CHARACTERISTICS OF IBA-1-IMMUNOPOSITIVE CELLS IN SKIN WOUNDS HEALING

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RATIONALE
Skin wound healing is a complex response of cells belonging to various differentiation groups and having different morphologies [1]. The cellular components of the connective tissues of the dermis play a special role in skin regeneration. This part of the organ contains fibroblasts, immune cells, macrophages, mast cells, blood vessels, and components of the extracellular matrix. The dermis supports trophic processes and maintains skin homeostasis. Dermal cellular differentiation groups are involved in all stages of regeneration after injury, and cutaneous macrophages play a key role among them [2, 3]. Macrophages are hematogenous in origin and are found in the tissues of various organs, forming a unified macrophage system in the body. It is known that impaired macrophage regulation can significantly slow the healing process and lead to the formation of chronic wounds [4]. Therefore, it is important to understand the heteromorphism and functional potential of this population during tissue repair [5]. Recent advances in histology have made it possible to identify the phenotypic diversity of representatives of this cellular differentiate and the molecular mechanisms of their involvement in wound healing using various methods [6]. In recent years, many studies have been published devoted to the study of this group of cells in animals [7, 8, 9]. In particular, it was found that various tissue macrophages are located in certain subtissue niches of the dermis (perivascular, perifollicular, perineural), but it is unknown whether these cell populations are homologs of human dermal macrophages. Previously, a significant limitation in the study of macrophages was the non-selectivity of the immunohistochemical markers that detect them. Until now, the most widely used marker of tissue macrophages was the immunohistochemical method based on the presence of the CD68 antigen. Sequencing of individual cells made it possible to identify several forms of CD68+ cells in the skin [10, 11, 12]. Researchers also use complex immunohistochemical reactions to determine macrophage phenotypes [13, 14]. Traditionally, human macrophages were identified based on the positive expression of HLA-DR, CD14, CD11b, CD1C, CD68, and colony-stimulating factor 1 receptor (CSF1R). There are isolated data showing that macrophage differentiated cells can be detected using rabbit monoclonal antibodies to the IBA-1 protein [15]. IBA-1 is a calcium-binding peptide that is a specific marker of all macrophages (including dendritic and interdigitating ones). As an immunohistochemical marker, IBA-1 was previously widely used to detect glial macrophages in nervous tissues, and only recently have data appeared on its use to detect macrophages in some other organs (liver, myocardium, lungs, and brain) [16]. This protein is uniformly distributed in the cytoplasm of cell bodies and their processes, allowing for the identification of these cells and morphometric studies. Currently, there is no information on IBA-1-immunopositive dermal cells in regenerative histogenesis.

Study Objective
The aim of the study was to use the immunohistochemical marker IBA-1 to identify and characterize connective tissue macrophages in healthy skin and in the perinecrotic wound region during the healing stages of a mechanical wound.
STUDY DESIGN
A single-center, controlled, randomized, unblinded study was conducted. The study subjects were thigh skin fragments from male Wistar rats at the healing stages following mechanical injury (deep incised wound). Paraffin sections obtained after standard histological processing were used for immunohistochemical reactions with antibodies to IBA-1. The resulting preparations were digitized for morphometric analysis (cell count per 10 fields of view, long and wide axis dimensions (µm), and nuclear eccentricity (e) calculation) and statistical processing.

STUDY CONDITIONS
The experimental portion of the study was conducted in the certified vivarium of the State Research and Testing Institute of Military Medicine of the Ministry of Defense of the Russian Federation (Scientific Cooperation Agreement No. 15 between the Kirov Military Medical Academy and the State Research Institute of Military Medicine of the Ministry of Defense of the Russian Federation, dated September 13, 2023). Morphometric and statistical analysis were performed at the Department of Histology with the Embryology Course of the Kirov Military Medical Academy of the Ministry of Defense of the Russian Federation.
ELIGIBILITY (SELECTION) CRITERIA
Forty-five male rats, aged 30-45 days, with an initial weight of 180-200 g, were included in the study.
Description of Eligibility Criteria
The animal groups specified above were randomly formed from individuals that had reached the required age. Participant selection into groups
The use of rats as experimental animals is based on their genetic similarity to humans, similar histological skin structure, short life cycle, and ease of care.

