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1.
 Figure 4. 

Figure 4.  . From: Corneal Sulfated Glycosaminoglycans and Their Effects on Trigeminal Nerve Growth Cone Behavior In Vitro: Roles for ECM in Cornea Innervation.

GAG distribution in pericorneal tissues at E14. Position of pericorneal nerve ring compared with distribution of sulfated GAGs in the pericorneal tissue within confocal stack projections following staining with monoclonal antibodies neuronal-specific Tuj1 (A), CS-56 for CSA/C (B), 7D4 for CS/DS (C), I22 for KS (D), and 5D4 also for KS (E), detected with AlexaFluor 488– or FITC-labeled secondary antibodies (green) at E14 days of incubation. Cell nuclei are stained blue with DAPI. Scale bars, 0.2 mm. co, cornea; nr, nerve ring; le, limbal epithelium; lm, limbal mesenchyme.

Tyler Schwend, et al. Invest Ophthalmol Vis Sci. 2012 December;53(13):8118-8137.
2.
 Figure 1. 

Figure 1.  . From: Corneal Sulfated Glycosaminoglycans and Their Effects on Trigeminal Nerve Growth Cone Behavior In Vitro: Roles for ECM in Cornea Innervation.

Schematic chemical structures of the repeating disaccharide unit of GAGs used in this study. (A) KS: [Gal-β-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 β-1,4 and β-1,3, and hydroxyl groups at the C-6 positions of Gal and GlcNAc residues may be sulfated. (B) CSC: [ΔUA-β-1,3-GalNAc(6S)]n. CSC repeating disaccharide unit is composed of N-acetylgalactosamine (GalNAc) residues alternating in β-1,3-glycosidic linkages with glucuronic acid (ΔUA) residues, and sulfated primarily at the C-6-position of GalNAc residue. (C) CSA: [ΔUA-β-1,3-GalNAc(4S)]n. CSA repeating disaccharide unit is composed of N-acetylgalactosamine (GalNAc) residues alternating in β-1,3-glycosidic linkages with glucuronic acid (ΔUA) residues, and sulfated primarily at the C-4-position of GalNAc residues. (D) DS: [IdoA-β-1,3-GalNAc(4S)]n. DS repeating disaccharide unit is composed of N-acetylgalactosamine (GalNAc) residues alternating in β-1,3-glycosidic linkages with iduronic acid (IdoA) residues, and sulfated primarily at the C-4-position of GalNAc residues.

Tyler Schwend, et al. Invest Ophthalmol Vis Sci. 2012 December;53(13):8118-8137.
3.
 Figure 8. 

Figure 8.  . From: Corneal Sulfated Glycosaminoglycans and Their Effects on Trigeminal Nerve Growth Cone Behavior In Vitro: Roles for ECM in Cornea Innervation.

Trigeminal neurite outgrowth in the presence of soluble GAGs. (A, B) Neurite outgrowth in the presence of soluble GAGs from trigeminal explants grown on PDL/laminin (A) or in a collagen I matrix gel (B) for 2 days. For a single explant within each culture condition, 8 to 10 of the longest neurites were identified and measured to derive a mean length (in mm) of the longest neurites for that explant. Histogram values are expressed as the mean neurite length (in mm) among a group of explants that were scored independently (n = 6–10 explants per condition). Error bars represent SD.

Tyler Schwend, et al. Invest Ophthalmol Vis Sci. 2012 December;53(13):8118-8137.
4.
 Figure 7. 

Figure 7.  . From: Corneal Sulfated Glycosaminoglycans and Their Effects on Trigeminal Nerve Growth Cone Behavior In Vitro: Roles for ECM in Cornea Innervation.

Growth cone morphology upon contact with immobilized GAG substrate. (AF) Time-lapse images of trigeminal growth cones contacting a water spot (control) (AC) or an area rich in DS (DF), while extending on a GAG-free, neuronal-supportive (PDL/laminin) substrate, on either 0 (A, D), 12 (B, E), or 24 (C, F) hours after the start of imaging. (AC) Brightfield, phase-contrast images of live explants and their neurite projections toward a water spot show that neurites advance over the spot unimpeded and growth cones migrate without pausing, or spreading (arrowhead denotes advancing, unspread growth cone). (DF) Brightfield, phase images of live explants and their neurite projections (DF, D′–F′) or fluorescent images of fixed explants and their neurite projections (D″–F″), toward a DS spot show that neurites advance unimpeded and growth cones are not spread out prior to contacting the DS spot (D′, D″, arrowheads), but that upon contacting DS-rich substrate, growth cones stop/stall and become spread out with extended filopodia (arrow denotes spreading growth cones), before turning and advancing along the DS border (arrowheads in E″, F′, F″). Dotted lines (D″–F″) represent the border between GAG-free substrate and GAG/control substrate.

