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Neonatal hypoxic-ischemic brain injury (HIBI) results in significant morbidity and mortality despite current standard therapies. MicroRNAs (miRNAs) are a promising therapeutic target; however, there is a paucity of data on endogenous miRNA expression of the brain after HIBI during the primary therapeutic window (6-72 h after injury).Postnatal day 9 mouse pups underwent unilateral carotid ligation+hypoxia (HIBI), sham surgery+hypoxia, or sham surgery+normoxia (controls). miRNA sequencing was performed on the ipsilateral brain of each of the three groups plus the contralateral HIBI brain at 24 and 72 h after injury. Findings were validated in eight key miRNAs by quantitative polymerase chain reaction.Hypoxia resulted in significant differential expression of 38 miRNAs at both time points. Mir-2137, -335, -137, and -376c were significantly altered by neonatal HIBI at 24 and 72 h, with 3 of the 4 demonstrating multiphasic expression (different direction of differential expression at 24 versus 72 h).Our global assessment of subacute changes in brain miRNA expression after hypoxia or HIBI will advance research into targeted miRNA-based interventions. It will be important to consider the multiphasic miRNA expression patterns after HIBI to identify optimal timing for individual interventions.This study is the first to comprehensively define endogenous brain microRNA expression changes outside of the first hours after neonatal hypoxic-ischemic brain injury (HIBI). Mir-2137, -335, -137, and -376c were significantly altered by neonatal HIBI and therefore deserve further investigation as possible therapeutic targets. The expression profiles described will support the design of future studies attempting to develop miRNA-based interventions for infants with HIBI. At 24 h after injury, contralateral HIBI miRNA expression patterns were more similar to ipsilateral HIBI than to controls, suggesting that the contralateral brain is not an appropriate "internal control" for miRNA studies in this model.

Aimed to investigate in an animal model the efficacy of humic acid by showing its antioxidant and anti-apoptotic effect comparing with the histopathological and neurological outcomes for the hypoxic-ischemic brain injury.28 Wistar-Albino rats who were on the 7th postnatal day and weighting between 9 and 19 g randomly divided into four groups with developed HIE model under the gas anesthesia. 20 mg/kg and 10 mg/kg intraperitoneal HA were given to Group I and II respectively. Saline was given to Group III and the sham group was Group IV. The brain tissues were stained with cresyl-violet histochemistry for grading neuronal cell injury and caspase immunohistochemistry.The neuronal cell injury was statistically lower in all neuroanatomical lands in HA treatment groups. The degree of ischemia was significantly smaller in HA groups. Caspase-3 immunoreactivity was decreased in the HA groups compared with the saline group. When the groups were compared, there were no serious neuronal injury in Group I.This is the first study which investigates the role of HA in HIE model. HA reduces apoptosis and neuronal injury in cerebral tissue of the rats. This findings suggest that HA may be viable protective agent against HIE.

In hypoxic-ischemic encephalopathy, the neural progenitors (NPs) of the developing brain fail to replenish the oligodendrocyte progenitor cells lost during hypoxic-ischemic injury (HII). In this study, we aim to examine the influence of HII on the vulnerability of human NPs derived from human embryonic stem cells with regard to cell survival and oxidative stress, followed by assessment of cellular deregulation through measuring glutathione levels, basal calcium, glutamate release, and intracellular calcium ([Ca

Background: Methylphenidate (MPH) is a stimulant drug mainly prescribed to treat cognitive impairments in attention-deficit/hyperactivity disorder (ADHD). We demonstrated that neonatal hypoxia-ischemia (HI) induced attentional deficits in rats and MPH administration reversed these deficits. However, MPH effects on memory deficits after the HI procedure have not been evaluated yet. Aims: We aimed to analyze learning and memory performance of young hypoxic-ischemic rats after MPH administration and associate their performance with brain-derived neurotrophic factor (BDNF) levels in the prefrontal cortex and hippocampus. Methods: Male Wistar rats were divided into four groups ( n=11–13/group): control saline (CTS), control MPH (CTMPH), HI saline (HIS) and HIMPH. The HI procedure was conducted at post-natal day (PND) 7 and memory tasks between PND 30 and 45. MPH administration (2.5 mg/kg, i.p.) occurred 30 min prior to each behavioral session and daily, for 15 days, for the BDNF assay ( n=5–7/group). Results: As expected, hypoxic-ischemic animals demonstrated learning and memory deficits in the novel-object recognition (NOR) and Morris water maze (MWM) tasks. However, MPH treatment did not improve learning and memory deficits of these animals in the MWM—and even disrupted the animals’ performance in the NOR task. Increased BDNF levels were found in the hippocampus of HIMPH animals, which seem to have been insufficient to improve memory deficits observed in this group. Conclusions: The MPH treatment was not able to improve memory deficits resulting from the HI procedure considering a dose of 2.5 mg/kg. Further studies investigating different MPH doses would be necessary to determine a dose–response relationship in this model.

Background Hypoxia-ischemia (HI) is the most common cause of brain injury in newborns and the survivors often develop cognitive and sensorimotor disabilities that undermine the quality of life. In the current study, we examined the effectiveness of flupirtine, a potassium channel opener, shown previously in an animal model to have strong anti-neonatal-seizure efficacy, to provide neuroprotection and alleviate later-life disabilities caused by neonatal hypoxic-ischemic injury. Methods The rats were treated with a single dose of flupirtine for four days following HI induction in 7-day-old rats. The first dose of flupirtine was given after the induction of HI and during the reperfusion period. The effect of treatment was examined on acute and chronic brain injury, motor functions, and cognitive abilities. Results Flupirtine treatment significantly reduced HI-induced hippocampal and cortical tissue loss at acute time-point. Furthermore, at chronic time-point, flupirtine reduced contralateral hippocampal volume loss and partially reversed learning and memory impairments, but failed to improve motor deficits. Conclusion The flupirtine treatment regimen used in the current study significantly reduced brain injury at acute time-point in an animal model of neonatal hypoxic-ischemic encephalopathy. However, these neuroprotective effects were not persistent and only modest improvement in functional outcomes were observed at chronic time-points.

This study explored the hypothesis that late gestational reduction of corticosteroids transforms the cerebrovasculature and modulates postnatal vulnerability to mild hypoxic-ischemic (HI) injury. Four groups of Sprague-Dawley neonates were studied: 1) Sham-Control, 2) Sham-MET, 3) HI-Control, and 4) HI-MET. Metyrapone (MET), a corticosteroid synthesis inhibitor, was administered via drinking water from gestational day 11 to term. In Shams, MET administration 1) decreased reactivity of the hypothalamic-pituitary-adrenal (HPA) axis to surgical trauma in postnatal day 9 (P9) pups by 37%, 2) promoted cerebrovascular contractile differentiation in middle cerebral arteries (MCAs), 3) decreased compliance ≤46% and increased depolarization-induced calcium mobilization in MCAs by 28%, 4) mildly increased hemispheric cerebral edema by 5%, decreased neuronal degeneration by 66%, and increased astroglial and microglial activation by 10- and 4-fold, respectively, and 5) increased righting reflex times by 29%. Regarding HI, metyrapone-induced fetal transformation 1) diminished reactivity of the HPA axis to HI-induced stress in P9/P10 pups, 2) enhanced HI-induced contractile dedifferentiation in MCAs, 3) lessened the effects of HI on MCA compliance and calcium mobilization, 4) decreased HI-induced neuronal injury but unmasked regional HI-induced depression of microglial activation, and 5) attenuated the negative effects of HI on open-field exploration but enhanced the detrimental effects of HI on negative geotaxis responses by 79%. Overall, corticosteroids during gestation appear essential for normal cerebrovascular development and glial quiescence but induce persistent changes that in neonates manifest beneficially as preservation of postischemic contractile differentiation but detrimentally as worsened ischemic cerebrovascular compliance, increased ischemic neuronal injury, and compromised neurobehavior.

