Rat Model Of Hypoxic-ischemic Brain Damage Research Articles

Protective mechanisms of quercetin in neonatal rat brain injury induced by hypoxic-ischemic brain damage (HIBD).

Neonatal hypoxic-ischemic brain damage (HIBD) is a leading cause of infant mortality worldwide. This study explored whether quercetin (Que) exerts neuroprotective effects in a rat model of HIBD. A total of 36 seven-day-old Sprague-Dawley rats were divided into control, Que, HI, and HI + Que groups. The Rice method was used to establish HIBD in HI and HI + Que rats, which were treated with hypoxia (oxygen concentration of 8%) for 2 h after ligation of the left common carotid artery. The rats in the HI + Que group were intraperitoneally injected with Que (30 mg/kg) 1 h before hypoxia, and the rats in the Que group were only injected with the same amount of Que. Brain tissues were harvested 24 h postoperation and assessed by hematoxylin and eosin staining, 2,3,5-triphenyltetrazolium chloride staining, and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay; relative gene and protein levels were evaluated by RT-qPCR, IHC, or western blot (WB) assay. Brain tissue morphologies were characterized by transmission electron microscopy (TEM); LC3B protein levels were assessed by immunofluorescence staining. Escape latencies and platform crossing times were significantly improved (p < .05) in HI + Que groups; infarct volume significantly decreased (p < .001), whereas the numbers of autophagic bodies and apoptotic cells increased and decreased, respectively. Meanwhile, NLRX1, ATG7, and Beclin1 expressions were significantly upregulated, and mTOR and TIM23 expressions, LC3B protein level, and LC 3II/LC 3I ratio were significantly downregulated. Que exerted neuroprotective effects in a rat model of HIBD by regulating NLRX1 and autophagy.

Dexmedetomidine Alleviates Hypoxic-Ischemic Brain Damage in Neonatal Rats Through Reducing MicroRNA-134-5p-Mediated NLRX1 Downregulation

BackgroundDexmedetomidine (Dex) is a safe and effective anesthetic adjunct which also has neuroprotective roles. This study aimed to validate the role of Dex in hypoxic-ischemic brain damage (HIBD) in neonatal rats and the functional molecules. MethodsA neonatal rat model of HIBD was established and treated with Dex. The learning and memory abilities of rats were determined by Morris water maze tests. The left-hemisphere encephalatrophy, pathological changes, neuronal apoptosis, and inflammation in rat hippocampal tissues were examined to evaluate the treating effects of Dex on HIBD. Differentially expressed microRNAs (miRNAs) in rats with HIBD were screened using microarray analysis. Potential downstream molecules mediated by miR-134-5p were predicted using bioinformatics analysis. Altered expression of miR-134-5p and NLR family member X1 (NLRX1) was induced in rats after Dex treatment for rescue experiments. ResultsDex treatment significantly enhanced the learning and memory abilities of rats and reduced encephalatrophy in rats. It also alleviated pathological changes, neuronal apoptosis, and the production of pro-inflammatory cytokines in rat hippocampal tissues. miR-134-5p was significantly upregulated in rats with HIBD. Dex treatment reduced the expression of miR-134-5p. NLRX1 was a target gene of miR-134-5p and it reduced the phosphorylation of IκBα and p65, namely the activation of NF-κB signaling. Overexpression of miR-134-5p blocked, whereas overexpression of NLRX1 strengthened the protective effects of Dex on neonatal rats. ConclusionThis study demonstrates that Dex treatment can alleviate HIBD in neonatal rats through restoring NLRX1 expression by suppressing miR-134-5p.

Dexmedetomidine alleviates the hypoxic-ischemic brain damage via miR-20a-5p/methionine adenosyltransferase 2B axis in rat pups.

