Thermal stress induces pyknosis in pigeon erythrocytes: digital image analysis

Abstract

Pyknosis or hypercondensation of chromatin is informative in the understanding of nucleosomal packing in translationally inactive chromatin and in the compression of cell death. However, mechanisms that result in the formation of avian erythrocytes with variant nuclear morphology are poorly understood. Purpose: In this work, we evaluated pyknosis in pigeon erythrocytes treated with thermal stress using Digital Image Analysis (DIA). Materials and methods: Pigeon erythrocytes were treated at thermal stress (33 °C, 43 °C, and 53 °C), and nuclear modifications were analyzed by DIA. Results: Our results showed that thermal stress induced DNA condensation. Based on DNA fluorescent staining and compaction, four subclasses with progressively more pyknotic nuclei each could be distinguished. Alkaline comet assay showed that the presence of pyknotic nuclei was associated with the DNA fragmentation typical of apoptosis. DIA analysis showed a decrease of nuclear area and a significant increase of fluorescence intensity with respect to non-pyknotic nucleus. Additionally we observed nuclear dissolution events associated with swell and loose membrane integrity. Conclusion: These findings can contribute to the evaluation of health and metabolic status in diagnostic cytology, especially in neoplastic conditions and infection by microorganisms.