This study was done to determine the effects of hypothermia on brain cell membrane function and energy metabolism after transient hypoxia-ischemia (HI) in the newborn piglet. Cerebral HI was induced by temporarily complete occlusion of bilateral common carotid arteries with surgical clips and simultaneous breathing with 8% oxygen for 30 min, followed by release of carotid occlusion and normoxic ventilation for 4 hr. 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 hr after HI. During HI, heart rate, glucose and lactate level in the blood and cerebrospinal fluid increased, and base excess, pH and blood pressure decreased significantly in both normothermic and hypothermic groups. After HI, these abnormalities returned to normal in normothermic group, but lactic acidosis persisted in hypothermic group. Decreased cerebral Na(+),K(+)- ATPase activity and increased lipid peroxidation products, indicative of HI- induced brain injury, were more profound in hypothermic group than in normothermic group. Brain ATP and phosphocreatine levels were not different between normothermic and hypothermic groups. In summary, hypothermia applied immediately after HI for 4 hr did not improve the recovery of brain cell membrane function and energy metabolism in the newborn piglet.
Animal ; Animals, Newborn ; Brain/cytology/*metabolism/physiology ; Cell Membrane/physiology ; Energy Metabolism ; Glucose/metabolism ; *Hypothermia, Induced ; Hypoxia-Ischemia, Brain/*metabolism/physiopathology/therapy ; Lactic Acid/metabolism ; Na(+)-K(+)-Exchanging ATPase/metabolism ; Swine

OBJECTIVETo investigate the effect of physical training on cerebral structure and spatial learning and memory in neonatal rats submitted to hypoxic-ischemic brain damage (HIBD).

METHODSForty-eight 7-day-old Sprague-Dawley rats were randomly divided into three groups: a group that was subjected to left carotid ligation followed by 2 hrs hypoxic stress (HIBD); a group that received physical training 2 weeks after the HIBD event; a control group that was subjected to a sham-operation without ligation and hypoxic stress. Following four weeks physical training, motor function test and water maze tasks were performed. Bilateral brain weight, cerebral morphology and left hippocampal ultrastructrue of the animals were examined. The expression levels of phosphor calmodulin-dependent protein kinase II (CaMKII) and brain derived neurotrophic factor (BDNF) were determined by immunohistochemistry.

RESULTSCompared with the control group, the motor function and the spatial learning and memory ability in the non-trained HIBD group were significantly decreased, whereas there was no significant difference between the trained-HIBD and the control groups. The left hemisphere weight and neurons in the left hippocampal CA1 zone of both HIBD groups decreased and the reduction was more significant in non-trained HIBD group. The ultrastructure of the left hippocampus was remarkably abnormal in the non-trained HIBD group, while no obvious abnormality was observed in the trained HIBD and the control groups. Phosphor-CaMKII and BDNF expression in the left hippocampus in the trained HIBD group increased significantly compared with that in the non-trained HIBD group.

CONCLUSIONSPhysical training can restrain brain damage and ameliorate spatial learning and memory impairments in rats with HIBD.

Animals ; Animals, Newborn ; Brain-Derived Neurotrophic Factor ; metabolism ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; metabolism ; Hypothalamus ; metabolism ; ultrastructure ; Hypoxia-Ischemia, Brain ; pathology ; physiopathology ; psychology ; therapy ; Maze Learning ; Memory ; Phosphorylation ; Physical Conditioning, Animal ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate whether desferoxamine (DFO) preconditioning can induce tolerance against cerebral ischemia and its effect on the expression of hypoxia inducible factor 1alpha (HIF-1alpha) and erythropoietin (EPO) in vivo and in vitro.

METHODSRat model of cerebral ischemia was established by middle cerebral artery occlusion with or without DFO administration. Infarct size was examined by TTC staining, and the neurological severity score was evaluated according to published method. Cortical neurons were cultured under ischemia stress which was mimicked by oxygen-glucose deprivation (OGD), and the neuron damage was assessed by MTT assay. Immunofluorescent staining was employed to detect the expressions of HIF-1alpha and EPO.

