Introduction. Computed X-ray microtomography (micro-CT) is a method enabling getting three-dimensional images of microscopic internal and external structures in the samples without damaging them, which makes it possible to study the objects inaccessible to other methods of analysis [1]. The method is based on obtaining a set of X-ray projection images by rotating a sample placed between an X-ray source and a detector (although other sample placements are also possible [2]). These images are then converted into a series of cross-sections using specialized reconstruction algorithms [3].

Micro-CT differs from traditional CT and targeted radiography by a number of parameters, including resolution, field of application, and radiation dose [3]. Micro-CT provides ultra-high resolution (up to 1 μm) and enables getting detailed three-dimensional images, making it highly valuable in scientific research, ex vivo tissue studies, and experimental oncology [3]. However, the use of the method is limited by long scanning time and high radiation exposure, which reduces its applicability in vivo. Traditional CT is widely used in clinical practice to diagnose pathologies of internal organs and the skeletal system. It enables rapid 3D imaging with a resolution of approximately 0.5–1 mm and using contrast agents for improved visualization of soft tissues [4-5]. Targeted radiography (bitewing X-ray) is used primarily in dentistry to detect caries and assess state of bone tissue and paradontium. This is a fast and low-dose method, however, it only provides a two-dimensional image and is not suitable for detailed examination of complex structures [4].

The choice of imaging method depends on the specific task. If the utmost possible detailed image of tissue is required, micro-CT is preferable. However, for clinical diagnostics and work with living patients, traditional CT and targeted radiography are more suitable. The characteristics of these methods are provided in more detail in Table 1.

The main advantages of microtomography are:

– high resolution: the technology enables the acquisition of images with the smallest discernible detail, less than one micron to tens of microns, ensuring clarity, detailing [5-6], and, if necessary, examination of quite large samples [6];

– a wide range of applications due to the ability to obtain 2D and 3D images: 2D images are used to measure length, thickness, and angles of curvature, while 3D helps to identify pathological changes such as osteolysis or bone hyperplasia [4], neoplasms [3], demineralization of dental tissue [4], etc.;

– digital data processing: digital images can easily be processed and rendered [7], which enables their rotation and analysis in three dimensions for a deeper understanding of the object’s condition [8].

High-quality images obtained using micro-CT allow researchers to visualize in detail the pathological processes in experimental animal models, thus, facilitating more accurate disease diagnostics, assessment of the therapeutic strategies’ efficiency, and better understanding the mechanisms of their development. Furthermore, the ability to study in detail pathological changes, such as fatty infiltration of tissue in hyperlipidemia, contributes to the expansion of scientific knowledge and improvement of treatment methods [5–6].

Over the past decades, micro-CT has become a powerful tool in biomedical research, widely used in studying bone morphology [3], animal phenotyping [1], and the investigation of internal organ pathologies [5]. Due to optimization of contrast agent development and administration, the capacities of the method had significantly increased. In vivo imaging enables assessment of vascular and soft tissue morphology, however ex vivo examination offers higher resolution due to increased scanning time and absence of limitations associated with anaesthesia and radiation [9].

In neurology, micro-CT is used to analyse the vascular anatomy of the brain, identify atherosclerotic lesions, and assess blood-brain barrier disruptions [9]. Furthermore, the technique enables longitudinal study to assess the brain tumour growth and the efficiency of antitumour therapy.

In cardiology, contrast-enhanced micro-CT is used to visualize the cardiac conduction system, diagnose congenital heart defects, and make quantitative assessment of pulmonary ventilation in pulmonary fibrosis [10].

Micro-CT is also actively used in cancer research. For example, in colorectal cancer models, the method demonstrates a high correlation between tumour volume and histological data [9]. The use of contrast agents makes it possible to differentiate healthy liver tissue from neoplasms, enabling quantitative assessment of metastases and the efficiency of experimental therapy [9].

Bone tissue studies using micro-CT cover a wide range of pathologies, including osteoporosis, fracture healing, osteogenesis, and bone resorption in tumours [2]. The technique is also used to assess interactions between soft tissues and implants, and to study angiogenesis and muscular dystrophy [9].

