Criteria for brain death in Denmark.

In 1990, the Danish brain death legislation was adopted by the Danish Parliament. Each year, around 100 patients in Denmark fulfil criteria for brain death/death by neurological criteria (BD/DNC). In this review of current Danish criteria including the indication for ancillary investigation, which in Denmark is digital subtraction angiography (DSA), we conclude that the time has come to revise the national BD/DNC criteria. We propose that visible anoxic-ischaemic encephalopathy on brain CT after cardiac arrest does not require evaluation by ancillary testing, and that CT-angiography can be used instead of DSA.

CXCL10 is a crucial chemoattractant for efficient intranasal delivery of mesenchymal stem cells to the neonatal hypoxic-ischemic brain.

BACKGROUND: Hypoxic-Ischemic Encephalopathy (HIE) is a leading cause of mortality and morbidity in newborns. Recent research has shown promise in using intranasal mesenchymal stem cell (MSC) therapy if administered within 10 days after Hypoxia-Ischemia (HI) in neonatal mice. MSCs migrate from the nasal cavity to the cerebral lesion in response to chemotactic cues. Which exact chemokines are crucial for MSC guidance to the HI lesion is currently not fully understood. This study investigates the role of CXCL10 in MSC migration towards the HI-injured brain. METHODS: HI was induced in male and female 9-day-old C57BL/6 mice followed by intranasal MSC treatment at day 10 or 17 post-HI. CXCL10 protein levels, PKH26-labeled MSCs and lesion size were assessed by ELISA, immunofluorescent imaging and MAP2 staining respectively. At day 17 post-HI, when CXCL10 levels were reduced, intracranial CXCL10 injection and intranasal PKH26-labeled MSC administration were combined to assess CXCL10-guided MSC migration. MSC treatment efficacy was evaluated after 18 days, measuring lesion size, motor outcome (cylinder rearing task), glial scarring (GFAP staining) and neuronal density (NeuN staining) around the lesion. Expression of the receptor for CXCL10, i.e. CXCR3, on MSCs was confirmed by qPCR and Western Blot. Moreover, CXCL10-guided MSC migration was assessed through an in vitro transwell migration assay. RESULTS: Intranasal MSC treatment at day 17 post-HI did not reduce lesion size in contrast to earlier treatment timepoints. Cerebral CXCL10 levels were significantly decreased at 17 days versus 10 days post-HI and correlated with reduced MSC migration towards the brain. In vitro experiments demonstrated that CXCR3 receptor inhibition prevented CXCL10-guided migration of MSCs. Intracranial CXCL10 injection at day 17 post-HI significantly increased the number of MSCs reaching the lesion which was accompanied by repair of the HI lesion as measured by reduced lesion size and glial scarring, and an increased number of neurons around the lesion. CONCLUSIONS: This study underscores the crucial role of the chemoattractant CXCL10 in guiding MSCs to the HI lesion after intranasal administration. Strategies to enhance CXCR3-mediated migration of MSCs may improve the efficacy of MSC therapy or extend its regenerative therapeutic window.

Menaquinone-4 alleviates hypoxic-ischemic brain damage in neonatal rats by reducing mitochondrial dysfunction via Sirt1-PGC-1α-TFAM signaling pathway.

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) is a major contributor to neonatal mortality and neurodevelopmental disorders, but currently there is no effective therapy drug for HIE. Mitochondrial dysfunction plays a pivotal role in hypoxic-ischemic brain damage(HIBD). Menaquinone-4 (MK-4), a subtype of vitamin K2 prevalent in the brain, has been shown to enhance mitochondrial function and exhibit protective effects against ischemia-reperfusion injury. However, the impact and underlying molecular mechanism of MK-4 in HIE have not been fully elucidated. METHODS: In this study, we established the neonatal rats HIBD model in vivo and oxygen-glucose deprivation and reperfusion (OGD/R) of primary neurons in vitro to explore the neuroprotective effects of MK-4 on HI damage, and illuminate the potential mechanism. RESULTS: Our findings revealed that MK-4 ameliorated mitochondrial dysfunction, reduced oxidative stress, and prevented HI-induced neuronal apoptosis by activating the Sirt1-PGC-1α-TFAM signaling pathway through Sirt1 mediation. Importantly, these protective effects were partially reversed by EX-527, a Sirt1 inhibitor. CONCLUSION: Our study elucidated the potential therapeutic mechanism of MK-4 in neonatal HIE, suggesting its viability as an agent for enhancing recovery from HI-induced cerebral damage in newborns. Further exploration into MK-4 could lead to novel interventions for HIE therapy.

