Group II Metabotropic Glutamate Receptors Reduce Apoptosis and Regulate BDNF and GDNF Levels in Hypoxic-Ischemic Injury in Neonatal Rats.

Birth asphyxia causes brain injury in neonates, but a fully successful treatment has yet to be developed. This study aimed to investigate the effect of group II mGlu receptors activation after experimental birth asphyxia (hypoxia-ischemia) on the expression of factors involved in apoptosis and neuroprotective neurotrophins. Hypoxia-ischemia (HI) on 7-day-old rats was used as an experimental model. The effects of intraperitoneal application of mGluR2 agonist LY379268 (5 mg/kg) and the specific mGluR3 agonist NAAG (5 mg/kg) (1 h or 6 h after HI) on apoptotic processes and initiation of the neuroprotective mechanism were investigated. LY379268 and NAAG applied shortly after HI prevented brain damage and significantly decreased pro-apoptotic Bax and HtrA2/Omi expression, increasing expression of anti-apoptotic Bcl-2. NAAG or LY379268 applied at both times also decreased HIF-1α formation. HI caused a significant decrease in BDNF concentration, which was restored after LY379268 or NAAG administration. HI-induced increase in GDNF concentration was decreased after administration of LY379268 or NAAG. Our results show that activation of mGluR2/3 receptors shortly after HI prevents brain damage by the inhibition of excessive glutamate release and apoptotic damage decrease. mGluR2 and mGluR3 agonists produced comparable results, indicating that both receptors may be a potential target for early treatment in neonatal HI.

Immuno-histological detection of resistant columnar units and vulnerable networks in the rat retina after asphyxia-induced transient cardiac arrest.

BACKGROUND: Stroke-related loss of vision is one of the residual impairments, restricting the quality of life. However, studies of the ocular manifestations of asphyxia cardiac arrest/resuscitation (ACA/R) have reported very heterogeneous results. OBJECTIVE: We aimed to evaluate the ACA/R-induced degeneration pattern of the different retinal cell populations in rats using different immuno-histological stainings. METHODS: The staining pattern of toluidine blue and the ganglion cell markers ß-III-tubulin and NeuN; the calcium-binding protein parvalbumin, indicating ganglion, amacrine, and horizontal cells; calretinin D28k, indicating ganglion and amacrine cells; calbindin, indicating horizontal cells; Chx 10, indicating cone bipolar cells; PKCα, indicating ON-type rod bipolar cells; arrestin, indicating cones; and rhodopsin, a marker of rods, as well as the glial cell markers GFAP (indicating astroglia and Müller cells) and IBA1 (indicating microglia), were evaluated after survival times of 7 and 21 days in an ACA/R rat model. Moreover, quantitative morphological analysis of the optic nerve was performed. The ACA/R specimens were compared with those from sham-operated and completely naïve rats. RESULTS: ACA/R-induced effects were: (i) a significant reduction of retinal thickness after long-term survival; (ii) ganglion cell degeneration, including their fiber network in the inner plexiform layer; (iii) degeneration of amacrine and cone bipolar cells; (iv) degeneration of cone photoreceptors; (v) enhanced resistance to ACA/R by rod photoreceptors, ON-type rod bipolar and horizontal cells, possibly caused by the strong upregulation of the calcium-binding proteins calretinin, parvalbumin, and calbindin, counteracting the detrimental calcium overload; (vi) significant activation of Müller cells as further element of retinal anti-stress self-defense mechanisms; and (vii) morphological alterations of the optic nerve in form of deformed fibers. CONCLUSIONS: Regardless of the many defects, the surviving neuronal structures seemed to be able to maintain retinal functionality, which can be additionally improved by regenerative processes true to the "use it or lose it" dogma.

Alzheimer's Disease Associated Presenilin 1 and 2 Genes Dysregulation in Neonatal Lymphocytes Following Perinatal Asphyxia.

