Hypoxia-ischemia Group Research Articles

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

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

Downregulation of Iduna is associated with AIF nuclear translocation in neonatal brain after hypoxia–ischemia

In adult stroke models, the neuroprotective protein, Iduna, inhibits poly (ADP-ribose) polymerase-1 (PARP-1)-dependent cell death by decreasing apoptosis-inducing factor (AIF) nuclear translocation. Because the PARP1-dependent pathway and Iduna, which promotes AIF degradation, contribute to hypoxic–ischemic (HI) brain damage in the immature brain, we examined the relationship between Iduna expression and AIF nuclear translocation in the cerebral cortex of postnatal day 7 rats after HI. Ninety rats were divided into three groups: sham, 1-h hypoxia and 2-h hypoxia. The HI insult was induced by permanent ligation of the left common carotid artery plus 1 or 2h of hypoxia. Brain damage pathological features were evaluated by hematoxylin and eosin staining, Nissl staining, transmission electron microscopy, TUNEL staining and immunofluorescence. Immunohistochemistry and western blot analysis were used to assess protein expression and ubiquitination status of AIF. The interaction between Iduna and AIF was tested by co-immunoprecipitation. Learning and memory were analyzed by the Morris water maze test. Compared with sham animals, the number of surviving neurons in the cerebral cortex decreased, and cell damage and DNA breakage were severe in rats with HI, with worse damage in the 2-h group. Iduna expression significantly decreased, whereas nuclear AIF expression increased. Furthermore, Iduna downregulation negatively correlated with nuclear AIF abundance in the 2-h HI group (r=−0.950; P<0.0001). Additionally, learning and memory ability decreased with hypoxic time. These results suggest that AIF nuclear translocation and neuronal cell death are associated with Iduna loss after severe HI in the immature brain.

Longer hypoxia–ischemia periods to neonatal rats causes motor impairments and muscular changes

Prematurity and hypoxia–ischemia (HI) can lead to movement disorders in infants. Considering that mild-moderate HI induced at postnatal day (PND) 3 has failed to produce motor disabilities similar to those seen in pre-term newborns, the main goal of the present study was to verify whether longer hypoxia periods would mimic motor function impairment, brain and muscle morphological alterations. Forty-nine Wistar rat pups of both sexes were randomly assigned to surgical control (CG) and HI groups. HI animals were submitted to the Levine-Rice model at PND 3, and exposed to 120 (HI-120′), 180 (HI-180′) or 210 (HI-210′) minutes of hypoxia (FiO2: 0.08). Sensorimotor function was assessed as from PND 35–45, by means of grasping strength, adhesive removal, cylinder and ladder walking tests. Histological staining was used to quantify the striatal volume and the cross-sectional area (CSA) of skeletal muscles. Cylinder and adhesive removal test evidenced that HI-180′ and HI-210′ groups had asymmetrical use of the forepaws when compared to controls. HI animals showed a decrease in the step placement quality and an increase in step errors when compared to CG (P⩽0.05). Reduction in striatal volume correlates with behavioral assessment, HI-180′ and HI-210′ groups presented lower biceps brachii and tibialis anterior CSA. These results show that rats exposed to longer hypoxic periods at PND3 have encephalic and sensorimotor impairments that mimic those observed in preterm infants. Morphological changes in muscle tissue evidence a new pathophysiological characteristic of the HI model that might be of relevance for the study of sensorimotor deficits.

Permeability and distribution of nerve growth factor in the brain of neonatal rats by periphery venous injection in hypoxic-ischemic state.

