Hypoxic-ischemic Brain Damage Group Research Articles

Gastrodin alleviates microglia-mediated inflammatory responses in neonatal mice with hypoxic-ischemic brain damage by regulating CCR5/AKT signaling

To investigate the mechanism behind the protective effects of gastrodin against microglia-mediated inflammatory responses following hypoxic-ischemic brain damage (HIBD) in neonatal mice. Thirty-six 10-day-old C57BL/6J mice were randomized into sham-operated group, HIBD (induced by ligation of the left common carotid artery followed by hypoxia for 40 min) group, and HIBD with gastrodin treatment groups (n=12). In gastrodin treatment group, 100 mg/kg gastrodin was injected intraperitoneally 1 h before and at 2 and 12 h after hypoxia. After the treatments, the expressions of CCR5, AKT, p-AKT, and TNF-α and the co-expression of IBA1 and CCR5 in the corpus callosum of the mice were detected with Western blotting and immunofluorescence double staining. In a BV2 microglial cell model of oxygen-glucose deprivation (OGD), the effects of pretreatment with gastrodin and Maraviroc (an CCR5 antagonist) on protein expressions of CCR5, AKT, p-AKT, TNF-α and IL-1β were evaluated using Western blotting and immunofluorescence double staining. The neonatal mice with HIBD showed significantly increased expressions of CCR5 and TNF-α with lowered p-AKT expression in the brain tissues, and GAS treatment obviously reversed these changes. HIBD also significantly increased the co-expression of IBA1 and CCR5 in the corpus callosum of the mice, which was obviously lowered by gastrodin treatment. In BV2 cells, OGD significantly increased the expressions of CCR5, TNF-α, and IL-1β and decreased the expression of p-AKT, and these changes were inhibited by treatment with gastrodin, Maraviroc or their combination; the inhibitory effect of the combined treatment did not differ significantly from that of gastrodin or Maraviroc alone. Gastrodin can produce neuroprotective effects in neonatal mice with HIBD by inhibiting inflammatory cytokine production and activate AKT phosphorylation via inhibiting CCR5.

Gastrodin improves microglia-mediated inflammatory response after hypoxic-ischemic brain damage in neonatal rats via PI3K/AKT pathway

To investigate the mechanism of gastrodin for inhibiting microglia-mediated inflammation after hypoxicischemic brain damage (HIBD) in neonatal rats. Thirty-nine 3-day-old SD rats were randomly divided into sham group, HIBD group and gastrodin treatment group. Western blotting was used to detect the expressions of TNF-α, IL-1β, IL-10 and TGF- β1 in the corpus callosum of the rats. The potential targets of gastrodin for treatment of HIBD were screened by network pharmacology analysis. The expressions of PI3K/AKT signaling pathway proteins following HIBD-induced microglial activation in the rats and in cultured microglial BV-2 cells with oxygen-glucose deprivation (OGD) were detected with Western blotting. The effects of LY294002 (a specific inhibitor of the PI3K/AKT pathway) and gastrodin on TNF-α and TGF-β1 mRNA levels in BV-2 cells with OGD was detected with RT-qPCR. In the neonatal rats with HIBD, gastrodin treatment significantly decreased TNF-α and IL-1β expressions and enhanced IL-10 and TGF-β1 expressions in the ischemic corpus callosum. Network pharmacology analysis showed significant enrichment of the PI3K/AKT signaling pathway and a strong binding between gastrodin and PI3K. Gastrodin significantly promoted PI3K and AKT phosphorylation in neonatal rats with HIBD and in BV-2 cells exposed to OGD. In BV-2 cells with OGD, gastrodin obviously suppressed OGD-induced increase of TNF-α and reduction of TGF-β1 mRNA expressions, and this effect was strongly attenuated by LY294002 treatment. Gastrodin can inhibit microglia-mediated inflammation in neonatal rats with HIBD by regulating the PI3K/AKT signaling pathway.

Identification and analysis of oxidative stress-related genes in hypoxic-ischemic brain damage using bioinformatics and experimental verification.

Oxidative stress (OS) plays a major role in the progress of hypoxic-ischemic brain damage (HIBD). This study aimed to investigate OS-related genes and their underlying molecular mechanisms in neonatal HIBD. Microarray data sets were acquired from the Gene Expression Omnibus (GEO) database to screen the differentially expressed genes (DEGs) between control samples and HIBD samples. OS-related genes were drawn from GeneCards and OS-DEGs in HIBD were obtained by intersecting with the DEGs. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were conducted to determine the underlying mechanisms and functions of OS-DEGs in HIBD. Moreover, the hub genes were screened using the protein-protein interactionnetwork and identified in the GSE144456 data set. CIBERSORTwas then performed to evaluate the expression of immunocytes in each sample and perform a correlation analysis of the optimal OS-DEGs and immunocytes. Finally,quantitative reverse transcription polymerase chain reaction(RT-qPCR) and immunohistochemistry were performed to validate the expression levels of the optimal OS-DEGs. In total, 93 OS-DEGs were identified. GO, KEGG, and GSEA enrichment analyses indicated that these genes were predominantly enriched in OS and inflammation. Four OS-related biomarker genes (Jun, Fos, Tlr2, and Atf3) were identified and verified. CIBERSORT analysis revealed the dysregulation of six types of immune cells in the HIBD group. Moreover, 47 drugs that might target four OS-related biomarker genes were screened. Eventually, RT-qPCR and immunohistochemistry results for rat samples further validated the expression levels of Fos, Tlr2, and Atf3. Fos, Tlr2 and Atf3 are potential OS-related biomarkers of HIBD progression. The mechanisms of OS are associated with those of neonatal HIBD.