Perinatal asphyxia is mainly a brain disease leading to the development of neurodegeneration, in which a number of peripheral lesions have been identified; however, little is known about the expression of key genes involved in amyloid production by peripheral cells, such as lymphocytes, during the development of hypoxic-ischemic encephalopathy. We analyzed the gene expression of the amyloid protein precursor, ß-secretase, presenilin 1 and 2 and hypoxia-inducible factor 1-α by RT-PCR in the lymphocytes of post-asphyxia and control neonates. In all examined periods after asphyxia, decreased expression of the genes of the amyloid protein precursor, ß-secretase and hypoxia-inducible factor 1-α was noted in lymphocytes. Conversely, expression of presenilin 1 and 2 genes decreased on days 1-7 and 8-14 but increased after survival for more than 15 days. We believe that the expression of presenilin genes in lymphocytes could be a potential biomarker to determine the severity of the post-asphyxia neurodegeneration or to identify the underlying factors for brain neurodegeneration and get information about the time they occurred. This appears to be the first worldwide data on the role of the presenilin 1 and 2 genes associated with Alzheimer's disease in the dysregulation of neonatal lymphocytes after perinatal asphyxia.

HSP70-mediated neuroprotection by combined treatment of valproic acid with hypothermia in a rat asphyxial cardiac arrest model.

It has been reported that valproic acid (VPA) combined with therapeutic hypothermia can improve survival and neurologic outcomes in a rat asphyxial cardiac arrest model. However, neuroprotective mechanisms of such combined treatment of valproic acid with hypothermia remains unclear. We hypothesized that epigenetic regulation of HSP70 by histone acetylation could increase HSP70-mediated neuroprotection suppressed under hypothermia. Male Sprague-Dawley rats that achieved return of spontaneous circulation (ROSC) from asphyxial cardiac arrest were randomized to four groups: normothermia (37°C ± 1°C), hypothermia (33°C ± 1°C), normothermia + VPA (300 mg/kg IV initiated 5 minutes post-ROSC and infused over 20 min), and hypothermia + VPA. Three hours after ROSC, acetyl-histone H3 was highly expressed in VPA-administered groups (normothermia + VPA, hypothermia + VPA). Four hours after ROSC, HSP70 mRNA expression levels were significantly higher in normothermic groups (normothermia, normothermia + VPA) than in hypothermic groups (hypothermia, hypothermia + VPA). The hypothermia + VPA group showed significantly higher HSP70 mRNA expression than the hypothermia group. Similarly, at five hours after ROSC, HSP70 protein levels were significantly higher in normothermic groups than in hypothermic groups. HSP70 levels were significantly higher in the hypothermia + VPA group than in the hypothermia group. Only the hypothermia + VPA group showed significantly attenuated cleaved caspase-9 levels than the normothermia group. Hypothermia can attenuate the expression of HSP70 at transcriptional level. However, VPA administration can induce hyperacetylation of histone H3, leading to epigenetic transcriptional activation of HSP70 even in a hypothermic status. Combining VPA treatment with hypothermia may compensate for reduced activation of HSP70-mediated anti-apoptotic pathway.

Multitarget neuroprotection by quercetin: Changes in gene expression in two perinatal asphyxia models.

Hypoxic-ischemic encephalopathy (HIE) causes mortality and long-term neurologic morbidities in newborns, affecting pathways related to energy failure, excitotoxicity and oxidative stress that often lead to cell death. The whole process of HIE injury is coupled to changes in the expression of a great array of proteins. A nanoliposomal preparation of the flavonoid quercetin has been shown to exert neuroprotective effects in perinatal asphyxia models. This study aimed to identify neonatal HIE markers and explore the effect of quercetin administration in two perinatal asphyxia models: newborn rats and piglets. In the rat model, nanoliposomal quercetin administration reduced mortality after asphyxia. In the piglet model, quercetin partially overrode the reduction of HIF-1α mRNA levels in the cortex induced by asphyxia. Quercetin administration also reduced increased level of HO-1 mRNA in asphyctic piglets. These results suggest that quercetin neuroprotection might be involved in the regulation of HIF-1α, HO-1 and their targets. A proteomic approach revealed that the glycolytic pathway is strongly regulated by quercetin in both species. We also identified a set of proteins differentially expressed that could be further considered as markers. In piglets, this set includes Acidic Leucine-rich nuclear phosphoprotein 32 (ANP32A), associated with nervous system differentiation, proteins related with death pathways and alpha-enolase which can be converted to neuron-specific enolase, a glycolytic enzyme that may promote neuroprotection. In newborn rats, other promising proteins associated with neurogenesis and neuroprotection emerged, such as dihydropyrimidinase-related proteins, catalytic and regulatory subunits of phosphatases and heterogeneous nuclear ribonucleoprotein K (hnRNPK). Our results show that a nanoliposomal preparation of quercetin, with protective effect in two HIE mammal models, modulates the expression of proteins involved in energy metabolism and other putative neuroprotective signals in the cortex. Identification of these signals could reveal potential molecular pathways involved in disease onset and the novel quercetin neuroprotective strategy.

