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Neonatal hypoxic‐ischemic encephalopathy (HIE) causes lifelong neurologic disability. Despite the use of therapeutic hypothermia, memory deficits and executive functions remain severely affected. Cholinergic neurotransmission from the basal forebrain to neocortex and hippocampus is central to higher cortical functions. We examined the basal forebrain by light microscopy and reported loss of choline acetyltransferase‐positive (ChAT)+ neurons, at postnatal day (P) 40, in the ipsilateral medial septal nucleus (MSN) after neonatal hypoxia‐ischemia (HI) in mice. There was no loss of ChAT+ neurons in the ipsilateral nucleus basalis of Meynert (nbM) and striatum. Ipsilateral striatal and nbM ChAT+ neurons were abnormal with altered immunoreactivity for ChAT, shrunken and crenated somas, and dysmorphic appearing dendrites. Using confocal images with 3D reconstruction, nbM ChAT+ dendrites in HI mice were shorter than sham (p =.0001). Loss of ChAT+ neurons in the MSN directly correlated with loss of ipsilateral hippocampal area. In the nbM and striatum, percentage of abnormal ChAT+ neurons correlated with loss of ipsilateral cerebral cortical and striatal area, respectively. Acetylcholinesterase (AChE) activity increased in adjacent ipsilateral cerebral cortex and hippocampus and the increase was linearly related to loss of cortical and hippocampal area. Numbers and size of cathepsin D+ lysosomes increased in large neurons in the ipsilateral nbM. After neonatal HI, abnormalities were found throughout the major cholinergic systems in relationship to amount of forebrain area loss. There was also an upregulation of cathepsin D+ particles within the nbM. Cholinergic neuropathology may underlie the permanent dysfunction in learning, memory, and executive function after neonatal brain injury. [ABSTRACT FROM AUTHOR]

Background: Disruption of brain oxygen delivery and consumption after hypoxic–ischemic injury contributes to neonatal mortality and neurological impairment. Measuring cerebral hemodynamic parameters, including cerebral blood flow (CBF), oxygen extraction fraction (OEF), and cerebral metabolic rate of oxygen (CMRO2), is clinically important. Purpose: Phase‐contrast (PC), velocity‐selective arterial spin labeling (VSASL), and T2‐relaxation‐under‐phase‐contrast (TRUPC) are magnetic resonance imaging (MRI) techniques that have shown promising results in assessing cerebral hemodynamics in humans. We aimed to test their feasibility in quantifying CBF, OEF, and CMRO2 in piglets. Study Type: Prospective. Animal Model: Ten neonatal piglets subacutely recovered from global hypoxia‐ischemia (N = 2), excitotoxic brain injury (N = 6), or sham procedure (N = 2). Field Strength/Sequence: VSASL, TRUPC, and PC MRI acquired at 3.0 T. Assessment: Regional CBF was measured by VSASL. Global CBF was quantified by both PC and VSASL. TRUPC assessed OEF at the superior sagittal sinus (SSS) and internal cerebral veins (ICVs). CMRO2 was calculated from global CBF and SSS‐derived OEF. End‐tidal carbon dioxide (EtCO2) levels of the piglets were also measured. Brain damage was assessed in tissue sections postmortem by counting damaged neurons. Statistical Tests: Spearman correlations were performed to evaluate associations among CBF (by PC or VSASL), OEF, CMRO2, EtCO2, and the pathological neuron counts. Paired t‐test was used to compare OEF at SSS with OEF at ICV. Results: Global CBF was 32.1 ± 14.9 mL/100 g/minute and 30.9 ± 8.3 mL/100 g/minute for PC and VSASL, respectively, showing a significant correlation (r = 0.82, P < 0.05). OEF was 54.9 ± 8.8% at SSS and 46.1 ± 5.6% at ICV, showing a significant difference (P < 0.05). Global CMRO2 was 79.1 ± 26.2 μmol/100 g/minute and 77.2 ± 12.2 μmol/100 g/minute using PC and VSASL‐derived CBF, respectively. EtCO2 correlated positively with PC‐based CBF (r = 0.81, P < 0.05) but negatively with OEF at SSS (r = −0.84, P < 0.05). Relative CBF of subcortical brain regions and OEF at ICV did not significantly correlate, respectively, with the ratios of degenerating‐to‐total neurons (P = 0.30, P = 0.10). Data Conclusion: Non‐contrast MRI can quantify cerebral hemodynamic parameters in normal and brain‐injured neonatal piglets. Level of Evidence: 1 Technical Efficacy Stage: 2 [ABSTRACT FROM AUTHOR]

