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.

Melatonin Modulates Cell Cycle Dynamics and Promotes Hippocampal Cell Proliferation After Ischemic Injury in Neonatal Rats.

Promoting neural cell proliferation may represent an important strategy for enhancing brain repair after developmental brain injury. The present study aimed to assess the effects of melatonin on cell proliferation after an ischemic injury in the developing hippocampus, focusing on cell cycle dynamics. After in vivo neonatal hypoxia-ischemia (HI), hippocampal cell cycle dynamics were assessed by flow cytometry, together with histological evaluation of dentate gyrus cellularity and proliferation. Melatonin significantly increased the number of proliferating cells in the G2/M phase as well as the proliferating cell nuclear antigen (PCNA) and doublecortin (DCX) labeling reduced by HI. In vivo BrdU labeling revealed a higher BrdU-positivity in the dentate gyrus of ischemic rats treated with melatonin, an effect followed by increased cellularity and preserved hippocampal tissue integrity. These results indicate that the protective effect of melatonin after ischemic injury in neonatal rats may rely on the modulation of cell cycle dynamics of newborn hippocampal cells and increased cell proliferation.

Therapeutic hypothermia modulates the neurogenic response of the newborn piglet subventricular zone after hypoxia-ischemia.

BACKGROUND: Neuroprotection combined with neuroregeneration may be critical for optimizing functional recovery in neonatal encephalopathy. To investigate the neurogenic response to hypoxia-ischemia (HI) followed by normothermia (38.5 °C) or three different hypothermic temperatures (35, 33.5, or 30 °C) in the subventricular zone (SVZ) of the neonatal piglet. METHODS: Following transient cerebral HI and resuscitation, 28 newborn piglets were randomized to: normothermia or whole-body cooling to 35 °C, 33.5 °C, or 30 °C during 2-26 h (all n = 7). At 48 h, piglets were euthanized and SVZ obtained to evaluate its cellularity, pattern of cell death, radial glia length, doublecortin (DCX, neuroblasts) expression, and Ki67 (cell proliferation) and Ki67/Sox2 (neural stem/progenitor dividing) cell counts. RESULTS: Normothermic piglets showed lower total (Ki67+) and neural stem/progenitor dividing (Ki67+Sox2+) cell counts when compared to hypothermic groups. Cooling to 33.5 °C obtained the highest values of SVZ cellularity, radial glia length processes, neuroblast chains area and DCX immunohistochemistry. Cooling to 30 °C, however, revealed decreased cellularity in the lateral SVZ and shorter radial glia processes when compared with 33.5 °C. CONCLUSIONS: In a neonatal piglet model, hypothermia to 33.5 °C modulates the neurogenic response of the SVZ after HI, highlighting the potential beneficial effect of hypothermia to 33.5 °C on endogenous neurogenesis and the detrimental effect of overcooling beyond this threshold. IMPACT: Neuroprotection combined with neuroregeneration may be critical for optimizing functional recovery in neonatal encephalopathy. Hypothermia may modulate neurogenesis in the subventricular zone (SVZ) of the neonatal hypoxic-ischemic piglet. Cooling to 33.5 °C obtained the highest values of SVZ cellularity, radial glia length processes, neuroblast chains area and doublecortin immunohistochemistry; cooling to 30 °C, however, revealed decreased cellularity and shorter radial glia processes. In a neonatal piglet model, therapeutic hypothermia (33.5 °C) modulates the neurogenic response of the SVZ after hypoxia-ischemia, highlighting also the detrimental effect of overcooling beyond this threshold.

The Long-Term Neuroprotective Effect of the Endocannabinoid 2-AG and Modulation of the SGZ's Neurogenic Response after Neonatal Hypoxia-Ischemia.