Neonatal hypoxic-ischemic encephalopathy (HIE) often leads to neonatal death or severe, irreversible neurological deficits. Pathologically, the occurrence of massive cell death and subsequent inflammation suggested that pyroptosis, an inflammation associated programed cell death, might play a role in HIE. Here, by measuring changes of key molecules in pyroptosis pathway in HIE patients, we discovered that their elevation levels tightly correlate with the severity of HIE. Next, we demonstrated that application of MCC950, a small molecule to inhibit NLRP3 inflammasome and thus pyroptosis, substantially alleviated pyroptosis and the injury severity in rats with neonatal hypoxic-ischemic brain damage (HIBD). Mechanistically, we showed that NLRP-3/caspase-1/GSDMD axis is required for microglia pyroptosis and activation. Our data demonstrated that microglia mediated pyroptosis played a crucial role in neonatal HIE, which shed lights into the development of intervention avenues targeting pyroptosis to treat HIE and traumatic brain injuries.

Background and Purpose: Despite the advances in treating neonatal hypoxic-ischemic encephalopathy (HIE) with induced hypothermia, the rates of severe disability are still high among survivors. Preclinical studies have indicated that cell therapies with hematopoietic stem/progenitor cells could improve neurological outcomes in HIE. In this study, we investigated whether the administration of AMD3100, a CXCR4 antagonist that mobilizes hematopoietic stem/progenitor cells into the circulation, has therapeutic effects in HIE. Methods: P10 Wistar rats of both sexes were subjected to right common carotid artery occlusion or sham procedure, and then were exposed to hypoxia for 120 minutes. Two subcutaneous injections of AMD3100 or vehicle were given on the third and fourth day after HIE. We first assessed the interindividual variability in brain atrophy after experimental HIE and vehicle treatment in a small cohort of rats. Based on this exploratory analysis, we designed and conducted an experiment to test the efficacy of AMD3100. Brain atrophy on day 21 after HIE was defined as the primary end point. Secondary efficacy end points were cognitive (T-water maze) and motor function (rotarod) on days 17 and 18 after HIE, respectively. Results: AMD3100 did not decrease the brain atrophy in animals of either sex. Cognitive impairments were not observed in the T-water maze, but male hypoxic-ischemic animals exhibited motor coordination deficits on the rotarod, which were not improved by AMD3100. A separate analysis combining data from animals of both sexes also revealed no evidence of the effectiveness of AMD3100 treatment. Conclusions: These results indicate that the subacute treatment with AMD3100 does not improve structural and functional outcomes in a rat HIE model.

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.

Maternal e-cigarette (e-cig) exposure is a pressing perinatal health concern. Emerging evidence reveals its potential adverse impacts on brain development in offspring, yet the underlying mechanisms are poorly understood. The present study tested the hypothesis that fetal e-cig exposure induces an aberrant DNA methylation profile in the developing brain, leading to alteration of autophagic flux signaling and programming of a sensitive phenotype to neonatal hypoxic-ischemic encephalopathy (HIE). Pregnant rats were exposed to chronic intermittent e-cig aerosol. Neonates were examined at the age of 9 days old. Maternal e-cig exposure decreased the body weight and brain weight but enhanced the brain-to-body weight ratio in the neonates. E-cig exposure induced a gender-dependent increase in hypoxic-ischemia-induced brain injury in male neonates associated with enhanced reactive oxygen species (ROS) activity. It differentially altered DNA methyltransferase expression and enhanced both global DNA methylation levels and specific CpG methylation at the autophagy-related gene 5 (ATG5) promoter. In addition, maternal e-cig exposure caused downregulations of ATG5, microtubule-associated protein 1 light chain 3β, and sirtuin 1 expression in neonatal brains. Of importance, knockdown of ATG5 in neonatal pups exaggerated neonatal HIE. In conclusion, the present study reveals that maternal e-cig exposure downregulates autophagy-related gene expression via DNA hypermethylation, leading to programming of a hypoxic-ischemic sensitive phenotype in the neonatal brain.

Neonatal hypoxic–ischemic encephalopathy is a notable cause of neonatal death and developmental disabilities. To achieve better outcomes, it is important in treatment strategy selection to categorize the degree of hypoxia ischemia and evaluate dose response. In an asphyxia piglet model with histopathological brain injuries that we previously developed, animals survived 5 days after insult and showed changes in cerebral blood volume (CBV) that reflected the severity of injuries. However, little is known about the relationship between changes in CBV during and after insult. In this study, an HI event was induced by varying the amount and timing of inspired oxygen in 20 anesthetized piglets. CBV was measured using near-infrared time-resolved spectroscopy before, during, and 6 h after insult. Change in CBV was calculated as the difference between the peak CBV value during insult and the value at the end of insult. The decrease in CBV during insult was found to correlate with the increase in CBV within 6 h after insult. Heart rate exhibited a similar tendency to CBV, but blood pressure did not. Because the decrement in CBV was larger in severe HI, the CBV increment immediately after insult is considered useful for assessing degree of HI insult.

Therapeutic hypothermia (TH) is a powerful neuroprotective strategy that has provided robust evidence for neuroprotection in pre-clinical studies of neurological disorders. Despite strong pre-clinical evidence, TH has not shown efficacy in clinical trials of most neurological disorders. The only successful trials employing therapeutic hypothermia were related to cardiac arrest in adults and hypoxic ischemic injury in neonates. Further investigations into the parameters of its use, and study design comparisons between pre-clinical and clinical studies, are warranted. This article demonstrates two methods of short-duration hypothermia induction. The first method allows for rapid hypothermia induction in rats using ethanol spray and fans. This method works by cooling the skin, which has been less commonly used in clinical trials and may have different physiological effects. Cooling is much more rapid with this technique than is achievable in human patients due to differences in surface area to volume ratio. Along with this, a second method is also presented, which allows for a clinically achievable cooling rate for short-duration hypothermia. This method is easy to implement, reproducible and does not require active skin cooling.

Ischemic stroke is a major cause of long-term disabilities, including vision loss. Neuronal and blood vessel maturation can affect the susceptibility of and outcome after ischemic stroke. Although we recently reported that exposure of neonatal mice to hypoxia–ischemia (HI) severely compromises the integrity of the retinal neurovasculature, it is not known whether juvenile mice are similarly impacted. Here we examined the effect of HI injury in juvenile mice on retinal structure and function, in particular the susceptibility of retinal neurons and blood vessels to HI damage. Our studies demonstrated that the retina suffered from functional and structural injuries, including reduced b-wave, thinning of the inner retinal layers, macroglial remodeling, and deterioration of the vasculature. The degeneration of the retinal vasculature associated with HI resulted in a significant decrease in the numbers of pericytes and endothelial cells as well as an increase in capillary loss. Taken together, these findings suggest a need for juveniles suffering from ischemic stroke to be monitored for changes in retinal functional and structural integrity. Thus, there is an emergent need for developing therapeutic approaches to prevent and reverse retinal neurovascular dysfunction with exposure to ischemic stroke.

