DNMT1-mediated PPARα methylation aggravates damage of retinal tissues in diabetic retinopathy mice

BACKGROUND: Peroxisome proliferator-activated receptor alpha (PPARα) is associated with diabetic retinopathy (DR), and the underlying mechanism is still unclear. Aim of this work was to investigate the mechanism of PPARα in DR. METHODS: Human retinal capillary pericytes (HRCPs) were treated with high glucose (HG) to induce DR cell model. DR mouse model was established by streptozotocin injection, and then received 5-Aza-2-deoxycytidine (DAC; DNA methyltransferase inhibitor) treatment. Hematoxylin-eosin staining was performed to assess retinal tissue damage. PPARα methylation was examined by Methylation-Specific PCR. Flow cytometry and DCFH-DA fluorescent probe was used to estimate apoptosis and reactive oxygen species (ROS). The interaction between DNA methyltransferase-1 (DNMT1) and PPARα promoter was examined by Chromatin Immunoprecipitation. Quantitative real-time PCR and western blot were performed to assess gene and protein expression. RESULTS: HG treatment enhanced the methylation levels of PPARα, and repressed PPARα expression in HRCPs. The levels of apoptotic cells and ROS were significantly increased in HRCPs in the presence of HG. Moreover, DNMT1 was highly expressed in HG-treated HRCPs, and DNMT1 interacted with PPARα promoter. PPARα overexpression suppressed apoptosis and ROS levels of HRCPs, which was rescued by DNMT1 up-regulation. In DR mice, DAC treatment inhibited PPARα methylation and reduced damage of retinal tissues. CONCLUSION: DNMT1-mediated PPARα methylation promotes apoptosis and ROS levels of HRCPs and aggravates damage of retinal tissues in DR mice. Thus, this study may highlight novel insights into DR pathogenesis.

Clinical studies using stem cells for treatment of retinal diseases: state of the art / Estudos clínicos com uso de células-tronco para tratamento de doenças da retina: estado da arte

ABSTRACT Degenerative retinal diseases such as retinitis pigmentosa, Stargardt's macular dystrophy, and age-related macular degeneration are characterized by irreversible loss of vision due to direct or indirect photoreceptor damage. No effective treatments exist, but stem cell studies have shown promising results. Our aim with this review was to describe the types of stem cells that are under study, their effects, and the main clinical trials involving them.

RESUMO As doenças degenerativas da retina, como retinose pigmentar, distrofia macular de Stargardt e degeneração macular relaciona à idade, são caracterizadas por perda irre versível da visão devido a danos diretos ou indiretos aos fotorreceptores. Não existem tratamentos eficazes, porém os estudos com células-tronco mostraram resultados promissores. Nosso objetivo com esta revisão foi descrever os tipos de células-tronco em estudo, seus efeitos e os principais ensaios clínicos que as envolvem.

Retinal ganglion cells in Strigidae raptors: distribution and morphometry / Células ganglionares de la retina en aves rapaces Strigidae: distribución y morfometría

The authors studied the morphometry and the topographical distribution of Retinal Ganglion Cells (RGCs) in four nocturnal raptors of the order of Strigiformes, family of Strigidae: little owl, tawny owl, scops owl, eared owl. In order to recognize specialized retinal vision areas (fovea and visual streak), the number of RGCs/mm2 and the soma size in the four retinal fields (dorsal, ventral, temporal and nasal) by the histological analysis of retinal radial sections were recorded. A temporal fovea was identified in little owl, tawny owl and eared owl while in scops owl this visual area was localized near the fundus oculi. A radial visual streak ventrally directed was pointed out in the retinas of the four raptors with different shape according to its width. The Authors linked the obtained data with the predatory behavior of nocturnal raptors in their habitat.

Se estudió la morfometría y la distribución topográfica de las células ganglionares de la retina (CGR) en cuatro aves rapaces nocturnas del orden de los Strigiformes, familia Strigidae: búho pequeño, mochuelo, autillo, y cárabo. Con el objetivo de definir las áreas de visión retiniana especializadas (fóvea y campo visual), se registró el número de CGRs/mm2 y el tamaño del soma en los cuatro campos retinianos (dorsal, ventral, temporal y nasal), mediante análisis histológico de las secciones radiales de la retina. Se identificó una fóvea temporal en mochuelo, búho leonado y búho pequeño, mientras que en el búho real, esta área visual se localizó cerca del fondo de ojo. Se observó un campo radial visual dirigido ventralmente en las retinas de las cuatro aves rapaces, con diferentes formas según su extensión. Se relacionaron los datos obtenidos con el comportamiento predatorio de aves rapaces nocturnas en su hábitat.