ObjectiveTo investigate the permeability of β-NGF through blood–brain-barrier (BBB) in neonatal and adult rats, and the spatial distribution of β-NGF in different brain regions in hypoxic-ischemic (HI) and normal neonatal rats.MethodsTo investigate the overall permeability of β-NGF through BBB, β-NGF labeled with I125 was injected into adult rats, neonatal rats and HI neonatal rats via tail vein. The radioactivity of brain tissue and blood was examined and analyzed 30 min after injection. Also, brain regions including the basal forebrain, frontal cortex, hippocampus, hypothalamus, cerebellum, bulbus olfactorius and hypophysis, of all the rats were dissected and radioactivity was examined to investigate the spatial specificity of NGF permeation through BBB.ResultsStatistically significant results were observed in I125-β-NGF contents in brain tissues of adult rats group, neonatal rats group and HI neonatal rats group (P < 0.05). Compared to the HI neonatal rats’ brain with the highest I125-β-NGF contents, normal neonatal rats ranks the second while the adult rats were the lowest. While for the spatial specificity examination part, I125-β-NGF in both HI group and control group were widely distributed in basal forebrain, frontal cortex, hippocampus, cerebellum and bulbus olfactorius. But the radioactivity in frontal cortex, hippocampus and cerebellum of HI groups are statistically higher than control groups (P < 0.05).Conclusionβ-NGF can more easily penetrate the BBB of newborn rats than adult rats via peripheral venous administration and this effect can be enhanced by HI insult. Also, this HI-induced permeation of β-NGF through BBB is more obvious in frontal cortex, hippocampus and cerebellum.

Temporal Profile of Circulating microRNAs after Global Hypoxia-Ischemia in Newborn Piglets

Background: There is a lack of reliable biomarkers that can identify and grade acute hypoxic-ischemic encephalopathy in newborns. MicroRNAs (miRNA) are short, non-coding strands of RNA that are released into the circulation in response to tissue stress and injury. Some miRNAs are highly tissue specific and thus may potentially be non-invasive biomarkers of neonatal hypoxic-ischemic brain injury. Objective: The aim of this study was to characterize the temporal expression of selected circulating miRNAs in a clinically relevant piglet model of neonatal hypoxia-ischemia (HI). Methods: A total of 13 anesthetized newborn piglets were randomized to either a control group (n = 5) or transient global HI group (n = 8). HI was achieved by ventilation with 8% oxygen until the point of severe acidosis (arterial base excess ≤-20 mmol/l) and/or hypotension (mean arterial blood pressure ≤20 mm Hg) was reached. Plasma was sampled at baseline, at the end of HI and 0.5, 3.5 and 9.5 h after HI. MiRNA expression was measured by qRT-PCR. Results: Compared to baseline, miR-374a increased during HI (p = 0.01), remained elevated at 0.5 h after HI (p = 0.02) and was downregulated at 9.5 h after HI (p = 0.02). MiR-210 increased during HI (p = 0.02) and rapidly normalized by 0.5 h after HI. MiR-124 and miR-125b did not exhibit significant alterations. Correlations were observed between miR-374a, arterial pH, base excess and lactate levels, and between miR-210 and pO₂ (p < 0.05). Conclusions: Our data suggest that miR-374a and miR-210 are important regulators in neonatal HI and might have a place as biomarkers in this setting.

Combined damage produced by multiple mild cerebral insults assessed using MRI in neonatal rats

PurposeTo determine whether damage to neonatal brain is exacerbated with multiple mild cerebral insults as detected with MRI and corroborated using histology. Materials and methodsThe combined brain injury produced by multiple procedures was compared in neonatal rats having: Sham surgery at P5, Sham surgery at P5 plus a diffuse mild transient unilateral cerebral hypoxia ischemia (HI) at P7, HI alone, and a minor photothrombotic (PT) stroke at P5 followed by HI. MRI after the ischemic insults was followed by final histology. ResultsPT produced lesions with increased T2 and decreased apparent diffusion coefficient for water (ADC) but no significant effects of a second HI. However, near the PT lesion/parietal cortex there were patchy areas of enhanced T2 and decreased ADC in 6/9, 3/8 and 0/8 animals in the PT+HI, Sham+HI and HI groups, respectively (P<0.05). Patches corresponded histologically to increased vacuolation and cell death and were more pronounced in the PT+HI and Sham+PT groups than the HI group. ConclusionThe extent of damage produced by a minor neonatal stroke followed by a diffuse HI two days later results in heterogeneous enhancement of T2, ADC and histological injury near the lesion. Surgical procedures including mechanical head manipulation followed by HI also produced some enhanced heterogeneity of hypoxic-ischemic injury affirming the need for sham controls.

