* University of Ottawa Eye Institute, Ottawa Health Research InstituteVision Centre, and Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, K1H 8L6, Canada, Ottawa Health Research InstituteDivision of Neuroscience, University of Ottawa, Ottawa, Ontario, K1Y 4E9, Canada,
Santen Pharmaceutical Company Ltd., Ikoma-shi, Nara, 630-0101, Japan
Received June 28, 2004; accepted August 25, 2004
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ABSTRACT |
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Key Words: innervation; tissue engineering; toxicology; 2-photon microscopy; cornea.
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INTRODUCTION |
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In addition to their proposed use for transplantation, TE substitutes also have the potential to provide new toxicology models. While tissues need to confer function as a replacement to a tissue or organ in transplantation, for use as an in vitro model for testing, TE tissues need to mimic key morphological, physiological, and biochemical properties of the natural tissue as closely as possible. Like TE constructs for transplantation, in vitro models may also require a sensory nerve supply to be mechanistically accurate. With the ban on animal testing for development of consumer products expected to expand from Europe (European Union Directive 76/768/EEC) into North America, the demand for in vitro methods for safety and efficacy testing (e.g., toxicology and drug testing) is expected to grow. At present, the response of many TE constructs to external stimuli is overly sensitive, possibly because of a lack of innervation (Griffith et al., 1999).
We recently described the fabrication of a fully innervated in vitro human corneal model that demonstrated basic anatomical and physiological similarities to the natural tissue in situ (Suuronen et al., 2004). In this article we demonstrated the importance of nervetarget cell interactions in the overall functioning of the engineered tissue. In the present study, we examined in greater detail the contributions of sensory innervation to the function of an engineered tissue. In particular, 2-photon confocal microscopy was used to examine in detail the reaction of fine, terminal sensory nerves to external stimuli.
The innervated TE cornea demonstrates the potential of TE substitutes to serve as replacements to animals for in vitro ocular toxicology testing or as models for the study of fine sensory nerves. The cornea's transparency, structural simplicity, and the importance of innervation for optimal corneal function make it an ideal tissue model for studying sensory nerve function. We observe that innervation affects the characteristics of the TE cornea and confers protection on its epithelium from chemical insult. The 2-photon imaging techniques allow for the visualization and study of the fine sensory axon fibers within the 3-dimensional tissue. We demonstrate differential responses of the nerves to chemical stimuli by changes in intracellular sodium as measured by 2-photon microscopy. This work demonstrates a role for innervation in the protective quality and function of the engineered tissue, and the potential to use the nerves themselves as indicators of the severity of an insult. These results are important to consider for the development of any optimized TE substitutes for in vitro use, either as toxicology models or for the study of peripheral sensory innervation.
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MATERIALS AND METHODS |
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For neurofilament staining, PFA-fixed whole tissue constructs (as above) were permeabilized by treatment with a detergent mix [150 mM NaCl, 1 mM ethylenediamine tetraacetic acid, 50 mM Tris, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (Brugge and Erikson, 1977)] for 20 min. Constructs were rinsed in Tris buffered saline (TBS), and incubated with the primary antibody, anti-neurofilament 200 [Sigma; diluted 1:40 in TBS containing 0.6% carrageenan and 0.3% Triton-X 100 (TCT)] overnight at 4°C. The constructs were then rinsed in TBS and incubated with a Cy3-conjugated secondary antibody (1:200 in TCT; Amersham, Baie D'Urfé, Canada) for 150 min at room temperature (RT). Negative controls were incubated without the primary antibody. Positive controls included staining for DRG and neural tube explants. Nerves were visualized under fluorescence confocal microscopy.
