Neonatal Hypoxic-ischemic Encephalopathy Model Research Articles

Evaluating a Targeted Bedside Measure of Cerebral Perfusion in a Nonhuman Primate Model of Neonatal Hypoxic‐Ischemic Encephalopathy

To compare ultrasound-derived resistive indices (RIs) obtained at the level of the thalamus via fast Doppler ultrasound with traditional anterior cerebral artery measures in a model of neonatal hypoxic-ischemic encephalopathy and to correlate each with clinical outcomes. Nine nonhuman primate neonates underwent no umbilical cord occlusion (n = 3), umbilical cord occlusion without hypothermia (n = 3), or umbilical cord occlusion with hypothermia (n = 3). The RI was measured in the anterior cerebral artery and thalamus on days 0, 1, and 4 of life. Magnetic resonance imaging with spectroscopy was performed on day 4. Mean thalamus and anterior cerebral artery RI values in the first 36 hours of life were statistically different in neonates who died (+0.13; P = .019) or developed cerebral palsy (-0.08; P = .003). Thalamic RI values showed stronger associations with serum and spectroscopic lactate values than those in the anterior cerebral artery. The umbilical cord occlusion-with-hypothermia group showed a significant increase in the RI in the thalamus but not the anterior cerebral artery. Resistive index measurements in the thalamus may eventually supplement other bedside measures for predicting outcomes in the HIE population, but further studies need to differentiate the effect of hypothermia from illness severity on thalamic perfusion.

Long-term cognitive effects of uridine treatment in a neonatal rat model of hypoxic-ischemic encephalopathy

Hypoxic-ischemic encephalopathy (HIE), is the most common brain disorder in neonates during the perinatal period, which, to date, can only be managed to some extent by hypothermia. Uridine is the principal circulating pyrimidine in humans which is utilized as a precursor for membrane phospholipid biosynthesis. Uridine has recently been shown to provide clinical benefit in treatment of Alzheimer’s disease due to its involvement in increasing number of brain synapses along with other phospholipid precursors. We previously showed that uridine treatment ameliorated brain damage by reducing apoptosis in a rat model of neonatal HIE. The aim of the present study was to investigate the effects of uridine administration on cognitive functions during periadolescent period in rats subjected to hypoxic-ischemic (HI) brain damage in neonatal period. Male newborn rats were subjected to HI insult on postnatal day 7 (P7) and were injected intraperitoneally with either saline or uridine (500mg/kg) for three consecutive days. Part of pups in each group were sacrificed on P10 to collect brain samples for active Caspase-3 analyses and the remaining pups were raised through P40 to evaluate early reflexes, sensorimotor coordination and learning and memory functions by Negative Geotaxis (NG), Beam Walking (BW) and Morris Water Maze (MWM) tasks, respectively. Confirming our previous findings, we showed that uridine administration reduced apoptotic cell damage on P10. No significant difference was observed between uridine and saline groups in early reflexes or sensorimotor coordination. On the other hand, rats receiving uridine displayed improved learning and memory in MWM during periadolescent period. We conclude that uridine treatment improves learning and memory in the long term by, probably, reducing apoptotic cell death in early newborn period. This is the first study to show beneficial cognitive effects of uridine in rats with brain damage.

Evaluations of Intravenous Administration of CD34+ Human Umbilical Cord Blood Cells in a Mouse Model of Neonatal Hypoxic-Ischemic Encephalopathy

Several cell therapies have been explored as novel therapeutic strategies for neonatal encephalopathy because the benefits of current treatments are limited. We previously reported that intravenous administration of human umbilical cord blood (hUCB) CD34⁺ cells (hematopoietic stem cells/endothelial progenitor cells) at 48 h after insult exerts therapeutic effects in neonatal mice with stroke, i.e., permanent middle cerebral artery occlusion. Although neonatal stroke and hypoxic-ischemic encephalopathy (HIE) are grouped under the term “neonatal encephalopathy,” their pathogenesis differs. However, little is known about the differences in the effects of the same treatment between these 2 diseases. In this study, we investigated whether the same treatment protocol exerts therapeutic effects in neonatal mice with HIE. The treatment significantly ameliorated the decreased cerebral blood flow in the ischemic penumbra. Although the cylinder and rotarod tests showed a trend of amelioration of behavioral impairments from the treatment, these were not statistically significant. Morphological brain injuries were not altered by treatment. The cell administration did not cause any adverse effects apart from hyperactivity in the open-field test. Some of these findings are consistent with the results obtained in our previous study using a stroke model, but others are not. This study suggests that the treatment protocol needs to be optimized for each pathological condition.

