Brain Damage In Neonatal Rats Research Articles

Resatorvid protects against hypoxic-ischemic brain damage in neonatal rats.

Secondary brain damage caused by hyperactivation of autophagy and inflammatory responses in neurons plays an important role in hypoxic-ischemic brain damage (HIBD). Although previous studies have implicated Toll-like receptor 4 (TLR4) and nuclear factor kappa-B (NF-κB) in the neuroinflammatory response elicited by brain injury, the role and mechanisms of the TLR4-mediated autophagy signaling pathway in neonatal HIBD are still unclear. We hypothesized that this pathway can regulate brain damage by modulating neuron autophagy and neuroinflammation in neonatal rats with HIBD. Hence, we established a neonatal HIBD rat model using the Rice-Vannucci method, and injected 0.75, 1.5, or 3 mg/kg of the TLR4 inhibitor resatorvid (TAK-242) 30 minutes after hypoxic ischemia. Our results indicate that administering TAK-242 to neonatal rats after HIBD could significantly reduce the infarct volume and the extent of cerebral edema, alleviate neuronal damage and neurobehavioral impairment, and decrease the expression levels of TLR4, phospho-NF-κB p65, Beclin-1, microtubule-associated protein l light chain 3, tumor necrosis factor-α, and interleukin-1β in the hippocampus. Thus, TAK-242 appears to exert a neuroprotective effect after HIBD by inhibiting activation of autophagy and the release of inflammatory cytokines via inhibition of the TLR4/NF-κB signaling pathway. This study was approved by the Laboratory Animal Ethics Committee of Affiliated Hospital of Yangzhou University, China (approval No. 20180114-15) on January 14, 2018.

Simvastatin preconditioning confers neuroprotection against hypoxia-ischemia induced brain damage in neonatal rats via autophagy and silent information regulator 1 (SIRT1) activation

Previous studies have shown that simvastatin (Sim) has neuroprotective effects in a neonatal model of hypoxia-ischemia (HI)-induced brain injury when administered before but not after HI, pointing to the preconditioning (PC)-like effects of the statin. The present study aimed to gain more insight into the PC-like effect of Sim by studying the role of autophagy and its modulation by mTOR and SIRT1 in neuroprotection. Sim potentiated the autophagy response induced by neonatal HI, as shown by the increased expression of both microtubule-associated protein 1 light chain 3 (LC3) and beclin 1, increased monodansylcadaverine (MDC) labeling, and reduced expression of p62. The autophagy inhibitor 3-methyladenine (3MA) completely blocked the neuroprotective effect of Sim. Two hours after HI, there was a reduction in the activity of mTORC1 and a concomitant increase in that of mTORC2. Sim preconditioning further decreased the activity of mTORC1, but did not affect that of mTORC2. However, 24 h after injury, mTORC2 activity was significantly preserved in Sim-treated rats. Sim preconditioning also prevented the depletion of SIRT1 induced by HI, an effect that was completely blocked by 3MA. These data show that Sim preconditioning may modulate autophagy and survival pathways by affecting mTORC1, mTORC2, and SIRT1 activities. This study provides further preclinical evidence of the PC-like effect of statins in brain tissue, supporting their beneficial effects in improving stroke outcome after prophylactic treatments.

Acoustic Radiation Force Impulse Imaging With Virtual Touch Tissue Quantification Enables Characterization of Mild Hypoxic-Ischemic Brain Damage in Neonatal Rats.

