Dimethyl Fumarate Strongly Ameliorates Gray and White Matter Brain Injury and Modulates Glial Activation after Severe Hypoxia-Ischemia in Neonatal Rats.

Neonatal hypoxia-ischemia is a major cause of infant death and disability. The only clinically accepted treatment is therapeutic hypothermia; however, cooling is less effective in the most severely encephalopathic infants. Here, we wanted to test the neuroprotective effect of the antioxidant dimethyl fumarate after severe hypoxia-ischemia in neonatal rats. We used a modified Rice-Vannucci model to generate severe hypoxic-ischemic brain damage in day 7 postnatal rats, which were randomized into four experimental groups: Sham, Sham + DMF, non-treated HI, and HI + DMF. We analyzed brain tissue loss, global and regional (cortex and hippocampus) neuropathological scores, white matter injury, and microglial and astroglial reactivity. Compared to non-treated HI animals, HI + DMF pups showed a reduced brain area loss (p = 0.0031), an improved neuropathological score (p = 0.0016), reduced white matter injuries by preserving myelin tracts (p < 0.001), and diminished astroglial (p < 0.001) and microglial (p < 0.01) activation. After severe hypoxia-ischemia in neonatal rats, DMF induced a strong neuroprotective response, reducing cerebral infarction, gray and white matter damage, and astroglial and microglial activation. Although further molecular studies are needed and its translation to human babies would need to evaluate the molecule in piglets or lambs, DMF may be a potential treatment against neonatal encephalopathy.

PHLDA1 contributes to hypoxic ischemic brain injury in neonatal rats via inhibiting FUNDC1-mediated mitophagy.

Hypoxia-ischemia (HI) is one of the main causes of neonatal brain injury. Mitophagy has been implicated in the degradation of damaged mitochondria and cell survival following neonatal brain HI injury. Pleckstrin homology-like domain family A member 1 (PHLDA1) plays vital roles in the progression of various disorders including the regulation of oxidative stress, the immune responses and apoptosis. In the present study we investigated the role of PHLDA1 in HI-induced neuronal injury and further explored the mechanisms underlying PHLDA1-regulated mitophagy in vivo and in vitro. HI model was established in newborn rats by ligation of the left common carotid artery plus exposure to an oxygen-deficient chamber with 8% O2 and 92% N2. In vitro studies were conducted in primary hippocampal neurons subjected to oxygen and glucose deprivation/-reoxygenation (OGD/R). We showed that the expression of PHLDA1 was significantly upregulated in the hippocampus of HI newborn rats and in OGD/R-treated primary neurons. Knockdown of PHLDA1 in neonatal rats via lentiviral vector not only significantly ameliorated HI-induced hippocampal neuronal injury but also markedly improved long-term cognitive function outcomes, whereas overexpression of PHLDA1 in neonatal rats via lentiviral vector aggravated these outcomes. PHLDA1 knockdown in primary neurons significantly reversed the reduction of cell viability and increase in intracellular reactive oxygen species (ROS) levels, and attenuated OGD-induced mitochondrial dysfunction, whereas overexpression of PHLDA1 decreased these parameters. In OGD/R-treated primary hippocampal neurons, we revealed that PHLDA1 knockdown enhanced mitophagy by activating FUNDC1, which was abolished by FUNDC1 knockdown or pretreatment with mitophagy inhibitor Mdivi-1 (25 µM). Notably, pretreatment with Mdivi-1 or the knockdown of FUNDC1 not only increased brain infarct volume, but also abolished the neuroprotective effect of PHLDA1 knockdown in HI newborn rats. Together, these results demonstrate that PHLDA1 contributes to neonatal HI-induced brain injury via inhibition of FUNDC1-mediated neuronal mitophagy.

Structural and Functional Effects of C5aR1 Antagonism in a Rat Model of Neonatal Hypoxic-Ischemic Encephalopathy.

