Trends of Bcl-2-positive lymphocytes in different zones of the splenic white pulp in rats after soft tissue injury of the thigh: a morphometric analysis

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Abstract

BACKGROUND: Soft tissue injuries constitute a substantial proportion of musculoskeletal damage and are frequently associated with complications related to immune response dysregulation, including secondary infections and delayed wound healing. Understanding systemic alterations in immune organs, particularly the spleen, is essential for developing strategies aimed at optimizing regeneration and preventing complications. Although the role of Bcl-2 (B-cell lymphoma 2) protein in the regulation of apoptosis is well established, data on its expression in the spleen following soft tissue injury remain limited. Most studies focus on local wound-site changes, whereas systemic effects, especially in lymphoid organs, have been insufficiently investigated.

AIM: To investigate changes in the number of Bcl-2-positive lymphocytes in different zones of the splenic white pulp in rats after soft tissue injury of the thigh using immunohistochemical and morphometric analysis.

METHODS: The study was performed on male outbred white rats weighing 180–200 g. Mechanical thigh injury comparable in kinetic energy to a 5.6-mm caliber bullet wound was induced using a specialized device (under ether anesthesia). Animals were euthanized by decapitation. Spleen specimens measuring 0.5 × 1.5 cm were collected at 1, 3, 7, and 14 days after injury for morphometric and immunohistochemical examination. Tissue sections were stained with hematoxylin and eosin. Immunohistochemistry was performed to detect cells expressing the antiapoptotic protein Bcl-2, T lymphocytes (CD3), and B lymphocytes (CD19), with Mayer’s hematoxylin nuclear counterstaining. Statistical analysis was conducted in R software (version 4.3.2) using the packages dplyr, tidyr, car, rstatix, pwr, ggplot2, and ggpubr.

RESULTS: Mechanical thigh injury in rats led to changes in the number of Bcl-2-positive lymphocytes in various splenic zones. A transient decrease in these cells was observed at 1 and 3 days post-injury, likely associated with lymphocyte migration to peripheral tissues, followed by gradual restoration and even increased Bcl-2 expression in the spleen by day 14.

CONCLUSION: An increase in Bcl-2-positive lymphocytes reflects a systemic immune response to local soft tissue injury of the thigh. This response may contribute to host defense against infections but could potentially delay wound healing when excessively activated. The association between local and systemic immune responses underscores the important regulatory role of the spleen and justifies further investigation to optimize therapeutic strategies for injury management.

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About the authors

Albina Kh. Lanicheva

Bashkir State Medical University

Author for correspondence.
Email: lanichevaa@mail.ru
ORCID iD: 0009-0004-0550-2760
SPIN-code: 9390-2689

MD, Cand. Sci. (Medicine), Assistant Professor

Russian Federation, Ufa

Valery V. Semchenko

Omsk State Agrarian University

Email: ivm_omgau_gistology@mail.ru
ORCID iD: 0009-0006-8933-9618
SPIN-code: 8269-3077

MD, Dr. Sci. (Medicine), Professor

Russian Federation, Omsk

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2. Fig. 1. White pulp of the rat spleen: a, control group; b, experimental group 1 day after mechanical trauma to the thigh soft tissues; hematoxylin and eosin staining; magnification: ×200; objective: ×20; scale bar: 100 μm.

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3. Fig. 2. Immunohistochemical detection of lymphocyte localization in the splenic white pulp of rats after mechanical trauma to the thigh soft tissues: a, CD3-positive T lymphocytes 3 days after trauma; b, Bcl-2-positive T lymphocytes 3 days after trauma; c, CD19-positive B lymphocytes 7 days after trauma; d, Bcl-2-positive B lymphocytes 7 days after trauma; magnification: ×200; objective: ×20; scale bar: 100 μm.

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4. Fig. 3. Number of Bcl-2-positive cells per 10,000 μm2 section area in different splenic zones of control rats and rats after mechanical trauma to the thigh soft tissues: the line indicates the median, and dots represent outliers; p < 0.0001, Kruskal–Wallis test for comparison across post-trauma time points.

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