Following X-ray irradiation, we observed an overall decrease in cell proliferation at 24 hours, a complete abolition of mitosis at 2 and 24 hours and a decrease in apoptosis at 2 hours. These data seem to indicate that the ON, and especially the active cycling zone of the ON, the basal lamina, has suffered a cell cycle arrest immediately after and as a consequence of ionizing radiation. X-ray irradiation has been shown before to induce permanent cell cycle arrest and the exhibition of senescence-like phenotypes in normal human cells  and to induce senescence in adult human mesenchymal stem cells . We do not believe these cells have entered senescence, a process in which cells permanently stop dividing and that can happen after DNA damage following X-ray irradiation, because both proliferation and apoptosis tend to return to normal levels after 2–5 weeks. We believe these cells have entered a reversible cell cycle arrest triggered by ionizing radiation-induced DNA damage, from which they recover after only 1–2 weeks, confirming the plasticity of the ON. Following X-ray irradiation, there are variations in radiation sensitivity of neural stem and progenitor populations in the developing mouse brain [34, 35]. This radiation sensitivity applies to cell proliferation and apoptosis. X-ray irradiation of the adult mouse nose did not cause increased cell death at any time points post-irradiation. 8 Gy of X-rays (used in this study) caused a significant increase in the numbers of apoptotic nuclei in the dentate subgranular zone following X-ray exposure to the brains of mice . A single 1 Gy dose of irradiation caused an increase in apoptosis within the subventricular zone however, no apoptosis was observed in other brain regions . Therefore different regions of the mouse central nervous system have varying levels of sensitivity to irradiation with respect to both the proliferation of neural precursors as well as cell death.
Contrary to other models of ON degeneration, which result in high morphological alterations and overall degeneration of the ON, our model seems to target specifically the proliferating cell populations, with minor morphological changes. We have found a disruption of the basal lamina at 1 and 2 weeks post-irradiation, but the overall gross morphology of the ON remained unaltered.
As expected, for all groups, the changes in proliferation and apoptosis mostly occurred in the basal lamina, indicating that this area is indeed actively cycling. This area is historically considered the locus for olfactory stem cells in the ON and the only zone for cell proliferation. Our results have also shown that apical sustentacular cells undergo proliferation and apoptosis. Following X-ray irradiation, there are greater numbers of cells in the apical layer of the ON expressing Ki67 compared to BrdU. It has been shown before that nestin, an intermediate filament protein expressed in progenitor cells of the neural lineage, in the ON is mainly expressed by endfeet and inferior processes of sustentacular cells . It has also been shown that during mouse ON development, in particular at E12.5, there are two populations of dividing progenitors, located respectively on the apical and basal sides of the ON . The proportion of basal progenitors then increases progressively, as development of the ON proceeds, and that of apical progenitors decreases [39, 40]. It has been suggested that these apical progenitors, which express Mash1, would give rise to more mature progenitors that migrate basally . Others have shown that apical progenitors also express the transcription factors Six1 and Hes1  and that they may be progenitors for sustentacular cells. It could be that different genes regulate neurogenesis differently during embryonic and established neurogenesis [37, 42, 43]. The endfoot of the sustentacular cell is adjacent to the basal lamina of the ON. We have shown that the basal lamina is disrupted following X-ray irradiation. It is possible that this process causes the sustentacular cell to undergo morphological changes. A study of the human ON showed that nestin expression in sustentacular cells was related to olfactory function however, olfactory impairment was not connected with abnormalities at the basal cell level . Overall, it seems that there might be indeed a role for sustentacular cells that is more complex that presently believed.