To investigate the application value of magnetic resonance diffusion kurtosis imaging (DKI) in hypoxic–ischemic brain damage (HIBD) in newborn piglets and to compare imaging and pathological results. Of 36 piglets investigated, 18 were in the experimental group and 18 in the control group. The HIBD model was established in newborn piglets by ligating the bilateral common carotid arteries and placing them into hypoxic chamber. All piglets underwent conventional MRI and DKI scans at 3, 6, 9, 12, 16, and 24 h postoperatively. Mean kurtosis (MK) and mean diffusivity (MD) maps were constructed. Then, the lesions were examined using light and electron microscopy and compared with DKI images. The MD value of the lesion area gradually decreased and the MK value gradually increased in the experimental group with time. The lesion areas gradually expanded with time; MK lesions were smaller than MD lesions. Light microscopy revealed neuronal swelling in the MK- and MD-matched and mismatched regions. Electron microscopy demonstrated obvious mitochondrial swelling and autophagosomes in the MK- and MD-matched region but normal mitochondrial morphology or mild swelling in the mismatched region. DKI can accurately evaluate early ischemic–hypoxic brain injury in newborn piglets.

Diagnosis of hypoxic-ischemic encephalopathy (HIE) is currently limited and prognostic biological markers are required for early identification of at risk infants at birth. Using pre-clinical data from our fetal sheep models, we have shown that micro-scale EEG patterns, such as high-frequency spikes and sharp waves, evolve superimposed on a significantly suppressed background during the early hours of recovery (0-6 h), after an HI insult. In particular, we have demonstrated that the number of micro-scale gamma spike transients peaks within the first 2-2.5 hours of the insult and automatically quantified sharp waves in this period are predictive of neural outcome. This period of time is optimal for the initiation of neuroprotection treatments such as therapeutic hypothermia, which has a limited window of opportunity for implementation of 6 h or less after an HI insult. Clinically, it is hard to determine when an insult has started and thus the window of opportunity for treatment. Thus, reliable automatic algorithms that could accurately identify EEG patterns that denote the phase of injury is a valuable clinical tool. We have previously developed successful machine-learning strategies for the identification of HI micro-scale EEG patterns in a preterm fetal sheep model of HI. This paper employs, for the first time, reverse biorthogonal Wavelet-Scalograms (WS) as the inputs to a 17-layer deep-trained convolutional neural network (CNN) for the precise identification of high-frequency micro-scale spike transients that occur in the 80-120Hz gamma band during first 2 h period of an HI insult. The rbio-WS-CNN classifier robustly identified spike transients with an exceptionally high-performance of 99.82%.Clinical relevance—The suggested classifier would effectively identify and quantify EEG patterns of a similar morphology in preterm newborns during recovery from an HI-insult.

Neonatal hypoxic-ischemic encephalopathy (HIE) evolves over different phases of time during recovery. Some neuroprotection treatments are only effective for specific, short windows of time during this evolution of injury. Clinically, we often do not know when an insult may have started, and thus which phase of injury the brain may be experiencing. To improve diagnosis, prognosis and treatment efficacy, we need to establish biomarkers which denote phases of injury. Our pre-clinical research, using preterm fetal sheep, show that micro-scale EEG patterns (e.g. spikes and sharp waves), superimposed on suppressed EEG background, primarily occur during the early recovery from an HI insult (0-6 h), and that numbers of events within the first 2 h are strongly predictive of neural survival. Thus, real-time automated algorithms that could reliably identify EEG patterns in this phase will help clinicians to determine the phases of injury, to help guide treatment options. We have previously developed successful automated machine learning approaches for accurate identification and quantification of HI micro-scale EEG patterns in preterm fetal sheep post-HI. This paper introduces, for the first time, a novel online fusion strategy that employs a high-level wavelet-Fourier (WF) spectral feature extraction method in conjunction with a deep convolutional neural network (CNN) classifier for accurate identification of micro-scale preterm fetal sheep post-HI sharp waves in 1024Hz EEG recordings, along with 256Hz down-sampled data. The classifier was trained and tested over 4120 EEG segments within the first 2 hours latent phase recordings. The WF-CNN classifier can robustly identify sharp waves with considerable high-performance of 99.86% in 1024Hz and 99.5% in 256Hz data. The method is an alternative deep-structure approach with competitive high-accuracy compared to our computationally-intensive WS-CNN sharp wave classifier.

Early diagnosis and prognosis of babies with signs of hypoxic-ischemic encephalopathy (HIE) is currently limited and requires reliable prognostic biomarkers to identify at risk infants. Using our pre-clinical fetal sheep models, we have demonstrated that micro-scale patterns evolve over a profoundly suppressed EEG background within the first 6 hours of recovery, post HI insult. In particular, we have shown that high-frequency micro-scale spike transients (in the gamma frequency band, 80-120Hz) emerge immediately after an HI event, with much higher numbers around 2-2.5 h of the insult, with numbers gradually declining thereafter. We have also shown that the automatically quantified sharp waves in this phase are predictive of neural outcome. Initiation of some neuroprotective treatments within this limited window of opportunity, such as therapeutic hypothermia, optimally reduces neural injury. In clinical practice, it is hard to determine the exact timing of the injury, therefore, reliable automatic identification of EEG transients could be beneficial to help specify the phases of injury. Our team has previously developed successful machine- and deep-learning strategies for the identification of post-HI EEG patterns in an HI preterm fetal sheep model.This paper introduces, for the first time, a novel online fusion approach to train an 11-layers deep convolutional neural network (CNN) classifier using Wavelet-Fourier (WF) spectral features of EEG segments for accurate identification of high-frequency micro-scale spike transients in 1024Hz EEG recordings in our preterm fetal sheep. Sets of robust features were extracted using reverse biorthogonal wavelet (rbio2.8 at scale 7) and considering an 80-120Hz spectral frequency range. The WF-CNN classifier was able to accurately identify spike transients with a reliable high-performance of 99.03±0.86%.Clinical relevance-Results confirm the expertise of the method for the identification of similar patterns in the EEG of neonates in the early hours after birth.

Premature babies are at high risk of serious neurodevelopmental disabilities, which in many cases are related to perinatal hypoxic&ndash, ischemic encephalopathy (HIE). Studies of neuroprotection in animal models consistently suggest that treatment must be started as early as possible in the first 6 h after hypoxia&ndash, ischemia (HI), the so-called latent phase before secondary deterioration, to improve outcomes. We have shown in preterm sheep that EEG biomarkers of injury, in the form of high-frequency micro-scale spike transients, develop and evolve in this critical latent phase after severe asphyxia. Real-time automatic identification of such events is important for the early and accurate detection of HI injury, so that the right treatment can be implemented at the right time. We have previously reported successful strategies for accurate identification of EEG patterns after HI. In this study, we report an alternative high-performance approach based on the fusion of spectral Fourier analysis and Type-I fuzzy classifiers (FFT-Type-I-FLC). We assessed its performance in over 2520 min of latent phase EEG recordings from seven asphyxiated in utero preterm fetal sheep exposed to a range of different occlusion periods. The FFT-Type-I-FLC classifier demonstrated 98.9 &plusmn, 1.0% accuracy for identification of high-frequency spike transients in the gamma frequency band (namely 80&ndash, 120 Hz) post-HI. The spectral-based approach (FFT-Type-I-FLC classifier) has similar accuracy to our previous reverse biorthogonal wavelets rbio2.8 basis function and type-1 fuzzy classifier (rbio-WT-Type-1-FLC), providing competitive performance (within the margin of error: 0.89%), but it is computationally simpler and would be readily adapted to identify other potentially relevant EEG waveforms.

