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Objective: To study the effect and mechanism of action of irisin on hypoxic-ischemic brain damage in neonatal rats., Methods: A total of 248 7-day-old Sprague-Dawley rats were randomly divided into a sham-operation group, a model group, and low- and high-dose irisin intervention groups (n=62 each). The rats in the model and irisin intervention groups were given hypoxic treatment after right common carotid artery ligation to establish a model of hypoxic-ischemic brain damage. Those in the sham-operation group were given the separation of the right common carotid artery without ligation or hypoxic treatment. The rats in the high- and low-dose irisin intervention groups were given intracerebroventricular injection of recombinant irisin polypeptide at a dose of 0.30 µg and 0.15 µg respectively. Those in the model and sham-operation groups were given the injection of an equal volume of PBS. The water maze test was used to compare neurological behaviors between groups. TTC staining, hematoxylin-eosin staining and TUNEL staining were used to observe histopathological changes of the brain. Western blot was used to measure the expression of the apoptosis-related molecules cleaved-caspase-3 (CC3), BCL-2 and BAX., Results: Compared with the sham-operation group, the model group had a significant increase in latency time and a significant reduction in the number of platform crossings (P<0.05). Compared with the model group, the high-dose irisin intervention group had a significant reduction in latency time and a significant increase in the number of platform crossings (P<0.05). Compared with the sham-operation group, the model group had massive infarction in the right hemisphere, with significant increases in karyopyknosis and karyorrhexis. Compared with the model group, the high-dose irisin intervention group had a smaller infarct area of the right hemisphere, with reductions in karyopyknosis and karyorrhexis. The model group had a significantly higher apoptosis rate of cells in the right cerebral cortex and the hippocampus than the sham-operation group. The high-dose irisin intervention group had a significantly lower apoptosis rate than the model group (P<0.05). At 24 and 48 hours after modeling, the sham-operation group had a significantly lower level of CC3 than the model group (P<0.05). Compared with the model group, the high-dose irisin intervention group had a significantly lower level of CC3 and a significantly higher BCL-2/BAX ratio (P<0.05). The low-dose irisin intervention group had similar laboratory markers and histopathological changes of the brain to the model group., Conclusions: Irisin can alleviate hypoxic-ischemic brain damage in neonatal rats in a dose-dependent manner, possibly by reducing cell apoptosis in the cerebral cortex and the hippocampus.

Objective: To investigate the expression of autophagic gene and circadian gene in the neurons of neonatal rats after hypoxic-ischemic brain damage and the mechanism of nerve injury induced by hypoxia/ischemia., Methods: Twelve Sprague-Dawley (SD) rats were randomly divided into hypoxic-ischemic (HI) group and sham-operation group, with 6 rats in each group. Ligation of the right common carotid artery and hypoxic treatment were performed to establish a model of hypoxic-ischemic brain damage. Western blot was used to measure the expression of the circadian protein Clock in the cortex and hippocampus. The neurons of the rats were cultured in vitro and randomly divided into oxygen glucose deprivation (OGD) group and control group. The neurons in the OGD group were treated with DMEM medium without glucose or serum to simulate ischemic state, and hypoxic treatment was performed to establish an in vitro model of hypoxic-ischemic brain damage. Western blot was used to measure the expression of autophagy-related proteins Beclin1 and LC3 and Clock protein at different time points. The changes in the expression of Beclin1 and LC3 were measured after the expression of Clock protein in neurons was inhibited by small interfering RNA technique., Results: The expression of autophagy-related proteins Beclin1 and LC3Ⅱ in neurons cultured in vitro displayed a rhythmic fluctuation; after OGD treatment, the expression of Beclin1 and LC3Ⅱ gradually increased over the time of treatment and no longer had a rhythmic fluctuation. Compared with the sham-operation group, the HI group had a significant reduction in the expression of Clock protein in the cortex and hippocampus (P<0.05). After OGD treatment, the neurons cultured in vitro had a significant reduction in the expression of Clock protein (P<0.05). Compared with the negative control group, the Clock gene inhibition group had significant reductions in the expression of Beclin1 and LC3Ⅱ (P<0.05)., Conclusions: Hypoxia/ischemia induces the disorder in the expression rhythm of autophagy-related proteins Beclin1 and LC3, and the mechanism may be associated with the fact that the circadian protein Clock participates in the regulation of the expression of Beclin1 and LC3.