Melatonin alleviates autophagy in cortical neurons of neonatal rats with hypoxic- ischemic brain damage via the PI3K/AKT pathway

To observe the effects of melatonin on autophagy in cortical neurons of neonatal rats with hypoxic-ischemic brain damage (HIBD) and to explore its mechanisms via the PI3K/AKT signaling pathway, aiming to provide a basis for the clinical application of melatonin. Seven-day-old Sprague-Dawley neonatal rats were randomly divided into a sham operation group, an HIBD group, and a melatonin group (n=9 each). The neonatal rat HIBD model was established using the classic Rice-Vannucci method. Neuronal morphology in the neonatal rat cerebral cortex was observed with hematoxylin-eosin staining and Nissl staining. Autophagy-related protein levels of microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 were detected by immunofluorescence staining and Western blot analysis. Phosphorylated phosphoinositide 3-kinase (p-PI3K) and phosphorylated protein kinase B (p-AKT) protein expression levels were measured by immunohistochemistry and Western blot. The correlation between autophagy and the PI3K pathway in the melatonin group and the HIBD group was analyzed using Pearson correlation analysis. Twenty-four hours post-modeling, neurons in the sham operation group displayed normal size and orderly arrangement. In contrast, neurons in the HIBD group showed swelling and disorderly arrangement, while those in the melatonin group had relatively normal morphology and more orderly arrangement. Nissl bodies were normal in the sham operation group but distorted in the HIBD group; however, they remained relatively intact in the melatonin group. The average fluorescence intensity of LC3 and Beclin-1 was higher in the HIBD group compared to the sham operation group, but was reduced in the melatonin group compared to the HIBD group (P<0.05). The number of p-PI3K+ and p-AKT+ cells decreased in the HIBD group compared to the sham operation group but increased in the melatonin group compared to the HIBD group (P<0.05). LC3 and Beclin-1 protein expression levels were higher, and p-PI3K and p-AKT levels were lower in the HIBD group compared to the sham operation group (P<0.05); however, in the melatonin group, LC3 and Beclin-1 levels decreased, and p-PI3K and p-AKT increased compared to the HIBD group (P<0.05). The correlation analysis results showed that the difference of the mean fluorescence intensity of LC3 and Beclin-1 protein in the injured cerebral cortex between the melatonin and HIBD groups was negatively correlated with the difference of the number of p-PI3K+ and p-AKT+ cells between the two groups (P<0.05). Melatonin can inhibit excessive autophagy in cortical neurons of neonatal rats with HIBD, thereby alleviating HIBD. This mechanism is associated with the PI3K/AKT pathway.

Mechanisms of ferroptosis in hypoxic-ischemic brain damage in neonatal rats

This study was to explore the mechanism of ferroptosis and hypoxic-ischemic brain damage in neonatal rats. The neonatal rat hypoxic-ischemic brain damage (HIBD) model was established using the Rice-Vannucci method and treated with the ferroptosis inhibitor liproxstatin-1. Cognitive assessment was performed through absentee field experiments to confirm the successful establishment of the model. Brain tissue damage was evaluated by comparing regional cerebral blood flow and quantifying tissue staining. Neuronal cell morphological changes in the rats' cortical and hippocampal regions were observed using HE and Nissl staining. ELISA was performed to determine GPX4, GSH and ROS expression levels in the rats' brain tissues, and Western blotting to assess the expression levels of 4-HNE, GPX4, GSS, ACSL4, SLC7A11, SLC3A2, TFRC, FHC, FLC, HIF-1α, and Nrf2 proteins in rat brain tissues. Compared to the Sham group, the HIBD group exhibited a significant decrease in cerebral blood perfusion, reduced brain nerve cells, and disordered cell arrangement. The use of the ferroptosis inhibitor effectively improved brain tissue damage and preserved the shape and structure of nerve cells. The oxidative stress products ROS and 4-HNE in the brain tissue of the HIBD group increased significantly, while the expression of antioxidant indicators GPX4, GSH, SLC7A11, and GSS decreased significantly. Furthermore, the expression of iron metabolism-related proteins TFRC, FHC, and FLC increased significantly, whereas the expression of the ferroptosis-related transcription factors HIF-1α and Nrf2 decreased significantly. Treatment with liproxstatin-1 exhibited therapeutic effects on HIBD and downregulated tissue ferroptosis levels. This study shows the involvement of ferroptosis in hypoxic-ischemic brain damage in neonatal rats through the System Xc−-GSH-GPX4 functional axis and iron metabolism pathway, with the HIF-1α and Nrf2 transcription factors identified as the regulators of ferroptosis involved in the HIBD process in neonatal rats.

