Induction of pluripotent stem cells by reprogramming human ocular fibroblasts under xeno-free conditions / Indução de células-tronco pluripotentes pela reprogramação de fibroblastos oculares humanos sob condições xeno-livre

ABSTRACT Purposes: To develop an efficient and xeno-free standard eye-derived induced pluripotent stem cell reprogramming protocol for use during induced pluripotent stem cell-based cell therapies in treating retinal degenerative diseases and to compare the relative effectiveness of both animal- and non-animal-derived culture systems in the generation of induced pluripotent stem cells. Methods: Primary cultured human pterygium fibroblasts and human Tenon's capsule fibroblasts were induced to induced pluripotent stem cells using a non-in­tegrated virus under two xeno-free systems; as part of this study, a traditional non-xeno-free reprogramming system was also assessed. Induced pluripotent stem cell clones were selected and counted by live staining. Reprogramming efficiencies were evaluated between the fibroblasts and among different culture systems. In a series of experiments, such as PCR and immunofluorescence staining, the induced pluripotent stem cells were characterized. Results: Human pterygium fibroblast- and human Tenon's capsule fibroblast-derived induced pluripotent stem cells were successfully established using different reprogramming systems, under which they exhibited properties of induced pluripotent stem cells. Reprogramming efficiencies of induced pluripotent stem cells using the cell therapy system, the traditional system, and the E6/E8 system were 0.014%, 0.028%, and 0.001%, respectively, and those of human pterygium fibroblast- and human Tenon's capsule fibroblast-derived induced pluripotent stem cells-using the aforementioned systems-were 0.018% and 0.017%, respectively. Conclusions: Sendai virus facilitates induced pluripotent stem cell reprogramming of ocular fibroblasts-both human pterygium and human Tenon's capsule fibroblasts being safe and efficient for induced pluripotent stem cell reprogramming. Although the reprogramming efficiencies of ocular-derived induced pluripotent stem cells under xeno-free conditions were not superior to those observed using the traditional reprogramming system, the cell therapy system reprogramming system is a good option when induced pluripotent stem cells are to be induced under xeno-free conditions.

RESUMO Objetivos: Desenvolver um protocolo padrão, eficiente e xeno-livre, para a reprogramação de células-tronco pluripotentes induzidas, que possa ser usado durante as terapias de células-tronco pluripotentes induzidas para o tratamento de doenças degenerativas da retina, e comparar a eficácia relativa de sistemas de cultivo de origem animal e de origem não animal na geração de células-tronco pluripotentes induzidas. Métodos: Cultivos primários de fibroblastos de pterígio humano e de fibroblastos da cápsula de Tenon humanos foram induzidos a células-tronco pluripotentes induzidas usando um vírus não integrado sob dois sistemas xeno-livres; um sistema tradicional de reprogramação não xeno-livre também foi avaliado como parte deste estudo. Os clones de células-tronco pluripotentes induzidas foram selecionados e contados por coloração de células vivas. As eficiências de reprogramação foram avaliadas entre os diferentes fibroblastos e entre os diferentes sistemas de cultivo. Uma série de experimentos, como o PCR e a coloração por imunofluorescência, foram conduzidos para caracterizar as células-tronco pluripotentes induzidas. Resultados: Célu­las-tronco pluripotentes induzidas derivadas de fibroblastos de pterígio humano e fibroblastos da cápsula de Tenon humanos foram estabelecidas com sucesso sob diferentes sistemas de reprogramação e exibiram propriedades de células-tronco pluripotentes induzidas. As eficiências de reprogramação das células-tronco pluripotentes induzidas usando o sistema de terapia celular, o sistema tradicional e o sistema E6/E8 foram 0,014, 0,028% e 0,001%, respectivamente. Além disso, as efi­ciências de reprogramação de células-tronco pluripotentes induzidas derivadas de fibroblastos de pterígio humano e de fibroblastos da cápsula de Tenon humanos usando todos os sistemas acima foram de 0,018% e 0,017%, respectivamente. Conclusões: O vírus Sendai pode ser usado para facilitar a reprogramação de fibroblastos oculares pelas células-tronco pluripotentes induzidas. Tanto os fibroblastos de pterígio humano quanto os fibroblastos da cápsula de Tenon humanos são seguros e eficientes para a reprogramação de células-tronco pluripotentes induzidas. Embora as eficiências de reprogramação das células-tronco pluripotentes induzidas de origem ocular sob condições xeno-livres não tenham sido superiores às eficiências observadas para o sistema tradicional de reprogramação, o sistema de reprogramação sistema de terapia celular é uma boa opção para a indução de células-tronco pluripotentes induzidas sob condições xeno-livres.