Tyler Schwend, et al. Invest Ophthalmol Vis Sci. 2012 December;53(13):8118-8137.
5.
 Figure 3. 

Figure 3.  . From: Corneal Sulfated Glycosaminoglycans and Their Effects on Trigeminal Nerve Growth Cone Behavior In Vitro: Roles for ECM in Cornea Innervation.

GAG distributions in corneal and pericorneal tissues during innervation. Position of corneal nerves compared to distribution of sulfated GAGs in developing eyefronts within confocal stack projections following staining with monoclonal antibodies neuronal-specific Tuj1 (A, F, K, P, U), CS-56 for CSA/C (B, G, L, Q, V), 7D4 for CS/DS (C, H, M, R, W), I22 for KS (D, I, N, S, X), and 5D4 also for KS (E, J, O, T, Y), detected with AlexaFluor 488– or FITC-labeled secondary antibodies (green) at E9 ([AE]; images show staining in pericorneal tissue and corneal edge), E10 ([FJ]; images show staining in corneal edge), E11 ([KO]; images show staining in mid-central cornea), E12 ([PT]; images show staining in central cornea), and E14 ([UY]; images show staining in central cornea) days of incubation. Cell nuclei are stained blue with DAPI. Open arrowheads in (F, K) delineate branching from main nerve trunk. Asterisks in (P, U) denote positive nerve staining in the epithelial layer of the cornea. Scale bars, 0.2 mm. co, cornea; nr, nerve ring; le, limbal epithelium; lm, limbal mesenchyme.

Tyler Schwend, et al. Invest Ophthalmol Vis Sci. 2012 December;53(13):8118-8137.
6.
 Figure 9. 

Figure 9.  . From: Corneal Sulfated Glycosaminoglycans and Their Effects on Trigeminal Nerve Growth Cone Behavior In Vitro: Roles for ECM in Cornea Innervation.

Trigeminal neurite behavior following elongation upon intermediate, permissive concentrations of immobilized GAG substrates. (AE) The strategy for generating culture substrates so that trigeminal neurons grow out on surfaces containing PDL/laminin and GAG or control spots was described in Figure 5A. A vertical line above (AC) or below (D, E) the image indicates the location of the boundary between the PDL/laminin only substrate (control, left) and the test substrate (GAG or control spot, right). Representative images are shown for neurites encountering a water control spot (A) or CSA (B), DS (C), CSC (D), or KS (E). Neurite behavior upon growing over CSA (B), DS (C), and CSC (D) is characterized by a marked shift from intermediate to thick fascicles (white arrows, fasciculation) and reduced defasciculation/branching. On the contrary, neurite behavior seen when growth cones migrate upon KS (E) is characterized as nonfasciculated (yellow arrows, nonfasciculated) with a high degree of branching (white arrowheads represent points of branching or defasciculation). (F) Percentages of neurites that grew as fasciculated or nonfasciculated under control and test conditions. (G) Percentages of branching points per neurite under control and test conditions. n values: water, 89 neurites; CSA, 127 neurites; DS, 114 neurites; CSC, 110 neurites; KS, 174 neurites.

Tyler Schwend, et al. Invest Ophthalmol Vis Sci. 2012 December;53(13):8118-8137.
7.
 Figure 6. 

Figure 6.  . From: Corneal Sulfated Glycosaminoglycans and Their Effects on Trigeminal Nerve Growth Cone Behavior In Vitro: Roles for ECM in Cornea Innervation.