Neural Stem and Progenitor Cells with High Proliferation Potential in Injured Areas in a Model of Neonatal Hypoxic-Ischemic Encephalopathy

Objective: Neonatal hypoxic-ischemic encephalopathy causes death and severe neurological sequelae including cerebral palsy, epilepsy, and mental retardation. Because hypothermia therapy is the only effective treatment for neonatal hypoxic-ischemic encephalopathy and its efficacy is limited in severe cases, a more effective treatment is needed. Neural stem and progenitor cells are important for neuroprotection and neuronal replacement following hypoxic-ischemic encephalopathy. The aim of this study was to confirm the presence of neural stem cells and the acceleration of their mitotic activity in injured areas in a model of neonatal hypoxic-ischemic encephalopathy. Method: Hypoxic-ischemic encephalopathy was induced in rat pups by left common carotid artery occlusion followed by hypoxia. Following ischemic injury, brains were sectioned, and columns of tissue were excised from infarct areas for cell culture using the neurosphere assay. Cell clusters were analyzed by immunocytochemistry and reverse transcription polymerase chain reaction. Clustered cells were also tested for their ability to differentiate into neurons, astrocytes, and oligodendrocytes. Results: Neurosphere-like cell clusters from infarct areas of ischemia-injured animals were dramatically increased compared to samples from sham-operated control animals. Clusters included cells immunopositive for bromodeoxyuridine, nestin, neural/glial antigen 2, β-III tubulin, glial fibrillary acidic protein, oligodendrocyte marker O4, vimentin, and ionizing calcium-binding adaptor molecule 1. Cell clusters also expressed mRNA for NANOG, (sex determining region Y)-box 2, octamer-binding transcription factor 3/4, and Rex1, similar to embryonic stem cells, and could be differentiated into neurons, astrocytes or oligodendrocytes that expressed mRNAs for nestin, β-III tubulin, glial fibrillary acidic protein, and myelin basic protein. Conclusion: We demonstrate the presence of many neural stem and progenitor cells with enhanced proliferative potential in infarct brain areas of a rat model of neonatal hypoxic-ischemic encephalopathy. Activation of neural stem and progenitor cells in the infarct brain area may contribute to neuroprotection and regeneration.

Combination treatment with ethyl pyruvate and IGF-I exerts neuroprotective effects against brain injury in a rat model of neonatal hypoxic-ischemic encephalopathy.

Neonatal hypoxic-ischemic (HI) brain injury causes severe brain damage in newborns. Following HI injury, rapidly accumulating oxidants injure neurons and interrupt ongoing developmental processes. The antioxidant, sodium pyruvate, has been shown to reduce neuronal injury in neonatal rats under conditions of oxygen glucose deprivation (OGD) and HI injury. In this study, we evaluated the effects of ethyl pyruvate (EP) and insulin-like growth factor-I (IGF-I) alone or in combination in a similar setting. For this purpose, we used an in vitro model involving primary neonatal rat cortical neurons subjected to OGD for 2.5 h and an in vivo model involving unilateral carotid ligation in rats on post-natal day 7 with exposure to 8% hypoxia for 2.5 h. The cultured neurons were examined by lactate dehydrogenase (LDH) and cell viability assays. For the in vivo experiments, behavioral development was evaluated by the foot fault test at 4 weeks of recovery. 2,3,5-Triphenyltetrazolium chloride monohydrate and cresyl violet staining were used to evaluate HI injury. The injured neurons were Fluoro-Jade B-labeled, new neuroprecursors were double labeled with bromodeoxyuridine (BrdU) and doublecortin, new mature neurons were BrdU-labeled and neuronal nuclei were labeled by immunofluorescence. Under conditions of OGD, the LDH levels increased and neuronal viability decreased. Treatment with 0.5 mM EP or 25 ng/ml IGF-I protected the neurons (P<0.05), exerting additive effects. Similarly, either the early administration of EP or delayed treatment with IGF-I protected the neonatal rat brains against HI injury and improved neurological performance and these effects were also additive. This effect may be the result of reduced neuronal injury, and enhanced neurogenesis and maturation. On the whole, our findings demonstrate that the combination of the early administration of EP with delayed treatment with IGF-I exerts neuroprotective effects against HI injury in neonatal rat brains.

