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Objective:To assess the severity of hypoxic-ischemic brain damage (HIBD) in neonatal rats and predict the occurrence of subsequent neurobehavioral abnormalities after brain injury by scoring and magnetic resonance imaging (MRI).Methods:7-day-old of 60 Sprague-Dawley (SD) rats were randomly divided into control group (14 rats), sham operation group (14 rats) and HIBD model group (32 rats). HIBD model was established by right common carotid artery dissection with Rice-Vannucci method and hypoxia. Within 24 h after modeling, the rats in the model group were evaluated by general condition score and Longa score, and the surviving rats with moderate and severe HIBD were selected for the experiment. 24 h after modeling, 5 rats of the model group were randomly selected for 2,3,5-triphenyltetrazole chloride staining to verify cerebral infarction. 1 week after modeling, 6 rats from each group were randomly selected for hematoxylin-eosin staining to observe HIBD brain injury. 4 weeks after modeling, 4 rats were randomly selected from the control group and the sham operation group, and 8 rats from the remaining model group were used to evaluate the volume of brain damage by MRI. 5-6 weeks after modeling, the remaining 8 rats from each group were subjected to the Cylinder test, and at 13 weeks, they underwent the Morris water maze test to evaluate their neurobehavior.Results:In HIBD model group, 19 rats with moderate to severe HIBD were selected from 32 rats. 24 h after modeling, cerebral infarction was verified in all rats, indicating moderate to severe HIBD. Brain tissue pathology observed 1 week after modeling revealed predominantly gray matter brain damage. MRI showed that 7 out of 8 rats had moderate to severe HIBD. Compared to the control and sham operation groups, the model group exhibited a significant decrease in the usage rate of the left forelimb in the Cylinder test at 5-6 weeks after modeling ( P<0.05), and the latency period in Morris water maze test was significantly prolonged at 13 weeks after modeling ( P<0.05), and the times of crossing platform quadrant were significantly reduced ( P<0.05). There was no significant difference in the right brain injury volume between 24 h and 4 weeks model group ( P>0.05). The brain injury volume in model group was negatively correlated with the usage rate of left forelimb in cylinder test at 5-6 weeks and the times of crossing platform quadrant in Morris water maze test at 13 weeks ( P<0.05), and positively correlated with latency period in Morris water maze test at 13 weeks ( P<0.05). Conclusions:Within 24 h of HIBD modeling, the severity of brain injury can be preliminarily predicted by general condition score and Longa score. 4 weeks after modeling, in the chronic phase of brain injury, MRI was proved to be an excellent predictor for mid-term and long-term neurobehavioral abnormalities in HIBD rats.
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Objective To explore the method of distinguishing the degree of hypoxic-ischemic brain damage ( HIBD) in living mice in early stage, so as to lay a foundation for the follow-up study of the molecular mechanism of different degrees of HIBD. Methods The modified Rice-Vannucci method was used to duplicate the HIBD model of C57BL/6 J mice. On the 1 day and 3 days after the model, the scalp of mice were cut and the brain tissue were observed to distinguish between mild and severe lesions in living mice, and then 2,3, 5-triphenyltetrazolium chloride (TTC) staining, laser speckle cerebral blood flow imaging, HE staining, Fluoro-Jade B ( FJB ) staining and body weight difference before and after operation were used to verify the reliability of observation in living mice. Results Through the gross observation of brain tissue in living mice, HIBD could be divided into mild injury (HI-M) group and severe injury (HI-S) group. On day 1 and day 3 after HIBD, a significant decrease in cerebral blood flow, obvious gray infarction and a large number of necrotic neurons were observed in the HI-S group, and the body weight was significantly lower than that before operation. In the HI-M group, the cerebral blood flow of the injured side decreased only on the 3rd day after HIBD, and the loose arrangement of neurons in the cortex and hippocampus of the injured side was observed morphologically. The body weight was lower than that before operation. Conclusion Gross observation of brain tissue by cutting the scalp is a reliable method to distinguish mild and severe brain injury in the early stage of HIBD in living mice.
