Corneal Sulfated Glycosaminoglycans and Their Effects on
Trigeminal Nerve Growth Cone Behavior In Vitro: Roles
for ECM in Cornea Innervation
Tyler Schwend,*,1Ryan J. Deaton,2Yuntao Zhang,1Bruce Caterson,3and Gary W. Conrad1
PURPOSE. Sensory trigeminal nerve growth cones innervate the
cornea in a highly coordinated fashion. The purpose of this
study was to determine if extracellular matrix glycosaminogly-
cans (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-b-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 immo-
bilized DS and CSA/C displayed increased fasciculation and
decreased branching, whereas KS caused decreased fascicula-
tion 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. (Invest Ophthalmol Vis Sci. 2012;53:8118–8137) DOI:
in the developing peripheral nervous system (PNS). These cues
may be growth-promoting, growth-permissive, or growth-
inhibitory, dependent on both the nature of the ECM
molecule(s) and the context in which they are presented in
the peripheral tissue to growth cones. Inhibitory ECM
molecules in the PNS serve as barriers to block and steer
axonal growth cones from projecting into inappropriate tissues
and keep them headed toward their target tissue, whereas
simultaneously, growth-promoting factors serve to positively
attract growth cones.1In this manner, sensorimotor innerva-
tion during early PNS development is dependent on transient
spatiotemporal distributions of ECM molecules with differen-
tial potentials for influencing nerve-growth cone behavior.
Proteoglycans (PGs) represent a class of glycoproteins that
carry sulfated polysaccharide side chains and are commonly
enriched in nonpermissive peripheral tissues that border the
pathways of growing axons during PNS development. PGs are
composed of a core protein covalently attached to glycosami-
noglycans (GAGs), long unbranched polysaccharide chains of
repeating disaccharide units. Each disaccharide unit contains
either D-glucuronic acid (GlcA), L-iduronic acid (IdoA), or D-
galactose (Gal) paired in each disaccharide with either D-N-
acetylglucosamine (GlcNAc) or D-N-acetylgalactosamine (Gal-
NAc), with one or both of the sugars in each disaccharide
sulfated, and thus highly charged ionically (Fig. 1). The adult
human corneal stroma contains (by weight) 65% keratan
sulfate (KS) and 30% chondroitin/dermatan sulfate (CS or DS),2
which were each first described as major corneal GAGs by
Meyer and colleagues.3,4
Chondroitin sulfate proteoglycans (CSPGs) influence many
biological functions, including cell adhesion, cell migration,
axonal guidance and pathfinding, and axonal fasciculation/
defasciculation behavior.5–7With respect to axonal growth
cone movement, CSPGs in the ECM have been shown to be
inhibitory both in vitro8–22and in vivo.23–29Interestingly, other
studies have reported positive effects of CSPGs on neurite
outgrowth.30–33Differential effects on neuronal growth likely
arise from the high degree of polymorphism of PGs and GAGs,5
their sulfation patterns in ‘‘hot spots’’ along GAG
chains,31,34–37their mode of presentation to neuronal growth
cones (i.e., immobilized versus soluble),38and their reversible
binding and release of growth factors, guidance cues, and
other ECM molecules of small and large molecular size and
he extracellular matrix (ECM) provides regulatory cues
that govern growth and guidance of axonal growth cones
Manhattan, Kansas; the
Illinois at Chicago, Chicago, Illinois; and3Connective Tissue Biology
Laboratories, School of Biosciences, Cardiff University, Cardiff,
Wales, United Kingdom.
Supported by an individual Ruth L. Kirschstein Postdoctoral
National Research Service Award/National Eye Institute/National
Institutes of Health (NEI/NIH) Grant F32 EY021708 (TS), NEI/NIH
Grant R01 EY000952 (GWC), funding from the Research Career
Development Core (Brychta) in the Division of Biology at Kansas
State University GOBO000657 (GWC), and by NIH-P20-RR017686 to
the Center of Biomedical Research Excellence Core B in the
Veterinary School at Kansas State University. The authors alone are
responsible for the content and writing of the paper.
Submitted for publication August 23, 2012; revised October 16,
2012; accepted October 27, 2012.
Disclosure: T. Schwend, None; R.J. Deaton, None; Y. Zhang,
None; B. Caterson, None; G.W. Conrad, None
Current affiliation: *Department of Comparative Biosciences,
College of Veterinary Medicine, University of Illinois at Urbana-
Champaign, 3411 Vet Med Basic Sciences, Urbana, Illinois.
