Neuroinflammation is a key contributor to the pathogenic cascades induced by hypoxic-ischemic (HI) insult in the neonatal brain. AD-16 is a novel anti-inflammatory compound, recently found to exert potent inhibition of the lipopolysaccharide-induced production of pro-inflammatory and neurotoxic mediators. In this study, we evaluated the effect of AD-16 on primary astrocytes and neurons under oxygen-glucose deprivation (OGD) in vitro and in mice with neonatal HI brain injury in vivo. We demonstrated that AD-16 protected against OGD-induced astrocytic and neuronal cell injury. Single dose post-treatment with AD-16 (1 mg/kg) improved the neurobehavioral outcome and reduced the infarct volume with a therapeutic window of up to 6 h. Chronic administration reduced the mortality rate and preserved whole-brain morphology following neonatal HI. The in vitro and in vivo effects suggest that AD-16 offers promising therapeutic efficacy in attenuating the progression of HI brain injury and protecting against the associated mortality and morbidity.
Animals ; Animals, Newborn ; Astrocytes/pathology* ; Brain/pathology* ; Brain Injuries/pathology* ; Glucose ; Hypoxia ; Hypoxia-Ischemia, Brain/drug therapy* ; Mice ; Neuroinflammatory Diseases ; Neuroprotective Agents/therapeutic use* ; Oxygen/therapeutic use*

BACKGROUNDRecombinant human-erythropoietin (rh-EPO) has therapeutic efficacy for premature infants with brain damage during the active rehabilitation and anti-inflammation. In the present study, we found that the rh-EPO was related to the promotion of neovascularization. Our aim was to investigate whether rh-EPO augments neovascularization in the neonatal rat model of premature brain damage through the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) signaling pathway.

METHODSPostnatal day 5 (PD5), rats underwent permanent ligation of the right common carotid artery and were exposed to hypoxia for 2 h. All the rat pups were randomized into five groups as follows: (1) control group; (2) hypoxia-ischemic (HI) group; (3) HI + LY294002 group; (4) HI + rh-EPO group; and (5) HI + rh-EPO + LY294002 group. The phospho-Akt protein was tested 90 min after the whole operation, and CD34, vascular endothelial growth factor receptor 2 (VEGFR2), and vascular endothelial growth factor (VEGF) were also tested 2 days after the whole operation.

RESULTSIn the hypoxic and ischemic zone of the premature rat brain, the rh-EPO induced CD34+ cells to immigrate to the HI brain zone (P < 0.05) and also upregulated the VEGFR2 protein expression (P < 0.05) and VEGF mRNA level (P < 0.05) through the PI3K/Akt (P < 0.05) signaling pathway when compared with other groups.

CONCLUSIONSThe rh-EPO treatment augments neovascularization responses in the neonatal rat model of premature brain damage through the PI3K/Akt signaling pathway. Besides, the endogenous EPO may exist in the HI zone of rat brain and also has neovascularization function through the PI3K/Akt signaling pathway.

Animals ; Animals, Newborn ; Antigens, CD34 ; metabolism ; Brain ; drug effects ; metabolism ; pathology ; Disease Models, Animal ; Erythropoietin ; genetics ; metabolism ; therapeutic use ; Female ; Humans ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; Neovascularization, Physiologic ; drug effects ; Phosphatidylinositol 3-Kinase ; metabolism ; Pregnancy ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; genetics ; metabolism ; therapeutic use ; Signal Transduction ; drug effects ; Vascular Endothelial Growth Factor A ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism

OBJECTIVETo examine the expression of calreticulin (CRT) and the changes of intracellular free calcium and neuronal apoptosis in the cerebral cortex of neonatal rats with hypoxic-ischemic brain damage (HIBD), and to investigate the intervention effects of Shenfu injection.

METHODSSeven-day-old rats were randomly assigned to three groups: control, hypoxic-ischemia (HI) and Shenfu-treated. Each group (n=50) was subdivided into 5 groups sacrificed at 3, 6, 12, 24 and 72 hours. Rat models of HIBD were prepared according to the Rice's method. Rats in the control group only underwent the separation of right common carotidartery. Shenfu injection was administered by intraperitoneal injections right after HI insults and then once daily at a dosage of 10 mL/kg for 3 days in the Shenfu-treated group. The expression of CRT in the cerebral cortex was detected by RT-PCR and Western blot. The free calcium concentrations were determined under a fluorescent microscope. The apoptosis rate was measured by the flow cytometry.