Sevoflurane postconditioning ameliorates cerebral hypoxia/reoxygenation injury in zebrafish involving the Akt/GSK-3ß pathway activation and the microtubule-associated protein 2 promotion.

Sevoflurane postconditioning has been shown to provide neuroprotection against cerebral hypoxia-ischemia injury, but the mechanisms remain elusive. Microtubule-associated protein 2 (MAP2) is implicated in early neuronal hypoxia-ischemia injury. This study aimed to investigate whether the neuroprotective effects of sevoflurane postconditioning are related to the Akt/GSK-3ß pathway and its downstream target MAP2 in zebrafish hypoxia/reoxygenation (H/R) model. Sevoflurane postconditioning or GSK-3ß inhibitor TDZD-8 were used to treat H/R zebrafish. The cerebral infarction, neuronal apoptosis, and mitochondrial changes were evaluated using TTC staining, TUNEL staining, and transmission electron microscopy, respectively. The distribution of MAP2 in the brain was determined by immunofluorescence imaging. The levels of Akt, p-Akt, GSK-3ß, p-GSK-3ß, and MAP2 proteins were evaluated by Western blotting. The neurobehavioral recovery of zebrafish was assessed based on optokinetic response behavior. Our results indicated that sevoflurane postconditioning and TDZD-8 significantly reduced the cerebral infarction area, suppressed cell apoptosis, and improved mitochondrial integrity in zebrafish subjected to H/R. Furthermore, sevoflurane postconditioning and TDZD-8 elevated the ratios of p-Akt/Akt and p-GSK-3ß/GSK-3ß. However, the neuroprotective effect of sevoflurane postconditioning was effectively abolished upon suppression of MAP2 expression. In conclusion, sevoflurane postconditioning ameliorated cerebral H/R injury and facilitated the restoration of neurobehavioral function through the activation of Akt/GSK-3ß pathway and promotion of MAP2 expression.

Neuronal autosis is Na+/K+-ATPase alpha 3-dependent and involved in hypoxic-ischemic neuronal death.

Macroautophagy (hereafter called autophagy) is an essential physiological process of degradation of organelles and long-lived proteins. The discovery of autosis, a Na+/K+-ATPase (ATP1)-dependent type of autophagic cell death with specific morphological and biochemical features, has strongly contributed to the acceptance of a pro-death role of autophagy. However, the occurrence and relevance of autosis in neurons has never been clearly investigated, whereas we previously provided evidence that autophagy mechanisms could be involved in neuronal death in different in vitro and in vivo rodent models of hypoxia-ischemia (HI) and that morphological features of autosis were observed in dying neurons following rat perinatal cerebral HI. In the present study, we demonstrated that neuronal autosis could occur in primary cortical neurons using two different stimulations enhancing autophagy flux and neuronal death: a neurotoxic concentration of Tat-BECN1 (an autophagy-inducing peptide) and a hypoxic/excitotoxic stimulus (mimicking neuronal death induced by cerebral HI). Both stimulations induce autophagic neuronal death (dependent on canonical autophagic genes and independent on apoptotic, necroptotic or ferroptotic pathways) with all morphological and biochemical (ATP1a-dependent) features of autosis. However, we demonstrated that autosis is not dependent on the ubiquitous subunit ATP1a1 in neurons, as in dividing cell types, but on the neuronal specific ATP1a3 subunit. We also provided evidence that, in different in vitro and in vivo models where autosis is induced, ATP1a3-BECN1 interaction is increased and prevented by cardiac glycosides treatment. Interestingly, an increase in ATP1a3-BECN1 interaction is also detected in dying neurons in the autoptic brains of human newborns with severe hypoxic-ischemic encephalopathy (HIE). Altogether, these results suggest that ATP1a3-BECN1-dependent autosis could play an important role in neuronal death in HI conditions, paving the way for the development of new neuroprotective strategies in hypoxic-ischemic conditions including in severe case of human HIE.