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy.

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world's 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials(1,2). The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (< 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Flaxseed mitigates brain mass loss, improving motor hyperactivity and spatial memory, in a rodent model of neonatal hypoxic-ischemic encephalopathy

Neonatal hypoxic-ischemic (HI) encephalopathy is a major cause of perinatal morbimortality. There is growing evidence that n-3 polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), attenuate brain injury. This study aimed to investigate the possible neuroprotective effect of maternal intake of flaxseed, rich in DHA׳s precursor α-linolenic acid, in the young male offspring subjected to perinatal HI. Wistar rats were divided in six groups, according to maternal diet and offspring treatment at day 7: Control HI (CHI) and Flaxseed HI (FHI); Control Sham and Flaxseed Sham; Control Control and Flaxseed Control. Flaxseed diet increased offspring׳s hippocampal DHA content and lowered depressive behavior. CHI pups presented brain mass loss, motor hyperactivity and poor spatial memory, which were improved in FHI rats. Maternal flaxseed intake may prevent depressive symptoms in the offspring and promote neuroprotective effects, in the context of perinatal HI, improving brain injury and its cognitive and behavioral impairments.

Sex differences in behavioral outcome following neonatal hypoxia ischemia: Insights from a clinical meta-analysis and a rodent model of induced hypoxic ischemic injury

Hypoxic ischemic encephalopathy (HIE) secondary to perinatal asphyxia leads to long-term visual disabilities. Dilated retinal exams in human newborns with HIE is an emerging diagnostic tool, but phenotypes of hypoxia ischemia (HI) related retinal vascular injury are unclear. 7,8-Dihydroxyflavone (7,8-DHF) is a TrkB agonist with protective effects on HI-related brain damage. We studied retinal vessels in a mouse model of neonatal HIE and the efficacy of 7,8-DHF in ameliorating HI-related retinal vascular injury.C57BL6/J mice at post-natal day (P) 9 received unilateral left carotid artery ligation followed by exposure to 10 % oxygen for 50 min. Phosphate buffered saline or 7,8-DHF (5 mg/kg) were administered daily for 7 days intraperitoneally. Control groups of naïve or carotid artery ligation only mice were studied. Fluorescein angiography was performed in acute (two weeks post-exposure) and chronic (four weeks post-exposure) time points. Retinal artery width, retinal vein width, and collateral vessel length were quantified.Ligation of the common carotid artery alone caused retinal artery dilation in acute and chronic time points, but had no effect on retinal veins. At acute time point, HI caused increased retinal artery vasodilation, but was reversed by 7,8-DHF. HI caused short collateral vessel formation in ipsilateral eyes, rescued by 7,8-DHF treatment.Retinal artery vasodilation and collateral vessel formation due to HI were rescued by 7,8-DHF treatment. Retinal and collateral vessel monitoring could be diagnostic biomarkers for HI severity. Studies to elucidate mechanisms of 7,8-DHF action on retinal vessels could aid development of therapies for neonatal HI.

Transplantation of Human Adipose Tissue-Derived Mesenchymal Stem Cells Restores the Neurobehavioral Disorders of Rats With Neonatal Hypoxic-Ischemic Encephalopathy.

Improving the effects of human adipose tissue-derived mesenchymal stem cells (ASCs) on the demyelination and neurobehavioral function was investigated in an experimental model of neonatal hypoxic-ischemic encephalopathy (HIE). Seven-day-old male rats were subjected to hypoxia-ischemia-lipopolysaccharide and intracerebroventricularly transplanted with human ASCs (4 × 10(5) cells/rat) once at postnatal day 10 (PND10) or repeatedly at PND10, 17, 27, and 37. Neurobehavioral abnormalities (at PND20, 30, and 40) and cognitive functions (at PND41-44) were evaluated using multiple test systems. Human ASCs recovered the using ratio of forelimb contralateral to the injured brain, improved locomotor activity, and restored rota-rod performance of HIE animals, in addition to showing a marked improvement of cognitive functions. It was confirmed that transplanted human ASCs migrated to injured areas and differentiated into oligodendrocytes expressing myelin basic protein (MBP). Moreover, transplanted ASCs restored production of growth and neurotrophic factors and expression of decreased inflammatory cytokines, leading to attenuation of host MBP loss. The results indicate that transplanted ASCs restored neurobehavioral functions by producing MBP as well as by preserving host myelins, which might be mediated by ASCs' anti-inflammatory activity and release of growth and neurotrophic factors.