RESULTSCompared with the control group, the expression levels of CRT in the HI and the Shenfu-treated groups were obviously up-regulated (P<0.05), and the expression levels of CRT in the Shenfu-treated group were notably higher than those in the HI group (P<0.05) at all time points. The concentrations of intracellular free calcium and the apoptosis rate of neurons in the cerebral cortex in the Shenfu-treated group were significantly reduced compared with those in the HI group (P<0.05), but increased significantly compared with those in the control group at all time points (P<0.05).

CONCLUSIONSShenfu injection may have neuroprotective effects against HIBD by up-regulation of CRT expression and relief of calcium overload.

Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Calcium ; metabolism ; Calreticulin ; analysis ; Cerebral Cortex ; metabolism ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Female ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; pathology ; Injections ; Male ; Neurons ; drug effects ; Rats, Sprague-Dawley

OBJECTIVETo study the effects of caffeine citrate on myelin basic protein (MBP) expression in the cerebral white matter of neonatal rats with hypoxic-ischemic brain damage (HIBD) and the related mechanism.

METHODSForty-eight seven-day-old Sprague-Dawley neonatal rats were randomly assigned to 3 groups: sham operation (n=16), HIBD (n=16) and HIBD+caffeine citrate (n=16). The rats in the HIBD and HIBD+caffeine citrate groups were subjected to left common carotid artery ligation, and then were exposed to 80 mL/L oxygen and 920 mL/L nitrogen for 2 hours to induce HIBD. The rats in the sham operation group were only subjected to a sham operation, without the left common carotid artery ligation or hypoxia exposure. Caffeine citrate (20 mg/kg) was injected intraperitoneally before hypoxia ischemia (HI) and immediately, 24 hours, 48 hours and 72 hours after HI. The other two groups were injected intraperitoneally with an equal volume of normal saline at the corresponding time points. On postnatal day 12, the expression of MBP in the left subcortical white matter was detected by immunohistochemistry, and the levels of adenosine A1 receptor mRNA and A2a receptor mRNA in the left brain were detected by real-time PCR.

RESULTSThe expression of MBP in the left subcortical white matter in the HIBD group was lower than in the sham operation group (P<0.05). The MBP expression in the HIBD+caffeine citrate group was significantly higher than in the HIBD group, but was still lower than the sham operation group (P<0.05). Real-time PCR showed that the adenosine A1 receptor mRNA expression was significantly higher in the HIBD group than in the sham operation group, and it was significantly lower in the HIBD+caffeine citrate group than in the HIBD group (P<0.05).

CONCLUSIONSCaffeine citrate can improve brain white matter damage following HIBD in neonatal rats and the protection mechanism might be related with the down-regulation of adenosine A1 receptor expression.

Animals ; Animals, Newborn ; Caffeine ; pharmacology ; Citrates ; pharmacology ; Female ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; pathology ; Male ; Myelin Basic Protein ; analysis ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptor, Adenosine A1 ; genetics ; Receptor, Adenosine A2A ; genetics ; White Matter ; chemistry

OBJECTIVETo observe the effect of Rg1-induced NSCs in treatment of neonatal rat model with hypoxiaischemia.

METHODThe neonatal rat model of HIE was established and assessed by using TTC staining and behavioral observation, then Rg1-induced NSCs was transplanted into the neonatal rat of HIE by lateral ventricle injection. Water maze test and somatosensory evoked potential were detected to observe brain function and the immunohistochemistry was done to assess growth and differentiation about transplanted NSCs a month after transplanted.

RESULTThe transplantation of Rg1-induced NSCs could significantly shorten incubation period, swimming distance, exploration time of target quadrants of water maze test and incubation period and amplitude of somatosensory evoked potentials. Additionally, the concentrated expression appeared in the hippocampus and grew around the ischemic injury area in transplantation group.

CONCLUSIONTransplantation of Rg1-induced NSCs play a better role in the treatment of neonatal HIE rats.