For visualization of live nerve fibers, acetoxymethyl (AM) ester (Calcein AM, Molecular Probes) was used. Calcein AM, provided at a concentration of 1 mg/ml in dimethylsulfoxide (DMSO), was diluted into 0.1 M phosphate-buffered solution (PBS) to a 1 µM working concentration. The dye was loaded into whole tissue for 30 min at RT, and then the constructs were rinsed several times in PBS prior to visualization with the 2-photon laser-scanning microscope. Imaging was performed on a Nikon C1 laser scanning microscope custom-modified for 2-photon operation (Risdale et al., 2004). Fluorophores were excited by a 800 nm pulsed beam from a Ti:sapphire laser (Tsunami, Spectra Physics, Mountainview, CA), and fluorescence collected by a 60x 1.0NA immersion objective. Images were analyzed using ImageTrak software written by PKS (http://www.ohri.ca/stys/imagetrak).
Effects of innervation on the epithelium. To investigate the effects of innervation on stratification of the epithelium and the production of its protective mucin layer, constructs with and without DRG were cultured. TE corneas were fixed in 4% PFA (as above) at 2, 4, 6, and 10 days, and subsequently sectioned. For examination of epithelial thickness, sections were processed for routine hematoxylin and eosin (H&E) staining. The thickness of the epithelium was measured in six random sections for each sample, and the mean was calculated for each time point. For mucin staining, sections were rinsed in TBS and incubated with the primary antibody, anti-MUC16 (CA125; Dako) at a dilution of 1:50 in TCT overnight at 4°C. The constructs were then rinsed in TBS and incubated with a Cy2-conjugated secondary antibody (1:200 in TCT; Amersham) for 150 min at RT. Negative controls were incubated without the primary antibody. Nerves were visualized under fluorescence confocal microscopy.
Live/dead staining. Innervated and non-innervated constructs were exposed to (1) artificial tears (Alcon) for 1 h; (2) a mixture of 8.5% Tween-80 surfactant and 1.5% ethanol in SHEM medium for 1 h; (3) 500 µM ouabain for 30 min; or (4) 0.5% sodium dodecyl sulfate (SDS) for 1 min. Controls consisted of untreated constructs with and without nerves. Quantification of the viability of epithelial cells was done using the acridine orange/ethidium bromide method combined with Hoescht staining for nuclear labeling. The dye mix consisted of 0.1 M PBS containing 100 µg/ml of acridine orange, 100 µg/ml of ethidium bromide, and 250 µg/ml of Hoescht dye. After the surfactant treatment, a volume of 10 µl of the dye mix was added to the inside of each insert, which contained 500 µl of medium. The constructs were stained for 5 min, and images were captured for epithelial cell counts. Dead cells (apoptotic or necrotic) stained red and live cells stained green. The numbers of live and dead epithelial cells were counted within five different areas per construct. Dividing the number of dead cells by the total number of cells, and multiplying by 100 determined the percentage of epithelial cells that were dead. A Two-way analysis of variance (ANOVA) was performed to determine statistical significance set at p < 0.05.
Sodium visualization. For these experiments, some modifications were made to the methods for cornea construction. In the experiments described above, the constructs were removed from their inserts for visualization by the 2-photon system. Because of the soft nature of the stromal matrix, there was a slow expansion of the tissue over time, which did not affect single image capture. However, for time series experiments, there is a requirement for tissue stability over time. The dimensions of the inserts described above do not allow for observation by the 2-photon system, and for this reason, constructs were transferred to organ tissue culture dishes (Becton-Dickinson) and surrounded by a ring of collagen-CS mixture, prepared as described above. This insert has dimensions suitable for use with the 2-photon system apparatus, and its use ensures that the field of interest is maintained over time. However, it cannot be used for long-term culture because of its lack of a membrane, essential for proper nutrient delivery within the corneal stroma.