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【Objective】 To explore the effects of ginsenoside Rg1 （ginsenoside Rg1） on cell apoptosis， oxidative stress and inflammation in young rats with hypoxic-ischemic brain damage （HIBD）. 【Methods】 Young Wistar rats were divided into healthy control group， model group （HIBD）， and HIBD+ginsenoside Rg1 group. Jumping test was used to detect the number of mistakes in each group of rats， brain tissue wet and dry weight method was used to calculate the brain water content and brain index of each group of rats. Hematoxylin-eosin （HE） staining was used to observe the degree of brain damage； TUNEL staining to observe the apoptosis level of brain tissue； Nissl body staining to detect neuronal cell apoptosis； Western blot to detect brain tissue Bax/Bcl-2， caspase-3， caspase-9 protein expressions； malondialdehyde （MDA） and superoxide dismutase （SOD） activity detection kit to detect MDA and SOD content， respectively； and ELISA to detect peripheral blood IL-6， iNOS， IL-4 contents. 【Results】 Compared with the model group， 20 mg/kg and 40 mg/kg ginsenoside Rg1 significantly reduced the number of mistakes in the jumping-off test of HIBD young rats， and brain index brain water rate and neuronal cell apoptosis （P<0.05）； alleviated brain tissue damage； down-regulated the protein expression levels of Bax/Bcl-2， cleaved cas9/cas9 and cleaved cas3/cas3 （P<0.05）； reduced IL-6 and iNOS protein content； and increased IL-4 protein content （P<0.05）. 【Conclusion】 Ginsenoside Rg1 improves brain tissue damage in young HIBD mice and inhibits neuronal cell apoptosis.
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Objective:To observe the long-term behavioral changes in movement, emotion, and learning and memory of newborn mice with hypoxic ischemic brain damage (HIBD). Methods:A total of 50 ten-day old newborn C57BL/6 mice were divided into control group (n = 20) and HIBD group (n = 30). The left common carotid artery was ligated in HIBD group and stayed in anoxic chamber for 45 minutes. All the mice were tested with suspension test, light/dark box test, elevated plus maze test, object recognition test and Y maze test two months after surgery. Results:There were 19 mice modeled successfully. Compared with the control group, the suspension test scores decreased (t = 2.785, P < 0.05); the time of latency of light/dark box test increased (t = -4.320, P < 0.001), the time and frequency in light box decreased (t > 2.603, P < 0.05); the time in open arm decreased (t = 4.576, P < 0.001) and the time in close arm increased (t = -3.287, P < 0.01) for the elevated plus maze test; the time nearing old object increased (t = -2.116, P < 0.05) and object recognition index decreased (t = 2.823, P < 0.05) for object recognition test; the time in the initial and novel arms decreased (t > 2.191, P < 0.05) for Y maze test in HIBD group. Conclusion:The long-term disorders of behavior may include disabilities of motor, learning and memory, and disorder of anxiety, in newborn mice with HIBD.
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OBJECTIVE: To investigate the effect of acupuncture stimulation of head acupoints "Jin San Zhen" (JIN's Three Acupuncture Needles Therapy) on behavior reactions, hippocampal neuronal autophagy and expression of autophagy associated proteins (Beclin-1 and light chain 3 Ⅱ/Ⅰ [LC 3 Ⅱ/Ⅰ]) in rats with hypoxic-ischemic brain damage (HIBD) due to fetal intrauterine distress, so as to reveal its underlying mechanisms in improving neonatal HIBD. METHODS: Pregnant SD rats were used in the present study. The HIBD model was established by delayed caesarean delivery and bilateral uterine arteries clipping for 10 minutes. The HIBD rats were randomly divided into model group and acupuncture groups (n＝9 rats in each group). The other 9 rats delivered naturally were used as the normal control group. On day 14 after delivery, the neonatal rats in the acupuncture group received acupuncture stimulation of head acupoints ("Nao San Zhen""Nie San Zhen" and "Zhi San Zhen") by twirling each needle leftward and rightward for 10 times, once a day for 14 d. The open field test and Morris water maze test were used to determine the locomotive activity and spatial learning-memory ability, respectively. The ultrastructure and autophagosomes in the hippocampal neurons were observed by transmission electron microscope. The contents and expression levels of Beclin-1 and LC3 Ⅱ/Ⅰ in the hippocampus tissues were detected by flow cytometry and Western blot, separately. RESULTS: Compared with the normal control group, the time to go out of the central region of open field test, and the escape latency and duration of first platform-quadrant-crossing