In veterinary medicine, micro-CT is becoming increasingly popular due to possibility to obtain high-quality three-dimensional images of tissues and structures, as well as possibility to examine anatomical features and pathological changes by a non-destructive method with a spatial resolution of up to 1 μm [10]: possibility of visualizing a biopsy specimen without cutting is particularly valuable for subsequent histological analysis [11].

Micro-CT can be used both in vivo and ex vivo: in vivo mode allows for visualization of tissue changes in small animals with a resolution of up to 30 μm, with a minimal radiation dose [11–13]. However, its implementation is limited due to low sensitivity to soft tissues, which can be compensated by the use of contrast agents. In ex vivo mode, micro-CT resolution can reach even higher values due to long exposures, which contributes to image detailing that surpasses the results of histological analysis.

Due to the possibility of 3D reconstruction and digital data processing, micro-CT provides researchers with a unique opportunity to analyse tissue morphology, develop virtual models, and even create precise physical 3D prototypes [14][15]. Moreover, the integration of micro-CT with traditional diagnostic methods, such as histopathology, expands its diagnostic potential by combining precise quantitative data with visual assessment of tissue structure [13]. In silico methods for micro-CT makes it possible to perform 3D reconstruction, segmentation, morphometric analysis, and tissue density calculations without destroying the specimens [16].

Despite the rapid development of modern imaging techniques, the potential of micro-CT for comprehensive analysis of the state of soft tissues and internal organs in animals remains understudied. The potential of this method for detailed quantitative assessment of morphological changes in internal organs caused by the pathological processes remains a relatively new area. For example, there are almost no studies referring to comprehensive analysis of renal tissue density and three-dimensional morphology in animal experimental models with diet-induced pathologies, such as hyperlipidemia. The kidneys were chosen as an object of study due to their vital role in metabolism and their high vulnerability to systemic disorders, such as hyperlipidemia. The resulting structural changes (fatty infiltration, fibrosis) directly affect renal tissue density, making micro-CT an ideal method for their detection and quantitative assessment.

The aim of the research is to evaluate the efficiency of using micro-CT for studying pathological changes of kidney tissue density and morphology in laboratory rats with different degrees of hyperlipidemia.

Materials and Methods. The experiment was conducted at Don State Technical University (Rostov-on-Don) from 2021 to 2024. 40 male Wistar rats were divided into 5 groups (4 experimental and 1 control), with 8 animals in each. The animals were kept in the DSTU vivarium on wood bedding, which was changed weekly.

All animals received a standard diet for the first 30 days. Afterwards, group 1 was withdrawn from the experiment by decapitation; in groups 2, 3, and 4, the diet was changed to the hyperlipidemic one with high cholesterol content; group 5 (control) received only standard feed throughout the entire period. On 120th , 150th , and 180th day, animals of groups 2, 3, and 4, respectively, were euthanized by decapitation. Group 5 was withdrawn from the experiment simultaneously with group 4 on 180th day. After this, an autopsy was performed to collect kidneys and prepare specimens for micro-CT scanning and subsequent data analysis.

After initial fixation in 10% formalin for 48 hours, all rat kidney specimens were sequentially dehydrated in 50%, 70%, 80%, 90%, 96%, and 100% ethanol (with incubation for 1 hour in each solution). The specimens were then stained with 1% iodine I₂ solution in absolute ethanol for 14 hours. Finally, the specimens were rinsed in absolute ethanol and placed in plastic containers filled with absolute ethanol for storage at 5°C.

Scanning was performed on an Xradia Versa 520 microtomograph (Carl Zeiss X-ray Microscopy, USA). The specimens were mounted vertically in plastic tubes filled with absolute ethanol and mounted in a collet holder located outside the detector’s field of view. The studies were performed in the normal air space of the instrument chamber, without the use of additional media.