Number and topographical distribution of retinal ganglion cells in diurnal and nocturnal raptors / Número y distribución topográfica de células ganglionares de la retina en rapaces diurnas y nocturnas

Retinal Ganglion Cells (RGCs) in two diurnal raptors, the buzzard (Buteo buteo) and kestrel (Falco tinnunculus), and in two nocturnal raptors, the little owl (Athene noctua) and tawny owl (Strix aluco), were studied in order to highlight their topographical distribution, taking into account the soma size. Cell density (RGCs/mm2) and median soma area from histological radial sections in four retinal fields (dorsal, ventral, temporal and nasal) were recorded in order to identify specialized retinal vision areas. The results showed a different RGCs distribution between diurnal and nocturnal raptors related to the location of the foveas and shape of the horizontal streak, confirming the bibliographic data. In diurnal raptors, a higher cell density was found in the temporal and central retinal fields as revealed by the presence of a temporal and a central fovea which showed a "horizontal streak". In nocturnal raptors the cell density was higher in the peripheral temporal field likely due to a temporal fovea. A peak in cell density observed in the ventral field, especially in the retina of little owls, might be linked to a more "radial visual streak". Comparing the data obtained we highlighted that the morphology and the number of RGCs are closely linked to the habitat and to the type of predation.

Se estudiaron las células ganglionares de la retina (RGC) en dos aves rapaces diurnas, el ratonero (Buteo buteo) y el cernícalo (Falco tinnunculus), y en dos aves rapaces nocturnas, el mochuelo (Athene noctua) y el búho leonado (Strix aluco), buscando resaltar su distribución topográfica, teniendo en cuenta el tamaño del soma. Se registraron la densidad celular (RGCs/mm2) y la media del área del soma de las secciones histológicas radiales en cuatro campos retinianos (dorsal, ventral, temporal y nasal) para identificar áreas de visión retinianas especializadas. Los resultados mostraron una distribución diferente de RGCs entre las rapaces diurnas y nocturnas relacionadas con la ubicación de las fóveas y la forma de la línea horizontal, lo que confirma los datos bibliográficos. En las aves rapaces diurnas, se encontró una densidad celular más alta en los campos de retina temporales y centrales como lo revela la presencia de una fóvea temporal y central que mostraba una "veta horizontal". En aves rapaces nocturnas, la densidad celular fue mayor en el campo temporal periférico probablemente debido a una fóvea temporal. Un aumento en la densidad celular, observado en el campo ventral, especialmente en la retina de los pequeños buhos, podría estar relacionado con una "línea visual radial". Comparando los datos obtenidos, resaltamos que la morfología y el número de RGC están estrechamente relacionados con el hábitat y el tipo de depredación.

Ganglionic cells apoptosis in retinal layer of rat offspring due to gestational diabetes / Células ganglionares apoptóticas en la capa de la retina de crías de ratas debido a la diabetes gestacional

Previous studies have shown the adverse effects of gestational diabetes on hippocampal neuronal density in animal models. This study was conducted to determine the effect of gestational diabetes on retinal ganglionic cell density, the thickness of the retinal layer and apoptotic ganglionic cell density in 28-day-old of rat offspring. In this experimental study, 10 Wistar rat dams were randomly allocated in control and diabetic groups. Gestational diabetes was induced by 40 mg/kg/body weight of streptozotocin at the first day of gestation, intraperitoneally, dams in control group received an equivalent volume normal saline. At postnatal day 28, six offspring of each gestational diabetes and controls were randomly selected, sacrificed and sections (6 micrometer) were taken from the eye and stained with hematoxylin and eosin. The density of ganglionic cells and the number of dUTP end-labeling (TUNEL)-positive cells were evaluated in 20000 mm2 area of ganglion layer of the retina. The ganglionic cells density were reduced (27.4%) in gestational diabetic offspring in compared to controls (22.5±1.5 vs. 31.0±0.9, P<0.01). The apoptotic ganglionic cells of retina in interventional group significantly increased in compared to controls (6.74±0.60 vs. 5.12±0.26, P<0.02). This study showed that the uncontrolled gestational diabetes can reduce the number of ganglionic cells and increase apoptotic ganglionic cells of retina layer in rat offspring.