of spatial exploration of Morris water maze tests were significantly increased (P<0.01，P<0.05，P<0.001), and the total distance and number of activities in the central region, and the target quadrant resistance time and number of platform-cros-sing remarkably decreased in the model group (P<0.01, P<0.05), suggesting a decline of both locomotor activity and learning-memory ability after modeling. The expression level (%) of Beclin-1 protein and ratio of LC3 Ⅱ/Ⅰ proteins were considerably increased in the model group (P<0.01). Following acupuncture interventions, the locomotor activity and spatial learning-memory ability were obviously increased (P<0.05，P<0.01，P<0.001), and the expression of Beclin-1 protein and ratio of LC3 Ⅱ/Ⅰ were further up-regulated relevant to the model group (P<0.001). Moreover, ultrastructural observation showed serrated change of nuclear membrane and widened perinuclear space, vacuolization in the mitochondria, dilation of endoplasmic reticulum and increase of autophagosomes in the hippocampal neurons in the model group. These situations were relatively milder in the acupuncture group. CONCLUSION: Acupuncture of head acupoints of "JIN San Zhen" may increase locomotor activity and learning-memory abi-lity in rats with HIBD due to fetal intrauterine anoxia, which is closely with its effect in promoting hippocampal neuronal autophagy via up-regulating the expression of Beclin-1 and LC3 Ⅱ/Ⅰ.
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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.
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Objective To observe the effects of 3 neuroprotective measures on the expressions of apoptosis-related factors and their ligands (Fas and FasL) in brain tissue of neonatal rats with hypoxic ischemic brain injury. Methods One hundred and twenty Wistar rats 7 days old were selected as experimental subjects, the rats were divided into four groups: neural stem cell, erythropoietin (EPO), ω-3 unsaturated fatty acid treatment groups and hypoxic ischemic brain damage model group according to random number table method, with 30 rats in each group. Neural stem cell group, EPO group and ω-3 unsaturated fatty acid group were respectively injected with neural stem cells, EPO and ω-3 unsaturated fatty acid, each 5 mL via tail vein after modeling; the hypoxic ischemic brain damage model group was given equal volume of normal saline. At 6, 12, 24, 48 and 72 hours after administration of drug, 6 rats were sacrificed in each group, brain tissue was taken, the mRNA expression levels of Fas/FasL, protein expression levels of Toll-like receptor 4 (TLR4), nuclear transcription factor-κB (NF-κB), tumor necrosis factor-α (TNF-α), interleukin (IL-1β, IL-6) and cell apoptotic rate in hippocampus tissue were measured. Results ① mRNA expressions: the mRNA expressions of Fas and FasL of the 3 experimental groups were significantly lower than those of the hypoxic ischemic brain damage model group, the degrees of descent after administration for 24 hours were the most significant, neural stem cell treatment group < EPO treatment group < ω-3 unsaturated fatty acid treatment group < hypoxic ischemic brain damage model group [Fas mRNA expression (2-ΔΔCt): 140.5±2.9, 156.4±2.5, 165.2±2.7 vs. 173.7±2.8, FasL mRNA expression (2-ΔΔCt): 143.1±4.3, 154.6±1.5, 160.7±1.4 vs. 174.7±2.8], the differences were statistically significant (all P < 0.05). ② Protein expressions: the protein expressions of TLR4, NF-κB, TNF-α, IL-1β, IL-6 of the 3 experimental groups were significantly lower than those of the hypoxic ischemic brain damage model group (TLR4/GAPDH: 0.7±0.2, 0.6±0.1, 0.2±0.1 vs. 1.4±0.1; NF-κB/GAPDH: 6.7±0.4, 5.3±0.1, 1.1±0.2 vs. 11.2±0.3; TNF-α/GAPDH: 14.3±1.4, 11.2±1.2, 3.2±2.1 vs. 23.2±0.5; IL-1β/GAPDH: 9.4±0.2, 7.4±0.3, 2.2±0.3 vs. 13.4±0.1; IL-6/GAPDH: 36.2±4.4, 39.3±1.5, 26.2±2.1 vs. 51.4±1.4, all P < 0.05), the protein expression levels of above indexes in neural stem cell treatment group < those of EPO treatment group < those of ω-3 unsaturated fatty acid treatment group < those of hypoxic ischemic brain damage model group. ③ Apoptotic rates:after drug administration, the apoptotic rates of the ω-3 unsaturated fatty acid group, EPO treatment group, neural stem cell treatment group were obviously lower than the rate of model group [(3.7±0.3)%, (3.4±0.2)%, (2.5±0.1)% vs. (5.5±0.4)%, all P < 0.05]. Conclusion The mRNA expressions of Fas/FasL in the brain of neonatal rats with hypoxic-ischemic brain damage are high, and the treatment with each of the following agents; neural stem cells, EPO and ω-3 unsaturated fatty acid can reduce the mRNA expressions of Fas/FasL in such rats' brain tissues.