Hypoxia ischemia results in blood brain barrier damage via AKT/GSK-3β/CREB pathway in neonatal rats

Previous studies have showed that the permeability of blood brain barrier (BBB) increased after hypoxia ischemia (HI). The current research uncovered the mechanism of altered BBB permeability after hypoxic-ischemic brain damage (HIBD) through AKT/GSK-3β/CREB signaling pathway in neonatal rats. Firstly, Magnetic resonance imaging (MRI) combined with hematoxylin-eosin (H&E) staining was used to assess brain injury. Initial findings showed abnormal signals in T2-weighted imaging (T2WI) and diffusion weighted imaging (DWI). Changes also happened in the morphology of nerve cells. Subsequently, we found that BBB damage is manifested as leakage of immunoglobulin G (IgG) and destruction of BBB-related proteins and ultrastructure. Meanwhile, the levels of matrix metalloproteinase-9 (MMP-9) significantly increased at 24 h after HIBD compared to a series of time points. Additionally, immunohistochemical (IHC) staining combined with Western blot (WB) was used to verify the function of the AKT/GSK-3β/CREB signaling pathway in BBB damage after HI in neonatal rats. Results showed that less Claudin-5, ZO-1, p-AKT, p-GSK-3β and p-CREB, along with more MMP-9 protein expression were visible on the damaged side of the cerebral cortex in the HIBD group in contrast to the sham and HIBD + SC79 groups. Together, our findings demonstrated that HI in neonatal rats might upregulate the levels of MMP-9 protein and downregulate the levels of Claudin-5 and ZO-1 by inhibiting the AKT/GSK-3β/CREB pathway, thus disrupting the BBB, which in turn aggravates brain damage after HI in neonatal rats.

Mfat-1 ameliorates cachexia after hypoxic-ischemic brain damage in mice by protecting the hypothalamus-pituitary-adrenal axis

AimsCachexia, a metabolic syndrome, affects 21 % of patients suffering from ischemic encephalopathy. However, the specific mechanism and prevention measures are still unclear. Omega-3 polyunsaturated fatty acids (n−3 PUFAs) have been proven to reduce inflammatory cytokine levels during ischemic events, but whether they have a protective effect against cachexia after hypoxic-ischemic brain damage (HIBD) remains unclear. Main methodsC57BL/6J wild-type and mfat-1 transgenic male mice were treated with and without HIBD. One day after HIBD, the epididymal white fat, gastrocnemius muscle and hypothalamus were weighed and analyzed the phenotypic changes. RNA sequencing was applied to gastrocnemius muscle to identify differential genes and pathways in HIBD groups. The effect of HPA axis on cachexia post-HIBD was examined via adrenalectomy, dexamethasone (0.1 mg/kg), and corticosterone injection (100 mg/kg). Key findingsThe results showed that the incidence of cachexia in mfat-1 mice, which produce high proportion of n−3 PUFAs, was significantly lower than that in wild-type mice post-HIBD. Cachexia-related factors, such as inflammation, muscle atrophy and lipid metabolism were significantly improved in mfat-1 HIBD. RNA sequencing revealed that catabolic and proteasome pathways were significantly downregulated. In hypothalamus, inflammatory cytokines, lipid peroxidation levels were reduced. Corticosterone, glucocorticoid receptor, and dexamethasone suppression test all showed that mfat-1 improved the dysfunction of the HPA axis post-HIBD. The present study elucidated for the first time that mfat-1 reduced HIBD-induced hyperactivation of the HPA axis in mice by reducing inflammation and oxidative stress and contributed to the reduction of metabolic imbalance in peripheral tissues. SignificanceOur study provides mechanistic information for the development of intervention strategies to prevent cachexia.

L-F001, a multifunctional fasudil-lipoic acid dimer, antagonizes hypoxic-ischemic brain damage by inhibiting the TLR4/MyD88 signaling pathway.

Neonatal hypoxic-ischemic brain damage (HIBD) is a serious inflammatory injury. At present, the standard treatment for this disease is hypothermia therapy, and the effect of drug intervention is still limited. L-F001 is a compound of fasudil and lipoic acid. Previous in vitro experiments have confirmed that L-F001 has anti-inflammatory neuroprotective functions. However, its therapeutic effect on neonates with HIBD remains unknown. This study was aimed at exploring the therapeutic effect of L-F001 on HIBD rats. The newborn rats were divided into three groups: Sham operation group, HIBD group, and HIBD+L-F001 group. HE staining, Nissil staining, the immunofluorescence of iNOS and COX-2, ELISA (IL-1β, IL-6, TNF-α, and IL-10), and western blotting analyses were performed to determine the therapeutic effect of L-F001. Finally, we evaluated the growth and development of each group by measuring body weight. The hippocampal structure of HIBD rats was disordered, and the Nissil body was small and shallow. The expressions of iNOS and COX-2 in HIBD rats were increased, whereas the expressions of IL-1β, IL-6, and TNF-α in plasma were upregulated, and the expression of IL-10 was decreased. L-F001 could improve the tissue structure and reduce the expression of iNOS and COX-2 in HIBD rats. Meanwhile, L-F001 could also reduce the expression of pro-inflammatory cytokines and restore the content of anti-inflammatory cytokines in plasma. We further found that the TLR4 pathway was activated after hypoxic-ischemia in neonatal rats. L-F001 could inhibit the activation of TLR4 pathway. Finally, we found that after L-F001 treatment, the body weight of HIBD rats increased significantly compared with the untreated group. L-F001 antagonizes the inflammatory response after hypoxic-ischemia by inhibiting the activation of the TLR4 signaling pathway, thus playing a neuroprotective role. L-F001 may be a potential therapeutic agent for neonatal HIBD.