Neuroprotective effects of uridine in a rat model of neonatal hypoxic–ischemic encephalopathy

Neonatal hypoxic–ischemic encephalopathy (HIE) is a major cause of neurological disability requiring newer therapeutic strategies. Uridine is the principal circulating pyrimidine in humans and a substrate for nucleotides and membrane phospholipids. The objective of this study was to investigate the effects of uridine in a neonatal rat model of HIE. Rat pups subjected to hypoxic–ischemic insult on postnatal day 7 were injected intraperitoneally with either saline or uridine (100, 300 or 500mg/kg) for three consecutive days and brains were collected for evaluation of brain infarct volume and apoptosis. Compared with Control group, uridine at 300 and 500mg/kg doses significantly reduced percent infarct volume, TUNEL(+) cell ratio and active Caspase-3 immunoreactivity in the cortex, as well as in CA1 and CA3 regions of the hippocampus. Uridine (300 and 500mg/kg) also decreased active Caspase-3 expression in the ipsilateral hemisphere. These data indicate that uridine dose-dependently reduces brain injury in a rat model of neonatal HIE by decreasing apoptosis.

Comprehensive Gene Expression Analysis of Cerebral Cortices from Mature Rats After Neonatal Hypoxic–Ischemic Brain Injury

Neonatal hypoxic-ischemic (HI) encephalopathy can lead to severe brain damage and is a common cause of neurological handicaps in adulthood. To elucidate the molecular events occurring in cerebral cortices of mature rats (8 weeks old) after neonatal HI brain insult, we performed comprehensive gene expression and gene network analyses using a DNA microarray system (Agilent 4x44K). A rat model of neonatal HI encephalopathy (Rice model) was obtained by unilateral ligation of the common carotid artery of 7-day-old rats with hypoxia (exposure to 8 % oxygen). Due to the HI insult-related breakdown of the ipsilateral hemisphere in the brain, RNAs were prepared from the contralateral cerebral cortices of 8-week-old rats and analyzed by DNA microarray. Biofunctional analysis of differentially regulated genes revealed that many upregulated genes were related to cell death signaling, such as the arachidonic acid cascade. In contrast, many downregulated genes were related to gene expression, reflecting progressive damage by the HI insult, even within the contralateral cerebral hemisphere.

Maternal docosahexaenoic acid-enriched diet prevents neonatal brain injury

Hypoxic-ischemic encephalopathy due to neonatal asphyxia is one of the most important causes of delayed neurological development. Prolonged neuronal apoptosis plays an important role in the processes contributing to neuronal degeneration. Docosahexaenoic acid (DHA), a major component of brain membrane phospholipids, prevents neuronal cell apoptosis and plays an important role as an anti-oxidant agent. We investigated the neuroprotective and anti-oxidant effects of maternal DHA supplementation during pregnancy in a model of neonatal hypoxic-ischemic encephalopathy. Pregnant rats were randomly assigned to two experimental groups: a control group or a DHA-enriched diet group. Hypoxic-ischemic encephalopathy was produced by left common carotid artery occlusion and exposure to 8% oxygen for 1.5 h. TUNEL assay, immunohistochemistry for caspase-3 and 8-hydroxy-deoxyguanosine (8-OHdG), and Western blot for caspase-3 were performed at postnatal days 8, 10 and 14. Fatty acid composition of brain was estimated on postnatal day 7. Maternal diet clearly influenced brain fatty acid composition in pups. Numbers of apoptotic neuronal cells and 8-OHdG immunoreactivity were significantly decreased in the DHA-enriched group. Our findings indicate that maternal DHA-enriched diet during pregnancy provides neuroprotection by inhibiting oxidative stress and apoptotic neuronal death. Dietary supplementation of DHA during pregnancy may thus be beneficial in preventing neonatal brain injury.

