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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 3, 2003 as doi:10.1096/fj.03-0043fje. |
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University of Ottawa Eye Institute, Ottawa Health Research Institute-Vision Centre, and Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, K1H 8L6, Canada;
* Santen Pharmaceutical Company Ltd., Ikoma-shi, Nara, Japan;
Department of Physiology, University of Tennessee Health Science Centre, Memphis, Tennessee, USA;
Ottawa Health Research Institute, Division of Neuroscience, University of Ottawa, Ottawa, Canada;
Department of Morphology, The Netherlands Ophthalmic Research Institute, Amsterdam, The Netherlands; and
Tokyo Dental College-Ichikawa General Hospital Cornea Centre, Ichikawa, Chiba, Japan
2Correspondence: University of Ottawa Eye Institute, Ottawa Health Research Institute-Vision Centre, and Department of Cellular and Molecular Medicine, University of Ottawa, 501 Smyth Rd., Ottawa, Ontario, K1H 8L6, Canada. E-mail: mgriffith{at}ohri.ca
SPECIFIC AIMS
A sensory nerve supply is crucial for optimal tissue function. Since the mechanisms for successful innervation and the signaling pathways between nerves and their target tissue are not fully understood, we developed engineered tissue substitutes with which to study tissue innervation and the associated interactions between nerves and their targets.
PRINCIPAL FINDINGS
1. Exogenous factors and in-growth of nerve fibers
Innervated tissue engineered (TE) corneas (Fig. 1
A) were fabricated by making modifications to a corneal tissue equivalent we had developed. Optimal concentrations of exogenous laminin and nerve growth factor (NGF) were added to the construct after individual tests without corneal cells for their ability to promote axonal growth from chick embryo dorsal root ganglia (DRG). The creation of a three-stepped laminin gradient within the stroma and the addition of NGF to the uppermost layer of corneal matrix were successful in guiding and promoting the growth of nerve processes.
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2. Nerve morphology and epithelial innervation
Immunofluorescence with a nerve-specific neurofilament antibody revealed nerve morphology similar to that of the natural human cornea. Nerve bundles originating from DRG within the scleral scaffold penetrated the cornea. These then progressed through the corneal stroma and into the overlying epithelium (Fig. 1B
). Stromal nerve bundles (Fig. 1C
) bifurcated to form a plexus (Fig. 1D
) below the basal epithelial cells. Beaded (Fig. 1E
) and smooth (Fig. 1F
) nerve fibers from the subepithelial network proceeded to target the epithelium. Transmission electron microscopy (TEM) revealed small fiber bundles within the basal epithelial layer. From these bundles, single nerve fibers containing secretory vesicles (Fig. 1G
) branched and established terminals in the epithelium. As described for natural human corneas, terminal nerve fibers were observed invaginating individual epithelial cells (Fig. 1H
).
3. Generation of nerve action potentials
Antibodies against sodium channels and neurofilaments showed colocalization within nerve fibers of the TE corneas. To determine whether these sodium channels could carry action potentials (APs), DRG were electrically stimulated. This electrical stimulation resulted in the generation of APs recorded from nerve terminals at the surface of the epithelium.
4. Substance P neurotransmitter release
Upon axonal stimulation, the neuropeptide SP is released from sensory nerve terminals in the epithelium of the cornea. To elicit a functional response such as SP release, innervated TE cornea constructs were treated with capsaicin or veratridine, both shown to cause SP release from corneal nerves. At all times after treatment, capsaicin elicited an increase in SP release over baseline leakage observed in controls (Fig. 2
A). Differential SP release was observed between innervated corneal constructs treated with capsaicin, veratridine, or drug vehicle only (Fig. 2B
). At 6 h post-treatment, only capsaicin elicited an increase in SP release; at 24 h both capsaicin and veratridine elicited increases compared with controls.
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5. Interaction among nerves and their target tissue
In testing the effects of laminin and NGF, a significant decrease in the number of neurites was observed over time. To test the possibility that this loss may be associated with the lack of a target tissue, epithelial cells were seeded onto the surface of the corneal matrix. With the addition of epithelial cells, no reduction in neuronal outgrowth was observed over the same culture period.