INTRODUCTION: The complement response activates upon reperfusion in neonatal hypoxic-ischemic encephalopathy (HIE) and contributes to excessive neuroinflammation and worse outcomes. C5a is a powerful anaphylatoxin central to each of the complement pathways, and its engagement with C5aR1 is directly tied to brain injury and neuronal death. Reasoning C5aR1 antagonism can decrease excessive neuroinflammation and thereby improve neurological and functional outcomes, we tested this hypothesis in a rat model of HIE with PMX205, a small molecule that inhibits C5a-C5aR1 interaction. METHODS: Term-equivalent pups (P10-12) were subjected to mild-moderate HIE by Vannucci's method and treated with PMX205. We compared motor and cognitive outcomes with two behavioral tests each (food handling and accelerod; novel object recognition [NOR] and open field) to improve the accuracy of our conclusions. RESULTS: Improvements were observed in fine motor function, balance, and exploratory behaviors, but little to no improvement in recognition memory and gross motor function. Lesion area and histological assessments showed robust cortical neuroprotection from treatment but persistent injury to the CA1 region of the hippocampus. Better structural and functional outcomes were seen within 1 day of treatment, suggesting C5aR1 antagonism beyond the latent injury phase may impair recovery. In a dose-response experiment, cerebral area loss from injury was improved only in female rats, suggesting underlying sexual dimorphisms in the complement response. CONCLUSION: These results demonstrate proof-of-concept for targeting C5aR1 signaling in neonatal HIE with PMX205 and underscore the role of sex in hypoxic-ischemic injury.

TSG-6-Mediated Extracellular Matrix Modifications Regulate Hypoxic-Ischemic Brain Injury.

Proteoglycans containing link domains modify the extracellular matrix (ECM) to regulate cellular homeostasis and can also sensitize tissues/organs to injury and stress. Hypoxic-ischemic (H-I) injury disrupts cellular homeostasis by activating inflammation and attenuating regeneration and repair pathways. In the brain, the main component of the ECM is the glycosaminoglycan hyaluronic acid (HA), but whether HA modifications of the ECM regulate cellular homeostasis and response to H-I injury is not known. In this report, employing both male and female mice, we demonstrate that link-domain-containing proteoglycan, TNFα-stimulated gene-6 (TSG-6), is active in the brain from birth onward and differentially modifies ECM HA during discrete neurodevelopmental windows. ECM HA modification by TSG-6 enables it to serve as a developmental switch to regulate the activity of the Hippo pathway effector protein, yes-associated protein 1 (YAP1), in the maturing brain and in response to H-I injury. Mice that lack TSG-6 expression display dysregulated expression of YAP1 targets, excitatory amino acid transporter 1 (EAAT1; glutamate-aspartate transporter) and 2 (EAAT2; glutamate transporter-1). Dysregulation of YAP1 activation in TSG-6-/- mice coincides with age- and sex-dependent sensitization of the brain to H-I injury such that 1-week-old neonates display an anti-inflammatory response in contrast to an enhanced proinflammatory injury reaction in 3-month-old adult males but not females. Our findings thus support that a key regulator of age- and sex-dependent H-I injury response in the mouse brain is modulation of the Hippo-YAP1 pathway by TSG-6-dependent ECM modifications.

Subventricular zone stem cell niche injury is associated with intestinal perforation in preterm infants and predicts future motor impairment.

Brain injury is highly associated with preterm birth. Complications of prematurity, including spontaneous or necrotizing enterocolitis (NEC)-associated intestinal perforations, are linked to lifelong neurologic impairment, yet the mechanisms are poorly understood. Early diagnosis of preterm brain injuries remains a significant challenge. Here, we identified subventricular zone echogenicity (SVE) on cranial ultrasound in preterm infants following intestinal perforations. The development of SVE was significantly associated with motor impairment at 2 years. SVE was replicated in a neonatal mouse model of intestinal perforation. Examination of the murine echogenic subventricular zone (SVZ) revealed NLRP3-inflammasome assembly in multiciliated FoxJ1+ ependymal cells and a loss of the ependymal border in this postnatal stem cell niche. These data suggest a mechanism of preterm brain injury localized to the SVZ that has not been adequately considered. Ultrasound detection of SVE may serve as an early biomarker for neurodevelopmental impairment after inflammatory disease in preterm infants.

Proteasome localization and activity in pig brain and in vivo small molecule screening for activators.

