Hypoxia-ischemia In Mice Research Articles

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury.

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci's method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence ("no cortical injury" group) or presence ("cortical injury" group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

How Does DHA Reach the Brain to Provide Neuroprotection after Hypoxic‐Ischemic Injury?

BackgroundPrevious papers have reported that intravenously injected radiolabeled docosahexaenoic acid (DHA) rapidly enters rat brain. Recent studies from our laboratory have shown that acute intraperitoneal (i.p) injection of triglyceride (TG) emulsions rich in n‐3 fatty acids, especially tri‐DHA provides neuroprotection after hypoxic‐ischemic (HI) injury in neonatal mice (PLoS One. 2016;11(8):e0160870).AIMTo determine how injected tri‐DHA released DHA and perhaps other DHA derivatives are delivered to brain to provide neuroprotection after HI.MethodologyLaboratory made tri‐DHA emulsions were labeled with non‐metabolizable [3H]‐cholesteryl hexadecyl ether. Neonatal naïve or HI mice (C57BL/6J) were injected with radiolabeled tri‐DHA emulsion intraperitoneally (0.375 mg TG/g bw). At 4 and 24 hrs after injection, blood, peritoneal fluid, lung, liver, heart, brain, kidney, muscle, bone, spleen and adipose tissue were harvested and levels of radiolabeled emulsion in each organ determined. Tissue uptake was expressed as percent of recovered injected dose. HI was produced by right carotid artery ligation followed by hypoxia (8% O2 at 37°C) for 15 mins.ResultsNo significant differences were observed between naïve or HI mice in uptake of tri‐DHA emulsion particles at 4 hrs after injection. Among naïve and HI mice, the percent of recovered dose of radioactivity in blood and intraperitoneal fluid at 4 hrs were found to be: blood‐ 7.7 ± 5.5% naïve vs 7.4 ± 0.81% HI; intraperitoneal fluid‐ 12.6 ± 8.8% naïve vs 17.8 ± 7.6% HI. Among all organs assayed, liver contained most of the recovered dose of radiolabeled emulsion (40.5 ± 8.9% naïve vs 42.7 ± 0.1% HI). Lowest emulsion particle uptake was found to be in the brain among naïve and HI mice (<0.1% of the recovered dose among both naïve and HI groups). Compared to 4 hrs, no significant changes were observed in the uptake of emulsion particles at 24 hrs after the injection.ConclusionsThe present data demonstrates that, neonatal mice efficiently clear radiolabeled emulsion from intraperitoneal fluid after i.p. injection. Uptake of emulsion particles was high in liver and very low in brain indicating that whole emulsion particle uptake by brain does not mediate neuroprotection after HI. This suggests the involvement of other pathways in DHA mediated neuroprotection in HI brain. We also hypothesize that local neuroprotection provided by DHA in HI brain is mediated, in part through DHA derivatives produced locally in brain and in other tissues.

Therapeutic Hypothermia Provides Variable Protection against Behavioral Deficits after Neonatal Hypoxia-Ischemia: A Potential Role for Brain-Derived Neurotrophic Factor

Background: Despite treatment with therapeutic hypothermia (TH), infants who survive hypoxic ischemic (HI) encephalopathy (HIE) have persistent neurological abnormalities at school age. Protection by TH against HI brain injury is variable in both humans and animal models. Our current preclinical model of hypoxia-ischemia (HI) and TH displays this variability of outcomes in neuropathological and neuroimaging end points with some sexual dimorphism. The detailed behavioral phenotype of this model is unknown. Whether there is sexual dimorphism in certain behavioral domains is also not known. Brain-derived neurotrophic factor (BDNF) supports neuronal cell survival and repair but may also be a marker of injury. Here, we characterize the behavioral deficits after HI and TH stratified by sex, as well as late changes in BDNF and its correlation with memory impairment. Methods: HI was induced in C57BL6 mice on postnatal day 10 (p10) (modified Vannucci model). Mice were randomized to TH (31°C) or normothermia (NT, 36°C) for 4 h after HI. Controls were anesthesia-exposed, age- and sex-matched littermates. Between p16 and p39, growth was followed, and behavioral testing was performed including reflexes (air righting, forelimb grasp and negative geotaxis) and sensorimotor, learning, and memory skills (open field, balance beam, adhesive removal, Y-maze tests, and object location task [OLT]). Correlations between mature BDNF levels in the forebrain and p42 memory outcomes were studied. Results: Both male and female HI mice had an approximately 8-12% lower growth rate (g/day) than shams (p ≤ 0.01) by p39. TH ameliorated this growth failure in females but not in males. In female mice, HI injury prolonged the time spent at the periphery (open field) at p36 (p = 0.004), regardless of treatment. TH prevented motor impairments in the balance beam and adhesive removal tests in male and female mice, respectively (p ≤ 0.05). Male and female HI mice visited the new arm of the Y-maze 12.5% (p = 0.05) and 10% (p = 0.03) less often than shams, respectively. Male HI mice also had 35% lower exploratory preference score than sham (p ≤ 0.001) in the OLT. TH did not prevent memory impairments found with Y-maze testing or OLT in either sex (p ≤ 0.01) at p26. At p42, BDNF levels in the forebrain ipsilateral to the HI insult were 1.7- to 2-fold higher than BDNF levels in the sham forebrain, and TH did not prevent this increase. Higher BDNF levels in the forebrain ipsilateral to the insult correlated with worse performance in the Y-maze in both sexes and in OLT in male mice (p = 0.01). Conclusions: TH provides benefit in specific domains of behavior following neonatal HI. In general, these benefits accrued to both males and females, but not in all areas. In some domains, such as memory, no benefit of TH was found. Late differences in individual BDNF levels may explain some of these findings.