Neurite outgrowth in response to varying concentrations of GAG substrates. (AI) High-magnification images show the areas surrounding the spot border. The strategy for generating culture substrates for trigeminal neurons that contain PDL/laminin and GAG or control spots was described in Figure 5A. A vertical line below the image indicates the location of the boundary between the PDL/laminin only substrate (control, left) and the test substrate (GAG or control spot, right). Test substrates and the molar concentration at which they were prepared are also provided below each image. Neurites encountering a GAG substrate of CSA (B, C), DS (D, E), CSC (F, G), or KS (H, I) are inhibited to different degrees, dependent on the type of GAG and concentration (1 μM vs. 2.5 μM), whereas neurites are not inhibited upon encountering a water control or heparin substrate (A). (J) Percentages of neurite density visible on test substrata for each GAG type and at various concentrations (right side of the vertical line) following 3 days of growth as compared with the neurite density visible on the control substrate (left side of the vertical line). Refer to the Methods section for further details about how the neurite density values were determined. Error bars represent SD. Scale bar, 0.5 mm.

Tyler Schwend, et al. Invest Ophthalmol Vis Sci. 2012 December;53(13):8118-8137.
8.
 Figure 5. 

Figure 5.  . From: Corneal Sulfated Glycosaminoglycans and Their Effects on Trigeminal Nerve Growth Cone Behavior In Vitro: Roles for ECM in Cornea Innervation.

Inhibitory potential of immobilized GAG substrates on trigeminal neurite growth. (A) Schematic of culture strategy used in this study. Neuronal explants containing primarily neural crest–derived neurons are prepared from the trigeminal ganglia harvested from E10 chick embryos. Trigeminal explants are seeded onto glass slides coated with PDL/laminin and adjacent to an area of the slide where GAG molecules have been spotted. Three explants could be positioned around a single spot. (B, C) When explants were positioned adjacent to a control spot of water or heparin, neurites from each trigeminal explant grew all around the explant (in a halo), including those neurites that grew from the edge of the explant nearest to the spots. Neurites were not impeded from growing over either spot. A dotted circle is used in these panels and throughout the figure to show the location of the control or GAG spots in relationship to explant/neurite positions. (DI) In contrast, neurites projecting from explants positioned adjacent to a spot of CSA (D), DS (F), or CSC (H) were inhibited from crossing over the spots. The inhibitory effect could be mitigated, first, by treating the spots with chondroitinase ABC enzyme, prior to seeding trigeminal explants (E, G, I). (J, K) Neurites from explants positioned adjacent to KS spots were partially inhibited (J), with a small subpopulation of neurites able to extend onto the spot (arrows), whereas the majority of neurites were inhibited; however, treating the spot with KSase II prior to neuronal seeding allowed a higher quantity of neurites to extend over the KS spot. Scale bar, 1.0 mm.

Tyler Schwend, et al. Invest Ophthalmol Vis Sci. 2012 December;53(13):8118-8137.
9.
 Figure 2. 

Figure 2.  . From: Corneal Sulfated Glycosaminoglycans and Their Effects on Trigeminal Nerve Growth Cone Behavior In Vitro: Roles for ECM in Cornea Innervation.

Cornea innervation by trigeminal sensory nerves during development. (AC) Nerves visualized in cornea whole-mounts (A, B) or sections (C) at E9 following staining with antineuronal β-tubulin-specific Tuj1 antibody, detected with AlexaFluor 488 (green), could be seen having grown around the cornea in the limbus, forming a pericorneal nerve ring. At this stage, individual nerves or nerve bundles/fascicles exited the nerve ring and advanced in a straight path, without branching (arrows in [B] show individual nerves), to the cornea. Dotted, curved line denotes corneal periphery. (DI) At E10 to E11, nerves that had penetrated the cornea began to branch/bifurcate ([E, F, H, I] open arrowheads show branch points) along their trunks and advancing terminii, whereas nerve trunks within the limbus remained unbranched ([E, H] solid arrows). Nerves in the cornea advanced exclusively within the anterior stroma, avoiding the posterior stroma and endothelium (F, I). (JO) Between E12 and E14, nerves continued to advance to the cornea's center (J), eventually reaching it by E14 (M). Increased branching along the corneal-lying nerve trunks increased the overall density of the neural network in the cornea (K, N). By E12, nerve growth cones had advanced anteriorly to penetrate among the epithelial cells (asterisks, [L, O]). Despite the extensive innervation of the chick eyefront during these stages, nerve staining never became visible in the limbal epithelium, the anterior limbal mesenchyme, or the posterior cornea layers. Scale bar, 0.5 mm. en, corneal endothelium; ep, corneal epithelium; co, cornea; nr, nerve ring; le, limbal epithelium; lm, limbal mesenchyme; st, corneal stroma.

Tyler Schwend, et al. Invest Ophthalmol Vis Sci. 2012 December;53(13):8118-8137.

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