Estudios previos en un modelo animal han demostrado los efectos adversos de la diabetes gestacional en la densidad neuronal del hipocampo. El objetivo fue determinar el efecto de la diabetes gestacional en la densidad de las células ganglionares de la retina, en el espesor de la capa de la retina y en la densidad de las células apoptóticas ganglionares, en crías de ratas de 28 días. En este estudio experimental, 10 ratas Wistar fueron asignadas aleatoriamente en grupos control y diabéticos. La diabetes gestacional se indujo a partir de la administración de 40 mg/kg/peso corporal de estreptozotocina en el primer día de la gestación, por vía intraperitoneal. Al grupo control se administró un volumen equivalente de solución salina normal. En el día 28 luego del nacimiento, se seleccionaron aleatoriamente seis crías procedentes de los grupos con diabetes gestacional y controles, se eutanasiaron y se tomaron muestras de los ojos, en forma de secciones de 6 micrómetros, las cuales se tiñeron con H & E. La densidad de las células ganglionares y el número final de células dUTP positivas (TUNEL) se evaluaron a nivel de la capa ganglionar de la retina, en un área de 20.000 mm2. La densidad de las células ganglionares se redujo un 27,4% en la descendencia con diabetes gestacional en comparación con los controles (22,5±1,5 vs. 31,0±0,9, P<0,01). Las células ganglionares apoptóticas de la retina en el grupo con diabetes gestacional aumentaron significativamente en comparación con los controles (6,74±0,60 vs. 5,12 ± 0,26, P <0,02). Este estudio demostró que la diabetes gestacional no controlada puede reducir el número de células ganglionares y aumentar el número de células ganglionares apoptóticas de la capa de la retina en las crías de las ratas con diabetes gestacional.

Effects of monocular deprivation on the dendritic features of retinal ganglion cells / Efectos de la privación monocular sobre las características dendríticas de las células ganglionares de la retina

Monocular deprivation results in anatomical changes in the visual cortex in favor of the non-deprived eye. Although the retina forms part of the visual pathway, there is scarcity of data on the effect of monocular deprivation on its structure. The objective of this study was to describe the effects of monocular deprivation on the retinal ganglion cell dendritic features. The study design was quasi-experimental. 30 rabbits (18 experimental, 12 controls) were examined. Monocular deprivation was achieved through unilateral lid suture in the experimental animals. The rabbits were observed for three weeks. Each week, 6 experimental and 3 control animals were euthanized, their retina harvested and processed for light microscopy. Photomicrographs of the retina were taken using a digital camera then entered into FIJI software for analysis. The number of primary branches, terminal branches and dendritic field area among the non-deprived eyes increased by 66.7%(p=0.385), 400%(p=0.002), and 88.4%(p=0.523) respectively. Non-deprived eyes had 114.3% more terminal dendrites (p=0.002) compared to controls. Among deprived eyes, all variables measured had a gradual rise in the first two weeks followed by decline with further deprivation. There were no statistically significant differences noted between the deprived and control eyes. Monocular deprivation results in increase in synaptic contacts in the non-deprived eye, with reciprocal changes occurring in the deprived eye.

La privación monocular de la visión resulta en cambios anatómicos en la corteza visual en favor del ojo no privado. Aunque la retina forma parte de la vía visual, hay escasez de datos sobre el efecto de la privación monocular en su estructura. El objetivo de esta investigación fue describir los efectos de la privación monocular en las características de las dendritas de las células ganglionares de la retina. Se diseñó un estudio cuasi-experimental. Se examinaron 30 conejos (18 experimentales, 12 controles). La privación monocular se logró a través de la sutura unilateral del párpado en los animales de experimentación. Los conejos fueron observados durante tres semanas. Cada semana, 6 animales experimentales y 3 control fueron eutanasiados, donde se obtuvo la retina y fue procesada para realizar microscopía óptica. Las microfotografías de la retina fueron tomadas con una cámara digital y luego se utilizó el software FIJI para su análisis. El número de dendritas primarias, terminales y el área del campo de dendritas en los ojos no privados aumentó un 66,7% (p=0,385), 400% (p=0,002), y 88,4% (p=0,523), respectivamente. Los ojos no privados, tenían 114,3% más dendritas terminales (p=0,002) en comparación con los controles. Entre los ojos privados, todas las variables medidas tuvieron un aumento gradual en las dos primeras semanas, seguido de descenso con mayor privación. No se observaron diferencias estadísticamente significativas entre los ojos privados y el grupo control. En conclusion, la privación monocular produce un aumento de los contactos sinápticos en los ojos no privados, con cambios recíprocos que se manifiestan en los ojos privados de la visión.