Protecting effects of 4-octyl itaconate on neonatal hypoxic-ischemic encephalopathy via Nrf2 pathway in astrocytes.

BACKGROUND: Neonatal hypoxic-ischemic encephalopathy (HIE) is one of the most common neurological problems occurring in the perinatal period. However, there still is not a promising approach to reduce long-term neurodevelopmental outcomes of HIE. Recently, itaconate has been found to exhibit anti-oxidative and anti-inflammatory effects. However, the therapeutic efficacy of itaconate in HIE remains inconclusive. Therefore, this study attempts to explore the pathophysiological mechanisms of oxidative stress and inflammatory responses in HIE as well as the potential therapeutic role of a derivative of itaconate, 4-octyl itaconate (4OI). METHODS: We used 7-day-old mice to induce hypoxic-ischemic (HI) model by right common carotid artery ligation followed by 1 h of hypoxia. Behavioral experiments including the Y-maze and novel object recognition test were performed on HI mice at P60 to evaluate long-term neurodevelopmental outcomes. We employed an approach combining non-targeted metabolomics with transcriptomics to screen alterations in metabolic profiles and gene expression in the hippocampal tissue of the mice at 8 h after hypoxia. Immunofluorescence staining and RT-PCR were used to evaluate the pathological changes in brain tissue cells and the expression of mRNA and proteins. 4OI was intraperitoneally injected into HI model mice to assess its anti-inflammatory and antioxidant effects. BV2 and C8D1A cells were cultured in vitro to study the effect of 4OI on the expression and nuclear translocation of Nrf2. We also used Nrf2-siRNA to further validate 4OI-induced Nrf2 pathway in astrocytes. RESULTS: We found that in the acute phase of HI, there was an accumulation of pyruvate and lactate in the hippocampal tissue, accompanied by oxidative stress and pro-inflammatory, as well as increased expression of antioxidative stress and anti-inflammatory genes. Treatment of 4OI could inhibit activation and proliferation of microglial cells and astrocytes, reduce neuronal death and relieve cognitive dysfunction in HI mice. Furthermore, 4OI enhanced nuclear factor erythroid-2-related factor (Nfe2l2; Nrf2) expression and nuclear translocation in astrocytes, reduced pro-inflammatory cytokine production, and increased antioxidant enzyme expression. CONCLUSION: Our study demonstrates that 4OI has a potential therapeutic effect on neuronal damage and cognitive deficits in HIE, potentially through the modulation of inflammation and oxidative stress pathways by Nrf2 in astrocytes.

The Temporal Relationship between Blood-Brain Barrier Integrity and Microglial Response following Neonatal Hypoxia Ischemia.

Blood-brain barrier (BBB) dysfunction and neuroinflammation are key mechanisms of brain injury. We performed a time-course study following neonatal hypoxia-ischemia (HI) to characterize these events. HI brain injury was induced in postnatal day 10 rats by single carotid artery ligation followed by hypoxia (8% oxygen, 90 min). At 6, 12, 24, and 72 h (h) post-HI, brains were collected to assess neuropathology and BBB dysfunction. A significant breakdown of the BBB was observed in the HI injury group compared to the sham group from 6 h in the cortex and hippocampus (p < 0.001), including a significant increase in albumin extravasation (p < 0.0033) and decrease in basal lamina integrity and tight-junction proteins. There was a decrease in resting microglia (p < 0.0001) transitioning to an intermediate state from as early as 6 h post-HI, with the intermediate microglia peaking at 12 h (p < 0.0001), which significantly correlated to the peak of microbleeds. Neonatal HI insult leads to significant brain injury over the first 72 h that is mediated by BBB disruption within 6 h and a transitioning state of the resident microglia. Key BBB events coincide with the appearance of the intermediate microglial state and this relationship warrants further research and may be a key target for therapeutic intervention.

TSG-6-Mediated Extracellular Matrix Modifications Regulate Hypoxic-Ischemic Brain Injury.