Assessment of the effect of intravitreal triamcinolone acetonide on the chorioretinal and vitreous inflammatory reaction to cryotherapy in rabbits / Avaliação do efeito da injeção intravítrea de triancionolonaacetonida sobre a reação inflamatória coriorretiniana e vítrea à crioterapia em coelhos

PURPOSE: To evaluate the inflammatory response in the choroid, retina and vitreous in rabbit eyes underwent cryotherapy followed by intravitreal triamcinolone acetonide and to compare with those underwent cryotherapy followed by intravitreal injection of saline solution. METHODS: This is a prospective case-control study. Surgical procedures were performed in eleven rabbits. Two animals were excluded because they did not complete the postoperative period or had intraoperative or postoperative complications. All rabbits underwent superior temporal peritomy and transscleralcryotherapy in both eyes. After cryotherapy, animals received intravitreal injection of triamcinolone acetonide in one eye and saline solution in the fellow eye. Animals were sacrificed seven days after the procedure and their eyes were enucleated. Histological sections of eyeballs were prepared and the vitreous humor was aspirated. The count of inflammatory cells was performed by light microscopy. RESULTS: Histological sections of both eyes of nine rabbits were analyzed. Inflammatory cells were found only in the choroid. There was no statistically significant difference in the number of inflammatory cells between the two groups, regardless of cell type analyzed. CONCLUSION: This study showed no statistically significant difference between the use or absence of intravitreal triamcinolone acetonide in the inflammatory response to cryotherapy in rabbit eyes. Studies with larger samples are needed to confirm the trend of this paper.

OBJETIVO: Avaliar a resposta inflamatória em coróide, retina e vítreo nos olhos de coelhos submetidos à crioterapia seguida de injeção intravítrea de triancinolona acetonida e comparar com os olhos de coelhos que foram submetidos à crioterapia seguida de injeção intravítrea de solução salina. MÉTODOS: Trata-se de um estudo prospectivo caso-controle. Foram realizados procedimentos cirúrgicos em 11 coelhos albinos, sendo excluídos os animais que não completaram o pós-operatório ou apresentaram complicações intra ou pós-operatórias. Todos os coelhos foram submetidos à peritomia temporal superior e aplicação de crioterapia transescleral em ambos os olhos. No final de cada procedimento, os animais receberam injeção intravítrea de triancinolona acetonida em um olho e de solução salina no olho contralateral. Após 7 dias de pós-operatório, os animais foram sacrificados e tiveram seus olhos enucleados. O humor vítreo desses olhos foi aspirado, e realizou-se a confecção de cortes histológicos dos globos oculares. Foi feita a contagem das células inflamatórias desses materiais em microscópio óptico. RESULTADOS: Foram analisados cortes histológicos dos olhos de 9 coelhos. Foram encontradas células inflamatórias apenas na coróide em todos os olhos. Não houve diferença estatisticamente significante no número de células inflamatórias entre os dois grupos estudados, independente do tipo celular analisado. CONCLUSÃO: O artigo em questão não demonstrou influência da triancinolona acetonida intravítrea na resposta inflamatória à crioterapia em coelhos. São necessários estudos com amostras maiores para confirmar a tendência deste trabalho.