Características morfológicas de la retina del calderón común (Globicephala melas; traill, 1809) y su relación con el hábitat / Morphological characteristics of pilot whales retina (Globicephala melas; traill, 1809) and their relationship to habitat

Existe una estrecha relación entre las características morfológicas de los sistemas sensoriales, su funcionamiento y el hábitat al que están adaptados los organismos. En este sentido, de todos los mamíferos marinos estudiados, los cetáceos son los que más profundamente han modificado su estructura y fisiología ocular por su estrecha adaptación a una vida exclusivamente acuática. Para aportar más datos a la literatura, el objetivo de este trabajo es describir morfológicamente la retina de la ballena piloto a través de técnicas de microscopia óptica, con el fin de relacionarla con su adaptación al medio acuático. Nuestros datos muestran que la retina de Globicephala melas se organiza de acuerdo al mismo plan básico de los vertebrados. Tiene un grosor medio alrededor de 330±23 µm en las zonas de alta densidad de células ganglionares y 175±2 µm en la zona periférica. La capa de los fotorreceptores se corresponde con el 45% del grosor de la retina total. Presenta largos segmentos externos. La capa más característica de cetáceos en general y de Globicephala melas en particular, es la capa de células ganglionares. Su grosor, de 77,76±37,26 siendo la más variable de toda la retina. Esta capa presenta baja densidad celular pero tamaños excepcionalmente grandes, de 10 a 75 µm (promedio de 33,5 µm), denominadas células ganglionares gigantes.

There is a close relationship between morphological features of sensory systems, their function and habitat to which these organisms are adapting. In this sense, of all marine mammals that have been studied, cetaceans are the ones that have profoundly changed structure and ocular physiology in their adaptation to an exclusively aquatic life. To add further data to the literature, the aim of this paper is to describe morphologically the retina of the pilot whale through optical microscopy and relate their adaptation to the aquatic environment. Our data show that the retina of the long-finned pilot whale is organized according to the same basic plan of vertebrates. It has an average thickness of about 330±23 microns in areas of high ganglion cell density of 175±2 microns in the peripheral zone. Photoreceptor layer corresponds to 45% of total thickness of the retina and has long outer segments. The most significant characteristic of cetaceans in general and long-finned pilot whale in particular, is the ganglion cell layer. Thickness of 77.76±37.26 being the most variable of the entire retina. This layer has a low density but exceptionally large cell size of 10 to 75 microns (average of 33.5 microns), known as giant ganglion cells.

Light-induced retinal injury enhanced neurotrophins secretion and neurotrophic effect of mesenchymal stem cells in vitro / Lesão de retina induzida por luz aumentou a secreção de neurotrofinas e o efeito neurotrófico das células primordiais mesenquimais in vitro

PURPOSE: To investigate neurotrophins expression and neurotrophic effect change in mesenchymal stem cells (MSCs) under different types of stimulation. METHODS: Rats were exposed in 10,000 lux white light to develop light-induced retinal injury. Supernatants of homogenized retina (SHR), either from normal or light-injured retina, were used to stimulate MSCs. Quantitative real time for polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were conducted for analysis the expression change in basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) in MSCs after stimulation. Conditioned medium from SHR-stimulated MSCs and control MSCs were collected for evaluation their effect on retinal explants. RESULTS: Supernatants of homogenized retina from light-injured rats significantly promoted neurotrophins secretion from MSCs (p<0.01). Conditioned medium from mesenchymal stem cells stimulated by light-injured SHR significantly reduced DNA fragmentation (p<0.01), up-regulated bcl-2 (p<0.01) and down-regulated bax (p<0.01) in retinal explants, displaying enhanced protective effect. CONCLUSIONS: Light-induced retinal injury is able to enhance neurotrophins secretion from mesenchymal stem cells and promote the neurotrophic effect of mesenchymal stem cells.