Assessment of long-term safety and efficacy of intranasal mesenchymal stem cell treatment for neonatal brain injury in the mouse.

Background:For clinical translation, we assessed whether intranasal mesenchymal stem cell (MSC) treatment after hypoxia–ischemia (HI) induces neoplasia in the brain or periphery at 14 mo. Furthermore, the long-term effects of MSCs on behavior and lesion size were determined.Method:HI was induced in 9-d-old mice. Pups received an intranasal administration of 0.5 × 106 MSCs or vehicle at 10 d post-HI. Full macroscopical and microscopical pathological analysis of 39 organs per mouse was performed. Sensorimotor behavior was assessed in the cylinder-rearing test at 10 d, 28 d, 6 mo, and 9 mo. Cognition was measured with the novel object recognition test at 3 and 14 mo post-HI. Lesion size was determined by analyzing mouse-anti-microtubule-associated protein 2 (MAP2) and mouse-anti-myelin basic protein (MBP) staining at 5 wk and 14 mo.Results:At 14 mo post-HI, we did not observe any neoplasia in the nasal turbinates, brain, or other organs of HI mice treated with MSCs. Furthermore, our results show that MSC-induced improvement of sensorimotor and cognitive function is long lasting. In contrast, HI-vehicle mice showed severe behavioral impairment. Recovery of MAP2- and MBP-positive area lasted up to 14 mo following MSC treatment.Conclusion:Our results provide strong evidence of the long-term safety and positive effects of MSC treatment following neonatal HI in mice.

Deficits of brainstem and spinal cord functions after neonatal hypoxia–ischemia in mice

Perinatal cerebral hypoxia-ischemia (HI) can lead to severe neurodevelopmental disorders. Studies in humans and animal models mainly focused on cerebral outcomes, and little is known about the mechanisms that may affect the brainstem and the spinal cord. Dysfunctions of neuromodulatory systems, such as the serotonergic (5-HT) projections, critical for the development of neural networks, have been postulated to underlie behavioral and motor deficits, as well as metabolic changes. The aim of this study was to investigate brainstem and spinal cord functions by means of plethysmography and sensorimotor tests in a neonatal Rice-Vanucci model of HI in mice. We also evaluated bioaminergic contents in central regions dedicated to the motor control of autonomic functions. Mice with cerebral infarct expressed motor disturbances and had a lower body weight and a decreased respiratory frequency than SHAM, suggesting defects of brainstem neural network involved in the motor control of feeding, suckling, swallowing, and respiration. Moreover, our study revealed changes of monoamine and amino acid contents in the brainstem and the spinal cord of HI mice. Our results suggest that monoaminergic neuromodulation plays an important role in the physiopathology of HI brain injury that may represent a good therapeutic target.

