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Abed Namavari, Shweta V. Chaudhary, Joy Sarkar, Lisette Yco, Ke Ma, Sandeep Jain; Nerve Regeneration in Murine Cornea. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1972.
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To determine the extent of nerve regeneration following surgical transection of corneal nerves, and to determine the expression of neurotrophins and nerve guidance proteins during nerve regeneration.
Hinged lamellar corneal flaps were fashioned surgically in Thy1-YFP neuro-fluorescent mice (n = 35). Sequential in vivo imaging of corneal nerves was performed at baseline and 2-week intervals for 12 weeks using stereo-fluorescence microscope. Maximum intensity projection was obtained from a Z stack of images at 5-micron thickness. Nerve fibers were traced by Neurolucida software and nerve fiber length density was calculated. To study the topography of regenerated pattern, 3D reconstruction of regenerated corneal nerves was created from confocal Z stack images of 1-micron thickness. The functional level of regenerated nerves was assessed using an electronic Von Frey filament. At 2 weeks, 4 weeks, 8 weeks (n > 5 for each time point) corneas were harvested. For each individual cornea, expression of neurotrophins (Ngf, Bdnf, Gdnf, Nt3, Nt5, Nrtn, Artn, and Pspn), nerve guidance proteins (Slit2, Sema3a, Sema6a, Sema7a, Epha3), and nerve regeneration-associated proteins (Tubb3, Gap43, and Sprr1a) was determined by pre-amplification followed by real-time quantitative PCR.
In normal mice, without surgery, corneal nerve density and pattern in each quadrant of the cornea, remained unchanged for the duration of study. According to histology (n = 10), the flap was reproducibly created with a thickness of 69% (SD = 9%) of the total corneal thickness. The mean nerve fiber length density was 18.2/mm at baseline, dropped to 10.1/mm at 2 weeks, and increased at a total rate of 1.7/mm/week (SD = 0.2/mm/week) over the duration of study. The regenerated nerves were predominantly located at sub-epithelial level. The morphology of sub-epithelial basal plexus of regenerated nerves showed an interlaced pattern with increased fiber thickness as opposed to normal hairpin pattern. RT-PCR showed increased (>2 folds) expression of Ngf, Bdnf, Sprr1a, Gap43, Slit2, Sema7a, and Tubb3. Bdnf and Ngf showed strong positive correlation with neurite extension between 2 weeks and 8 weeks.
Corneal nerves do not follow the normal pattern during regeneration. Ngf and Bdnf are the major neurotrophins, and Slit2 and Sema7a are the major nerve guidance proteins expressed in the cornea during nerve regeneration. Sprr1a, a regeneration-associated protein, is robustly expressed during nerve regeneration.
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