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Objective:To investigate the effects of enriched environment on hippocampal neuronal apoptosis and brain-derived neurotrophic factor (BDNF)/tyrosine kinase receptor B (TrkB) signaling pathway in neonatal rats with hypoxic-ischemic brain damage. Methods:Forty-eight newborn Wistar rats aged seven days were randomly divided into sham operation group, model group and enriched environment group, each group was divided in to 14 days group and 28 days group, with eight in each subgroup. The model was established with the Rice method. The sham operation group and the model group did not receive any intervention, and the enriched environment group received enriched environment stimulation 24 hours after modeling. Fourteen days and 28 days after modeling, the levels of neuronal apoptosis in hippocampus were detected by TUNEL and double immunofluorescence staining; BDNF and TrkB proteins in hippocampus were detected by immunohistochemical staining. Results:Fourteen days and 28 days after modeling, the numbers of TUNEL positive cells, double immunofluorescence positive cells, BDNF and TrkB positive cells were significantly more in the model group than in the sham operation group (t > 27.214, P < 0.001), while the numbers of TUNEL positive cells, double immunofluorescence positive cells were significantly less in the enriched environment group than in the model group (t > 12.687, P < 0.001); and the number of BDNF and TrkB positive cells were significantly more in the enriched environment group than in the model group 28 days after modeling (t > 137.998, P < 0.001). Conclusion:Enriched environmental stimulation could reduce the apoptosis of hippocampal neurons, and up-regulate the expression of BDNF and TrkB proteins in the neonatal rats with hypoxic-ischemic brain damage.
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Targeted temperature management (TTM) is referred to as the reducing of the core body temperature to a specific temperature to repair or mitigate tissue damage caused by inadequate blood perfusion. It is a promising treatment method. However, as it is widely used in clinical practice, more and more disputes have been made about the scope and effect of TTM. This paper will review the mechanism of TTM,the method of its implementation and its application in the disease, so as to provide references for further understanding of TTM and optimizing the clinical application of TTM.
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Objective@#To investigate the role of granulocyte-colony stimulating factor (G-CSF) on the regulation of inflammatory cytokines in neonatal hypoxic-ischemic brain damage(HIBD) rat model, and to explore the possible mechanism involved in G-CSF neuroprotective effect via the mammalian target of Rapamycin/p70 ribosomal S6 protein kinase (mTOR/p70S6K) signaling pathway.@*Methods@#A group of postnatal day 7 (P7) Sprague-Dawley rat pups (90 cases) were randomly divided into sham-operated group, hypoxia-ischemia(HI) group, G-CSF group, Rapamycin (RAP) group and control group, and the improved Rice method was used to establish a neonatal rat model of HIBD.One hour before HI induction, Rapamycin was administered intraperitoneally with a dose of 250 μg/kg, and the control group was given equal volume of ethanol injected intraperitoneally.One hour after HI, a dose of 50 μg/kg of G-CSF was injected intraperitoneally into the G-CSF group, Rapamycin group and control group.The same volume of normal saline was injected intraperitoneally into HI group and sham-operated group.Forty-eight hours after HI, Western blot was used to detect the protein levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-10, and the mTOR/p70S6K signaling pathway in brain tissue.Neuron injury of the hippocampal CA1 region and the cortex was assessed by Nissl staining, and infarct volume detected by 2, 3, 5-triphenyltetrazolium chloride staining.