Post-Ischemic Long-Term Neuronal Changes in the Sensorimotor Cortex in the Experimental Setting

The aim of the study was to identify the characteristics of dynamic reactive neuronal changes in sensorimotor cortex (SMC) layers III and V of the rat brain in the remote period (up to 270 days) following bilateral common carotid arteries ligation (CCAL).

Methods. The experiment was conducted on 66 male Wistar rats (a prospective cohort study with sequential terminal outcomes): intact control (n = 6) and groups with survival at 30, 90, 150, 210, and 270 days post-bilateral CCAL (n = 6 in each group after adjusting for mortality). Histological methods (Nissl and hematoxylin-eosin staining) were used to assess the total neuronal numerical density (NND), the density of four types of reactive altered cells (hyperchromatic shriveled cells — HCSC; hyperchromatic non-shriveled cells — HCNSC; hypochromatic cells — HCC; shadow cells — SC) and the neuroglial index (NGI). Paired and multiple comparison methods, linear mixed models (LMM) with random intercept for the animal (to account for the paired data structure), Jonckheere–Terpstra test, quadratic trend test, correlation analysis, and ∆-analysis were applied.

Results. A biphasic reduction in NND was observed in layer III of the SMC (maximum of -44.6% at 30 days, p < 0.001) with a wave-like dynamics (quadratic trend: F = 16.4, p < 0.001). HCNSC peak at 30 days (+683%) followed by a decrease, while HCC showed a delayed peak at 150 days (+500%); both parameters showed a mixed pattern (steadily increasing trend: Z = 1.88, p=0.030 for HCNSC; Z = 2.45, p = 0.007 for HCC; quadratic component: F = 8.2 and 7.4, p < 0.01). SCs demonstrated non-monotonous dynamics (quadratic trend: F = 19.2, p < 0.001) with a delayed peak at 210 days. NGI peaked at 30 days (transient gliosis, +95.5%, steadily decreasing trend, Z = –4.92, p < 0.001). The decrease in NND was less pronounced in layer V of the SMC, (–11.6% at 150 days, p = 0.048), but there was an extreme increase in HCSC at 30 days (+945% from control, with a continuously decreasing trend, Z = –2.94, p = 0.002), a monotonous depletion of HCC (linear increasing trend, Z = 4.82, p < 0.001), and a prolonged building up gliosis (NGI +42% by 270 days, mixed pattern: steadily increasing trend Z = 4.15, p < 0.001; quadratic component F = 5.1, p = 0.028). LMM confirmed a significant «Layer × Time» interaction for all parameters (F = 24.1–71.2; p < 0.001). A correlational analysis revealed moderate positive correlations between the layers (for SCs: r = 0.58; r_partial = 0.52; p = 0.004; for HCSC: r = 0.52; p = 0.008; for NGI: r = 0.44; p = 0.016). The ∆-analysis showed consistency in changes for SC, HCSC, and NGI. The extremum moments for SC (210 days) and HCSC (30 days) were synchronous in both layers. Temporal precedence of layer III was found for NGI (early peak of gliosis at 30 days compared to 270 days in layer V), as well as for HCSC and HCC (30–150 days compared to 90 days in layer V). Temporal precedence of layer V was identified for the first peak of SC (90 days compared to 210 days in layer III).

Conclusion. Chronic ischemia induces various damage scenarios: layer III is characterized by a biphasic reduction in NND, asynchronous peaks of HCSC (30 days) and HCC (150 days), a delayed SC peak (210 days), and transient gliosis (a model of damage with delayed degeneration); layer V exhibits an extreme early increase in HCSC (30 days), early peak of SC (90 days), and prolonged gliosis (a model of progressive degeneration). Correlation and ∆-analysis indicate moderate synchrony in the degeneration processes; no convincing evidence of cascading damage propagation from superficial layers to deeper ones was obtained.

1. Iadecola C. The pathobiology of vascular dementia. Neuron. 2013; 80 (4): 844–866. DOI: 10.1016/j.neuron.2013.10.008. PMID: 24267647.