Mild Hypoxia Postconditioning Induces Hypoxia-Inducible Factor 1 Alpha Expression and Improves the Neurobehavioral Abilities of Rats with Hypoxic-Ischemic Brain Damage

Background: While severe hypoxia is known to contribute to neurotoxicity and lead to abnormal behavior, mild hypoxia may have beneficial effects mediated through endogenous adaptive responses. Objectives: The present study aimed to investigate the effects of mild hypoxia postconditioning and long-term neurobehavioral ability rehabilitation after hypoxic-ischemic brain damage (HIBD). Methods: Seven-day-old rats underwent left carotid ligation followed by 2 hours of hypoxia stress. Rats received different protocols of mild hypoxia postconditioning for 5 days and underwent neurobehavioral testing during the last week of the study. Hypoxia-inducible factor 1 alpha (HIF-1a) expression was assessed, and neurobehavioral ability assays were performed. Results: Compared with the HIBD group, rats postconditioning with mild hypoxia showed increased HIF-1a expression, and their brain functions were better in neurobehavioral analyses. The rehabilitation of brain functions may be associated with high HIF-1a expression and better behavioral performance. Conclusions: Our findings indicate that mild hypoxia postconditioning improves neurobehavioral ability, and HIF-1a may be a potential mediator of the observed effects. Our findings suggest that there may be clinical implications for treating infants with HIBD.

Ferroptosis is Involved in Hypoxic-ischemic Brain Damage in Neonatal Rats

Ferroptosis is an iron-dependent form of regulated cell death, which is driven by loss of activity of the lipid repair enzyme glutathione peroxidase 4 (GPX4) and subsequent accumulation of lipid peroxidation. Ferroptosis is implicated in various diseases involving neuronal injury. However, the role of ferroptosis in hypoxic-ischemic brain damage (HIBD) has not been elucidated. The objectives of this study were to evaluate whether ferroptosis is involved in hypoxic-ischemic brain damage and its mechanisms through the HIBD model. A 7-day-old male Sprague-Dawley neonatal rat HIBD model was established by blocking the left common carotid artery. Laser speckle contrast imaging, immunohistochemical staining, transmission electron microscopy were used to measure the effects of ferroptosis on HIBD. Brain tissue on the damaged side in the HIBD group showed atrophied, even liquefied, glial cells increased, and blood perfusion was significantly reduced. The HIBD group insult significantly increased reactive oxygen species levels, as well as the protein levels of iron metabolism-related proteins transferrin receptor (TFRC), ferritin heavy chain (FHC), and ferritin light chain (FLC), while reducing the levels of Solute Carrier Family 7 Member 11 (SLC7A11), glutathione (GSH), and GPX4. These changes resulted in diminished cellular antioxidant capacity and mitochondrial damage, causing neuronal ferroptosis in the cerebral cortex. We conclude that ferroptosis plays a role in HIBD in neonatal rats. Ferroptosis-related mechanisms such as abnormalities in iron metabolism, amino acid metabolism, and lipid peroxidation regulation play important roles in HIBD.

The TXNIP/Trx-1/GPX4 pathway promotes ferroptosis in hippocampal neurons after hypoxia-ischemia in neonatal rats