Background Hypoxic ischemic (HI) brain injury is a significant cause of childhood neurological deficits. Preclinical rodent models are often used to study these deficits; however, no preclinical study has determined which behavioral tests are most appropriate for long-term follow up after neonatal HI. Methods HI brain injury was induced in postnatal day (PND) 10 rat pups using the Rice-Vannucci method of unilateral carotid artery ligation. Rats underwent long-term behavioral testing to assess motor and cognitive outcomes between PND11-50. Behavioral scores were transformed into Z-scores and combined to create composite behavioral scores. Results HI rats showed a significant deficit in three out of eight behavioral tests: negative geotaxis analysis, the cylinder test and the novel object recognition test. These individual test outcomes were transformed into Z-scores and combined to create a composite Z-score. This composite z-score showed that HI rats had a significantly increased behavioral burden over the course of the experiment. Conclusion In this study we have identified tests that highlight specific cognitive and motor deficits in a rat model of neonatal HI. Due to the high variability in this model of neonatal HI brain injury, significant impairment is not always observed in individual behavioral tests, but by combining outcomes from these individual tests, long-term behavioral burden can be measured.

Purpose: To evaluate the effects of HBO (Hyperbaric oxygen) and NGF (Nerve growth factor) on the long-term neural behavior of neonatal rats with HIBD (Neonatal hypoxic ischemic brain damage). Methods: The HIBD model was produced by ligating the right common carotid artery of 7 days old SD (Sprague-Dawley) rats followed by 8% O2 + 92% N2 for 2h. Totally 40 rats were randomly divided into 5 groups including sham-operated group, HIBD control group, HBO treated group, NGF treated group and NGF + HBO treated group. The learning and memory ability of these rats was evaluated by Morris water maze at 30 days after birth, and sensory motor function was assessed by experiments of foot error and limb placement at 42 days after birth. Results: The escape latency of HBO treated group, NGF treated group and NGF + HBO treated group was shorter than that of HIBD control group (p

This review analyzes recent data on mechanisms of cerebral hypoxia and the protective methods of hypoxic and ischemic postconditioning, as well as their interrelationship with the key mechanisms responsible for neuroprotection and neuroplasticity. Upregulation of expression of antiapoptotic factors and neurotrophins and modulation of activity of several protein kinases and transcription factors such as hypoxia-inducible factor-1 (HIF-1) are considered as the most important aspects in the neuroprotective potential of postconditioning. The presented information indicates substantial transformative promise of the noninvasive techniques of hypoxic postconditioning as well as significant similarity between the adaptive pathways activated by various postconditioning methods, which are far from being fully understood.

Infection and inflammation are known risk factors for neonatal brain injury. Mycoplasma and Gram-positive bacteria, for which Toll-like receptor 2 (TLR2) plays a key role in recognition and inflammatory response, are among the most common pathogens in the perinatal period. Here, we report that systemic activation of TLR2 by Pam3CSK4 (P3C) increases neural tissue loss and demyelination induced by subsequent hypoxia–ischemia (HI) in neonatal mice. High-resolution respirometry of brain isolated mitochondria revealed that P3C suppresses ADP-induced oxidative phosphorylation, the main pathway of cellular energy production. The results suggest that infection and inflammation might contribute to HI-induced energy failure.

The aim of the present study was to evaluate the role of polygalasaponin F on ischemic myocardial injury in neonatal rats with hypoxic-ischemic brain damage. A primary in-vitro myocardial cell oxygen-glucose deprivation/reperfusion model and an in-vivo middle cerebral artery occlusion model were established. The results demonstrated that polygalasaponin F protects myocardium in hypoxic-ischemic brain injury. The mechanisms of its protective effect involved in (1) reducing oxidant stress injury, (2) reducing the apoptosis rate of myocardial cells through increasing the Bcl-2 protein level and decreasing the Cyt-C and Bax values, and (3) alleviating liver and kidney damage caused by cerebral hypoxia and ischemia via reducing the damage markers. The results of the present study may contribute toward the development of novel strategies for clinical cardioprotection with hypoxic-ischemic brain damage.

Maternal cigarette smoking is a major perinatal insult that contributes to an increased risk of cardiovascular and neurodevelopmental diseases in offspring. Our previous studies revealed that perinatal nicotine exposure reprograms a sensitive phenotype in neonatal hypoxic-ischemic encephalopathy (HIE), yet the underlying molecular mechanisms remain largely elusive. The present study tested the hypothesis that perinatal nicotine exposure impacts autophagy signaling in the developing brain, resulting in enhanced susceptibility to neonatal HIE. Nicotine was administered to pregnant rats via subcutaneous osmotic minipumps. Neonatal HIE was conducted in 9-day-old male rat pups. Protein kinase B/glycogen synthase kinase- 3β/mammalian target of rapamycin (Akt/GSK-3β/mTOR) signaling and key autophagy markers were determined by Western blotting analysis. Rapamycin and MK2206 were administered via intracerebroventricular injection. Nicotine exposure significantly inhibited autophagy activities in neonatal brain tissues, characterized by an increased ratio of phosphoylated (p-) to total mTOR protein expression but reduced levels of autophagy-related 5, Beclin 1, and LC3βI/II. Treatment with mTOR inhibitor rapamycin effectively blocked nicotine-mediated autophagy deficiency and, more importantly, reversed the nicotine-induced increase in HI brain infarction. In addition, nicotine exposure significantly upregulated p-Akt and p-GSK-3β. Treatment with the Akt selective inhibitor MK2206 reversed the enhanced p-Akt and p-GSK-3β, restored basal autophagic flux, and abolished nicotine-mediated HI brain injury. These findings suggest that perinatal nicotine-mediated alteration of Akt/GSK-3β/mTOR signaling plays a key role in downregulation of autophagic flux, which contributes to the development of hypoxia/ischemia-sensitive phenotype in the neonatal brain.