DESCRIPTION OF THE INTERVENTION
Animals were housed in separate, marked cages corresponding to the time of withdrawal from the experiment. The procedures were performed in the morning. Each animal was anesthetized (with a mixture of Zoletil®; Vibrac, France, dose 10 mg/kg, in combination with xylazine, 10 mg/kg; Pharmamagist Ltd., Hungary). A deep, long (approximately 2 cm) transverse skin incision was made on the right thigh (in the middle third) with a scalpel, without antiseptic preparation. After the injury, the animals were returned to their cages. Skin fragments for study were obtained from animals at successive stages of wound healing: 12 hours, 1 day, and on days 2, 3, 6, 10, 15, and 25 from the start of the experiment. Skin from intact rats (the middle third of the right thigh) was used as a control. STUDY OUTCOMES
Primary outcome of the study. In this study, the immunohistochemical labeling method used enabled the identification of connective tissue macrophages in the dermis, which was confirmed by electron microscopy. As a result, when processing digital images of IHC-derived specimens, it became possible to use automated cell counting (Zen 2.3 software, SlideViewer software) in the perinecrotic area at various stages of the wound healing process and determine cell morphometric parameters: shape and size, the true number of nuclei in the section, and their location.

Methods for recording outcomes.
Immunohistochemical reactions were performed using rabbit monoclonal antibodies (Biocare medical (USA) at a dilution of 1:1000 in order to detect a marker corresponding to the expression of the IBA-1 protein. Skin samples were fixed in a buffered 10% formalin solution. After 24 hours, they were transferred to 96% ethyl alcohol. Then, after passing all the processing stages, the material was embedded in paraffin blocks placed on special plastic cassettes. Paraffin sections were made using a Sakura manual sled microtome (Japan). Sections 5 μm thick were placed on polylysine-coated slides. To dry, the slides with sections were placed in a thermostat for 12 hours at t = 37o C. Next, the preparations were deparaffinized and rehydrated in two changes of xylene (10 min each), the sections were rehydrated in alcohols of descending concentration (two changes 96% ethanol for 5 min, one change of 80% ethanol - 5 min) and washed in distilled water (5 min). The preparations were placed in citrate buffer S1700 (Dako, Denmark), preheated in a thermostat to 60 ° C. Thermal unmasking of the antigen was carried out in a steamer for 20-25 minutes. The sections were washed in distilled water for 5 minutes and placed in a 3% hydrogen peroxide solution (H2O2) for 10 minutes, then, after washing, they were placed in a solution of 0.01 M phosphate-buffered saline (PBS, Biolot, Russia) (pH 7.4) for 5 minutes. To block antibodies, a blocking solution was used (Protein Block DP-125, Spring Bioscience, USA) and left at room temperature for 10 minutes. After removing the blocking solution, a solution of primary antibodies was applied. The preparations were placed into humid chambers and incubated at 27°C. After incubation in primary antibodies, the preparations were washed in two changes of PBS and anti-rabbit secondary reagents conjugated with polymer and horseradish peroxidase were applied to bind the corresponding primary antibodies (from the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (cat. No. S008, R&D Systems, USA). Incubation was carried out in humid chambers in a thermostat at 27°C for 30-40 min. After washing in PBS, the sections were applied with the required amount of working solution of 3,3-diaminobenzidine tetrahydrochloride (Dako, Denmark) to detect the product of the immunohistochemical reaction. The formation of a colored reaction product occurred within 1-3 min. After achieving optimal staining of the sections, which was controlled under a microscope, the preparations were washed in a 3% solution of hydrogen peroxide and distilled water (3 changes of 5 min each). The preparations were washed in distilled water, dehydrated in isopropyl alcohol and a mixture of isopropyl alcohol and xylene (1:1), cleared in xylene, and embedded in Cytoseal 60 permanent medium (Thermo Scientific, USA). A Zeiss Axio Scope.A1 light microscope with a built-in Zeiss Axiocam ERc 5s camera (Zeiss, Germany) and a Pannoramic MIDI digital scanner (3DHISTECH, Hungary) for layer-by-layer scanning of the preparations were used to visualize the immunohistochemical reaction.