The specific cytopathology that causes abnormal fractional anisotropy (FA) and mean diffusivity (MD) from diffusion tensor imaging (DTI) after neonatal hypoxia‐ischemia (HI) is not completely understood. The panoply of cell types in the brain might contribute differentially to changes in DTI metrics. Because glia are the predominant cell type in brain, we hypothesized that changes in FA and MD would signify perturbations in glial microstructure. Using a 3‐Tesla clinical scanner, we conducted in vivo DTI MRI in nine neonatal piglets at 20–96 h after excitotoxic brain injury from striatal quinolinic acid injection or global HI. FA and MD from putamen, caudate, and internal capsule in toto were correlated with astrocyte swelling, neuronal excitotoxicity, and white matter injury. Low FA correlated with more swollen astrocytes immunophenotyped by aquaporin‐4 (AQP4), glial fibrillary acidic protein (GFAP), and glutamate transporter‐1 (GLT‐1). Low FA was also related to the loss of neurons with perineuronal GLT‐1+ astrocyte decorations, large myelin swellings, lower myelin density, and oligodendrocyte cell death identified by 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase, bridging integrator‐1, and nuclear morphology. MD correlated with degenerating oligodendrocytes and depletion of normal GFAP+ astrocytes but not with astrocyte or myelin swelling. We conclude that FA is associated with cytotoxic edema in astrocytes and oligodendrocyte processes as well as myelin injury at the cellular level. MD can detect glial cell death and loss, but it may not discern subtle pathology in swollen astrocytes, oligodendrocytes, or myelin. This study provides a cytopathologic basis for interpreting DTI in the neonatal brain after HI. [ABSTRACT FROM AUTHOR]

Background: Diffusion MRI is routinely used to evaluate brain injury in neonatal encephalopathy. Although abnormal mean diffusivity (MD) is often attributed to cytotoxic edema, the specific contribution from neuronal pathology is unclear.Purpose: To determine whether MD from high-resolution diffusion tensor imaging (DTI) can detect variable degrees of neuronal degeneration and pathology in piglets with brain injury induced by excitotoxicity or global hypoxia-ischemia (HI) with or without overt infarction.Study Type: Prospective.Animal Model: Excitotoxic brain injury was induced in six neonatal piglets by intrastriatal stereotaxic injection of the glutamate receptor agonist quinolinic acid (QA). Three piglets underwent global HI or a sham procedure. Piglets recovered for 20-96 hours before undergoing MRI (n = 9).Field Strength/sequence: 3.0T MRI with DTI, T1 - and T2 -weighted imaging.Assessment: MD, fractional anisotropy (FA), and qualitative T2 injury were assessed in the putamen and caudate. The cell bodies of normal neurons, degenerating neurons (excitotoxic necrosis, ischemic necrosis, or necrosis-apoptosis cell death continuum), and injured neurons with equivocal degeneration were counted by histopathology.Statistical Tests: Spearman correlations were used to compare MD and FA to normal, degenerating, and injured neurons. T2 injury and neuron counts were evaluated by descriptive analysis.Results: The QA insult generated titratable levels of neuronal pathology. In QA, HI, and sham piglets, lower MD correlated with higher ratios of degenerating-to-total neurons (P < 0.05), lower ratios of normal-to-total neurons (P < 0.05), and greater numbers of degenerating neurons (P < 0.05). MD did not correlate with abnormal neurons exhibiting nascent injury (P > 0.99). Neuron counts were not related to FA (P > 0.30) or to qualitative injury from T2 -weighted MRI.Data Conclusion: MD is more accurate than FA for detecting neuronal degeneration and loss during acute recovery from neonatal excitotoxic and HI brain injury. MD does not reliably detect nonfulminant, nascent, and potentially reversible neuronal injury.Evidence Level: 1 TECHNICAL EFFICACY: Stage 2 J. Magn. Reson. Imaging 2020;52:1216-1226. [ABSTRACT FROM AUTHOR]

