Comparison of the immunohistochemical and ultrastructural studies of the white rats sensorimotor cortex synaptic terminals reaction to common carotid arteries ligation

The aim of the research was to study the structural and functional changes in axonal terminals in layers I, III, and V of the sensorimotor cortex (SMC) of the brain of Wistar rats after the common carotid artery (CCA) bilateral ligation using immunohistochemical and apparatus-microscopic methods.

Material and methods. Incomplete cerebral ischemia was modeled by bilateral ligation of the common carotid arteries (CCA - 2-vessel model of global ischemia without hypotension) in white Wistar rats (n=36). SMC was studied in the control (intact rats, n=6), 1, 3, 7, 14 and 30 days (n=30) after POCA. Nissl, hematoxylin-eosin, immunohistochemical reactions for p38, and electron microscopy were used. The total number density and relative area of axonal terminals were determined. Statistical hypotheses were tested using nonparametric methods for pairwise and multiple comparisons using the Statistica 8.0 program.

Results. After CCA bilateral ligation, the content of degeneratively altered neurons in the rat brain SMC increased. Changes in the SMC neurons were accompanied by neuropil hyperhydration and reactive astrogliosis. The total number density of terminals in all SMC layers decreased statistically significantly after 1 day (by 28.6% in layer I, 46.9% in layer III, and 46.4% in layer V) and remained approximately at this level throughout the entire observation period. . The relative area of synaptic terminals differed in the compared SMC layers. In layers I and III of the SMC, the values of this indicator first (days 1 and 3) decreased, and then (days 7, 14, and 30) they increased.

In layer V of the SMC, activation of the expression of this protein occurred already in the acute period (days 1 and 3), decreased after 7 and 14 days, and increased again after 30 days. Ultrastructural examination revealed more small terminal axonal branches. However, the general trend of changes in the number of terminals was similar.

Conclusion. After CCA bilateral ligation, destructive and compensatory-restorative changes in axonal terminals were revealed in layers I, III, and V of the rat MMC. The reorganization of interneuronal relationships occurred against the background of pronounced manifestations of neuropil hyperhydration. The maximum destruction of synaptic terminals was noted in layer III of the SMC, and their adaptive changes were observed in layer V. The results of immunohistochemical and ultrastructural studies are comparable and complement each other. All this can probably be considered as a structural basis for changes in the integrative-starting activity of the brain after CCA bilateral ligation.

1. Borovikov V. Statistica. Iskusstvo analiza dannykh na komp'yutere. 2-oe izd. Saint-Petersburg: Piter; 2003 (in Russian).

2. Makar’eva LM, Akulinin VA, Stepanov SS, Shoronova AYu, Avdeev DB, Korzhuk MS. Morphological and morphometric description of neurons in the sensorimotor cortex of the rat brain after ligation of the common carotid arteries. Journal of Anatomy and Histopathology. 2022 Mar 30;11(1):49–58 (in Russian). EDN: HWPPMD. doi: 10.18499/2225-7357-2022-11-1-49-58

3. Makar’eva LM, Korzhuk MS, Akulinin VA, Stepanov SS, Shoronova AYu, Avdeev DB. Neuroglial relationships and structures of interneuronal communication of the white rat sensorimotor cortex layer v after the common carotid artery ligation. Journal of Anatomy and Histopathology. 2022 Jun 30;11(2):43–51] (in Russian). EDN: UWTZLX. doi 10.18499/2225-7357-2022-11-2-43-51

4. Piters A, Palei S, Uebster G. Ul'trastruktura nervnoi sistemy: Per. s angl. 1972 (in Russian).

5. Semchenko VV, Stepanov SS, Bogolepov NN. Sinapticheskaya plastichnost' golovnogo mozga (fundamental'nye i prikladnye aspekty). Omsk: Omskaya oblastnaya tipografiya; 2008 (in Russian).

6. Stepanov AS. Comparative characteristics of the white rats neocortex, hippocampus and amygdale complex synaptoarchitectonics in norm and after acute ischemia. Journal of Anatomy and Histopathology. 2017 Dec 12;6(4):47–54] (in Russian). EDN: ZXWOWN. doi: 10.18499/2225-7357-2017-6-4-47-54

7. Stepanov AS, Akulinin VA, Stepanov SS, Avdeev DB, Gorbunova AV. Neurons Communication in the Hippocampus of Field CA3 of the White Rat Brain after Acute ischemia. General Reanimatology. 2018 Oct 28;14(5):38–49 (in Russian). EDN: VJVWPO. doi: 10.15360/1813-9779-2018-5-38-49

8. Bergsman JB, Krueger SR, Fitzsimonds RM. Automated criteria-based selection and analysis of fluorescent synaptic puncta. Journal of Neuroscience Methods. 2006 Apr;152(1-2):32–9. doi: 10.1016/j.jneumeth.2005.08.008