Helium preconditioning protects the brain against hypoxia/ischemia injury via improving the neurovascular niche in a neonatal rat model

This study aimed to investigate whether helium preconditioning (He-PC) is able to exert neuroprotective effects via improving focal neurovascular niche in a neonatal rat hypoxia/ischemia (HI) brain injury model. Seven day old rat pups were divided into control group, HI group and He-PC group. HI was induced by exposure to 8% oxygen for 90min one day after preconditioning with 70% helium—30% oxygen for three 5-min periods. At 3 and 7 days, the brain was collected for the detection of inflammation related factors (tumor necrosis factor α [TNF-α], interleukin-1β [IL-1β], IL-10) and growth/neurotrophic factors (brain-derived neurotrophic factor [BDNF], basic fibroblast growth factor [bFGF] and nerve growth factor [NGF]); at 7 days, neurobehaviors were evaluated, and the brain was collected for the detection of mRNA expression of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) by PCR, protein expression of angiogenesis related molecules (VEGF, Ang-1, Tie-2 and Flt-1) by Western blotting and microvessel density (MCD) by immunohistochemistry for vWF. Results showed He-PC was able to reduce TNF-α and IL-1β, further increase IL-10, BDNF, bFGF and NGF, elevate the mRNA expression of VEGF and Ang-1, increase the protein expression of VEGF, Ang-1, Tie-2 and Flt-1, promote angiogenesis and improve neurobehaviors as compared to HI group. These findings suggest that He-PC may improve the post-stroke neurovascular niche to exert neuroprotective effects on neonatal HI brain injury.

Insights into the role of iron in immature rat model of hypoxic-ischemic brain injury.

This study aimed to investigate the role of iron in the occurrence and development of hypoxic-ischemic brain injury (HIBI) in immature rat models using 3-day-old Sprague Dawley rats. Normal control (NC), hypoxic-ischemic (HI), anemia, HI + ischemia, early iron treatment and late iron treatment groups were established. Rat brain tissue sections were stained with hematoxylin and eosin and pathologically evaluated. Iron content and mRNA expression levels of iron regulatory protein 2 (IRP2) and transferrin receptor in the brain tissues were measured. Ultrastructural changes in the actin, microtubules, myelin and mitochondria of oligodendrocytes and axons were examined by electron microscopy. Numbers of viable myelin sheaths and oligodendrocytes in the periventricular area were also observed. Pathological damage of brain tissue in the HI group was markedly increased compared with that in the NC group. Furthermore, there was a higher iron content and reduced number of viable oligodendrocytes in the periventricular area of the HI group compared with the NC group. No significant difference in iron content was observed between the HI + anemia and NC groups. The number of viable oligodendrocytes in the HI + anemia group was increased compared with that in the HI group, and the number in the HI + anemia group with late iron treatment was lower compared with that in the NC group and increased compared with that in the HI + anemia group. Electron microscopy revealed a significantly higher number of myelin sheaths in the HI + anemia group than in the HI group. IRP2 mRNA expression levels in the brain tissues were significantly decreased in the HI + anemia group compared with the HI group. The results suggest that anemia may reduce the rate of increase of iron content of the brain following HI. However, the early occurrence of anemia may protect against HIBI.