The neuroprotective effect of dexmedetomidine (DEX) has been demonstrated in hypoxic-ischemic brain damage (HIBD) animal models, the mechanism of which will be the foothold in this work. After establishment of HIBD rat model, the rats were treated with DEX, miR-20a-5p agomir and adenoviral methionine adenosyltransferase 2B (MAT2B) overexpression vector, and then their brain tissues were harvested. The infarction volume and pathological changes of these brain tissues were measured using the triphenyl tetrazolium chloride (TTC), Nissl and hematoxylin-eosin (HE) stainings. The levels of miR-20a-5p, Bcl-2, Bax and MAT2B in these brain tissues were detected by Real-Time PCR (RT-PCR) and western blot. The binding sites of MAT2B and miR-20a-5p were predicted using the TargetScan and verified using the dual-luciferase reporter assay. The memory deficits and spatial learning of rat pups were assessed by Morris water maze test. MiR-20a-5p expression was upregulated, while MAT2B expression was downregulated in rats with HIBD. MAT2B was targeted by miR-20a-5p. DEX treatment improved the neurons and hippocampal tissue damage and decreased miR-20a-5p level in brain tissues of rats with HIBD. MiR-20a-5p overexpression overturned the protective effect of DEX on brain tissues and learning and memory abilities in rats with HIBD. Moreover, DEX promoted Bcl-2 level while inhibiting Bax level in HIBD rats' brain tissues. Besides, overexpressed MAT2B reversed the effect of overexpressed miR-20a-5p on the levels of MAT2B, Bcl-2 and Bax, brain tissue damage, as well as the learning and memory abilities in rats with HIBD. DEX alleviated HIBD via the miR-20a-5p/MAT2B axis in rats.

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.

High-mobility Group Box 1 Contributes to Hypoxic-Ischemic Brain Damage by Facilitating Imbalance of Microglial Polarization through RAGE-PI3K/Akt Pathway in Neonatal Rats

High mobility group box 1 (HMGB1) is a damage-associated molecular pattern integral for hypoxic-ischemic brain damage (HIBD) in neonatal rats since it regulates the phenotypic polarization of microglia, as depicted in our previous studies. Since this mechanism is not clear, this study establishes an oxygen-glucose deprivation (OGD) model of highly aggressively proliferating immortalized microglia while modulating the expression of HMGB1 by plasmid transfection. The M1/M2 microglial phenotype and receptor for advanced glycation end products-phosphoinositide 3-kinase/Akt (RAGE-PI3K/Akt) activation were evaluated, showing that HMGB1 promoted the polarization of microglia to the M1 phenotype under OGD conditions. Meanwhile, RAGE, which is the main receptor of HMGB1, was activated, and phosphorylation of PI3K/Akt was upregulated. However, knockdown or inhibition of HMGB1 can weaken the activation of RAGE and phosphorylation of PI3K/Akt. The inhibition of HMGB1 or RAGE-PI3K/Akt attenuated microglial polarization to the M1 phenotype and promoted M2 microglial polarization instead, reducing the release of pro-inflammatory factors. In the neonatal HIBD rat model, the RAGE-PI3K/Akt pathway was activated seven days after hypoxic-ischemic (HI) exposure, and the activation was partly inhibited after pretreatment with the HMGB1 inhibitor. Concurrently, inhibition of the HMGB1-RAGE-PI3K/Akt pathway alleviated neuronal damage in the hippocampus. These findings verified that HMGB1 could lead to an imbalance in M1/M2 microglial polarization through activation of the RAGE-PI3K/Akt signaling pathway under OGD conditions. Obstructing this pathway may attenuate the imbalanced polarization of microglia, enabling its utilization as a therapeutic strategy against brain injury in HIBD.

Overexpression of Long Noncoding RNA H19 Inhibits Cardiomyocyte Apoptosis in Neonatal Rats with Hypoxic-Ischemic Brain Damage Through the miR-149-5p/LIF/PI3K/Akt Axis.