Sensory innervation is important for optimal tissue function and known to promote proliferation and to enhance wound healing of its target tissue. The presence of nerves in the TE cornea promoted greater epithelial stratification (up to twofold) and increased the number of keratocytes present within the stroma (
twofold) vs. noninnervated controls. To test the effect of nerves on wound healing in our system, epithelial wounds were created in TE corneas constructed with and without nerves and wound closure rates were measured. During the first 18 h, wound closure was faster in innervated corneal constructs, but had equalized by 24 h. Incorporation of bromodeoxyuridine (BrdU, a mitotic indicator) at 0, 6, and 24 h after wounding revealed that proliferation in innervated constructs was 2.5-fold greater in innervated constructs at all time points. This demonstrated that the presence of nerves in the TE cornea had a positive influence on the proliferation of epithelial cells and early epithelial wound healing.
CONCLUSIONS
We developed an in vitro model for studying innervation and nerve-target cell interactions and have demonstrated accurate key morphological and functional aspects of the nerves within this model. Whereas the addition of laminin and NGF promoted increased initial neurite outgrowth from the DRG into the TE cornea, the number of extending axons decreased over time. This loss of nerve outgrowth could be attributed to a lack of target tissue since addition of epithelial cells to the TE cornea reversed this trend, suggesting the target epithelial cells were essential for neuronal survival.
Whereas target tissues promote the survival of nerves, the sensory nerve supply in turn influences phenotypic characteristics important to the maintenance of a healthy tissue. As in the human cornea, TEM results demonstrate that nerve fibers invaginated individual epithelial cells in the TE cornea. Direct innervation might help modulate corneal responses to insult or injury. The presence of nerves in TE corneas promoted proliferation and enhanced the stratification of epithelial cells and resulted in a greater number of keratocytes within the stroma. In wounded TE corneas, innervation allowed significantly higher wound closure rates during the first 24 h of healing than noninnervated controls. However, there was no difference in the time for complete wound closure. Early enhanced resurfacing of the wound in the innervated TE corneas could be attributable to increased pressure exerted due to the increased epithelial cell proliferation, a phenomenon also observed in mouse corneal epithelium. The lack of a difference in the time for complete wound closure was likely due to the differential stratification of the epithelium. It has been shown that as epithelial cells stratify, their ability to migrate can decrease by as much as 50%. Therefore, cells from the less stratified epithelium of noninnervated constructs may have an increased ability to migrate, allowing for equal time for full wound closure, despite the initial enhanced wound closure of innervated TE corneas. To our knowledge, a 3-dimensional model for studying the relationship between nerves and their target cells has not been developed. This model may provide an excellent in vitro system for investigating the complex pathways of communication between nerves and their target tissues.
Direct electrophysiological recording confirmed that in-grown nerve bundles were able to conduct APs. These APs exhibited configuration and amplitude similar to those recorded from native guinea pig cornea polymodal nociceptors, the most abundant class of neuron in the cornea. The generation of APs, which can trigger the release of SP, is important to corneal nerve function in the epithelium. SP stimulates corneal epithelial cell proliferation and migration and enhances wound healing. We triggered SP release in our innervated TE corneas by treatment with capsaicin and veratridine. Capsaicin is a neurotoxin that depletes SP from nerve terminal stores via an action potential-independent mechanism. Veratridine, however, causes SP release from nerve terminals by opening sodium channels and depolarizing the membrane. Sodium channel-dependent and independent mechanisms of SP release were observed in our model. Differential levels of neuropeptide release in response to treatment with capsaicin, veratridine, or drug vehicle may suggest that the nerves are able to distinguish among different chemicals. Similar to native nerve processes, the present results show that nerves growing into our TE corneas respond to chemical stimuli and contribute to the maintenance of the tissue.
In the short term, these functional, innervated corneas could be used as research models or as in vitro alternatives to animal-based eye tests for chemical or drug safety and efficacy testing. The Draize and the low-volume eye test are rabbit ocular irritancy tests used for measuring potential ocular toxicity. Differences in ocular irritancy are related to differences in the extent of initial injury. The degree of cell death, levels of neurotransmitter release, and the AP response could all be used to ascertain the toxicity of potential ocular irritants in our model.
In summary, we report the development of an in vitro tissue engineered cornea complete with functional innervation and demonstrate nerve-target cell interaction. This model provides a useful tool for studying corneal innervation and cell-cell interactions, with the advantage of a controlled environment and natural transparency for imaging. Although we focused on the cornea, these results may have broader implications for the field of tissue engineering by providing some insight into nerve regeneration and the innervation of other engineered organ or tissue systems designed for transplantation, both major goals in tissue engineering.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0043fje 
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