CD15-positive olfactory glandular cells migrate from the lamina propria into the basal layer of the ON corresponding to a severe disruption in the basal lamina at 1 and 2 weeks post-X-ray irradiation. A previous study showed that degeneration of the ON during normal aging spatially co-localizes with lesions in the Bowman's gland in the mouse olfactory mucosa . We have observed activation/migration of CD15-positive glandular cells coinciding with an increase in olfactory neuron markers (carnosine and OMP), an increase in cell proliferation and an overall surge of activation/regeneration in the ON. CD15 is considered a marker for glandular cells but it has been shown previously to label adult neural stem cells [46, 47]. The previous demonstration that olfactory cell populations marked by CD15 give rise to neurospheres , indicates that glandular cells marked by CD15 might have neurogenic potential. It has been proposed that there is a mesenchymal stem cell population in the lamina propria, which is able to cross to the ON [48, 49]. The disruption of the basal lamina and the opportunity for CD15-positive glandular cell to migrate across the basal lamina as a consequence of X-ray irradiation, leads us to hypothesize that there may be a fluidic interaction between Bowman's gland in the lamina propria and the ON.
Mash1 is a transcription factor involved in neurogenic cell fate [50–52], and is necessary for the differentiation of OSNs in the ON . Pax6 is a transcription factor expressed in embryonic and adult stem cells and involved in determination of neurogenic cell fate [53, 54]. In the olfactory system Pax6 has been shown to be involved in the development of olfactory supporting cells . Both Mash1 and Pax6 were expressed in cells throughout the ON (including sustentacular cells in the apical layer). The numbers of Pax6- and Mash1-positive nuclei are at their highest levels 24 hours post-irradiation and then decline from 1–5 weeks post-irradiation. This increase in transcription factors subsequently led to a greater number of mature OSNs 1 week post-irradiation. We know from previous experiments that an increase in the numbers of Mash1-positive cells followed a reduction in OSNs after bulbectomy . Following methyl bromide lesion, expression of the transcription factor p63 is no longer evident in horizontal basal cells . It is re-expressed 3 days post-lesion and by 7 days post-lesion, ORNs are evident . However, another cue for regulating transcription factors is driven by the apoptosis of olfactory precursors. Over expression of the apoptosis signaling regulating kinase-1 (ASK1) in adult hippocampus-derived progenitor cells activated the Mash1 promoter .
X-ray irradiation has undoubtedly resulted in impairment in the olfactory function, measured by both the Buried Food Pellet test and the Habituation/Dishabituation test. It is evident at 24 hours post-irradiation that there is olfactory impairment. As olfactory bulbs were protected by a lead shield during irradiation, we do not think the olfactory impairment is due to a deficit in olfactory bulb function, OSN signaling via axons or odorant detection by olfactory neuron cilia. The examination of βIIITubulin expression in the cell soma, nerve fibres and cilia of OSNs in the ON and the nerve fibres, glomerular and mitral cells of the bulbs showed no change following irradiation. Previous studies have shown that βIIITubulin forms microtubules that form neurites  and that it is specific for the cilia, dendrites, somata and axons of OSNs [59, 60]. Following axonal degeneration caused by bulbectomy in the rat, βIIITubulin expression is lost in OSNs . This did not occur in our irradiated mice. In the Habituation/Dishabituation test one of the odours (strawberry) could still be identified after 24 hours and impairment for this odour was only seen 1 week post-irradiation. The repertoire of OSNs in the mouse ON is highly complex [61, 62], so that the identification of each odour probably relies on the use of distinct or partially distinct OSN families, differently affected by our irradiation protocol. The Buried Food Pellet test relies on the animal’s natural tendency to use olfactory cues for foraging whereas the Habituation/Dishabituation test relies on the animal’s tendency to investigate novel smells and discriminate between different odours. The irradiation of the ON led to an immediate disruption of olfactory responses. Detection of general odorants by the vomeronasal organ has been reported [63, 64]. Examination of the vomeronasal organs of the irradiated mice compared to the control group showed no histological aberrations. Therefore we do not attribute the loss of olfactory function to a defect in the vomeronasal organs of irradiated mice. However, we were able to show that the mucous layer as shown by CD15 expression was absent at 24 hours post-irradiation. Nagashima & Touhara  demonstrated that enzyme inhibitors in the mucosa cause the enzymatic conversion of odours fast enough to affect recognition of the odorant at the levels of the OSNs. Therefore, following X-ray irradiation the detection and discrimination of odours is affected by the differential presence of nasal mucous.