Metabolomics improves the histopathological diagnosis of asphyxial deaths: an animal proof-of-concept model.

The diagnosis of mechanical asphyxia remains one of the most difficult issues in forensic pathology. Asphyxia ultimately results in cardiac arrest (CA) and, as there are no specific markers, the differential diagnosis of primitive CA and CA secondary to asphyxiation relies on circumstantial details and on the pathologist experience, lacking objective evidence. Histological examination is currently considered the gold standard for CA post-mortem diagnosis. Here we present the comparative results of histopathology versus those previously obtained by 1H nuclear magnetic resonance (NMR) metabolomics in a swine model, originally designed for clinical purposes, exposed to two different CA causes, namely ventricular fibrillation and asphyxia. While heart and brain microscopical analysis could identify the damage induced by CA without providing any additional information on the CA cause, metabolomics allowed the identification of clearly different profiles between the two groups and showed major differences between asphyxiated animals with good and poor outcomes. Minute-by-minute plasma sampling allowed to associate these modifications to the pre-arrest asphyxial phase showing a clear correlation to the cellular effect of mechanical asphyxia reproduced in the experiment. The results suggest that metabolomics provides additional evidence beyond that obtained by histology and immunohistochemistry in the differential diagnosis of CA.

Model for the prediction of mechanical asphyxia as the cause of death based on four biological indexes in human cardiac tissue.

Determination of mechanical asphyxia as the cause of death has always been difficult for forensic pathologists, particularly when signs of asphyxia are not obvious on the body. Currently, depending on only physical examination of corpses, pathologists must be cautious when making cause-of-death appraisals. In a previous study, four biomarkers-dual-specificity phosphatase 1 (DUSP1), potassium voltage-gated channel subfamily J member 2 (KCNJ2), miR-122, and miR-3185-were screened in human cardiac tissue from cadavers that died from mechanical asphyxia compared with those that died from craniocerebral injury, hemorrhagic shock, or other causes. Expression of the markers correlated with death from mechanical asphyxia regardless of age, environmental temperature, and postmortem interval. However, a single biological index is not an accurate basis for the identification of the cause of death. In this study, receiver operating characteristic curves of the ΔCq values of the four indexes were generated. The diagnostic accuracy of the indexes was judged according to their area under the curve (DUSP1: 0.773, KCNJ2: 0.775, miR-122: 0.667, and miR-3185: 0.801). Finally, a nomogram was generated, and single blind experiment was conducted to verify the cause of death of mechanical asphyxia.

Neonatal Mesenchymal Stem Cell Treatment Improves Myelination Impaired by Global Perinatal Asphyxia in Rats.

The effect of perinatal asphyxia (PA) on oligodendrocyte (OL), neuroinflammation, and cell viability was evaluated in telencephalon of rats at postnatal day (P)1, 7, and 14, a period characterized by a spur of neuronal networking, evaluating the effect of mesenchymal stem cell (MSCs)-treatment. The issue was investigated with a rat model of global PA, mimicking a clinical risk occurring under labor. PA was induced by immersing fetus-containing uterine horns into a water bath for 21 min (AS), using sibling-caesarean-delivered fetuses (CS) as controls. Two hours after delivery, AS and CS neonates were injected with either 5 µL of vehicle (10% plasma) or 5 × 104 MSCs into the lateral ventricle. Samples were assayed for myelin-basic protein (MBP) levels; Olig-1/Olig-2 transcriptional factors; Gglial phenotype; neuroinflammation, and delayed cell death. The main effects were observed at P7, including: (i) A decrease of MBP-immunoreactivity in external capsule, corpus callosum, cingulum, but not in fimbriae of hippocampus; (ii) an increase of Olig-1-mRNA levels; (iii) an increase of IL-6-mRNA, but not in protein levels; (iv) an increase in cell death, including OLs; and (v) MSCs treatment prevented the effect of PA on myelination, OLs number, and cell death. The present findings show that PA induces regional- and developmental-dependent changes on myelination and OLs maturation. Neonatal MSCs treatment improves survival of mature OLs and myelination in telencephalic white matter.

Exploring metabolic alterations associated with death from asphyxia and the differentiation of asphyxia from sudden cardiac death by GC-HRMS-based untargeted metabolomics.

