A Minimally Invasive, Scalable and Reproducible Neonatal Rat Model of Severe Focal Brain Injury

Mondal, V.; Ross-Munro, E.; Balasuriya, G. K.; Kumari, R.; Shearer, I. K.; Micic, A.; Sohag, A. A. M.; Shi, A.; Barresi, M.; Nisbet, D. R.; King, G. F.; Williams, R. J.; Gressens, P.; Wong, F. Y.-w.; Cheong, J.; Walker, D. W.; Tolcos, M.; Fleiss, B.

doi:10.1101/2025.11.10.687751

bioRxiv · 10.1101/2025.11.10.687751

A Minimally Invasive, Scalable and Reproducible Neonatal Rat Model of Severe Focal Brain Injury

Mondal, V.·Ross-Munro, E.·Balasuriya, G. K.·Kumari, R.·Shearer, I. K.·Micic, A.·Sohag, A. A. M.·Shi, A.·Barresi, M.·Nisbet, D. R.·King, G. F.·Williams, R. J.·Gressens, P.·Wong, F. Y.-w.·Cheong, J.·Walker, D. W.·Tolcos, M.·Fleiss, B.

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Abstract

BackgroundNeonatal brain injuries such as stroke cause focal ischemic lesions that often result in lifelong neurological disabilities, as treatment options are limited. To speed up the discovery of potential therapies, early-phase screening with models that reliably reproduce brain injury, with scalable injury volume and minimal confounders, such as varying anaesthesia duration and painful procedures, is essential. MethodsPostnatal day 10 Sprague-Dawley rats of both sexes, with four litters per group and timepoint, were randomly allocated to delivery of intraperitoneal Rose Bengal (25, 40, or 60 mg/kg) and 10 minutes of light-emitting diode illumination through the intact scalp and skull. Infarct progression and reproducibility were assessed at 24 hours, 7 days, and 14 days post-injury. Outcomes included infarct volume and sensorimotor function, and cleaved caspase-3, glial fibrillary acidic protein (GFAP), and ionised calcium-binding adaptor molecule 1 (Iba1) immunoreactivity, with analysis of sex differences. Data were analysed using one-way or two-way ANOVA with Sidaks post-hoc tests. ResultsThere was no mortality due to the infarct, and procedure time was approximately 19 minutes across all groups; the lesion was consistent and supported scalability. The 25 mg/kg dose produced a reproducible cortical infarct (3.74 {+/-} 0.58 mm3; CV = 31%). Lesion size increased with dose and decreased over time (11.15 {+/-} 0.63 mm3 at 60 mg/kg versus 0.05 {+/-} 0.007 mm3 at 14 days; p < 0.0001). Cleaved caspase-3 and glial activation persisted for 14 days, indicating ongoing apoptosis and gliosis. No sex-dependent effects were observed in lesion volume, behaviour, or gliosis. ConclusionsThis refined neonatal photothrombotic ischaemia model is reproducible, scalable, and ethically improved, requiring no skin incision. Its minimal surgical burden, absence of mortality, consistent histopathology, and measurable functional outcomes make it an ideal platform for preclinical screening of neuroprotective and reparative interventions in the developing brain.

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Mondal, V., Ross-Munro, E., Balasuriya, G. K., Kumari, R., Shearer, I. K., Micic, A., Sohag, A. A. M., Shi, A., Barresi, M., Nisbet, D. R., King, G. F., Williams, R. J., Gressens, P., Wong, F. Y.-w., Cheong, J., Walker, D. W., Tolcos, M., Fleiss, B.. 2025-11-13. A Minimally Invasive, Scalable and Reproducible Neonatal Rat Model of Severe Focal Brain Injury. https://doi.org/10.1101/2025.11.10.687751

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