The aim of this study was to investigate whether the measurement of brain tissue stiffness using acoustic radiation force impulse (ARFI) elastography with virtual touch tissue quantification can improve the early detection of neonatal hypoxic-ischemic brain damage in rats. Seven-day-old Sprague-Dawley rats were randomly assigned to 3 groups: the mild asphyxia (n = 30), moderate asphyxia (n = 30), and sham control (n = 10) groups. Rats in the mild and moderate asphyxia groups were exposed to 8% oxygen (hypoxia) for 30 and 60 minutes, respectively, at 1 hour after ligation of the right common carotid artery. An ultrasound diagnostic instrument was used to obtain 2-dimensional ultrasound images, and ARFI with virtual touch tissue quantification was used to measure shear wave velocity preoperatively and at 12, 24, 48, and 72 hours postoperatively. Hematoxylin-eosin staining was used to evaluate brain damage. Two-dimensional ultrasound imaging detected swelling and increased echogenicity at 48 to 72 hours in the mild asphyxia group and at 24 to 72 hours in the moderate asphyxia group. The shear wave velocity substantially increased from 0.65 ± 0.04 m/s preoperatively to 0.78 ± 0.07 m/s at 72 hours in the moderate asphyxia group and from 0.64 ± 0.04 m/s preoperatively to 0.70 ± 0.03 m/s at 72 hours in the mild asphyxia group. The changes in the shear wave velocity coincided with the histopathologic changes in the brain, which included neuronal demyelination, hyperplasia, and necrosis; edema around vascular structures; and hemorrhage in the ependymal and periventricular areas. Shear wave velocity data obtained with the virtual touch tissue quantification technique may be used for early diagnosis of neonatal hypoxic-ischemic brain damage.

Chlorogenic Acid Prevents Alcohol-induced Brain Damage in Neonatal Rat.

The present investigation evaluates the neuroprotective effect of chlorogenic acid (CA) in alcohol-induced brain damage in neonatal rats. Ethanol (12 % v/v, 5 g/kg) was administered orally in the wistar rat pups on postnatal days (PD) 7-9. Chlorogenic acid (100 and 200 mg/kg, p.o.) was administered continuously from PD 6 to 28. Cognitive function was estimated by Morris water maze (MWM) test. However, activity of acetylcholinesterase, inflammatory mediators, parameters of oxidative stress and activity of caspase-3 enzyme was estimated in the tissue homogenate of cerebral cortex and hippocampus of ethanol-exposed pups. It has been observed that treatment with CA attenuates the altered cognitive function in ethanol-exposed pups. There was a significant decrease in the activity of acetylcholinesterase in the CA treated group compared to the negative control group. However, treatment with CA significantly ameliorates the increased oxidative stress and concentration of inflammatory mediators in the brain tissues of ethanol-exposed pups. Activity of caspase-3 enzyme was also found significantly decreased in the CA treated group compared to the negative control group. The present study concludes that CA attenuates the neuronal damage induced in alcohol exposed neonatal rat by decreasing the apoptosis of neuronal cells.

Neuroprotective Effect of Chitosan Oligosaccharide on Hypoxic-Ischemic Brain Damage in Neonatal Rats.

Neonatal hypoxic-ischemic brain damage (HIBD) is one of the leading causes of neonatal mortality and permanent neurological disability worldwide and the effective treatment strategies are not yet available. It has been demonstrated that Chitosan oligosaccharide (COS) exerts protective effect in vitro ischemic brain injury. However, no information is available on possible effects of COS on neonatal HIBD. To investigate the hypothesis of the potential neuroprotective effect of COS on the brain injury due to HIBD, 7-day-old Sprague-Dawley rats were treated with left carotid artery ligation followed by exposure to 8% oxygen (balanced with nitrogen) for 2.5h at 37 °C. After COS treatment, the cerebral damage was measured by behavior tasks, 2,3,5-triphenyltetrazolium chloride(TTC), Hematoxyline-Eosin(HE), Nissl and Fluoro-Jade B(FJB)staining. In addition, the oxidative stress were assayed with ipsilateral hemisphere homogenates. Immunofluorescence staining were used to examine the activation of the astrocyte and microglia. Expression of inflammatory-related proteins were analyzed by western-blot analysis. In this study we found that administration of COS ameliorated early neurological reflex behavior, significantly reduce brain infarct volume and attenuated neuronal cell injury and degeneration. Furthermore, COS markedly decreased the level of MDA, lactic acid and increased SOD, GSH-Px and T-AOC. COS attenuated hypoxic-ischemic induced up-regulation of expressions of interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), meanwhile it dramatically increased the interleukin-10 (IL-10). These results suggest that COS exerts neuroprotection on hypoxic-ischemic brain damage in neonatal rats, it implies COS might be a potential therapeutic for the treatment of HIBD.

