title: In vivo serial imaging of regenerating corneal nerves after surgical transection in transgenic thy1-YFP mice.
abstract: OBJECTIVE
  To determine the effect of lamellar transection surgery on the nerve fiber density (NFD) and pattern of nerve regeneration in the cornea of thy1-YFP transgenic mice.
  
  
  METHODS
  Wide-field stereo fluorescence microscopy was used to obtain serial images of nerves in live thy1-YFP mice, which express a fluorescent protein in their axons. NFD (mm/mm(2)) was calculated from maximum intensity projection images as the total length of fibers within the area of the contour in which nerves were traced. Whole-mount confocal microscopy was performed to analyze the arrangement of nerves and the types of regenerating fibers.
  
  
  RESULTS
  NFD in normal corneas was 35.3 ± 1.8 mm/mm(2). Stereo fluorescence microscopy revealed the presence of a subbasal hairpin nerve layer and an intrastromal nerve trunk layer. After surgery, regenerative sprouting was observed from transected distal ends of intrastromal nerve trunks. NFD also increased, with this increase being maximal between 4 and 6 weeks after surgery. NFD approximated baseline values at 6 weeks and did not change any further at 8 weeks. Regenerated nerves did not readopt the normal corneal nerve arrangement. A dense interlacing network of regenerated nerves was present in the corneal bed. Branches from this network traversed the flap to innervate the epithelium. Immunofluorescence staining revealed that regenerating fronds contained peptidergic nociceptive fibers (positive for calcitonin gene-related peptide and substance P) and myelinated non-nociceptive fibers (positive for neurofilament 200).
  
  
  CONCLUSIONS
  Although corneal NFD recovers to normal levels by 8 weeks after nerve transection, the arrangement of regenerated nerves is abnormal.
authors: Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, College of Medicine, Chicago, Illinois 60612, USA.
authors: Abed
authors: A
authors: Namavari
authors: Shweta
authors: S
authors: Chaudhary
authors: Joy
authors: J
authors: Sarkar
authors: Lisette
authors: L
authors: Yco
authors: Kunal
authors: K
authors: Patel
authors: Kyu Yeon
authors: KY
authors: Han
authors: Beatrice Y
authors: BY
authors: Yue
authors: Jin-Hong
authors: JH
authors: Chang
authors: Sandeep
authors: S
authors: Jain
mesh_terms: D000818:Animals; D019771:Axotomy; D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism; D002452:Cell Count; D003315:Cornea / Q000294:innervation* / Q000601:surgery; D003319:Corneal Stroma / Q000294:innervation; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009413:Nerve Fibers, Myelinated / Q000502:physiology; D009416:Nerve Regeneration / Q000502:physiology*; D016900:Neurofilament Proteins / Q000378:metabolism; D009619:Nociceptors / Q000378:metabolism; D009882:Ophthalmic Nerve / Q000601:surgery; D013373:Substance P / Q000378:metabolism; D018800:Thy-1 Antigens / Q000235:genetics; D013997:Time Factors; D012668:Trigeminal Ganglion / Q000473:pathology* / Q000502:physiology* / Q000601:surgery
chemical_list: D008164:Luminescent Proteins; D016900:Neurofilament Proteins; D018800:Thy-1 Antigens; C051774:neurofilament protein H; D013373:Substance P; D015740:Calcitonin Gene-Related Peptide