Neonatal hypoxia-ischemia (HI) often causes hypoxic-ischemic encephalopathy (HIE), a neurological condition that can lead to overall disability in newborns. The only treatment available for affected neonates is therapeutic hypothermia; however, cooling is not always effective to prevent the deleterious effects of HI, so compounds such as cannabinoids are currently under research as new therapies. Modulating the endocannabinoid system (ECS) may reduce brain damage and/or stimulate cell proliferation at the neurogenic niches. Further, the long-term effects of cannabinoid treatment are not so clear. Here, we studied the middle- and long-term effects of 2-AG, the most abundant endocannabinoid in the perinatal period after HI in neonatal rats. At middle-term (postnatal day 14), 2-AG reduced brain injury and increased SGZ's cell proliferation and the number of neuroblasts. At post-natal day 90, the treatment with the endocannabinoid showed global and local protection, suggesting long-lasting neuroprotective effects of 2-AG after neonatal HI in rats.

Effects of Cannabidiol, Hypothermia, and Their Combination in Newborn Rats with Hypoxic-Ischemic Encephalopathy.

Therapeutic hypothermia is well established as a standard treatment for infants with hypoxic-ischemic (HI) encephalopathy but it is only partially effective. The potential for combination treatments to augment hypothermic neuroprotection has major relevance. Our aim was to assess the effects of treating newborn rats following HI injury with cannabidiol (CBD) at 0.1 or 1 mg/kg, i.p., in normothermic (37.5°C) and hypothermic (32.0°C) conditions, from 7 d of age (neonatal phase) to 37 d of age (juvenile phase). Placebo or CBD was administered at 0.5, 24, and 48 h after HI injury. Two sensorimotor (rotarod and cylinder rearing) and two cognitive (novel object recognition and T-maze) tests were conducted 30 d after HI. The extent of brain damage was determined by magnetic resonance imaging, histologic evaluation, magnetic resonance spectroscopy, amplitude-integrated electroencephalography, and Western blotting. At 37 d, the HI insult produced impairments in all neurobehavioral scores (cognitive and sensorimotor tests), brain activity (electroencephalography), neuropathological score (temporoparietal cortexes and CA1 layer of hippocampus), lesion volume, magnetic resonance biomarkers of brain injury (metabolic dysfunction, excitotoxicity, neural damage, and mitochondrial impairment), oxidative stress, and inflammation (TNFα). We observed that CBD or hypothermia (to a lesser extent than CBD) alone improved cognitive and motor functions, as well as brain activity. When used together, CBD and hypothermia ameliorated brain excitotoxicity, oxidative stress, and inflammation, reduced brain infarct volume, lessened the extent of histologic damage, and demonstrated additivity in some parameters. Thus, coadministration of CBD and hypothermia could complement each other in their specific mechanisms to provide neuroprotection.

Prolactin and morning cortisol concentrations in antipsychotic naïve first episode psychosis: A systematic review and meta-analysis.

IMPORTANCE: Alterations in prolactin and cortisol levels have been reported in antipsychotic naïve patients with first episode psychosis (FEP). However, it has been studied in very small samples, and inter-group variability has never been studied before. OBJECTIVE: To provide estimates of standardized mean differences (SMD) and inter-group variability for prolactin, cortisol awakening response (CAR) and morning cortisol concentrations in antipsychotic naïve FEP (AN-FEP) patients and healthy controls (HC). DATA SOURCES: BIOSIS, KCI, MEDLINE, Russian Science Citation Index, SciELO, Cochrane, PsycINFO, Web of Science were searched from inception to February 28, 2022. STUDY SELECTION: Peer-reviewed cohort studies that reported on prolactin or cortisol blood concentrations in AN- FEP patients and HC were included. DATA EXTRACTION AND SYNTHESIS: Study characteristics, means and standard deviations (SD) were extracted from each article. Inter group differences in magnitude of effect were estimated using Hedges g. Inter-group variability was estimated with the coefficient of variation ratio (CVR). In both cases estimates were pooled using random-effects meta-analysis. Differences by study-level characteristics were estimated using meta-regression. PRISMA guideline was followed (No. CRD42022303555). MAIN OUTCOMES AND MEASURES: Prolactin, CAR and morning cortisol blood concentrations in AN-FEP group in relation to HC group. RESULTS: Fourteen studies for prolactin (N = 761 for AN-FEP group, N = 687 for HC group) and twelve studies for morning cortisol (N = 434 for AN-FEP group, N = 528 for HC group) were included. No studies were found in CAR in AN-FEP patients. Mean SMD for prolactin blood concentration was 0.88 (95% CI 0.57, 1.20) for male and 0.56 (95% CI 0.26, 0.87) for female. As a group, AN-FEP presented greater inter-group variability for prolactin levels than HC (CVR=1.28, 95% CI 1.02, 1.62). SMD for morning cortisol concentrations was non-significant: 0.34 (95% CI -0.01, 0.69) and no inter-group variability significant differences were detected: CVR= 1.05 (95% CI 0.91, 1.20). Meta-regression analyses for age and quality were non-significant. Funnel plots did not suggest a publication bias. CONCLUSIONS AND RELEVANCE: Increased prolactin levels were found in AN-FEP patients. A greater inter-group variability in the AN-FEP group suggests the existence of patient subgroups with different prolactin levels. No significant abnormalities were found in morning cortisol levels. Further research is needed to clarify whether prolactin concentrations could be used as an illness biomarker.