Constructs within organ culture dishes were loaded with the Na+-indicator dye, sodium-binding benzofuran-isophthalate (SBFI; 10 µM), and the solubilization facilitator Pluronic F-127 (both from Molecular Probes), at a ratio of 1:1, prepared just before use. Loading time was 4 h, and the tissues were rinsed in PBS several times after loading. The construct was transferred to a custom-built chamber and immersed in artificial cerebral spinal fluid (aCSF; in mM): NaCl, 126; KCl, 3.0; MgSO4, 2.0; NaHCO3, 26; NaH2PO4, 1.25; CaCl2, 2.0; and dextrose, 10). Axons within the construct were imaged before and 10 min after application of the following treatments: aCSF, 14 µM dimethyl sulfoxide (DMSO), 50 µM veratridine, 500 µM ouabain, or 50 µM veratridine + 500 µM ouabain at RT. Because DMSO is used at a final concentration of 14 µM to reconstitute veratridine and ouabain, its effect on Na+ retention was also tested to examine the possibility that it contributes to any observed response in the treatment groups. The signal from the Na+-insensitive background was subtracted from the Na+-dependent signal to give the Na+ fluorescence intensity. The subtraction of fluorescence intensity at time = 0 from the intensity at time = 10 min, and multiplication by 100, yielded the percent change of Na+ fluorescence intensity over time. Data were then normalized (relative percentage increase for each test group over the aCSF control).
Statistical analyses. Statistical analysis of epithelial thickness was performed using two-way multivariate analysis of variance (2-way ANOVA) procedures. Independent samples (n = 3) were used for each time point. The endpoint included in the 2-way ANOVA was number of cell layers, and significant differences between group means were analyzed using Tukey's Studentized Range Test (Tukey's test). For epithelial cell viability, a 2-way ANOVA was performed with independent samples for each treatment condition (n = 4). The endpoint consisted of the percentage of the epithelial cells that were dead and Tukey's test was used to analyze significant differences between group means. For analysis of Na+ concentration within the nerves (n = 4), a 1-way ANOVA was performed with the normalized relative change in SBFI fluorescence as the endpoint and with Tukey's test to analyze significant differences between group means. For all comparisons, p < 0.05.
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RESULTS |
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DISCUSSION |
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The tissue engineered in vitro human cornea model we developed addresses the issue of innervation. This is important, considering that the optimal function, maintenance, and wound healing of many tissues are dependent, in part, on proper peripheral sensory innervation (Anand et al., 1996; Gover et al., 2003
; Holzer, 1988
). The effect of nerves on the properties of the epithelial and stromal cells in our model was previously demonstrated (Suuronen et al., 2004
). Here, we provide evidence that these effects contribute to the overall function of the model and its response to chemical irritation. We showed that the presence of nerves in the TE cornea served to protect the epithelium from chemical irritation. After exposure to a surfactant/ethanol mixture, ouabain, or SDS, epithelial cell death was approximately 4, 2, and 2.5 times greater, respectively, in non-innervated corneas compared to innervated constructs. These findings were consistent with the demonstrated role for nerves in the homeostasis of corneal epithelial cells in the human cornea (Araki-Sasaki et al., 2000
). Sodium dodecyl sulfate, the harshest of the treatment conditions, elicited the greatest degree of cell death within the constructs. In addition, ouabain and surfactant/ethanol, both considered milder irritants, caused greater cell death in the epithelium than in the TE corneas after application of medium or artificial tears, both non-irritants. Therefore, these data demonstrated differential cell death in response to different treatment substances, showing evidence of sensitivity of the innervated TE cornea. The nerves affected several properties of the epithelium, which in turn may affect the response of the TE cornea to chemical irritation.
One property of the epithelium that can play a role in its protective function is its thickness. Differences in ocular irritancy are related to differences in the extent of initial injury (Maurer et al., 1999) and a thicker epithelium will provide greater protection from insult. The thickness of the epithelium in innervated TE corneas was greater compared to non-innervated constructs over a culture period of 10 days. Trigeminal denervation of the rabbit cornea has been previously observed to cause thinning of the epithelium (Araki et al., 1992
) and increased permeability (Beuerman and Schimmelpfennig, 1980
). Therefore, it is possible that the significantly greater epithelial cell death observed in non-innervated corneas upon chemical treatment could be due to greater permeation of the toxicant through the epithelium as a result of its compromised thickness. The thinning of the epithelium in denervated corneas has been associated with impairment of epithelial cell adhesion (Araki et al., 1994
). We observed compromised adhesion in non-innervated TE corneas after prolonged chemical assault (data not shown). These differential adhesive properties between innervated and non-innervated constructs may also have been a factor in the response of the cornea to chemical insult. Overall, the presence of nerves in the TE cornea model promoted an increase in epithelial thickness, which can potentially reduce permeability and enhance the resistance to chemical irritation.