Introduction: Loss of proteasome function, proteinopathy, and proteotoxicity may cause neurodegeneration across the human lifespan in several forms of brain injury and disease. Drugs that activate brain proteasomes in vivo could thus have a broad therapeutic impact in neurology. Methods: Using pigs, a clinically relevant large animal with a functionally compartmental gyrencephalic cerebral cortex, we evaluated the localization and biochemical activity of brain proteasomes and tested the ability of small molecules to activate brain proteasomes. Results: By Western blotting, proteasome protein subunit PSMB5 and PSMA3 levels were similar in different pig brain regions. Immunohistochemistry for PSMB5 showed localization in the cytoplasm (diffuse and particulate) and nucleus (cytoplasm < nucleus). Some PSMB5 immunoreactivity was colocalized with mitochondrial (voltage-gated anion channel and cyclophilin D) and cell death (Aven) proteins in the neuronal soma and neuropil in the neocortex of pig and human brains. In the nucleus, PSMB5 immunoreactivity was diffuse, particulate, and clustered, including perinucleolar decorations. By fluorogenic assay, proteasome chymotrypsin-like activities (CTL) in crude tissue soluble fractions were generally similar within eight different pig brain regions. Proteasome CTL activity in the hippocampus was correlated with activity in nasal mucosa biopsies. In pilot analyses of subcellular fractions of pig cerebral cortex, proteasome CTL activity was highest in the cytosol and then ~50% lower in nuclear fractions; ~15-20% of total CTL activity was in pure mitochondrial fractions. With in-gel activity assay, 26S-singly and -doubly capped proteasomes were the dominant forms in the pig cerebral cortex. With a novel in situ histochemical activity assay, MG132-inhibitable proteasome CTL activity was localized to the neuropil, as a mosaic, and to cell bodies, nuclei, and centrosome-like perinuclear satellites. In piglets treated intravenously with pyrazolone derivative and chlorpromazine over 24 h, brain proteasome CTL activity was modestly increased. Discussion: This study shows that the proteasome in the pig brain has relative regional uniformity, prominent nuclear and perinuclear presence with catalytic activity, a mitochondrial association with activity, 26S-single cap dominance, and indications from small molecule systemic administration of pyrazolone derivative and chlorpromazine that brain proteasome function appears safely activable.

Neuroactive metabolites and bile acids are altered in extremely premature infants with brain injury.

The gut microbiome is associated with pathological neurophysiological evolvement in extremely premature infants suffering from brain injury. The exact underlying mechanism and its associated metabolic signatures in infants are not fully understood. To decipher metabolite profiles linked to neonatal brain injury, we investigate the fecal and plasma metabolome of samples obtained from a cohort of 51 extremely premature infants at several time points, using liquid chromatography (LC)-high-resolution mass spectrometry (MS)-based untargeted metabolomics and LC-MS/MS-based targeted analysis for investigating bile acids and amidated bile acid conjugates. The data are integrated with 16S rRNA gene amplicon gut microbiome profiles as well as patient cytokine, growth factor, and T cell profiles. We find an early onset of differentiation in neuroactive metabolites between infants with and without brain injury. We detect several bacterially derived bile acid amino acid conjugates in plasma and feces. These results provide insights into the early-life metabolome of extremely premature infants.

Neonatal brain injury unravels transcriptional and signaling changes underlying the reactivation of cortical progenitors.

Germinal activity persists throughout life within the ventricular-subventricular zone (V-SVZ) of the postnatal forebrain due to the presence of neural stem cells (NSCs). Accumulating evidence points to a recruitment for these cells following early brain injuries and suggests their amenability to manipulations. We used chronic hypoxia as a rodent model of early brain injury to investigate the reactivation of cortical progenitors at postnatal times. Our results reveal an increased proliferation and production of glutamatergic progenitors within the dorsal V-SVZ. Fate mapping of V-SVZ NSCs demonstrates their contribution to de novo cortical neurogenesis. Transcriptional analysis of glutamatergic progenitors shows parallel changes in methyltransferase 14 (Mettl14) and Wnt/ß-catenin signaling. In agreement, manipulations through genetic and pharmacological activation of Mettl14 and the Wnt/ß-catenin pathway, respectively, induce neurogenesis and promote newly-formed cell maturation. Finally, labeling of young adult NSCs demonstrates that pharmacological NSC activation has no adverse effects on the reservoir of V-SVZ NSCs and on their germinal activity.

Long-term recruitment of peripheral immune cells to brain scars after a neonatal insult.