Effects of discoidin domain receptor 1 mediated phosphorylation of protein Tau on hypoxic-ischemic brain damage in neonatal rats

Objective To study the effects of discoidin domain receptor 1 (DDR1) mediated phosphorylation of protein Tau on hypoxic-ischemic brain damage (HIBD) in neonatal rats and its possible mechanism. Methods Sixty-four seven-day-old male specific-pathogen-free Wistar rats were randomly divided into four groups with sixteen in each: Sham, HIBD, HIBD with normal saline (HIBD+NS) and HIBD with DDR1 inhibitor (HIBD+DI) groups. A rat model of HIBD was established by subjecting the rats to left common carotid artery ligation, followed by exposing them to hypoxia for two hours. In HIBD+DI group, the inhibitor of DDR1 was immediately injected into lateral cerebroventricles of the rats following modeling. Forty-eight hours after injection, tissues of left cerebral cortex were collected from each rat to evaluate histopathological changes with HE staining. Western-blotting was used to assess the phosphorylation levels of DDR1 and protein Tau. Enzyme-linked immunosorbent assay was performed to detect the concentrations of acetylcholine. Analysis of variance or t test were used for statistical analysis. Results (1) Damages in cerebral cortex: Percentages of abnormal neurons in the rats of HIBD group were higher than those in Sham group [(80.28±4.51)% vs (10.40±2.17)%, t=39.491, P<0.01]. Pyknotic or necrotic neurons in the rats of HIBD+DI group were less than those in HIBD+NS group [(31.91±3.05)% vs (82.01±7.20)%, t=18.123, P<0.01]. (2) Phosphorylation of DDR1 and protein Tau: Levels of phosphorylated DDR1 in the cerebral cortexes of rats in HIBD group were higher than those in Sham group (0.922±0.199 vs 0.095±0.023, t=10.379, P<0.01), and those levels in HIBD+NS group were higher than those in HIBD+DI group (1.200±0.171 vs 0.255±0.111, t=11.901, P<0.01). The phosphorylation of protein Tau was similar to that of DDR1 (0.919±0.228 vs 0.194±0.224 in HIBD and Sham groups, t=7.347; 1.100±0.167 vs 0.291±0.210 in HIBD+NS and HIBD+DI groups, t=9.447; both P<0.01). (3) Levels of acetylcholine: Levels of acetylcholine in cerebral cortexes of rats in HIBD group were lower than those in Sham group [(3.685±0.472) vs (7.429±0.861) ng/g protein, t=10.781, P<0.01], and that levels in HIBD+DI group were higher than those in HIBD+NS group [(7.058±0.915) vs (2.521±0.723) ng/g protein, t=10.989, P<0.01]. Conclusions Activation of DDR1 plays a key role in enhancing the phosphorylation of protein Tau and in reducing the secretion of acetylcholine in cerebral cortexes of rats with HIBD. Inhibitor of DDR1 could protect neonatal rats from HIBD through the decreasing of protein Tau phosphorylation and increasing of acetylcholine release by inhibiting the activation of DDR1. Key words: Hypoxia-ischemia,brain; Discoidin domain receptor 1; Tau proteins; Phosphorylation; Animals, newborn; Rats

RETRACTED ARTICLE: RIPK3 interactions with MLKL and CaMKII mediate oligodendrocytes death in the developing brain

