✍🏼 Joseph Vithayathil, Anjali Shankar, Ginger L Milne, Frances E Jensen, Delia M Talos, Joshua L Dunaief

🏠 Division of Neurology, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA

📑BioRxiv (2025)

Abstract
Iron accumulation and lipid peroxidation are pathophysiologic mechanisms that drive neonatal hypoxic-ischemic (HI) brain injury. Characterization of spatiotemporal changes in these processes will help elucidate their role in ischemic neuronal injury as an initial step towards developing targeted interventions.

HI was induced in post-natal day 9 mice using the modified-Vannucci model. Hippocampal tissue from ipsilateral HI exposed, contralateral hypoxia exposed and sham animals was collected at 6h, 24h, 72h and 7d post-HI. Tissue was subsequently evaluated for markers of cell death (TUNEL), intracellular iron changes (FerroOrange, fluorescent in situ and immunofluorescence), and lipid peroxidation (real time PCR, Gpx4 immunofluorescence and mass spectrometry). Mass spectrometry measured isoprostanes (15-F2t-IsoP) and neuroprostanes (4-F4t-NP) as lipid peroxidation markers of arachidonic (ARA) and docosahexaenoic acid (DHA), respectively.

Compared to sham, the HI hippocampus showed increased intracellular labile iron levels that was maximal at 6h post-HI with subsequent elevation in only neuroprostanes at 24h post-HI. TUNEL labeling peaked at 24h post-HI. At 72h, labile iron levels and lipid peroxidation declined corresponding with peak infiltration of ferritin positive microglia/macrophages and the start of TUNEL staining decline. In addition, surviving neurons had increased expression of Gpx4 peaking at 72h post-HI that normalized by 7d post-HI.

These findings suggest that following HI, an acute increase in labile iron and DHA peroxidation are correlated with markers of cell death that peak at 24h post-HI. Microglial/macrophage iron sequestration and neuronal antioxidant responses may ameliorate further injury and represent targets for neuroprotective therapies.

How the WOLF was used in this study
In the bioRxiv preprint (10.1101/2025.07.02.662653v1), the authors used the WOLF Nanocellect flow cytometer to perform fluorescence-activated cell sorting (FACS) of microglial and immune cells from dissociated mouse brain tissue. Specifically, after preparing single-cell suspensions and staining with an anti-CD11b antibody to label microglia/macrophage populations and a viability dye to exclude dead cells, they ran the samples through the WOLF instrument to isolate viable CD11b⁺ cells. The sorted populations (on the order of 20,000–40,000 cells per sample) were then collected for downstream RNA isolation and gene expression analyses, enabling the researchers to assess cell-specific changes in response to hypoxic-ischemic injury with greater purity and reduced background from non-target or dead cells.