Environmental enrichment attenuates the blood brain barrier dysfunction induced by the neonatal hypoxia-ischemia

Environmental enrichment (EE) is considered an efficient neuroprotector against neonatal hypoxia-ischemia (HI). Nevertheless, the mechanisms involved are not yet clear. In this context, the aim of this study was to investigate the effects of neonatal HI and environmental stimulation in the hippocampus of rats at 3 different time points (PND 8, 22 and 60), evaluating some aspects of BBB structure and function. Seven-day-old Wistar rats were divided into four groups: a control group maintained in a standard environment (CTSE), a control group maintained in an enrichment environment (CTEE), an HI group maintained in a standard environment (HISE) and an HI group maintained in an enrichment environment (HIEE). At the 7th postnatal day (PND), rats were submitted to the Levine-Rice model of neonatal HI. This method consists of permanent occlusion of the right common carotid artery with subsequent exposure to hypoxia. Rats from CTEE and HIEE were stimulated with environmental enrichment. The EE protocol started 24h after HI, in which pup rats with their dams were stimulated in a maintained EE (PND 8–22). Subsequently, animals were submitted to daily EE (1h/day, PND 23–60). The expression of some proteins involved in BBB structure (β-catenin, occludin, connexin-43, aquaporin-4, glut-1 and GFAP) were quantified by western blotting in the hippocampi of rats in three periods, at PND 8, 22 and 60. The BBB permeability and integrity was assessed by Evans blue staining and the immunohistochemistry for GFAP in the CA1 region of the hippocampus were also performed. The results showed an HI-induced decreased occludin expression at PND 22 and low levels of occludin, β-catenin and GFAP at PND 60 in the hippocampus of the hypoxic-ischemic rats. Interestingly, in young and adult rats, EE reversed these effects. Evans blue extravasation into the brain parenchyma confirmed the BBB dysfunction brought on by HI. No differences were observed at PND 8, probably due to the immaturity of the BBB at this age. The present study makes an important contribution to understanding the mechanism of the hypoxic-ischemic brain damage and also to presents, for the first time, the recovery of BBB dysfunction as a possible pathway for the protective effect of EE.

Neuroprotective actions of taurine on hypoxic-ischemic brain damage in neonatal rats

Taurine is an abundant amino acid in the nervous system, which has been proved to possess antioxidation, osmoregulation and membrane stabilization. Previously it has been demonstrated that taurine exerts ischemic brain injury protective effect. This study was designed to investigate whether the protective effect of taurine has the possibility to be applied to treat neonatal hypoxic-ischemic brain damage. Seven-day-old Sprague-Dawley rats were treated with left carotid artery ligation followed by exposure to 8% oxygen to generate the experimental group. The cerebral damage area was measured after taurine post-treatment with 2,3,5-triphenyltetrazolium chloride (TTC) staining, Hematoxyline-Eosin (HE) staining and Nissl staining. The activities of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), myeloperoxtidase (MPO), ATP and Lactic Acid productions were assayed with ipsilateral hemisphere homogenates. Western-blot and immunofluorescence assay were processed to detect the expressions of AIF, Cyt C, Bax, Bcl-2 in brain. We found that taurine significantly reduced brain infarct volume and ameliorated morphological injury obviously reversed the changes of SOD, MDA, GSH-Px, T-AOC, ATP, MPO, and Lactic Acid levels. Compared with hypoxic-ischemic group, it showed marked reduction of AIF, Cyt C and Bax expressions and increase of Bcl-2 after post-treatment. We conclude that taurine possesses an efficacious neuroprotective effect after cerebral hypoxic-ischemic damage in neonatal rats.

Effect of HET0016 combined with mild hypothermia on expression of m-calpain protein in cerebral neurons of neonatal piglets with hypoxic-ischemic encephalopathy