Abstract: Delayed hippocampal injury and memory impairments follow neonatal hypoxia‐ischemia (HI) despite the use of therapeutic hypothermia (TH). Death of hippocampal pyramidal cells occurs acutely after HI, but characterization of delayed cell death and injury of interneurons (INs) is unknown. We hypothesize that injury of INs after HI is: (i) asynchronous to that of pyramidal cells, (ii) independent of injury severity, and (iii) unresponsive to TH. HI was induced in C57BL6 mice at p10 with unilateral right carotid ligation and 45 min of hypoxia (FiO2 = 0.08). Mice were randomized to normothermia (36 °C, NT) or TH (31 °C) for 4 hr after HI and anesthesia‐exposed shams were use as controls. Brains were studied at 24 hr (p11) or 8 days (p18) after HI. Vglut1, GAD65/67, PSD95, parvalbumin (PV) and calbindin‐1 (Calb1) were measured. Cell death was assessed using cresyl violet staining and TUNEL assay. Hippocampal atrophy and astroglyosis at p18 were used to assess injury severity and to correlate with number of PV + INs. VGlut1 level decreased by 30% at 24 hr after HI, while GAD65/67 level decreased by ∼50% in forebrain 8 days after HI, a decrease localized in CA1 and CA3. PSD95 levels decreased in forebrain by 65% at 24 hr after HI and remained low 8 days after HI. PV + INs increased in numbers (per mm2) and branching between p11 and p18 in sham mice but not in NT and TH mice, resulting in 21–52% fewer PV + INs in injured mice at p18. Calb1 protein and mRNA were also reduced in HI injured mice at p18. At p18, somatodendritic attrition of INs was evident in all injured mice without evidence of cell death. Neither hippocampal atrophy nor astroglyosis correlated with the number of PV + INs at p18. Thus, HI exposure has long lasting effects in the hippocampus impairing the development of the GABAergic system with only partial protection by TH independent of the degree of hippocampal injury. © 2018 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Purpose We investigated whether oscillating gradient diffusion MRI (dMRI) can provide information on brain microstructural changes after formaldehyde fixation and after hypoxic-ischemic (HI) injury beyond that provided by conventional dMRI. Methods Pulsed gradient spin echo (PGSE) and oscillating gradient spin echo (OGSE) dMRI of the adult mouse brain was performed in vivo (50-200 Hz, b = 600 mm2/s), and a similar protocol was applied to neonatal mouse brains at 24 h after unilateral hypoxia-ischemia. Animals were perfusion fixed with 4% paraformaldehyde for ex vivo dMRI and histology. Results Apparent diffusion coefficients (ADCs) measured in the live adult mouse brain presented tissue-dependent frequency-dependence. In vivo OGSE-ADC maps at high oscillating frequencies (>100 Hz) showed clear contrast between the molecular layer and granule cell layer in the adult mouse cerebellum. Formaldehyde fixation significantly altered the temporal diffusion spectra in several brain regions. In neonatal mouse brains with HI injury, in vivo ADC measurements from edema regions showed diminished edema contrasts at 200 Hz compared with the PGSE results. Histology showed severe tissue swelling and necrosis in the edema regions. Conclusion The results demonstrate the unique ability of OGSE-dMRI in delineating tissue microstructures at different spatial scales. Magn Reson Med 72:1366-1374, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Purpose To investigate if frequency-dependent contrasts using oscillating gradient diffusion MRI (dMRI) can detect hypoxia-ischemia (HI) -induced neurodegeneration in the neonatal mouse hippocampus. Methods Pulsed- and oscillating-gradient dMR images (at 50, 100, and 150 H z) were acquired from postmortem fixed brains of mice exposed to neonatal HI using the Rice-Vanucci model. MRI data were acquired at 1, 4, and 8 days following HI, and compared with histological data from the same mice for in situ histological validation of the MRI findings. Results The rate of change of apparent diffusion coefficient with gradient frequency (ΔfADC) revealed unique layer-specific contrasts in the neonatal mouse hippocampus. ΔfADC measurements were found to show a significant decrease in response to neonatal HI injury, in the pyramidal (Py) and granule (GrDG) cell layers compared with contralateral regions. The areas of reduced intensity in the ΔfADC maps corresponded to regional neurodegeneration seen with H&E and Fluoro-Jade C stainings, indicating that alterations in ΔfADC contrasts are sensitive to early microstructural changes due to HI-induced neurodegeneration in the studied regions. Conclusion The findings show that the frequency-dependence of ADC measurements with oscillating-gradient dMRI can provide a sensitive contrast to detect HI-induced neurodegeneration in neuronal layers of the neonatal mouse hippocampus. Magn Reson Med 72:829-840, 2014. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2012) 121, 168-179. Abstract 20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450 metabolite of arachidonic acid that that contributes to infarct size following focal cerebral ischemia. However, little is known about the role of 20-HETE in global cerebral ischemia or neonatal hypoxia-ischemia (H-I). The present study examined the effects of blockade of the synthesis of 20-HETE with N-hydroxy- N′-(4- n-butyl-2-methylphenyl) formamidine (HET0016) in neonatal piglets after H-I to determine if it protects highly vulnerable striatal neurons. Administration of HET0016 after H-I improved early neurological recovery and protected neurons in putamen after 4 days of recovery. HET0016 had no significant effect on cerebral blood flow. cytochrome P450 4A immunoreactivity was detected in putamen neurons, and direct infusion of 20-HETE in the putamen increased phosphorylation of Na+,K+-ATPase and NMDA receptor NR1 subunit selectively at protein kinase C-sensitive sites but not at protein kinase A-sensitive sites. HET0016 selectively inhibited the H-I induced phosphorylation at these same sites at 3 h of recovery and improved Na+,K+-ATPase activity. At 3 h, HET0016 also suppressed H-I induced extracellular signal-regulated kinase 1/2 activation and protein markers of nitrosative and oxidative stress. Thus, 20-HETE can exert direct effects on key proteins involved in neuronal excitotoxicity in vivo and contributes to neurodegeneration after global cerebral ischemia in immature brain. [ABSTRACT FROM AUTHOR]

Despite the introduction of therapeutic hypothermia, neonatal hypoxic ischemic (HI) brain injury remains a common cause of developmental disability. Development of rational adjuvant therapies to hypothermia requires understanding of the pathways of cell death and survival modulated by HI. The conceptualization of the apoptosis-necrosis "continuum" in neonatal brain injury predicts mechanistic interactions between cell death and hydrid forms of cell death such as programmed or regulated necrosis. Many of the components of the signaling pathway regulating programmed necrosis have been studied previously in models of neonatal HI. In some of these investigations, they participate as part of the apoptotic pathways demonstrating clear overlap of programmed death pathways. Receptor interacting protein (RIP)-1 is at the crossroads between types of cellular death and survival and RIP-1 kinase activity triggers formation of the necrosome (in complex with RIP-3) leading to programmed necrosis. Neuroprotection afforded by the blockade of RIP-1 kinase following neonatal HI suggests a role for programmed necrosis in the HI injury to the developing brain. Here, we briefly review the state of the knowledge about the mechanisms behind programmed necrosis in neonatal brain injury recognizing that a significant proportion of these data derive from experiments in cultured cell and some from in vivo adult animal models. There are still more questions than answers, yet the fascinating new perspectives provided by the understanding of programmed necrosis in the developing brain may lay the foundation for new therapies for neonatal HI. [ABSTRACT FROM AUTHOR]

The article offers information on a medical research carried out on neuronal cell death in neonatal hypoxia-ischemia. It is believed that perinatal hypoxic-ischemic encephalopathy (HIE) is a significant cause of mortality and morbidity in infants and young children. So, accurate identification of the various cell death chreodes and their mechanisms unfolding within the immature brain matrix is needed for developing meaningful therapies for neonatal and pediatric HIE.