@*Results@#The G-CSF group and control group were associated with significantly reduced infarction volume compared to the HI group [(12.87±1.54)%, (11.90±1.31)% vs.(24.21±3.28)%], and the differences were statistically significant(P<0.05). There was an increased positive neuron cell number in the ipsilateral hemispheres of the hippocampal CA1 region in the G-CSF group and the control group [(61.00±4.90) cell/field and (61.67±6.40) cell/field] and cortex [(92.67±6.68) cell/field and (90.17±4.45) cell/field] compared with those in HI group [(42.62±4.46) cell/field and (70.83±6.97) cell/field], and the differences were all statistically significant (all P<0.05). The expression levels of TNF-α and IL-1β were significantly decreased in the G-CSF group and the control group, compared with those in HI group(TNF-α: 0.67±0.07, 0.55±0.05 vs.0.86±0.05; IL-1β: 0.65±0.06, 0.52±0.10 vs.0.86±0.06), and the differences were all statistically significant (all P<0.05). There was increased expression levels of IL-10, p-mTOR/mTOR and p-p70S6K/p70S6K in the G-CSF group and the control group, compared with those in HI group (IL-10: 0.68±0.04, 0.62±0.05 vs.0.34±0.02; p-mTOR/mTOR: 0.53±0.02, 0.51±0.01 vs.0.26±0.01; p-p70S6K/p70S6K: 0.89±0.03, 0.90±0.03 vs.0.55±0.02), and the differences were all statistically significant(all P<0.05). There was an increased infarct volume in Rapamycin group [(25.70±1.50)%], compared with the G-CSF group and the control group, and there were decreased number of positive neuron cell count in the hippocampal CA1 region [(40.67±3.50) cell/field] and cortex [(68.33±8.17) cell/field], increased expression levels of TNF-α and IL-1β (0.97±0.06 and 0.98±0.10, respectively), decreased expression levels of IL-10, p-mTOR/mTOR and p-p70S6K/p70S6K (0.21±0.02, 0.30±0.01 and 0.55±0.01, respectively) in the Rapamycin group, and the differences were all statistically significant (all P<0.05).@*Conclusions@#G-CSF may inhibit inflammatory responses after HIBD by up-regulating the mTOR/p70S6K signaling pathway in neonatal HI encephalopathy.
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Neonatal hypoxic-ischemic encephalopathy (HIE) is a leading cause of infant morbidity and mortality,often causing irreversible brain damage,and eventually leading to neurodevelopmental and cognitive dysfunction.Previous studies have shown that some commonly used inhaled anesthetics has certain neuroprotective effects on hypoxic-ischemic brain damage(HIBD),but its mode of action and the mechanisms are not clear.In this review,the protective effect of inhaled anesthetics on HIBD in neonates and its related mechanisms are described.
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Aim To explore the protective effects of naringin on hypoxic ischemic brain injury in neonatal rats and its mechanism. Methods Ninety-six healthy 7-day neonatal SD rats were randomly divided into sham operation group, hypoxic-ischemic brain damage group (HIBD group),HIBD with low-dose naringenin group(50 mg·kg-1, NG-L) and HIBD with high-dose naringenin group(100 mg·kg-1,NG-H). Neu-rological deficit, HE staining and brain water content were measured 48h after operation. Immunoblotting was used to detect the expressions of NOD2,RIP2 and NF-κB. Enzyme linked immunosorbent assay(ELISA) method was adopted to detect TNF-α and IL-1β ex-pression. Results Compared with HIBD group, the neurological deficit score decreased, the pathological damage was reduced, and the water content of brain tissues markedly decreased by naringenin(50,100 mg ·kg-1) treatment(P<0.05). Western blot revealed the down-regulation of NOD2,RIP2 and NF-κB by na-ringenin (50,100mg·kg-1) treatment (P<0.05). The content of TNF-α and IL-1β in brain tissues was lower than that of HIBD group (P <0.05). Conclu-sion Naringenin is likely to exert a protective role in neonatal rats of hypoxic ischemic brain injury perhaps through decreasing the expression of NOD2, RIP2 and NF-κB,and reducing the secretion of TNF-α and IL-1β.