Objective To evaluate the effect of HET0016 combined with mild hypothermia on the expression of m-calpain protein in cerebral neurons of neonatal piglets with hypoxic-ischemic encephalopathy (HIE). Methods Thirty-six healthy neonatal piglets (1-2 days after birth), were randomly divided into 4 groups using a random number table: sham operation group (S group, n=6), hypoxia-ischemia and normothermia group (HI+ NT group, n=6), hypoxia-ischemia and mild hypothermia group (HI+ MH group, n=12), and hypoxia-ischemia and HET0016 combined with mild hypothermia group (HI+ HET+ MH group, n=12). The model of HIE was established by using inhalation of the low concentration of oxygen and asphyxia under anesthesia.In HI+ MH and HI+ HET+ MH groups, the body temperature was cooled down to 34 ℃ by using the ice packs at 3 h after establishment of the HIE model.In group HI+ HET+ MH, HET0016 1 mg/kg was infused intravenously at 3 min after cardiopulmonary resuscitation.At 10 days after independent feeding, the animals were sacrificed, brains were removed, and brain tissue specimens were obtained for determination of the expression of m-calpain protein in the putamen, sensory cortex, hippocampal CA3 region, and ventroposterolateral nucleus of the thalamus using the immunohistochemical staining. Results Compared with group S, the expression of m-calpain protein in different brain regions was significantly up-regulated in the other groups (P<0.05). Compared with group HI+ NT, the expression of m-calpain protein in different brain regions was significantly down-regulated in HI+ MH and HI+ HET+ MH groups (P<0.05). Compared with group HI+ MH, the expression of m-calpain protein in different brain regions was significantly down-regulated in group HI+ HET+ MH (P<0.05). Conclusion The mechanism by which HET0016 combined with mild hypothermia inhibits neuronal apoptosis may be related to down-regulation of the expression of m-calpain protein in cerebral neurons of neonatal piglets with HIE. Key words: Hypothermia, induced; Hypoxia-ischemia, brain; Infant, newborn; Neurons; Calpain; HET0016

Effect of obstructive sleep apnea hypoxia on learning memory capacity after cerebral ischemia-reperfusion in rats

To investigate the effect of obstructive sleep apnea hypoxia on learning memory capacity in rat after ischemia. Eighty healthy male wister rats were randomly divided into: sham operation group (SO group, n=20), merely ischemia group (I/R group, n=20), and obstructive sleep apnea hypoxia for 7 days ischemia group (IH7+ I/R group, n=20), obstructive sleep apnea hypoxia for 21 days ischemia group (IH21+ I/R group, n=20). Obstructive sleep apnea hypoxia ischemia groups were respectively given obstructive sleep apnea hypoxia for 7 days and 21 days. Ischemia animals were prepared cerebral ischemia-reperfusion model by improved pulsinelli four vessels block (4-VO), the morphological changes of hippocampus nerve cells of rat brain were detected with HE, neuron pathology in hippocampal regin was observed using electron microscope, and learning memory capacity of rats were assessed by the Morris water maze test. Compared with the SO group, the I/R group demonstrated shortened escaping latency, increased frequency of crossing the platform in the water maze test, decreased survival rate of neurons, and increased apoptotic cells and ultrastructure damages(P<0.05). Compared with the I/R group, obstructive sleep apnea hypoxia ischemia groups showed shortened escaping latency, increased frequency of crossing the platform, decreased survival rate of neurons, and increased apoptotic cells and ultrastructure damages(P<0.05), especially in the IH21+ I/R group(P<0.05). Obstructive sleep apnea hypoxia can increase the damage of learning memory capacity. This damage is related to hippocampus nerve loss and ultrastructure injury from obstructive sleep apnea hypoxia.

Impacts of Erythropoietin on Vascular Endothelial Growth Factor Receptor 2 by the Extracellular Signal-regulated Kinase Signaling Pathway in a Neonatal Rat Model of Periventricular White Matter Damage