The Synthetic Cannabinoid URB447 Exerts Antitumor and Antimetastatic Effect in Melanoma and Colon Cancer.

The endocannabinoid system is widespread through the body and carries out a wide variety of functions. However, its involvement in other pathologies, such as cancer, still needs further attention. We aim to investigate the role of CB2 receptor during melanoma and colorectal cancer (CRC) aggressiveness and metastatic growth in the liver. We used the synthetic cannabinoid URB447, a known CB2 agonist and CB1 antagonist drug, and studied prometastatic ability of mouse B16 melanoma and MCA38 CRC cells, by means of proliferation, apoptosis, cell cycle, migration and matrix degradation in vitro upon URB447 treatment. We reported a dose-dependent viability decrease in both tumor types. This result is partly mediated by apoptotic cell death and cell cycle arrest in G1/G0 phase, as observed through flow cytometry. Melanoma and CRC cell migration was affected in a dose-dependent fashion as observed through scratch assay, whereas the secretion of matrix degrading proteins metalloprotease 2 (MMP2) and 9 (MMP9) in tumor cells did not significantly change. Moreover, daily treatment of tumor bearing mice with URB447 decreased the development of liver metastasis in a melanoma model in vivo. This proof of concept study points out to the synthetic cannabinoid URB447 as a potential candidate for deeper studies to confirm its potential as antitumor therapy and liver metastasis treatment for CRC and melanoma.

The Prevention of Ischemia-Reperfusion Injury in Elderly Rats after Lower Limb Tourniquet Use.

Background: Lower limb ischemia-reperfusion injury (IRI-LL) is a common major complication of orthopedic surgery, especially in elderly patients. It has previously been demonstrated that folinic acid (FA) reduced IRI-LL damage in 3−4-month-old rats. This current work analyses the effect of FA in the prevention of IRI-LL in elderly animals. Methods: Forty-two 18-month-old male WAG/RijHsd rats were subjected to 3 h of ischemia. Eighteen animals received FA (2.5 mg/kg, ip) 20 min before the end of the ischemia period, while the other half received the same volume of saline solution. The animals were sacrificed after 3 h, 24 h, and 14 days of reperfusion for biochemical (tissue damage markers and electrolytes), histopathological studies of the gastrocnemius muscle and the daily assessment of the limb function by the Rota Rod test, respectively. Results: The administration of FA prior to the end of the ischemia period reduced the increase in LDH and CK observed in non-treated animals by 30−40% (p < 0.0001). When the histological sections were analyzed, FA was found to have reduced the number of damaged muscle fibers per field by 20% (60 ± 17.1 vs. 80.7 ± 16.4, p < 0.0001). The functional test revealed that FA also led to an improvement in the muscle function, assessed by the length of time that the animals kept running on the rod, compared to untreated animals. Conclusions: The administration of FA, prior to the end of the ischemic period, decreases the damage induced by IRI-LL, also achieving a faster recovery of mobility.

