Figure 1.
Peripheral nerve damage following corneal transplantation.
Corneal transplantation in humans invovles a 360 degree full thickness incision of the cornea (A), and in vivo confocal micorscopy reveals recipient nerve fibers (white arrow) extending to the donor-host junction (arrow heads)(B). None of the 6 patients exmined demonstrated signs of nerve regerenation within the donor at 3 months. A murine transplantation model was developed by transplanting 2 mm wild-type donors into P0-Cre/Floxed-EGFP hosts (C) Sutures were removed after 7 days to avoid excessive inflammation (D). Peripheral nerves can be observed extending to the donor host juntion (arrow heads in E) by positive βIII tubulin staining and GFP in a magnified view (F). Dotted line in (F) shows the border of the donor and recipient cornea. Scale bar = 50 µm in B, 500 µm in E and 100 µm in F.
Figure 2.
SM-345431 enhances nerve regeneration into the donor cornea.
Peripheral nerves in the cornea of P0-Cre/Floxed-EGFP mice can be observed by GFP fluorescence (A), and immunohistochemisty of a 40 µm-thick frozen section shows the expression of Sema3A in the basal and suprabasal layers of the corneal epithelium (B, red) and GFP-positive major nerve fibers running through the corneal stromal layer under the epithelium (B, Green). Scale bar = 500 µm in A, 100 µm in B. GFP and βIII tubulin double-positive nerve fibers extending into the donor cornea were traced on an image processing software. The SM-345431-treated group showed a more robust network of regenerating nerves (D, F) compared to vehicle control (C, E).
Figure 3.
SM-345431 enhances the corneal blink reflex.
The extension of nerve regeneration in the SM-345431 treated-group was significantly greater than control as measured by the total pixel count of traced axonal growth (A). In order to semi-quantitatively measure corneal sensation, a Cochet-Bonnet esthesiometer was used to measure the length of filament required to elicit a blink reflex. Three weeks postoperatively, the Sema3A inhibitor-treated group showed significant improvement in corneal sensitivity compared to the control group (B).
Figure 4.
SM-345431 does not induce angiogenesis.
Sema3A is also known to suppress VEGF-induced neovascularization. In order to examine possible angiogenesis by inhibiting Sema3A, neovascularization in the transplanted grafts were quantified by anti-CD31 immunostaining (A, B) and image tracing (C, D). There was no significant difference in vascular area between the two groups (E). Scale bar = 500 µm in A, B.
Figure 5.
SM-345431 inhibits epithelial cell proliferation but does not affect cell viability or Sema3A production.
Since the corneal epithelium was the predominant source of Sema3A in the cornea, the effect of SM-345431 on cultured murine corneal epithelial cells were observed. There was a slight dose-dependent inhibition on cell proliferation (A) (n = 5, *, **P<0.05, One way ANOVA followed with Scheffe's F test). However, there was no effect on Sema3A production observed by western blot (B, C) or on cell viability under the dose used in the in vivo experiments.