Hypoxic-ischemic brain damage (HIBD) is a leading cause of fatality and neural system injury in neonates. This study aims to explore the effect of long noncoding RNA H19 on cardiomyocyte apoptosis in neonatal rats with HIBD. The neonatal rat model of HIBD was established. The cerebral infarction volume and apoptosis index of cardiomyocyte increased, while H19 expression decreased in neonatal rats with HIBD. After the lentivirus vector of overexpressed H19 was injected into neonatal rats with HIBD, the cardiomyocyte apoptosis was suppressed; levels of inflammatory factors and oxidative stress injury of myocardial tissues were reduced. The binding relationships between H19 and miR-149-5p, and miR-149-5p and leukemia inhibitory factor (LIF) were predicted by a bioinformatics website and verified using the dual-luciferase reporter gene assay. H19 competitively bound to miR-149-5p to upregulate LIF expression and activate the PI3K/Akt pathway. Moreover, a functional rescue experiment was carried out. Injection of Wortmannin reversed the inhibitory effect of H19 overexpression on cardiomyocyte apoptosis in neonatal rats with HIBD. It could be concluded that H19 competitively bound to miR-149-5p to upregulate LIF expression and activate the PI3K/Akt pathway, thus reducing cardiomyocyte apoptosis in neonatal rats with HIBD. This study may offer new insights for HIBD treatment.

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.

MiR-375-3p mediates reduced pineal function in hypoxia-ischemia brain damage

The functional roles of microRNAs (miRNAs) have been studied in various diseases, including hypoxic-ischemic brain damage (HIBD). However, changes in the expression of miRNAs and the underlying mechanisms in the pineal gland during HIBD remain unknown. Based on the previous study by microRNA array, hundreds of miRNAs showed altered expression patterns in the pineal gland in a rat model of HIBD. MiR-375-3p was found to be significantly upregulated and abundant in the pineal gland. Further investigation in an in vitro HI model of pinealocytes showed that miRNA-375 exacerbated the damage to pineal function. After oxygen-glucose deprivation / reoxygenation (OGD/R), miR-375-3p expression increased, while aralkylamine N-acetyltransferase (AANAT) expression and melatonin (MT) secretion decreased. Overexpression of miRNA-375 in pinealocytes aggravated the influence of OGD/R on AANAT expression and MT secretion. Because miRNA-375 overexpression in pinealocytes induced decreased rasd1 mRNA and protein expression, rasd1 may mediate the effect of miR-375-3p on pineal function. Furthermore, miR-375-3p aggravated the cognitive impairment caused by HIBD in rats, as observed by Morris water maze test, and also affected emotion and circadian rhythm in HIBD-treated rats. Thus, miR-375-3p may be a key regulatory molecule in the pineal gland following HIBD, and targeting of miR-375-3p may represent a new strategy for the treatment of 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.

The role of pineal microRNA-325 in regulating circadian rhythms after neonatal hypoxic-ischemic brain damage.

Circadian rhythm disorder is a common, but often neglected, consequence of neonatal hypoxic-ischemic brain damage (HIBD). However, the underlying molecular mechanisms remain largely unknown. We previously showed that, in a rat model of HIBD, up-regulation of microRNA-325 (miR-325) in the pineal gland is responsible for the suppression of Aanat, a key enzyme involved in melatonin synthesis and circadian rhythm regulation. To better understand the mechanism by which miR-325 affects circadian rhythms in neonates with HIBD, we compared clinical samples from neonates with HIBD and samples from healthy neonates recruited from the First Affiliated Hospital of Soochow University (Dushuhu Branch) in 2019. We found that circulating miR-325 levels correlated positively with the severity of sleep and circadian rhythm disorders in neonates with HIBD. Furthermore, a luciferase reporter gene assay revealed that LIM homeobox 3 (LHX3) is a novel downstream target of miR-325. In addition, in miR-325 knock-down mice, the transcription factor LHX3 exhibited an miR-325-dependent circadian pattern of expression in the pineal gland. We established a neonatal mouse model of HIBD by performing double-layer ligation of the left common carotid artery and exposing the pups to a low-oxygen environment for 2 hours. Lhx3 mRNA expression was significantly down-regulated in these mice and partially rescued in miR-325 knockout mice subjected to the same conditions. Finally, we showed that improvement in circadian rhythm-related behaviors in animals with HIBD was dependent on both miR-325 and LHX3. Taken together, our findings suggest that the miR-325-LHX3 axis is responsible for regulating circadian rhythms and provide novel insights into the identification of potential therapeutic targets for circadian rhythm disorders in patients with neonatal HIBD. The clinical trial was approved by Institutional Review Board of Children's Hospital of Soochow University (approval No. 2015028) on July 20, 2015. Animal experiments were approved by Animal Care and Use Committee, School of Medicine, Soochow University, China (approval No. XD-2016-1) on January 15, 2016.

