Tomografía de coherencia óptica de segmento anterior de alta resolución como método de diagnóstico diferencial entre neoplasia intraepitelial córneo-conjuntival y pterygium / High resolution anterior segment optical coherence tomography for differential diagnosis between corneo-conjunctival intraepithelial neoplasia and pterygium

OBJETIVO: Valorar si la tomografía de coherencia óptica de segmento anterior (OCT-SA) es un método de diagnóstico no invasivo adecuado para diferenciar lesiones córneo-conjuntivales benignas (pterygium) de las premalignas (neoplasia intraepitelial córneo-conjuntival [CIN]). MATERIAL Y MÉTODOS: Estudio observacional, analítico y transversal de 22 ojos con diagnóstico de sospecha de pterygium y CIN durante 2 años. Con la OCT-SA se estudiaron las características morfológicas y se compararon espesores epiteliales (EE) y grado de extensión sobre la superficie corneal (GIC). Posteriormente se confirmó el diagnóstico con el estudio histopatológico tras exéresis quirúrgica. RESULTADOS: La edad media de los pacientes con pterygium (n = 18) fue de 52,67 ± 15 años y 74 ± 12 años en los sujetos con CIN (n = 4) (p < 0,021). En los pterygium, la OCT-SA mostró EE normal, adelgazado o levemente aumentado (77,4 ± 26 μm), además de aumento del tejido subepitelial en forma de cuña. Los CIN presentaron un aumento del EE (262,5 ± 124 μm), que era fuertemente hiperreflectivo, con transición abrupta entre epitelio sano y patológico. El análisis de los EE en OCT-SA entre pterygium y CIN reveló diferencias estadísticamente significativas (p < 0,002). La curva ROC reveló una sensibilidad y especificidad del 100% de la OCT-SA en la diferenciación entre CIN y pterygium, utilizando EE de 141μm como punto de corte. CONCLUSIÓN: La OCT-SA es una herramienta útil para la diferenciación entre pterygium y CIN, ya que proporciona características morfológicas típicas. Un aumento del espesor del EE córneo-conjuntival mayor de 141 μm en OCT-SA sugiere una sensibilidad y especificidad del 100% para diagnosticar CIN

OBJECTIVE: To assess if anterior segment optical coherence tomography (AS-OCT) is a noninvasive diagnostic method suitable to differentiate benign corneo-conjunctival lesions (pterygium) from premalignant lesions (corneo-conjunctival intraepithelial neoplasia, CIN). MATERIAL AND METHODS: An observational, analytical and cross-sectional study was conducted in 22 eyes with conjunctival lesions clinically suspicious for pterygium and CIN during two years. Morphological differences between both lesions were studied with AS-OCT; epithelial thicknesses (EE) and extension length on corneal surface (GIC) were compared between both groups. A surgical excision of the lesion was performed for histopathological diagnosis. RESULTS: Mean age of patients with pterygium (n = 18) was 52.67 ± 15 y.o and 74 ± 12 y.o in subjects with CIN (n = 4) (p < 0.021). In pterygia, AS-OCT showed typical features (normal, thinning or slightly thickened EE; 77.4 ± 26 μm), in addition to an increase in wedge-shaped subepithelial tissue. Patients with CIN had a mean thickened EE (262.5 ± 124μm) and strongly hyperreflective, with abrupt transition between normal and pathological epithelium. Analysis of EE between subjects with pterygium and CIN revealed statistically significant differences (p < 0.002). ROC curve revealed a 100% sensitivity and specificity of OCT-SA in differentiation between CIN and pterygium, using 141μm as cutoff point of EE. CONCLUSION: AS-OCT is a useful tool for the differentiation between pterygium and CIN able to provide typical morphological characteristics. An EE greater than 141 μm in AS-OCT suggests a sensitivity and specificity of 100% for the diagnosis of CIN

High resolution anterior segment optical coherence tomography for differential diagnosis between corneo-conjunctival intraepithelial neoplasia and pterygium. / Tomografía de coherencia óptica de segmento anterior de alta resolución como método de diagnóstico diferencial entre neoplasia intraepitelial córneo-conjuntival y pterygium.