Animals ; Cell Differentiation ; drug effects ; Evoked Potentials ; Female ; Ginsenosides ; pharmacology ; Hippocampus ; pathology ; physiopathology ; Hypoxia-Ischemia, Brain ; pathology ; physiopathology ; therapy ; Male ; Maze Learning ; Neural Stem Cells ; cytology ; drug effects ; transplantation ; Rats ; Rats, Sprague-Dawley ; Recovery of Function ; physiology

OBJECTIVETo observe the therapeutic effect of Yangxue Qingnao Granule (, YXQNG) on cognitive impairment induced by chronic cerebral hypoperfusion and to investigate its impact on oxidative stress, apoptosis, and the cholinergic system.

METHODSAdult male Wistar rats were subjected to chronic cerebral hypoperfusion by permanent occlusion of bilateral common carotid arteries (2-VO). Thirty rats were randomly assigned to one of the five treatment groups in a 1:1:1:1:1 ratio: sham operation plus normal saline treatment, 2-VO plus normal saline treatment, 2-VO plus YXQNG at a dose of 2 g·kg(-1)·d(-1) or 4 g·kg(-1)·d(-1), or 2-VO plus rivastigmine 2 mg·kg(-1)·d(-1). The Morris water maze test was used to assess the spatial memory retrieval. Apoptosis, total antioxide capacity (T-AOC), acetylcholine esterase (AchE) and choline acetyl transferase (ChAT) activities in the hippocampus and the cortex were investigated.

RESULTSIn the chronic cerebral hypoperfusion model, the 2-VO plus saline treatment resulted in impaired special learning as shown by the significantly prolonged escape latency and shorter swim time in the first quadrant as compared to the sham operation. The impairment was associated with apoptosis and significant decreases in T-AOC, AchE and ChAT activities in the hippocampus and the cortex. Treatment with YXQNG at either 2 g·kg(-1)·d(-1) or 4 g·kg(-1)·d(-1) dose, or rivastigmine resulted in significantly shorter escape latencies and longer swim time in the first quadrant. YXQNG at both doses, but not rivastigmine, had significant reduction in apoptosis, and significant increases in T-AOC and ChAT activity in both the hippocampus and the cortex. Unlike rivastigmine, neither dose of YXQNG showed significant reduction in AchE activity.

CONCLUSIONSYXQNG ameliorated cognitive impairment induced by chronic cerebral hypoperfusion. The protective effect may be mediated through its regulation of apoptosis and activities of T-AOC and ChAT in the hippocampus and cortex of the rats in the chronic cerebral hypoperfusion model, a mechanism that is different from rivastigmine.

Animals ; Apoptosis ; drug effects ; Brain ; drug effects ; pathology ; Chemistry, Pharmaceutical ; Chronic Disease ; Cognition Disorders ; drug therapy ; etiology ; pathology ; Disease Models, Animal ; Drug Evaluation, Preclinical ; Drugs, Chinese Herbal ; administration & dosage ; therapeutic use ; Hypoxia-Ischemia, Brain ; complications ; drug therapy ; pathology ; Male ; Maze Learning ; drug effects ; Rats ; Rats, Wistar ; Spatial Behavior ; drug effects ; Task Performance and Analysis

OBJECTIVEImmunosuppressant tacrolimus (FK506) has shown neuroprotective effects on hypoxic-ischemic brain damage (HIBD) in the adult animal model. This study investigated whether FK506 has a protection against HIBD in neonatal rats by examining growthjassociated protein-43 (GAP-43) expression in the hippocampus.

METHODSNinety-six seven-day-old Sprague-Dawley rats were randomly divided into three groups: sham-operation, HIBD and FK506 intervention group. HIBD was induced in the later two groups. The FK506 intervention group was intraperitoneally injected with FK506 immediately after HIBD, at a dosage of 1 mg/kg daily, for three days. The HIBD group was injected with normal saline. Immunohistochemical technical was applied to examine GAP-43 expression in the hippocampus 24 and 72 hrs and 7 and 14 days after HIBD.

RESULTSCompared with the HIBD group, hematoxylin-eosin staining showed attenuated neuronal necrosis in the FK506 intervention group. In the HIBD group, the expression of GAP-43 increased significantly 72 hrs, and 7 and 14 days after HIBD compared with that in the sham-operation group. The GAP-43 expression in the FK506 intervention group was significantly higher than that in the HIBD group 72 hrs and 7 days after HIBD.

CONCLUSIONSFK506 might have neuroprotective effects against HIBD in neonatal rats.