Proteoglycans containing link domains modify the extracellular matrix (ECM) to regulate cellular homeostasis and can also sensitize tissues/organs to injury and stress. Hypoxic-ischemic (H-I) injury disrupts cellular homeostasis by activating inflammation and attenuating regeneration and repair pathways. In the brain, the main component of the ECM is the glycosaminoglycan hyaluronic acid (HA), but whether HA modifications of the ECM regulate cellular homeostasis and response to H-I injury is not known. In this report, employing both male and female mice, we demonstrate that link-domain-containing proteoglycan, TNFα-stimulated gene-6 (TSG-6), is active in the brain from birth onward and differentially modifies ECM HA during discrete neurodevelopmental windows. ECM HA modification by TSG-6 enables it to serve as a developmental switch to regulate the activity of the Hippo pathway effector protein, yes-associated protein 1 (YAP1), in the maturing brain and in response to H-I injury. Mice that lack TSG-6 expression display dysregulated expression of YAP1 targets, excitatory amino acid transporter 1 (EAAT1; glutamate-aspartate transporter) and 2 (EAAT2; glutamate transporter-1). Dysregulation of YAP1 activation in TSG-6-/- mice coincides with age- and sex-dependent sensitization of the brain to H-I injury such that 1-week-old neonates display an anti-inflammatory response in contrast to an enhanced proinflammatory injury reaction in 3-month-old adult males but not females. Our findings thus support that a key regulator of age- and sex-dependent H-I injury response in the mouse brain is modulation of the Hippo-YAP1 pathway by TSG-6-dependent ECM modifications.

Cognitive outcome and its neural correlates after cardiorespiratory arrest in childhood.

Hypoxia-ischaemia (HI) can result in structural brain abnormalities, which in turn can lead to behavioural deficits in various cognitive and motor domains, in both adult and paediatric populations. Cardiorespiratory arrest (CA) is a major cause of hypoxia-ischaemia in adults, but it is relatively rare in infants and children. While the effects of adult CA on brain and cognition have been widely studied, to date, there are no studies examining the neurodevelopmental outcome of children who suffered CA early in life. Here, we studied the long-term outcome of 28 children who suffered early CA (i.e., before age 16). They were compared to a group of control participants (n = 28) matched for age, sex and socio-economic status. The patient group had impairments in the domains of memory, language and academic attainment (measured using standardised tests). Individual scores within the impaired range were most commonly found within the memory domain (79%), followed by academic attainment (50%), and language (36%). The patient group also had reduced whole brain grey matter volume, and reduced volume and fractional anisotropy of the white matter. In addition, lower performance on memory tests was correlated with bilaterally reduced volume of the hippocampi, thalami, and striatum, while lower attainment scores were correlated with bilateral reduction of fractional anisotropy in the superior cerebellar peduncle, the main output tract of the cerebellum. We conclude that patients who suffered early CA are at risk of developing specific cognitive deficits associated with structural brain abnormalities. RESEARCH HIGHLIGHTS: Our data shed light on the long-term outcome and associated neural mechanisms after paediatric hypoxia-ischaemia as a result of cardiorespiratory arrest. Patients had impaired scores on memory, language and academic attainment. Memory impairments were associated with smaller hippocampi, thalami, and striatum. Lower academic attainment correlated with reduced fractional anisotropy of the superior cerebellar peduncle.

Sudden death due to butane abuse - An overview.

Volatile Solvents Abuse (VSA) poses major health risks, especially for young people and those living in precarious socio-economic conditions. Such substances can in fact bring about psychoactive effects such as euphoria, and even lead to sudden death from cardiac arrhythmias, respiratory depression, myocardial infarction, laryngospasm, encephalopathy, and rhabdomyolysis. The present case report is centered around a 23-year-old man who died in prison due to inhalation of a cooker gas mixture (n-butane, propane, and isobutane) inside a plastic bag. External examination and autopsy showed non-specific signs of asphyxia associated with edema and brain swelling. Histological signs of early myocardial damage and hypoxic-ischemic injury (HII) were highlighted in the brain and cerebellum, as well as activated macrophages and anthracotic-like material in the lungs. Toxicological investigations revealed the presence of propane, isobutane and n-butane in liquids and biological samples. Besides the cardiotoxic effect, there was an asphyctic component due to the plastic bag that may have facilitated death. The assessment of cerebral HII and cardiopulmonary damage in acute cases is very important to prove death by butane inhalation. In the forensic field, it may be useful to shed more light on intoxications, deaths, and butane encephalopathies, as the latter can be mistaken for a hypoxic-ischemic encephalopathy.

