To evaluate whether inhibition of ADAM17 correlated with better healing after NM exposure, the four hydroxamates were individually applied to three to five sets of corneas at 2 hours post NM exposure. The 3-nmol dose for the hydroxamates was used, delivered in 20 μL four times over the course of the 22 hours after the 2-hour exposure. This dose for each was chosen because it was in the linear range of inhibition for each hydroxamate, being optimal for NDH4417. In addition, this dose gave the best spread for the levels of inhibition of the four hydroxamates, and it would therefore give a range of ADAM17 inhibition that should affect the resulting degree of epithelial–stromal separation. Our first measure of attenuated injury was from calculating the preservation of epithelial–stromal integrity at 24 hours post NM exposure from the histology.
Figure 6A demonstrates that NDH4385, a treatment that gave the least ADAM17 inhibition at 3-nmol doses after NM exposure, did not effectively help retain epithelial–stromal integrity. When compared to NM-exposed corneas with no subsequent treatment (compare to
Fig. 2C), there was little difference between an exposed cornea receiving no therapy and one receiving four 3-nmol doses of NDH4385 over a 24-hour period. With NDH4409 (Fig. 6B) and NDH4450 (
Fig. 6D) there was some improvement in epithelial–stromal integrity as observed by more areas where the cell layers remained attached. As indicated by the ADAM17 activity assays, corneas treated with 3-nmol doses of NDH4417 four times over a 22-hour period showed the highest level of epithelial–stromal integrity at 24 hours post NM exposure (
Fig. 6C). Higher magnifications of other H&E-stained corneas, untreated or treated with the hydroxamates, are shown in
Figure 7. These are accompanied by immunofluorescence analyses of sections reacted with the R&D ADAM17 antibody, which detects the active enzyme on sections. The series of corneal sections in
Figure 7A across the top of the figure represent corneas that receive no hydroxamate (indicated as “no drug”). The two corneas in the left two panels were not exposed to NM and showed 100% epithelial–stromal integrity in the H&E section and no green fluorescence in the companion section, suggesting ADAM17 activity was minimal. The two panels on the right side of
Figure 7A were exposed to NM and received no hydroxamate. The H&E section showed approximately 85% detachment (15% attachment) of the epithelium at 24 hours after NM exposure. Sections of these exposed but untreated corneas reacted with the ADAM17 antibody, showing intense green fluorescence at the epithelial–stromal junction across most of the cornea.
Figures 7B through 7E follow the same setup, with two unexposed cornea sections that were treated with a hydroxamate on the left side, and NM-exposed corneas treated with the same hydroxamate on the right side. The H&E-stained sections in
Figure 7B indicate that treatment with NDH4385 after NM exposure has some small ability to prevent total cell layer separation 24 hours later. The immunofluorescence (IF) images of both the unexposed and NM-exposed samples in
Figure 7B show green fluorescence at the epithelial–stromal junction, albeit less in the unexposed corneas. This suggests that NDH4385 alone may slightly activate ADAM17 activity at this dose. Comparing the NM-exposed corneas receiving no treatment and those receiving NDH4385, however, it is clear that ADAM17 immunofluorescence does indicate that the enzyme was somewhat attenuated with NDH4385 treatment. In the
Figure 7C panels are corneas treated with NDH4409. Here the unexposed corneas appear normal, but after NM exposure, the H&E and IF samples have the typical appearance of epithelial and stromal layers that are likely to detach. In the
Figure 7D panels, the corneas were treated with NDH4417, the best ADAM17 inhibitor as evaluated by ADAM17 activity assays. These corneas showed a good outcome. The nuclei did not rise in the basal epithelial cells of the NM-exposed corneas treated with NDH4417, and the immunofluorescent signal for ADAM17 was attenuated compared to that of exposed corneas receiving treatment with other hydroxamates. The NDH4450-treated corneas (
Fig. 7E, panels) have some green fluorescence in both the unexposed and NM-exposed corneas. In the unexposed cornea, this fluorescence was close to the basal cell nuclei, unlike the NM-exposed cornea, where the fluorescence was distant from the basal cell nuclei. This was supported by the H&E panels of the unexposed and NM-exposed corneas, which showed nuclei higher in the basal epithelial cells after NM exposure. The fluorescence in unexposed corneas suggests NDH4450 may also activate ADAM17 to some degree, and therefore it would not be an ideal therapy with this dosing regimen. The best result was clearly obtained with NDH4417 (
Fig. 7D). At 24 hours post NM exposure, the basal epithelial cell nuclei remained in place in the H&E sections and the ADAM17 antibody produced a minor, patchy fluorescent signal at the epithelial–stromal border, much attenuated compared to NM-exposed corneas receiving no hydroxamate treatment.