Dose-Dependent Onset of Regenerative Program in Neutron Irradiated Mouse Skin
Figure 6
Oxydative DNA damage determination by 8-oxo-G reaction and proliferation rate by Ki67 immunohistochemistry.
Representative images of 8-oxo-G reaction and Ki67 immunohistochemistry are reported in Panel A, and C respectively. Scale bar and abbreviations are identical to Fig. 3 for Ki67. Negative control N for 8-oxo-G reactivity is obtained with omission of the probe mix. Reactivity of the 8-oxo-G was nuclear in all samples. Worth noting, at 24 hours after irradiation, 1 Gy sample showed higher reactivity and a spread localization of damaged nuclei across stratified epithelia. On the contrary, 24 hours after 0.2 Gy exposure showed faintly positive nuclei that were localized along the basal layer. Arbitrary densitometric units of positive fluorescent 8-oxo-G reaction are plotted as whisker box in Panel B. Statistical analysis showed a significantly (p<0,05) higher reactivity for 1 Gy in the 6 hours mice (*) with respect to all other tested mice. The 0,2 Gy, in the 6 hour treated mice and 1 Gy, in the 24 hour treated mice were grouped to show a significantly (p<0,05) higher reactivity as compared with sham irradiated and 0.2 Gy, 24 hours group of mice (O). The 8-oxo-G reaction showed limited individual variation within the groups. Proliferation was determined by Ki67 nuclear localization in irradiated samples. Positive nuclei were scored following contrast phase microscopy visualization in oil immersion at 100x magnification, as shown as an inset in all samples. The frequency of positive Ki67 labeled cells are plotted as whisker box in Panel D. Statistical analysis showed a significantly (p<0,05) higher reactivity in the 0.2 Gy, 24 hours mice (*) with respect to all others treated mice. The 0.2 Gy, 6 hours treated mice and the 1 Gy, 6 hours were grouped to show a significantly (p<0,05) higher reactivity level with respect to sham irradiated and 1 Gy, 24 hours mice (O) (Panel C). The Ki67 immunohistochemistry reaction showed limited individual variation within the groups.