Neonatal Hypoxia-Ischemia alters Brain-Derived Contactin-2-Positive Extracellular Vesicles in the Mouse Plasma.

Neonatal encephalopathy (NE) impairs white matter development and results in long-term neurodevelopmental deficits. Leveraging prior findings of altered neuronal proteins carried by brain-derived extracellular vesicles (EVs) that are marked by a neural-specific cell surface glycoprotein Contactin-2 (CNTN2) in NE infants, the present study aimed to determine the correlation between brain and circulating CNTN2+-EVs and whether NE alters circulating CNTN2+-EV levels in mice. Brain tissue and plasma were collected from postnatal day (P)7, 10, 11, 15 mice to determine the baseline CNTN2 correlation between these two compartments (n = 4-7/time point/sex). NE was induced in P10 pups. Brain and plasma samples were collected at 1, 3, 6, 24, and 120 h (n = 4-8/time point/sex). CNTN2 from brain tissue and plasma EVs were quantified using ELISA. ANOVA and linear regression analyses were used to evaluate changes and correlations between brain and plasma CNTN2+-EVs. In baseline experiments, CNTN2 in brain tissue and plasma EVs peaked at P10 with no sex-difference. Brain and plasma CNTN2+-EV showed a positive correlation across early postnatal ages. NE pups showed an elevated CNTN2 in brain tissue and EVs at 1 h and only in brain tissue at 24 h. NE also abolished the positive plasma-brain correlation. The findings establish a link for central CNTN2 and its release into circulation during early postnatal life. The immediate elevation and release of CNTN2 following NE highlight a potential molecular response shortly after a brain injurious event. Our findings further support the utility of circulating brain-derived EVs as a possible bioindicator of NE.

Bone Marrow Nucleated Cells and Bone Marrow-Derived CD271+ Mesenchymal Stem Cell in Treatment of Encephalopathy and Drug-Resistant Epilepsy.

The broad spectrum of brain injuries in preterm newborns and the plasticity of the central nervous system prompts us to seek solutions for neurodegeneration to prevent the consequences of prematurity and perinatal problems. The study aimed to evaluate the safety and efficacy of the implantation of autologous bone marrow nucleated cells and bone marrow mesenchymal stem cells in different schemes in patients with hypoxic-ischemic encephalopathy and immunological encephalopathy. Fourteen patients received single implantation of bone marrow nucleated cells administered intrathecally and intravenously, followed by multiple rounds of bone marrow mesenchymal stem cells implanted intrathecally, and five patients were treated only with repeated rounds of bone marrow mesenchymal stem cells. Seizure outcomes improved in most cases, including fewer seizures and status epilepticus and reduced doses of antiepileptic drugs compared to the period before treatment. The neuropsychological improvement was more frequent in patients with hypoxic-ischemic encephalopathy than in the immunological encephalopathy group. Changes in emotional functioning occurred with similar frequency in both groups of patients. In the hypoxic-ischemic encephalopathy group, motor improvement was observed in all patients and the majority in the immunological encephalopathy group. The treatment had manageable toxicity, mainly mild to moderate early-onset adverse events. The treatment was generally safe in the 4-year follow-up period, and the effects of the therapy were maintained after its termination.

URB447 Is Neuroprotective in Both Male and Female Rats after Neonatal Hypoxia-Ischemia and Enhances Neurogenesis in Females.