The determination of cause of death is one of the most important tasks in forensic practice. However, asphyxia is a difficult cause of death to determine, especially when the deceased has an underlying disease that can lead to a sudden unexpected death, such as coronary atherosclerotic heart disease (CAHD, which is the leading cause of sudden cardiac death, SCD), because its determination is currently still based on an exclusion strategy. In this study, gas chromatography coupled with high-resolution mass spectrometry (GC-HRMS)-based untargeted metabolomics was employed to obtain the pulmonary metabolic profiles of rats who died from asphyxia and SCD. First, fourteen metabolites were identified to investigate the mechanism of death from asphyxia, and we proposed some explanations that may account for these metabolic alterations, including the perturbation of amino acid metabolism, lipid metabolism, and energy metabolism (TCA cycle). Second, we discovered eight potential biomarkers to differentiate between asphyxia and SCD as the cause of death. The excellent classification performances of the eight individual biomarkers and their combination in fresh lung tissue were observed. Third, we also explored the relative change in the concentration of the eight metabolites and their classification performance in decomposed tissue (at 24 h postmortem). Lactic acid, pantothenic acid, and the combination of the eight biomarkers can be recognized as perfect classifiers to discriminate asphyxia from SCD even when decomposition has occurred. Our results showed that GC-HRMS-based untargeted metabolomics can be used as a promising tool to explore the metabolic alterations of the death process and to determine the cause of death.

Morphological and biochemical changes in the pancreas associated with acute systemic hypoxia.

This study aimed to investigate the changes associated with acute systemic hypoxia in the endocrine system, particularly in pancreatic tissues. The investigation was based on macroscopic, pathohistological, biochemical, and molecular biological findings in cell lines and human cadavers. The results showed that cases of death due to asphyxia more frequently showed severe subcapsular/interstitial hemorrhage versus the other causes of death. Histological examination showed that asphyxia cases were associated with severe morphological changes. Although measured insulin levels in the asphyxia were higher compared to other causes of death, no differences were noted for the glucagon and amylase levels with regard to the cause of death. Increased blood insulin levels were not associated with macro- and micromorphological changes, and did not show any association with glucose or cortisol levels. The experiment conducted under hypoxic conditions in cultured cells demonstrated that insulin mRNA expression and insulin protein levels peaked at 10 min after hypoxia exposure. However, there were no changes in either the amylase mRNA or protein levels. Corticosterone level peaked at 120 min after exposure to hypoxic conditions. Overall, acute systemic hypoxic conditions can directly affect the mechanisms involved in pancreatic insulin secretion.

Cardiac Arrest Induced by Asphyxia Versus Ventricular Fibrillation Elicits Comparable Early Changes in Cytokine Levels in the Rat Brain, Heart, and Serum.

Background Current postresuscitative care after cardiac arrest (CA) does not address the cause of CA. We previously reported that asphyxial CA (ACA) and ventricular fibrillation CA (VFCA) elicit unique injury signatures. We hypothesized that the early cytokine profiles of the serum, heart, and brain differ in response to ACA versus VFCA. Methods and Results Adult male rats were subjected to 10 minutes of either ACA or VFCA. Naives and shams (anesthesia and surgery without CA) served as controls (n=12/group). Asphyxiation produced an ≈4-minute period of progressive hypoxemia followed by a no-flow duration of ≈6±1 minute. Ventricular fibrillation immediately induced no flow. Return of spontaneous circulation was achieved earlier after ACA compared with VFCA (42±18 versus 105±22 seconds; P<0.001). Brain cytokines in naives were, in general, low or undetectable. Shams exhibited a modest effect on select cytokines. Both ACA and VFCA resulted in robust cytokine responses in serum, heart, and brain at 3 hours. Significant regional differences pinpointed the striatum as a key location of neuroinflammation. No significant differences in cytokines, neuron-specific enolase, S100b, and troponin T were observed across CA models. Conclusions Both models of CA resulted in marked systemic, heart, and brain cytokine responses, with similar degrees of change across the 2 CA insults. Changes in cytokine levels after CA were most pronounced in the striatum compared with other brain regions. These collective observations suggest that the amplitude of the changes in cytokine levels after ACA versus VFCA may not mediate the differences in secondary injuries between these 2 CA phenotypes.

Perinatal Infection: A Major Contributor to Efficacy of Cooling in Newborns Following Birth Asphyxia.