Edaravone Inhibits DNA Peroxidation and Neuronal Cell Death in Neonatal Hypoxic-Ischemic Encephalopathy Model Rat

Neonatal hypoxic-ischemic encephalopathy (HIE) is the most frequent neurologic disease in the perinatal period. Its major cause is oxidative stress, which induces DNA peroxidation and apoptotic neuronal death. We examined 8-hydroxy-2'-deoxyguanosine (8-OHdG) expression to evaluate brain damage in neonatal HIE and the therapeutic effect of edaravone, a free radical scavenger. Using HPLC and immunohistochemistry, the 8-OHdG levels of neonatal HIE model Sprague-Dawley rats that were subjected to left common carotid artery ligation and 2-h hypoxia significantly increased after 24-48 h of hypoxic-ischemic (HI) insult, but decreased after 72 h. Moreover, the number of apoptotic cells with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and karyorrhexis significantly increased after 24-72 h of HI insult. In a therapeutic experiment, edaravone was administered i.p. (9 mg/kg) after HI insult every 24 h. Edaravone reduced both the apoptotic neuronal cell number and 8-OHdG expression after 24-48 h of HI. From a double immunofluorescent study, DNA peroxidation occurred in apoptotic neuronal cells with 8-OHdG expression. Edaravone may inhibit the number of apoptotic neuronal cells and 8-OHdG expression within 48 h after HI insult.

Brainstem monoamine pathology of neonatal hypoxic–ischemic brain damage: A model of acute stage of neonatal asphyxia

Neonatal hypoxic–ischemic encephalopathy (HIE) is one of the most severe perinatal diseases and leads to high mortality and sometimes severe neurological sequelae. At the acute stage of HIE, it is thought to be the damage of catecholaminergic system in the brainstem. And then, HIE reflects mental development throughout the norepinephrine and serotonin systems, which mainly originates in the brainstem. Therefore, we studied both systems in the brainstem of neonatal HIE model rats with tyrosine hydroxylase (TH) and tryptophan hydroxylase (TpH) immunohistochemistry and a high-performance liquid column (HPLC) to measure norepinephrine and serotonin and their metabolism. As a result, the TH-positive and TpH-positive cell numbers significantly decreased 2 days after hypoxic–ischemic (HI) insult ( n = 10). However, 7 days after insult ( n = 10), the TH-positive and TpH-positive cell numbers had recovered in most regions. HPLC demonstrated a significant difference in the norepinephrine concentration 2 days after HI insult, but not in the other monoamines.

Developing a long-term surviving piglet model of neonatal hypoxic-ischemic encephalopathy

Objective: This study was designed to develop a piglet model of neonatal hypoxic-ischemic encephalopathy, which would allow for serial assessments of long-term neurodevelopmental impairment.Methods: In 12 newborn piglets, we produced hypoxia by 8% oxygen breathing for 5–91 minutes. We combined ischemia by reversible bilateral common carotid artery occlusion for varying times. Outcome measures were clinical neurological evaluation, magnetic resonance spectroscopy studies and brain histology.Results: Those animals which received intravenous sedation and no mechanical ventilation showed poor tolerance to hypoxia-ischemia and died early in the course of the experiments. The use of inhalation anesthesia during surgical procedures and mechanical ventilation during hypoxia-ischemia was associated with long-term survival. Seven of eight animals that survived ≥ 48 hr showed clinical neurological abnormalities, that later resolved. Magnetic resonance spectroscopy measurements did not change significantly following hypoxia-ischemia. None of the animals had histopathological brain lesions.Conclusion: When subjected to acute hypoxia-ischemia, piglets were likely to survive only if they were given such supportive measures as anesthesia and mechanical ventilation. Even with hypoxic-ischemic injury sufficient to produce acute signs of neurological dysfunctions, longterm, stable survival with no evident brain histopathological abnormalities was possible.

Hypothermia does not explain MK-801 neuroprotection in a rat model of neonatal hypoxicischemic encephalopathy

Hypothermia does not explain MK-801 neuroprotection in a rat model of neonatal hypoxicischemic encephalopathy