The need for new and effective treatments for neonates suffering from hypoxia-ischemia is urgent, as the only implemented therapy in clinics is therapeutic hypothermia, only effective in 50% of cases. Cannabinoids may modulate neuronal development and brain plasticity, but further investigation is needed to better describe their implication as a neurorestorative therapy after neonatal HI. The cannabinoid URB447, a CB1 antagonist/CB2 agonist, has previously been shown to reduce brain injury after HI, but it is not clear whether sex may affect its neuroprotective and/or neurorestorative effect. Here, URB447 strongly reduced brain infarct, improved neuropathological score, and augmented proliferative capacity and neurogenic response in the damaged hemisphere. When analyzing these effects by sex, URB447 ameliorated brain damage in both males and females, and enhanced cell proliferation and the number of neuroblasts only in females, thus suggesting a neuroprotective effect in males and a double neuroprotective/neurorestorative effect in females.

Acute seizure activity in neonatal inflammation-sensitized hypoxia-ischemia in mice.

OBJECTIVE: To examine acute seizure activity and neuronal damage in a neonatal mouse model of inflammation-sensitized hypoxic-ischemic (IS-HI) brain injury utilizing continuous electroencephalography (cEEG) and neurohistology. METHODS: Neonatal mice were exposed to either IS-HI with Escherichia coli lipopolysaccharide (LPS) or HI alone on postnatal (p) day 10 using unilateral carotid artery ligation followed by global hypoxia (n = 10 [5 female, 5 male] for IS-HI, n = 12 [5 female, 7 male] for HI alone). Video cEEG was recorded for the duration of the experiment and analyzed for acute seizure activity and behavior. Brain tissue was stained and scored based on the degree of neuronal injury in the hippocampus, cortex, and thalamus. RESULTS: There was no significant difference in acute seizure activity among mice exposed to IS-HI compared to HI with regards to seizure duration (mean = 63 ± 6 seconds for HI vs mean 62 ± 5 seconds for IS-HI, p = 0.57) nor EEG background activity. Mice exposed to IS-HI had significantly more severe neural tissue damage at p30 as measured by neuropathologic scores (mean = 8 ± 1 vs 23 ± 3, p < 0.0001). INTERPRETATION: In a neonatal mouse model of IS-HI, there was no significant difference in acute seizure activity among mice exposed to IS-HI compared to HI. Mice exposed to IS-HI did show more severe neuropathologic damage at a later age, which may indicate the presence of chronic inflammatory mechanisms of brain injury distinct from acute seizure activity.

NKG2D knockdown improves hypoxic-ischemic brain damage by inhibiting neuroinflammation in neonatal mice.

Hypoxic-ischemic brain damage (HIBD) is a leading cause of neonatal death and neurological dysfunction. Neuroinflammation is identified as one of the crucial pathological mechanisms after HIBD, and natural killer group 2 member D (NKG2D) is reported to be implicated in the pathogenesis of immunoinflammatory diseases. However, the role of NKG2D in neonatal HIBD is seldomly investigated. In this study, a neonatal mice model of HIBD was induced, and the role of the NKG2D in neuroinflammation and brain injury was explored by intracerebroventricular injection of lentivirus to knockdown NKG2D in neonatal mice with HIBD. The results showed that a significant increase in NKG2D protein level in the brain of neonatal mice with HIBD. The NKG2D knockdown in the brain significantly alleviated cerebral infarction, neurobehavioral deficits, and neuronal loss in neuronal HIBD. Moreover, the neuroprotective effect of NKG2D knockdown was associated with inhibition of the activation of microglia and astrocytes, expression of NKG2D ligands (NKG2DLs) and DAP10, and the nuclear translocation of NF-κB p65. Our findings reveal NKG2D knockdown may exert anti-inflammatory and neuroprotective effects in the neonatal mice with HIBD through downregulation of NKG2D/NKG2DLs/DAP10/NF-κB pathway. These results suggest that NKG2D may be a potential target for the treatment of neonatal HIBD.

Hypothermia Treatment after Hypoxia-Ischemia in Glutathione Peroxidase-1 Overexpressing Mice.

