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.

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.

Neonatal hypoxic-ischemic encephalopathy is the most common cause of long-term disability in term neonates, and white matter injury is the primary cause of cerebral palsy. Therapies that focus on the neuroprotection of myelination and oligodendrocyte proliferation could potentially ameliorate long-lasting neurological impairments after hypoxic-ischemic encephalopathy. Clemastine, a histamine H1 antagonist, has been shown to exert neuroprotective effects in multiple sclerosis and spinal cord injury by promoting oligodendrogenesis and re-myelination. In this study, we demonstrated the neuroprotective effects of clemastine in our rat model of neonatal hypoxic-ischemic brain injury. Animals received a single intraperitoneal injection of either vehicle or clemastine (10 mg/kg) for 6 consecutive days. Our results showed a significant reduction in white matter loss after treatment, with a clear effect of clemastine on oligodendrocytes, showing a significant increase in the number of Olig2+ cells. We characterized the MAPK/ERK pathway as a potential mechanistic pathway underlying the neuroprotective effects of clemastine. Altogether, our results demonstrate that clemastine is a potential compound for the treatment of hypoxic-ischemic encephalopathy, with a clear neuroprotective effect on white matter injury by promoting oligodendrogenesis.

Neonatal hypoxic–ischemic encephalopathy (HIE) is a major cause of neonatal mortality and long-term neurological disability, affecting 1–3 per 1000 live births in developed countries and occurring at substantially higher rates in developing countries. Neuroinflammation is a key contributor to disease progression, with growing evidence implicating the activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome following hypoxic–ischemic (HI) injury. In this study, we investigated the role and regulation of the NLRP3 inflammasome in neonatal HIE using in vitro and in vivo models. Primary microglial cultures subjected to oxygen–glucose deprivation and the Vannucci neonatal rat model of HI were used to characterize NLRP3 activation and its contribution to injury. We demonstrated that HI induces NLRP3 inflammasome activation, whereas pharmacological inhibition of NLRP3 enhances cell viability and attenuates brain damage. Our findings identify microglia as a central mediator of NLRP3-driven neuroinflammation and highlight microglial NLRP3 signaling as a promising therapeutic target for neuroinflammatory diseases. Collectively, this study provides an integrated view of NLRP3 regulation in neonatal HIE and supports inflammasome-directed strategies for neuroprotection following neonatal HI injury.