Although brain scars in adults have been extensively studied, there is less data available regarding scar formation during the neonatal period, and the involvement of peripheral immune cells in this process remains unexplored in neonates. Using a murine model of neonatal hypoxic-ischemic encephalopathy (HIE) and confocal microscopy, we characterized the scarring process and examined the recruitment of peripheral immune cells to cortical and hippocampal scars for up to 1 year post-insult. Regional differences in scar formation were observed, including the presence of reticular fibrotic networks in the cortex and perivascular fibrosis in the hippocampus. We identified chemokines with chronically elevated levels in both regions and demonstrated, through a parabiosis-based strategy, the recruitment of lymphocytes, neutrophils, and monocyte-derived macrophages to the scars several weeks after the neonatal insult. After 1 year, however, neutrophils and lymphocytes were absent from the scars. Our data indicate that peripheral immune cells are transient components of HIE-induced brain scars, opening up new possibilities for late therapeutic interventions.

Quantification of Diffusion Magnetic Resonance Imaging for Prognostic Prediction of Neonatal Hypoxic-Ischemic Encephalopathy.

Neonatal hypoxic-ischemic encephalopathy (HIE) is the leading cause of acquired neonatal brain injury with the risk of developing serious neurological sequelae and death. An accurate and robust prediction of short- and long-term outcomes may provide clinicians and families with fundamental evidence for their decision-making, the design of treatment strategies, and the discussion of developmental intervention plans after discharge. Diffusion tensor imaging (DTI) is one of the most powerful neuroimaging tools with which to predict the prognosis of neonatal HIE by providing microscopic features that cannot be assessed by conventional magnetic resonance imaging (MRI). DTI provides various scalar measures that represent the properties of the tissue, such as fractional anisotropy (FA) and mean diffusivity (MD). Since the characteristics of the diffusion of water molecules represented by these measures are affected by the microscopic cellular and extracellular environment, such as the orientation of structural components and cell density, they are often used to study the normal developmental trajectory of the brain and as indicators of various tissue damage, including HIE-related pathologies, such as cytotoxic edema, vascular edema, inflammation, cell death, and Wallerian degeneration. Previous studies have demonstrated widespread alteration in DTI measurements in severe cases of HIE and more localized changes in neonates with mild-to-moderate HIE. In an attempt to establish cutoff values to predict the occurrence of neurological sequelae, MD and FA measurements in the corpus callosum, thalamus, basal ganglia, corticospinal tract, and frontal white matter have proven to have an excellent ability to predict severe neurological outcomes. In addition, a recent study has suggested that a data-driven, unbiased approach using machine learning techniques on features obtained from whole-brain image quantification may accurately predict the prognosis of HIE, including for mild-to-moderate cases. Further efforts are needed to overcome current challenges, such as MRI infrastructure, diffusion modeling methods, and data harmonization for clinical application. In addition, external validation of predictive models is essential for clinical application of DTI to prognostication.

Neuroprotective Action of Tacrolimus before and after Onset of Neonatal Hypoxic-Ischaemic Brain Injury in Rats.

(1) Background: Neonatal brain injury can lead to permanent neurodevelopmental impairments. Notably, suppressing inflammatory pathways may reduce damage. To determine the role of neuroinflammation in the progression of neonatal brain injury, we investigated the effect of treating neonatal rat pups with the immunosuppressant tacrolimus at two time points: before and after hypoxic-ischaemic (HI)-induced injury. (2) Methods: To induce HI injury, postnatal day (PND) 10 rat pups underwent single carotid artery ligation followed by hypoxia (8% oxygen, 90 min). Pups received daily tacrolimus (or a vehicle) starting either 3 days before HI on PND 7 (pre-HI), or 12 h after HI (post-HI). Four doses were tested: 0.025, 0.05, 0.1 or 0.25 mg/kg/day. Pups were euthanised at PND 17 or PND 50. (3) Results: All tacrolimus doses administered pre-HI significantly reduced brain infarct size and neuronal loss, increased the number of resting microglia and reduced cellular apoptosis (p < 0.05 compared to control). In contrast, only the highest dose of tacrolimus administered post-HI (0.25 mg/kg/day) reduced brain infarct size (p < 0.05). All doses of tacrolimus reduced pup weight compared to the controls. (4) Conclusions: Tacrolimus administration 3 days pre-HI was neuroprotective, likely mediated through neuroinflammatory and cell death pathways. Tacrolimus post-HI may have limited capacity to reduce brain injury, with higher doses increasing rat pup mortality. This work highlights the benefits of targeting neuroinflammation during the acute injurious period. More specific targeting of neuroinflammation, e.g., via T-cells, warrants further investigation.