Fallo multiorgánico neonatal tras asfixia perinatal / Neonatal multiple organ failure after perinatal asphyxia

La asfixia perinatal es un acontecimiento con efectos de gran alcance, pudiendo conducir no solo al desarrollo de encefalopatía neonatal, sino también a un fallo multiorgánico (FMO). Esta afectación posiblemente se deba a la redistribución del flujo sanguíneo, mediante el cual se conserva la irrigación de órganos vitales como el corazón, el cerebro y las glándulas suprarrenales, a expensas de su disminución en otros órganos.El objetivo del presente trabajo fue conocer la incidencia y la etiopatogenia de los órganos más frecuentemente afectados en el FMO neonatal tras la asfixia perinatal.Se realizó una búsqueda bibliográfica sistemática en las bases de datos Pubmed, Scopus y The Cochrane Library empleando los términos MeSH (ischemia AND hypoxia AND multiorgan dysfunction AND neonat*), (asphyxia AND multiorgan dysfunction AND neonat*) y (liver / kidney / digestive OR gastrointestinal / heart injury AND ischemia AND hypoxia AND neonat*). Se incluyeron trabajos clínicos y preclínicos posteriores al año 2000 y se excluyeron series de casos, cartas al director, cohortes sin grupo comparador y abstracts.En el presente trabajo describimos que el fallo multiorgánico asociado a la asfixia perinatal es un fenómeno frecuente y relevante en la morbimortalidad del neonato, pudiendo llegar a producir no solo alteraciones en riñón, hígado y tracto gastrointestinal, sino también miocardiopatía si el fenómeno se prolonga o es de elevada gravedad. (AU)

Perinatal asphyxia is an event with far-reaching consequences that can lead not only to the development of neonatal encephalopathy, but also to multiple organ failure (MOF). This ailment may result from the redistribution of blood flow, which would preserve the perfusion of vital organs such as the heart, brain and adrenal glands at the expense of other organs.The objective of the study was to determine the incidence and aetiopathogenesis of failure in the organs most frequently involved in neonatal MOF following perinatal asphyxia.We conducted a systematic literature search in the PubMed, Scopus and Cochrane Library databases using the MeSH terms (ischemia AND hypoxia AND multiorgan dysfunction AND neonat*), (asphyxia AND multiorgan dysfunction AND neonat*) and (liver/kidney/digestive OR gastrointestinal/heart injury AND ischemia AND hypoxia AND neonat*). We selected clinical and preclinical studies published after 2000 and excluded case series, letters to the editor, cohort studies without comparison groups and abstracts.In this study, we found that MOF associated with perinatal asphyxia is a frequent phenomenon with a relevant impact on neonatal morbidity and mortality, as it can cause changes not only in the kidney, liver and gastrointestinal tract, but also cardiomyopathy if the ailment is protracted or severe. (AU)

Neonatal multiple organ failure after perinatal asphyxia.

Perinatal asphyxia is an event with far-reaching consequences that can lead not only to the development of neonatal encephalopathy, but also to multiple organ failure (MOF). This ailment may result from the redistribution of blood flow, which would preserve the perfusion of vital organs such as the heart, brain and adrenal glands at the expense of other organs. The objective of the study was to determine the incidence and aetiopathogenesis of failure in the organs most frequently involved in neonatal MOF following perinatal asphyxia. We conducted a systematic literature search in the PubMed, Scopus and Cochrane Library databases using the MeSH terms (ischemia AND hypoxia AND multiorgan dysfunction AND neonat*), (asphyxia AND multiorgan dysfunction AND neonat*) and (liver/kidney/digestive OR gastrointestinal/heart injury AND ischemia AND hypoxia AND neonat*). We selected clinical and preclinical studies published after 2000 and excluded case series, letters to the editor, cohort studies without comparison groups and abstracts. In this study, we found that MOF associated with perinatal asphyxia is a frequent phenomenon with a relevant impact on neonatal morbidity and mortality, as it can cause changes not only in the kidney, liver and gastrointestinal tract, but also cardiomyopathy if the ailment is protracted or severe.

