Hypoxic-ischemic encephalopathy (HIE) resulting from asphyxia in the peripartum period is the most common cause of neonatal brain damage and can result in significant neurologic sequelae, including cerebral palsy. Currently therapeutic hypothermia is the only accepted treatment in addition to supportive care for infants with HIE, however, many additional neuroprotective therapies have been investigated. Of these, glucocorticoids have previously been shown to have neuroprotective effects. HIE is also frequently compounded by infectious inflammatory processes (sepsis) and as such, the infants may be more amenable to treatment with an anti-inflammatory agent. Thus, the present study investigated dexamethasone and hydrocortisone treatment given after hypoxic-ischemic (HI) insult in neonatal rats via intracerebroventricular (ICV) injection and intranasal administration. In addition, we examined the effects of hydrocortisone treatment in HIE after lipopolysaccharide (LPS) sensitization in a model of HIE and sepsis. We found that dexamethasone significantly reduced rat brain infarction size when given after HI treatment via ICV injection; however it did not demonstrate any neuroprotective effects when given intranasally. Hydrocortisone after HI insult also significantly reduced brain infarction size when given via ICV injection; and the intranasal administration showed to be protective of brain injury in male rats at a dose of 300 µg. LPS sensitization did significantly increase the brain infarction size compared to controls, and hydrocortisone treatment after LPS sensitization showed a significant decrease in brain infarction size when given via ICV injection, as well as intranasal administration in both genders at a dose of 300 µg. To conclude, these results show that glucocorticoids have significant neuroprotective effects when given after HI injury and that these effects may be even more pronounced when given in circumstances of additional inflammatory injury, such as neonatal sepsis.

Cerebral edema, primarily caused by disruption of the blood-brain barrier (BBB), is one of the serious complications associated with brain injury in neonatal hypoxic-ischemic encephalopathy (HIE). Our recent study demonstrated that the hypoxic-ischemic (HI) treatment significantly increased microRNA-210 (miR-210) in the neonatal rat brain and inhibition of miR-210 provided neuroprotection in neonatal HI brain injury. The present study aims to determine the role of miR-210 in the regulation of BBB integrity in the developing brain. miR-210 mimic was administered via intracerebroventricular injection (i.c.v.) into the brain of rat pups. Forty-eight hours after the injection, a modified Rice-Vannucci model was conducted to produce HI brain injury. Post-assays included cerebral edema analysis, western blotting, and immunofluorescence staining for serum immunoglobulin G (IgG) leakage. The results showed that miR-210 mimic exacerbated cerebral edema and IgG leakage into the brain parenchyma. In contrast, inhibition of miR-210 with its complementary locked nucleic acid oligonucleotides (miR-210-LNA) significantly reduced cerebral edema and IgG leakage. These findings suggest that miR-210 negatively regulates BBB integrity i n the neonatal brain. Mechanistically, the seed sequences of miR-210 were identified complementary to the 3′ untranslated region (3′ UTR) of the mRNA transcripts of tight junction protein occludin and adherens junction protein β-catenin, indicating downstream targets of miR-210. This was further validated by in vivo data showing that miR-210 mimic significantly reduced the expression of these junction proteins in rat pup brains. Of importance, miR-210-LNA preserved the expression of junction proteins occludin and β-catenin from neonatal HI insult. Altogether, the present study reveals a novel mechanism of miR-210 in impairing BBB integrity that contributes to cerebral edema formation after neonatal HI insult, and provides new insights in miR-210-LNA mediated neuroprotection in neonatal HI brain injury.

Strokes are one of the leading causes of mortality and chronic morbidity in the world, yet with only limited successful interventions available at present. Our previous studies revealed the potential role of the glucocorticoid receptor (GR) in the pathogenesis of neonatal hypoxic-ischemic encephalopathy (HIE). In the present study, we investigate the effect of GR knockdown on acute ischemic brain injuries in a model of focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO) in adult male CD1 mice. GR siRNAs and the negative control were administered via intracerebroventricular (i.c.v.) injection 48 h prior to MCAO. The cerebral infarction volume and neurobehavioral deficits were determined 48 h after MCAO. RT-qPCR was employed to assess the inflammation-related gene expression profiles in the brain before and after MCAO. Western Blotting was used to evaluate the expression levels of GR, the mineralocorticoid receptor (MR) and the brain-derived neurotrophic factor/tropomyosin receptor kinase B (BDNF/TrkB) signaling. The siRNAs treatment decreased GR, but not MR, protein expression, and significantly enhanced expression levels of pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α) in the brain. Of interest, GR knockdown suppressed BDNF/TrkB signaling in adult mice brains. Importantly, GR siRNA pretreatment significantly increased the infarction size and exacerbated the neurobehavioral deficits induced by MCAO in comparison to the control group. Thus, the present study demonstrates the important role of GR in the regulation of the inflammatory responses and neurotrophic BDNF/TrkB signaling pathway in acute ischemic brain injuries in adult mice, revealing a new insight into the pathogenesis and therapeutic potential in acute ischemic strokes.