Neonatal encephalopathy (NE) is a global burden, as more than 90% of NE occurs in low- and middle-income countries (LMICs). Perinatal infection seems to limit the neuroprotective efficacy of therapeutic hypothermia. Efforts made to use therapeutic hypothermia in LMICs treating NE has led to increased neonatal mortality rates. The heat shock and cold shock protein responses are essential for survival against a wide range of stressors during which organisms raise their core body temperature and temporarily subject themselves to thermal and cold stress in the face of infection. The characteristic increase and decrease in core body temperature activates and utilizes elements of the heat shock and cold shock response pathways to modify cytokine and chemokine gene expression, cellular signaling, and immune cell mobilization to sites of inflammation, infection, and injury. Hypothermia stimulates microglia to secret cold-inducible RNA-binding protein (CIRP), which triggers NF-κB, controlling multiple inflammatory pathways, including nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasomes and cyclooxygenase-2 (COX-2) signaling. Brain responses through changes in heat shock protein and cold shock protein transcription and gene-expression following fever range and hyperthermia may be new promising potential therapeutic targets.

Apoptosis and necroptosis occur in the different brain regions of hippocampus in a rat model of hypoxia asphyxia.

AIM OF THE STUDY: Hypoxic-ischemic encephalopathy (HIE) is a major cause of newborn brain injury. Apoptosis and necroptosis are two forms of cell death which may occur in HIE but reported data are yet limited. This study investigates the expression of receptor interacting protein kinase (RIPK) 1 and 3, and caspase3, the key modulators of necroptosis and apoptosis, respectively, in a model of HIE to determine whether both forms of cell death occur in the corresponding brain regions. MATERIALS AND METHODS: Postneonatal day 7 Sprague-Dawley rats were subjected to right carotid artery ligation followed by hypoxia or subjected to skin incision under surgical anesthesia without ligation and hypoxia. Neuroglioma (H4) cell was cultured and subjected to 24 h hypoxic insults. Necrostatin-1, a RIPK1 inhibitor, was administered in both in vivo and in vitro settings before insult. RESULTS: After hypoxic-ischemic insults, both RIPK1 and RIPK3 expression were significantly increased in the region of hippocampal dentate gyrus in the injurious hemisphere. However, cleaved caspase3 was significantly increased in the hippocampal cornu ammonis 1 region in the injurious hemisphere. After hypoxic insults, RIPK1 and RIPK3 expression was also found in H4 cells. In addition, it was identified that the increased RIPK1 and RIPK3 can be inhibited by necrostatin-1 in both in vivo and in vitro. CONCLUSIONS: These data indicated that apoptosis and necroptosis occur in different brain regions of hippocampus in a model of HIE which may suggest that strategies to prevent each form of neuronal death is valuable to be developed.

Nitric oxide and the brain. Part 2: Effects following neonatal brain injury-friend or foe?

Nitric oxide (NO) has critical roles in a wide variety of key biologic functions and has intricate transport mechanisms for delivery to key distal tissues under normal conditions. However, NO also plays important roles during disease processes, such as hypoxia-ischemia, asphyxia, neuro-inflammation, and retinopathy of prematurity. The effects of exogenous NO on the developing neonatal brain remain controversial. Inhaled NO (iNO) can be neuroprotective or toxic depending on a variety of factors, including cellular redox state, underlying disease processes, duration of treatment, and dose. This review identifies key gaps in knowledge that should prompt further investigation into the possible role of iNO as a therapeutic agent after injury to the brain. IMPACT: NO is a key signal mediator in the neonatal brain with neuroprotective and neurotoxic properties. iNO, a commonly used medication, has significant effects on the neonatal brain. Dosing, duration, and timing of administration of iNO can affect the developing brain. This review article summarizes the roles of NO in association with various disease processes that impact neonates, such as brain hypoxia-ischemia, asphyxia, retinopathy of prematurity, and neuroinflammation. The impact of this review is that it clearly describes gaps in knowledge, and makes the case for further, targeted studies in each of the identified areas.

Excess cerebral oxygen delivery follows return of spontaneous circulation in near-term asphyxiated lambs.