OBJECTIVE: To assess if anterior segment optical coherence tomography (AS-OCT) is a noninvasive diagnostic method suitable to differentiate benign corneo-conjunctival lesions (pterygium) from premalignant lesions (corneo-conjunctival intraepithelial neoplasia, CIN). MATERIAL AND METHODS: An observational, analytical and cross-sectional study was conducted in 22 eyes with conjunctival lesions clinically suspicious for pterygium and CIN during two years. Morphological differences between both lesions were studied with AS-OCT; epithelial thicknesses (EE) and extension length on corneal surface (GIC) were compared between both groups. A surgical excision of the lesion was performed for histopathological diagnosis. RESULTS: Mean age of patients with pterygium (n=18) was 52.67±15 y.o and 74±12 y.o in subjects with CIN (n=4) (p<0.021). In pterygia, AS-OCT showed typical features (normal, thinning or slightly thickened EE; 77.4±26µm), in addition to an increase in wedge-shaped subepithelial tissue. Patients with CIN had a mean thickened EE (262.5±124µm) and strongly hyperreflective, with abrupt transition between normal and pathological epithelium. Analysis of EE between subjects with pterygium and CIN revealed statistically significant differences (p<0.002). ROC curve revealed a 100% sensitivity and specificity of OCT-SA in differentiation between CIN and pterygium, using 141µm as cutoff point of EE. CONCLUSION: AS-OCT is a useful tool for the differentiation between pterygium and CIN able to provide typical morphological characteristics. An EE greater than 141µm in AS-OCT suggests a sensitivity and specificity of 100% for the diagnosis of CIN.

Retinal ganglion cell death after acute retinal ischemia is an ongoing process whose severity and duration depends on the duration of the insult.

In adult Sprague-Dawley rats we have investigated retinal ganglion cell survival after transient intervals of retinal ischemia of 30, 45, 60, 90 or 120 min duration, induced by ligature of the ophthalmic vessels. Animals were killed 5, 7, 14, 21, 30, 60, 90 or 180 days later and densities of surviving retinal ganglion cells were estimated in retinal whole mounts by counting cells labelled with diAsp. This dye was applied, 3 days prior to death, to the ocular stump of the intraorbitally transected optic nerve. We found that retinal ganglion cell loss after retinal ischemia proceeds for different lengths of time. All the ischemic intervals induced loss of retinal ganglion cells whose severity and duration was related to the length of the ischemic interval. Following 30 or 45 min of ischemia, cell loss lasted 14 days and caused the death of 46 or 50%, respectively, of the population of retinal ganglion cells. Sixty, 90 or 120 min of retinal ischemia were followed by a period of cell loss that lasted up to 90 days and caused the death of 75%, 87% or 99%, respectively, of the population of retinal ganglion cells. We conclude that retinal ganglion cell loss after retinal ischemia is an ongoing process that may last up to 3 months after the injury and that its severity and duration are determined by the ischemic interval.

Expression of netrin-1 and its receptors DCC and UNC-5H2 after axotomy and during regeneration of adult rat retinal ganglion cells.

Netrins are a family of chemotropic factors that guide axon outgrowth during development; however, their function in the adult CNS remains to be established. We examined the expression of the netrin receptors DCC and UNC5H2 in adult rat retinal ganglion cells (RGCs) after grafting a peripheral nerve (PN) to the transected optic nerve and following optic nerve transection alone. In situ hybridization revealed that both Dcc and Unc5h2 mRNAs are expressed by normal adult RGCs. In addition, netrin-1 was found to be constitutively expressed by RGCs. Quantitative analysis using in situ hybridization demonstrated that both Dcc and Unc5h2 were down-regulated by RGCs following axotomy. In the presence of an attached PN graft, Dcc and Unc5h2 were similarly down-regulated in surviving RGCs regardless of their success in regenerating an axon. Northern blot analysis demonstrated expression of netrin-1 in both optic and sciatic nerve, and Western blot analysis revealed the presence of netrin protein in both nerves. Immunohistochemical analysis indicated that netrin protein was closely associated with glial cells in the optic nerve. These results suggest that netrin-1, DCC, and UNC5H2 may contribute to regulating the regenerative capacity of adult RGCs.

Retinal ganglion cell and nonneuronal cell responses to a microcrush lesion of adult rat optic nerve.

