Features of NeuN distribution in the layers of the human cerebral cortex in the acute phase of subarachnoid hemorrhage

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Abstract

Background. Neuronal damage is an important component of the pathogenesis of hemorrhagic stroke, but cellular and molecular markers of neuronal changes during the acute phase of the disease in humans have been poorly studied. One neuronal marker is the nuclear protein NeuN. Opinions on the use of NeuN as a marker of neuronal damage are contradictory. The heterogeneity of immunohistochemical (IHC) staining described in studies under various pathological and physiological conditions creates the basis for studying NeuN as a marker of the functional state of neurons in various diseases. This study aims to identify the distribution patterns of NeuN in the layers of the human cerebral cortex during the acute phase of hemorrhagic stroke. Aims — to identify the features of NeuN distribution in the layers of the human cerebral cortex in the acute phase of hemorrhagic stroke. Methods. A retrospective analysis of forensic medical examination materials was conducted from February to September 2019 and from January to February 2022. The study included cases in which the causes of death were: 1) non-traumatic subarachnoid hemorrhage (SAH group); 2) sudden cardiac death (SCD) or coronary artery disease (CAD) (control group). Standard histological processing was then performed with embedding in paraffin blocks, from which sections were prepared and stained with hematoxylin and eosin. Morphological examination of neurons was performed using immunohistochemistry with antibodies to the NeuN protein. Morphometry was performed on micrographs obtained using a scanner. Results. 16 cases were selected for the IHC study, 10 of which were SAH and 6 were SCD/CHD. In the SAH group there were 5 men and 5 women, median age — 62.5 (57.0–76.5), in all observations SAH was of basal localization. The control group included 3 men and 3 women, median age — 58.5 (46.0–73.2), in all observations the cause of death was sudden coronary death. As a result of comparison of SAH and control groups, the following significant results were obtained: in the first layer, there were more fully stained neurons in the SAH group than in the control group (22.9 ([13.6–46.4) vs. 0 [(0–6.9); p = 0.001); in the third layer, there were more unstained neurons in the SAH group than in the control group (22.7 ([16.8–37.8) vs. 5.4 [(0–9.5); p = 0.005). In the fifth layer, there were more neurons with stained nuclei but unstained cytoplasm in the control group than in the SAH group (42.3 ([28.1–73.6) vs. 22.4 [(8.6–35.8); p = 0.031). Conclusion. This study is the first to demonstrate an association between subarachnoid hemorrhage and changes in NeuN staining of neurons in human cerebral cortex. These results confirm that the use of NeuN immunohistochemical staining should not be limited to the detection of intact neurons.

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About the authors

Maksim A. Lyubomudrov

Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology

Author for correspondence.
Email: mlyubomudrov@fnkcrr.ru
ORCID iD: 0000-0002-1735-592X
SPIN-code: 4022-5096

MD

Russian Federation, Moscow

Anastasiya S. Babkina

Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology

Email: ababkina@fnkcrr.ru
ORCID iD: 0000-0003-1780-9829
SPIN-code: 2918-0460

MD, PhD

Russian Federation, Moscow

Arkady M. Golubev

Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology

Email: arkadygolubev@mail.ru
SPIN-code: 7890-2597

MD, PhD, Professor

Russian Federation, Moscow

Andrey V. Grechko

Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology

Email: avgrechko@fnkcrr.ru
ORCID iD: 0000-0003-3318-796X
SPIN-code: 4865-8723

MD, PhD, Professor, Academician of the RAS

Russian Federation, Moscow

Artem N. Kuzovlev

Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology

Email: artem_kuzovlev@fnkcrr.ru
ORCID iD: 0000-0002-5930-0118
SPIN-code: 8648-3771

MD, PhD, Professor

Russian Federation, Moscow

Viktor V. Moroz

Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology

Email: vmoroz@fnkcrr.ru
ORCID iD: 0000-0002-8880-7364
SPIN-code: 5152-6626

MD, PhD, Professor, Corresponding Member of the RAS

Russian Federation, Moscow

Dmitriy V. Sundukov

Peoples’ Friendship University of Russia Named after Patrice Lumumba

Email: sundukov.1958@mail.ru
ORCID iD: 0000-0001-8173-8944
SPIN-code: 2968-7961

MD, PhD

Russian Federation, Moscow

Zoya I. Tsokolaeva

Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology

Email: tsokolaevazoya@mail.ru
ORCID iD: 0000-0003-2219-5315
SPIN-code: 5100-0242

PhD in Biology

Russian Federation, Moscow

Sergey V. Shigeev

Bureau of Forensic Medical Examination of the Department of Healthcare of the City of Moscow

Email: shigeev@mail.ru
SPIN-code: 3116-2928

MD, PhD

Russian Federation, Moscow

Denis E. Shumeyko

Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology

Email: d.e.shumeyko@gmail.com
ORCID iD: 0000-0002-5449-8444
SPIN-code: 4469-2931

MD

Russian Federation, Moscow

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2. Fig. 1. NeuN staining patterns: A — neurons with stained nucleus and cytoplasm; B — neurons with stained nucleus but unstained cytoplasm; C — unstained neurons; D — neurons with stained cytoplasm but unstained nucleus. Images were taken on a Nikon Eclipse Ni-E microscope. NeuN staining is counterstained with hematoxylin. Magnification 60×

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3. Fig. 2. Images of preparations from both groups: A, B — control; C, D — SAH. Hematoxylin and eosin staining (A, C), NeuN IHC staining (B, D). Roman numerals indicate layers.

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4. Fig. 3. Distribution of neurons stained for NeuN in the cerebral cortex layers in the control and SAH groups. Note: * — significant differences between groups (p < 0.05).

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