BACKGROUND: Neonatal hypoxic-ischemic encephalopathy disproportionately affects low- and middle-income countries, where ≈96% of affected infants reside. The current standard of care, therapeutic hypothermia, is frequently ineffective in this setting, likely because injury may be occurring earlier during labor. Here, we studied the pharmacokinetics, safety, and efficacy of perinatal caffeine administration in near-term lambs following global ischemic injury to support the development of earlier treatment strategies targeting the fetus in utero as well as the infant postnatally. METHODS: Ewes were randomly assigned to receive either 1 g IV caffeine citrate or placebo before delivery and placental transport assessed. Near-term lambs (141–143 days) of both sexes were subjected to severe global hypoxia-ischemia utilizing an acute umbilical cord occlusion model. Lambs that received caffeine in utero also received 20 mg/kg IV caffeine citrate following resuscitation and 10 mg/(kg·d) IV for 2 days. An additional cohort received 60 mg/kg followed by 30 mg/(kg·d) (low dose versus high dose) postnatally. Biochemical, histological, and neurological outcome measures in lambs were assessed over a 6-day period. RESULTS: Perinatal caffeine administration demonstrated excellent placental transport kinetics and was well tolerated with lamb plasma levels comparable to those targeted in neonates with apnea of prematurity. Caffeine administration resulted in a systemic immunomodulatory effect, evidenced by significant reductions in proinflammatory IP-10 levels. Treated lambs demonstrated improved neurodevelopmental outcomes, while histological analysis revealed that caffeine reduced gray matter injury and attenuated inflammation in the cingulate and parasagittal cortex. This neuroprotective effect was greater and via a different mode of action than we previously reported for azithromycin. A higher caffeine dosing regimen demonstrated significant toxicity. CONCLUSIONS: Perinatal caffeine administration is well tolerated, attenuates systemic and brain inflammation, and contributes to improvements in histological and neurological outcomes in an ovine model of neonatal hypoxic-ischemic encephalopathy.

BACKGROUND: Hypoxic-ischemic brain injury/encephalopathy affects about 1.15 million neonates per year, 96% of whom are born in low- and middle-income countries. Therapeutic hypothermia is not effective in this setting, possibly because injury occurs significantly before birth. Here, we studied the pharmacokinetics, safety, and efficacy of perinatal azithromycin administration in near-term lambs following global ischemic injury to support earlier treatment approaches. METHODS: Ewes and their lambs of both sexes (n=34, 141–143 days) were randomly assigned to receive azithromycin or placebo before delivery as well as postnatally. Lambs were subjected to severe global hypoxia-ischemia utilizing an acute umbilical cord occlusion model. Outcomes were assessed over a 6-day period. RESULTS: While maternal azithromycin exhibited relatively low placental transfer, azithromycin-treated lambs recovered spontaneous circulation faster following the initiation of cardiopulmonary resuscitation and were extubated sooner. Additionally, peri- and postnatal azithromycin administration was well tolerated, demonstrating a 77-hour plasma elimination half-life, as well as significant accumulation in the brain and other tissues. Azithromycin administration resulted in a systemic immunomodulatory effect, demonstrated by reductions in proinflammatory IL-6 (interleukin-6) levels. Treated lambs exhibited a trend toward improved neurodevelopmental outcomes while histological analysis revealed that azithromycin supported white matter preservation and attenuated inflammation in the cingulate and parasagittal cortex. CONCLUSIONS: Perinatal azithromycin administration enhances neonatal resuscitation, attenuates neuroinflammation, and supports limited improvement of select histological outcomes in an ovine model of hypoxic-ischemic brain injury/encephalopathy.

Originally approved by the U.S. Food and Drug Administration (FDA) for its antihistamine properties, clemastine can also promote white matter integrity and has shown promise in the treatment of demyelinating diseases such as multiple sclerosis. Here, we conducted an in-depth analysis of the feasibility, safety, and neuroprotective efficacy of clemastine administration in near-term lambs (n = 25, 141–143 days) following a global ischemic insult induced via an umbilical cord occlusion (UCO) model. Lambs were randomly assigned to receive clemastine or placebo postnatally, and outcomes were assessed over a six-day period. Clemastine administration was well tolerated. While treated lambs demonstrated improvements in inflammatory scores, their neurodevelopmental outcomes were unchanged.