To explore the impacts of erythropoietin on vascular endothelial growth factor receptor 2 (VEGFR2) by the extracellular signal-regulated kinase (ERK) signaling pathway in a neonatal rat model of periventricular white matter damage. All of postnatal day 4 rats were randomized into three groups: the sham group [without hypoxia-ischemia (HI)], the HI group (HI with saline administration), and the erythropoietin (EPO) group [HI with recombinant human erythropoietin (rh-EPO) administration]. Rat pups underwent permanent ligation of the right common carotid artery, followed by 6% O2 for 2 hours or sham operation and normoxic exposure. Immediately after the HI, rats received a single intraventricular injection of rh-EPO (0.6 IU/g body mass) or saline. ERK and phosphorylation-ERK were examined at 60 minutes and 90 minutes after operation, and VEGFR2 were detected at 2 and 4 days after operation by using Western blot. At 60 minutes and 90 minutes after operation, the proteins of phosphorylation-ERK were significantly higher in HI rats than in the sham rats and significantly higher in HI+EPO rats than in the HI rats (P<0.05). Two days after operation, VEGFR2 was not significantly different between sham and HI rats. However, the proteins of VEGFR2 were increased after administration of rh-EPO (P<0.05). Four days after operation, the proteins of VEGFR2 were significantly higher in HI rats than in the sham rats and significantly higher in HI+EPO rats than in the HI rats (P<0.05). EPO may regulate VEGFR2 expression by affecting the intracranial ERK signaling pathways.

Effect of hypoxic-ischemic time on reduction of hypoxic-ischemic brain injury by sevoflurane postconditioning in neonatal rats

Objective To evaluate the effect of hypoxic-ischemic time on reduction of hypoxic-ischemic brain injury by sevoflurane postconditioning in neonatal rats. Methods Two hundred and ten 7-day-old Sprague-Dawley rats (105 male, 105 female), weighing 13-17 g, were randomly divided into 7 groups (n=30 each) using a random number table: sham operation group (group Sham), hypoxia-ischemia group (group HI), and sevoflurane postconditioning at different hypoxic-ischemic time point groups (P0, P3, P6, P12 and P24 groups). Immediately after ligation of the left common carotid artery, and at 3, 6, 12 and 24 h after ligation, the rats inhaled the mixed gas containing 2% sevoflurane for 30 min in P0, P3, P6, P12 and P24 groups, respectively.The fatality was recorded within 7 days after establishment of the model.At 7 days after establishment of the model, the rats were sacrificed, the brains were removed, and the right and left cerebral hemispheres were weighed separately, and the left/right cerebral hemisphere weight ratio was calculated.The hippocampal CA1 region and posterior cingulate gyrus were isolated, and the ratio of density of normal neurons in the left to the right was calculated. Results Compared with group Sham, the left cerebral hemisphere weight, left/right cerebral hemisphere weight ratio, and ratio of density of normal neurons were significantly decreased, and the fatality rate was increased in the other six groups (P 0.05). Compared with group P6, the left cerebral hemisphere weight, left/right cerebral hemisphere weight ratio, and ratio of density of normal neurons were significantly increased in P0 and P3 groups (P 0.05). Conclusion Sevoflurane postconditioning performed within 6 h of hypoxia-ischemia can reduce hypoxic-ischemic brain injury, and it provides no cerebral protection if exceeding 12 h. Key words: Time factors; Anesthetics, inhalation; Hypoxia-ischemia, brain; Infant, newborn; Ischemic postconditioning

Isoflurane postconditioning induces concentration- and timing-dependent neuroprotection partly mediated by the GluR2 AMPA receptor in neonatal rats after brain hypoxia-ischemia.

