Dark Neurons of the Sensorimotor Cortex of White Rats after Acute Incomplete Ischemia in Terms of Artifacts Fixation and Neuroglial Relationships
Authors: Avdeev D. B. , Stepanov S. S. , Gorbunova A. V. , Shoronova A. Yu. , Makar'eva L. M. , Akulinin V. A. , Korzhuk M. S. , Zabolotnykh M. V. 
Published: July 2021

The aimof research was to study morphofunctional signs of the sensorimotor "dark neurons" formation in the cerebrum of sexually mature white rats under normal conditions and in 40 minutesafter the common carotid arteries (CCA) occlusion.

Material and methods.Acute ischemia was simulated in white Wistar rats by a 40-min CCA occlusion. The brain was fixed by immersion and perfusion methods. A comparative morphometric assessment of cyto-and gliocytoarchitectonics of the neocortex was carried out under normal conditions (n = 6), 1 (n = 6), 3 (n= 6), and in 7 days (n = 6) after the common carotid arteries (CCA) occlusion. The Nissl staining, hematoxylin and eosin staining, immunohistochemical typing NSE, MAP-2, HSP-70, p38, CASP3, GFAP and AIF1 were applied. The numerical density of normochromic and hyperchromic (dark) pyramidal neurons, oligodendrocytes, microgliocytes, and the relative area of edema –swelling zones were determined. Statistical hypotheses were tested by nonparametric methods using Statistica 8.0 software.

Results.The method of the brain fixation significantly influenced the content of the artifact dark neurons,which are similar in morphology and tinctorial properties to degeneratively altered dark neurons. The appearance of reversibly and irreversibly degeneratively altered dark neurons in the sensorimotor cortex after the CCA occlusion was accompanied by an increase in the relative area of edema  –swelling zones (control  –5.4%, 1st day  –17.6%). The maximum content of degeneratively altered dark neurons (53%) was found in layer V in 7 days after ischemia. According to the findings of immunohistochemical typing NSE, MAP-2, HSP-70, p38, CASP3, the specific proteins of the most degeneratively altered dark neurons were retained, ensuring their restoration and the neural network functioning. The total number density of sensorimotor neurons decreased by 26.4% (p =0.001) in layer III, and by 18.5% in layer V (Mann –Whitney U Test; p = 0.01) in 7 days after the intervention. The revealed changes were of a diffuse focal character. In the zones of degeneratively altered dark neurons accumulation, the content of astrocytes, microgliocytes and oligodendrocytes increased. The neuro-astroglial index (control  –1.62) increased to 2.72 in 3 days after the CCA occlusion. The peak in the number density of microgliocyte s was noted in 1 day, and the oligodendrocytes in 7 days after acute incomplete ischemia (Mann –Whitney U Test; p0.001).

Conclusion.After a 40-minute common carotid arteries occlusion, dark neurons were detected in layers III and V of the sensorimotor cortex with underlying hydropic dystrophy; their cytomorphological pro perties indicated the dynamics of their in vivo degenerative changes. Restoration of degeneratively altered dark neurons was accompanied  by  an  increase  in  the  number  of  satellite  oligodendrocytes,  astrocytes  and  microgliocytes. The  revealed changes were considered as one of the variants of reversible changes in neurons in response to moderate ischemic damage.