Oligodendrocyte progenitor cells (OPCs) death is a key contributor to cerebral white matter injury (WMI) in the developing brain. A previous study by our group indicated that receptor-interacting proteins (RIPs) are crucial in mediating necroptosis in developing neurons. However, whether this mechanism is involved in OPCs death is unclear. We aimed to explore the mechanisms of RIP-mediated oligodendrocytes (OLs) death in the developing brain. Oligodendrocytes necroptosis was induced by oxygen-glucose deprivation plus caspase inhibitor zVAD treatment (OGD/zVAD) in vitro. Western blotting and immunofluorescence were used to detect RIPK1, RIPK3, mixed lineage kinase domain-like protein (MLKL), and Ca2+ and calmodulin-dependent protein kinase IIδ (CaMKIIδ). Immunoprecipitation was used to assess the interactions between RIPK3 and RIPK1, MLKL, and CaMKIIδ. Necrostatin-1 was used to disturb the RIPK3–RIPK1 interaction, and siRNA was used to inhibit RIPK3 or MLKL expression. Oligodendrocytes death was examined using PI staining, EM, and cell membrane leakage assays. In vivo, brain damage in neonatal rats was induced by hypoxia–ischemia (HI). This was followed by an examination of myelin development. We found that OGD/zVAD treatment upregulates the expression of RIPK3 and the interaction of RIPK3 with RIPK1, MLKL, and CaMKIIδ. Inhibition of the RIPK3-MLKL or RIPK3-CaMKIIδ interaction attenuates OLs death induced by OGD/zVAD. These protective mechanisms involve the translocation of MLKL to the OLs membrane, and the phosphorylation of CaMKIIδ. However, inhibition of the RIPK3–RIPK1 interaction did not protect OLs death induced by OGD/zVAD. In vivo studies indicated that the disrupted development of myelin was attenuated after the inhibition of RIPK3-MLKL or RIPK3-CaMKIIδ interaction. Taken together, our data indicate that RIPK3 is a key factor in protection against OLs death and abnormal myelin development via its interaction with MLKL and CaMKIIδ after HI. This suggests that RIPK3 may be a potential target for the treatment of WMI in neonates.

Amelioration by glycine of brain damage in neonatal rat brain following hypoxia–ischemia

Glycine protected adult brains against injury in an experimental model of stroke, but, because the ischemic response of neonatal brains differs from that of adult brains, we examined the neuroprotective efficacy of glycine and associated mechanisms in an experimental model of neonatal hypoxic-ischemic (HI) encephalopathy. Neonatal (postnatal day 7) Wistar rats were randomly divided into an untreated group (non-HI) and two HI groups that were treated with left common carotid artery ligation and saline control or glycine. After recovery, pups that received surgery were injected i.p. with saline or glycine (800 mg/kg; optimal dose determined in pilot experiments) and were placed in a controlled 8% O2 chamber for 120 min. Brains were harvested at various times after return to normoxia (several hours-days after HI) for analysis of infarct area, glial activation, cell apoptosis, and tumor necrosis factor-α (TNF-α) expression on histology and reverse transcription-polymerase chain reaction. Glycine injections induced large (approx. 15-fold) but brief (approx. 2 h) increases in cerebrospinal fluid concentrations. In particular, the glycine group had a >70% decrease in infarct areas compared with controls at 7 days after HI. Glycine also significantly reduced astrocyte reactive transformation, microglia activation, and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive (apoptotic) cell numbers in peri-lesional areas at 3 days after HI, and TNF-α mRNA expression in the injured hemisphere at 12 and 24 h after HI. Glycine protected neonatal rat brains against HI, in part by inhibiting TNF-α-induced inflammation and gliosis. Hence, systemic glycine infusions may have clinical utility for the treatment of HI injury in human newborns.

Neuroprotective actions of pterostilbene on hypoxic-ischemic brain damage in neonatal rats through upregulation of heme oxygenase-1

Neonatal hypoxic–ischemic (HI) brain damage causes acute mortality and morbidity in newborns and long-term neurological disorders in the survivors. Pterostilbene (PTE) is a natural compound possessing various biological and pharmacological activities. In the present study, we aimed to investigate the effect of PTE on neonatal HI brain damagein P7 rat model and to explore the possible mechanisms. Neonatal HI brain damage was induced in rat pups (P7). Prior to the induction of HI injury, PTE was injected with or without zinc protoporphyrin IX (ZnPP), an inhibitor of heme oxygenase-1 (HO-1). ZnPP was used to test whether abnormal changes of HO-1 expression were involved in the effect of PTE. The results showed that PTE exhibited excellent neuroprotective effects against neonatal HI brain injury, as evidenced by the decrease of brain infarct volume, brain edema, neurological score, and improvement in motor coordination motor deficit and working memory deficit. PTE pretreatment decreased the expression of several proinflammatory cytokines, including TNFα, IL-1β, IL-6, and key transcription factor p65 NF-κB, and reduced the number of TUNEL-stained neurons, indicating the inhibition of inflammation and programmed cell death. Moreover, PTE pretreatment decreased thiobarbituric acid reactive substances content, increased superoxide dismutase activity and decreased reactive oxygen species level, indicating that PTE played an important antioxidant role. Furthermore, ZnPP was able to inhibit PTE-induced suppression of oxidative stress, programmed cell death, inflammation and brain damage. In conclusion, PTE pretreatment prevented HI-induced brain injury in newborns through HO-1-mediated reduction of oxidative stress, programmed cell death, and inflammation, and final improvement of histological and functional injury. Overall, the data that obtained in rat model provide novel insights into the pathogenesis of neonatal HI brain injury and may be translational to human clinical intervention for HI-associated brain injury in newborns.