Hypoxic-ischemic encephalopathy (HIE) resulting from asphyxia is the most common cause of neonatal brain damage and results in significant neurological sequelae, including cerebral palsy. The current therapeutic interventions are extremely limited in improving neonatal outcomes. The present study tests the hypothesis that the suppression of endogenous glucocorticoid receptors (GRs) in the brain increases hypoxic-ischemic (HI) induced neonatal brain injury and worsens neurobehavioral outcomes through the promotion of increased inflammation. A mild HI treatment of P9 rat pups with ligation of the right common carotid artery followed by the treatment of 8% O₂ for 60 min produced more significant brain injury with larger infarct size in female than male pups. Intracerebroventricular injection of GR siRNAs significantly reduced GR protein and mRNA abundance in the neonatal brain. Knockdown of endogenous brain GRs significantly increased brain infarct size after HI injury in male, but not female, rat pups. Moreover, GR repression resulted in a significant increase in inflammatory cytokines TNF-α and IL-10 at 6 h after HI injury in male pups. Male pups treated with GR siRNAs showed a significantly worsened reflex response and exhibited significant gait disturbances. The present study demonstrates that endogenous brain GRs play an important role in protecting the neonatal brain from HI induced injury in male pups, and suggests a potential role of glucocorticoids in sex differential treatment of HIE in the neonate.

Nicotine exposure either from maternal cigarette smoking or e-cigarette vaping is one of the most common risk factors for neurodevelopmental disease in offspring. Previous studies revealed that perinatal nicotine exposure programs a sensitive phenotype to neonatal hypoxic-ischemic encephalopathy (HIE) in postnatal life, yet the underlying mechanisms remain undetermined. The goal of the present study was to determine the regulatory role of H19/miR-181a/ATG5 signaling in perinatal nicotine exposure-induced development of neonatal brain hypoxic-ischemic sensitive phenotype. Nicotine was administered to pregnant rats via subcutaneous osmotic minipumps. All experiments were conducted in offspring pups at postnatal day 9 (P9). Perinatal nicotine exposure significantly enhanced expression of miR-181a but attenuated autophagy-related protein 5 (ATG5) mRNA and protein levels in neonatal brains. Of interest, miR-181a mimicking administration in the absence of nicotine exposure also produced dose-dependent increased hypoxia/ischemia (H/I)-induced brain injury associated with a decreased ATG5 expression, closely resembling perinatal nicotine exposure-mediated effects. Locked nucleic acid (LNA)-miR-181a antisense reversed perinatal nicotine-mediated increase in H/I-induced brain injury and normalized aberrant ATG5 expression. In addition, nicotine exposure attenuated a long non-coding RNA (lncRNA) H19 expression level. Knockdown of H19 via siRNA increased the miR-181a level and enhanced H/I-induced neonatal brain injury. In conclusion, the present findings provide a novel mechanism that aberrant alteration of the H19/miR-181a/AGT5 axis plays a vital role in perinatal nicotine exposure-mediated ischemia-sensitive phenotype in offspring and suggests promising molecular targets for intervention and rescuing nicotine-induced adverse programming effects in offspring.

Adult hematopoietic stem cells (HSCs) and bone marrow (BM) mesenchymal stem/stromal cells (MSCs) are essential for lifelong hematopoiesis, skeletal homeostasis, immune competence, and tissue regeneration. The use of electronic cigarettes (E-cigs) among women of reproductive age continues to rise, raising concerns about potential adverse developmental effects; however, the long-term consequences of maternal E-cig vaping on offspring BM stem cell function and hematopoietic homeostasis remain incompletely understood. Here, using a rat model of maternal E-cig exposure (containing nicotine) during gestation, combined with longitudinal in vivo analyses and complementary ex vivo studies of human cells, we show that prenatal E-cig exposure is associated with persistent alterations in offspring BM stem cell function and lineage commitment. Gestational E-cig exposure was associated with expansion of the CD11b/c⁺ myeloid-enriched compartment, increased CD90⁺ stromal cells, and impaired osteogenic differentiation in rat offspring. Complementary experiments using primary human cells showed that nicotine exposure was associated with reduced T-cell proliferation and impaired cytotoxic activity in a proof-of-principle co-culture assay. Mechanistically, transcriptomic profiling followed by Gene Ontology and pathway enrichment analyses identified alterations in molecular programs associated with KLF4–Notch1 signaling, mitochondrial biogenesis, inflammation, and stem cell regulation in the BM of E-cig-exposed rat offspring. Changes in CCL11, FTO, and RUNX2 were additionally associated with an inflammatory and aging-related molecular phenotype that persisted from early life into adulthood, although these findings do not establish a causal CCL11–FTO–RUNX2 signaling axis or direct cellular senescence. Collectively, our study provides a phenotypic and mechanistic framework for understanding how maternal E-cig exposure may influence long-term offspring hematopoietic, skeletal, and immune health while highlighting the need for further studies to establish causal molecular mechanisms and determine their relevance to maternal E-cig use in humans.