The developing brain is uniquely susceptible to oxidative stress, and endogenous antioxidant mechanisms are not sufficient to prevent injury from a hypoxic-ischemic challenge. Glutathione peroxidase (GPX1) activity reduces hypoxic-ischemic injury. Therapeutic hypothermia (HT) also reduces hypoxic-ischemic injury, in the rodent and the human brain, but the benefit is limited. Here, we combined GPX1 overexpression with HT in a P9 mouse model of hypoxia-ischemia (HI) to test the effectiveness of both treatments together. Histological analysis showed that wild-type (WT) mice with HT were less injured than WT with normothermia. In the GPX1-tg mice, however, despite a lower median score in the HT-treated mice, there was no significant difference between HT and normothermia. GPX1 protein expression was higher in the cortex of all transgenic groups at 30 min and 24 h, as well as in WT 30 min after HI, with and without HT. GPX1 was higher in the hippocampus of all transgenic groups and WT with HI and normothermia, at 24 h, but not at 30 min. Spectrin 150 was higher in all groups with HI, while spectrin 120 was higher in HI groups only at 24 h. There was reduced ERK1/2 activation in both WT and GPX1-tg HI at 30 min. Thus, with a relatively moderate insult, we see a benefit with cooling in the WT but not the GPX1-tg mouse brain. The fact that we see no benefit with increased GPx1 here in the P9 model (unlike in the P7 model) may indicate that oxidative stress in these older mice is elevated to an extent that increased GPx1 is insufficient for reducing injury. The lack of benefit of overexpressing GPX1 in conjunction with HT after HI indicates that pathways triggered by GPX1 overexpression may interfere with the neuroprotective mechanisms provided by HT.

Quantification of Diffusion Magnetic Resonance Imaging for Prognostic Prediction of Neonatal Hypoxic-Ischemic Encephalopathy.

Neonatal hypoxic-ischemic encephalopathy (HIE) is the leading cause of acquired neonatal brain injury with the risk of developing serious neurological sequelae and death. An accurate and robust prediction of short- and long-term outcomes may provide clinicians and families with fundamental evidence for their decision-making, the design of treatment strategies, and the discussion of developmental intervention plans after discharge. Diffusion tensor imaging (DTI) is one of the most powerful neuroimaging tools with which to predict the prognosis of neonatal HIE by providing microscopic features that cannot be assessed by conventional magnetic resonance imaging (MRI). DTI provides various scalar measures that represent the properties of the tissue, such as fractional anisotropy (FA) and mean diffusivity (MD). Since the characteristics of the diffusion of water molecules represented by these measures are affected by the microscopic cellular and extracellular environment, such as the orientation of structural components and cell density, they are often used to study the normal developmental trajectory of the brain and as indicators of various tissue damage, including HIE-related pathologies, such as cytotoxic edema, vascular edema, inflammation, cell death, and Wallerian degeneration. Previous studies have demonstrated widespread alteration in DTI measurements in severe cases of HIE and more localized changes in neonates with mild-to-moderate HIE. In an attempt to establish cutoff values to predict the occurrence of neurological sequelae, MD and FA measurements in the corpus callosum, thalamus, basal ganglia, corticospinal tract, and frontal white matter have proven to have an excellent ability to predict severe neurological outcomes. In addition, a recent study has suggested that a data-driven, unbiased approach using machine learning techniques on features obtained from whole-brain image quantification may accurately predict the prognosis of HIE, including for mild-to-moderate cases. Further efforts are needed to overcome current challenges, such as MRI infrastructure, diffusion modeling methods, and data harmonization for clinical application. In addition, external validation of predictive models is essential for clinical application of DTI to prognostication.

Long-term effect of indomethacin on a rat model of neonatal hypoxia ischemic encephalopathy through behavioral tests.