Corresponding author: Gary W. Conrad, Division of Biology,
Ackert Hall, Kansas State University, Manhattan, KS 66506;
1Division of Biology, Kansas State University,
2Department of Pathology, University of
Investigative Ophthalmology & Visual Science, December 2012, Vol. 53, No. 13
Copyright 2012 The Association for Research in Vision and Ophthalmology, Inc.
charge.39–41Moreover, the developmental context of the cell–
ECM interaction, such as the origin and history of the neuron
and the presence of other ECM molecules in the substratum,
likely also influences the behavior of neuronal growth cones
traversing the ECM. Keratan sulfate proteoglycans (KSPGs) also
are generally considered to constitute inhibitory barriers to
axonal growth.42–47Based on findings from many of the above
studies, which studied the biological function of the GAG chain
independently of the PG core protein, it is now generally
believed that the sulfated GAG moiety contributes substantially,
if not wholly, to the nerve-influencing function of the CS/
KSPGs. In support of this, recent reports indicate that animals
treated with chondroitinase ABC, which degrades sulfated
CSA/CSC/CSB(DS) GAG chains but leaves PG core proteins
intact, display enhanced axonal growth, regeneration, and
function following nervous system injury.24,27,48–53Moreover,
digestion of CS–GAG chains in the notochord54,55or head56
during embryonic development leads to growing sensory and
motor axons projecting into tissue locations that normally do
not support axon growth.
The cornea is the most densely innervated tissue on the
surface of the body.57The vast majority of those nerves are
sensory,57,58derived from neural crest–derived neurons
residing in the ophthalmic branch (OpV) of the trigeminal
ganglion (TG).59Chick cornea innervation from the TG is
highly regulated. Beginning at embryonic day (E) 4, TG-derived
(trigeminal) growth cones grow directionally toward the
cornea, approaching its periphery by E5, yet trigeminal growth
cones are then repelled from entering the cornea for several
days. Trigeminal nerve bundles encountering the margin of the
cornea bifurcate into a dorsal and a ventral stream at the
cornea, yet stay close to its edge, as though still positively
attracted into the stroma, thus eventually forming a complete
pericorneal nerve ring.60During later corneal development, at
E9 to E10, the cornea becomes permissive to a subset of the
trigeminal nerve growth cones that are exclusively of neural
crest origin, whereas ectodermal placode–derived trigeminal
nerve growth cones remain associated with the pericorneal
nerve ring.59Axons then send growth cones toward the edge
of the corneal stroma from all along the circumference of the
nerve ring61and extend exclusively into the anterior layers of
the developing corneal stroma, navigating steadily toward the
corneal epithelium,62the outermost cell layer on the surface of
the eye. In this manner, growth cones avoid innervating
posterior stromal regions and the corneal endothelium, lining
the innermost surface of the cornea. Currently, the mecha-
nism(s) controlling these axon guidance decisions remain
unclear. Increased levels of sulfated GAGs have been reported
coincident with nerves beginning to invade the corneal
stoma.63–67GAGs localized in different anterior–posterior
positions in the corneal stroma during innervation may serve
to provide specific positive or negative signals to incoming
growth cones. In this manner, corneal GAGs may provide
selective barriers to neuronal growth cone extension and
guidance to ensure that they are guided toward the outermost
surface of the cornea, finally penetrating the basement
membrane and extensively bifurcating and branching among
the epithelial cells.68
Despite the robust presence of GAGs in the developing
cornea, their potential in guiding corneal nerve growth cones
during innervation has not been studied. Moreover, few studies
have considered the effects of individual GAG moieties on
sensory outgrowth, nor have the effects of GAGs on sensory
neurons of the cornea (i.e., neural crest–derived neurons
derived from OpV TG) been investigated to date. In the current
study we examined differences in the neurite behavior of
trigeminal nerve growth cones when challenged with different
GAG molecules that are known to be present in the developing
corneal stroma. Among the GAGs that were examined herein
are KS and DS, the predominant corneal stroma ECM–GAGs, as
well as CSA and CSC that are detectable in the limbal
mesenchyme and cornea during development. The distribution
of each GAG in the cornea was examined by immunohisto-
chemistry at stages concurrent with trigeminal growth cones
projecting from the mesenchyme region containing the limbal
nerve ring toward the periphery of the corneal stroma (E9), up
to a later stage wherein growth cones have progressed to the
center of the cornea and anteriorly into the epithelium (E14).