Neurogenesis Is Reduced at 48 h in the Subventricular Zone Independent of Cell Death in a Piglet Model of Perinatal Hypoxia-Ischemia.

Cellular and tissue damage triggered after hypoxia-ischemia (HI) can be generalized and affect the neurogenic niches present in the central nervous system. As neuroregeneration may be critical for optimizing functional recovery in neonatal encephalopathy, the goal of the present work was to investigate the neurogenic response to HI in the neurogenic niche of the subventricular zone (SVZ) in the neonatal piglet. A total of 13 large white male piglets aged <24 h were randomized into two groups: i) HI group (n = 7), animals submitted to transient cerebral HI and resuscitation; and ii) Control group (n = 6), non-HI animals. At 48 h, piglets were euthanized, and the SVZ and its surrounding regions, such as caudate and periventricular white matter, were analyzed for histology using hematoxylin-eosin staining and immunohistochemistry by evaluating the presence of cleaved caspase 3 and TUNEL positive cells, together with the cell proliferation/neurogenesis markers Ki67 (cell proliferation), GFAP (neural stem cells processes), Sox2 (neural stem/progenitor cells), and doublecortin (DCX, a marker of immature migrating neuroblasts). Hypoxic-ischemic piglets showed a decrease in cellularity in the SVZ independent of cell death, together with decreased length of neural stem cells processes, neuroblast chains area, DCX immunoreactivity, and lower number of Ki67 + and Ki67 + Sox2 + cells. These data suggest a reduction in both cell proliferation and neurogenesis in the SVZ of the neonatal piglet, which could in turn compromise the replacement of the lost neurons and the achievement of global repair.

The Endocannabinoid System as a Target for Neuroprotection/Neuroregeneration in Perinatal Hypoxic-Ischemic Brain Injury.

The endocannabinoid (EC) system is a complex cell-signaling system that participates in a vast number of biological processes since the prenatal period, including the development of the nervous system, brain plasticity, and circuit repair. This neuromodulatory system is also involved in the response to endogenous and environmental insults, being of special relevance in the prevention and/or treatment of vascular disorders, such as stroke and neuroprotection after neonatal brain injury. Perinatal hypoxia-ischemia leading to neonatal encephalopathy is a devastating condition with no therapeutic approach apart from moderate hypothermia, which is effective only in some cases. This overview, therefore, gives a current description of the main components of the EC system (including cannabinoid receptors, ligands, and related enzymes), to later analyze the EC system as a target for neonatal neuroprotection with a special focus on its neurogenic potential after hypoxic-ischemic brain injury.

The Synthetic Cannabinoid URB447 Reduces Brain Injury and the Associated White Matter Demyelination after Hypoxia-Ischemia in Neonatal Rats.

The number of functions controlled by the endocannabinoid system in health and disease continues growing over the years. In the brain, these include the modulation of harmful events such as glutamate excitotoxicity, oxidative stress, and inflammation, mainly regulated by activation/blockade of CB1/CB2 cannabinoid receptors. In the present work, we evaluated the capacity of the CB1 antagonist/CB2 agonist synthetic cannabinoid URB447 on reducing neurodegeneration after brain injury. By using a model of hypoxia-ischemia (HI) in neonatal rats, we found that URB447 strongly reduced brain injury when administered before HI. A comparable effect was observed with the CB1 antagonist SR141716A, whereas the CB1 agonist WIN-55,212-2 reduced the effect of URB447. When administered 3 h after HI, which is considered a clinically feasible therapeutic window to treat perinatal brain injury in humans, URB447 reduced neurodegeneration and white matter damage. Markers of astrogliosis and microglial activation also appeared reduced. These results confirm the important role played by the endocannabinoid system in the neurodegenerative process and strongly encourage further research into the mechanisms of URB447-induced neuroprotection.

Cannabinoid-mediated Modulation of Oxidative Stress and Early Inflammatory Response after Hypoxia-Ischemia.

