ANALYSIS OF THE VOLUME OF BONE MARROW CELL NUCLEI FOR ASSESSMENT OF CYTOSTATIC MYELOSUPPRESSION AND ITS PREVENTION BY ACTIVATED CARBON

Abstract

Bone marrow suppression (myelosuppression) is a frequent complication of chemotherapy, and the need for monitoring and managing this side effect is still in great demand. Aim: to study the changes in the volume of nuclei in bone marrow cells of chemotherapy drug-treated laboratory animals with or without enterosorption by activated carbon (AC). Object and methods: both flow cytometry and confocal microscopy were used to analyze acridine orange (AO)-stained bone marrow samples of intact, doxorubicin (DOX)-, and DOX+AC-treated rats. Confocal Z-series that represent sequential scans of cell nuclei directed from top to bottom at 0.5-μm step size were acquired at 40× magnification using argon laser (488 nm) for excitation of AO. Green fluorescence emitted by DNA-bound AO was detected through a 505–530 nm band-pass filter, allowing distinct visualization of nuclei and their boundaries. Z-series were further processed and analyzed with ImageJ software to quantify the values of nuclear volumes. Results: there were apparent differences between the nuclear volumes in the bone marrow samples of intact, DOX-treated, and DOX+AC-treated rats. A significant increase of the volume of nuclei in DOX+AC-treated rats, compared with those in DOX-treated (1.42-fold) and intact rats (1.14-fold), is likely due to an active DNA replication, suggesting an ongoing recovery of the pool of nucleated cells. Notably, in these three groups, the populations of bone marrow nucleated cells, as estimated by flow cytometry, correlated well with the aforementioned values of nuclear volumes. However, the volume of nuclei may not necessarily correlate with the height of Z-series representing the thickness of nuclei, providing a clue that can help to delineate the role of nuclear deformability. Conclusion: analysis of the volume of bone marrow cell nuclei proposed in this work is important in terms of obtaining supplementary information in the study of the course of induced myelosuppression and of the ways of its prevention.