2. Morgan A. E., Mc Auley M. T. Vascular dementia: from pathobiology to emerging perspectives. Ageing Res Rev. 2024; 96: 102278. DOI: 10.1016/j.arr.2024.102278. PMID: 38513772.

3. Kalaria R.N. The pathology and pathophysiology of vascular dementia. Neuropharmacol. 2018; 134 (Pt B): 226–239. DOI: 10.1016/j.neuropharm.2017.12.030. PMID: 29273521.

4. Zhang X., Chang S., Liu J., Wang D. Combined metabolomics and proteomics analysis of vascular cognitive impairment in hypertensive rats induced by endothelial injury. PLoS One. 2025; 20 (10): e0332827. DOI: 10.1371/journal.pone.0332827. PMID: 41066429.

5. Schaechter J.D., Perdue K.L. Enhanced corticospinal tract fiber integrity and motor recovery following stroke evidenced by diffusion tensor imaging. Neurorehabil Neural Repair. 2008; 22 (6): 758–772. DOI: 10.1177/1545968308317530.

6. DeFelipe J. The anatomical problem posed by brain complexity and size: a potential solution. Front Neuroanat. 2015; 9: 104. DOI: 10.3389/fnana.2015.00104. PMID: 26347617.

7. Zhang S., Murphy T.H. Imaging the impact of cortical microcirculation on synaptic structure and sensory-evoked hemodynamic responses in vivo. PLoS Biol. 2007; 5 (5): e119. DOI: 10.1371/journal.pbio.0050119. PMID: 17456007.

8. Carmichael S.T. Brain excitability in stroke: the yin and yang of stroke progression. Arch Neurol. 2012; 69 (2): 161–167. DOI: 10.1001/archneurol.2011.1175. PMID: 21987395.

9. Kalinichenko S.G., Korobtsov A.V., Matveeva N.Yu., Pushchin I.I. Structural and chemical changes in glial cells in the rat neocortex induced by constant occlusion of the middle cerebral artery. Acta Histochem. 2020; 122 (5): 151573. DOI: 10.1016/j.acthis.2020.151573. PMID: 32622419.

10. Zhang X., Wei M., Fan J., Yan W., Zha X., Song H., Wan R., Yin Y., Wang W. Ischemia-induced upregulation of autophagy preludes dysfunctional lysosomal storage and associated synaptic impairments in neurons. Autophagy. 2021; 17 (6): 1519–1542. DOI: 10.1080/15548627.2020.1840796. PMID: 33111641.

11. Avdeev D.B., Stepanov S.S., Gorbunova A.V., Shoronova A.Yu., Makaryeva L.M., Akulinin V.A., Korzhuk M.S., et al. Dark neurons of the sensorimotor cortex of white rats after acute incomplete ischemia in terms of fixation artifacts and neuroglial relationships. Journal of Anatomy and Histopathology = Zhurnal Anatomiya i Gistopatologiya. 2021; 10 (2): 9-22. (in Russ.). DOI: 10.18499/2225-7357-2021-10-2-9-22.

12. Pekny M., Pekna M. Astrocyte reactivity and reactive astrogliosis: costs and benefits. Physiol Rev. 2014; 94 (4): 1077–1098. DOI: 10.1152/physrev.00041.2013. PMID: 25287860.

13. Zimatkin S. M., Bon E. I. Dark neurons of the brain. Morphollogy = Morfologiya. 2017; 152 (6): 81–86. (in Russ.). DOI: 10.17816/morph.398200.

14. Brown G.C. Neuronal loss after stroke due to microglial phagocytosis of stressed neurons. Int J Mol Sci. 2021; 22 (24): 13442. DOI: 10.3390/ijms222413442. PMID: 34948237.

15. Chen S., Shao L., Ma L. Cerebral edema formation after stroke: emphasis on blood–brain barrier and the lymphatic drainage system of the brain. Front Cell Neurosci. 2021; 15: 716825. DOI: 10.3389/fncel.2021.716825. PMID: 34483842.

16. Kalinichenko S.G., Pushchin I.I., Matveeva N.Yu. Neurotoxic and cytoprotective mechanisms in the ischemic neocortex. J Chem Neuroanat. 2023; 128: 102230. DOI: 10.1016/j.jchemneu.2022.102230. PMID: 36603664.

