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Dev Dyn
2019 Jul 01;2487:530-544. doi: 10.1002/dvdy.42.
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The role of sensory innervation in cornea-lens regeneration.
Perry KJ
,
Hamilton PW
,
Sonam S
,
Singh R
,
Henry JJ
.
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BACKGROUND: Numerous sensory nerves in the cornea contribute to normal tissue homeostasis. Interestingly, cells within the basal corneal epithelium can regenerate new lenses in the frog, Xenopus. In this study, we investigated whether cornea sensory nerves or their neuropeptides are important for supporting cornea-lens regeneration.
RESULTS: Attempts to sever the trigeminal nervetrunk, which provides sensory nerve branches to the cornea, did not inhibit lens regeneration. However, using this approach we found that it was not possible to completely disrupt sensory innervation, as these nerves are able to quickly regenerate back to the cornea. On the other hand, attenuation of neuropeptide levels with capsaicin was found to significantly inhibit lens regeneration, as visualized by a reduction of Substance P. These treatments also led to a reduction of cornea sensory innervation. Interestingly, inhibition of the Substance P-preferred receptor NK-1 with Spantide II did not affect lens-regeneration rates.
CONCLUSIONS: This study provides evidence that cornea nerves support cornea-lens regeneration, which could occur through the release of various neurotrophic factors. Substance P, however, does not appear to be the critical component of this signaling pathway. Further studies are needed to investigate what role other known neurotrophic factors may play in this process.
Figure 1
Severing of the TGN and its effects on lens regeneration. A: Normal innervation of the larval Xenopus cornea (st 52) labeled with anti‐acetylated alpha‐tubulin antibody (green fluorescence). B: Schematic diagram of Xenopus cranial innervation on the left side of the head. The TGN branches from the brain and travels through the head region, where some branches serve the cornea (red branches; see Perry et al., 2013 for full description). The site containing the main nervetrunk that was severed for nerve cut experiments is noted with scissors. C: Time line of the TGN cutting experiments in conjunction with the lentectomy procedure to remove the lens. Time (days) is represented along the horizontal axis. D: Xenopus cornea labeled with anti‐acetylated alpha‐tubulin antibody (green fluorescence) at the end of the TGN cutting/lens regeneration experiment outlined in C. Note reduced labeling of main nerve branches and significantly reduced nerve structures in the central cornea region, as compared to A. Some fibers from both the mandibular nerve and the recurrent branch of the nasociliary nerve are seen. E: Representative transverse section from eye presented in D. The regenerated lens is labeled with anti‐lens antibody (labeled with ln; red fluorescence). F: A second case showing an anti‐acetylated alpha‐tubulin‐labeled (green fluorescence) cornea at the end of the TGN cutting/lens regeneration experiment outlined in C. Note little if any innervation from the mandibular nerve on the posterior side (left), but some innervation from the nasociliary nerve is present on the anterior side (right). G: Representative transverse section from eye presented in E, where the regenerated lens has been labeled with anti‐lens antibody (labeled with ln; red fluorescence). H: Graphical representation of lens regeneration rates following TGN cuts compared to control animals without TGN cuts. Abbreviations: co, cornea; ion, infraorbital nerve; ln, lens; mn, mandibular nerve; mnb, mandibular nerve branch; on, optic nerve; rb, recurrent nerve branch; rt, retina; TGN, trigeminal nerve. P value = 0.2606. Error bars represent SD. Scale bar in A,D,F = 105 μm. Scale bar in E,G = 140 μm
Figure 2
Effects of capsaicin treatment on Xenopus corneal innervation. Acetylated tubulin labeling is visualized with red fluorescence, and Substance P labeling with green fluorescence. A‐C: Central area of corneaepithelium. A: Anti‐acetylated tubulin‐labeled nerves of control corneaepithelium. B: Corresponding image to A showing subset of Substance P‐labeled nerves. C: Merged image from A and B, showing both acetylated tubulin‐ and Substance P‐labeled nerves. D: Acetylated tubulin‐labeled control cornea epithelial pelt, showing the entire region that overlies the eye. Large nerve trunks radiate finer branches toward the central cornea region (the latter noted with asterisk). E: Capsaicin‐treated cornea epithelial pelt (two