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Objective To explore the pathophysiology and MRI features of hypoxic-ischemic brain damage in premature and the correlation with the degree of hypoxia,and to improve the understanding of this disease.Methods Thirty patients with clinically diagnosed hypoxic-ischemic brain damage in preterm infants were examined by MRI and the MRI findings were analyzed. Results Of the 30 children, 7 cases suffered from single subventricular hemorrhage; 6 cases had subventricular hemorrhage combined with a small amount of hemorrhage in the periventricular white matter; 4 cases had subarachnoid hemorrhage and intraventricular hemorrhage;2 cases had subarachnoid hemorrhage;9 cases had periventricular white matter injury; 2 cases had hemorrhagic cerebral infarction. Conclusion MRI findings of hypoxic-ischemic brain damage in premature infants are complex and diverse,and are associated with multiple signs.There is no specific relationship between MRI signs and the degree of perinatal hypoxia.MRI can accurately detect the lesion,and provide a reliable imaging basis for clinical diagnosis and treatment.
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Objectives To explore the effect of exogenous erythropoietin (EPO) on the expression of glial fibrillary acidic protein (GFAP) in hippocampal CA1 region and 5- bromide -2- uracil (BrdU) in hippocampal DG region in neonatal Wistar rats with hypoxic-ischemic brain damage (HIBD). Methods Forty-eight Wistar rats aged 7 days were randomly divided into HIBD model group and EPO experimental group, and another 24 rats as sham operated group. The HIBD model was established by ligating the right common carotid artery and inhaling hypoxia gas mixture (8％ O2 and 92％ N2) for 2 h. The expression of GFAP in hippocampal CA1 region and the number of BrdU positive cells in the hippocampus were detected by immunohistochemical method on at 14 d, 21 d, and 28 d after operation and compared among three groups. Results On 14 d and 21 d after operation, the expression of GFAP in CA1 region and the number of BrdU positive cells were statistically different among three groups (P<0.01) with EPO experimental group having the highest, HIBD model group having the second highest and sham operation group having the lowest in both, . On 28 d after operation, there was no difference in the expression of GFAP and the number of BrdU positive cells in the DG among three groups (P>0.05). At different time point (14 d, 21 d, 28 d) in every group, the expression of GFAP in CA1 region and the number of BrdU positive cells in DG region were all statistically different (P<0.01), all with the highest on 14 d after operation, second highest on 21 d, and the lowest on 28 d. Conclusions Early administration of exogenous EPO can promote the expression of GFAP in hippocampal CA1 region and increase the number of BrdU positive cells in DG region, which indicates that EPO can promote the proliferation and regeneration of damaged neurons. EPO had neuroprotective effect on neonatal rats with hypoxic-ischemic brain damage.
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@#The enriched environment is an artificial environment for animal models of rodentia. In the enriched environment, model animals may improve synaptic plasticity, inhibit apoptisis and regulate autophage after hypoxic-ischemic brain damage, that promote the recovery.
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Notoginsenoside R1(NGR1),a critical compound in traditional herb Panax notoginseng, is a kind of estrogen receptor agonist.It is reported to exhibit anti-apoptotic,anti-oxidative and anti-inflammatory properties activity, so it is widely used for treatment of various diseases.In order to investigate the potential neuroprotective effect of NGR1 in hypoxic-ischemic brain damage(HIBD), primary cortical neurons were used in this study to establish oxygen-glucose deprivation/reoxygenation(OGD/R) injury models. They were treated with NGR1 and estrogen receptor inhibitor ICI-182780 respectively, then the neuronal survival, cell membrane integrity and apoptosis were assessed by MTT assay,lactate dehydrogenase test(LDH) and Hoechst 33342 stain respectively, while the protein expression levels of ATF6α,p-Akt,Akt,Bax and Cleaved Caspase-3 were measured by Western blotting. Results indicated that as compared with the blank control group,OGD/R could induce cell injury and apoptosis(P<0.05), reduce relative integrity of cell membrane(P<0.05), decrease protein expression of ATF6α,p-Akt(P<0.05), and increase protein expression of Bax and Cleaved Caspase-3(P<0.05) in the primary cortical cells. After NGR1 treatment, the expression levels of ATF6α,p-Akt were obviously increased, and the expression levels of Bax and Cleaved Caspase-3 and the apoptosis of neuron were decreased(P<0.05). However, these neuroprotective properties of NGR1 against ODG/R-induced cell damage could be blocked by ICI-182780. This finding indicated that NGR1 may protect the primary cortical neurons against OGD/R induced injury,and the mechanism may be associated with accelerating the activation of the ATF6/Akt signaling pathway via estrogen receptors.