Histología y morfometría del ojo del pez dulceacuícola Paracheirodon axelrodi (Characiformes: Characidae)

Histology and morphometry of the eye in the freshwater Paracheirodon axelrodi (Characiformes: Characidae). The visual system of Teleost fishes has a wide range of adaptations. The eye has suffered modifications, mainly towards better sensitivity and visual resolution according to specific environmental conditions. In that way, this organ is so important for survival. In spite of being the most commercialized Orinoquía species, and one of the most important ornamental fish worldwide, there are almost no studies on the communication systems of P. axelrodi (including vision). We studied the eye in ten individuals of P. axelrodi, with a mean total length of 2.7 cm (SD= 0.2, n= 20). We measured eye relative size (TRO) after sacrifice. Heads were processed for hematoxylin-eosin, and 8 μm thick sections were obtained. Images were digitalized for histological identification and morphometrics. The relation between TRO and body length was 9.8% (SD= 1.1, n= 20); lens was spherical with a relative size of 64.3% (SD= 4.9, n= 20). Rete mirabilis choroid was composed mainly by capillaries, while sclera was composed of cartilaginous tissue. Retina with eight layers and two membranes. An areae centrales was observed in the posterior retina. Iris covered by a simple cubic epithelium. Cornea of endothelial tissue with two membranes. Pupil almost circular, with a naso-caudal elongation. Measures of retina thickness were as follows: lateral retina 144.5 μm (SD= 5.4, n= 20), temporal retina 210.4 μm (SD= 14.8, n= 20). Lens measurements were: dorso- ventral 759.6 μm (SD= 31.7, n= 20), antero-posterior 763.4 μm (SD= 30.7, n= 20). Cornea thickness was 27.6 μm (SD= 4.3, n= 20); iris thickness was 18.9 μm (SD= 1.9, n= 20); rete mirabilis choroid 22.2 μm (SD= 3.2, n= 20) and sclera: 15.9 μm (SD= 1.2, n= 20). This species has a typical Teleost eye conformation. The retina was thicker in the posterior area, probably related to the presence of neuron groups. This modification allows for a better sharpness and visual resolution. The TRO, areae centrales in the retina, and thin cornea, suggests the need of a well developed visual system resulting from a predatory diet. Rev. Biol. Trop. 57 (4): 1107-1118. Epub 2009 December 01.

El pez dulceacuícola P. axelrodi es de gran importancia comercial en la Orinoquía Colombiana y a nivel mundial, pero carece de estudios acerca de su visión. Se utilizaron diez individuos, con longitud total promedio de 2.7 cm (SD= 0.2, n= 20). Se realizaron cortes a 8 μm y se tiρeron con H-E. El tamaρo relativo del ojo es 9.8% (SD= 1.1, n= 20); el cristalino tiene un tamaρo relativo de 64.3% (SD= 4.9, n=20). Una esclerótica con tejido cartilaginoso. La retina está constituida por ocho capas y dos membranas, hay areae centrales en la zona posterior, por lo tanto tiene mayor espesor que la zona lateral. El espesor promedio de las estructuras es: retina lateral: 144.5 μm (SD= 5.4, n= 20), retina temporal: 210.4 μm (SD= 14.8, n= 20); cristalino (esfιrico): dorso-ventral 759.6 μm (SD= 31.7, n= 20) y antero- posterior 763.4 μm (SD= 30.7, n= 20) y cσrnea: 27.6 μm (SD= 4.3, n= 20). El tamaρo relativo del ojo, relacionado con la presencia de areae centrales y una córnea delgada, muestra las necesidades visuales del neón cardenal (cazador) para adaptarse al hábitat, lo que hace necesario tener un sistema visual desarrollado que le permita tener mejor sensibilidad y resolución visual.