9. Black MM. Axonal transport: The orderly motion of axonal structures. Methods in Cell Biology. 2016;(131):1–19. doi: 10.1016/bs.mcb.2015.06.001

10. Calhoun ME, Jucker M, Martin LJ, Thinakaran G, Price DL, Mouton PR. Comparative evaluation of synaptophysin-based methods for quantification of synapses. Journal of Neurocytology. 1996 Jan;25(1):821–8. doi: 10.1007/BF02284844

11. Chen KS, Masliah E, Mallory M, Gage FH. Synaptic loss in cognitively impaired aged rats is ameliorated by chronic human nerve growth factor infusion. Neuroscience. 1995 Sep;68(1):19–27. doi: 10.1016/0306-4522(95)00099-5

12. Clare R, King VG, Wirenfeldt M, Vinters HV. Synapse loss in dementias. Journal of Neuroscience Research. 2010 Apr 5;88(10):2083–90. doi: 10.1002/jnr.22392

13. Gordon-Weeks PR, Fournier AE. Neuronal cytoskeleton in synaptic plasticity and regeneration. Journal of Neurochemistry. 2013 Nov 11;129(2):206–12. doi: 10.1111/jnc.12502

14. Guillamón-Vivancos T, Gómez-Pinedo U, Matías-Guiu J. Astrocitos en las enfermedades neurodegenerativas (I): función y caracterización molecular. Neurología. 2015 Mar;30(2):119–29. doi: 10.1016/j.nrl.2012.12.007

15. Herold J, Schubert W, Nattkemper TW. Automated detection and quantification of fluorescently labeled synapses in murine brain tissue sections for high throughput applications. Journal of Biotechnology. 2010 Sep 15;149(4):299–309. doi: 10.1016/j.jbiotec.2010.03.004

16. Hu W, An C, Chen WJ. Molecular Mechanoneurobiology: An Emerging Angle to Explore Neural Synaptic Functions. BioMed Research International. 2015 Apr 14;2015:e486827. doi: 10.1155/2015/486827

17. Ippolito DM, Eroglu C. Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number. Journal of Visualized Experiments. 2010 Nov 16;(45):2270. doi: 10.3791/2270

18. Jing Z, Shi C, Zhu L, Xiang Y, Chen P, Xiong Z, et al. Chronic Cerebral Hypoperfusion Induces Vascular Plasticity and Hemodynamics but Also Neuronal Degeneration and Cognitive Impairment. Journal of Cerebral Blood Flow & Metabolism. 2015 Apr 8;35(8):1249–59. doi: 10.1038/jcbfm.2015.55

19. Knott G, Marchman H, Wall D, Lich B. Serial Section Scanning Electron Microscopy of Adult Brain Tissue Using Focused Ion Beam Milling. Journal of Neuroscience. 2008 Mar 19;28(12):2959–64. doi: 10.1523/JNEUROSCI.3189-07.2008

20. Koizumi S, Hirayama Y, Morizawa YM. New roles of reactive astrocytes in the brain; an organizer of cerebral ischemia. Neurochemistry International. 2018 Oct;119(10):107–14. Doi: 10.1016/j.neuint.2018.01.007

21. Kreshuk A, Straehle CN, Sommer C, Koethe U, Cantoni M, Knott G, et al. Automated Detection and Segmentation of Synaptic Contacts in Nearly Isotropic Serial Electron Microscopy Images. Barnes S, editor. PLoS ONE. 2011 Oct 21;6(10):e24899. Doi: 10.1371/journal.pone.0024899

22. Merchan-Pérez A, Rodriguez J-R, Alonso-Nanclares L, Schertel A, DeFelipe J. Counting synapses using FIB/SEM microscopy: a true revolution for ultrastructural volume reconstruction. Frontiers in Neuroanatomy. 2009;3(18):1–14. doi: 10.3389/neuro.05.018.2009

23. Mishchenko Y. Automation of 3D reconstruction of neural tissue from large volume of conventional serial section transmission electron micrographs. Journal of Neuroscience Methods. 2009 Jan;176(2):276–89. doi: 10.1016/j.jneumeth.2008.09.006

24. Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 5-th ed. Amsterdam, Boston: Elsevier Academic Press; 2005.

25. Uyeda A, Muramatsu R. Molecular Mechanisms of Central Nervous System Axonal Regeneration and Remyelination: A Review. International Journal of Molecular Sciences. 2020 Oct 30;21(21):8116. doi: 10.3390/ijms21218116

26. Wu C-C, Reilly JF, Young WG, Morrison JH, Bloom FE. High-throughput Morphometric Analysis of Individual Neurons. Cerebral Cortex. 2004 May;14(5):543–54. Doi: 10.1093/cercor/bhh016

27. Xing Y, Bai Y. A Review of Exercise-Induced Neuroplasticity in Ischemic Stroke: Pathology and Mechanisms. Molecular Neurobiology. 2020 Jul 20;57(10):4218–31. doi: 10.1007/s12035-020-02021-1