Studies on cerebral protection of digoxin against hypoxic-ischemic brain damage in neonatal rats.

Hypoxic-ischemic brain damage (HIBD) is a major cause of neonatal acute deaths and chronic nervous system damage. Our present study was designed to investigate the possible neuroprotective effect of digoxin-induced pharmacological preconditioning after hypoxia-ischemia and underlying mechanisms. Neonatal rats were assigned randomly to control, HIBD, or HIBD+digoxin groups. Pharmacological preconditioning was induced by administration of digoxin 72 h before inducing HIBD by carotid occlusion+hypoxia. Behavioral assays, and neuropathological and apoptotic assessments were performed to examine the effects; the expression of Na/K ATPase was also assessed. Rats in the HIBD group showed deficiencies on the T-maze, radial water maze, and postural reflex tests, whereas the HIBD+digoxin group showed significant improvements on all behavioral tests. The rats treated with digoxin showed recovery of pathological conditions, increased number of neural cells and proliferative cells, and decreased number of apoptotic cells. Meanwhile, an increased expression level of Na/K ATPase was observed after digoxin preconditioning treatment. The preconditioning treatment of digoxin contributed toward an improved functional recovery and exerted a marked neuroprotective effect including promotion of cell proliferation and reduction of apoptosis after HIBD, and the neuroprotective action was likely associated with increased expression of Na/K ATPase.

Transplantation of vascular endothelial growth factor-modified neural stem/progenitor cells promotes the recovery of neurological function following hypoxic-ischemic brain damage.

Neural stem/progenitor cell (NSC) transplantation has been shown to effectively improve neurological function in rats with hypoxic-ischemic brain damage. Vascular endothelial growth factor (VEGF) is a signaling protein that stimulates angiogenesis and improves neural regeneration. We hypothesized that transplantation of VEGF-transfected NSCs would alleviate hypoxic-ischemic brain damage in neonatal rats. We produced and transfected a recombinant lentiviral vector containing the VEGF165 gene into cultured NSCs. The transfected NSCs were transplanted into the left sensorimotor cortex of rats 3 days after hypoxic-ischemic brain damage. Compared with the NSCs group, VEGF mRNA and protein expression levels were increased in the transgene NSCs group, and learning and memory abilities were significantly improved at 30 days. Furthermore, histopathological changes were alleviated in these animals. Our findings indicate that transplantation of VEGF-transfected NSCs may facilitate the recovery of neurological function, and that its therapeutic effectiveness is better than that of unmodified NSCs.

Involvement of the JNK/FOXO3a/Bim Pathway in Neuronal Apoptosis after Hypoxic-Ischemic Brain Damage in Neonatal Rats.

c-Jun N-terminal kinase (JNK) plays a key role in the regulation of neuronal apoptosis. Previous studies have revealed that forkhead transcription factor (FOXO3a) is a critical effector of JNK-mediated tumor suppression. However, it is not clear whether the JNK/FOXO3a pathway is involved in neuronal apoptosis in the developing rat brain after hypoxia-ischemia (HI). In this study, we generated an HI model using postnatal day 7 rats. Fluorescence immunolabeling and Western blot assays were used to detect the distribution and expression of total and phosphorylated JNK and FOXO3a and the pro-apoptotic proteins Bim and CC3. We found that JNK phosphorylation was accompanied by FOXO3a dephosphorylation, which induced FOXO3a translocation into the nucleus, resulting in the upregulation of levels of Bim and CC3 proteins. Furthermore, we found that JNK inhibition by AS601245, a specific JNK inhibitor, significantly increased FOXO3a phosphorylation, which attenuated FOXO3a translocation into the nucleus after HI. Moreover, JNK inhibition downregulated levels of Bim and CC3 proteins, attenuated neuronal apoptosis and reduced brain infarct volume in the developing rat brain. Our findings suggest that the JNK/FOXO3a/Bim pathway is involved in neuronal apoptosis in the developing rat brain after HI. Agents targeting JNK may offer promise for rescuing neurons from HI-induced damage.

