Análisis de nervios estromales en pacientes con queratocono / Comparison of stromal corneal nerves between normal and keratoconus patients using confocal microscopy

OBJETIVO: Evaluar las diferencias de los nervios del estroma de la córnea entre sujetos normales y pacientes con queratocono. MÉTODOS: Un total de 140 ojos de 70 sujetos normales (grupo A) y 122 ojos de 87 pacientes con queratocono (grupo B), fueron evaluados con el microscopio confocal, realizando un rastreo central del espesor total de la córnea. La morfología y el espesor de los nervios fueron evaluados utilizando el programa Navis V. 3.5.0. El espesor de los nervios se obtuvo del promedio de la porción más delgada y la más gruesa de cada nervio. RESULTADOS: Los nervios del estroma se observaron como estructuras lineales de alta reflexión e irregulares, con porciones gruesas y angostas en todos los casos. El promedio del espesor de los nervios en el grupo A fue de 5,7 ± 1,7 (rango de 3,3 a 10,4 μ), en el grupo B fue de 7,2 ± 1,9 (rango de 3,5 a 12,0 μ). La diferencia en el espesor de los nervios entre el grupo A y el grupo B fue estadísticamente significativa (p < 0,05). CONCLUSIONES: La morfología de los nervios del estroma de la córnea fue similar en ambos grupos; el espesor de estos fue mayor en los pacientes con queratocono

OBJECTIVE: To evaluate the differences in stromal corneal nerves between normal patients and keratoconus patients. MATERIAL AND METHODS: A total of 140 eyes of 70 normal patients (group A) and 122 eyes of 87 keratoconus patients (group B) were examined with the confocal microscope, with a central scan of the total corneal thickness being taken. The morphology and thickness of the corneal stromal nerves were evaluated by using the Navis V. 3.5.0. software. Nerve thickness was obtained from the mean between the widest and the narrowest portions of each stromal nerve. RESULTS: Corneal stromal nerves were observed as irregular linear hyper-reflective structures with wide and narrow portions in all cases. Mean corneal stromal nerves thickness in group A was 5.7 ± 1.7 (range from 3.3 to 10.4 μ), mean corneal stromal nerves thickness in group B was 7.2 ± 1.9 (range from 3.5 to 12.0 μ). There was a statistical significant difference (P<0.05) in stromal corneal nerves thickness between group A and group B. CONCLUSION: Stromal corneal nerves morphology was similar in both groups, but stromal nerves were thicker in keratoconus patients

Regeneración de la superficie ocular: stem cells/células madre y técnicas reconstructivas / Regeneration of the ocular surface: stem cells and reconstructive techniques

La córnea es un tejido transparente constituido microscópicamente por 5 capas bien diferenciadas. El epitelio corneal es esencial para la transparencia corneal y se encuentra en continua renovación a lo largo de la vida a partir de la población de células madre limbocorneales. La localización de estas células madre limbocorneales parece residir en las capas basales del epitelio limbocorneal, de vital importancia para mantener el microambiente de estas células madre limbocorneales, que depende de una variedad de factores intrínsecos y extrínsecos. La insuficiencia límbica se produce cuando ocurre una pérdida parcial o total de estas células madre limbocorneales. Este cuadro lleva a una opacificación corneal con la consiguiente pérdida de visión. En estos casos, el trasplante corneal supone únicamente un reemplazo temporal del epitelio corneal; es necesario llevar a cabo un tratamiento previo con trasplante de limbo autólogo o alogénico, que permita regenerar la población de células limbocorneales dañadas. Para disminuir el riesgo que supone el trasplante de limbo en el ojo donante, se han propuesto técnicas de cultivo de células limbocorneales a partir de pequeñas biopsias limbocorneales (AU)

The cornea is a transparent tissue microscopically constituted by 5 well differentiated layers. The corneal epithelium is essential for corneal transparency and is found in a state of constant renovation throughout life on the basis of the population of limbocorneal stem cells. The localisation of these limbocorneal stem cells seems to be in the basal layers of the limbocorneal epithelium, of vital importance for maintaining the micro-environment of these limbocorneal stem cells, which depend on a variety of intrinsic and extrinsic factors. Limbic insufficiency occurs when there is a partial or total loss of these limbocorneal stem cells. These clinical features lead to a corneal clouding with a resulting loss of vision. In these cases, corneal transplant only represents a temporary replacement of the corneal epithelium; it is necessary to carry out a prior treatment involving transplant of the autologous or allogeneic limbus, which enables regeneration of the population of damaged limbocorneal cells. To reduce the risk involved in the transplant of the limbus of the donor eye, techniques of cultivation of limbocorneal cells on the basis of small limbocorneal biopsies are proposed (AU)

Catecholaminergic nerve fibres in normal and alkali-burned rabbit cornea.

BACKGROUND: In recent years anatomic research has demonstrated the presence of various types of nerve fibre in the cornea. The purpose of this study was to investigate the distribution of catecholaminergic fibres in various corneal layers and to study the effects of an experimental superficial corneal lesion on the pattern of catecholaminergic nerve fibre distribution in the various corneal layers. METHODS: Three weeks after the creation of an alkali burn in the centre of the right cornea of five albino rabbits, the animals were killed, and histologic sections from the cornea of both eyes were stained for observation of catecholaminergic nerve fibres and photographed on black-and-white film. The photographs were examined using the Quantimet image analyser (Leica). RESULTS: Catecholaminergic nerve fibres were observed in the corneal epithelium and the deep stromal layers. Sections from injured cornea showed a drastic reduction in epithelial and superficial stromal catecholaminergic nerve fibres, whereas the nerve fibres in the endothelium and deep stroma were not damaged. INTERPRETATION: Although catecholaminergic nerve fibres were observed in all corneal layers, the pattern of catecholaminergic fibres following the creation of a superficial lesion of the cornea seems to suggest that the superficial and deep nerve fibres may have a different distribution.

Computer analysis of corneal innervation density using a novel double stain in rat corneal whole mounts.

Computerised morphometry and a double stain technique were utilised to examine the corneal nerves in whole mounts. This novel stain combines the nonspecific acetylcholinesterase (NsAchE) and gold chloride (AuCl) procedures to enhance staining contrast and facilitate computerised detection of corneal nerves. Fresh rat corneas were dissected, and the Descemet's membrane-endothelium complex was removed. Then the corneas were fixed in 4% paraformaldehyde with 50 mM Na-K phosphate buffer (pH 7.2) and 8% sucrose for 30 min. They were rinsed and stained singly with NsAchE or AuCl, or were double stained using NsAchE followed by AuCl. Between NsAchE and AuCl staining the corneas were stored frozen in OCT compound at -70 degrees C. Flat mounts of whole corneas were photographed before and after the second staining. Measurable stromal innervation density (mean +/- S.D.) in age-matched corneas stained with AuCl (3.90 +/- 0.36 mm/mm2) was not significantly different from that of NsAchE stained corneas. However, double staining compared with NsAchE staining of the same corneas revealed a 48 +/- 27% increase in demonstrable innervation density of the subepithelial nerve plexus (7.95 +/- 0.86 mm/mm2 vs 5.52 +/- 1.31 mm/mm2, respectively). Improved visualisation of epithelial nerves and their fine ramifications (leashes) was also obtained by double staining. This novel combination of 2 procedures enhances the detection of corneal nerves for analysis by computerised morphometry and provides a more representative estimate of total corneal innervation density.