Melatonin improves hypoxic-ischemic brain damage through the Akt/Nrf2/Gpx4 signaling pathway

Melatonin (Mel) has neuroprotective effects; however, its roles in hypoxic-ischemic brain damage (HIBD) and the underlying mechanisms remain unknown. We aimed to explore its roles and mechanisms in a HIBD rat model. We found that exogenous Mel treatment ameliorated HIBD-induced pathological changes, inhibited neuronal ferroptosis, and promoted hippocampal neuronal survival. Moreover, Mel improved the learning and memory abilities of the HIBD rats. Further, we found that glutathione peroxidase 4 (Gpx4) inhibition with RSL3, Akt inhibition with LY29400, and nuclear factor erythroid-2-related factor 2 (Nrf2) inhibition with ML385 abolished the Mel protective effects in HIBD. Our findings indicate that exogenous Mel treatment has a protective effect on HIBD via the Akt/Nrf2/Gpx4 pathway.

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.

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/.

Effect of erythropoietin on Fas/FasL expression in brain tissues of neonatal rats with hypoxic-ischemic brain damage.

Hypoxic-ischemic brain damage (HIBD) occurs due to intrauterine hypoxia ischemia influencing the energy supply for fetal brain cells, which affects the metabolism of the brain to make the brain suffer a severe damage. Erythropoietin (EPO), which regulates hemacytopoiesis, is a kind of cytokine. EPO is sensitive to hypoxia ischemia. In this study, we aimed to investigate the effect of EPO on the expression of Fas/FasL in brain tissues of neonatal rats with HIBD. Neonatal rats were assigned randomly to sham, HIBD, and EPO groups. Five time points for observation were 6, 12, 24, 48, and 72 h after the HIBD rat model had been established, respectively. In the HIBD group, Fas/FasL expression began to rise at 6 h, reached the peak at 12–24 h, and dropped from 24 h. In the EPO group, the expression of Fas/FasL was lower than those in HIBD group at 12, 24, and 48 h (P<0.05). Our findings suggest that EPO may reduce cell apoptosis after hypoxic-ischemic damage through reduction of the expression of Fas and FasL, and that optimal therapeutic time window is 6–24 h after HIBD.

Tanshinone I alleviates motor and cognitive impairments via suppressing oxidative stress in the neonatal rats after hypoxic-ischemic brain damage

Neonatal hypoxia-ischemia is one of the main reasons that cause neuronal damage and neonatal death. Several studies have shown that tanshinone I (TsI), one of the major ingredients of Danshen, exerts potential neuroprotective effect in adult mice exposed to permanent left cerebral ischemia. However, it is unclear whether administration of TsI has neuroprotective effect on neonatal hypoxic-ischemic brain damage (HIBD), and if so, the potential mechanisms also remain unclear. Here, we reported that treatment with TsI (5 mg/kg, i.p.) significantly alleviated the deficits of myodynamia and motor functions as well as the spatial learning and memory in the rat model of HIBD. These behavioral changes were accompanied by a significant decrease in the number of neuronal loss in the CA1 area of hippocampus. Moreover, ELISA assay showed that TsI significantly increased the production of antioxidants including total antioxidant capacity (T-AOC), glutathione (GSH), total superoxide dismutase (T-SOD) and catalase (CAT), and reduced the production of pro-oxidants including hydrogen peroxide (H2O2), total nitric oxide synthase (T-NOS) and inducible nitric oxide synthase (iNOS). Taken together, these results indicate that TsI presents potential neuroprotection against neuronal damage via exerting significantly antioxidative activity and against pro-oxidant challenge, thereby ameliorating hypoxia-ischemia-induced motor and cognitive impairments in the neonatal rats, suggesting that TsI may be a potential therapeutic agent against HIBD.