Injury of the optic nerve has served as an important model for the study of cell death and axon regeneration in the CNS. Analysis of axon sprouting and regeneration after injury by anatomical tracing are aided by lesion models that produce a well-defined injury site. We report here the characterization of a microcrush lesion of the optic nerve made with 10-0 sutures to completely transect RGC axons. Following microcrush lesion, 62% of RGCs remained alive 1 week later, and 28% of RGCs, at 2 weeks. Optic nerve sections stained by hematoxylin-based methods showed a thin line of intensely stained cells that invaded the lesion site at 24 h after microcrush lesion. The lesion site became increasingly disorganized by 2 weeks after injury, and both macrophages and blood vessels invaded the lesion site. The microcrush lesion was immunoreactive for chondroitin sulfate proteoglycans (CSPG), and an adjacent GFAP-negative zone developed early after the lesion, disappearing by 1 week. Luxol fast blue staining showed a myelin-free zone at the lesion site, and myelin remained distal to the lesion at 8 weeks. To study the axonal response to microcrush lesion, anterograde tracing was used. Within 6 h after injury all RGC axons retracted back from the site of lesion. By 1 week after injury, axons regrew toward the lesion, but most stopped abruptly at the injury scar. The few axons that were able to cross the injury site did not extend further in the optic nerve white matter by 8 weeks postlesion. Our observations suggest that both the CSPG-positive scar and the myelin-derived growth inhibitory proteins contribute to the failure of RGC regeneration after injury.

Death and neuroprotection of retinal ganglion cells after different types of injury.

In adult Sprague-Dawley rats, retinal ganglion cell survival was investigated after intraorbital optic nerve section and after transient ischemia of the retina induced by elevation of the intraocular pressure or by selective ligature of the ophthalmic vessels. The thickness of the inner nuclear and inner plexiform layers was also assessed after transient periods (120 min) of retinal ischemia induced by selective ligature of the ophthalmic vessels. In addition, we have also investigated the neuroprotective effects of different substances in these paradigms. The intraocular injection of brain-derived neurotrophic factor increased RGC survival after retinal ischemia induced by elevation of the intraocular pressure or by selective ligature of the ophthalmic vessels. The caspase-inhibitor Z-DEVD increased retinal ganglion cell survival after optic nerve section and also after 90 min of retinal ischemia induced by selective ligature of the ophthalmic vessels. The peptide Bcl-2 did not increase retinal ganglion cell survival after optic nerve section but increased retinal ganglion cell survival after 60 or 90 min of retinal ischemia induced by selective ligature of the ophthalmic vessels. Finally, BDNF, nifedipine, naloxone and bcl-2 prevented in part the decrease in thickness of the inner nuclear layer and inner plexiform layer induced by selective ligature of the ophthalmic vessels. Our results suggest that retinal ganglion cell loss induced by different types of injury, may be prevented by substances with neuroprotective effects, by altering steps of the cascade of events leading to cell death.

Inactivation of Rho signaling pathway promotes CNS axon regeneration.

Regeneration in the CNS is blocked by many different growth inhibitory proteins. To foster regeneration, we have investigated a strategy to block the neuronal response to growth inhibitory signals. Here, we report that injured axons regrow directly on complex inhibitory substrates when Rho GTPase is inactivated. Treatment of PC12 cells with C3 enzyme to inactivate Rho and transfection with dominant negative Rho allowed neurite growth on inhibitory substrates. Primary retinal neurons treated with C3 extended neurites on myelin-associated glycoprotein and myelin substrates. To explore regeneration in vivo, we crushed optic nerves of adult rat. After C3 treatment, numerous cut axons traversed the lesion to regrow in the distal white matter of the optic nerve. These results indicate that targeting signaling mechanisms converging to Rho stimulates axon regeneration on inhibitory CNS substrates.

Retinal ganglion cell death after different transient periods of pressure-induced ischemia and survival intervals. A quantitative in vivo study.

PURPOSE: To quantitate in vivo retinal ganglion cell (RGC) survival after transient periods of pressure-induced ischemia of the rat retina and after different survival intervals. METHODS: In adult rats, RGCs were labeled with fluorogold applied to their main targets in the brain. Seven days later, in several groups of rats, the left retinas were subjected to transient periods of ischemia of 30, 45, 60, 75, 90, 105, or 120 minutes, respectively, by increasing the intraocular pressure (IOP) of the left eye above systolic values. Five, 7, 14, and 30 days later, the rats were killed, and their retinas were prepared as wholemounts for examination under fluorescence microscopy to estimate RGC survival. RESULTS: The authors found that periods of ischemia of 30 and 45 minutes do not induce RGC death; longer periods of transient ischemia induce the death of a proportion of RGCs, and the proportion increases with the duration of the ischemia; RGC death, which can be observed as early as 5 days after ischemia, continues during the 30-day study period; and periods of ischemia that last 90 minutes or more cause the death of approximately 95% of the RGC population 30 days later. CONCLUSIONS: Increases of the IOP above systolic levels for periods of 60 minutes or more result in RGC loss in the rat retina. Both the duration of the initial transient period of ischemia and the duration of the survival period influence the proportion of RGC death.