To obtain high-quality data, three segments of each specimen were scanned, starting with the bottom and finishing with the top one. General scanning parameters:

– objective magnification: 0.4×,

– source voltage: 80 kV,

– power: 6.5 W,

– voxel size: 20 µm,

– rotation: 360°,

– exposure time: 1 s,

– number of projections: 1601 for each segment.

Projection data collection was performed using software (Carl Zeiss AG, Germany) and resulted in reconstruction of micro-CT images. To improve contour clarity, center offset parameters were manually adjusted. Final processing of the reconstructed data was performed in VGStudio MAX 3.5 (Volume Graphics GmbH, Germany):

– media around the specimen was removed manually using a density bar chart;

– volume rendering based on Phong model [17] was used for visualization;

– segmentation of structures was performed using manual, automatic, and semiautomatic tools. Each region of interest (ROI) was marked with a pseudocolor.

The efficiency of micro-CT was assessed based on the quality of visualization and reconstruction, i.e., the ability of the method to clearly highlight the cortex and medulla, vessels, and other renal structures in a 3D model with high detail enhancement with minimal artefact introduction. Quantitative analysis of tissue changes—density measurements using gray values—was also taken into account. (MGV, mean gray value).

Results. High-quality microtomograms of the kidneys made it possible to clearly distinguish the organ’s structural components: the cortex and renal artery (Fig. 1); the medulla (Fig. 2); and the vascular network including large arterial and venous blood vessels, as well as small capillaries (Fig. 3). In reconstructed 3D kidney models, the cortex, medulla, and vessels were highlighted as ROIs based on mean gray values (MGV). A uniform “plasma” colour scheme was used to clearly visualize changes of density.

Conducting experiment in several experimental groups, enabled tracking progression of pathological changes in time dynamics (in particular, tissue density increase) in response to the duration of hyperlipidemic treatment. Density indicators, expressed as mean gray values (MGVs) for each group, are presented in Table 2

The data in the table demonstrate a clear dynamics of density changes in renal tissue depending on the duration of the hyperlipidemic diet. The most significant increase in density across all the structures was observed in group 2 (3 months of diet), which obviously reflects the peak of fatty infiltration and inflammatory response development. By the end of 5th month of the diet (4th group), the density values approached the control levels, which might indicate the development of fibrosis and tissue sclerosis also attributed with the density increase that however develops by a different mechanism and requires further histological confirmation.

Analysis of the vascular network revealed thickening of the vessel walls and loss of elasticity, indicating reduced blood supply and an increased risk of ischemic tissue damage. The obtained data are consistent with the previous studies on cavernous body fibrosis in a castrated rabbit model, where micro-CT enabled quantitative assessment of changes in radiodensity at various stages of the disease [18].

Discussion and Conclusion. The conducted micro-CT examination resulted in high-resolution images of the kidneys, which enabled detailed visualization of their structure. The method proved to be highly accurate in determining density values of renal tissue in hyperlipidemia, which may be useful for assessing structural changes in various pathologies. Density analysis allows for the quantitative assessment of such changes and their association with clinical manifestations of the disease.

Among the limitations of the study are: the small sample size, the use of just Wistar breed male rats, which restricts possibility of generalizing the results, and the potential influence of experiment timing and diet on the development of pathologies and data variability. Specimen preparation (fixation, dehydration, staining) could have introduced the artefacts affecting the accuracy of tissue density measurements. Limitations of the micro-CT method itself should also be considered: the high cost of equipment and consumables, low sensitivity to soft tissues without contrast agents, limitations of in vivo studies (anaesthesia, radiation doses, scanning time), and the need for specialised software and skills for data processing and analysis.

Further research may focus on the use of contrast agents to enable a more detailed study of the renal vasculature and processes associated with their perfusion. It is also necessary to establish the efficiency of micro-CT in identification and quantitative assessment of structural abnormalities and fibrosis by direct comparison with the results of histopathology. Such an approach will foster more accurate detection of changes in cells and tissues and expand the potential for micro-CT application in clinical practices for disease diagnosis and monitoring.