BACKGROUND: Many medical experts prescribe indomethacin because of its anti-inflammatory, analgesic, tocolytic, and duct closure effects. This article presents an evaluation of the enduring impact of indomethacin on neonatal rats with hypoxic-ischemic (HI) insults, employing behavioral tests as a method of assessment. METHODS: The experiment was conducted on male Wistar-Albino rats weighing 10 to 15 g, aged between seven and 10 days. The rats were divided into three groups using a random allocation method as follows: hypoxic ischemic encephalopathy (HIE) group, HIE treated with indomethacin group (INDO), and Sham group. A left common carotid artery ligation and hypoxia model was applied in both the HIE and INDO groups. The INDO group was treated with 4 mg/kg intraperitoneal indomethacin every 24 h for 3 days, while the Sham and HIE groups were given dimethylsulfoxide (DMSO). After 72 h, five rats from each group were sacrificed and brain tissue samples were stained with 2,3,5-Triphenyltetrazolium chloride (TCC) for infarct-volume measurement. Seven rats from each group were taken to the behavioral laboratory in the sixth postnatal week (PND42) and six from each group were sacrificed for the Evans blue (EB) experiment for blood-brain barrier (BBB) integrity evaluation. The open field (OF) test and Morris water maze (MWM) tests were performed. After behavioral tests, brain tissue were obtained and stained with TCC to assess the infarct volume. RESULTS: The significant increase in the time spent in the central area and the frequency of crossing to the center in the INDO group compared with the HIE group indicated that indomethacin decreased anxiety-like behavior (p < 0.001, p < 0.05). However, the MWM test revealed that indomethacin did not positively affect learning and memory performance (p > 0.05). Additionally, indomethacin significantly reduced infarct volume and neuropathological grading in adolescence (p < 0.05), although not statistically significant in the early period. Moreover, the EB experiment demonstrated that indomethacin effectively increased BBB integrity (p < 0.05). CONCLUSIONS: In this study, we have shown for the first time that indomethacin treatment can reduce levels of anxiety-like behavior and enhance levels of exploratory behavior in a neonatal rat model with HIE. It is necessary to determine whether nonsteroidal anti-inflammatory agents, such as indomethacin, should be used for adjuvant therapy in newborns with HIE.

Long-term recruitment of peripheral immune cells to brain scars after a neonatal insult.

Although brain scars in adults have been extensively studied, there is less data available regarding scar formation during the neonatal period, and the involvement of peripheral immune cells in this process remains unexplored in neonates. Using a murine model of neonatal hypoxic-ischemic encephalopathy (HIE) and confocal microscopy, we characterized the scarring process and examined the recruitment of peripheral immune cells to cortical and hippocampal scars for up to 1 year post-insult. Regional differences in scar formation were observed, including the presence of reticular fibrotic networks in the cortex and perivascular fibrosis in the hippocampus. We identified chemokines with chronically elevated levels in both regions and demonstrated, through a parabiosis-based strategy, the recruitment of lymphocytes, neutrophils, and monocyte-derived macrophages to the scars several weeks after the neonatal insult. After 1 year, however, neutrophils and lymphocytes were absent from the scars. Our data indicate that peripheral immune cells are transient components of HIE-induced brain scars, opening up new possibilities for late therapeutic interventions.

A New Approach for Exploring Reperfusion Brain Damage in Hypoxic Ischemic Encephalopathy.

Reperfusion is an essential pathological stage in hypoxic ischemic encephalopathy (HIE). Although the Rice-Vannucci model is widely used in HIE research, it remains difficult to replicate HIE-related reperfusion brain injury. The purpose of this study is to establish a rat model of hypoxia ischemia reperfusion brain damage (HIRBD) using a common carotid artery (CCA) muscle bridge in order to investigate the mechanisms of cerebral resistance to hypoxic-ischemic and reperfusion brain damage. Random assignment of Sprague-Dawley (SD) rats to the Sham, HIRBD, and Rice-Vannucci groups. Changes in body weight, mortality rate, spontaneous alternation behavior test (SAB test), and dynamic changes in cerebral blood flow (CBF) were detected. The damaged cerebral cortices were extracted for morphological comparison, transcriptomic analysis, and quantitative real-time PCR. Harvesting the hippocampus for transmission electron microscopy (TEM) detection. As a result, CCA muscle bridge could effectively block CBF, which recovered after the muscle bridge detachment. Pathological comparison, the SAB test, and TEM analysis revealed that brain damage in Rice-Vannucci was more severe than HIRBD. Gpx1, S100a6, Cldn5, Esr1, and Gfap were highly expressed in both HIRBD and Rice-Vannucci. In conclusion, the CCA muscle bridge-established HIRBD model could be used as an innovative and dependable model to simulate pathological process of HIRBD.