Objective Clinical magnetic resonance studies show delayed and ongoing neurodegeneration after neonatal hypoxia-ischemia (HI), but the mechanisms and timing of this neurodegeneration remain unclear. We used ex vivo diffusion tensor imaging (DTI) and brain neuropathology to determine whether selective injury to white matter tracts occurs after neonatal HI in mice resulting in neural system-associated attrition in remote regions and at delayed times. Methods The Rice-Vannucci model (unilateral carotid ligation + 45 minutes of hypoxia FiO2 = 0.08) was used to cause brain injury in postnatal day 7 (p7) C57BL6 mice, and ex vivo DTI and correlative neuropathology were performed at p8, p11, p15, p21, p28, and p42. Results DTI provides excellent contrast visualization of unmyelinated white matter in the immature mouse brain. Severe ipsilateral injury to the hippocampus is seen with both histopathology and diffusion-weighted magnetic resonance imaging 24 hours after injury. Injury to axons is evident 24 hours after HI in the hippocampal alveus. By p11 and continuing until p28, the ipsilateral fimbria fornix degenerates. At p15, there is injury and loss of axons entering the ipsilateral septal nucleus followed by ipsilateral septal atrophy. Volume loss in the hippocampus is rapid and severe, but is subacute and significantly slower in the ipsilateral septum. Neonatal HI also interrupts the normal developmental increase in fractional anisotropy in the ipsilateral fimbria but not in the contralateral fimbria from p8 to p42. Interpretation In neonatal brain, there is progressive systems-preferential injury after HI. DTI allows unparalleled visualization of this neural network-associated attrition so that it can be followed longitudinally in developing brain. Ann Neurol 2008;64:535-546 [ABSTRACT FROM AUTHOR]

Abstract: The basal ganglia of newborns are extremely vulnerable to hypoxic ischemia (HI). Striatal neurons undergo prominent necrosis after HI. The mechanisms for this degeneration are not well understood. Postasphyxic hypothermia ameliorates the striatal necrosis, but the mechanisms of hypothermia-induced neuroprotection are not known. We used a newborn piglet model of hypoxic-asphyxic cardiac arrest to test the hypotheses that N-methyl-d-aspartate receptor activation and free radical damage coexist, prior to neurodegeneration, early after resuscitation, and that these changes are attenuated with hypothermia. Piglets were subjected to 30min of hypoxia followed by 7min of airway occlusion, causing asphyxic cardiac arrest, and then were resuscitated and survived normothermically for 5min, 3h, or 6h, or hypothermically for 3h. By 6h of normothermic recovery, 50% of neurons in putamen showed ischemic cytopathology. Striatal tissue was fractionated into membrane or soluble proteins and was assayed by immunoblotting for carbonyl modification, phosphorylation of the N-methyl-d-aspartate receptor subunit NR1, and neuronal nitric oxide synthase. Significant accumulation of soluble protein carbonyls was present at 3h (196% of control) and 6h (142% of control). Phosphorylation of serine-897 of NR1 was increased significantly at 5min (161% of control) and 3h (226% of control) after HI. Phosphorylation of serine-890 of NR1 was also increased after HI. Membrane-associated neuronal nitric oxide synthase was increased by 35% at 5min. Hypothermia attenuated the oxidative damage and the NR1 phosphorylation in striatum. We conclude that neuronal death signaling in newborn striatum after HI is engaged rapidly through N-methyl-d-aspartate receptor activation, neuronal nitric oxide synthase recruitment, and oxidative stress. Postasphyxic, mild whole body hypothermia provides neuroprotection by suppressing N-methyl-d-aspartate receptor phosphorylation and protein oxidation. [Copyright &y& Elsevier]