For morphometric analysis of digitized images from a Zeiss Axio Scope.A1 light microscope, licensed Zen 2.3 software was used. SlideViewer was used to process the scanned slides. Immunopositive cells were visualized in 10 fields of view in the wound channel, primary necrosis, and perinecrotic areas. Their number was automatically counted in each slide using the "Dots" option in Zen 2.3, followed by the calculation of the average count across the 10 fields of view. Next, the dimensions (µm) of the long and wide axes of cell nuclei were calculated using the built-in "Ruler" option. To reliably determine the deviation of the cell nucleus from a perfect circle (the degree of elongation), the eccentricity (e) of macrophage nuclei was calculated:
e = c/a,
where c is the focal length and a is the semiaxis length. Cell morphology was also refined by calculating the average optical cross-sectional area of ​​the nucleus (V), using the formula:
V = π/6 LB2,
where L is the long diameter of the nucleus and B is the short diameter of the nucleus. To standardize the data, the true number of complete nuclei in the section was calculated based on their apparent number (Ntrue):
Ntrue = NT/T + D,
where N is the apparent number of nuclei, T is the section thickness, and D is the nuclear diameter. The numerical data were entered into Excel spreadsheets for statistical processing and graph generation.

SUBGROUP ANALYSIS
Animals were divided into 9 groups: the control group consisted of intact animals (n=5); the remaining groups corresponded to the time points after mechanical skin trauma: 12 hours, 24 hours, 2, 3, 6, 10, 15, and 25 days (n=5 in each group).
STATISTICAL PROCEDURES
Planned Sample Size
The sample size was not pre-calculated.
Statistical Methods
The resulting numerical data were processed using Excel and Statistica 10.0 (StatSoft, USA). When comparing cell groups, the distribution type was determined using the Kolmogorov-Smirnov test with Lilliefors correction. For normally distributed data, the Student's t-test was used, and values ​​were presented as the mean and standard error. For non-normal distributions, the Mann-Whitney U test was used for two groups. Differences were considered statistically significant at p ≤ 0.05.
RESULTS
SAMPLE CHARACTERISTICS
Skin fragments from the thighs of male Wistar rats at different stages of healing following mechanical injury—a deep incised wound. Samples, 1.5–2.0 cm in size, included the wound channel, the primary necrotic zone, and the perinecrotic area.
MAIN RESULTS
The results of the study indicate that immunohistochemical labeling for the IBA-1 protein allows for the detection of dermal macrophages and the determination of the size and shape of their nuclei. Macrophages in the connective tissues of the skin are a heteromorphic population. Within the perinecrotic area of ​​the wound, various forms of these cells are found, which were divided into 3 groups: small macrophages (nucleus along the longitudinal axis 6-8 μm); medium macrophages (nucleus along the longitudinal axis 8-10 μm); large macrophages (nucleus along the longitudinal axis 11 μm and more). Regarding the phenotype of the identified macrophages, it can be said that they are mainly oval, oblong or irregularly shaped cells with large nuclei (from round to ellipsoid) (Fig. 1). In the dermis of the skin of intact rats, an average of 100±0.12 macrophages were detected. Some of them are determined in the composition of the outer epithelial sheath of the hair follicle.

Fig.1. Intact rat skin. Reticular dermis. Large macrophages with vesicles. Immunohistochemical reaction for IBA-1. Ob. 70, e. 10

The average cell size along the longitudinal axis was 24.06±0.17 μm, the average optical cross-sectional area of ​​the nucleus V = 8±1.12 (μm2), i.e. all the cells belong to the group of large macrophages. The nuclei are oval (esp = 0.51). The Ntrue index = 83, which means that among the visible nuclei there are 83 complete ones. In the preparations 2 days after the injury, an average of 410.8±6.2 macrophages are determined, which is 310% more than in the preparations of the skin of intact rats. The true number of complete nuclei in the dermis is 340. At this time, macrophages of all designated groups (large, medium, small) are present in the perinecrotic area of ​​the wound. Among them there were 18±0.2 large macrophages (elarge = 0.56), 172±1.18 medium cells (eaverage = 0.58) and 220±1.25 small (emal = 0.28). The average optical cross-sectional area of ​​their nuclei at this time was V = 10±5.15 (μm2). By the 6th day of the experiment, 230.2±2.1 immunopositive cells were detected (Ntrue = 181). This is 130% more than in the intact skin, however, the number of cells relative to the 2nd day of the experiment statistically significantly decreases by 44% (p = 0.03). Among the large forms, 170±1.13 (eaver = 0.53) were detected, medium - 11±0.32 (eaver = 0.64), small - 49±0.54 (eaver = 0.29). The average optical cross-sectional area of ​​the nucleus V = 11±3.03 (μm2). In the preparations from the 15th day of the experiment, 780.1±7.6 cells were detected in 10 fields of view, Ntru = 632. A statistically significant (p = 0.01) increase in the number of macrophages compared to the 6th day of the experiment by 239% was observed. The average optical cross-sectional area of ​​the nucleus of dermal macrophages in the regenerate on the 15th day V = 7±3.65 (μm2). The group of medium macrophages (359±2.21 (eaver = 0.68)) quantitatively prevails over the groups of large (122±3.36 (elarge = 0.53)) and small (299±0.24 (elarge = 0.29)).

