The morphometric method is a way to demonstrate cell death in the laboratory. Morphometric measurement provides the result of cell death as a volume, size, weight and length of tissue, organ and the whole organism that compares with before and after the occurrence of cell death. This method was observed by Attalah and Johnson who used electronic particle analyses to determine cell viability.
Histochemical and autoradiographical techniques Another indicator of cell death is acid hydrolysis, which is released from digestion during
phagocytosis of dead cells by
macrophages or neighboring cells, and the intravital dye is a marker of secondary phagocytosis.
Histological and cytochemical techniques To demonstrate cell death in some cases a
vital dye is used to detect when cellular function is disrupted. This procedure uses living tissue that is immersed in diluted 1:0000 solution of Nile blue sulphate in
saline. The measurement of cell death by using this dye is observing a change of color or the formation of
fluorescence. When the cell died the nucleus went through destruction stages, one of them pyknosis, which lead to the release of a basic
histone group and this happened when the irreversible condensation of chromatins occurred. The phagocytosis process took place in secondary lysosomes and the autophagy and heterophagy controlled the dead cell by acid hydrolysis activity. The techniques used to explained this is by the detection of (6-3H)-thymidine and acid phosphates' activity in cryostat.
Procedure Specimen was injected with (6-3H)-thymidine, and then the tissue was sacrificed, removed after 1 hour and quenched in liquid nitrogen. Then 4 μm
cryostat sections were cut and mounted on clean cover slips, the cover slips were held with sections in cryostat, fixed in cold analar acetone for 10 minutes and the cover slips were rinsed in buffer-incubated in acid phosphate medium (15 minutes).
Naphthol AS TR phosphate was used as the substrate and hexazonium paraosaniline as coupler. Again the sections were rinsed thoroughly in distilled water and the sections were dipped in autoradiography emulsion (I1ford L4 diluted 1:5). Preparations were exposed to be (0-4 °C) 2 to 3 weeks in dark room. The photographical slide was processed, counterstained in haematoxylin and mounted for
microscopy.
Results The result of this experiment is the red color which is caused by
azo-dye technique done above and this is an indicator that cell autolysis occurred. This was the major aim in the morphometric method. Another thing is the production of silver grains in the
photographic emulsion.
Discussion This change in color is due to fine
homogeneous red reaction of acid phosphatase activity. In the lysosome there's a lot of indication of cell death like the free hydrolase. Incorporate tritiated thymidine gives silver grains in the photographic emulsion which happened in the cell nuclei. The ideal tissue for this procedure is thymus tissue. This discussion focuses on two changes that occur in the thymus of mouse as an example study. The first change is the ratio of cells that are dying (diffusing acid phosphatase) and the second is the thymidine incorporating cells (cells synthesizing DNA). The results are compared according to the age of the mouse. After measuring the ratios and numbers, the conclusion is that the level of cell death in involuting thymus doubled in comparison to the young thymus and the thymidine decreased in the older thymus compared to the young thymus. To further show these results it was observed that some thymocytes contained lysosomal sites of acid phosphatase activity. When the macrophages engulfed the dying cells the levels of acid phosphatase increased.
Autoradiography technique with histological staining Lewis employed autoradiographic incorporation of 3H-thymidine to calculate
mitotic indices and estimate pyknotic indices. This technique can be used to study the tissue kinetics of tumors and has applications in the
scanning electron microscope.
Scanning electron microscopy The scanning electron microscope was employed by Hodges and Muir (1975) to study autoradiograph. This approach was combined with the cytochemical method for demonstrating free acid phosphate and
cell lysis. Dying cells which are rich in free acid phosphate will contain a brominated reaction product and will give a characteristic signal for bromine when subjected to
x-ray microanalysis. Fine structural studies There are common fine-structural changes occurring in dying cells. This was concluded after some attempts from the scientists like Kerr (1972) who proposed the general concept of apoptosis in vertebrates, While Scheweichel and Merker (1973) described induced and physiological cell death in prenatal mouse tissues. Using fine structural distinctions, it is possible to recognize and differentiate between the types of cell death, Acid phosphate and cell deletion: Acid phosphate is an enzyme or group of iso-enzymes which can be used in demonstrating cell death and this can be easily observed by the electron microscope. P-nitrophoenyl phosphate activity can be used as a good marker for cell death. This marker has been used to localize the cell death in cells during embryological development, and the result noticed was the release of exoplasmic nonlysosomal acid phosphate. This appears as a sign of cell death. There are many experiments showing that the ectoplasmic p-nitrophoenyl phosphate which was released is related to
ribosomes not to lysosomes. ==DNA, RNA and protein synthesis in cell death==