IMMUNOHISTOCHEMICAL FEATURES OF BAX-DEPENDENT APOPTOSIS IN THE TROPHOBLAST OF PLACENTAL CHOROIDAL VILLI AT DELIVERY AFTER 40 WEEKS

Abstract

The relevance of studying the molecular mechanisms that regulate apoptosis in placental tissues, especially in the later stages of pregnancy, is significant for understanding this organ's normal and pathological functioning. Apoptosis, or programmed cell death, is essential for maintaining tissue homeostasis, ensuring normal placental function, and maintaining adequate metabolism between mother and fetus. One of the main proteins that regulate apoptosis is BAX – a molecule that is actively involved in the initiation of this process through the activation of caspases.

The study aimed to determine the features of BAX-dependent apoptosis in the trophoblast of the placental chorionic villi by immunohistochemical method at delivery after 40 weeks.

Materials and methods: The placental material (pieces from the intermediate zone from the fetal plate to the basal plate) was fixed for 22–24 hours in a neutral buffered (Lilly) 10% aqueous formalin solution. Then, the fixed placental material was dehydrated in an ascending alcohol series (from 50 degrees to “absolute” alcohol) and embedded in paraffin at 58 °C.

Histological serial sections of 5 μm thickness were obtained on a sledge microtome MS-2. After deparaffinization of the histological sections, hematoxylin and eosin staining was performed, and immunohistochemical techniques were used on other serial sections in accordance with the manufacturer’s protocols (DAKO). In particular, immunohistochemical reactions with primary antibodies against the pro-apoptotic BAX protein and the anti-apoptotic protein Bcl-2 were performed. Visualization of primary antibodies was carried out using a polymeric system to visualize reaction results with diaminobenzidine dye (it gives a clear brown color to the locations of the studied antigens).

Along with the descriptive method of histopathological examination, we also performed computer morphometry on previously obtained digital copies of optical microscopic tissue images (Delta Optical Evolution 100 microscope – planachromatic objectives in accordance with the required optical magnification – and Olympus SP550UZ digital camera with an adapter).

Digital copies of the image were analyzed using a legal copy of the ImageJ v1.53t computer program, specialized for digital histometric studies. In particular, the hematoxylin and eosin-stained sections were subjected to a scoring test (repeated counting, which provides data on the proportion of villi with “syncytial nodules”).

The digital copies of images with immunohistochemical staining were assessed for specific staining intensity by computerized microdensitometry. To do this, we used the built-in standard tools of the ImageJ v1.53t computer program to obtain the computerized value of color brightness in an 8-bit color analysis system (with a gradation from zero to 255) using the microprobe method. Further, this value was converted to relative optical density (in optical density units) by logarithmic transformation. The value obtained in this way was convenient for interpreting the intensity of color as it ranged from zero (absolute transparency) to one (absolute opacity).

The obtained digital data were processed using statistical methods. In particular, with the help of a legal copy of the computer program for statistical calculations PAST v 4.17, a preliminary test for the normality of the distribution was performed using the Shapiro–Wilk test. According to this criterion, the hypothesis of normality of distribution was not rejected (at p=0.05) for all the studied statistical samples; thus, parametric statistical analysis methods were used: calculation of the arithmetic mean and its error, unpaired two-sided Student's test. In addition to the Student's test, the non-parametric Mann–Whitney test was also used, but statistical significance was reported only for the Student's test.

Results: Thus, from the point of view of molecular regulation of apoptosis in the trophoblast of the placental chorionic villi at delivery after 40 weeks vs. physiological pregnancy and delivery, conditions are created for enhanced trophoblast death by BAX-dependent apoptosis, since there is an increase in the concentration of the pro-apoptotic BAX-protein, while the concentration of its main antagonist, Bcl-2, decreases markedly.

Conclusions: At delivery after 40 weeks, the molecular processes of BAX-dependent apoptosis are activated in the placental trophoblast, and the proportion of villi with syncytial nodules increases accordingly.