Parameters of Delayed Neuroinflammation Following Focal Ischemic Stroke in the Mouse Cerebral Cortex Depend on Lesion Size at the Beginning of the Subacute Stage

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Vyacheslav I. Alekseev^1,2,a*, Evgenia N. Kislukhina², Natalia V. Lizunova², Alexander M. Surin³, Tatiana V. Lipina¹, Kirill V. Savostyanov², and Zanda V. Bakaeva^2,4,5

¹Department of Cell Biology and Histology, Faculty of Biology, Lomonosov Moscow State University, 119991 Moscow, Russia

²National Medical Research Center for Children’s Health, Ministry of Health of the Russian Federation, 119991 Moscow, Russia

³Research Institute of General Pathology and Pathophysiology, 125315 Moscow, Russia

⁴I. M. Sechenov First Moscow State Medical University (Sechenov University), 119991 Moscow, Russia

⁵B. B. Gorodovikov Kalmyk State University, 358000 Elista, Russia

^* To whom correspondence should be addressed.

Received: November 11, 2025; Revised: April 22, 2026; Accepted: April 23, 2026

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Dynamics of glial activity changes in the subacute and chronic stages of ischemic stroke after small focal injuries remains poorly understood due to complexity of the long-term animal monitoring and data interpretation. The aim of this study was to assess relationship between the delayed morphological changes in nervous tissue after experimental stroke and lesion parameters determined in vivo at various time points. For this purpose, photothrombotic ischemia of the cerebral cortex was induced in the C57BL/6J-Tg(Thy1-GCaMP6f)GP5.17Dkim/J mice, which express fluorescent calcium sensor protein GCaMP6f in cortical neurons. Lesion (ischemic core) size was determined using wide-field optical imaging (WFOI) through a cranial window via the GCaMP6f fluorescence at 3 min, 1 day, and 7 days post-photothrombosis. On day 19, brain sections were analyzed using Nissl staining and immunohistochemistry for microglial (Iba1) and astrocytic (GFAP) markers. It was found that the signs of neuroinflammation – changes in glial cell morphology and quantity – persist in the perifocal region even 19 days after ischemia induction, despite the small lesion volume. A significant linear relationship between microglial nuclear area and lesion size on day 7 was identified. Conversely, no significant correlation was found between the lesion sizes determined in the hyperacute phase (3 min) and acute phase (1 day) and cellular parameters (cell count, morphometric parameters). This indicates that the lesion formation in the acute phase is dynamic, and only the lesion size after its stabilization influences long-term stroke outcomes. Absence of a correlation between the delayed glial changes and ischemic core size during the hyperacute and acute phases suggests that therapeutic window for interventions modulating glial activity may extend to the later period after stroke, even with small lesion size. The results also allow us to conclude that it is not necessary to make an amendment for the initial lesion size in the studies of delayed neuroglial processes in preclinical models. In turn, the correlation between the lesion size on day 7 and microglial cell nucleus area on day 19 demonstrates that the lesion size at the end of the acute phase may be one of the prognostic factors for effectiveness of the post-stroke therapy.

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KEY WORDS: ischemic stroke, photothrombosis, microglia, Iba1 (AIF1, IRT1), immunohistochemical analysis, neuroinflammation, fluorescent Ca²⁺ sensor GCaMP6f, wide-field optical imaging, astrocytes

DOI: 10.1134/S0006297925604009

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