In the process of neonatal encephalopathy, oxidative stress and neuroinflammation have a prominent role after perinatal asphyxia. With the exception of therapeutic hypothermia, no therapeutic interventions are available in the clinical setting to target either the oxidative stress or inflammation, despite the high prevalence of neurological sequelae of this devastating condition. The endocannabinoid system (ECS), recently recognized as a widespread neuromodulatory system, plays an important role in the development of the central nervous system (CNS). This study aims to evaluate the potential effect of the cannabinoid (CB) agonist WIN 55,212-2 (WIN) on reactive oxygen species (ROS) and early inflammatory cytokine production after hypoxia-ischemia (HI) in fetal lambs. Hypoxic-ischemic animals were subjected to 60 min of HI by partial occlusion of the umbilical cord. A group of lambs received a single dose of 0.01 µg/kg WIN, whereas non-asphyctic animals served as controls. WIN reduced the widespread and notorious increase in inflammatory markers tumor necrosis factor (TNF)-α and interleukin (IL)-1ß and IL-6 induced by HI, a modulatory effect not observed for oxidative stress. Our study suggests that treatment with a low dose of WIN can alter the profile of pro-inflammatory cytokines 3 h after HI.

Terapia combinada frente a la encefalopatía hipóxico-isquémica neonatal / Combined therapy in neonatal hypoxic-ischaemic encephalopathy

La encefalopatía hipóxico-isquémica neonatal como consecuencia de la falta de oxígeno en el cerebro del recién nacido aparece asociada a secuelas neurológicas severas como la parálisis cerebral, pudiendo incluso desencadenar la muerte del recién nacido asfíctico. Actualmente, la hipotermia es la única terapia empleada en las unidades de cuidados intensivos neonatales, gracias, principalmente, a su capacidad para disminuir la acumulación de aminoácidos excitatorios y a su efecto antioxidante, antiinflamatorio y antiapoptótico. Sin embargo, la hipotermia no presenta beneficio clínico significativo en aproximadamente la mitad de los casos, haciéndose necesario encontrar u optimizar una terapia eficaz que disminuya las secuelas neurológicas derivadas de la asfixia perinatal, mediante su aplicación en solitario o en combinación con la hipotermia. Dentro de estas terapias con potencial terapéutico demostrado destacan la melatonina, el alopurinol y la eritropoyetina, con ensayos clínicos en marcha, y otras como las células madre, la N-acetilcisteína o los gases nobles, con trabajos preclínicos publicados. La melatonina desarrolla un notable efecto antioxidante y antiinflamatorio, actuando como secuestrador de radicales libres y regulando diversos mediadores proinflamatorios. Por su parte, el alopurinol ejerce una potente función antioxidante mediante la inhibición de la enzima xantina oxidasa. La eritropoyetina, en cambio, tiene su principal diana terapéutica en la muerte celular y en la estimulación de la neurogénesis. Teniendo en consideración las diferentes dianas y estrategias terapéuticas, el futuro del tratamiento de la encefalopatía hipóxico-isquémica podría hallarse en la combinación de uno o varios de estos tratamientos junto con la hipotermia terapéutica

Neonatal hypoxic-ischaemic encephalopathy due to the lack of oxygen at birth can have severe neurological consequences, such as cerebral palsy, or even the death of the asphyxiated newborn. Hypothermia is currently the only therapy included in intensive care neonatal units. This shows a clinical benefit in neonates suffering from hypoxic-ischaemic encephalopathy, mainly because of its ability to decrease the accumulation of excitatory amino acids and its anti-inflammatory, antioxidant, and anti-apoptotic effects. However, hypothermia is not effective in half of the cases, making it necessary to search for new, or to optimize current therapies, with the aim on reducing asphyxia-derived neurological consequences, either as single treatments or in combination with cooling. Within current potential therapies, melatonin, allopurinol, and erythropoietin stand out among the others, with clinical trials on the way. While, stem cells, N-acetylcysteine and noble gases have obtained promising pre-clinical results. Melatonin produces a powerful antioxidant and anti-inflammatory effect, acting as free radical scavenger and regulating pro-inflammatory mediators. Through the inhibition of xanthine oxidase, allopurinol can decrease oxidative stress. Erythropoietin has cell death and neurogenesis as its main therapeutic targets. Keeping in mind the whole scenario of current therapies, management of neonates suffering from neonatal asphyxia could rely on the combination of one or some of these treatments, together with therapeutic hypothermia

[Combined therapy in neonatal hypoxic-ischaemic encephalopathy]. / Terapia combinada frente a la encefalopatía hipóxico-isquémica neonatal.