Animals ; Animals, Newborn ; GAP-43 Protein ; analysis ; Hippocampus ; chemistry ; drug effects ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; pathology ; Immunosuppressive Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Tacrolimus ; pharmacology

The aim of this study was to investigate the effect of erythropoietin (EPO) on histological brain injury, subventricular zone (SVZ) expansion, and sensorimotor function deficits induced by hypoxia-ischemia (HI) in newborn rat pups. Seven-day-old male rat pups were divided into six groups: normoxia control, normoxia EPO, hypoxia control, hypoxia EPO, HI control, and HI EPO group. Sham surgery or HI was performed in all animals. HI was induced by ligation of the right common carotid artery followed by 90 min of hypoxia with 8% oxygen. Recombinant human EPO 3 U/g or saline was administered intraperitoneally, immediately, at 24- and 48-hr after insult. At two weeks after insult, animals were challenged with cylinder-rearing test for evaluating forelimb asymmetry to determine sensorimotor function. All animals were then sacrificed for volumetric analysis of the cerebral hemispheres and the SVZ. The saline-treated HI rats showed marked asymmetry by preferential use of the non-impaired, ipsilateral paw in the cylinder-rearing test. Volumetric analysis of brains revealed significantly decreased preserved ipsilateral hemispheric volume and increased ipsilateral SVZ volume compared with the sham-operated animals. Treatment of EPO significantly improved forelimb asymmetry and preserved ipsilateral hemispheric volume along with decreased expansion of ipsilateral SVZ following HI compared to the saline-treated HI rats. These results support the use of EPO as a candidate drug for treatment of neonatal hypoxic-ischemic brain damage.
Animals ; Animals, Newborn ; Carotid Artery, Common ; Cerebral Ventricles/*pathology ; Erythropoietin, Recombinant/*pharmacology ; Female ; Hypoxia-Ischemia, Brain/*drug therapy/*pathology ; Ligation ; Male ; Motor Activity/drug effects ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; Recovery of Function/drug effects

OBJECTIVETo study the effects of androgen on the expression of aromatase cytopigment P450 (AROM) and nerve growth factor (NGF) in the brain and brain ultrastructure in neonatal rats with hypoxic-ischemic brain damage (HIBD) in order to investigate the mechanism underlying the protective effect of androgen against HIBD.

METHODSNinety-six seven-day-old Sprague-Dawley rats were randomly divided into three groups: sham-operation, HIBD and androgen treatment (n=32 each). HIBD was induced by the ligation of left common carotid artery and hypoxia exposure. The rats in the androgen treatment and the HIBD groups were injected intraperitoneally with testosterone propionate (25 mg/kg) and arachis oil respectively immediately after hypoxia-ischemia (HI). After 24 and 72 hrs and 7 and 10 days of HI, AROM and NGF expression in the cortex and the hippocampus was detected with the immunohistochemical method. The ultrastructural changes of neurons in the cortex and the hippocampus were observed under a transmission electron microscope.

RESULTSNerve cells of the HIBD group showed obvious injuries including cell organ decreasing, cellularoedema, nuclear swelling, chromatic agglutination, mitochondria decreasing and swelling, as well as an increase in apoptotic cells. Compared with the HIBD group, the nerve cells in the androgen treatment group had integrated nuclear membrane, well-distributed chromatin and abundant cell organs, and less cell apoptosis and increased axon regeneration. There was a positive expression of NGF and AROM in the brain cortex and the hippocampus in the HIBD group 24 hrs after HI. The expression of NGF and AROM increased significantly 72 hrs after HI, peaked 7 days after HI and then began to decrease but remained at a higher level than that in the sham-operation group 10 days after HI. The NGF and AROM expression in the cortex and the hippocampus in the androgen treatment group was significantly higher than that in the sham-operation and the HIBD groups 72 hrs, and 7 and 10 days after HI.

CONCLUSIONSAndrogen treatment can promote axon regeneration and morphous recovery of neurons and decrease neural apoptosis in neonatal rats with HIBD. The neuroprotection of androgen is produced possibly through an increase in the expression of NGF and AROM in the brain.

Androgens ; therapeutic use ; Animals ; Animals, Newborn ; Aromatase ; analysis ; Brain ; enzymology ; Female ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; pathology ; Immunohistochemistry ; Male ; Nerve Growth Factor ; analysis ; Neurons ; ultrastructure ; 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