ADVERSE EVENTS
No adverse events. DISCUSSION
SUMMARY OF STUDY RESULTS
Immunohistochemical reaction with antibodies to the IBA-1 protein is a reliable method for identifying macrophages in dermal connective tissue. In an experimental study of wound healing, it allowed us to confirm and clarify the manifestation of heteromorphy and heterochrony during the stages of connective tissue regeneration in the skin. Morphometry of the resulting immunohistochemical preparations suggests significant heterogeneity among the macrophages involved in the reparative histogenesis of rat dermal connective tissue. This is primarily manifested in the variability in morphology and karyometric characteristics of the identified cells. A distinct numerical dynamic is observed between macrophage groups at different stages of the wound healing process (Fig. 2).

Fig. 2. Dynamics of the number of different forms of IBA-1-immunopositive cells at the stages of skin wound healing

Thus, the greatest number of small macrophages within the wound process is detected during the inflammatory phase, while the number of medium-sized macrophages, along with isolated large immunopositive cells, peaks during this period. We detected the greatest number of medium-sized macrophages during the differentiation phase. It is worth noting that this period is characterized by the highest overall concentration of cells of this type of differon; these preparations also showed the highest numbers of small and large macrophages.
INTERPRETATION OF STUDY RESULTS
Using an immunohistochemical reaction with antibodies to the IBA-1 protein, cells of the macrophage differon were detected in the connective tissue of the skin. They are localized in both the superficial and deep dermis. Cells of this immunophenotype were also detected during regeneration after mechanical skin wounding. In the necrosis phase, small macrophages predominate in the perinecrotic zone of the wound, in the inflammation phase – small and medium forms, in the differentiation phase – medium ones. As a result of the initial count of immunopositive cells, the highest number of IBA-1 immunopositive cells was detected in the inflammation and differentiation phases of tissue structures, which corresponded to the 2nd, 6th and 15th days of the experiment. Based on the literature data, it can be stated that studies based on the immunohistochemical reaction with antibodies to the IBA-1 protein confirm the relationship between the processes of regenerative histogenesis and the previously identified dynamics of the number of macrophages in the perinecrotic area of ​​a gunshot musculocutaneous wound [17]. In the connective tissue of the deep layers of intact skin, only large macrophages similar to each other in shape were found. Apparently, this is a population of resident macrophages - histiocytes. Young animals (1.5–2 months) were used in the experiment. Macrophages from these individuals are known to have a well-developed secretory apparatus, as confirmed by the reaction performed. Numerous granules are clearly visible in most cells (Fig. 3).

Fig. 3. IBA-1-immunopositive cells in the dermis of an intact rat. Immunohistochemical reaction to IBA-1 protein. Ob. 40, e. 10

During the inflammatory process, in the first 24 hours after skin injury, monocytes become the dominant cells of the inflammatory infiltrate. They migrate to the wound, attracted by fragments of extracellular matrix proteins, transforming growth factor beta (TGF-β), and monocyte chemotactic protein 1, and upon arrival, transform into macrophages [18]. Compared to the control group, during the wound healing process in the inflammatory phase, the majority of macrophages identified belong to the medium and small groups. This is likely due to a slowdown in monocyte migration to the site of inflammation and increased differentiation of previously arriving cells. The immunohistochemical marker used allows us to detect, at the light-optical level, the presence of intense granulation in the cytoplasm of medium and large cells and a uniform staining of the cytoplasm among small forms, without distinct granules. According to the immunohistochemical study, it can be assumed that small and medium macrophages belong to monocytoid and transitional forms, while large ones belong to mature ones, which make up the main part of the macrophage population in intact skin, and then have a relatively constant number at the stages of proliferation and differentiation, as well as during the period of adaptation of tissue structures.