MiR-126 and miR-146a as Melatonin-Responsive Biomarkers for Neonatal Brain Ischemia.

Despite advances in obstetric and neonatal care, challenges remain in early identification of neonates with encephalopathy due to hypoxia-ischemia who are undergoing therapeutic hypothermia. Therefore, there is a deep search for biomarkers that can identify brain injury. The aims of this study were to investigate the serum and brain expressions of two potential biomarkers, miR-126/miR-146a, in a preclinical model of hypoxia-ischemia (HI)-induced brain injury, and to explore their modulation during melatonin treatment. Seven-day-old rats were subjected to permanent ligation of the right carotid artery followed by 2.5 h hypoxia (HI). Melatonin (15 mg/kg) was administered 5 min after HI. Serum and brain samples were collected 1, 6 and 24 h after HI. Results show that HI caused a significant increase in the circulating levels of both miR-126 and miR-146a during the early phase of ischemic brain damage development (i.e. 1 h), with a parallel and opposite pattern in the ischemic cerebral cortex. These effects are not observed 24 h later. Treatment with melatonin restored the HI-induced effects on miR-126/miR-146a expressions, both in the cerebral cortex and in serum. We conclude that miR-126/miR-146a are promising biomarkers of HI injury and demonstrate an associated change in concentration following melatonin treatment.

Tactile stimulation facilitates functional recovery and dendritic change following neonatal hemidecortication in rats.

After large neocortical lesions, such as hemidecortication, children can show significant motor and cognitive impairments. It thus is of considerable interest to identify treatments that might enhance long-term functional outcome. We have previously shown that tactile stimulation enhances recovery from perinatal focal cortical lesions in rats, so the goal of the present experiment was to explore the effectiveness of postlesion tactile stimulation in reducing functional deficits associated with neonatal hemidecortication. Rats were given hemidecortications on postnatal day 10 (P10). Half of the group was then exposed to a daily tactile stimulation treatment for 15 min, three times a day for eleven days following the surgery. All groups were then tested on a number of behavioural tasks (Morris water task, skilled reaching, forelimb placing during spontaneous vertical exploration, and a sunflower seed opening task) beginning at P 120. The brains of the male animals were prepared for Golgi-Cox staining and subsequent analysis of dendritic arborisation and spine density. There were two main findings in this experiment: 1) Tactile stimulation improved cognitive ability and some motor performance after P 10 hemidecortication; and, 2) Tactile stimulation altered cortical organization after P10 hemidecortication. Tactile stimulation may provide an important noninvasive therapy after hemispherectomy in children.

EEG functional connectivity after perinatal stroke.

Impaired cognitive functioning after perinatal stroke has been associated with long-term functional brain network changes. We explored brain functional connectivity using a 64-channel resting-state electroencephalogram in 12 participants, aged 5-14 years with a history of unilateral perinatal arterial ischemic or haemorrhagic stroke. A control group of 16 neurologically healthy subjects was also included-each test subject was compared with multiple control subjects, matched by sex and age. Functional connectomes from the alpha frequency band were calculated for each subject and the differences in network graph metrics between the 2 groups were analyzed. Our results suggest that the functional brain networks of children with perinatal stroke show evidence of disruption even years after the insult and that the scale of changes appears to be influenced by the lesion volume. The networks remain more segregated and show a higher synchronization at both whole-brain and intrahemispheric level. Total interhemispheric strength was higher in children with perinatal stroke compared with healthy controls.

Biomarkers of hypoxic-ischemic encephalopathy: a systematic review.

BACKGROUND: Current diagnostic criteria for hypoxic-ischemic encephalopathy in the early hours lack objective measurement tools. Therefore, this systematic review aims to identify putative molecules that can be used in diagnosis in daily clinical practice (PROSPERO ID: CRD42021272610). DATA SOURCES: Searches were performed in PubMed, Web of Science, and Science Direct databases until November 2020. English original papers analyzing samples from newborns > 36 weeks that met at least two American College of Obstetricians and Gynecologists diagnostic criteria and/or imaging evidence of cerebral damage were included. Bias was assessed by the Newcastle-Ottawa Scale. The search and data extraction were verified by two authors separately. RESULTS: From 373 papers, 30 met the inclusion criteria. Data from samples collected in the first 72 hours were extracted, and increased serum levels of neuron-specific enolase and S100-calcium-binding protein-B were associated with a worse prognosis in newborns that suffered an episode of perinatal asphyxia. In addition, the levels of glial fibrillary acidic protein, ubiquitin carboxyl terminal hydrolase isozyme-L1, glutamic pyruvic transaminase-2, lactate, and glucose were elevated in newborns diagnosed with hypoxic-ischemic encephalopathy. Moreover, pathway analysis revealed insulin-like growth factor signaling and alanine, aspartate and glutamate metabolism to be involved in the early molecular response to insult. CONCLUSIONS: Neuron-specific enolase and S100-calcium-binding protein-B are potential biomarkers, since they are correlated with an unfavorable outcome of hypoxic-ischemic encephalopathy newborns. However, more studies are required to determine the sensitivity and specificity of this approach to be validated for clinical practice.