Hypoxic-ischaemia renders the neonatal brain susceptible to early secondary injury from oxidative stress and impaired autoregulation. We aimed to describe cerebral oxygen kinetics and haemodynamics immediately following return of spontaneous circulation (ROSC) and evaluate non-invasive parameters to facilitate bedside monitoring. Near-term sheep fetuses [139 ± 2 (SD) days gestation, n = 16] were instrumented to measure carotid artery (CA) flow, pressure, right brachial arterial and jugular venous saturation (SaO2 and SvO2, respectively). Cerebral oxygenation (crSO2) was measured using near-infrared spectroscopy (NIRS). Following induction of severe asphyxia, lambs received cardiopulmonary resuscitation using 100% oxygen until ROSC, with oxygen subsequently weaned according to saturation nomograms as per current guidelines. We found that oxygen consumption did not rise following ROSC, but oxygen delivery was markedly elevated until 15 min after ROSC. CrSO2 and heart rate each correlated with oxygen delivery. SaO2 remained > 90% and was less useful for identifying trends in oxygen delivery. CrSO2 correlated inversely with cerebral fractional oxygen extraction. In conclusion, ROSC from perinatal asphyxia is characterised by excess oxygen delivery that is driven by rapid increases in cerebrovascular pressure, flow, and oxygen saturation, and may be monitored non-invasively. Further work to describe and limit injury mediated by oxygen toxicity following ROSC is warranted.

Stearic acid methyl ester affords neuroprotection and improves functional outcomes after cardiac arrest.

Cardiac arrest causes neuronal damage and functional impairments that can result in learning/memory dysfunction after ischemia. We previously identified a saturated fatty acid (stearic acid methyl ester, SAME) that was released from the superior cervical ganglion (sympathetic ganglion). The function of stearic acid methyl ester is currently unknown. Here, we show that SAME can inhibit the detrimental effects of global cerebral ischemia (i.e. cardiac arrest). Treatment with SAME in the presence of asphyxial cardiac arrest (ACA) revived learning and working memory deficits. Similarly, SAME-treated hippocampal slices after oxygen-glucose deprivation inhibited neuronal cell death. Moreover, SAME afforded neuroprotection against ACA in the CA1 region of the hippocampus, reduced ionized calcium-binding adapter molecule 1 expression and inflammatory cytokines/chemokines, with restoration in mitochondria respiration. Altogether, we describe a unique and uncharted role of saturated fatty acids in the brain that may have important implications against cerebral ischemia.

High-Dose Melatonin and Ethanol Excipient Combined with Therapeutic Hypothermia in a Newborn Piglet Asphyxia Model.

With the current practice of therapeutic hypothermia for neonatal encephalopathy, disability rates and the severity spectrum of cerebral palsy are reduced. Nevertheless, safe and effective adjunct therapies are needed to optimize outcomes. This study's objective was to assess if 18 mg/kg melatonin given rapidly over 2 h at 1 h after hypoxia-ischemia with cooling from 1-13 h was safe, achieved therapeutic levels within 3 h and augmented hypothermic neuroprotection. Following hypoxia-ischemia, 20 newborn piglets were randomized to: (i) Cooling 1-13 h (HT; n = 6); (ii) HT+ 2.5% ethanol vehicle (HT+V; n = 7); (iii) HT + Melatonin (HT+M; n = 7). Intensive care was maintained for 48 h; aEEG was acquired throughout, brain MRS acquired at 24 and 48 h and cell death (TUNEL) evaluated at 48 h. There were no differences for insult severity. Core temperature was higher in HT group for first hour after HI. Comparing HT+M to HT, aEEG scores recovered more quickly by 19 h (p < 0.05); comparing HT+V to HT, aEEG recovered from 31 h (p < 0.05). Brain phosphocreatine/inorganic phosphate and NTP/exchangeable phosphate were higher at 48 h in HT+M versus HT (p = 0.036, p = 0.049 respectively). Including both 24 h and 48 h measurements, the rise in Lactate/N-acetyl aspartate was reduced in white (p = 0.030) and grey matter (p = 0.038) after HI. Reduced overall TUNEL positive cells were observed in HT+M (47.1 cells/mm2) compared to HT (123.8 cells/mm2) (p = 0.0003) and HT+V (97.5 cells/mm2) compared to HT (p = 0.012). Localized protection was seen in white matter for HT+M versus HT (p = 0.036) and internal capsule for HT+M compared to HT (p = 0.001) and HT+V versus HT (p = 0.006). Therapeutic melatonin levels (15-30mg/l) were achieved at 2 h and were neuroprotective following HI, but ethanol vehicle was partially protective.

Inhibition of PAR-2 Attenuates Neuroinflammation and Improves Short-Term Neurocognitive Functions Via ERK1/2 Signaling Following Asphyxia-Induced Cardiac Arrest in Rats.