The "oligovascular unit" is a dynamic structural complex composed of endothelial cells (ECs) and oligodendrocyte progenitor cells (OPCs)/oligodendrocytes. By improving the microenvironment of OPCs in the "oligovascular unit" and promoting the proliferation and differentiation of OPCs, both myelination and white matter injury can be repaired. However, it is unclear what characteristic changes occur in the microenvironment of the "oligovascular unit" after preterm white matter injury (PWMI). Here, we demonstrate the changes in the "oligovascular unit" induced by hypoxia-ischemia (HI) and its underlying mechanism in PWMI mice. White matter injury and inhibited production of myelin from OPCs were observed by histopathological staining in HI neonatal mice. We further observed that the proliferation of OPCs and angiogenesis were increased after HI, which is considered the response of the body and cells to HI. HI-induced oligogenesis occurs around the vessels, indicating that "oligovascular units" exist and promote the proliferation and differentiation of OPCs after HI in the short term. We also determined that angiogenesis and oligogenesis induced by HI in the white matter are related to the PI3K/AKT/mTOR pathway. Furthermore, the myelin sheaths were shown to be disordered on the side of the surgery, and the myelin-dense layer was poorly developed at P14 and P28. Different degrees of damage to the vascular ECs and basement membrane on the surgical side were detected beginning at P4, indicating that EC injury is an early phenomenon that subsequently affects oligogenesis. Taken together, our findings indicate that the proliferation of OPCs and angiogenesis in white matter are increased in the early stage of HI involving PI3K/AKT/mTOR pathway activation. Promoting vascular endothelial function and angiogenesis may increase the proliferation and survival of OPCs via the "oligovascular unit," which suggests a potential method to repair injured white matter in the early stage of PWMI.

We have recently demonstrated that micro-scale Sharp waves in the first few hours EEG of asphyxiated preterm fetal sheep models are the reliable prognostic biomarkers for Hypoxic-Ischemic Encephalopathy (HIE). Higher number of sharp waves within the first 2 hours from a hypoxic insult is shown to be significantly correlated to subcortical neuronal survival in caudate nucleus of striatum. Cerebral therapeutic hypothermia is also shown to be optimally neuroprotective only if initiated as soon as possible during a short window of opportunity within the first 2-3 hours of HI insult, called the latent phase. Therefore there is an urgent necessity for reliable automated algorithms to robustly identify such biomarkers to help early diagnosis of HIE, in real time at birth, before the optimal window of opportunity for treatment is missed.We have previously introduced successful automated signal processing strategies based on the fusion of wavelet and fuzzy techniques, for real-time identification and quantification of sharp waves along a profoundly suppressed EEG/ECoG background, post HI-insult, during the latent phase of sheep models. This work, in particular, for the first time represents a novel online fusion strategy based on the combination of a deep Convolutional Neural Network (CNN) in conjunction with Wavelet Scalogram (WS) for the real-time identification and classification of micro-scale sharp wave biomarkers within the 1024Hz high resolution ECoG recordings as well as the down-sampled 256Hz signals, from in utero preterm fetal sheep. The WS-CNN classifier highlights ability in the identification of HI sharp waves with remarkable high accuracies of 95.34% for 1024Hz and 94.62% for 256Hz data tested over one hour HI ECoG within the most important interval during the first 2 hours of the latent phase, where experiments have suggested hypothermia is optimally effective.

Hypoxic–ischemic brain damage (HIBD) is one of the leading causes of brain injury in infant with high risk of mortality and disability; therefore, it is important to explore more feasible and effective treatment strategies. Here, we assessed the neuroprotective effects of different hydrogen inhalation times for the treatment of HIBD. We induced hypoxia–ischemia in Sprague–Dawley rats (postnatal day 7, both sexes), followed by treatment with hydrogen inhalation for 30, 60, or 90 min. Morphological brain injury was assessed by Nissl and TUNEL staining. Acute inflammation was evaluated by examining the expression of interleukin-1β (IL-1β) and NF-κB p65, as well as Iba-1 immunofluorescence in the brain. Neural apoptosis was evaluated by examining the expression of P-JNK and p53 as well as NeuN immunofluorescence. Neurobehavioral function of rats was evaluated by Morris water maze test at 36 days after surgery. The results showed that hypoxia–ischemia injury induced the inflammatory response of microglia; however, these changes were inhibited by hydrogen inhalation. The inhibitory effects became more apparent as the treatment duration increased ( P Impact statement Oxidative stress is known to be involved in the main pathological progression of neonatal hypoxic–ischemic brain damage (HIBD). Hydrogen (H2) is an antioxidant that can be used to treat HIBD; however, the mechanism by which hydrogen may be used as a promising treatment for neonates with HIBD is not very clear. This study demonstrated that inhaled H2 is neuroprotective against HIBD in SpragueDawley rats by inhibiting the brain’s inflammatory response and neuronal apoptosis or damage and protecting against spatial memory decline. Further, this study showed that inhaled H2 has potential as a therapeutic approach for HIBD. This is relevant to clinical treatment protocols when hypoxia–ischemia is suspected in neonates.

Neonatal hypoxia-ischemia and resulting encephalopathies are of significant concern. Intrapartum asphyxia is a leading cause of neonatal death globally. Among surviving infants, there remains a high incidence of hypoxic-ischemic encephalopathy due to neonatal hypoxic-ischemic brain injury, manifesting as mild conditions including attention deficit hyperactivity disorder, and debilitating disorders such as cerebral palsy. Various animal models of neonatal hypoxic brain injury have been implemented to explore cellular and molecular mechanisms, assess the potential of novel therapeutic strategies, and characterize the functional and behavioural correlates of injury. Each of the animal models has individual advantages and limitations. The present review looks at several widely-used and alternative rodent models of neonatal hypoxia and hypoxia-ischemia; it highlights their strengths and limitations, and their potential for continued and improved use.

Hypoxic-ischemic (HI) brain injury in newborns results in serious damage. Magnesium sulfate has been clinically used as a cyto-protective agent against HI brain injury in newborns in some countries, including Japan. However, it is not clear how magnesium exerts this effect and how it acts on the individual types of cells within the newborn brain. In this study, we exposed cultured rat oligodendrocyte precursor cells to magnesium sulfate during the period when they differentiate into oligodendrocytes, and showed that magnesium-exposed oligodendrocytes exhibited more resistance to hypoxic-ischemic injury. Our data may support the use of magnesium sulfate in the clinical setting.

Hypoxia leads to activation of many cellular adaptive processes which are regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 consists of HIF-1α and HIF-1ß subunits and levels of HIF-1α protein are regulated by HIF prolyl-hydroxylase enzymes (PHD1, 2, 3). The aim of the current study was to investigate the expression of HIF-1α and PHDs at various time points after hypoxia-ischemia (HI), using a neonatal rat model of HI brain injury. Sprague-Dawley rat pups (postnatal day 7) were anaesthetized and underwent right carotid artery occlusion and were then exposed to 6 % oxygen for 2.5 h at 37 °C. HI injured animals demonstrated a significant reduction in the size of the ipsilateral hemisphere, compared to sham controls. Protein analysis using western blotting and enzyme-linked immunosorbent assay showed that 24 h after HI, there was a significant increase in PHD3 protein and an increase of HIF-1α compared to controls. At the 72 h time point, there was a reduction in PHD3 protein, which appeared to relate to cellular loss. There were no changes in PHD1 or PHD2 protein levels after HI when compared to age-matched controls. Further studies are necessary to establish roles for the HIF-1 regulatory enzyme PHD3 in brain injury processes.