A six-rhabdomere, open rhabdom arrangement in the eye of the chrysanthemum beetle Phytoecia rufiventris: some ecophysiological predictions based on eye anatomy

We are describing a rhabdom organization of the eye of the chrysanthemum beetle Phytoecia rufiventris that to date has not been described from any other insect. In cerambycid beetles free rhabdomeres, forming a circular, open rhabdom, surround a central rhabdom made up of the rhabdomeres of one or two cells. In Phytoecia rufiventris the central rhabdomeres are missing throughout the eye and the microvilli of the outer 6 rhabdomeres are regularly oriented in three directions. Following the classification of rhabdom types suggested by Wachmann (1979), we suggest to name the rhabdom arrangement seen in the retina of Phytoecia rufiventris [quot ]Grundmuster 3[quot ]. This pattern ought to facilitate polarization sensitivity and movement perception, features that agree with the behavioural repertoire of Phytoecia rufiventris.

GAL4 causes developmental defects and apoptosis when expressed in the developing eye of Drosophila melanogaster

The UAS/GAL4 ectopic expression system is widely used in Drosophila melanogaster for the overexpression of transgenes. This system operates under the assumption that the yeast transcription factor, GAL4, is inactive in D. melanogaster. Thus, GAL4 can be expressed under the control of D. melanogaster -specific promoters with little effect upon the organism. We have shown that expression of GAL4 in the developing eye under the control of the glass multiple reporter (GMR) promoter element does have an effect on eye development. Although GMR-GAL4 heterozygotes appear normal when raised at 25 degrees C, the homozygotes have a highly disorganized ommatidial array. In addition, the levels of apoptosis in the third-instar larval eye imaginal disc (where GAL4 is expressed) are slightly higher in GMR-GAL4 heterozygotes, and much higher in GMR-GAL4 homozygotes when compared to wild type discs. The morphological eye defects caused by GMR-GAL4 are significantly enhanced when flies are raised at 29 degrees C (presumably due to the higher activity of GAL4 at this temperature); however, the levels of apoptosis appear to be similar at these two temperatures. Taken together, these data suggest that GAL4 can have adverse effects on D. melanogaster development, especially at high expression levels. In addition, GAL4 appears to induce apoptosis even in the absence of any visible morphological defects. Thus, despite the benefits of the UAS/GAL4 ectopic expression system, one must use caution in the design and interpretation of experiments

Microanatomical characteristics of marginal ommatidia in three different size-classes of the semi-terrestrial isopod Ligia exotica (Crustacea / Isopoda)

The aims of this paper have been (a) to characterize marginal ommatidia from different eye regions through a detailed description of their distinct ultrastructural features in three different size-classes of L. exotica, and (b) to compare microanatomical characteristics of the marginal ommatidia with those of ommatidia of the same eye, but located further centrally. On the basis of transverse as well as longitudinal sections we conclude that new ommatidia are added from a crescentic dorso-anterio-ventral edge of the eye and that maturing ommatidia go through a sequence in which originally the nuclei of cone-, pigment-, and retinula cells are arranged in three separate layers. At the beginning of the microvillar development, the organization of the corresponding rhabdomeres is still quite different (much less regular) from that of those rhabdomeres that make up the mature rhabdom. Marginal ommatidia always possess smaller diameters than more centrally located ones and retinal screening pigment granules are most apparent in the retinula cells only after the first microvilli have appeared. The diameters of rhabdom microvilli (approx. 55 nm) do not differ in ommatidia from the five investigated eye regions in small specimens (< 1.5 cm body length), but show a tendency to be slightly wider in the anterior (= frontal or rostral) regions of the eye (approx. 65 nm) in larger specimens (> 2.0 cm body length).

Fisiologia da acomodaçäo e presbiopia / Physiology of accommodation and presbyopia

A presbiopia é um dos mais precoces sinais do envelhecimento natural e a fisiopatologia básica envolvida no seu desenvolvimento tem sido um tema de controvérsia durante séculos. Este artigo discute vários aspectos da presbiopia através de uma revisäo literária das alteraçöes que ocorrem no olho durante este processo, e que já foram descritas previamente por diversos autores.