Human Dental Pulp-Derived Stem Cells Protect Against Hypoxic-Ischemic Brain Injury in Neonatal Mice

Perinatal hypoxia-ischemia (HI) has high rates of neurological deficits and mortality. So far, no effective treatment for HI brain injury has been developed. In this study, we investigated the therapeutic effects of stem cells from human exfoliated deciduous teeth (SHED) for the treatment of neonatal HI brain injury. Unilateral HI was induced in postnatal day 5 (P5) mice. Twenty-four hours later, SHED, human skin fibroblasts, or serum-free conditioned medium derived from these cells was injected into the injured brain. The effects of cell transplantation or conditioned medium injection on the animals' neurological and pathophysiological recovery were evaluated. Transplanted SHED, but not fibroblasts, significantly reduced the HI-induced brain-tissue loss and improved neurological function. SHED also improved the survival of the HI mice. The engrafted SHED rarely differentiated into neural lineages; however, their transplantation inhibited the expression of proinflammatory cytokines, increased the expression of anti-inflammatory ones, and significantly reduced apoptosis. Notably, the intracerebral administration of SHED-conditioned medium also significantly improved the neurological outcome, inhibited apoptosis, and reduced tissue loss. SHED transplantation into the HI-injured brain resulted in remarkable neurological and pathophysiological recovery. Our findings indicate that paracrine factors derived from SHED support a neuroprotective microenvironment in the HI brain. SHED graft and SHED-conditioned medium may provide a novel neuroprotective therapy for HI.

Decreased vulnerability of hippocampal neurons after neonatal hypoxia–ischemia in bis‐deficient mice

The Bcl-2-interacting death suppressor (Bis) protein is involved in antiapoptosis and antistress pathways. However, its roles after neonatal hypoxia-ischemia remain obscure. Therefore, we investigated the effects of Bis deletion on hippocampal cell death following neonatal hypoxia-ischemia. We transected the right common carotid artery of bis(+/+) and bis(-/-) mice at postnatal Day 7 and subjected them to hypoxia for 35 min. Cresyl violet staining showed that hypoxia-ischemia induced progressive cell death in the hippocampi of bis(+/+) mice. Moreover, Bis was expressed in astrocytes, not microglia, in sham-manipulated hippocampi of bis(+/+) mice, and was markedly enhanced after hypoxia-ischemia. Immunoblotting showed that Bis expression significantly increased 3 and 7 days following hypoxia-ischemia. Unexpectedly, 7 days after hypoxia-ischemia, the number of hippocampal NeuN-positive cells was higher in the bis(-/-) mice than in the bis(+/+) mice. We subsequently performed transcriptomic analysis and quantitative real time polymerase chain reaction to search for the underlying genes responsible for resistance to hypoxia-ischemia in the bis(-/-) hippocampus. These studies showed that 6 h after hypoxia-ischemia, galectin 3 and filamin C levels increased to a lesser extent in the bis(-/-) hippocampi compared with the bis(+/+) hippocampi. Finally, our in vitro hypoxia-ischemia model, using A172 glioma cells and primary astrocytes, showed that downregulation of Bis blocked the enhanced expression of galectin 3 after oxygen-glucose deprivation. This study demonstrated that Bis was upregulated in the astrocytes after hypoxia-ischemia. In addition, we showed that hippocampal neurons are less vulnerable to hypoxia-ischemia in mice lacking Bis, possibly because of the modulation of galectin 3 induction.

Mesenchymal stem cells restore cortical rewiring after neonatal ischemia in mice

A study was undertaken to investigate the effect of neonatal hypoxic-ischemic (HI) brain damage and mesenchymal stem cell (MSC) treatment on the structure and contralesional connectivity of motor function-related cerebral areas. Brain remodeling after HI±MSC treatment in neonatal mice was analyzed using diffusion tensor magnetic resonance imaging, immunohistochemistry, anterograde tracing with biotinylated dextran amine (BDA), and retrograde tracing with fluorescent pseudorabies virus (PRV). MSC treatment after HI reduced contralesional rewiring taking place after HI. Following MSC treatment, fractional anisotropy values, which were increased in both ipsi- and contralesional cortices and decreased in the corpus callosum (CC) after HI, were normalized to the level observed in sham-operated mice. These results were corroborated by myelin basic protein intensity and staining pattern in these areas. Anterograde tracing of ipsilesional motor neurons showed that after MSC treatment, fewer BDA-positive fibers crossed the CC and extended into the contralesional motor cortex compared to HI mice. This remodeling was functional, because retrograde labeling showed increased connectivity between impaired (left) forepaw and the contralesional (left) motor cortex after HI, whereas MSC treatment reduced this connection and increased the connection between the impaired (left) forepaw and the ipsilesional (right) motor cortex. Finally, the extent of contralesional rewiring measured with BDA and PRV tracing was related to sensorimotor dysfunction. This is the first study to describe MSC treatment after neonatal HI markedly reducing contralesional axonal remodeling induced by HI brain injury.