It has been demonstrated that preconditioning with 1.5% isoflurane reduces hypoxia/ischemia (HI)-induced brain loss/injury in neonatal rats. Ca(2+) influx mediated by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) is involved in HI-induced neuronal death. Here, we investigated the effective concentrations and time windows for neuroprotection by isoflurane postconditioning in neonatal rats after brain HI and determined whether GluR2-containing AMPARs mediate this neuroprotection. Seven-day-old Sprague-Dawley (SD) rats were randomly divided into eight groups (n=40 in each). The rats underwent left common carotid arterial ligation (brain HI) or sham surgery, followed by exposure to 8% oxygen for 2h at 37°C in a thermoregulated environment. Post-conditioning with 1, 1.5, or 2% isoflurane for 30min was performed immediately after brain HI. Others were post-treated with 1.5% isoflurane for 30min at 3, 6, and 12h after brain HI. The weight ratio, neuronal density ratio in the ventral posteromedial thalamic nucleus, and retrosplenial granular cortex of left to right cerebral hemispheres at 7days after brain HI were evaluated in all groups. Cerebral hemispheres were harvested for Western-blot analysis of GluR2 on the cellular membranes 24h after HI or sham surgery in neonatal rats from the sham group, the HI group, and the HI+immediate exposure to the 1.5% isoflurane group. In another experiment, the function of learning and memory were assessed in adolescence (4weeks) using Morris water maze. Compared with the control (sham) group, brain HI decreased the weight ratio and the neuronal density ratio in the ventral posteromedial thalamic nucleus and the retrosplenial granular cortex of the left to right cerebral hemispheres (p<0.05). These effects of brain HI were reduced by postconditioning with 1.5 or 2% isoflurane for 30min within 6h of HI, which coincided with the results of Morris water maze. GluR2 protein expression on cellular membranes was reduced after HI compared with sham surgery group (p<0.05); this down-regulation was attenuated by isoflurane postconditioning. Postconditioning with 1.5 and 2% isoflurane affords neuroprotection in neonatal rats. The time window for isoflurane postconditioning to be effective against neonatal HI-induced brain injury was 0-6h after HI. This protection may be mediated by GluR2-containing AMPARs.

Helium preconditioning protects against neonatal hypoxia–ischemia via nitric oxide mediated up-regulation of antioxidases in a rat model

This study aimed to investigate the role of nitric oxide (NO) in the neuroprotective effects of helium preconditioning (He-PC) in a neonatal hypoxia/ischemia (HI) rat model. Seven-day old rat pups were divided into normal control group, He-PC group, HI group, He-PC+HI group, L-NAME+HI group and L-NAME+He-PC+HI group. HI was induced by exposure to 80% oxygen for 90min. He-PC was conducted with 70% helium—30% oxygen for three 5-min periods. Three hours after He-PC, animals in control group and He-PC group were sacrificed, and the brain was collected for the detection of NO content. At 24h after HI, animals in control group, HI group, He-PC+HI group, and L-NAME+He-PC+HI group were sacrificed, and the brain was collected for detection of infarct ratio, antioxidases (SOD, HO-1 and Nrf2), DNA binding activity of Nrf2 and TUNEL staining. Three weeks later, the neurological function and brain atrophy were determined. Results showed pretreatment with L-NAME alone failed to exert protective effect on HI. He-PC significantly increased NO content, reduced the brain infarct area, increased anti-oxidases expression and DNA binding activity of Nrf2, decreased the apoptotic cells, and improved the neurological function and brain atrophy. In addition, this protection was markedly inhibited by L-NAME (a non-selective NOS inhibitor). These findings suggest that the He-PC may induce NO production to activate Nrf2, exerting neuroprotective effect on neonatal HI.

Mechanisms of neurodegeneration after severe hypoxic-ischemic injury in the neonatal rat brain

PurposeApoptosis is implicated in mild-moderate ischemic injury. Cell death pathways in the severely ischemic brain are not characterized. We sought to determine the role of apoptosis in the severely ischemic immature brain. MethodsSeven-day old rats were randomly assigned to mild-moderate or severe cerebral hypoxia-ischemia (HI) group. After ligating the right common carotid artery, animals were subjected to hypoxia for 90min in the mild-moderate HI or 180min in the severe HI. The core and peri-infarct area were measured in H&E stained brain sections using NIS Elements software. Brain sections were processed for caspase-3, AIF and RIP3 immuno-staining. Number of positive cells were counted and compared between the two groups. ResultsThe core constituted a significantly higher proportion of the ischemic lesion in the severely compared to the moderately injured brain (P<0.04) up to 7 days post-injury. Apoptotic cell death was significantly higher (P<0.05) in the core than the peri-infarct of the severe HI brain. In the peri-infarct area of severe HI, AIF-induced cell death increased over time and caspase-3 and AIF equally mediated neuronal death. Necroptosis was significantly higher (P=0.02) in the peri-infarct of the severe HI lesion compared to the moderate HI lesion. In males, but not in females, apoptosis was higher in moderate compared to severe HI. ConclusionsCaspase-independent cell death plays an important role in severe ischemic injury. Injury severity, timing of intervention post-injury and sex of the animal are important determinants in designing neuroprotective intervention for the severely ischemic immature brain.