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Objective To investigate the effect of erythropoietin (EPO) on the expression of integrin-β1 in hippocampus of neonatal rats after hypoxic-ischemic brain damage (HIBD) and to discuss the neuroprotective mechanisms for EPO.Methods Neonatal Sprague-Dawley rats of 7 days old were randomly divided into 3 groups by random number table method (24 cases in each group):sham-operated group,HIBD group and EPO treated group(EPO group),then each group was further divided into 4 subgroups (n=6) based on different time points following the injection of 9 g/L or EPO (6 h,24 h,3 d,7 d).The expressions of integrin-β1 and integrin-β1 gene in hippocampus were determined by Western blot and real-time fluorescent quantitative PCR(RT-qPCR).Results By Western blot method:integrin-β1 had low expression in the hippocampus of the sham-operated group at each time point,which had no significant difference(0.47±0.22,0.45±0.18,0.54±0.24,0.53±0.22,F=.223,P=0.879).The expression of integrin-β1 in HIBD group was increased at first and then decreased,which had a significant difference(0.62±0.16,1.42±0.32,1.13±0.37,0.81±0.21,F=11.809,P=0.000).In the EPO group the expression of integrin-β1 was rising significantly at 24 h and 7 d and showed two peaks (0.64±0.21,1.10±0.37,0.96±0.30,1.19±0.31,F=3.741,P=0.028).Compared with the sham-operated group at corresponding time points,the integrin-β1 protein expression showed significant increase at 24 h and on 3 d in HIBD group and 24 h,3 d,and 7 d in EPO group (all P<0.05).By RT-qPCR:there was no significant difference in the gene expression of integrin-β1 in ippocampus of the sham-operated group at different time points (0.31±0.13,0.33±0.16,0.34±0.15,0.32±0.17,F=0.036,P=0.990).Gene expression of integrin-β1 in HIBD group was increased at first and then decreased and the summit was at 24 h point(0.72±0.35,1.39±0.33,0.95±0.24,0.61±0.25).The difference between each time point was statistically significant (F=8.022,P=0.001).Integrin-β1 gene expression of EPO group showed two peaks at 24 h and on 7 d(0.77±0.30,1.09±0.27,0.90±0.38,1.17±0.36),and there was a significant difference between 6 h group and 7 d group (P<0.05).Compared with the sham-operated group at corresponding time points,the expression of integrin-β1 gene in between HIBD group and EPO group was extremely higher(all P<0.05).Besides,the gene expression of integrin-β1 in EPO group was higher than that in the HIBD group on 7 d with statistically significant difference (P<0.05).Conclusion Erythropoietin can upregulate the expression of integrin-β1 in the recovery phase of HIBD,which may be one of the beneficial effects of EPO for HIBD.
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Aim To study the therapeutic effect of CpG-ODN, an agonist of Toll-like receptor 9 (TLR9), on hypoxic/ischemic encephapathy in neonatal rats and investigate the mechanisms.Methods Fifty healthy 7-day-old neonatal Wistar rats (in either gender, weighing 12~17g) were randomly divided into sham operation group, HIBD group, and CpG-ODN low group(0.35 mL·kg-1), CpG-ODN middle group(1.40 mL·kg-1), CpG-ODN high group(5.60 mL·kg-1).The neurological function was scored after 48h operation;ten rats of each group was executed respectively and brains tissue was taken;HE staining was used to observe the brain pathological changes.Western blot assay was used to detect the expressions of TLR9 and phosphor-p38 mitogen-activated protein kinases(p-p38 MAPK), and enzyme linked immunosorbent assay (ELISA) method was adopted to detect TNF-α expression.Results The CpG-ODN low, middle group were improved in impairment significantly compared with the HIBD group, and the brain pathological change was lessened, while the CpG-ODN high group was impaired significantly compared with the HIBD group (P<0.05), and brain pathological change was sharpened.Western blot showed the up-regulation in TLR9 and p-p38 MAPK and a significant increase of the expression of TNF-α in the brain tissue in CpG-ODN group with statistical difference in HIBD group and sham operation group(P<0.05).Conclusions The neuro-behavioral score and nervous system function can be improved and the hypoxic/ischemic brain damage can be reduced in neonatal rats in the CpG-ODN low, middle group.The protective mechanisms may be suitably via activating p38 MAPK signaling pathway to promote p38 MAPK phosphory1ation and up-regulation of the expression of TNF-α in the brain tissue of rats.