Trace Fear Conditioning Detects Hypoxic-Ischemic Brain Injury in Neonatal Mice

Trace fear conditioning is a well-established test for the assessment of learning deficits in rodents. The aim of this study was to determine whether hypoxia-ischemia (HI) on postnatal day 9 (P9) in mice prevents the acquisition and expression of cued and contextual fear learning in early adulthood. Brain injury was induced in mice on P9 by 30 min of HI. On P49 and P50, animals were tested for: (1) trace fear conditioning with a short delay (2 s) between a shock-paired tone plus light and shock, (2) trace fear conditioning with a longer delay (20 s) between a shock-paired tone and shock, and (3) trace fear conditioning with a 2-second delay between a shock-paired tone and shock with additional visual, olfactory and tactile contextual cues in the fear conditioning apparatus. Outcome was assessed as percent of time spent freezing during a 2-min test. Histological assessment of the hippocampus and amygdala was performed on P51 to determine the extent of HI injury. Both shock-paired tone plus light with a short delay and shock-paired tone with a short delay plus additional contextual cues enhanced tone-induced freezing behavior in a nonhandled control group, but not in the HI group. For trace fear conditioning with a 20-second delay between the tone and the shock, freezing behavior did not differ significantly between nonhandled control and HI animals. Dorsal hippocampal and amygdala volumes were smaller in the ischemic hemispheres of the HI mice that displayed impaired fear memory with shock-paired tone plus light. In summary, we have shown that trace fear conditioning is a sensitive method for detecting memory impairments in adolescent mice following mild HI injury during the neonatal period. Combining a discrete conditioned stimulus (shock-paired tone plus light) with a short trace delay was the most sensitive method for using the fear conditioning paradigm to detect mild HI damage to the hippocampus and amygdala.

Protective Effects of a Water-Soluble Extract from Culture Medium of Lentinus Edodes Mycelia against Neuronal Damage after Hypoxia-Ischemia in Mice

Protective Effects of a Water-Soluble Extract from Culture Medium of Lentinus Edodes Mycelia against Neuronal Damage after Hypoxia-Ischemia in Mice

Mild neonatal hypoxia–ischemia induces long-term motor- and cognitive impairments in mice

To understand and potentially treat the lifelong cognitive and motor deficits in humans resulting from perinatal mild cerebral hypoxic–ischemic (HI) events, valid animal models are of high importance. Nowadays the murine model of neonatal cerebral HI-injury (unilateral carotid artery occlusion followed by hypoxia) is applied more frequently. In the present study we investigated motor, behavioral and cognitive functioning in mice with mild cerebral HI-injury (45 min of hypoxia; HI-45) in comparison to mice exposed to severe HI (HI-75) and sham-control mice. Lateralizing motor disturbances as measured using the cylinder rearing test developed in both HI-45 and HI-75 mice and was significantly more severe in HI-75 animals. To assess behavior and cognitive functions, we used the modified hole board (mHB) test in two stages. First, the ability of the animals to find the three food rewards in cued holes over time was determined. The results revealed an overall learning impairment in HI-75 mice, while HI-45 mice were not different from sham controls. In the second stage, a reversal test was performed with rewarded cylinders being non-cued and non-rewarded cylinders being cued. This reversal-task revealed impairments in cognitive flexibility in HI-45 mice as compared to sham-control animals. Our data indicate that both the cylinder rearing task and the two stages of the mHB are suitable behavioral approaches to differentiate consequences of neonatal mild and severe brain damage on executive functioning.

マウス低酸素脳虚血障害に対する霊芝菌糸体培養培地抽出物の保護効果

脳虚血では，局所における過剰の活性酸素種の産生によりアポトーシスが誘発される．本研究では，酸化ストレスによる神経障害に対する霊芝菌糸体培養培地抽出物 (WER) の保護効果について，培養細胞およびマウス脳虚血モデルを用いて検討した． PC12 細胞に H2O2 処理を行い，誘発される細胞死に対する WER の作用を MTT assay および TUNEL 法により評価した．また，マウスに WER (1 g/kg) を単回または 7 日間経口投与した後，低酸素脳虚血処置を行い，神経症状，脳梗塞巣体積およびアポトーシスの評価を行った． PC12 細胞を用いた実験において，WER は H2O2 処理後の細胞生存率を上昇させ，アポトーシス陽性細胞数を減少させた．WER を 7 日間経口投与したマウス群では，対照群と比較し，虚血後の神経症状の改善が認められ，また脳梗塞巣体積およびアポトーシス陽性細胞数が有意に減少した．これらの結果から，WER は酸化ストレスによるアポトーシスを抑制し，脳虚血障害に対して保護作用を示すことが示唆された．