Interleukin-10 release from astrocytes suppresses neuronal apoptosis via the TLR2/NFκB pathway in a neonatal rat model of hypoxic-ischemic brain damage.

The biological function of interleukin-10 (IL-10) and the relationship between IL-10 secretion and the Toll-like receptor 2 (TLR2) expression levels in the central nervous system following hypoxic-ischemic brain damage (HIBD) are poorly understood. Here, we intend to elucidate the biological function and mechanism of IL-10 secretion following HIBD. In this study, we used a neonatal rat model of HIBD and found that rats injected with adeno-associated virus-IL-10-shRNA (short hairpin RNA) exhibited partially impaired learning and memory function compared to rats administered adeno-associated virus-control-shRNA. In vitro oxygen-glucose deprivation (OGD) induced IL-10 release from astrocytes but not from neurons. Pretreatment with exogenous recombinant IL-10 alleviated OGD-mediated apoptosis of neurons but not astrocytes. In addition, we also observed that hypoxicinjury induced a marked increase in IL-10 expression in astrocytes as a result of activation of the TLR2/phosphorylated nuclear factor kappa B (p-NFκB) p65 signaling cascade; furthermore, this effect disappeared upon small interfering RNA targeting rat TLR2 gene (siTLR2) treatment. Pyrrolidinedithiocarbamate, an inhibitor of NFκB activation, reduced the IL-10 expression levels in both OGD-injured astrocytes invitro and the hippocampi of HIBD rats invivo but did not significantly affect TLR2 expression. Furthermore, a luciferase assay revealed that p-NFκB p65 could bind the -1700/-1000bp proximal region of the IL-10 gene promoter to regulate IL-10 secretion from astrocytes and that this interaction could be controlled by OGD treatment. These data suggest that HIBD induces IL-10 secretion from astrocytes to exert a paracrine-induced anti-apoptotic effect on injured neurons via the TLR2/NFκB signaling pathway, which may improve learning and memory dysfunction after ischemic injury.

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

The research about bcl-2/bax/fas/fasL mRNA of splenic lymphocytes in HIBD rat model