To observe the change in ferroptosis in hippocampal neurons after hypoxia-ischemia (HI) in neonatal rats and investigate the related mechanism based on the TXNIP/Trx-1/GPX4 signaling pathway. Healthy neonatal Sprague-Dawley rats, aged 7 days, were randomly divided into three groups: sham-operation (n=30), hypoxic-ischemic brain damage (HIBD) (n=30) and siRNA (TXNIP siRNA) (n=12). The classic Rice-Vannucci method was used to establish a neonatal rat model of HIBD. At 6 hours, 24 hours, 72 hours, and 7 days after modeling, Western blot was used to measure the protein expression of GPX4 in the hippocampal tissue at the injured side; at 24 hours after modeling, laser speckle imaging combined with hematoxylin-eosin staining was used to determine whether the model was established successfully; NeuN/GPX4 and GFAP/GPX4 immunofluorescence staining combined with Western blot and other methods was used to measure the protein expression of GPX4 and the signal molecules TXNIP and Trx-1 in the hippocampal tissue at the injured side; the kits for determining the content of serum iron and tissue iron were used to measure the change in iron content; quantitative real-time PCR was used to measure the mRNA expression of TXNIP, Trx-1, and GPX4. At 6 hours, 24 hours, 72 hours, and 7 days after modeling, the HIBD group had a significantly lower protein expression level of GPX4 than the sham-operation group (P<0.05). At 24 hours after modeling, the HIBD group had a significantly lower cerebral blood flow of the injured side than the sham-operation group (P<0.05), with loose and disordered arrangement and irregular morphology of hippocampal CA1 neurons at the injured side. Compared with the sham-operation group, the HIBD group had a significantly higher number of TXNIP+ cells and significantly lower numbers of Trx-1+ cells and NeuN+GPX4+/NeuN+ cells in the hippocampal CA1 region at the injured side (P<0.05), with almost no GFAP+GPX4+ cells in the hippocampal CA1 region. Compared with the sham-operation group, the HIBD group and the siRNA group had significantly higher levels of serum iron and tissue iron in the hippocampus at the injured side (P<0.05). Compared with the HIBD group, the siRNA group had significantly lower levels of serum iron and tissue iron in the hippocampus at the injured side (P<0.05). The HIBD group and the siRNA group had significantly higher mRNA and protein expression levels of TXNIP than the sham-operation group (P<0.05), and the siRNA group had significantly lower expression levels than the HIBD group (P<0.05). The HIBD group and the siRNA group had significantly lower mRNA and protein expression levels of Trx-1 and GPX4 in the hippocampus at the injured side than the sham-operation group (P<0.05), and the siRNA group had significantly higher expression levels than the HIBD group (P<0.05). HI induces ferroptosis of hippocampal neurons in neonatal rats by activating the TXNIP/Trx-1/GPX4 pathway, thereby resulting in HIBD.

Inhibition of immunoproteasome subunit low molecular mass polypeptide 7 with ONX-0914 improves hypoxic-ischemic brain damage via PI3K/Akt signaling.

The immunoproteasome subunit low molecular mass polypeptide 7 (LMP7) leads to brain injuries, such as autoimmune neuritis and ischemic stroke, by activating inflammation. However, the roles and mechanisms of LMP7 in hypoxic-ischemic brain damage (HIBD) remain unclear. This study explored these issues in a rat model of HIBD. Pathology was evaluated using hematoxylin-eosin staining. LMP7 expression was detected using western blot analysis, reverse transcription-quantitative PCR (RT-qPCR), and immunohistochemical staining. The presence of proinflammatory cytokines, including tumor necrosis factor-a, interleukin-6, and interleukin-1β, was tested using ELISA and RT-qPCR. Behavioral performance was evaluated using a short-term neurological function score and the Morris water maze test. Compared to those in the Sham group, the HIBD group exhibited obvious upregulated LMP7 and pro-inflammatory cytokine levels. HIBD rats exhibited severe pathological and behavioral damage. LMP7 inhibition with ONX-0914 reduced proinflammatory cytokine expression, attenuated pathological damage, and enhanced behavioral performance of rats with HIBD. Inhibition of phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling with LY29400 increased LMP7 expression and abolished the protective effects of ONX-0914 in HIBD rats. Our findings indicate that LMP7 aggravates brain injury by triggering inflammatory responses in HIBD rats. LMP7 inhibition with ONX-0914 exerts protective effects on HIBD rats, possibly via PI3K/Akt signaling.

Effect of astragaloside IV on the expression of NOD-like receptor protein 3 inflammasome in neonatal rats with hypoxic-ischemic brain damage

OBJECTIVE To study the effect of astragaloside IV (AS-IV) on NOD-like receptor protein 3 (NLRP3) inflammasome in neonatal rats with hypoxic-ischemic brain damage (HIBD). METHODS A total of 24 Sprague-Dawley rats, aged 7 days, were randomly divided into a sham-operation group, an HIBD group, and an AS-IV treatment group, with 8 rats in each group. After 24 hours of modeling, brain tissue was collected for hematoxylin-eosin staining, yo-PRO-1 staining, and EthD-2 immunofluorescent staining in order to observe the cerebral protection effect of AS-IV in vivo. HT22 cells were used to prepare a model of oxygen-glycogen deprivation (OGD), and a concentration gradient (50-400 μmol/L) was established for AS-IV. CCK-8 assay was used to measure the viability of HT22 cells. RT-PCR and Western blot were used to observe the effect of different concentrations of AS-IV on the mRNA and protein expression of NLRP3, gasdermin D (GSDMD), caspase-1, and interleukin-1β (IL-1β). RESULTS Yo-Pro-1 and EthD-2 staining showed that compared with the sham-operation group, the HIBD group had an increase in pyroptotic cells with a small number of necrotic cells, and the AS-IV group had reductions in both pyroptotic and necrotic cells. Compared with the sham-operation group, the HIBD group had significantly higher protein expression levels of NLRP3, IL-1β, caspase-1, and GSDMD (P < 0.05). Compared with the HIBD group, the AS-IV group had significant reductions in the protein expression levels of NLRP3, caspase-1, and GSDMD (P < 0.05). HT22 cell experiment showed that compared with the OGD group, the AS-IV group had inhibited mRNA and protein expression of NLRP3, GSDMD, caspase-1, and IL-1β, with the best therapeutic effect at the concentration of 200 μmol/L (P < 0.05). CONCLUSIONS AS-IV may alleviate HIBD in neonatal rats by inhibiting the expression of NLRP3, GSDMD, caspase-1, and IL-1β.