RESULTSThe protective effect induced by DFO (decreasing the infarction volume and ameliorating the neurological function) appeared at 2 d after administration of DFO (post-DFO), lasted until 7 d and disappeared at 14 d (P < 0.05); the most effective action was observed at 3 d post-DFO. DFO induced tolerance of cultured neurons against OGD: neuronal viability was increased 23%, 34%, 40%, 48% and 56% at 8 h, 12 h, 24 h, 36 h, and 48 h, respectively, post-DFO (P < 0.05). Immunofluorescent staining found that HIF-1alpha and EPO were upregulated in the neurons of rat brain at 3 d and 7 d post-DFO; increase of HIF-1alpha and EPO appeared in cultured cortex neurons at 36 h and 48 h post-DFO.

CONCLUSIONDFO induced tolerance against focal cerebral ischemia in rats, and exerted protective effect on OGD cultured cortical neurons. DFO significant induced the expression of HIF-1alpha and EPO both in vivo and in vitro. DFO preconditioning can protect against cerebral ischemia, which may be associated with the synthesis of HIF-1alpha and EPO.

Animals ; Brain Ischemia ; drug therapy ; metabolism ; physiopathology ; Cells, Cultured ; Cerebral Infarction ; drug therapy ; metabolism ; physiopathology ; Deferoxamine ; pharmacology ; therapeutic use ; Disease Models, Animal ; Erythropoietin ; metabolism ; Fluorescent Antibody Technique ; Hypoxia-Inducible Factor 1, alpha Subunit ; drug effects ; metabolism ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; physiopathology ; Infarction, Middle Cerebral Artery ; drug therapy ; metabolism ; physiopathology ; Iron ; metabolism ; Ischemic Preconditioning ; methods ; Nerve Degeneration ; drug therapy ; metabolism ; physiopathology ; Neurons ; drug effects ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley ; Siderophores ; pharmacology ; therapeutic use ; Time Factors ; Treatment Outcome ; Up-Regulation ; drug effects ; physiology

OBJECTIVENogo-A antibody IN-1 can neutralize Nogo-A, a neurite growth inhibitory protein, promoting axonal regeneration following lesions of the central nervous system (CNS) in adult rats. This study aimed to examine the effect of ventricle injection of Nogo-A antibody on neuronal regeneration in neonatal rats following hypoxic-ischemic brain damage (HIBD).

METHODSA model of neonatal HIBD was prepared by the ligation of the left common carotid artery, followed by 8% hypoxia exposure. Forty HIBD rats were randomly given a ventricle injection of 10 microL Nogo-A antibody IN-1 (IN-1 group) or 10 microL artificial cerebrospinal fluid (artificial CSF group) (n=20 each). Another 20 neonatal rats were sham-operated, without hypoxia-ischemia, and were used as the controls. The levels of Nogo-A and GAP-43 protein in the brain were measured by immunohistochemistry.

RESULTSThe number of immunohistory positive cells of Nogo-A in the brain in the IN-1 group (28.61+/-1.70) was obviously less than that in the artificial CSF (39.52 +/-1.40) and the sham-operated groups (32.78 +/- 1.87) (both P < 0.01). There were significant differences in the Nogo-A protein expression between the artificial CSF and the sham-operated groups (P < 0.01). The GAP-43 protein expression in the IN-1 group (31.14 +/- 1.88) was noticeably higher than that in the artificial CSF group (27.73 +/- 1.43 ) (P < 0.01). Both the IN-1 and the artificial CSF groups showed lower GAP-43 protein levels than the sham-operated groups (33.64 +/- 1.24) (both P < 0.01).

CONCLUSIONSNogo-A antibody can reduce the expression of Nogo-A protein in the brain and thus promote neuronal regeneration in neonatal rats following HIBD. An increased GAP-43 protein expression in the brain after Nogo-A antibody administration shows an enhanced neuronal regeneration in the neonatal rats following HIBD.