Metallothionein I,II deficient mice do not exhibit significantly worse long-term behavioral outcomes following neonatal hypoxia–ischemia: MT-I,II deficient mice have inherent behavioral impairments

Metallothionein I and II are small metal binding proteins with a high affinity for zinc. They are found in the CNS and are thought to play a role in modulating the effects of free zinc. We hypothesized that MT-I,II deficient mice would have more neurological deficits both functionally and anatomically following a neonatal hypoxic-ischemic (HI) insult than wild-type mice subjected to the same insult. Forty wild-type and 40 MT-I,II deficient C57 X 129T2 F1 P10 mice were randomized to either 45 min of HI or sham HI. Beginning on P50, the mice were given a series of behavioral tests including locomotor activity, novel object recognition, Morris water maze (cued, hidden platform, reduced platform), a 2-week-delayed probe trial and an apomorphine-induced rotation test. At the conclusion of testing, the brains were removed for histological analysis including staining with NeuN and GFAP to assess neuronal loss and reactive gliosis. There were no significant differences in functional or anatomic measures between the wild-type HI mice and the MT-I,II deficient HI mice. The MT-I,II deficient mice exhibited an impaired rate of learning in the spatially oriented mazes but once learned retained the information as well as the wild-type mice. The absence of functional MT-I,II proteins does not result in significantly worse injury following 45 min of HI on P10. The MT-I,II deficient mice have baseline impairments in spatial learning but not retention.

Apoptosis-inducing factor is a major contributor to neuronal loss induced by neonatal cerebral hypoxia-ischemia

Nine-day-old harlequin (Hq) mice carrying the hypomorphic apoptosis-inducing factor (AIF)(Hq) mutation expressed 60% less AIF, 18% less respiratory chain complex I and 30% less catalase than their wild-type (Wt) littermates. Compared with Wt, the infarct volume after hypoxia-ischemia (HI) was reduced by 53 and 43% in male (YX(Hq)) and female (X(Hq)X(Hq)) mice, respectively (P<0.001). The Hq mutation did not inhibit HI-induced mitochondrial release of cytochrome c or activation of calpain and caspase-3. The broad-spectrum caspase inhibitor quinoline-Val-Asp(OMe)-CH(2)-PH (Q-VD-OPh) decreased the activation of all detectable caspases after HI, both in Wt and Hq mice. Q-VD-OPh reduced the infarct volume equally in Hq and in Wt mice, and the combination of Hq mutation and Q-VD-OPh treatment showed an additive neuroprotective effect. Oxidative stress leading to nitrosylation and lipid peroxidation was more pronounced in ischemic brain areas from Hq than Wt mice. The antioxidant edaravone decreased oxidative stress in damaged brains, more pronounced in the Hq mice, and further reduced brain injury in Hq but not in Wt mice. Thus, two distinct strategies can enhance the neuroprotection conferred by the Hq mutation, antioxidants, presumably compensating for a defect in AIF-dependent redox detoxification, and caspase inhibitors, presumably interrupting a parallel pathway leading to cellular demise.

Late measures of brain injury after neonatal hypoxia-ischemia in mice.

This work was undertaken to determine to what degree long-term neurofunctional outcome of neonatal hypoxic-ischemic (HI) brain injury in mice correlates with anatomical extent of cerebral damage assessed by magnetic resonance imaging (MRI) and histopathology. On postnatal day 7, mice were subjected to HI. At 7 to 9 weeks after HI neurofunctional outcome was assessed by water-maze, rota-rod, and open-field test performance, followed by cerebral MRI and histopathology evaluation. At 10 weeks after HI, MRI revealed ipsilateral brain atrophy alone or with porencephalic cyst formation and contralateral ventriculomegaly. Adult HI-affected mice, especially those that developed a porencephalic cyst, demonstrated significant neurofunctional deficit compared with age-matched naïve mice. HI-affected mice with ipsilateral cerebral atrophy but without porencephaly demonstrated no or an intermediate level of neurofunctional deficit. Neurobehavioral assessment of mice subjected to HI insult revealed a strong correlation between degree of brain injury and functional neurohandicap. This is the first study to demonstrate that long-term neurofunctional outcome in mice after a neonatal HI correlates tightly with anatomical pattern/extent of cerebral damage, defined by MRI and histopathology.