Oxymatrine attenuated hypoxic-ischemic brain damage in neonatal rats via improving antioxidant enzyme activities and inhibiting cell death

Oxymatrine (OMT), an active constituent of Chinese herb Sophora flavescens Ait, has been proved to possess anti-tumor, anti-oxidant, anti-inflammatory, and anti-apoptotic activities. Previous study has demonstrated that OMT had protective roles on multiple in vitro and in vivo brain injury models including regulation of apoptosis-related proteins caspase-3, Bax and Bcl-2. In this study, we investigated whether this protective effect could apply to neonatal hypoxic-ischemic brain damage. Seven-day-old Sprague–Dawley rats were treated with the left carotid artery ligation followed by exposure to 8% oxygen (balanced with nitrogen) for 2.5 h at 37 °C. In sham group rats, neither ligation nor hypoxia was performed. After two successive days intraperitoneal injection with OMT (30, 60 and 120 mg/kg), Nimodipine (1 mg/kg), and saline, brain infarct volume was estimated, histomorphology changes were performed by hematoxylin–eosin (HE) staining as well as electron microscopy. In addition, the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and total antioxidant capacity (T-AOC), as well as production of malondialdehyde (MDA) were assayed in ipsilateral hemisphere homogenates to evaluate the redox status after hypoxic-ischemic. Expression of apoptosis-related proteins Caspase-3, Bax and Bcl-2 in brain were analyzed by western-blot analysis and immunofluorescence. Administration of OMT significantly decreased brain infarct volume and the percentage of injured cells, and ameliorated histopathology and morphological injury as well. Furthermore, OMT obviously increased the activities of SOD, GSH-Px, CAT and T-AOC, and decreased MDA content. Western-blot analysis showed a marked decrease in Caspase-3 expression and increase in the ratio of Bcl-2/Bax after OMT (120 mg/kg) post-treatment as compared with hypoxic-ischemic group. These results suggest that OMT exerts a neuroprotective effect against hypoxic-ischemic brain damage in neonatal rats, which is likely to be mediated through increasing anti-oxidant enzyme activities and inhibiting cell death.

Effect of ischemic postconditioning on cerebral edema and the AQP4 expression following hypoxic-eschemic brain damage in neonatal rats.

A rat model for neonatal hypoxic-ischemic brain damage (HIBD) was established to observe the effect of ischemic postconditioning (IPostC) on cerebral edema and the AQP4 expression following HIBD and to verify the neuroprotection of IPostC and the relationship between changes of AQP4 expression and cerebral edema. Water content was measured with dry-wet method, and AQP4 transcription and the protein expression of the lesions were detected with real-time PCR and immunohistochemistry staining, respectively. Within 6-48 hours, the degree of ipsilateral cerebral edema was significantly lower in IPostC-15 s/15 s group than in HIBD group. Similar to the HIBD group, the AQP4 transcription and expression in the IPostC group showed a downward and then upward trend. But the expression was still more evident in the HIBD group than in the IPostC-15 s/15 s group. From 24 to 48 hours, IPostC-15 s/15 s decreased the slowing down expression of AQP4. IPostC has neuroprotective effect on neonatal rats with HIBD and it may relieve cerebral edema by regulating the expression of AQP4.