The production of the mucin layer is another protective mechanism of the epithelium in several tissues, including the cornea. Mucins are glycoproteins produced by the surface epithelia (Gipson et al., 1995) that lubricate the surface to provide a barrier for the cell membrane and protect the cornea from desiccation (Argueso and Gipson, 2001
). Mucin 16, in particular, is expressed by corneal surface epithelia (Argueso et al., 2003
), and its expression is altered in the conjunctival epithelia of patients who suffer from dry eye syndrome (Danjo et al., 1998
), a condition related to compromised innervation (Stern et al., 1998
). In the TE cornea model, innervation promoted greater production of the mucin layer by the surface epithelia, as determined by MUC16 staining. This demonstrated that innervation plays a role in the expression of mucin in the cornea, as had already been observed in the surrounding conjunctiva (Danjo et al., 1998
), and that this model could be used to study the pathology of dry eye syndrome. In addition, the lessened mucin production in non-innervated TE corneas may contribute to the observed increase in epithelial cell death. Without a protective surface layer of mucin, the permeation of the chemical assault may be deeper and cause greater damage to the epithelial tissue of the cornea. Although not investigated, deep chemical penetration could also affect stromal cell viability and alter its interaction with the epithelium, itself important for the health of the epithelial cells (Wilson et al., 1999
).
Overall, the presence of nerves within the TE cornea promoted greater epithelial thickness, improved production of the protective mucin layer, and as was shown previously, increased epithelial and stromal cell proliferation, and it also had an effect on epithelial wound healing (Suuronen et al., 2004). These are important considerations in the development of tissue engineered models for toxicology testing, as they have an effect on the resistance and protective qualities of the epithelial surface, and hence can alter sensitivity of the model. The lack of innervation may be responsible, in part, for the failure of the current alternative toxicology models to provide undisputed, suitable, replacements for rabbit eye tests.
While innervation affected the protective quality and function of the engineered tissue, we also investigated the effects of irritation on the nerves themselves. Upon stimulation, the nerve transmits its message by the generation of action potentials (APs), which propagate via the axon to the central nervous system to cause the sensation of pain (Brock et al., 1998), and to the nerve terminals to cause the release of neuropeptides (Unger, 1990
). Action potential generation occurs by the opening of Na+ channels, and the subsequent Na+ current and depolarization of the membrane. Variability of the Na+ current has been correlated with the amplitude of the nerve response (Johansson, 1994
). In the TE cornea, differential changes in internal Na+ concentration [Na+]i were observed within the nerve fibers when treated with different chemicals, including veratridine and ouabain. While veratridine operates by a Na+ channeldependent mechanism, ouabain does not, yet both elicited responses that can be detected by changes in [Na+]i. Ouabain, in addition to its role as a Na+ regulator, has been shown to induce cell death of epithelial cells, including those of renal and lens origin (Lichstein et al., 1999
; Pchejetski et al., 2003
) and of neuronal cells (Xiao et al., 2002
). Although further study of the relationship between sensory neuronal toxicity and tissue irritancy is needed, these results provide proof of the concept that this technique could be used to detect the effects of irritants that do and do not act via Na+ channels. As discussed, the health of the epithelium is dependent on innervation; therefore, differential [Na+]i responses within nerves could be used as indicators of the severity of an insult in this model, by both the assessment of intensity and depth of permeation.