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Objective To explore the protective effects of Quercetin on hypoxic ischemic brain damage (HIBD). Methods Forty-eight 7-day-old SD rats were randomly divided into sham-operation group, HIBD group and Que treatment group, 16 rats each. HIBD group and Que treatment group were treated by ligation of right common carotid artery to make anoxia and build HIBD model; sham-operation group had the separation of the right common carotid artery only. Que treatment group were injected intraperitoneally with quercetin (40 mg/kg) once a day for 7 days immediately after modeling while sham-operation group and HIBD group received equivalent normal saline at the same time. The rats in each group were scored of neurological function at 1 h after the last administration, and the ability of spatial learning-memory was tested by Morris water maze at the age of 28 days. After performing the test above, all rats were decapitated and the brains were taken. Pathological changes of hippocampus were observed by HE staining; the expression of brain-derived neurotrophic factor (BDNF) and nerve growth associated protein (GAP-43) in hippocampus CA1 area were detected by immunohistochemistry. Results There were significant differences in neurological deficit score and learning-memory ability among the three groups (P<0.01), and neurological deficit score was the highest and the learning-memory ability was the lowest in HIBD group. Pathological examination showed that the structure of hippocampal neurons was intact in sham-operation group. It was loose and disorder, and even loss of neurons in HIBD group. Compared with the HIBD group, the loose in the structure of hippocampal was lighter, and the number of neurons was increased in the Que treatment group. There was statistical difference in the positive expression of BDNF and GAP-43 in the hippocampal CA1 area among the three groups (P<0.01), with those in HIBD group being lower than in Que treatment group and sham-operation group and those in treatment group being lower than in sham-operation group (P<0.01). Conclusions Quercetin can enhance the expression of BDNF and GAP-43 in hippocampal CA1 region, promote nerve regeneration, improve the long-term learning-memory ability of HIBD neonatal rats, and protect the brain.
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Objective To explore the effects of environmental enrichment (EE) on learning and memory ability and the expression of brain-derived neurotrophic factor (BDNF) and synaptophysin in hippocampus of neonatal rats with hypoxic-ischemic brain damage(HIBD).Methods Forty Wistar neonatal male rats aged 7 days were randomly divided into EE intervention for 6 hours(6 h EE) group (n =10),EE intervention for 12 hours (12 h EE) group (n =10),model group (n =10) and sham group (n =10).The first 3 groups were performed with HIBD.The 6 h EE and 12 h EE group received EE stimuli for 6 h and 12 h respectively,once a day for 14 days.Learning and memory of the rats were tested by using Morris water maze.The expression levels of BDNF and synaptophysin in hippocampus were determined with Western blot.Results The escape latency of all groups gradually reduced with the increase of training days,but there was no significant difference in the escape latency among the 4 groups (F =0.237,P ＞ 0.05).The rats in the 6 h EE group,12 h EE group and model group spent less time in the target quadrant and showed a significant reduction of BDNF and synaptophysin(6 h EE group:0.529 ± 0.038,0.889 ± 0.027;12 h EE group:0.660 ± 0.034,1.114 ± 0.037;model group:0.225 ± 0.015,0.672 ± 0.057) in the hippocampus compared with the sham group (0.803 ± 0.026,1.347 ± 0.092) (all P ＜ 0.01).In the 6 h EE group and 12 h EE group,the rats significantly increased the time spent in target quadrant and aggrandized the expression of BDNF and synaptophysin in hippocampus compared with the model group.Moreover,the 12 h EE group had a better performance than the 6 h EE group in the space exploration and the expression of BDNF and synaptophysin.Conclusion EE is helpful for improving learning and memory ability in neonatal rats with HIBD,which may be associated with up-regulating the expression of BDNF and synaptophysin in hippocampus.