Fig.4. Mechanical trauma of rat skin. 6th day after injury. Large and medium macrophages with vesicles (a) and small macrophages (b) in the dermis. Immunohistochemical reaction for IBA-1. Ob. 70, e. 10

During the proliferative phase, functionally mature macrophages remain dominant in the connective tissues of the dermis. However, the number of small macrophages increases, indicating a slow replenishment of the pool of monocytic macrophages migrating into granulation tissue. Notably, during the differentiation phase of tissue structures, an explosive increase in the number of IBA-1 immunopositive cells in the dermis occurs. This is likely due to a slight increase in the number of monocytic macrophages and a large population of functioning mature macrophages due to delayed cell death in the regenerate. Furthermore, given their morphological characteristics, the small macrophage forms we detected may be dying cells that are also labeled with IBA-1 antibodies and, therefore, are counted in the overall population.
The data obtained from the immunohistochemical study are generally consistent with data on the correlation between the phases of the wound process and the population size of macrophages of various forms [19]. This is explained by the fact that the perinecrotic region is the primary migration zone for cells necessary for tissue regeneration.

LIMITATIONS OF THE STUDY
Immunohistochemical reactions with antibodies to IBA-1 allow for the selective detection of macrophage differentiated cells, but are not reliable for characterizing cell morphology to determine the degree of differentiation. The number of visible nuclei differs significantly from the actual number of nuclei in the section, which also reduces the reliability of karyometry. We believe that a systematic approach combining classical histological examination methods, immunohistochemical labeling, and mathematical statistics is required to fully determine intradifferential macrophage heteromorphism in wound healing.

CONCLUSION
The connective tissue of the skin, both in normal skin and in the perinecrotic region of a mechanical skin wound, is a polydifferentiated structure [20]. One of its elements is IBA-1-immunopositive cells, which, according to research, are classified as macrophages [16, 17]. Taking into account the histion concept of regenerative histogenesis, actively developed by histologists at the Military Medical Academy, the regenerative histion of the dermis is represented not only by cells of various differons, but also by components of the intercellular substance, blood vessels, and nerve elements [20]. The composition of the regenerative histion changes depending on the phase of regeneration [19]. At all stages of skin wound healing, the histion contains IBA-1-immunopositive cells, which contribute to regenerative histogenesis.
CONTRIBUTIONS OF THE AUTHORS
I.A. Odintsova – study management, project administration, methodology development, concept definition, and validation. All authors approved the manuscript (the version to be published) and agreed to be accountable for all aspects of this work, ensuring that any questions related to the accuracy and integrity of any part of it are appropriately reviewed and resolved.
T.I. Berezovskaya – concept definition, data management, data analysis, study execution, study support, validation, visualization, manuscript drafting, manuscript revision, and editing.

ETHICAL REVIEW
The conditions under which the experimental animals were cared for complied with the principles of bioethics and the "Rules for Conducting Work Involving Experimental Animals" (Declaration of Helsinki, 2000), the Federal Law "On the Protection of Animals from Cruelty to Animals" (Chapter V, Article 104679-GD of December 1, 1999), Order No. 267 of the Russian Ministry of Health dated June 19, 2003, and Order No. 199n of the Russian Ministry of Health dated April 1, 2016, "On Approval of the Rules of Good Laboratory Practice." The study protocol was approved by the independent local ethics committee of the S.M. Kirov Military Medical Academy of the Ministry of Defense of the Russian Federation. Extract from the minutes of the Local Ethics Committee meeting dated October 17, 2023 (minutes no. 283).

Sources of Funding.

None.

REVIEW AND REVIEW
The authors declare no relationships, activities, or interests over the past three years with third parties (commercial or non-commercial organizations) whose interests may be affected by the content of this article.

ORIGINALITY STATEMENT
The authors did not use previously obtained and published information (data, text, or illustrations) in conducting the research and creating this article.

DATA ACCESS
All data obtained in this study are presented in the article.

GENERATIVE ARTIFICIAL INTELLIGENCE
No generative artificial intelligence technologies were used in the creation of this article.

REVIEW AND PEER-REVIEW
This work was submitted to the journal on an unsolicited basis and was reviewed according to the standard review procedure. Two external reviewers, a member of the editorial board, and the scientific editor participated in the review process.

About the authors

Irina Alekseevna Odintsova

Military Medical Academy of S.M.Kirov

Email: odintsova-irina@mail.ru
ORCID iD: 0000-0002-0143-7402
SPIN-code: 1523-8394

Доктор медицинских наук, профессор

Russian Federation, St. Petersburg, Russian Federation, Academician Lebedev str., 6

Tatiana Berezovskaia

S.M.Kirov Military Medical Academy of the Ministry of the Russian Federation

Author for correspondence.
Email: lapi2@yandex.ru
ORCID iD: 0009-0009-1591-9152
SPIN-code: 2508-7042

Преподаватель кафедры гистологии с курсом эмбриологии

Russian Federation

References

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