Melatonin ameliorates brain injury induced by systemic lipopolysaccharide in neonatal rats

Our previous study showed that lipopolysaccharide (LPS)-induced brain injury in the neonatal rat is associated with nitrosative and oxidative stress. The present study was conducted to examine whether melatonin, an endogenous molecule with antioxidant properties, reduces systemic LPS-induced nitrosative and oxidative damage in the neonatal rat brain. Intraperitoneal (i.p.) injection of LPS (2mg/kg) was administered to Sprague–Dawley rat pups on postnatal day 5 (P5), and i.p. administration of melatonin (20mg/kg) or vehicle was performed 5min after LPS injection. Sensorimotor behavioral tests were performed 24h after LPS exposure, and brain injury was examined after these tests. The results show that systemic LPS exposure resulted in impaired sensorimotor behavioral performance, and acute brain injury, as indicated by the loss of oligodendrocyte immunoreactivity and a decrease in mitochondrial activity in the neonatal rat brain. Melatonin treatment significantly reduced LPS-induced neurobehavioral disturbances and brain damage in neonatal rats. The neuroprotective effect of melatonin was associated with attenuation of LPS-induced nitrosative and oxidative stress, as indicated by the decreased nitrotyrosine- and 4-hydroxynonenal-positive staining in the brain following melatonin and LPS exposure in neonatal rats. Further, melatonin significantly attenuated LPS-induced increases in the number of activated microglia in the neonatal rat brain. The protection provided by melatonin was also associated with a reduced number of inducible nitric oxide synthase (iNOS)+ cells, which were double-labeled with ED1 (microglia). Our results show that melatonin prevents the brain injury and neurobehavioral disturbances induced by systemic LPS exposure in neonatal rats, and its neuroprotective effects are associated with its impact on nitrosative and oxidative stress.

Creating rat model for hypoxic brain damage in neonates by oxygen deprivation.

Current study explores the feasibility of using a non-surgical method of oxygen deprivation to create Hypoxic brain damage in neonatal rats for medical studies. 7-day-old Sprague Dowley (SD) rats were kept in a container with low oxygen level (8%) for 1.5h. A second group had bilateral cephalic artery ligation before the 1.5h-low oxygen treatment, a method similar to the popular Rice method, to expose the brain to both hypoxic and ischemic situations. Short term neural functions and brain water weights were evaluated 1 day after the hypoxic treatment. Brain pathology and histology were also examined at 1 day and 3 days after the hypoxic treatment. Both groups showed impaired neural functions and increased brain water weight compared to the controls. Histology studies also revealed injuries in the subcortex, hippocampus and lateral ventricle in the brains from both groups. There is no significant difference in the degree of brain damages observed in the two groups. Our work demonstrated that oxygen deprivation alone is sufficient to cause brain damages similar to those seen in Hypoxic-ischemic brain disease (HIBD). Because this method avoids the invasive surgical procedure and therefore reduces the stress and mortality of laboratory animals during the experiment, we recommend it to be the favorable method for creating rat models for HIBD studies.

Resveratrol for prenatal-stress-induced oxidative damage in growing brain and its consequences on survival of neurons

Prenatal-stress-induced neuronal damage in offspring is multifactorial, including oxidative damage in the developing brain. Resveratrol is known to exert its neuroprotective potentials by upregulating several antioxidant systems. Hence, the study was undertaken to evaluate the neuroprotective effect of resveratrol against prenatal-stress-induced hippocampal damage and oxidative damage in neonate rat brains. Pregnant rats were subjected to restraint stress during early or late gestational period. Another set of rats received resveratrol during the entire gestational period along with early or late gestational stress. The study parameters included several antioxidant studies directly from rat brain homogenate on the 40th postnatal day and hippocampal neuronal assay on the 21st postnatal day. Early as well as late gestational stress resulted in a significant increase in lipid peroxidation and advanced oxidation protein products and decrease in total antioxidant activity and nitric oxide levels in rat brain homogenate. The neurons of the dentate gyrus were severely affected in early and late gestational stress, and only the neurons of the CA3 region were adversely affected in late gestational stress. Administration of resveratrol reversed the prenatal-stress-induced oxidative damage and neurons of dentate gyrus but not the CA3 hippocampal neurons. These results show the neuroprotective abilities of resveratrol against prenatal-stress-induced oxidative damage in neonatal rat brain.