Animals ; Animals, Newborn ; Antibodies ; administration & dosage ; Brain Chemistry ; Female ; GAP-43 Protein ; analysis ; Hypoxia-Ischemia, Brain ; metabolism ; physiopathology ; therapy ; Immunohistochemistry ; Injections, Intraventricular ; Male ; Myelin Proteins ; analysis ; antagonists & inhibitors ; immunology ; Nerve Regeneration ; Nogo Proteins ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study changes in paired-immunoglobulin-like receptor B (PirB) expression after hypoxic-ischemic brain damage (HIBD) as well as the role for targeted inhibition of PirB activity in nerve regeneration in rats.

METHODSNewborn Sprague-Dawleyrats rats were divided into: a sham operation group (n=30), a HIBD group (n=30), and an anti PirB antibody treatment group (n=6). In the HIBD group, HIBD was induced by right carotid artery ligature and subsequent exposure to hypoxia (8% O2) for 3 hours. In the sham operation group, right carotid artery was dissected as in the HIBD group but no ligature and hypoxic exposure was not applied. In the two groups, 6 animals were sacrificed at 0, 6, 12, 24 and 72 hours after the operation and hypoxic exposure. In the antibody treatment group, after carotid artery ligation and hypoxia exposure as in the HIBD group, an anti PirB antibody was injected intracerebrally and animals were sacrificed 72 hours after the injection. Immediately after sacrifice of the animals at designated time points, brain tissue specimens were collected. The presence and content of PirB protein were assessed by immunohistochemistry and Western blot analysis respectively, the abundance of PirB mRNA was determined by RT-PCR, and the Rho kinase (Rock) activity was determined by immunoprecipitation.

RESULTSAt 72 hours after operation, PirB mRNA abundance and protein content in the brain were significantly increased as compared with the measurements at 0 hour after operation in the HIBD group (P<0.05); ROCK activity was significantly increased in the HIBD group as compared with the sham operation and anti PirB antibody groups (P<0.05).

CONCLUSIONSPirB might be involved in HIBD through a Rho-ROCK-dependent mechanism and antibody-mediated neutralization of PirB in the brain may offer a novel therapeutic strategy for HIBD.

Animals ; Animals, Newborn ; Hypoxia-Ischemia, Brain ; physiopathology ; therapy ; Nerve Regeneration ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptors, Immunologic ; antagonists & inhibitors ; genetics ; physiology ; Signal Transduction ; rho-Associated Kinases ; metabolism

OBJECTIVENeonatal hypoxic-ischemic encephalopathy (HIE) harms the lives and health of newborn infants and children severely. Given the absence of effective therapies for HIE, it is important to derive new strategies. Neural stem cells (NSCs) have great potential as a therapeutic tool for the repair of a number of central nervous system disorders that involve cell loss. This study was designed to transplant the neural stem cells derived from human fetal brain (hNSCs) into cerebral ventricle of neonatal rat following hypoxic-ischemic injury and to investigate their survival, migration and differentiation in rat brain.

METHODSCells obtained from the forebrain of a 12-week old fetus were cultured in the presence of epidermal growth factor, basic fibroblast growth factor and leukemia inhibitory factor for 11 days. Animal models were built in 7-day-postnatal Wistar rats, 3-days after hypoxia-ischemia (HI), 5 microl suspension containing 5.0 x 10(5) hNSCs was injected into the left cerebral ventricle of each HIE rat by using stereotactic instrument. No immunosuppression therapy was given to the animals. At 1, 2, 4 weeks and 3 months after transplantation, the rats were sacrificed and brain tissues were harvested and were then examined by H-E staining and immunohistochemical analysis.

RESULTSImplanted cells expressing human nuclear protein (hNP) migrated form the subventricular zone (SVZ) along corpus callosum to the damaged areas, especially to the injured side of cortex and hippocampus. In different areas, the implanted hNSCs differentiated into different cell types which were similar to the host cells. The 85% implanted cells in cortex consisted of hNuc-NF or hNuc-Tublin double positive cells, while in the migratory way, 60% implanted cells differentiated into hNuc-GFAP double positive cells. Compared with the 1-week time point, an increased number of hNP-positive cells were observed at 2-weeks, but the number of these cells greatly decreased at 4-weeks and 3 months.