The effect of magnetic guiding BMSCs on hypoxic-ischemic brain damage via magnetic resonance imaging evaluation

Hypoxic-ischemic brain damage (HIBD) is a critical disease in pediatric neurosurgery with high mortality rate and frequently leads to neurological sequelae. The role of bone marrow mesenchymal stem cells (BMSCs) in neuroprotection has been recognized. However, using the imaging methods to dynamically assess the neuroprotective effects of BMSCs is rarely reported. In this study, BMSCs were isolated, cultured and identified. Flow cytometry assay had shown the specific surface molecular markers of BMSCs, which indicated that the cultivated cells were purified BMSCs. The results demonstrated that CD29 and CD90 were highly expressed, whilst CD45 and CD11b were negatively expressed. Further, BMSCs were transplanted into Sprague Dawley (SD) rats established HIBD via three ways, including lateral ventricle (LV) injection, tail vein (TV) injection, and LV injection with magnetic guiding. Magnetic resonance imaging (MRI) was used to monitor and assess the treatment effect of super paramagnetic iron oxide (SPIO)-labeled BMSCs. The mean kurtosis (MK) values from diffusion kurtosis imaging (DKI) exhibited the significant differences. It was found that the MK value of HIBD group increased compared with that in Sham. At the meantime, the MK values of LV + HIBD, TV + HIBD and Magnetic+LV + HIBD groups decreased compared with that in HIBD group. Among these, the MK value reduced most significantly in Magnetic+LV + HIBD group. MRI illustrated that the treatment effect of Magnetic+LV + HIBD group was best. In addition, HE staining and TUNEL assay measured the pathological changes and apoptosis of brain tissues, which further verified the MRI results. All data suggest that magnetic guiding BMSCs, a targeted delivery way, is a new strategic theory for HIBD treatment. The DKI technology of MRI can dynamically evaluate the neuroprotective effects of transplanted BMSCs in HIBD.

Effect of different melatonin treatment regimens on white matter damage in neonatal rats with hypoxic-ischemic brain damage

OBJECTIVE To study the effect of different melatonin treatment regimens on long-term behavior and white matter damage in neonatal rats with hypoxic-ischemic brain damage (HIBD), and to seek an optimal melatonin treatment regimen. METHODS Healthy Sprague-Dawley rats, aged 7 days, were randomly divided into four groups: sham-operation, HIBD, single-dose immediate treatment (SDIT), and 7-day continuous treatment (7DCT), with 8 rats in each group. A neonatal rat model of HIBD was prepared according to the classical Rice-Vannucci method. On day 21 after HIBD, the Morris water maze test was used to evaluate spatial learning and memory abilities. On day 70 after HIBD, immunofluorescence assay was used to measure the expression of neuronal nuclear antigen (NeuN) in the cerebral cortex and the hippocampal CA1 region of neonatal rats, and double-label immunofluorescence was used to measure the expression of myelin basic protein (MBP) and neurofilament 200 (NF200) in the corpus striatum and the corpus callosum. RESULTS The results of the Morris water maze test showed that the SDIT and 7DCT groups had a significantly shorter mean escape latency than the HIBD group, and the 7DCT group had a significantly shorter mean escape latency than the SDIT group (P < 0.05). The results of immunofluorescence assay for NeuN showed that the SDIT and 7DCT groups had a significantly higher number of NeuN+ cells in the cerebral cortex and the hippocampal CA1 region than the HIBD group, and the 7DCT group had a significantly higher number than the SDIT group (P < 0.05). MBP/NF200 double-label immunofluorescence showed that compared with the HIBD group, the SDIT group and the 7DCT group had significantly higher fluorescence intensities of MBP and NF200 in the corpus striatum, and the 7DCT group had significantly higher fluorescence intensities than the SDIT group (P < 0.05); the 7DCT group had significantly higher fluorescence intensities of MBP and NF200 in the corpus callosum than the SDIT and HIBD groups (P < 0.05). CONCLUSIONS Both SDIT and 7DCT can improve long-term behavior and reduce white matter damage in neonatal rats with HIBD, and 7DCT is more effective than SDIT.

Intranasal administration of Cytoglobin modifies human umbilical cord‑derived mesenchymal stem cells and improves hypoxic‑ischemia brain damage in neonatal rats by modulating p38 MAPK signaling‑mediated apoptosis.