Neonatal hypoxic-ischaemic encephalopathy due to the lack of oxygen at birth can have severe neurological consequences, such as cerebral palsy, or even the death of the asphyxiated newborn. Hypothermia is currently the only therapy included in intensive care neonatal units. This shows a clinical benefit in neonates suffering from hypoxic-ischaemic encephalopathy, mainly because of its ability to decrease the accumulation of excitatory amino acids and its anti-inflammatory, antioxidant, and anti-apoptotic effects. However, hypothermia is not effective in half of the cases, making it necessary to search for new, or to optimize current therapies, with the aim on reducing asphyxia-derived neurological consequences, either as single treatments or in combination with cooling. Within current potential therapies, melatonin, allopurinol, and erythropoietin stand out among the others, with clinical trials on the way. While, stem cells, N-acetylcysteine and noble gases have obtained promising pre-clinical results. Melatonin produces a powerful antioxidant and anti-inflammatory effect, acting as free radical scavenger and regulating pro-inflammatory mediators. Through the inhibition of xanthine oxidase, allopurinol can decrease oxidative stress. Erythropoietin has cell death and neurogenesis as its main therapeutic targets. Keeping in mind the whole scenario of current therapies, management of neonates suffering from neonatal asphyxia could rely on the combination of one or some of these treatments, together with therapeutic hypothermia.

Melatonin as an adjunct to therapeutic hypothermia in a piglet model of neonatal encephalopathy: A translational study.

Therapeutic hypothermia is only partially protective for neonatal encephalopathy; there is an urgent need to develop treatments that augment cooling. Our objective was to assess safety, efficacy and pharmacokinetics of 5 and 15 mg/kg/24 h melatonin (proprietary formulation) administered at 2 h and 26 h after hypoxia-ischemia (HI) with cooling in a piglet model. Following moderate cerebral HI, 30 piglets were eligible and randomized to: i) Hypothermia (33.5 °C, 2-26 h) and vehicle (HT + V;n = 13); b) HT and 5 mg/kg melatonin over 6 h at 2 h and 26 h after HI (HT + Mel-5;n = 4); c) HT and 15 mg/kg melatonin over 6 h at 2 h and 26 h after HI (HT + Mel-15;n = 13). Intensive care was maintained for 48 h; brain MRS was acquired and cell death (TUNEL) evaluated at 48 h. Comparing HT + V with HT + Mel-5 and HT + Mel-15, there was no difference in blood pressure or inotropic support needed, brain Lactate/N Acetylaspartate at 24 h and 48 h was similar, ATP/phosphate pool was higher for HT + Mel-15 versus HT + V at 24 h (p = 0.038) but not 48 h. A localized reduction in TUNEL positive cell death was observed in the sensorimotor cortex in the 15 mg/kg melatonin group (HT + Mel-15 versus HT + V; p < 0.003) but not in the 5 mg/kg melatonin group (HT + Mel-5 versus HT + V; p = 0.808). Putative therapeutic melatonin levels were reached 8 h after HI (104 increase from baseline; ~15-30 mg/l). Mean ±â€¯SD peak plasma melatonin levels after the first infusion were 0.0014 ±â€¯0.0012 mg/l in the HT + V group, 3.97 ±â€¯1.53 mg/l in the HT + Mel-5 group and 16.8 ±â€¯8.3 mg/l in the HT + Mel-15 group. Protection was dose dependent; 15 mg/kg melatonin started 2 h after HI, given over 6 h, was well tolerated and augmented hypothermic protection in sensorimotor cortex. Earlier attainment of therapeutic plasma melatonin levels may optimize protection by targeting initial events of reperfusion injury. The time window for intervention with melatonin, as adjunct therapy with cooling, is likely to be narrow and should be considered in designing future clinical studies.

