María Carmen Martínez-García

Differential expression and localization of transient receptor potential vanilloid 1 in rabbit and human eyes

INTRODUCTION The superfamily of transient receptor potential (TRP) cation channels is involved in nociception. Members of this family, such as the vanilloid receptor type 1 (TRPV1) channel, are activated by a wide range of stimuli including heat (⟩43°C), low pH (⟨6.5), hypoxia, and hypertonicity. Here we report TRPV1 expression in rabbit and human eyes. MATERIAL AND METHODS We analyzed the expression of TRPV1 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and protein by immunohistochemistry in eyes of New Zealand White rabbits and humans. RESULTS In rabbit and human eyes, TRPV1 protein was present in all layers of the corneal epithelium, but only in the basal layer of the conjunctiva. It was also in the ciliary and lens epithelia of both species as well as in the secretory cells of the rabbit lacrimal gland. The retinal pigment epithelium was positive for this protein in both species. TRPV1 was also present in rabbit Müller cells, where it had a similar pattern of expression to vimentin intermediate filaments. Analysis by qRT-PCR showed that TRPV1 mRNA was found in all of the structures where the protein was present. The highest level was in the lens and the lowest in the retina. CONCLUSION TRPV1 is expressed in cells that are particularly active in Ca²⁺ exchange as well as in cells with significant water transport activity. Because TRPV1 is a Ca²⁺ channel, it probably functions in the regulation of both water and Ca²⁺ movements in ocular tissues.

Measurement of correlation between transmission and scattering during wound healing in hen corneas

The aim of this work is to provide experimental data for corneal transparency and scattering to help create a more complete model of corneal transparency. The scattered light in 96 healing hen corneas was measured for three wavelengths by a scatterometer constructed in the Optics Laboratory (The University of Valladolid, Spain). With the help of mirrors and beamsplitters, the light from the three lasers is directed toward the cell containing the sample to be measured. The measured scattered light varies between six orders of magnitude. Corneal transmissivity, mean cosine of a scattering angle, and angular distribution of scattered light were all computed. The total transmitted light remained practically constant over a wide range of light values transmitted in a forward direction (direct transmissivity). The value of the mean cosine of the scattering direction is very close to the unit ( ), even in corneas with high opacities. The behavior of g indicates that even damaged corneas evidence extremely small scattering, compared to other biological tissues. The transmission reduction of each cornea is related to an increase in scattered light. In all cases, scattered light is concentrated at very small angles. This behavior is acceptable in corneas that are healthy or which evidence small lesions, but remains in corneas that are severely injured.

Clinical, Refractive and Histological Reversibility of Corneal Additive Surgery in Deep Stroma in an Animal Model.

ABSTRACT Purpose: The aim was to evaluate the reversibility of the clinical and histological changes induced in the corneas of an animal model after removing an intracorneal ring segment (ICRS). Methods: Surgery for this study was performed in 38 eyes of an experimental animal model (Gallus domesticus) for ICRS surgery (Ferrara technique). The animals without complications were randomized to two groups; in all of them, 1 segment was implanted in each eye and later removed at different times (1 and 3 months after implantation). In each group, after explantation, corneas were processed at different times for histological analysis with hematoxylin and eosin (H&E) stain and electronic microscopy. The refractive state of the eyes was also measured. Results: In corneas without complications (88.23%), explantation was performed correctly. During the first few days, around the area where the ICRS was implanted we observed deposits of cells and a moderate degree of corneal opacity (haze). These signs decreased progressively without disappearing completely. Histologically, at 7 days, we observed hyperplasia and abnormal arrangement of collagen fibers. Later, these findings also decreased in both groups, albeit at a faster rate in group 1. Minimal changes were observed in electron microscopy up to the end of the study in both groups. Preoperative refractive state was achieved at 1 month after explantation in both groups. Conclusions: ICRS can safely be explanted from the cornea. Refractive reversibility was achieved at 1 month after explantation. However, the clinical and histological findings after ICRS explantation depend on the time from implantation to explantation.

Validation of an experimental animal model for corneal additive surgery

Purpose: To assess the hen cornea as a model for training and future wound healing studies after implantation of intrastromal corneal ring segments (ICRS) by clinical and optical outcomes. Setting: University of Valladolid, Valladolid, Spain. Design: Experimental study. Methods: One 90°, 150-μm thick polymethyl methacrylate Ferrara ICRS segment was manually implanted at 70-80% depth of 192 Gallus domesticus corneas. Clinical follow-up for 6 months included monitoring corneal thickness, epithelial wound closure, edema, haze, and the location and severity of deposits. The refractive state was also measured. After each animal was euthanized, corneas were processed for direct transmittance and histological analysis. Results: Complications were present in 16% of the eyes. Epithelial wound closure was completed at 3 ± 2 days. A slight corneal edema in the channel site was present for the first 15 days. All corneas had deposits by 4 months located along the inner, outer curvatures and under the segments. Corneal haze was present only at the incision site. ICRS induced hyperopic changes in the refractive state without changes in direct transmitance of central cornea. New cells and extracellular matrix were present around the segment where deposits were seen on clinical follow-up. Conclusions: With hen as an animal model, ICRS were implanted in a precise and reproducible way after a learning curve. Similar to humans, the follow-up period during the first 6 months after implantation showed fast wound closure, deposits, and haze at the incision site. ICRS in hens also reduced the refractive power without affecting the central cornea.

Comparación de las características corneales en gallina y codorniz como modelos experimentales de cirugía refractiva

AIM To compare the histological, morphological and the biophysical measurements between hen and quail corneas, in order to determine which of them were better suited for use as an animal model for research into corneal refractive surgery. MATERIAL AND METHODS A study was performed using the biophysical measurements of the cornea (curvature, thickness, refraction, and axial length) of 20 animals (10 hens and 10 quails). The corneas were then prepared for histological analysis under microscopy light. RESULTS The analysis showed that both groups have the same number of corneal layers as the human cornea and with an evident Bowmans layer. The thickness of the hen cornea and axial length of the eye, 225.3±18.4μm and 12.8±0.25mm, respectively, were larger than that of the quail (P<.01 and P<.001, respectively). The radius of curvature for the hen central cornea, 3.65±0.08mm, was greater than that for the quail (P<.001), but the refractive power of each cornea was similar. The proportion of total corneal thickness of the hen stroma, 82.6%, was more similar to that of the human than was the quail stroma, 72.5%. Within the hen stroma, the density of keratocytes, 8.57±1.49 per 5,000μm(2), was about half that in the quail stroma (P<.005). CONCLUSIONS Because of the large size of the hen cornea, the stromal thickness and proportional similarity of the corneal layers with human cornea, the hen maybe better than the quail as an alternative species suitable for use in studies of corneal refractive surgery.