Objective: To investigate the effect of telomerase activation on biological behaviors of neural stem cells after hypoxic-ischemic insults., Methods: The neural stem cells passaged in vitro were divided into four groups: control, oxygen-glucose deprivation (OGD), OGD+cycloastragenol (CAG) high concentration (final concentration of 25 μM), and OGD+CAG low concentration (final concentration of 10 μM). The latter three groups were subjected to OGD. Telomerase reverse transcriptase (TERT) expression level was evaluated by Western blot. Telomerase activity was detected by telomerase repeat amplification protocol (TRAP). Cell number and neural sphere diameter were measured under a microscope. The activity of lactate dehydrogenase (LDH) was examined by chemiluminescence. Cell proliferation rate and apoptosis were detected by flow cytometry., Results: After OGD insults, obvious injury of neural stem cells was observed, including less cell number, smaller neural sphere, more dead cells, lower proliferation rate and decreased survival rate. In CAG-treated groups, there were higher TERT expression level and telomerase activity compared with the control group (P<0.05). In comparison with the OGD group, CAG treatment attenuated cell loss (P<0.05) and neural sphere diameter decrease (P<0.05), promoted cell proliferation (P<0.05), and increased cell survival rate (P<0.05). Low and high concentrations of CAG had similar effects on proliferation and survival of neural stem cells (P>0.05). In the normal cultural condition, CAG treatment also enhanced TERT expression (P<0.05) and increased cell numbers (P<0.05) and neural sphere diameter (P<0.05) compared with the control group., Conclusions: Telomerase activation can promote the proliferation and improve survival of neural stem cells under the state of hypoxic-ischemic insults, suggesting telomerase activators might be potential agents for the therapy of hypoxic-ischemic brain injury.

Objective: To study the effect of telomerase reverse transcriptase (TERT) on cell apoptosis in neonatal rat brains after hypoxic-ischemic brain injury (HIBD)., Methods: A total of 72 neonatal rats were divided into sham, vehicle, HIBD and TERT groups. HIBD was induced by Rice method in the later three groups. The neonatal rats in the vehicle and TERT groups were injected with plasmids containing mock or full length TERT by an intracerebroventricular injection 30 minutes after hypoxic-ischemic (HI) injury. Pathological changes of brain tissue were observed by hematoxylin and eosin (HE) staining. Western blot was used to detect the protein expression of TERT, apoptosis-inducing factor (AIF) and cleaved caspase 3 (CC3). Apoptotic cells were detected by TUNEL staining., Results: Western blot showed that TERT protein was dramatically increased in the vehicle, HIBD and TERT groups compared with the sham group. Compared with the vehicle and HIBD groups, TERT protein in the TERT group was significantly upregulated. Compared with the sham group, there was a significant increase in apoptotic index and expression of AIF and CC3 proteins in the vehicle and HIBD groups (p<0.01). The TERT group showed decreased expression of AIF and CC3 proteins and apoptotic index compared with the vehicle and HIBD groups (p<0.01)., Conclusions: TERT can inhibit cell apoptosis induced by HI and might have a neuroprotective role in developing brain with HIBD.

Long non-coding RNAs (lncRNAs) are transcripts with a complex structure and a length of >200 nt which are unable to encode proteins. The lncRNAs interact with DNA, mRNA, and proteins and regulate gene expression through various mechanisms, thus participating in the regulation of various biological processes. Studies have shown that lncRNAs play important roles in neural development and the pathogenesis of diseases. This article reviews the roles of lncRNAs in hypoxic-ischemic brain damage.

Objective: To study the effect of PINK1 (phosphatase and tensin homolog deleted on chromosome ten induced putative kinase 1) gene on cell apoptosis and cell autophagy in neonatal mice with hypoxic-ischemic brain damage (HIBD)., Methods: Seventy-two wild-type C57BL/6 mice and 72 PINK1 gene knockout neonatal C57BL/6 mice were randomly divided into four groups: sham-operated wild-type (SWT), HIBD model wild-type (MWT), sham-operated knockout (SKO) and HIBD model knockout (MKO). HIBD model was prepared by low oxygen exposure for 2.5 hours after right carotid artery ligation. After 24 hours of hypoxia-ischemia treatment, TTC (2,3,5-triphenyl four azole nitrogen chloride) staining was used to measure brain infarct volume. The immunohistochemical staining was used to measure the expression of cell apoptosis protein cleaved-caspase-3 (CC3) in brain tissues. The TUNEL method was used to measure cell apoptosis. The immunofluorescence staining and Western blot were used to measure the expression of cell autophagy protein LC3., Results: Compared with the MWT group, the infarct volume of brain tissues was markedly reduced in the MKO group (P<0.05), the number of apoptotic cells and the cell apoptosis index were markedly decreased in the MKO group (P<0.05), the expression of apoptosis protein CC3 was significantly reduced in the MKO group (P<0.05), the expression of cell autophagy protein LC3 was significantly decreased in the MKO group, and the autophagy indicator LC3II/LC3I was also markedly reduced in the MKO group (P<0.05)., Conclusions: PINK1 gene knockout can protect neonatal mice from HIBD.

