Neutrophil Extracellular Traps Release following Hypoxic-Ischemic Brain Injury in Newborn Rats Treated with Therapeutic Hypothermia.

The peripheral immune system plays a critical role in neuroinflammation of the central nervous system after an insult. Hypoxic-ischemic encephalopathy (HIE) induces a strong neuroinflammatory response in neonates, which is often associated with exacerbated outcomes. In adult models of ischemic stroke, neutrophils infiltrate injured brain tissue immediately after an ischemic insult and aggravate inflammation via various mechanisms, including neutrophil extracellular trap (NETs) formation. In this study, we used a neonatal model of experimental hypoxic-ischemic (HI) brain injury and demonstrated that circulating neutrophils were rapidly activated in neonatal blood. We observed an increased infiltration of neutrophils in the brain after exposure to HI. After treatment with either normothermia (NT) or therapeutic hypothermia (TH), we observed a significantly enhanced expression level of the NETosis marker Citrullinated H3 (Cit-H3), which was significantly more pronounced in animals treated with TH than in those treated with NT. NETs and NLR family pyrin domain containing 3 (NLRP-3) inflammasome assembly are closely linked in adult models of ischemic brain injury. In this study, we observed an increase in the activation of the NLRP-3 inflammasome at the time points analyzed, particularly immediately after TH, when we observed a significant increase in NETs structures in the brain. Together, these results suggest the important pathological functions of early arriving neutrophils and NETosis following neonatal HI, particularly after TH treatment, which is a promising starting point for the development of potential new therapeutic targets for neonatal HIE.

Comparing the efficacy in reducing brain injury of different neuroprotective agents following neonatal hypoxia-ischemia in newborn rats: a multi-drug randomized controlled screening trial.

Intrapartum hypoxia-ischemia leading to neonatal encephalopathy (NE) results in significant neonatal mortality and morbidity worldwide, with > 85% of cases occurring in low- and middle-income countries (LMIC). Therapeutic hypothermia (HT) is currently the only available safe and effective treatment of HIE in high-income countries (HIC); however, it has shown limited safety or efficacy in LMIC. Therefore, other therapies are urgently required. We aimed to compare the treatment effects of putative neuroprotective drug candidates following neonatal hypoxic-ischemic (HI) brain injury in an established P7 rat Vannucci model. We conducted the first multi-drug randomized controlled preclinical screening trial, investigating 25 potential therapeutic agents using a standardized experimental setting in which P7 rat pups were exposed to unilateral HI brain injury. The brains were analysed for unilateral hemispheric brain area loss after 7 days survival. Twenty animal experiments were performed. Eight of the 25 therapeutic agents significantly reduced brain area loss with the strongest treatment effect for Caffeine, Sonic Hedgehog Agonist (SAG) and Allopurinol, followed by Melatonin, Clemastine, ß-Hydroxybutyrate, Omegaven, and Iodide. The probability of efficacy was superior to that of HT for Caffeine, SAG, Allopurinol, Melatonin, Clemastine, ß-hydroxybutyrate, and Omegaven. We provide the results of the first systematic preclinical screening of potential neuroprotective treatments and present alternative single therapies that may be promising treatment options for HT in LMIC.

Impact of Hypoxia-Ischemia on Neurogenesis and Structural and Functional Outcomes in a Mild-Moderate Neonatal Hypoxia-Ischemia Brain Injury Model.

Hypoxic-ischemic encephalopathy (HIE) is a common type of brain injury caused by a lack of oxygen and blood flow to the brain during the perinatal period. The incidence of HIE is approximately 2−3 cases per 1000 live births in high-income settings; while in low- and middle-income countries, the incidence is 3−10-fold higher. Therapeutic hypothermia (TH) is the current standard treatment for neonates affected by moderate−severe HIE. However, more than 50% of all infants with suspected HIE have mild encephalopathy, and these infants are not treated with TH because of their lower risk of adverse outcomes. Despite this, several analyses of pooled data provide increasing evidence that infants who initially have mild encephalopathy may present signs of more significant brain injury later in life. The purpose of this study was to expand our knowledge about the effect of mild−moderate hypoxia-ischemia (HI) at the cellular, structural, and functional levels. An established rat model of mild−moderate HI was used, where postnatal day (P) 7 rats were exposed to unilateral permanent occlusion of the left carotid artery and 90 min of 8% hypoxia, followed by TH or normothermia (NT) treatment. The extent of injury was assessed using histology (P14 and P42) and MRI (P11 and P32), as well as with short-term and long-term behavioral tests. Neurogenesis was assessed by BrdU staining. We showed that mild−moderate HI leads to a progressive loss of brain tissue, pathological changes in MRI scans, as well as an impairment of long-term motor function. At P14, the median area loss assessed by histology for HI animals was 20% (p < 0.05), corresponding to mild−moderate brain injury, increasing to 55% (p < 0.05) at P42. The data assessed by MRI corroborated our results. HI led to a decrease in neurogenesis, especially in the hippocampus and the lateral ventricle at early time points, with a delayed partial recovery. TH was not neuroprotective at early time points following mild−moderate HI, but prevented the increase in brain damage over time. Additionally, rats treated with TH showed better long-term motor function. Altogether, our results bring more light to the understanding of pathophysiology following mild-moderate HI. We showed that, in the context of mild-moderate HI, TH failed to be significantly neuroprotective. However, animals treated with TH showed a significant improvement in motor, but not cognitive long-term function. These results are in line with what is observed in some cases where neonates with mild HIE are at risk of neurodevelopmental deficits in infancy or childhood. Whether TH should be used as a preventive treatment to reduce adverse outcomes in mild-HIE remains of active interest, and more research has to be carried out in order to address this question.

Temporal Characterization of Microglia-Associated Pro- and Anti-Inflammatory Genes in a Neonatal Inflammation-Sensitized Hypoxic-Ischemic Brain Injury Model.

Hypoxic-ischemic encephalopathy (HIE) mainly affects preterm and term newborns, leading to a high risk of brain damage. Coexisting infection/inflammation and birth asphyxia are key factors associated with intracerebral increase of proinflammatory cytokines linked to HIE. Microglia are key mediators of inflammation during perinatal brain injury, characterized by their phenotypic plasticity, which may facilitate their participation in both the progression and resolution of injury-induced inflammation. The purpose of this study was to investigate the temporal expression of genes associated with pro- and anti-inflammatory cytokines as well as the nucleotide-binding domain, leucine-rich repeat protein (NLRP-3) inflammasome from microglia cells. For this purpose, we used our established neonatal rat model of inflammation-sensitized hypoxic-ischemic (HI) brain injury in seven-day-old rats. We assessed gene expression profiles of 11 cytokines and for NLRP-3 using real-time PCR from sorted CD11b/c microglia of brain samples at different time points (3.5 h after LPS injection and 0, 5, 24, 48, and 72 hours post HI) following different treatments: vehicle, E. coli lipopolysaccharide (LPS), vehicle/HI, and LPS/HI. Our results showed that microglia are early key mediators of the inflammatory response and exacerbate the inflammatory response following HI, polarizing into a predominant proinflammatory M1 phenotype in the early hours post HI. The brains only exposed to HI showed a delay in the expression of proinflammatory cytokines. We also demonstrated that NLRP-3 plays a role in the inflammatory resolution with a high expression after HI insult. The combination of both, a preinfection/inflammation condition and hypoxia-ischemia, resulted in a higher proinflammatory cytokine storm, highlighting the significant contribution of acute inflammation sensitizing prior to a hypoxic insult on the severity of perinatal brain damage.