Does TUNEL staining during peri- and post-natal development of the mouse inner ear indicate apoptosis?

Terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labelling (TUNEL) is being used more frequently to investigate programmed cell death (PCD). We have applied this method in order to examine how PCD is involved in the development of the mouse inner ear. In a series of studies, we identified a population of TUNEL-positive cells in the perinatal mouse ear that could not be regarded as apoptosis based upon morphological features of the nuclei. Theoretically, TUNEL detects DNA fragmentation, which can also occur in necrosis. Other authors regard TUNEL-positive cells in the sensory epithelia of the rat equilibrium organs between gestational day (GD) 19 and 7 days after birth (DAB) as apoptosis. We determined whether or not cells in the inner ear of perinatal and post-natal mice were TUNEL-positive due to apoptosis. We stained the inner ears of BALB/c mice aged GD17.5-4 weeks by the TUNEL method and analysed morphology by light microscopy and transmission electron microscopy (TEM). TUNEL-positive cells were distinct in the saccule from DAB3, and in the cochlea from DAB8. The number of TUNEL-positive cells in the hair cells of the saccule and in the cochlea increased with age, and seemed to reach a plateau just before 2 weeks of age. However, morphological analyses did not reveal findings characteristic of apoptosis. We conclude that these TUNEL-positive cells were labelled not because of apoptosis, but due to necrosis or post-mortem autolysis. We surmise that TUNEL staining can identify vulnerable cells of the inner ear that consume high levels of oxygen and easily undergo autolysis.