The effect of red light on the expression of apoptosis factors during acute hypoxic-ischemic brain damage in neonatal rats

Objective To investigate the effect of red light on the expression of caspase-8 and caspase-3 mRNA and protein during the acute stage of hypoxic-ischemic brain damage (HIBD) in neonatal rats.Methods Forty-five seven-day-old Sprague-Dawley rats were randomly divided into a sham group,an HIBD model group (model group) and an irradiation group.The rats in the model and irradiation groups were subject to HIBD induced using the Rice-Vannucci method.The irradiation group was treated by irradiation with red light on the forehead immediately after the establishment of the HIBD model for 30 min/d on 3 consecutive days,while the sham group and the model group received no treatment.On the 3rd day after the operation,10 rats from each group were sacrificed by cervical dislocation and the left hippocampus was rapidly isolated and snap-frozen in liquid nitrogen for mRNA and protein examination.Another 5 rats in each group were used for immunofluorescence testing to localize and semi-quantify the expression of caspase-8 and caspase-3.Results In the model group,expression of caspase-8 and caspase-3 mRNA and protein in the left hippocampus was higher than in the sham group on the 3rd day.After red light irradiation,expression of caspase-8 and caspase-3 mRNA and protein decreased significantly in the irradiation group compared with the HIBD group.In the CA 1 region of the hippocampus,the levels of caspase-8 and caspase-3 in the irradiation group were significantly lower than in the model group.Conclusion Red light irradiation can decrease the expression of such apoptosis factors as caspase-8 and caspase-3 in hypoxic-ischemic brain damage at the acute stage and inhibit neural cell apoptosis so as to exert therapeutic effects for hypoxic-ischemic brain damage. Key words: Hypoxia; Ischemia; Brain damage; Red light; Apoptosis

Involvement of the Akt/GSK-3β/CRMP-2 pathway in axonal injury after hypoxic–ischemic brain damage in neonatal rat

Akt has been demonstrated as a survival kinase in brain after hypoxia–ischemia (HI). Previous studies have shown that glycogen synthase kinase-3β (GSK-3β)/collapsin response mediator protein 2 (CRMP-2) signaling pathway could be regulated by Akt for axonal–dendritic polarity. CRMP-2 is associated also with microtubule-mediated trafficking. However, whether Akt could regulate GSK-3β/CRMP-2 pathway and the possible effects of this regulation is unclear in developing brain after HI. In this study, we detected the expression of total and phosphorylated Akt, GSK-3β, and CRMP-2, as well as the axonal injury marker amyloid precursor protein (APP) by utilizing an HI model in postnatal 10-day rats. Axonal loss was determined by Bielschowsky silver impregnation, and histological injury was evaluated by hematoxylin and eosin (H&E) staining. We found that the phosphorylation of Akt was accompanied by phosphorylation of GSK-3β and dephosphorylation of CRMP-2 after HI. Furthermore, Akt inhibition significantly decreased the phosphorylation of GSK-3β and dephosphorylation of CRMP-2. Moreover, the down-regulation of dephosphorylated CRMP-2 was associated with increased axonal injury (increased APP expression and axonal loss). Our findings suggest that the Akt/GSK-3β/CRMP-2 pathway mediates axonal injury in neonatal rat brain after HI.

Dexamethasone and betamethasone protect against lipopolysaccharide-induced brain damage in neonatal rats

The aim of this study is to test whether dexamethasone (Dex) and betamethasone (Beta), two of the most commonly used corticosteroids, protect against lipopolysaccharide (LPS)-induced white matter damage and neurobehavioral dysfunction. LPS or sterile saline was injected into the brain white matter of rat pups at postnatal day 5 (P5) and Dex or Beta was given intraperitoneally to the rat pups 1 h before the LPS microinjection. Brain inflammatory response, brain damage, and myelination were examined at P6, P8 and P14. Neurobehavioral tests were performed from P3 through P22. Our results demonstrate that Dex and Beta markedly diminish the LPS-induced brain inflammatory response, restore myelin basic protein (MBP) expression and alleviate lateral ventricle dilation. Both corticosteroids demonstrate significant protection against most of LPS-induced behavioral deficits, including those in rearing, vibrissa-elicited forelimb-placing, beam walking, learning and elevated plus-maze test. Notably, only Beta improved the locomotion and stereotype dysfunction. In contrast to their beneficial effects, neither drug prevented LPS-induced delay in body weight gain from P6 through P21. Our study suggests that if their adverse effects are minimized, corticosteroids may be the potential candidate drugs to prevent brain damage in premature infants.