OBJECTIVE: Global cerebral ischemia-induced neuroinflammation causes neurofunctional impairment following cardiac arrest. Previous studies have demonstrated that the activation of protease-activated receptor-2 (PAR-2) contributes to neuroinflammation. In the present study, we aimed to determine the potential treatment effect of PAR-2 inhibition against neuroinflammation in the setting of asphyxial CA (ACA) in rats. METHODS: A total of 116 adult, male Sprague-Dawley rats were randomly divided into Sham (n = 18) and ACA (n = 98) groups. Time course, short-term outcome, and mechanism studies were conducted. All drugs were delivered intranasally. The effect of PAR-2 inhibitor FSLLRY-NH2 on neurocognitive functions was assessed by neurologic deficit score, number of seizures, and T-maze test, while hippocampal neuronal degeneration was evaluated by Fluoro-Jade C staining after ACA. Western blotting was performed for the mechanism study at 24 h following ACA. Selective PAR-2 agonist (AC55541) and ERK1/2 inhibitor (PD98059) were used for intervention. RESULTS: Inhibition of PAR-2 decreased neuroinflammation, reduced the number of degenerating hippocampal neurons and improved neurocognitive functions following ACA. PAR-2 activator alone exerted opposite effects to PAR-2 inhibitor. PAR-2 mediated the augmented brain levels of proinflammatory cytokines by promoting the phosphorylation of ERK1/2. CONCLUSIONS: PAR-2 inhibition diminished neuroinflammation and thereby reduced hippocampal neuronal degeneration and neurocognitive impairment following ACA. This effect was at least partly mediated via the PAR-2/ERK1/2 signaling.

Novel application of amino-acid buffered solution for neuroprotection against ischemia/reperfusion injury.

Ischemic neuron loss contributes to brain dysfunction in patients with cardiac arrest (CA). Histidine-tryptophan-ketoglutarate (HTK) solution is a preservative used during organ transplantation. We tested the potential of HTK to protect neurons from severe hypoxia (SH) following CA. We isolated rat primary cortical neurons and induced SH with or without HTK. Changes in caspase-3, hypoxia-inducible factor 1-alpha (HIF-1α), and nicotinamide adenine dinucleotide phosphate oxidase-4 (NOX4) expression were evaluated at different time points up to 72 h. Using a rat asphyxia model, we induced CA-mediated brain damage and then completed resuscitation. HTK or sterile saline was administered into the left carotid artery. Neurological deficit scoring and mortality were evaluated for 3 days. Then the rats were sacrificed for evaluation of NOX4 and H2O2 levels in blood and brain. In the in vitro study, HTK attenuated SH- and H2O2-mediated cytotoxicity in a volume- and time-dependent manner, associated with persistent HIF-1α expression and reductions in procaspase-3 activation and NOX4 expression. The inhibition of HIF-1α abrogated HTK's effect on NOX4. In the in vivo study, neurological scores were significantly improved by HTK. H2O2 level, NOX4 activity, and NOX4 gene expression were all decreased in the brain specimens of HTK-treated rats. Our results suggest that HTK acts as an effective neuroprotective solution by maintaining elevated HIF-1α level, which was associated with inhibited procaspase-3 activation and decreased NOX4 expression.

Acute LPS sensitization and continuous infusion exacerbates hypoxic brain injury in a piglet model of neonatal encephalopathy.

Co-existing infection/inflammation and birth asphyxia potentiate the risk of developing neonatal encephalopathy (NE) and adverse outcome. In a newborn piglet model we assessed the effect of E. coli lipopolysaccharide (LPS) infusion started 4 h prior to and continued for 48 h after hypoxia on brain cell death and systemic haematological changes compared to LPS and hypoxia alone. LPS sensitized hypoxia resulted in an increase in mortality and in brain cell death (TUNEL positive cells) throughout the whole brain, and in the internal capsule, periventricular white matter and sensorimotor cortex. LPS alone did not increase brain cell death at 48 h, despite evidence of neuroinflammation, including the greatest increases in microglial proliferation, reactive astrocytosis and cleavage of caspase-3. LPS exposure caused splenic hypertrophy and platelet count suppression. The combination of LPS and hypoxia resulted in the highest and most sustained systemic white cell count increase. These findings highlight the significant contribution of acute inflammation sensitization prior to an asphyxial insult on NE illness severity.