Neonatal hypoxic-ischemic brain damage (HIBD) is a common clinical syndrome in newborns. Hypothermia is the only approved therapy for the clinical treatment; however, the therapeutic window of hypothermia is confined to 6 h after birth and even then, >40% of the infants either die or survive with various impairments, including cerebral palsy, seizure disorder and intellectual disability following hypothermic treatment. The aim of the present study was to determine whether nasal transplantation of Cytoglobin (CYGB) genetically modified human umbilical cord-derived mesenchymal stem cells (CYGB-HuMSCs) exhibited protective effects in neonatal rats with HIBD compared with those treated without genetically modified CYGB. A total of 120 neonatal Sprague-Dawley rats (postnatal day 7) were assigned to either a Sham, HIBD, HuMSCs or CYGB-HuMSCs group (n = 30 rats/group). For HIBD modeling, rats underwent left carotid artery ligation and were exposed to 8% oxygen for 2.5 h. A total of 30 min after HI, HuMSCs (or CYGB-HuMSCs) labeled with enhanced-green fluorescent protein (eGFP) were intranasally administered. After modeling for 3, 14 and 29 days, five randomly selected rats were sacrificed in each group, and the expression levels of CYGB, ERK, JNK and p38 in brain tissues were determined. Nissl staining of the cortex and hippocampal Cornu Ammonis 1 area of rats in each group were compared after 3 days of modeling. TUNEL assay and immunofluorescence were performed 3 days after modeling. Long term memory in rats was assessed using a Morris-water maze 29 days after modeling. The HIBD group demonstrated significant deficiencies compared with the Sham group based on Nissl staining, TUNEL assay and the Morris-water maze test. HuMSC treated rats exhibited improvement on in all the tests, and CYGB-HuMSCs treatment resulted in further improvements. PCR and western blotting results indicated that the CYGB mRNA and protein levels were increased from day 3 to day 29 after transplantation of CYGB-HuMSCs. Furthermore, it was identified that CYGB-HuMSC transplantation suppressed p38 signaling at all experimental time points. Immunofluorescence indicated the scattered presence of HuMSCs or CYGB-HuMSCs in damaged brain tissue. No eGFP and glial fibrillary acidic protein or eGFP and neuron-specific enolase double-stained positive cells were found in the brain tissues. Therefore, CYGB-HuMSCs may serve as a gene transporter, as well as exert a neuroprotective and antiapoptotic effect in HIBD, potentially via the p38 mitogen-activated protein kinase signaling pathway.

Transplantation of umbilical cord blood mononuclear cells attenuates the expression of IL-1β via the TLR4/NF-κB pathway in hypoxic-ischemic neonatal rats

Objective:This study aims to observe the effects of transplantation of umbilical cord blood mononuclear cells (UCBMCs) on the expression of interleukin (IL)-1β and explore the mechanism via the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) pathway in hypoxic-ischemic neonatal rats.Methods:Seven-day-old Sprague-Dawley neonatal rats were randomly divided into Sham, hypoxic-ischemic brain damage (HIBD), and UCBMC groups. The HIBD model was prepared by Rice-Vannucci method, and UCBMC were transplanted 24 h after HIBD in the UCBMC group. At 7 days after transplantation, changes in neurons and the TLR4 protein were examined by neuronal nuclei (NeuN)/TLR4 immunofluorescence staining. The expression of pNF-κB and IL-1β proteins was detected by immunohistochemical staining and enzyme linked immunosorbent assay (ELISA).Results:The percentage of NeuN+DAPI+ cells in the injured cortex in the UCBMC group was significantly higher than that in the HIBD group and lower than that in the Sham group (P &lt; 0.05). The number of NeuN+TLR4+DAPI+cells in the UCBMC group was significantly lower than that in the HIBD group (P &lt; 0.05) but higher than that in the Sham group (P &lt; 0.05). More pNF-κB+ cells were observed in the HIBD group than in Sham and UCBMC groups (P &lt; 0.05), and more pNF-κB+ cells were observed in the UCBMC group than in the Sham group (P &lt; 0.05). ELISA results showed that the IL-1β expression in the injured cerebral cortex in the UCMBC group was significantly lower than that in the HIBD group but remained higher than that in the Sham group (P &lt; 0.05).Conclusions:UCBMC transplantation could inhibit the IL-1β protein expression in the injured cortex, thereby alleviating HIBD in neonatal rats. The underlying mechanism might be associated with the down- regulation of TLR4 and pNF-κB proteins.

Role of miR-326 in neonatal hypoxic-ischemic brain damage pathogenesis through targeting of the \u03b4-opioid receptor

Hypoxic-ischemic brain damage (HIBD) is a relatively common malignant complication that occurs in newborn infants, but promising therapies remain limited. In this study, we focused on the role of miR-326 and its target gene δ-opioid receptor (DOR) in the pathogenesis of neonatal HIBD. The expression levels of miR-326 and DOR after hypoxic-ischemic injury were examined both in vivo and in vitro. The direct relationship between miR-326 and DOR was confirmed by a dual-luciferase reporter assay. Further, effects of miR-326 on cell viability and apoptosis levels under oxygen glucose deprivation (OGD) were analyzed. The expression levels of miR-326 were significantly lower and DOR levels were significantly higher in the HIBD group than the control group both in vivo and in vitro. Overexpression of miR-326 downregulated the expression of DOR, while suppression of miR-326 upregulated the expression of DOR. The dual-luciferase reporter assay further confirmed that DOR could be directly targeted and regulated by miR-326. MiR-326 knockdown improved cell survival and decreased cell apoptosis by decreasing the expression levels of Caspase-3 and Bax and increasing Bcl-2 expression in PC12 cells after exposure to OGD. Moreover, DOR knockdown rescued the effect of the improved cell survival and suppressed cell apoptosis induced by silencing miR-326. Our findings indicated that inhibition of miR-326 may improve cell survival and decrease cell apoptosis in neonatal HIBD through the target gene DOR.