17. Hu C., Chen X., Wang M., Zhang L., Gao D., Zang L. Analgecine protects against cerebral ischemia-reperfusion through apoptosis inhibition and anti-neuroinflammation in rats. Neuropeptides. 2022; 93: 102230. DOI: 10.1016/j.npep.2022.102230. PMID: 35378359.

18. Behrouzifar S., Esmaily H. The biological efficacy of Apelin against focal transient cerebral ischemia-reperfusion injury. A systematic review and meta-analysis of animal studies. Brain Res. 2024; 15: 1833: 148887. DOI: 10.1016/j.brainres.2024.148887. PMID: 38552935.

19. Sofroniew M.V. Astrocyte barriers to neurotoxic inflammation. Nat Rev Neurosci. 2015; 16 (5): 249–263. DOI: 10.1038/nrn3898. PMID: 25891508.

20. Wilson D.M., Cookson M.R., Van Den Bosch L., Zetterberg H., Holtzman D.M., Dewachter I. Hallmarks of neurodegenerative diseases. Cell. 2023; 186: 693–714. DOI: 10.1016/j.cell.2022.12.032. PMID: 36803602.

21. Delgado-Martín S., Martínez-Ruiz A. The role of ferroptosis as a regulator of oxidative stress in the pathogenesis of ischemic stroke. FEBS Lett. 2024; 598 (17): 2160–2173. DOI: 10.1002/18733468.14894. PMID: 38676284.

22. Wang X., Zhang X.Y., Liao N. Q., He Z.H., Chen Q.F. Identification of ribosome biogenesis genes and subgroups in ischaemic stroke. Front Immunol. 2024; 15: 1449158. DOI: 10.3389/fimmu.2024.1449158. PMID: 39290696.

23. Golubev A.M. Morphological classification of neuronal damage. General Reanimatology = Obshchaya Reanimatologiya. 2025; 21 (5): 4–14. (in Russ. & Eng.). DOI: 10.15360/1813-9779-2025-5-2580.

24. Sheikh A., Meng X., Kao J.P.Y, Kanold P.O. Neonatal hypoxia-ischemia causes persistent intracortical circuit changes in layer 4 of rat auditory cortex. Cereb Cortex. 2022; 32 (12): 2575–2589. DOI: 10.1093/cercor/bhab365. PMID: 34729599.

25. Chen J.-M., Shi G., Yu Lu-Lu., Shan W., Sun J.-Yu, Guo A.-C., Wu J.-P., Tang T.-S., Zhang X.-J., Wang Q. 3-HKA promotes vascular remodeling after stroke by modulating the activation of A1/A2 reactive astrocytes. Adv Sci (Weinh). 2025; 12 (11): e2412667. DOI: 10.1002/advs.202412667. PMID: 39854137.

26. Yanumula A., Cusick J. K. Biochemistry, extrinsic pathway of apoptosis. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025. PMID: 32809646 Free Books & Documents.

27. Bon E.I., Maksimovich N.E., Karnyushko O.A., Zimatkin S.M., Novak A.A. Changes in content of bcl-2 in the parietal cortex and hippocampus of rats with cerebral ischemia of varying severity News of Biomedical Sciences=Novosti Mediko-biologicheskih Nauk. 2025; 25 (2): 42–47. (in Russ.).

28. Boeva E.A., Kalabushev S.N., Varnakova L.A., Lyubomudrov M.A., Tsokolayeva Z.I., Kuzovlev A.N., Moroz V.V., et al. Argon-oxygen mixture as a multisystem therapy after circulatory arrest: an experimental study. General Reanimatology = Obshchaya reanimatologiya. 2026; 22 (1): 26–40. (in Russ. & Eng.). DOI: 10.15360/1813-9779-2026-1-2618.

29. Song W., Teng L., Wang H., Pang R., Liang R., Zhu Z. Exercise preconditioning increases circulating exosome miR-124 expression and alleviates apoptosis in rats with cerebral ischemia-reperfusion injury. Brain Res. 2025; 15: 1851: 149457. DOI: 10.1016/j.brainres.2025.149457. PMID: 39824375.