BackgroundHypoxic-ischemic encephalopathy (HIE) is a major cause of neonatal morbidity and mortality. Therapeutic hypothermia (TH) is the only available intervention, but neuroprotection is incomplete and variable. Seizures are common in infants with HIE undergoing TH and may worsen outcome. Phenobarbital (PB) is sometimes added, although use of prophylactic PB is controversial in the neonate. We hypothesize that prophylactic PB will not reduce, and may enhance, the neuroprotective effects of TH on brain injury and motor outcomes in the postnatal day 10 (P10) hypoxic-ischemic (HI) rat.MethodsP10 rat pups were subjected to unilateral HI and 4 h recovery with: normothermia (N); hypothermia (TH); and hypothermia with phenobarbital (TH+PB). Brain damage was assessed longitudinally at 24 h and 2 weeks using brain magnetic resonance imaging and 12 weeks using histochemical analysis. Motor function was assessed with the beam walk and cylinder tests.ResultsTH and TH+PB induced neuroprotection, as measured by global brain damage score and improved motor function. Exploratory analyses suggest that TH+PB may confer enhanced protection, especially to the extent of damage.ConclusionProphylactic PB with TH is not deleterious and may provide additional long-term neuroprotection, including improvement of motor outcomes following HI in the term-equivalent, neonatal rat.

In membrane physiology, as in other fields, myths or speculations may be repeated so often and so widely that they are perceived as facts. To some extent, this has occurred with regard to gap junctions, hemichannels, pannexin channels and P2X7 (ionotropic receptors), especially concerning the interpretation of the individual role of these channels in hypoxic-ischemic CNS since these channels may be closed by the same pharmacological blockers. Significance of existing controversial data are highlighted and contradictory views from different groups are critically discussed herein.

BackgroundGender is increasingly recognized as an important influence on brain development, disease susceptibility, and response to pharmacologic/rehabilitative treatments. In regenerative medicine, it remains entirely unknown whether there is an interaction between transplanted stem cells and host gender that might bias efficacy and safety in some patients but not others.MethodsWe examined the role of recipient gender in a neonatal rat hypoxic-ischemic injury (HII) model, treated with female human neuronal stem cells (hNSCs), labeled with superparamagnetic iron oxide particles implanted into the contralateral cerebral ventricle. We monitored HII evolution (by magnetic resonance imaging, histopathology, behavioral testing) and hNSC fate (migration, replication, viability).ResultsRecipient gender after implantation did not influence the volume or location of ischemic injury (1, 30, or 90 d) or behavior (90 d). Superparamagnetic iron oxide labeling did not influence HII evolution. Implantation had its greatest benefit on mild/moderate injuries, which remained stable rather than increasing as in severe HII as is the natural history for such lesions.ConclusionOur results suggest that hNSC treatment (including using hNSCs that are prelabeled with iron to allow tracking in real time by magnetic resonance imaging) would be equally safe and effective for male and female human newborns with mild-to-moderate HII.

Neonatal hypoxic ischemia encephalopathy (HIE) leads to major deficits in language development. While clinically there is a known correlation in the degree of HIE injury and subsequent language disability, there are no treatments beyond speech and language therapy; therefore, experimental studies with a HIE animal model to test new interventions and therapeutics are warranted. Neonatal rodents normally ultrasonically vocalize at postnatal day 7 (PND 7) to PND 14 in response to removal from their mothers. At 6–8 weeks of age juvenile male rodents ultrasonically vocalize in response to exposure to a mature female mouse. Changes in ultrasonic vocalization (USV) production after neonatal brain injury, such as hypoxic ischemia (HI), have not been studied. This study examines the acute and long-term ultrasonic vocalization ability of mice after HI at PND 10. Pups were subjected to HI, sham, or naïve conditions; where in HI and sham surgeries the right common carotid artery was exposed, in the HI this artery was double ligated. The HI and sham pups were then exposed to 60 minutes of hypoxia. Naïve pups did not undergo surgery and were subjected to 60 minutes of room air. At 3 days following surgery, HI and sham pups vocalize less than nonsurgical naïve controls; yet “juvenile” mice of 6–8 weeks old that underwent HI at PND 10 vocalize less than sham and naïve mice. We conclude that HI injury has significant impact on later adult vocalization.

The timing and mechanisms of protection by hyperbaric oxygenation (HBO) in hypoxic-ischemic brain damage (HIBD) have only been partially elucidated. We monitored the effect of HBO on the mitochondrial function of neuronal cells in the cerebral cortex of neonatal rats after HIBD. Neonatal Sprague-Dawley rats (total of 360 of both genders) were randomly divided into normal control, HIBD, and HIBD+HBO groups. The HBO treatment began immediately after hypoxia-ischemia (HI) and continued once a day for 7 consecutive days. Animals were euthanized 0, 2, 4, 6, and 12 h post-HI to monitor the changes in mitochondrial membrane potential (ΔΨm) occurring soon after a single dose of HBO treatment, as well as 2, 3, 4, 5, 6, and 7 days post-HI to study ΔΨm changes after a series of HBO treatments. Fluctuations in ΔΨm were observed in the ipsilateral cortex in both HIBD and HIBD+HBO groups. Within 2 to 12 h after HI insult, the ΔΨm of the HIBD and HIBD+HBO groups recovered to some extent. A secondary drop in ΔΨm was observed in both groups during the 1-4 days post-HI period, but was more severe in the HIBD+HBO group. There was a secondary recovery of ΔΨm observed in the HIBD+HBO group, but not in the HIBD group, during the 5-7 days period after HI insult. HBO therapy may not lead to improvement of neural cell mitochondrial function in the cerebral cortex in the early stage post-HI, but may improve it in the sub-acute stage post-HI.

© 2016 Liu et al. Background: Periventricular leukomalacia (PVL) is the leading cause of neurological disabilities including motor and cognitive deficits in premature infants. Periventricular leukomalacia is characterized by damage to the white matter in the immature brain, but the mechanisms by which damage to immature white matter results in widespread deficits of cognitive and motor function are unclear. The thalamocortical system is crucial for human consciousness and cognitive functions, and impaired development of the cortico-thalamic projections in the neonatal period is implicated to contribute importantly to abnormalities of cognitive function in children with PVL. Results: In this study, using a mouse model of PVL, we sought to test the hypothesis that PVL-like injury affects the different components of the thalamocortical circuitry that can be defined by vesicular glutamate transporters 1 and 2 (vGluT1 and vGluT2), both of which are required for glutamatergic synaptic transmission in the central nervous system. We combined immunocytochemistry and immuno-electron microscopy to investigate changes in cortico-thalamic synapses which were specifically identified by vGluT1 immunolabeling. We found that a drastic reduction in the density of vGluT1 labeled profiles in the somatosensory thalamus, with a reduction of 72-74% in ventroposterior (VP) nucleus and a reduction of 42-82% in thalamic reticular nucleus (RTN) in the ipsilateral side of PVL mice. We further examined these terminals at the electron microscopic level and revealed onefold-twofold decrease in the sizes of vGluT1 labeled corticothalamic terminals in VP and RTN. The present study provides anatomical and ultrastructural evidence to elucidate the cellular mechanisms underlying alteration of thalamic circuitry in a mouse model of PVL, and reveals that PVL-like injury has a direct impact on the corticothalamic projection system. Conclusions: Our findings provide the first set of evidence showing that the thalamocortical circuitry is affected and vulnerable in PVL mice, supporting a working model in which vGluT1 defined corticothalamic synapses are altered in PVL mice, and vGluT2 defined thalamocortical synapses are associated with such changes, leading to the compromised thalamocortical circuitry in the PVL mice. Our study demonstrates that the thalamocortical circuitry is highly vulnerable to hypoxia-ischemia in the PVL model, thus identifying a novel target site in PVL pathology.