Effect of granulocyte-colony stimulating factor on endoplasmic reticulum stress in neonatal rats after hypoxic-ischemic brain damage

Objective To evaluate the protective effect of granulocyte-colony stimulating factor(G-CSF) on neonatal rats after hypoxic-ischemic brain damage(HIBD)and its effect on endoplasmic reticulum (ER) stress. Methods According to the random number table, a total of 54 Sprague-Dawley (SD) rats aged 7 days were divided into 3 groups(18 rats in each group): Sham group, HIBD group and G-CSF group, and the improved Rice method was used to establish a neonatal rat model of HIBD.A dose of 50 μg/kg of G-CSF was administered intraperitoneally 1 hour after HIBD (G-CSF group), while the rats in HIBD group and Sham group received saline only.At 24 hours of HIBD, pups were euthanized to quantify brain infarct volume by using 2, 3, 5-Triphenyltetrazolium chloride.Hematoxylin-Eosin (HE) staining was used to observe the changes of brain structure.Neuronal cell death was determined by using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Then the expressions of glucose-regulated protein 78 (GRP78), cysteinyl aspartate specific proteinase 12 (Caspase-12), CCAAT/enhancer binding-protein homologous protein (CHOP) were assessed by Western blot and immunofluorescence staining. Results Twenty-four hours after operation, HE staining showed that no significant neuronal damage was observed in Sham group.The brain tissue structure of rats in the HIBD group was significantly damaged, while some improvement was observed in the G-CSF group.The infarction volume in HIBD group[(25.40±5.15)%] increased compared with that in the Sham group[(0.31±0.15)%] and the G-CSF group[(16.36±4.97)%], and the differences were statistically significant(all P<0.05). There was increased positive expression of GRP78 protein in HIBD group, compared with that in the Sham group and the G-CSF group[(49.38±10.06)% vs.(9.12±4.50)%, (30.61±6.35)%], and the differences were statistically significant (all P<0.05). The percentage of apoptosis in the hippocampal CA1 region and conex in HIBD group [(44.84±11.54)%, (48.23±14.07)%] were higher than those in the G-CSF group [(17.87±7.20)%, (26.18±9.96)%], and the differences were statistically significant (all P<0.05). The expression of GRP78, CHOP and Caspase-12 in the HIBD group (0.63±0.24, 0.72±0.21, 0.68±0.25) were higher than those in the Sham group (0.20±0.08, 0.28±0.08, 0.23±0.07), and the G-CSF group (0.39±0.13, 0.51±0.18, 0.48±0.16), and the differences were statistically significant (all P<0.05). Conclusions G-CSF exerts neuroprotective effect on the neonatal rats after HIBD.G-CSF may be an effective treatment of HIBD by reducing ER stress-induced neuronal apoptosis. Key words: Infant, newborn; Hypoxic-ischemic brain damage; Granulocyte-colony stimulating factor; Endoplasmic reticulum stress

Analysis of long non-coding RNA expression profiles in neonatal rats with hypoxic-ischemic brain damage.

Hypoxic-ischemic brain damage (HIBD) which is a common cause of acute mortality and neurological dysfunction in neonates still lacks effective therapeutic methods. Long non-coding RNAs (lncRNAs) were demonstrated to play a crucial role in many diseases. To give a foundation for subsequent functional studies of lncRNAs in HIBD, we investigated the profiling of lncRNAs and messenger RNAs (mRNAs) using neonatal HIBD rat model. Six neonatal rats were divided into sham-operated group (n=3) and HIBD group (n=3) randomly. Deep RNA sequencing was implemented to find out the meaningful lncRNAs and mRNAs. Quantitative real-time PCR was used to validate expressions of lncRNAs and mRNAs. The Gene Ontology (GO) and kyoto encyclopedia of genes a genomes (KEGG) database were used to predict functions of lncRNAs. A total of 328 differentially expressed lncRNAs (177 down-regulated vs 151 up-regulated) and 7157 differentially expressed mRNAs (2552 down-regulated vs 4605 up-regulated) were identified. The Quantitative real-time PCR results showed significant differential expressions of five lncRNAs and five mRNAs which were consistent with the RNA-Seq data. Gene ontology and KEGG analysis showed these lncRNAs and their expression-correlated mRNAs were closely related to the Janus tyrosine kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, NF-kappa B signaling pathway, Toll-like receptor signaling pathway, calcium signaling pathway, Notch signaling pathway, mitogen activated protein kinase signaling pathway, neuroactive ligand-receptor interaction pathway and more. The results of our study identified the characterization and expression profiles of lncRNAs in neonatal HIBD and may be a basis for further therapeutic research. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* and *Open Data* because it provided all relevant information to reproduce the study in the manuscript and because it made the data publicly available. The data can be accessed at https://osf.io/yf3da/. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.