Capsaicin attenuates excitotoxic-induced neonatal brain injury and brain mast cell-mediated neuroinflammation in newborn rats.

Excitotoxicity and neuroinflammation are key contributors to perinatal brain injuries. Capsaicin, an active ingredient of chili peppers, is a potent exogenous agonist for transient receptor potential vanilloid 1 receptors. Although the neuroprotective and anti-inflammatory effects of capsaicin are well-documented, its effects on excitotoxic-induced neonatal brain injury and neuroinflammation have not previously been investigated. The aim of this study was to investigate the effects of capsaicin on brain damage, brain mast cells, and inflammatory mediators in a model of ibotenate-induced excitotoxic brain injury in neonatal rats. P5 rat-pups were intraperitoneally injected with vehicle, 0.2-, 1-, and 5-mg/kg doses of capsaicin, or the NMDA (N-methyl-d-aspartate) receptor antagonist MK-801 (dizocilpine), 30 min before intracerebral injection of 10 µg ibotenate. The naive-control group received no substance administration. The rat pups were sacrificed one or five days after ibotenate injection. Levels of activin A and interleukin (IL)-1ß, IL-6, and IL-10 in brain tissue were measured using the enzyme-linked immunosorbent assay method. Cortex and white matter thicknesses, white matter lesion size, and mast cells were evaluated in brain sections stained with cresyl-violet or toluidine-blue. Capsaicin improved ibotenate-induced white matter lesions and cerebral white and gray matter thicknesses in a dose-dependent manner. In addition, it suppressed the degranulation and increased number of brain mast cells induced by ibotenate. Capsaicin also reduced the excitotoxic-induced production of neuronal survival factor activin A and of the pro-inflammatory cytokines IL-1ß, and IL-6 in brain tissue. However, IL-10 levels were not altered by the treatments. MK-801, as a positive control, reversed all these ibotenate-induced changes, further confirming the success of the model. Our findings provide, for the first time, evidence for the therapeutic effects of capsaicin against excitotoxic-induced neonatal brain injury and brain mast cell-mediated neuroinflammation. Capsaicin may therefore be a promising candidate in the prevention and/or reduction of neonatal brain damage.

Alpha1-antitrypsin protects the immature mouse brain following hypoxic-ischemic injury.

Introduction: Preterm brain injury often leads to lifelong disabilities affecting both cognitive and motor functions, and effective therapies are limited. Alpha1-antitrypsin (AAT), an endogenous inhibitor of serine proteinases with anti-inflammatory, anti-apoptotic, and cytoprotective properties, might be beneficial in treating preterm brain injury. The aim of this study was to investigate whether AAT has neuroprotective effects in a mouse preterm brain injury model. Methods: Preterm brain injury was induced on postnatal day 5, and mouse pups' right common carotid arteries were cut between two ligations followed by hypoxia induction. Brain injury was evaluated through immunohistochemistry staining and magnetic resonance imaging. Fluoro-Jade B and immunohistochemistry staining were performed to investigate the neuronal cell death and blood-brain barrier (BBB) permeability. The motor function and anxiety-like behaviors were revealed by CatWalk gait analysis and the open field test. Results: After hypoxia-ischemia (HI) insult, brain injury was alleviated by AAT treatment, and this was accompanied by reduced BBB permeability, reduced neuronal cell death and caspase-3 activation, and inhibition of microglia activation. In addition, AAT administration significantly improved HI-induced motor function deficiencies in mice. The neuroprotective effect of AAT was more pronounced in male mice. Conclusion: AAT treatment is neuroprotective against preterm brain injury in neonatal mice, and the effect is more pronounced in males.