days of treatment) showing a reduced number of acetylated tubulin‐labeled nerve branches radiating into the central cornea (asterisk), as well as reduction in the nerve trunks around the periphery. F: Higher magnification of acetylated tubulin labeling within the control corneaepithelium. G: Acetylated tubulin labeling of the capsaicin‐treated cornea (two days of treatment). Note reduced density of labeled fibers. H: Substance P‐labeled control cornea, corresponding to the same region shown in F. The white arrowheads point out Substance P–labeled fibers that overlap with acetylated tubulin–labeled nerve fibers. I: Substance P labeling of the two‐day capsaicin–treated cornea, corresponding to the same region shown in G. Substance P is visibly reduced along the acetylated tubulin–labeled nerve fiber shown in G (noted with white arrowhead). J–M: Representative corneas from the conclusion of the capsaicin treatment experiment (eight days of treatment). J: Acetylated tubulin–labeled control cornea pelt. K: Capsaicin‐treated cornea pelt, labeled with acetylated tubulin. Note the reduced density of fibers in the central region (compared with J). L: Substance P–labeled control corneaepithelium. M: Greatly reduced Substance P labeling of a capsaicin‐treated corneaepithelium (compared with L). N: Western blot analysis of control vs five‐day capsaicin‐treated corneas. An observable reduction was noted in protein levels of alpha‐tubulin (50kD) and Substance P (18kD) proteins. Beta‐catenin (94kD) was used as a loading control. Scale bar in A–C = 24 μm. Scale bar in D,E,J,K = 50 μm. Scale bar in F–I,L,M = 12 μm
Figure 3
A–F: Effects of capsaicin treatment and ΔNp63 labeling of central cornea epithelium. A: ΔNp63‐labeled (red fluorescence) cells of the basal layer of the cornea epithelium. B: Corresponding image to that shown in A of DAPI‐labeled nuclei (blue fluorescence). C: Merged image from A and B. Note the overlapping ΔNp63 and DAPI nuclear labeling in basal epithelial cells. D–F: Central region of capsaicin‐treated cornea epithelium. D: Normal‐appearing ΔNp63‐labeled cells (red fluorescence) in the basal epithelial layer. E: Corresponding image to that shown in D of DAPI nuclear label (blue fluorescence). F: Merged image of D and E, with overlapping nuclear and ΔNp63 labeling in the basal cells, which appears similar to the control shown in A–C. Scale bar in F = 20 μm
Figure 4
Capsaicin treatment inhibits Xenopus lens regeneration. A: Timeline of the capsaicin treatments in conjunction with the lentectomy procedure to remove the lens. Time (days) is represented along the horizontal axis. B: Graphical results of the lens regeneration rates following capsaicin treatment, as compared to control larvae. C‐D: Transverse section of eye region of a control, untreated tadpole undergoing lens regeneration (st 52). C: Anti‐lens antibody labeling of control regenerated lens (red fluorescence). D: Corresponding DIC image of that shown in A. E–H: Transverse section of eye region from capsaicin‐treated tadpoles (st 52). E: Anti‐lens antibody labeling of representative capsaicin‐treated larvae challenged to regenerate the lens, following original lens removal. As seen in most cases, no lens regeneration is apparent. F: Corresponding DIC image to that shown in D. G: Anti‐lens antibody labeling of capsaicin‐treated larvae that were able to regenerate a small lens (red fluorescence). H: DIC image corresponding to section shown in G. Abbreviations: co, cornea; ln, lens; rt, retina. * = P value <0.01. Scale bar in C–H = 140 μm
Figure 5
Ex vivo Xenopus lens regeneration assay results following capsaicin or Spantide II treatment. A: Summary timeline of treatments in conjunction with the lentectomy procedure to remove the lens. Capsaicin treatment (Exp 1) is represented in orange and Spantide II treatment (Exp 2) is represented in blue. Time (days) is represented along the horizontal axis. B: Transverse section of control, untreated eye showing merged images of regenerated lens body stained with anti‐lens antibody (red fluorescence) and nuclei stained with DAPI (blue fluorescence). C: Graphical representation of the results of the ex vivo lens regeneration experiments in controls vs capsaicin‐treated cultures. D: Transverse section of larva treated with capsaicin, showing merged image of regenerated lens body. E: Graphical representation of ex vivo lens regeneration experiments in controls vs Spantide II–treated cultures. F: Transverse section of ex vivo cultured eye in Spantide II containing media, showing a regenerated lens body. Abbreviations: ln, lens; rt, retina. Scale bar in B,D,F = 140 μm