Dexmedetomidine Combined with Therapeutic Hypothermia Is Associated with Cardiovascular Instability and Neurotoxicity in a Piglet Model of Perinatal Asphyxia.

The selective α2-adrenoreceptor agonist dexmedetomidine has shown neuroprotective, analgesic, anti-inflammatory, and sympatholytic properties that may be beneficial in neonatal encephalopathy (NE). As therapeutic hypothermia is only partially effective, adjunct therapies are needed to optimize outcomes. The aim was to assess whether hypothermia + dexmedetomidine treatment augments neuroprotection compared to routine treatment (hypothermia + fentanyl sedation) in a piglet model of NE using magnetic resonance spectroscopy (MRS) biomarkers, which predict outcomes in babies with NE, and immunohistochemistry. After hypoxia-ischaemia (HI), 20 large White male piglets were randomized to: (i) hypothermia + fentanyl with cooling to 33.5°C from 2 to 26 h, or (ii) hypothermia + dexmedetomidine (a loading dose of 2 µg/kg at 10 min followed by 0.028 µg/kg/h for 48 h). Whole-brain phosphorus-31 and regional proton MRS biomarkers were assessed at baseline, 24, and 48 h after HI. At 48 h, cell death was evaluated over 7 brain regions by means of transferase-mediated d-UTP nick end labeling (TUNEL). Dexmedetomidine plasma levels were mainly within the target sedative range of 1 µg/L. In the hypothermia + dexmedetomidine group, there were 6 cardiac arrests (3 fatal) versus 2 (non-fatal) in the hypothermia + fentanyl group. The hypothermia + dexmedetomidine group required more saline (p = 0.005) to maintain blood pressure. Thalamic and white-matter lactate/N-acetylaspartate did not differ between groups (p = 0.66 and p = 0.21, respectively); the whole-brain nucleotide triphosphate/exchangeable phosphate pool was similar (p = 0.73) over 48 h. Cell death (TUNEL-positive cells/mm2) was higher in the hypothermia + dexmedetomidine group than in the hypothermia + fentanyl group (mean 5.1 vs. 2.3, difference 2.8 [95% CI 0.6-4.9], p = 0.036). Hypothermia + dexmedetomidine treatment was associated with adverse cardiovascular events, even within the recommended clinical sedative plasma level; these may have been exacerbated by an interaction with either isoflurane or low body temperature. Hypothermia + dexmedetomidine treatment was neurotoxic following HI in our piglet NE model, suggesting that caution is vital if dexmedetomidine is combined with cooling following NE.

Surgery increases cell death and induces changes in gene expression compared with anesthesia alone in the developing piglet brain.

In a range of animal species, exposure of the brain to general anaesthesia without surgery during early infancy may adversely affect its neural and cognitive development. The mechanisms mediating this are complex but include an increase in brain cell death. In humans, attempts to link adverse cognitive development to infantile anaesthesia exposure have yielded ambiguous results. One caveat that may influence the interpretation of human studies is that infants are not exposed to general anaesthesia without surgery, raising the possibility that surgery itself, may contribute to adverse cognitive development. Using piglets, we investigated whether a minor surgical procedure increases cell death and disrupts neuro-developmental and cognitively salient gene transcription in the neonatal brain. We randomly assigned neonatal male piglets to a group who received 6h of 2% isoflurane anaesthesia or a group who received an identical anaesthesia plus 15 mins of surgery designed to replicate an inguinal hernia repair. Compared to anesthesia alone, surgery-induced significant increases in cell death in eight areas of the brain. Using RNAseq data derived from all 12 piglets per group we also identified significant changes in the expression of 181 gene transcripts induced by surgery in the cingulate cortex, pathway analysis of these changes suggests that surgery influences the thrombin, aldosterone, axonal guidance, B cell, ERK-5, eNOS and GABAA signalling pathways. This suggests a number of novel mechanisms by which surgery may influence neural and cognitive development independently or synergistically with the effects of anaesthesia.