Background Neonatal hypoxia-ischemia (HI) remains a major cause of severe brain damage and is often associated with high mortality and lifelong disability. Immature brains are extremely sensitive to hypoxia-ischemia, shown as prolonged mitochondrial neuronal death. Sodium pyruvate (SP), a substrate of the tricarboxylic acid cycle and an extracellular antioxidant, has been considered as a potential treatment for hypoxic-ischemic encephalopathy (HIE), but its effects have not been evaluated in appropriate animal models for hypoxic-ischemic encephalopathy (HIE). Methods This investigation employed primary cortical neuron cultures derived from neonatal rats subjected to oxygen and glucose deprivation (OGD) and a well-established neonatal rat hypoxia-ischemia model. Results HI caused brain tissue loss and impaired sensorimotor function and spatial memory while SP significantly reduced brain damage and improved neurological performance. These neuroprotective effects of SP are likely the result of improved cerebral metabolism as demonstrated by maintaining ATP levels and preventing an increase in intracellular reactive oxygen species (ROS) levels. SP treatment also decreased levels of Bax, a death signal for immature neurons, blocked caspases-3 activation, and activated a key survival signaling kinase, Akt, both in vitro and in vivo. Conclusion SP protected neonatal brain from hypoxic-ischemic injury through maintaining cerebral metabolism and mitochondrial function.

Perinatal hypoxia-ischemia remains the single most important cause of brain injury in the newborn, leading to death or lifelong sequelae. Because of the fact that there is still no specific treatment for perinatal brain lesions due to the complexity of neonatal hypoxic-ischemic pathophysiology, the search of new neuroprotective therapies is of great interest. In this regard, therapeutic possibilities of the endocannabinoid system have grown lately. The endocannabinoid system modulates a wide range of physiological processes in mammals and has demonstrated neuroprotective effects in different paradigms of acute brain injury, acting as a natural neuroprotectant. Concerning perinatal asphyxia, the neuroprotective role of this endogenous system is emerging these years. The present review mainly focused on the current knowledge of the cannabinoids as a new neuroprotective strategy against perinatal hypoxic-ischemic brain injury.

Purpose: To develop and compare an automated detection system for ischemic lesions in a neonatal model of bilateral carotid artery occlusion with hypoxia (BCAO-H) from T2 weighted MRI (T2WI) to the currently used “gold standard” of manual segmentation. Materials and Methods: Forty-three P10 BCAO-H rat pups and 8 controls underwent T2WI at 1 day and 28 days. A computational imaging method, Hierarchical Region Splitting (HRS), was developed to automatically and rapidly detect and quantify 3D lesion and normal appearing brain matter (NABM) volumes. Results: HRS quantified lesion and NABM volumes within 15 s in comparison to 3 h for its manual counterpart, with a high correlation for injury (r2 = 0. 95; P = 8.6 × 10−7) and NABM (r2 = 0. 92; P = 1.4 × 10−22). Average lesion volumes for mild, moderate, and severe injuries were 3.85%, 28.85%, and 52.98% for HRS and 0.51%, 24.22%, and 48.74% for manual detection. Lesion volumes and locations were similar for both methods (sensitivity: 0.82, specificity: 0.86, and similarity: 1.47). Conclusion: HRS is an accurate, objective, and rapid method to quantify injury evolution in neonatal hypoxic ischemic injury models. J. Magn. Reson. Imaging 2011;33:772–781. © 2011 Wiley-Liss, Inc.

Two-day-old (P2) rat pups were subjected to either a global hypoxia or to electrocoagulation of the right carotid artery followed by 2.5. h hypoxia. Cellular and regional injury in the cerebellum (CB) was studied at 1, 2 and 19. days using immunohistology. Following hypoxia and hypoxia-ischemia, all neuronal populations of the CB were damaged in a subset of Purkinje cells. The decrease in the number of interneurons, as well as the thickness of molecular and granular layers was significant following hypoxia. Diffuse white matter damage, with loss of preoligodendrocytes was more severe following hypoxia than hypoxia-ischemia. Global hypoxia in the rat at P2 produces extensive damage to many cell types in different areas of the CB. The addition of unilateral forebrain ischemia does not increase the severity of these changes. Our data provide insight into the mechanisms of the changes observed in the CB of premature newborns. © 2010 Elsevier Inc.

The hypoxic-ischemic encephalopathy caused by peripartum asphyxia is a serious disease in newborn infants, and effective therapies need to be developed to reduce injury-related disorders. We evaluated the effects of NEP1-40 and fasudil on Nogo-A expression in neonatal hypoxic-ischemic brain damage (HIBD) rats. Seven-day-old Wistar rats were randomly divided into control, HIBD, NEP1-40, and fasudil groups. NEP1-40 and fasudil groups were injected intraperitoneally with these compounds. Rat brains at 6, 24, 72 h, and 7 days after HIBD were collected to determine histopathological damage and the expression levels of Nogo-A. Histopathological damage was reduced in NEP1-40 and fasudil groups compared with the untreated HIBD group. The expression of Nogo-A in the HIBD group was significantly higher than that in control, NEP1-40 and fasudil groups at the same times. Compared with the fasudil group, the expression levels of Nogo-A were significantly reduced in the NEP1-40 group. We conclude that NPE1-40 and fasudil have potential for neuroprotective effects in the neonatal rat HIBD model, mediated by inhibiting Nogo-A/ Rho pathways.

Background: Perinatal hypoxia-ischemia is a major cause of mortality and long-term neurological deficits. Objectives: The objective of this study was to compare the effects of two neuroprotective agents; magnesium sulfate and melatonin, administered alone or in combination, on brain infarct volume and TUNEL positivity in a neonatal hypoxic-ischemic (HI) rat model. Methods: After being anesthetized, 7-day-old pups (n = 80) underwent ischemia followed by exposure to hypoxia for 2 h. The pups were then divided equally and randomly into 4 groups in order to receive the vehicle (saline, control group), magnesium sulfate, melatonin or a combination of magnesium sulfate and melatonin. Treatments were administered intraperitoneally three times; the first being just before ischemia, the second after hypoxia and the third 24 h after the second dose. The pups were sacrificed on postnatal day 10, their brains harvested and evaluated for infarct volume and neuronal apoptosis. Results: Percent infarcted brain volume was significantly reduced in pups receiving the drugs (either magnesium sulfate, melatonin or their combination) compared with those receiving the vehicle. In addition, TUNEL staining showed markedly reduced numbers of TUNEL-positive cells per unit area in the CA1, CA3 and dentate gyrus regions of the hippocampus and in the cortex. However, no statistically significant differences were found regarding percent infarcted brain volume and number of TUNEL-positive cells among the drug-treated groups. Conclusions: Magnesium sulfate and melatonin, two agents acting at different stages of HI brain damage, administered either alone or in combination, significantly reduced the percent infarcted brain volume and TUNEL positivity, suggesting that these agents may confer a possible benefit in the treatment of infants with HI encephalopathy.

Seven-day old rats were subjected to unilateral ligation of the common carotid artery followed by 2-h exposure to a gas mixture consisting of 8% oxygen and 92% nitrogen. Locomotor function of rats was monitored weekly. Functional deficit in these animals persisted for at least 3 months. The exercise tests of rotarod, hanging, and narrowing track were most informative. Our results can be used in preclinical studies of new drugs for the therapy of perinatal brain injury.

