BACKGROUND: Neonatal hypoxic-ischemic (HI) brain injury is one of the leading causes of long-term neurological morbidity in newborns. Current treatment options for HI brain injury are limited, but mesenchymal stem cell (MSC) therapy is a promising strategy to boost neuroregeneration after injury. Optimization strategies to further enhance the potential of MSCs are under development. The current study aimed to test the potency of hypoxic preconditioning of MSCs to enhance the therapeutic efficacy in a mouse model of neonatal HI injury. METHODS: HI was induced on postnatal day 9 in C57Bl/6 mouse pups. MSCs were cultured under hypoxic (hypoxic-preconditioned MSCs [HP-MSCs], 1% O ₂ ) or normoxic-control (normoxic-preconditioned MSCs [NP-MSCs], 21% O ₂ ) conditions for 24 hours before use. At 10 days after HI, HP-MSCs, NP-MSCs, or vehicle were intranasally administered. Gold nanoparticle–labeled MSCs were used to assess MSC migration 24 hours after intranasal administration. At 28 days post-HI, lesion size, sensorimotor outcome, and neuroinflammation were assessed by hematoxylin and eosin staining, cylinder rearing task, and ionized calcium-binding adapter molecule 1 (IBA1) staining, respectively. In vitro, the effect of HP-MSCs was studied on transwell migration, neural stem cell differentiation and microglia activation, and the MSC intracellular proteomic content was profiled using quantitative Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). RESULTS: Intranasally administered HP-MSCs were superior to NP-MSCs in reducing lesion size and sensorimotor impairments post-HI. Moreover, hypoxic preconditioning enhanced MSC migration in an in vitro set-up, and in vivo to the lesioned hemisphere after intranasal application. In addition, HP-MSCs enhanced neural stem cell differentiation into more complex neurons in vitro but had similar anti-inflammatory effects compared with NP-MSCs. Lastly, hypoxic preconditioning led to elevated abundances of proteins in MSCs related to extracellular matrix remodeling. CONCLUSIONS: This study shows for the first time that hypoxic preconditioning enhanced the therapeutic efficacy of MSC therapy in a mouse model of neonatal HI brain injury by increasing the migratory and neuroregenerative capacity of MSCs.

Hypoxic-ischemic (HI) brain injury is associated with high mortality and severe long-term neurodevelopmental impairments in term newborns. Intranasal mesenchymal stem cell (MSC) therapy is a promising strategy to boost neurorepair after injury, and optimization strategies to further enhance its therapeutic potential are under development. In this study, we explored whether 24 h preconditioning of MSCs with 1000 ng/mL of osteopontin (OPN) could enhance MSC properties in vitro and in vivo. OPN-preconditioned MSCs (OPN-MSCs) showed increased activation of the ERK transcription pathway at 1 h during preconditioning and enhanced migration compared to naïve-MSCs. OPN preconditioning also altered gene expression of neurotrophic and immunomodulatory factors in MSCs. In vitro assessment of MSC potency showed that while OPN-MSCs were as effective as naïve-MSCs in reducing microglia activation, OPN preconditioning enhanced the potency of MSCs to boost neural stem cell differentiation into more complex neurons. However, in vivo, OPN-MSCs were not superior to naïve-MSCs in reducing lesion size in mice when applied at 3 days post-HI. Altogether, OPN preconditioning enhanced the migratory and neurotrophic properties of MSCs in vitro but not in vivo, highlighting its potential to optimize MSC function while underscoring the need for further research to refine in vivo translation and to evaluate functional outcomes for therapeutic efficacy.

Background: Hypoxic–ischemic (HI) brain injury due to perinatal asphyxia causes long‐term neurological morbidity in newborns. HI affects brain areas including the cortex, hippocampus, and basal ganglia. More recently the mammillary bodies (MBs), critical for memory, were also shown to be affected in patients, despite treatment with therapeutic hypothermia (TH). Here we characterized the kinetics and extent of MB damage in an established neonatal HI mouse model and evaluated the therapeutic potential of TH or a novel regenerative intervention: intranasal mesenchymal stem cell (MSC) therapy. Methods: HI was induced in 9‐day‐old mouse pups; structural and cellular MB damage was assessed at days 1 to 3 and day 28 post HI by immunohistochemistry. For TH, mice were placed at 32 °C for 3 hours directly post HI, and MB damage was assessed at day 10. For MSC therapy, mice received intranasally 0.5×10 ⁶ MSCs at day 10 post HI and MB damage was assessed at day 28 post HI. Results: At days 1 to 3 post HI, mice showed cell death, microglia activation, and early gliosis in the ipsilateral MB. Glial scarring was still present at day 28 post HI with concomitant ipsilateral MB atrophy. Additionally, HI induced mammillothalamic tract atrophy and reduced the expression of calbindin ⁺ but not parvalbumin ⁺ cells. While TH and intranasal MSCs both reduced the HI‐induced cortical/hippocampal lesions, neither treatment reduced MB damage. Conclusions: Our novel findings demonstrate early onset and long‐lasting MB damage in a neonatal HI mouse model, mirroring newborn patients. TH and MSCs therapy failed to protect the MBs, highlighting the importance for future research into treatment strategies to protect or repair this vulnerable structure.