Objective: To study the role and mechanisms of STAT3 signaling pathway in hypoxic-ischemic brain damage (HIBD) of neonatal rats., Methods: Eighty 7-day-old Sprague-Dawley rats were randomly divided into two groups: HI and sham-operated (n=40 each). The rats in the HI group were subjected to right carotid artery ligation and subsequent hypoxia exposure (8% O2) for 2.5 hours, and the rats in the sham-operated group underwent the right carotid artery dissection without subsequent ligation or hypoxia treatment. Brain tissue samples were collected at 4, 6, 8, 12 and 24 hours after operation and hypoxic exposure. Immunohistochemistry and Western blot were used to detect the expression of STAT3, phosphorylated STAT3 (p-STAT3) and vascular endothelial growth factor (VEGF) proteins. TUNEL staining was used to detect apoptotic cells., Results: No significant difference in STAT3 expression was observed at all time points between the HI and sham-operated groups (P>0.05). Compared with the sham-operated group, the expression of p-STAT3 protein in the HI group was significantly upregulated at 4, 6, 8, 12 hours after operation and hypoxic exposure, and peaked at 6 hours (P<0.01). The VEGF expression in the HI group was higher than that in the sham-operated group at all time points, which peaked at 8 hours (P<0.05). TUNEL staining showed that the apoptotic cells increased significantly in a time-dependent manner compared with the sham-operated group (P<0.01)., Conclusions: HI may lead to phosphorylation of STAT3 which probably induces the VEGF expression in the brain of neonatal rats. The activated STAT3 signaling pathway may be involved in the apoptosis regulation of nerve cells, and related to apoptosis inhibition of nerve cells.

Objective: To investigate the effects of dimethyloxalyl glycine on hypoxic-ischemic brain damage in newborn rats., Methods: Forty eight postnatal day 10 SD rats were divided into 3 groups, including sham surgery group, hypoxic-ischemic group and DMOG treated group. The brain tissues were collected at 4, 8, 24 and 72 hours after the hypoxic-ischemic treatment. The expressions of hypoxia inducible factor-1alpha (HIF-1alfa) protein and anti apoptoticprotein cleaved caspase 3 (CC3) were detected by immunohistochemistry. The apoptotic cells were detected by TUNEL staining., Results: The expression level of HIF-1alpha was significantly higher in DMOG treated group than in hypoxic-ischemic group. While the expression level of CC3 was lower and the number of tunel positive cells was fewer in DMOG treated group than that in hypoxic-ischemic group., Conclusion: Dimethyloxalyl glycine may play a neuro-protective role in hypoxic-ischemic brain damage in newborn rats by stabilizing HIF-1alpha.

Objective: To investigate the relationship between the expression of glucose transporter protein 1 and the apoptosis of neuron during hypoxic-ischemia brain damage in neonatal rats., Methods: Total 120 10-day old SD rats were divided into normal group, sham control group and hypoxic-ischemia (HI) group. In HI group, hypoxic-ischemia brain damage (HIBD) were generated according to Rice-Vannucci method, brain tissues were harvested at 2, 8, 24, 48 and 72 h after HI. The brain samples were also collected at the same time points in normal group and sham control group. The pathological changes was observed by hematoxylin-eosin (HE) staining, the mRNA expression of glucose transporter 1 (GLUT1) was detected by reverse transcriptase-polymerase chain reaction (RT-PCR), the protein expressions of GLUT1 and cleaved caspase-3 (CC3) were detected by immunohistochemistry, the apoptosis of neuron was measured by TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining., Results: HE staining showed that the degree of brain cell damage increased with time after HI, the loss of neuronal cells peaked at 48 h, while the cells in control group apperanted in an orderly and normal morphology. The mRNA and protein expressions of GLUT1 were increased after HI, which began to increase at 2 h, and reach the peak at 24 h. and the expression levels at each time points were statistically higher (P< 0.01) than those in control group. CC3 protein expression also began to increase at 2 h, peaked at 48 h after HI, which was higher than that of control group (P<0.01). The number of positive cells was significantly increased after HI,with a peak at 48 h., Conclusion: The mRNA and protein expression of GLUT1 in brain tissue increased significantly after hypoxic-ischemia, and the peak time was earlier than that of CC3 protein and cellular apoptosis. This suggests that GLUT1 expression upregulation may be a certain degree of inhibition on neuronal apoptosis.

Purpose: To compare the apparent diffusion coefficients (ADCs) and histologic changes in young rats subjected to cerebral ischemic hypoxia (IH)., Materials and Methods: Fifteen 3-week-old rats were subjected to a 30-minute IH insult (unilateral common carotid arterial ligation and exposure to 8% oxygen) and were examined at diffusion-weighted magnetic resonance imaging and light and electron microscopy on cessation of the insult (n = 5), 60 minutes after resuscitation (n = 5), or 48 hours after resuscitation (n = 5). Twelve control rats either underwent unilateral common carotid arterial ligation or were subjected to hypoxia., Results: The experimental rats showed primary ADC reduction during the insult, transient ADC recovery after resuscitation, and secondary ADC reduction 48 hours after the insult. Histologic examination revealed dendritic swelling and mild swelling of the perivascular astrocytic end-feet during the primary ADC reduction phase, dark neurons and pronounced swelling of the perivascular astrocytic end-feet during the transient ADC recovery phase, and severely retracted dark neurons and extensive swelling of the astrocytic end-feet during the secondary ADC reduction phase., Conclusion: Transient ADC normalization after cerebral IH does not necessarily mean that histologic normalization has occurred. The transient ADC recovery phase appeared to have limited potential for neuronal salvage.