamus and thalamus, then is DG. There was no time-dependent trend in numbers of FJB+ cells observed in brains collected from 6 h to 7 days after the last hypoglycemic insult (F_3,20=1.3600, p=0.2836). The fluorescence intensity of positive cells decreased from 7d after injection of insulin. The ultrastructural changes in brains of INS-S, INS-P and FAS animals were minimal.2. The mechanism of brain injury induced by repetitive and profound neonatal hypoglycemia.â‘ Changes of the mitochondrialΔΨm. The fluorescence intensity of Rho 123 in suspensions of neuron of INS-P rats was declined significantly (p<0.01). The fluorescence intensity only was 38.36% of that of CON.â‘¡The level of cyt c in neuronal endochylema. The level of cyt c in neuronal plasmosin of the brain following repetitive neonatal hypoglycemia expressed highly at 2h and 6h, decreased at 3d after the third insulin injection.â‘¢To detect activated caspase-3. Sporadic of caspase-3 P20 positive cells, with normal appearance of cell nucleus stained by DAPI, were observed in brains of INS-P rats at 2h after hypoglycemia insult and CON rats at every time point. In the brains collected 6 h or more after hypoglycemic insult of INS-P rats, the number and the fluorescence intensity of caspase-3 P20 positive cells increased at 6h after treatments, reached the peak at 3d and decreased at 7d. The regions with the greatest numbers of caspase-3 P20 positive cells are the parasagittal cortex, piriform cortex, DG of hippocampus, hypothalamus and thalamus. There were only small amounts of caspase-3 P20 positive cells detected in CA1 and CA3. The caspase-3 P20 positive cells co-located with NeuN. At 14d after hypoglycemic insult, many caspase-3 P20 positive cells showed morphological signs of apoptosis.3. The neuroprotectection of pyruvate to repetitive and profound neonatal hypoglycemic brain injury. The numer of FJB positive dead neuron in each brain region of INS-PP rats decreased significantly compared with that of INS-P rats, and no FJB positive cell was detected in brain sections of CON rats. Six weeks after the last challenge of hypoglycemia, in Morris water maze test, rats of INS-P showed a significant impairment in their ability to locate the platform when compared with rats of INS-PP and CON. Performance of the rats of INS-P was significantly better than rats of INS-P (P <0.05) and not significantly different than the CON group.Conclusions:1. Insulin (15 U/kg) hypodermic injections can induce consistent hypoglycemia in newborn rats. New born rat has good sensitiveness and tolerance to insulin injection. Fasting for 12h also can induce mild hypoglycemia in new born rats. 2. Repetitive and profound neonatal hypoglycemia can result in extensive neurodegeneration, and the neurons of cortex, thalamus, hypothalamus and dentate gyrus are more vulnerable to hypoglycemic insult in newborn rats.3. FJB staining is a useful method of marking neuronal degeneration in neonatal rat following hypoglycemic brain damage.4. The procedure of apoptosis induced by mitochondria plaies a key role in the mechanism of brain injury induced by repetitive and profound neonatal hypoglycemia.5. Pyruvate administration can provid a neuroprotectective effect to rat suffered repetitive and profound neonatal hypoglycemia. Pyruvate may be an effective intervention for patients with severe hypoglycemia.