Abstract The mechanisms of injury- and disease-associated apoptosis of neurons within the CNS are not understood. We used a model of cortical injury in rat and mouse to induce retrograde neuronal apoptosis in thalamus. In this animal model, unilateral ablation of the occipital cortex induces apoptosis of corticopetal projection neurons in the dorsal lateral geniculate nucleus (LGN), by 7 days post-lesion, that is p53 modulated and Bax dependent. We tested the hypothesis that this degenerative process is initiated by oxidative stress and early formation of DNA damage and is accompanied by changes in the levels of pro-apoptotic mediators of cell death. Immunoblotting revealed that the protein profiles of Bax, Bak and Bad were different during the progression of neuronal apoptosis in the LGN. Bax underwent a subcellular redistribution by 1 day post-lesion, while Bak increased later. Bad showed an early sustained increase. Cleaved caspase-3 was elevated maximally at 5 and 6 days. Active caspase-3 underwent a subcellular translocation to the nucleus. A dramatic phosphorylation of p53 was detected at 4 days post-lesion. DNA damage was assessed immunocytochemically as hydroxyl radical adducts (8-hydroxy-2-deoxyguanosine) and single-stranded DNA. Both forms of DNA damage accumulated early in target-deprived LGN neurons. Transgenic overexpression of superoxide dismutase-1 provided significant protection against the apoptosis but antioxidant pharmacotreatments with trolox and ascorbate were ineffective. We conclude that overlapping and sequential signaling pathways are involved in the apoptosis of adult brain neurons and that DNA damage generated by superoxide derivatives is an upstream mechanism for p53-regulated, Bax-dependent apoptosis of target-deprived neurons. [ABSTRACT FROM AUTHOR]

Acidosis is thought to play an important role in cerebral ischemic injury. In an adult stroke model, inhibition of the Ca2+-permeable acid-sensing ion channels (ASICIa) by psalmotoxin-I (PcTX) reduced injury. We postulated that PcTX attenuates neuronal damage in selectively vulnerable striatum in a global model of neonatal hypoxic-ischemic (HI) encephalopathy. H-I consisted of 40 min of hypoxia (9.8% O2) followed by 7 min of asphyxia in 5-7 day-old piglets. PcTX (1 µg) or CSF vehicle (50 µl) was injected into each lateral ventricle 20 min before H-I. No significant differences occurred at 37 min of hypoxia in PaO2 (21±3 Torr both groups; ±SD) or arterial pressure (MABP; CSF=94±16; PcTX=82±18 mmHg). During subsequent asphyxia, arterial O2 saturation decreased to 4±4% and 5±3%, arterial pH decreased to 6.82±0.07 and 6.85+0.09, and MABP decreased to 22±17 and 25±9 mmHg in the corresponding CSF and PcTX groups. The density of viable neurons in putamen 4 d later was increased from 20±6% (% of sham) in the CSF group to 54+28% in the PcTX group. We conclude that blocking ASICIa activation with PcTX protected the highly vulnerable putamen neurons from ischemic death in immature brain. [ABSTRACT FROM AUTHOR]