Isabelle K. Shearer

Kynurenine pathway (KP) metabolite imbalance is implicated in perinatal brain injury, and KP metabolites are measured in human plasma and cerebrospinal fluid as markers of neuroinflammation. Methods validated in neonatal brain tissue or glial culture supernatant to the same standard are scarce, limiting comparisons across preclinical and clinical measurements. We optimised LC-MS/MS detection of tryptophan (TRYP), kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), quinolinic acid (QA), anthranilic acid and picolinic acid after protein precipitation and Enhanced Matrix Removal-Lipid clean-up, validated it against Clinical and Laboratory Standards Institute (CLSI) acceptance criteria in brain tissue homogenate and cell culture supernatant, and applied it to a rat model of neonatal stroke and immune-activated BV2 microglia, reporting KYN/TRYP, KYNA/KYN, 3-HK/3-HAA and QA/KYNA ratios. The lower limit of quantification was 6.53-34.17 nM; imprecision was 0.57-11.47%, accuracy was 84.9-108.3%, recovery was 83.2-112.3%, and matrix effect was 87.8-108.8%, with linear calibration (R ² > 0.99) over 6.5-2000 nM. KYN/TRYP and QA/KYNA ratios were higher in the stroke hemisphere than in control brain at 1, 7 and 14 days (p<0.001), and KYNA/KYN was lower; lipopolysaccharide- and cytokine-stimulated BV2 microglia showed the same change in all four ratios (p<0.01). The assay quantifies eight KP metabolites and four pathway-balance ratios in brain tissue and microglial supernatant within clinical acceptance limits, allowing direct comparison with KP ratios reported in human plasma and cerebrospinal fluid. Changes in these ratios after neonatal stroke identify KYN/TRYP and QA/KYNA as candidate targets for neonatal biofluid studies.

Microglia play dynamic roles in the developing brain and are central mediators of injury responses in perinatal brain injury. In mice, microglial gene regulatory and transcriptional programes reveal progression through early, pre-mature and mature stages. In contrast, microglial maturation has not been systematically characterised in the large-brained species most widely used to model human perinatal brain injury, particularly sheep and pigs. These large-animal models are indispensable as they share gyrencephaly, an expanded subplate, and clinically relevant physiology with the human infant brain. Here, we integrate established mouse and human frameworks of microglial maturation with a critical re-analysis of available sheep and pig datasets to assess whether rodent-derived insights into microglial development extend to large-animal models. Current sheep datasets lack sufficient resolution to infer maturation states, whereas pig data, although limited, reveal stage-dependent patterns consistent with late-gestation human development. This review also briefly considers emerging data on microglial development in non-human primates and the extent to which microglial gene expression programes appear conserved across species. Overall, microglial transitions are most dynamic during fetal and early postnatal life, underscoring the importance of developmentally aligned benchmarks for interpreting injury responses and informing microglia-targeted neuroprotective strategies. Impact: This article provides the first structured comparison of microglial maturation across human and mouse and uses these frameworks to benchmark large-brain animal models of perinatal brain injury, addressing a key translational gap. It shows that while existing sheep datasets lack sufficient resolution to define microglial maturation states, available pig data align closely with human late-gestation microglial development, supporting their use for developmental benchmarking. The work highlights that failure to account for microglial developmental stage risks misinterpretation of injury responses and underscores the need for developmentally aligned microglial markers in large-animal and non-human primate models to guide microglia-targeted neuroprotective strategies.

Neonatal brain injuries, such as stroke, cause focal ischaemic lesions that often result in lifelong neurological disabilities, yet effective treatments remain limited. Early-phase therapeutic screening requires models that can reliably reproduce injury severity while minimising confounding variables, including prolonged or variable anaesthesia, surgical stress, and invasive procedures that themselves affect injury progression. Existing models of neonatal focal ischaemia often exhibit high mortality, technical complexity, and substantial variability in lesion location and volume. As a result, there is a critical need for a rapid, ethically refined, and scalable neonatal model that produces consistent cortical injury suitable for screening neuroprotective, biomaterial-based, and regenerative therapies. We established a minimally invasive photothrombotic ischaemia model in postnatal day 10 rats by administering intraperitoneal Rose Bengal (25, 40, or 60 mg/kg) and activating it with a fixed 10-minute exposure to 565-nm light through the intact scalp and skull. This incision-free protocol allowed a total procedure duration of 19 min. We characterized dose-dependent effects on infarct volume and anatomical distribution, cortical atrophy, ventricular enlargement, apoptosis (cleaved caspase-3), astrocytic and microglial reactivity (glial fibrillary acidic protein, GFAP; ionized calcium-binding adapter molecule 1, Iba1), and sensorimotor outcomes (wire hang, cylinder rearing, adhesive tape removal) at 1, 7, and 14 days after injury. Additional analyses assessed the reproducibility of lesion size across litters and explored sex-specific differences. A 25 mg/kg dose induced a reliable and well-localized motor cortex infarct with no mortality. Higher doses of Rose Bengal produced proportionally larger infarcts with greater subcortical involvement and more pronounced secondary atrophy. Across all groups, apoptotic signalling and glial reactivity remained elevated through 14 days, indicating persistent tissue injury. Sensorimotor impairments were robust at all stages, with deficits in forepaw use, endurance, and tactile response correlating with lesion volumes in the 25 mg/kg group. No significant sex differences were observed for any histological or behavioural outcomes. This refined neonatal photothrombotic model provides a reproducible, simple, scalable, and ethically optimized platform for inducing severe focal cortical injury. The model’s stable injury territory, short, standardized procedure, and consistent functional readouts fill a major gap in current research tools and provide a practical foundation for early-phase testing of neuroprotective and regenerative interventions.

Perinatal brain injuries, including encephalopathy related to fetal growth restriction, encephalopathy of prematurity, neonatal encephalopathy of the term neonate, and neonatal stroke, are a major cause of neurodevelopmental disorders. They trigger cellular and molecular cascades that lead in many cases to permanent motor, cognitive, and/or behavioral deficits. Damage includes neuronal degeneration, selective loss of subclasses of interneurons, blocked maturation of oligodendrocyte progenitor cells leading to dysmyelination, axonopathy and very likely synaptopathy, leading to impaired connectivity. The nature and severity of changes vary according to the type and severity of insult and maturation stage of the brain. Microglial activation has been demonstrated almost ubiquitously in perinatal brain injuries and these responses are key cell orchestrators of brain pathology but also attempts at repair. These divergent roles are facilitated by a diverse suite of transcriptional profiles and through a complex dialogue with other brain cell types. Adding to the complexity of understanding microglia and how to modulate them to protect the brain is that these cells have their own developmental stages, enabling them to be key participants in brain building. Of note, not only do microglia help build the brain and respond to brain injury, but they are a key cell in the transduction of systemic inflammation into neuroinflammation. Systemic inflammatory exposure is a key risk factor for poor neurodevelopmental outcomes in preterm born infants. Based on these observations, microglia appear as a key cell target for neuroprotection in perinatal brain injuries. Numerous strategies have been developed experimentally to modulate microglia and attenuate brain injury based on these strong supporting data and we will summarize these.