Hypoxic-ischemic encephalopathy (HIE) is the leading cause of neonatal morbidity and mortality worldwide. Approximately 1 million infants born with HIE each year survive with cerebral palsy and/or serious cognitive disabilities. While infants born with mild and severe HIE frequently result in predictable outcomes, infants born with moderate HIE exhibit variable outcomes that are highly unpredictable. Here, we describe an umbilical cord occlusion (UCO) model of moderate HIE with a 6-day follow-up. Near-term lambs (n = 27) were resuscitated after the induction of 5 min of asystole. Following recovery, lambs were assessed to define neurodevelopmental outcomes. At the end of this period, lambs were euthanized, and brains were harvested for histological analysis. Compared with prior models that typically follow lambs for 3 days, the observation of neurobehavioral outcomes for 6 days enabled identification of animals that recover significant neurological function. Approximately 35% of lambs exhibited severe motor deficits throughout the entirety of the 6-day course and, in the most severely affected lambs, developed spastic diparesis similar to that observed in infants who survive severe neonatal HIE (severe, UCOs). Importantly, and similar to outcomes in human neonates, while initially developing significant acidosis and encephalopathy, the remainder of the lambs in this model recovered normal motor activity and exhibited normal neurodevelopmental outcomes by 6 days of life (improved, UCOi). The UCOs group exhibited gliosis and inflammation in both white and gray matters, oligodendrocyte loss, neuronal loss, and cellular death in the hippocampus and cingulate cortex. While the UCOi group exhibited more cellular death and gliosis in the parasagittal cortex, they demonstrated more preserved white matter markers, along with reduced markers of inflammation and lower cellular death and neuronal loss in Ca3 of the hippocampus compared with UCOs lambs. Our large animal model of moderate HIE with prolonged follow-up will help further define pathophysiologic drivers of brain injury while enabling identification of predictive biomarkers that correlate with disease outcomes and ultimately help support development of therapeutic approaches to this challenging clinical scenario.

Background: Hypoxic–ischemic (HI) brain injury triggers a dynamic, multi-phase response involving early microglial efferocytosis followed by extracellular matrix (ECM) deposition and scar formation. Arginase-1 (ARG1), a key enzyme in tissue repair, is implicated in both processes, yet its role in neonatal microglia remains poorly defined. We characterize ARG1-linked pathways in neonatal microglia, identifying distinct efferocytic and fibrotic phases post-HI. Methods: HI was induced in P9 mice using the Vannucci model, and brains were collected at 24 h (D1) and 5 days (D5). Spatially resolved single-cell transcriptomics (seqFISH) was performed using a targeted panel enriched for microglial, ARG1-pathway, efferocytosis, and profibrotic genes. Cell segmentation, clustering, and spatial mapping were conducted using Navigator and Seurat. Differential expression, GSEA, and enrichment analyses were used to identify time- and injury-dependent pathways. Results: Spatial transcriptomics identified 12 transcriptionally distinct cell populations with preserved neuroanatomical organization. HI caused the expansion of microglia and astrocytes and the loss of glutamatergic neurons by D5. Microglia rapidly activated regenerative and profibrotic programs—including TGF-β, PI3K–Akt, cytoskeletal remodeling, and migration—driven by early DEGs such as Cd44, Reln, TGF-β1, and Col1a2. By D5, microglia adopted a collagen-rich fibrotic state with an upregulation of Bgn, Col11a1, Anxa5, and Npy. Conclusion: Neonatal microglia transition from early efferocytic responses to later fibrotic remodeling after HI, driven by the persistent activation of PI3K–Akt, TGF-β, and Wnt/FZD4 pathways. These findings identify microglia as central regulators of neonatal scar formation and highlight therapeutic targets within ARG1-linked signaling.

Introduction: Neonatal hypoxic-ischemic (HI) brain injury is a major cause of mortality and long-term neurological disability, yet effective neuroprotective strategies remain limited. Microglia are central mediators of injury and repair, with arginase 1 (ARG1) marking anti-inflammatory, reparative states. However, the functional roles of ARG1+ microglia in tissue remodeling after HI are poorly understood. Methods: Neonatal mice (P10) underwent HI using the Vannucci procedure. ARG1 activity was inhibited pharmacologically using N-omega-hydroxy-nor-L-arginine (nor-NOHA). ARG1 expression, microglial morphology, efferocytosis, tissue scar, and injury volume were assessed via immunohistochemistry, Western blotting, and arginase activity assays at 1 and 5 days post-injury. Results: ARG1+ microglia rapidly engaged apoptotic neurons, exhibiting phagocytic activity confirmed by CD68 expression. Nor-NOHA treatment reduced ARG1 enzymatic activity, impaired microglial process extension, attenuated efferocytosis, and increased injury volume. ARG1+ microglia persisted in the glial scar and colocalized with collagen I alpha-1 (Col1a1), suggesting a role in extracellular matrix (ECM) deposition. Inhibition of ARG1 decreased Col1a1 expression, highlighting its contribution to tissue remodeling. Conclusions: ARG1+ microglia are pivotal in neonatal HI, mediating early efferocytosis and later ECM remodeling, thereby limiting injury and shaping scar architecture. Pharmacological blockade of ARG1 exacerbates injury, underscoring its reparative function. These findings establish ARG1 as a critical regulator of microglial-mediated neuroprotection and tissue repair, providing a potential therapeutic target for neonatal HI brain injury.