Objective To obtain the change of bcl-2/bax/fas/fasL in splenic lymphoctyes with different lasting time of hypoxicischemic brain damage (HIBD). Methods The newborn rat were divided into 6 groups by the time of being HIBD model randomly, includes 1/6/12/24/48/72 hour(s) (8 for every group),and control groups were established at the same time point. The following four apoptosis related genes bcl-2/bax/fas/fasL were tested by real time PCR. Results ( 1 ) bcl-2: the mRNA expressions of HIBD groups were lower than control groups at the same time ( P<0.01 ). Eliminated the control effects, the mRNA expressions of HIBD groups were differernt by the modeling time(P ＜0.01 ). (2)bax: the mRNA expressions of HIBD groups were higher than control groups at the same time( P ＜0.01 ), and in control group the expression of 6 h was much higher than any other groups (P<0.01 ). Eliminated the control effects, the mRNA expressions of H IBD groups were different by the modeling time( P<0.01 ). (3)bcl-2/bax: the ratios of HIBD groups were lower than control groups at the same time( P ＜0.05 ), the ratios in control groups were higher than 1 ( except for 1 h); while in HIBI) groups the ratios were lower than 1; Eliminated the control effects, the ratios were different in all the groups. (4)fas: the mRNA expressions of HIBD groups were higher than control groups at the same time ( P ＜0.01 ), and both were maximum at 6 h. (5)fasL: the mRNA expressions of HIBD groups were higher than control groups in 1 h and 6 h ( P<0.01 ), while lower than control group at other time points( P<0.01 ),the expression of 24 h was the maximum of control groups and 12 h was the maximum of HIBD groups. (6)fas/fasL: the ratios of HIBD groups were higher than control groups( P ＜0.01 ) (except for 6 h), and the ratios in control groups were lower than 1 ( P<0.01 ) ( except for 6 h), and not concentrated, while in HIBD groups were higher than 1 ( except for 24 h), between 0.69 to 5.65. Conclusion Pro-apoptosis genes ( include bax/fas/fasL) were promoted by HIBD, while anti-apoptosis gene(bcl-2) was inhibited. The maximum of pro-apoptosis genes became early in HIBD. Both the pro- and anti-apoptosis genes got their maximum at 6 h and 12 h of HIBD. The apoptosis suppression was the main effects in control groups from the ratio of bcl-2/bax, which was lower than 1. The apoptosis promotion was the main effects in HIBD groups from the ratio of bcl-2/bax, which was higher than 1, especially at 12 h. Thefas/fasL effect which is the major way of lymphocytes apoptosis was strengthened in HIBD. Key words: Hypoxicischemic brain damage (HIBD); bcl-2; bax; fas; fasL

AP2α transcriptional activity is essential for retinoid-induced neuronal differentiation of mesenchymal stem cells

Pre-activation of the retinoid signaling pathway by all-trans retinoic acid facilitates neuronal differentiation of mesenchymal stem cells. Using protein/DNA based screening assays, we identified activator protein 2α as an important downstream target of all-trans retinoic acid. Although all-trans retinoic acid treatment significantly increased activator protein 2α transcriptional activity, it did not affect its expression. Inhibition of activator protein 2α with dominant-negative mutants reduced ATRA-induced differentiation of mesenchymal stem cells into neurons and reversed its associated functional recovery of memory impairment in the cell-based treatment of a hypoxic-ischemic brain damage rat model. Dominant-negative mutants of activator protein 2α inhibited the expression of neuronal markers which were induced by retinoic acid receptor β activation. All-trans retinoic acid treatment increased phosphorylation of activator protein 2α and resulted in its nuclear translocation. This was blocked by siRNA-mediated knockdown of retinoic acid receptor β. Furthermore, we found that retinoic acid receptor β directly interacted with activator protein 2α. In summary, the regulation of all-trans retinoic acid on activator protein 2α transcriptional activity was mediated by activation of retinoic acid receptor β and subsequent phosphorylation and nuclear translocation of activator protein 2α. Our results strongly suggest that activator protein 2α transcriptional activity is essential for all-trans retinoic acid-induced neuronal differentiation of mesenchymal stem cells.

Dedifferentiation-Reprogrammed Mesenchymal Stem Cells with Improved Therapeutic Potential

Stem cell transplantation has been shown to improve functional outcome in degenerative and ischemic disorders. However, low in vivo survival and differentiation potential of the transplanted cells limits their overall effectiveness and thus clinical usage. Here we show that, after in vitro induction of neuronal differentiation and dedifferentiation, on withdrawal of extrinsic factors, mesenchymal stem cells (MSCs) derived from bone marrow, which have already committed to neuronal lineage, revert to a primitive cell population (dedifferentiated MSCs) retaining stem cell characteristics but exhibiting a reprogrammed phenotype distinct from their original counterparts. Of therapeutic interest, the dedifferentiated MSCs exhibited enhanced cell survival and higher efficacy in neuronal differentiation compared to unmanipulated MSCs both in vitro and in vivo, with significantly improved cognition function in a neonatal hypoxic-ischemic brain damage rat model. Increased expression of bcl-2 family proteins and microRNA-34a appears to be the important mechanism giving rise to this previously undefined stem cell population that may provide a novel treatment strategy with improved therapeutic efficacy.