We found the distributions for each GAG to be developmen-
tally regulated. CS/DS was localized between corneal cell layers
that were mostly avoided by trigeminal axonal growth cones,
whereas KS was more broadly localized throughout the cornea
in positions both occupied and unoccupied by axonal growth
cones. To examine the potential biological relevance of GAGs
in cornea innervation, embryonic trigeminal neurons were
cultured with purified substratum-bound, or soluble, CSA, DS,
CSC, or KS and neurite outgrowth behaviors were analyzed
qualitatively and quantitatively. We show that trigeminal
neurites displayed a growth response when they encountered
GAG molecules bound to substrate, but not when GAGs were
presented in a soluble fashion. High concentrations of
substrate-bound KS, DS, and CSA/C inhibited neurite growth
to varying degrees, with KS showing the weakest inhibitory
potential among the GAGs tested. Moreover, neurites travers-
unit of GAGs used in this study. (A) KS: [Gal-b-1,4-GlcNAc(6S)]n. KS
repeating disaccharide unit is composed of alternating residues of D-
galactose (Gal) and N-acetyl-D-glucosamine (GlcNAc) linked b-1,4 and b-
1,3, and hydroxyl groups at the C-6 positions of Gal and GlcNAc
residues may be sulfated. (B) CSC: [DUA-b-1,3-GalNAc(6S)]n. CSC
repeating disaccharide unit is composed of N-acetylgalactosamine
(GalNAc) residues alternating in b-1,3-glycosidic linkages with glucur-
onic acid (DUA) residues, and sulfated primarily at the C-6-position of
GalNAc residue. (C) CSA: [DUA-b-1,3-GalNAc(4S)]n. CSA repeating
disaccharide unit is composed of N-acetylgalactosamine (GalNAc)
residues alternating in b-1,3-glycosidic linkages with glucuronic acid
(DUA) residues, and sulfated primarily at the C-4-position of GalNAc
residues. (D) DS: [IdoA-b-1,3-GalNAc(4S)]n. DS repeating disaccharide
unit is composed of N-acetylgalactosamine (GalNAc) residues alternat-
ing in b-1,3-glycosidic linkages with iduronic acid (IdoA) residues, and
sulfated primarily at the C-4-position of GalNAc residues.
Schematic chemical structures of the repeating disaccharide
IOVS, December 2012, Vol. 53, No. 13
Roles for ECM in Cornea Innervation 8119
ing over lower, permissive concentrations of immobilized DS
and CSA/C displayed increased fasciculation and decreased
branching, whereas neurites that traversed similar KS concen-
trations were mainly nonfasciculated and displayed a high
degree of branching. Collectively, these data implicate corneal
ECM–GAGs as being involved in cornea innervation and reveal
that each type of GAG triggers a unique set of effects on
trigeminal neurite growth cone behavior. Understanding the
roles of individual GAGs present in the cornea on neuronal
behavior should improve our understanding of axon guidance
in the cornea during embryonic development and may shed
insight into the general failure of injured axonal stumps to
regenerate growth cones capable of penetrating the stroma
and epithelium following cornea transplantation/penetrating
keratoplasty, trauma, or elective surgeries in adult corneas.
Chick Embryo Husbandry
Fertile White Leghorn chicken eggs (Nelson’s Hatchery, Manhattan, KS)
were stored at 158C for up to a week before being transferred to a 388C
humidified poultry incubator on E0 and incubated at 45% humidity for
up to 14 days.
Trigeminal Neuron Preparation
Trigeminal ganglia (TG) were dissected from E10 chick embryos into
sterile Howard Ringer’s saline solution (7.2 g NaCl, 0.17 g CaCl2?2H2O,
0.37 g KCl in 1 L distilled water, pH 7.3) and the proximal region
(containing mostly neural crest–derived neurons59) of each ganglion
was cut into tissue explants using a tungsten needle (Fine Science
Tools, Foster City, CA). Explants were cultured in Dulbecco’s modified
Eagle’s medium (DMEM; Gibco, Carlsbad, CA) supplemented with 10%
fetal bovine serum, antibiotics (100 units penicillin, 0.1 mg/mL
streptomycin; Sigma, St. Louis, MO), and 25 ng/mL nerve growth
factor (NGF; Sigma) to support neurogenesis specifically of the neural
crest–derived cells of the trigeminal explants,69,70rather than those of
the ectodermal-placode–derived neuroblasts that also reside in TG.
Trigeminal explants were positioned on prepared tissue chamber slides
and incubated at 378C in a humidified, CO2incubator for 72 hours.
After incubation, neuronal explants were fixed in 4% paraformaldehyde
and neuronal growth was visualized by immunostaining.
