The ELISA analysis in 8-, 26-, and 52-week-old mice corneas showed a reduction of SP levels in older mice (
Fig. 1A, −56%,
P = 0.0045, 8 vs. 52 weeks; −43%,
P = 0.004, 26 vs. 52 weeks). Corneal total nerve length was measured in mice with different ages using an anti-β3 tubulin antibody (
pan-neuronal marker). Older mice showed a decreased total nerve length (TNL;
Fig. 1B,
1C, −18%,
P = 0.018, 8 weeks vs. 26 weeks; −36%,
P = 0.0001, 8 weeks vs. 52 weeks; −22%,
P = 0.02, 26 weeks vs. 52 weeks). Interestingly, SP-KO mice showed the same TNL as Wild type (WT) mice at 8 weeks of age, but a lower TNL at 26 weeks (−19%
P = 0.047) when compared with age-matched WT animals (
Fig. 1B,
1C). When compared with 8-week-old KO mice, 26-week-old KO mice showed a greater TNL reduction (−39%,
P = 0.0016) when compared with the TNL reduction observed in aging WT mice (
Fig. 1B). In the oldest group of WT mice, the reduction of SP concentration related to a reduction of cornea sensitivity after NaCl topical application (
Fig. 1D, −30.5%,
P = 0.007, 8 weeks vs. 52 weeks). The age-related reduction of sensitivity did not reach the reduction caused by the total absence of SP in KO mice (
Fig. 1E, −50.4%,
P < 0.0001, WT vs. KO). Interestingly, the treatment of 8-week-old KO mice with topical SP was able to restore sensitivity levels to those observed in WT mice (
Fig. 1E,
P = 0.021 KO vs. KO+SP; not significant, WT vs. KO+SP). Cross-section staining of 8-week WT mouse cornea showed a colocalization of SP with the neuronal marker β3 tubulin (
Fig. 1F).