One mechanism by which sensory nerves exert their influence on their environment is by the release of neuropeptides. This may include the release of substance P (SP) neuropeptide (Maggi, 1991), which can be regulated by Na+ channel-dependent mechanisms. The differential changes in [Na+]i observed in nerves of the TE cornea could translate to differential responses of the nerve (Johansson, 1994
), including release of SP. Therefore, the release of SP can be correlated to [Na+]i, which can be regulated by the influx of Na+ into the fibers through the Na+ channels of the axonal membrane. Unlike the highly focal distribution of Na+ channels in myelinated axons, Na+ channels present in the TE cornea were distributed continuously along the membrane of the nerve. This pattern is typical of nonmyelinated sensory axons (Black et al., 2002
), the type of axons found in the cornea, and it supports continuous (as opposed to saltatory) conduction along these axons. We previously observed differential release of SP upon treatment with various chemicals, including veratridine (Suuronen et al., 2004
). Veratridine causes the release of SP by opening Na+ channels and depolarizing the membrane (Neubert et al., 2000
). Therefore, it appeared as though the nerves within the TE corneal model behaved in a manner typical of native corneal nerves. Described simply, the application of a chemical irritant to the innervated TE cornea caused changes in [Na+]i, and through the depolarizing effects, SP neuropeptide was released, which then could affect epithelial properties (Gallar et al., 1990
; Nishida et al., 1996
). The magnitude of the response would depend on the level of irritation. As mentioned above, some chemicals operate by Na+ channel-independent mechanisms, but can still result in the release of neuropeptides, including SP (Keen et al., 1982
). This mechanism of SP release was also observed in the TE cornea in previous work (Suuronen et al., 2004
). Therefore, our innervated TE cornea model could be developed for use as an alternative to animals in ocular toxicity testing. The degree of cell death, the levels of neurotransmitter release, and the response of the nerves themselves could all be used to ascertain the toxicity of potential ocular irritants.
Knowledge of sensory nerve function has come largely from monolayer or thin tissue slice culture systems that do not fully replicate the in vivo environment. The emergence of tissue engineering is providing new, more relevant models for the study of tissues and the function of their constituent cells. The re-creation of both the natural environment and the appropriate interactions between nerves and their target tissues make tissue-engineered systems superior to traditional cell culture, and more accessible and easier to manipulate than in vivo models for the study of innervation. However, the investigation of sensory nerve fibers, in particular, and their terminals and interactions with their target tissue is difficult in whole, 3-dimensional tissue because of their miniature size. As such, their study has been limited and many of their functional properties remain unclear. The technique of 2-photon microscopy has significantly enhanced the visualization and study of fine nerve fibers (Malone et al., 2002; Rose et al., 1999
). It has the advantage of high resolution imaging at substantial depths in intact tissue, despite the scattering effects of thick tissues (Denk and Svoboda, 1997
), and it minimizes the effects of photobleaching and photodamage (Denk et al., 1995
). Combining the technologies of tissue engineering and 2-photon microscopy presents methodologies that can improve existing knowledge of nerve behavior and function, using more accurate, 3-dimensional models.
Nerves within the TE cornea, stained with anti-NF 200 (fixed tissue) or Calcein AM (live tissue), were easily visualized in the epithelium and the stroma via the 2-photon system. Typical of corneal nerves (Müller et al., 1997), both smooth and beaded fibers migrated within the epithelium. The 2-photon system allowed observation of the nerves from just below the epithelial surface to a depth of 200 µm within the stroma. The retention of the Calcein AM dye, an indicator of cell viability, provided evidence that both the nerve and epithelial cells were alive and healthy. Therefore, 2-photon microscopy enables the study of the fine sensory structures within the TE cornea and their interactions with their target tissue. These results emphasize the potential of combining the technologies of tissue engineering and 2-photon microscopy to improve current knowledge of nerve behavior and function.
In summary, we demonstrate the importance of nerves in an engineered tissue with respect to its function. The presence of nerves affects several properties of the TE cornea, including its epithelial thickness, its production of the protective mucin layer, and its proliferation of epithelial and stromal cells, all of which can affect both its maintenance and its sensitivity. These are important considerations for the development of any optimized TE substitutes for transplantation that promote the growth of nerves from the host tissue, or for in vitro use, either as toxicology models or for the study of innervation.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be sent at University of Ottawa Eye Institute, Ottawa Health Research InstituteVision Centre, and Department of Cellular and Molecular Medicine, University of Ottawa, 501 Smyth Road, Ottawa, Ontario K1H 8L6, Canada. Fax: (613) 739-6070. E-mail: mgriffith{at}ohri.ca
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