Extremely preterm infants commonly show brain injury with long-term structural and functional consequences. Three-day-old (P3) rat pups share some similarities in terms of cerebral development with the very preterm infant (born at 24-28 weeks of gestation). The aim of this study was to assess longitudinally the cerebral structural and metabolic changes resulting from a moderate neonatal hypoxic ischemic injury in the P3 rat pup using high-field (9.4 T) MRI and localized H-1 magnetic resonance spectroscopy techniques. The rats were scanned longitudinally at P3, P4, P11, and P25. Volumetric measurements showed that the percentage of cortical loss in the long term correlated with size of damage 6 h after hypoxia-ischemia, male pups being more affected than female. The neurochemical profiles revealed an acute decrease of most of metabolite concentrations and an increase in lactate 24 h after hypoxia-ischemia, followed by a recovery phase leading to minor metabolic changes at P25 in spite of an abnormal brain development. Further, the increase of lactate concentration at P4 correlated with the cortical loss at P25, giving insight into the early prediction of long-term cerebral alterations following a moderate hypoxia-ischemia insult that could be of interest in clinical practice. Magn Reson Med 65:305-312, 2011. (C) 2010 Wiley-Liss, Inc.

The aim of this study was to evaluate the hypothesis that cerebral hemoglobin (Hb) oxygenation is related to phosphorylation potential during primary and secondary cerebral energy failure in newborn infants who have experienced birth asphyxia. We subjected newborn piglets to severe transient cerebral hypoxic-ischemia followed by resuscitation and examined cerebral energy metabolism by 31P-magnetic resonance spectroscopy and evaluated changes in cerebral Hb oxygen saturation (ScO2) using full-spectrum near-infrared spectroscopy before, during, and up to 54 h after the hypoxic-ischemic insult. ScO2 was significantly decreased during the hypoxic-ischemic insult compared with baseline values. During secondary energy failure, piglets were separated based on the relationship between the ratio of phosphocreatine to inorganic phosphate and ScO2; those with a negative correlation were less injured than those with a positive correlation. These results indicate that changes in ScO2 as measured by near-infrared spectroscopy are related to phosphorylation potential during secondary energy failure in asphyxiated infants.

Animal models are often presumably the first step in determining mechanisms underlying disease, and the approach and effectiveness of therapeutic interventions. Perinatal brain damage, however, evolves over months of gestation, during the rapid maturation of the fetal and newborn brain. Despite marked advances in our understanding of these processes and technologic advances providing an improved window on the timing and duration of injury, neonatal brain injury remains a "moving target" regarding our ability to "mimic" its processes in an animal model. Moreover, interfering with normal processes of development as part of a therapeutic intervention may do "more harm than good." Hence, controversy continues over which animal model can reflect human disease states. Numerous models have provided information regarding the pathophysiology of brain damage in term and preterm infants. Our challenges consist of identifying infants at greatest risk for permanent injury, identifying the timing of injury, and adapting therapies that provide more benefit than harm. A combination of appropriately suitable animal models to conduct these studies will bring us closer to understanding human perinatal damage and the means to treat it.

Study the effect of ephedrine on neural plasticity of hypoxic ischemic brain damaged (HIBD) in neonatal rats, and explore the underlying molecular mechanism.60 Sprats suffered from HIBD (7 days old) were randomly divided into ephedrine group, D-amphetamine (D-AMPH) group, cytidine triphosphate (CTP) group, ganglioside (GM1) group, and spontaneous recovery group. Using immunohistochemical method to test the expression of growth-associated protein-43(GAP-43) and synaptophysin (SYP) on one side of hippocampal CA3 area, then, 4 weeks later, Morris Water Maze test was performed for five days.(1) The expression levels of GAP-43 and SYP on hippocampal CA3 area in ephedrine group were higher than that in spontaneous recovery group (P0.05). However, there was no statistical difference in ephedrine groups, CTP group, and D-AMPH group. (2) The average time of escape latency was significantly shorter in treating groups than that in spontaneous recovery group (P0.05), and the frequency of original platform passing was higher than that in spontaneous recovery group (P0.01). The average time of escape latency was longer in ephedrine group than that in GM1 group. The frequency of original platform passing was significantly less in ephedrine group than that in GM1 group, No statistical difference found in ephedrine groups, CTP group, and D-AMPH group.Ephedrine may enhance memory, the abilities of spatial orientation and learning in HIBD rats. This protective effect may be associated with increasing synaptic connectivity, as assessed by increased expression of GAP-43 and SYP after HIBD. Ephedrine triggered similar protection against HIBD as treatment of D-AMPH and CTP. However, the amelioration of ability of spatial orientation, learning and memory by ephedrine on HIBD rats in later stage is slightly weaker than that by GM1, which may be related with ephedrine dosage.

Brain injury occurring in the perinatal period is an important etiology of subsequent neurodevelopmental disabilities. Magnetic resonance imaging (MRI) is a tool that is used to evaluate the nature of brain injury in the human infant. MRI techniques have also been applied to various animal models of perinatal injury. The most commonly used model is the immature rat, but there have also been imaging studies in mice, rabbit kits and piglets. The studies have been carried out using MR systems of various magnetic field strengths, ranging from 1.5 to 11.7tesla (T), with applications for quantification of infarct volume, T1 measurements, T2 measurements, proton and phosphorus spectroscopy and diffusion imaging. The MR findings are then related to histopathology and, in a few cases, behavioral evaluations. There is also a growing number of studies utilizing MRI in evaluating the efficacy of neuroprotective treatments, such as hypothermia.

There is approximately a 6-8 hour window that exists from when a hypoxic-ischemic insult occurs, in utero, before significant irreversible brain injury occurs in new born infants. The focus of our work is to determine through the electroencephalogram (EEG) if such a hypoxic-ischemic insult has occurred such that neuro-protective treatment can be sought within this period. At present, there are no defined biomarkers in the EEG that are currently being used to help classify if a hypoxic ischemia insult has occurred. However, micro-scale transients in the form of spikes, sharps and slow waves exists that could provide precursory information whether a hypoxic-ischemic insult has occurred or not. In our previous studies we have successfully automatically identified spikes with high sensitivity and selectivity in the conventional 64Hz sampled EEG. This paper details the significant advantage that can be obtained in using high frequency 1024Hz sampled EEG for sharp wave detection over the typically employed 64Hz sampled EEG. This advantage is amplified when a combination of wavelet Type-2 Fuzzy Logic System (WT-Type-2-FLS) classifiers are used to identify the sharp wave transients. By applying WT-Type-2-FLS to the 1024Hz EEG record and to the same down-sampled 64Hz EEG record we demonstrate, how the sharp wave transients detection increases significantly for high resolution 1024Hz EEG over 64Hz EEG. The WT-Type-2-FLS algorithm performance was assessed over 3 standardised time periods within the first 8 hours, post occlusion of a fetal sheep, in utero. 1024Hz EEG results demonstrate the algorithm detected sharps with overall performance rates of 85%, 92%, and 87% in the Early/Mid and Late-latent phases of injury, respectively as compared to 25%, 55% and 31% in the 64Hz EEG. These results demonstrate the power of Wavelet Type-2 Fuzzy Logic System at detecting sharp waves in 1024Hz EEG and suggest that there should be a movement toward recording high frequency EEG for analysis of hypoxic ischemic micro-scale transients that does not occur at present.