CONCLUSIONThe implanted hNSCs could extensively survive, migrate in the brain of neonatal rat with HIE and could differentiate into neurons and astrocytes in a regionally specific manner.

Animals ; Animals, Newborn ; Brain ; pathology ; Carotid Artery, Common ; surgery ; Cell Differentiation ; Cell Movement ; Disease Models, Animal ; Fetal Stem Cells ; transplantation ; Humans ; Hypoxia ; complications ; physiopathology ; Hypoxia-Ischemia, Brain ; pathology ; physiopathology ; therapy ; Immunohistochemistry ; Injections, Intraventricular ; methods ; Ligation ; methods ; Neurons ; Nuclear Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stem Cell Transplantation ; methods ; Survival Analysis ; Time Factors

OBJECTIVETo investigate the protective effects of Huperzine A, a potent acetylcholinesterase inhibitor, against the hypoxic ischemic brain damage (HIBD) of the cognitive and morphology in the neonatal rats.

METHODSPostnatal 7 days old rats were given vehicle or Huperzine A (0.05 mg/kg or 0.1 mg/kg, i.p.) following HIBD (unilateral carotid artery ligation followed by hypoxia) or sham operation, and then tested the learning ability and memory in the Morris water maze (MWM) from 36 to 40 postnatal days. The performance in MWM (escape latency, probe time) were recorded to evaluate the learning and memory dysfunction. At the end of MWM trials, the rats were decapitated and their brains were histologically analyzed. The tissue loss in different brain regions including striatum, cortex, and hippocampus were analyzed by image analysis system. The CA(1) subfield neurons numbers were counted to evaluate the brain damage. The acetylcholinesterase histochemistry staining was used to determine the activity of acetylcholinesterase in different brain regions.

RESULTSCompared with sham-operated group, HIBD rats with the vehicle treatment displayed significant tissue losses in the hippocampus (including CA(1) neurons), cortex, and striatum, as well as severe spatial memory deficits (escape latency: 44 s vs 30 s, P < 0.05, probe time: 14 s vs 40 s, P < 0.01). Huperzine A treatment (0.1 mg/kg) resulted in significant protection against both HI-induced brain tissue losses and spatial memory impairments (mean escape latency: 34 s vs 44 s, P < 0.05, probe time: 35 s vs 14 s,P < 0.01). However, Huperzine A treatment (0.05 mg/kg) did not show any significant improvement of spatial memory impairments (mean escape latency: 45 s vs 44 s, P > 0.05, probe time: 17 s vs 14 s, P > 0.05), but moderate to severe brain tissue losses. There was a pronounced reduction of CA(1) neuron density in ipsilateral hemisphere of vehicle-treated group and 0.05 mg/kg Huperzine A group compared with contralateral hemisphere or ipsilateral hemisphere of sham-operated group and 0.1 mg/kg Huperzine A group (72 vs 232, P < 0.01, 72 vs 229, P < 0.01, respectively). There was a close linear correlation between the CA(1) neurons cell number and the mean escape latency for 5 d acquisition trials (r = 0.777, P < 0.01).

CONCLUSIONThe unilateral HI brain injury in a neonatal rat model was associated with cognitive deficits, and that Huperzine A treatment may be protective against both brain injury and spatial memory impairment. Huperzine A showed a therapeutic potential for the treatment of hypoxic-ischemic encephalopathy (HIE) caused by the perinatal asphyxia.

Acetylcholinesterase ; metabolism ; Alkaloids ; Animals ; Animals, Newborn ; Cerebral Cortex ; drug effects ; enzymology ; pathology ; Cognition Disorders ; drug therapy ; physiopathology ; Corpus Striatum ; drug effects ; enzymology ; pathology ; Female ; Hippocampus ; drug effects ; enzymology ; pathology ; Hypoxia-Ischemia, Brain ; drug therapy ; Male ; Maze Learning ; drug effects ; Neuroprotective Agents ; administration & dosage ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Sesquiterpenes ; administration & dosage ; therapeutic use ; Treatment Outcome