Prenatal Hypoxia Ischemia Increases Male Rat Sexual Behavior.

Research consistently indicates an association between prenatal hypoxia-ischemia (HI) and mortality and chronic neurological diseases in newborns. HI can cause permanent effects, including mental retardation, motor impairment, learning disabilities, epilepsy, and cerebral palsy. Moreover, little is known about the relationship between HI and sexual behavior. The aims of this study are to examine whether HI is associated with changes in sexual behavior. HI was induced by clamping the uterine arteries of pregnant rats. The arteries were clamped for 45 minutes on the 18th day of gestation (HI group). Shams received laparotomy and anesthesia only. Pups were born at term. At 90 days of age, sexual behavior was evaluated. Statistical analysis was performed using two-way analysis of variance and post hoc Bonferonni correction. The main outcome measures of sexual response were standard sexual behavior, homosexual behavior, and sexual attempt on nonreceptive females. The stimulatory effect of HI on male rat sexual behavior has been shown in various experimental models; these animals showed reduced mount, intromission and ejaculation latencies; increased copulatory efficiency; and homosexual mounting. Additionally, there was an increase in fighting in trying to mount an unreceptive female. Our results indicate that HI had a long-term effect on sexual behavior despite exhibiting motor skill impairment. Accordingly, injuries during the fetal period may cause behavioral problems in adulthood.

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

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

Recombinant human erythropoietin promotes angiogenesis in preterm rat models with periventricular white matter damage

Objective To evaluate whether recombinant human erythropoietin (rh-EPO)could increase the angiogenic responses in preterm rat models with periventricular white matter damage (PWMD). Methods Three-day postnatal rats were divided into 3 groups randomly: the sham group, the hypoxic-ischemic (HI)group and the recombinant human erythropoietin(rh-EPO)treatment group.Rat pups underwent permanent ligation of the right common carotid artery followed by 60 mL/L O2 for 4 h or sham operation and normoxic exposure.Immediately after the HI, rats received a single intraperitoneal injection of rh-EPO (5 U/g)or saline.Angiogenesis-related cells (CD34+ cells), microvessel density (MVD)and arteriovenous related genes (ephrinB2 and EphB4)were examined at 48 h and 96 h after operation. Results At 48 h after operation, the proteins of CD34 in HI rats increased compared with the sham rats (HI group vs Sham group: 0.54±0.05 vs 0.42±0.05, P 0.05). The proteins of CD34, the mRNA of ephrinB2 and EphB4 increased after rh-EPO treatment compared with HI rats.However, the MVD did not increase.As the proteins of CD34 increased further at 96 h after operation(HI group vs Sham group: 0.85±0.06 vs 0.62 ±0.06, P<0.05), the MVD(3.14±1.21 vs 1.50±1.04), ephrinB2 (7.51±1.89 vs 1.28±0.24)and EphB4(4.58±0.82 vs 1.21±0.22) also increased (all P<0.05). And the proteins of CD34 increased after administration of rh-EPO(EPO group vs HI group: 0.98±0.07 vs 0.85±0.06, P<0.05) and MVD(EPO group vs HI group: 4.71±1.38 vs 3.14±1.21, P<0.05) were increased after administration of rh-EPO.However, the ephrinB2 and EphB4 did not increased after administration of rh-EPO at 96h time point. Conclusion Endogenous regenerative response is triggered in the damaged tissues and rh-EPO increases the angiogenesis in model rats with PWMD. Key words: Erythropoietin; Periventricular white matter damage; Hypoxia-ischemia; Immature rat, new born