Tissue culture chamber slides (one- or two-chambered, Lab-Tek;
Thermo Fisher Scientific, Waltham, MA) were coated with poly-D-lysine
(PDL; 70,000–150,000 KD, 100 lg/mL in borate buffer, pH 8.4; Sigma)
overnight at 48C, washed three times with water, and stored dry at 48C
for up to 2 weeks prior to use. To perform neurite guidance spot assays
in which neurite growth cones extending from trigeminal explants
encounter immobilized GAG on an otherwise homogeneous growth-
supporting substrate, GAGs were immobilized to the PDL-coated slides
by depositing 4-lL drops of five different types of GAGs at varying
molar concentrations (0.1–5.0 lM in sterile water). GAGs were allowed
to adsorb to the slide surface in a humidified chamber at 378C for 2
days, then the chamber slides were washed three times with sterile
water to remove any unbound GAG chains. Deposited GAGs included
CSA (C-8529, from bovine trachea; Sigma), DS (previously known as
chondroitin sulfate B; C-3788, from porcine intestinal mucosa; Sigma),
CSC (C-4384, from shark cartilage; Sigma), KS (from bovine cornea;
Seikagaku Corp., Tokyo, Japan), or heparin (H-3393, from porcine
intestinal mucosa; Sigma). Water spots served as a control. The exact
positions of the control and test spots were outlined on the bottom of
the chamber slide using a permanent marker. Prior to adding trigeminal
explants, the entire surface of the chamber slide, including the GAG
spots and control spots, was further coated with laminin (25 lg/mL in
water; Sigma) for 3 hours at room temperature, resulting in a
homogeneous layer of laminin on the slide, covering both GAG-bound
and GAG-free regions. At the onset of the study we prepared additional
substrates, where laminin and GAG solution were mixed together and
allowed to adhere to the PDL surface simultaneously, followed by
whole-slide coating with laminin. This latter substrate preparation was
performed to ensure that changes in neurite growth cone behavior at
the GAG spot interface was not simply due to less available binding
spots for laminin to PDL following GAG adsorption. Neurite behavior in
control and test conditions did not vary between the two different
substrate preparations described above; thus, data presented herein are
from GAG solutions deposited alone as isolated drops allowed to
adhere to PDL, followed by coating the entire culture surface with
Soluble GAG Assay
For growth on laminin substrates, tissue culture chamber slides (four-
chambered, Lab-Tek; Thermo Fisher Scientific) were coated with PDL
and laminin as described above. CSA/C, DS, KS, or heparin was
reconstituted in NGF-containing DMEM culture media in a final volume
of 0.5 mL at final GAG molarities ranging between 5 and 20 lM. GAG-
containing media were added to chamber slides along with trigeminal
explants, which were then incubated at 378C in a humidified, CO2
incubator for 48 hours. For growth in 3-dimensional collagen matrices,
neuronal explants first were suspended in soluble collagen solu-
tion69,71,72that had been mixed with GAGs to generate final GAG
molarities ranging between 5 and 20 lM. After orienting the tissues in
the collagen solutions, the collagen was allowed to solidify at 378C
under sterile conditions prior to addition of NGF-containing media on
top of such gels. After incubation at 378C in a humidified, CO2
incubator for 48 hours, neuronal explants were fixed in 4%
paraformaldehyde and neuronal growth was visualized by immuno-
Enzymatic Digestion of GAG Chains
In some experiments, GAG spots on chamber slides were pretreated
with enzyme solutions prior to serving as substrates for trigeminal
neurons. Chondroitinase ABC (chABC, from Proteus vulgaris, protease
free, EC 184.108.40.206; Sigma) was used at 1 mU/mL, pH 8.0, in 0.1 M
ammonium acetate buffer to cleave CSA, CSC, and DS while leaving
other GAGs unaltered.67,73Keratanase II (from Bacilus sp.; Seikagaku
America, Falmouth, MA) was used at 0.5 mU/mL, pH 6.0, in 0.1 M
ammonium acetate buffer to digest KS at sites of sulfated N-acetyl-
glucosamine moieties,67,74while leaving other GAGs unaltered.
Enzyme treatments were carried out overnight in a humidified
chamber at 378C. In control experiments, GAG spots were treated in
a similar fashion with enzyme-free ammonium acetate buffer.
Nerves were visualized in (1) fixed whole-mount anterior eyefronts,
comprised of the cornea and surrounding limbus and sclera tissues that
were freed from the adherent lens, iris, retina, and more posterior eye
structures; and in (2) fixed trigeminal neuronal explants in culture with
antineuronal b-tubulin–specific antibody75–77(Tuj1; R&D Systems,
Minneapolis, MN) used at a 1:100 dilution in antibody block solution
(PBS, 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na2HPO4, 0.24 g/L KH2PO4, pH
7.2 [PBS], containing 10% goat serum, 1% bovine serum albumin [BSA],
0.1% Triton X-100), either for 48 hours, when staining nerves in whole-
mount tissues, or overnight, when staining trigeminal neuronal
explants or dissociated trigeminal neurons, with all staining occurring
at 48C with mild rocking. Fixation was carried out in 4% paraformal-
dehyde, overnight at 48C with mild agitation for whole anterior
eyefronts and for 1 to 2 hours at room temperature with mild rocking
for trigeminal explants in culture. Following three washes in PBS-T
(PBS with 0.1% Triton X-100), tissue was incubated for 2 to 3 hours at
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