Fig 1.
Dissimilarity in corneal nerve patterns.
Stereoflourescent microscope image of cornea shows nerve patterns are dissimilar within the same mouse (OS and OD; A1 and A2 respectively) and between littermates (OS of two littermates; B1 and B2 respectively). (C—E) Stereoflourescent microscope images of corneal nerves showing different branching patterns. (C) Dichotomously branching trunks terminate near the corneal apex (red dotted line) and interconnected nerve trunks (green dotted line) span limbus to limbus. (D) Interconnected pattern (green dotted line). (E) Dichotomously branching pattern (red dotted line). S: Superior; I: Inferior; N: Nasal; T: Temporal position; Black scale bars in Panels A—E: 500 μm.
Fig 2.
Fluorescent microscope image of in situ nerve transection.
After a single laser pulse, loss of nerve trunk fluorescence is noticed in the ablated area. Red arrow shows location of laser ablation. White scale bar: 20 μm.
Fig 3.
(A) In vitro neurite transection. Intact neurite (A1), transected neurite (A2), and disrupted neurite (A3) in TG neuronal cell culture. Red arrows show location of laser ablation. (B) In situ nerve transection. In vivo stereofluorescent images of corneal nerve before (B1) and after (B2) laser transection (inset: a dichotomously branching nerve trunk showing the ablation site that is marked with green arrows). (B3-B5) Confocal fluorescent microscope images of corneal whole mounts showing discontinuity in YFP fluorescence at the site of laser ablation (B3). Immunostaining for beta-III tubulin shows corresponding discontinuity in the nerve trunk (B4); thus confirming structural discontinuity in the nerve trunk (B5). White scale bars in Panels (A1- A3): 250 μm; Black scale bars in panels (B1-B2): 500 μm; White scale bars in Panels (B3 –B5): 50 μm.
Fig 4.
Nerve healing in interconnected nerves after single point transection.
(A): Stereofluorescent image of cornea prior to nerve transection (red arrow indicates point of planned transection; green dotted lines mark the interconnected nerves. (B-E) Wide-field fluorescent image showing nerve prior to transection (B), immediately after transection (C), four weeks after transection (D) and six weeks after transection (E). (F) Confocal image of transected area.
Fig 5.
Nerve healing after point transection.
(A): Magnified area of a stereofluorescent image of cornea two weeks after point transection (green dotted lines mark the transected area with cells; red dotted lines mark intact area of a normal untransected region along the nerve (B) inset shows magnified view of normal untransected nerve with cells oriented parallel along the nerve fibers (C) inset shows magnified view of transected area two weeks after ablation with cells oriented in perpendicular direction to the nerve.
Fig 6.
Nerve healing in dichotomous nerves after single point transection.
Dichotomous nerves before transection (A) and six weeks after transection (B). Red mark indicates point of transection; blue dotted lines mark the zone around the transected dichotomous nerve. Insets show magnified view of the dichotomous nerve before transection (A1) and disappearance of the nerve fluorescence six weeks after transection (B1)., Panels A and B; Panels A1 and B1 are magnified insets of A and B respectively.
Fig 7.
Nerve degeneration in interconnected corneal nerves after two point transection.
Nerve transection at 2 points (red arrow heads indicate point of transection) caused loss of YFP fluorescence and disappearance of nerve segment that was enclosed within the cut ends. The red arrow heads depict position of the 2 cuts along the nerve segment (Panel A) and green dotted oval denotes the region of the cut nerve. Panels B, C, D and E are magnified images of the transected nerve at different time points (Days 0, 14, 28 and 56 respectively). In 2-point nerve transections, the nerve segment enclosed within the two laser cuts degenerated and did not result in nerve healing. Black scale bar in Panel A: 500 μm; AT refers to “after transection”.
Fig 8.
Annular nerve transection causes nerve degeneration and regeneration.
Stereolumar image of annular nerve transection shows disappearance, reappearance or retention of nerve fluorescence. The yellow arrow heads depict positions of the nerve cuts within an area demarcated by the red circle. Panels B, C, D and E show magnified area within the red circle showing nerve changes corresponding to different time points after surgery (Days 0, 14, 28 and 56 respectively). The red arrow heads (Panel B) show nerve degeneration at different time points (Panels C–E). The yellow dotted area inset shows area of aberrant nerve regeneration; Black scale bar in Panel A: 500 μm; AT refers to “after transection”.