Morphological Analysis of the olfactory epithelium at day 2 after Triton-X application.

<p>(A,B) Representative photographs of hematoxylin and eosin-stained sagittal sections of the OE, two days after intranasal injection of Triton-X in 6 month old (A) G3 <i>mTerc^−/−</i> and (B) <i>mTerc^+/+</i> mice. There are no significant differences between the two cohorts, both showing strong damage to 80–90% of the OE. The histograms show the percentage of the chemically damaged olfactory epithelium in <i>mTerc^+/+</i> and G3 <i>mTerc^−/−</i> mice at two days after Triton-X induced injury: (C) percentage of damaged epithelium of 0–4 cell layer thickness (P = 0.7887), (D) percentage of completely damaged epithelium of 0–2 cell layer thickness (P = 0.8208).</p

Telomere shortening does not affect homeostasis of the olfactory epithelium in aging mice.

<p>(A, B): Representative photographs of hematoxylin and eosin-stained longitudinal sections of the OE from 2–3 month old (A) <i>mTerc^+/+</i> and (B) G3 <i>mTerc^−/−</i> mice, and 10–12 month old (C) <i>mTerc^+/+</i> and (D) G3 <i>mTerc^−/−</i> mice. (E-J) Immunohistological analysis of longitudinal sections of the OE 10–12 month old (E, G, I) <i>mTerc^+/+</i> and (F, H, J) G3 <i>mTerc^−/−</i> mice: (E, F) Olfactory marker protein (OMP), (G, H) GAP43 and (I, J) proliferating cell nuclear antigen (PCNA). White arrows point to PCNA positive cells (G, H). (K) Histogram showing percentage of PCNA-positive cells in the OE of 10–12 month old <i>mTerc^+/+</i> and G3 <i>mTerc^−/−</i> mice (n = 10 mice per group, P = 0.4580).</p

Telomere shortening impairs regeneration of the olfactory epithelium in response to injury.

<p>(A, B) Representative photographs of hematoxylin and eosin-stained sagital sections of the nasal cavity, seven days after intranasal injection of Triton-X in 6 month old (A) G3 <i>mTerc^−/−</i> and (B-E) <i>mTerc^+/+</i> mice. Dotted line in A and B marks incompletely regenerated epithelium of 0–2 cell layer thickness, double line marks incompletely regenerated epithelium of 3–4 cell layer thickness, dot/bar line marks completely regenerated epithelium of 5–6 cell layer thickness. Representative high-power photographs of G3 <i>mTerc^−/−</i> mice showing (C) incompletely regenerated epithelium with 0–2 cell layer thickness (dotted line), (D) 3–4 cell layer thickness (double linier) (E) completely regenerated olfactory epithelium (E). (F, G) The histograms show the percentage of the olfactory epithelium with incomplete regeneration in <i>mTerc^+/+</i> and G3 <i>mTerc^−/−</i> mice at seven days after Triton-X induced injury: (F) percentage of incompletely regenerated epithelium of 0–2 cell layer thickness, (G) percentage of incompletely regenerated epithelium of 0–4 cell layer thickness.</p

Limited proliferation potential of the OE in telomere deficient mice.

<p>(A,B) Representative photographs of BrdU-stained longitudinal sections of the olfactory epithelium, 7 days after Triton-X treatment in (A) <i>mTerc^+/+</i> and (B) G3 <i>mTerc^−/−</i> mice. (C) Histogram showing BrdU positive cells in the OE of G3 <i>mTerc^+/+</i> and <i>mTerc^+/+</i> mice. Note that there is no significant difference of the ratio of BrdU positive cells v.s. negative cells between <i>mTerc^+/+</i> and G3 <i>mTerc^−/−</i> mice in injured olfactory epithelium of one cell layer thickness (P = 0.216) but there was a significant reduction of BrdU positive cells in G3 <i>mTerc^−/−</i> compared to <i>mTerc^+/+</i> mice in injured olfactory epithelium of three cell layer thickness (P = 0.008) and 5–6 cell layer thickness (P = 0.0293), n = 5 mice per group. (D) The histogram shows the percentage of the olfactory epithelium with incomplete regeneration (0–2 cell layer thickness) in 6–8 month mice of the indicated genotypes at 7 days after Triton-X induced injury. Note that <i>p21</i> deletion rescues regenerative defects in G3 <i>mTerc^−/−</i> mice. The cohorts in this experiment show an overall higher rate of tissue damage compared to the previous experiment depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027801#pone-0027801-g003" target="_blank">Figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027801#pone-0027801-g004" target="_blank">4</a>.</p

Shortened telomeres in the olfactory epithelium of G3 <i>mTerc^−/−</i> mice.

<p>(A, B): Distribution of the mean telomere fluorescence intensity (TFI) of cells of the olfactory epithelium in 6–8 month old <i>mTerc^+/+</i> mice (A) and of G3 <i>mTerc^−/−</i> mice (B) (n = 4 mice per group). The dotted line shows the mean TFI of olfactory epithelial cells in mice of the two cohorts. Note that the olfactory epithelium of G3 <i>mTerc^−/−</i> have shorter telomeres than <i>mTerc^+/+</i> mice (P = 0.0207).</p