title: Corneal sulfated glycosaminoglycans and their effects on trigeminal nerve growth cone behavior in vitro: roles for ECM in cornea innervation.
abstract: OBJECTIVE
  Sensory trigeminal nerve growth cones innervate the cornea in a highly coordinated fashion. The purpose of this study was to determine if extracellular matrix glycosaminoglycans (ECM-GAGs), including keratan sulfate (KS), dermatan sulfate (DS), and chondroitin sulfate A (CSA) and C (CSC), polymerized in developing eyefronts, may provide guidance cues to nerves during cornea innervation.
  
  
  METHODS
  Immunostaining using antineuron-specific-β-tubulin and monoclonal antibodies for KS, DS, and CSA/C was performed on eyefronts from embryonic day (E) 9 to E14 and staining visualized by confocal microscopy. Effects of purified GAGs on trigeminal nerve growth cone behavior were tested using in vitro neuronal explant cultures.
  
  
  RESULTS
  At E9 to E10, nerves exiting the pericorneal nerve ring grew as tight fascicles, advancing straight toward the corneal stroma. In contrast, upon entering the stroma, nerves bifurcated repeatedly as they extended anteriorly toward the epithelium. KS was localized in the path of trigeminal nerves, whereas DS and CSA/C-rich areas were avoided by growth cones. When E10 trigeminal neurons were cultured on different substrates comprised of purified GAG molecules, their neurite growth cone behavior varied depending on GAG type, concentration, and mode of presentation (immobilized versus soluble). High concentrations of immobilized KS, DS, and CSA/C inhibited neurite growth to varying degrees. Neurites traversing lower, permissive concentrations of immobilized DS and CSA/C displayed increased fasciculation and decreased branching, whereas KS caused decreased fasciculation and increased branching. Enzymatic digestion of sulfated GAGs canceled their effects on trigeminal neurons.
  
  
  CONCLUSIONS
  Data herein suggest that GAGs may direct the movement of trigeminal nerve growth cones innervating the cornea.
authors.affiliation: Division of Biology, Kansas State University, Manhattan, Kansas 66506, USA.
authors.forename: Tyler
authors.initials: T
authors.lastname: Schwend
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Deaton
authors.forename: Yuntao
authors.initials: Y
authors.lastname: Zhang
authors.forename: Bruce
authors.initials: B
authors.lastname: Caterson
authors.forename: Gary W
authors.initials: GW
authors.lastname: Conrad
mesh_terms: D000818:Animals; D002642:Chick Embryo; D002809:Chondroitin Sulfates / Q000378:metabolism; D003315:Cornea / Q000196:embryology* / Q000294:innervation*; D003871:Dermatan Sulfate / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006025:Glycosaminoglycans / Q000378:metabolism*; D020439:Growth Cones / Q000502:physiology*; D007632:Keratan Sulfate / Q000378:metabolism; D018613:Microscopy, Confocal; D009474:Neurons / Q000502:physiology; D014276:Trigeminal Nerve / Q000196:embryology*
chemical_list: D006025:Glycosaminoglycans; D003871:Dermatan Sulfate; C013786:A73025; D002809:Chondroitin Sulfates; D007632:Keratan Sulfate