OBJETIVO: Investigar a expressão de neurotrofinas e mudança no efeito neurotrófico de células-tronco mesenquimais (MSCs) sob diferentes tipos de estimulação. MÉTODOS: Os ratos foram expostos em 10.000 lux de luz branca para desenvolver a lesão da retina induzida por luz. Os sobrenadantes de homogeneizado de retina (SHR) quer a partir de retina normal ou da lesada por luz, foram usados para estimular as células-tronco mesenquimais. O RT-PCR quantitativa e ELISA foram realizados para análise da alteração de expressão do fator básico de crescimento de fibroblastos (bFGF), do fator neurotrófico derivado do cérebro (BDNF) e do fator neurotrófico ciliar (CNTF) em MSCs após a estimulação. O meio condicionado de células-tronco mesenquimais estimuladas por SHR e controles foram coletadas para avaliação de seu efeito sobre os explantes de retina. RESULTADOS: SHR de retinas de rato lesadas por luz promoveram aumento significativo de secreção de neurotrofinas em MSCs (p<0,01). O meio condicionado de SHR lesado por luz reduziu significativamente a fragmentação do DNA de MSCs (p<0,01), elevação de Bcl-2 (p<0,01) e redução de bax (p<0,01) em explantes de retina, mostrando um aumento do efeito protetor. CONCLUSÕES: A lesão da retina induzidos pela luz é capaz de aumentar a secreção de neurotrofinas e promover o efeito neurotrófico de células-tronco mesenquimais.

Multifocal electroretinogram in normal emmetropic subjects: Correlation with optical coherence tomography.

Aim of the Study: To establish the normative database for multifocal electroretinogram (mfERG) parameters in a normal emmetropic population. To correlate the data so obtained with the central macular thickness obtained using the optical coherence tomography (OCT) scan. Materials and Methods: mfERG data were obtained from 222 eyes of 111 emmetropic subjects. The amplitude (nv/deg2) and implicit times (ms) of the first-order kernel mfERG responses (N1, P1, and N2 waves) were obtained and grouped into five rings (Ring 1: Central 2°, Ring 2: 2–5°, Ring 3: 5–10°, Ring 4: 10–15°, Ring 5: >15°). The central macular thickness (CMT) was obtained using the macular thickness scan protocol of the OCT. Results: The mfERG data obtained were used to create a normative database. The amplitudes of the mfERG waves were maximum in the fovea and progressively decreased with increasing eccentricity (P = 0.0001). The latencies of the P1 and N2 waves were longest in the central ring and progressively shortened with eccentricity (P = 0.0001). No statistically significant correlations were observed between central ring 1 parameters and the CMT. Conclusion: This study establishes normative database for mfERG parameters in an emmetropic population. No statistically significant correlation was noted between CMT and mfERG parameters.

Effect of Amiloride to Retinal Toxicity Induced by Tissue Plasminogen Activator

PURPOSE: The effects of amiloride on cellular toxicity caused by tissue plasminogen activator (tPA) in mouse primary retinal cells were investigated. METHODS: Primary retinal cell cultures were maintained using glial conditioned medium. Commercial tPA and L-arginine were added, and the level of cyclic guanosine monophosphate (cyclic-GMP) in the culture supernatant was assessed using an ELISA assay. We measured the cell viability of cultured retinal cells pretreated with three different concentrations of amiloride (1, 10, and 100 microm) in addition to commercial tPA or L-arginine treatment. RESULTS: After exposing the cultured mouse retinal cells to tPA plus L-arginine or L-arginine alone, cyclic-GMP concentrations were 61.9 +/- 5.1 pmole/mL and 63.1 +/- 6.1 pmole/mL, respectively. However, the control group had a significantly lower concentration of cyclic-GMP (37.2 +/- 3.4 pmole/mL, p < 0.01). The cyclic GMP-dissolved solution did not cause retinal cell death. In the control group and the group treated with 1 microm amiloride and tPA containing L-arginine, the cell viability was 43.7% and 44.5%, respectively. However, cell viability increased to 70.6% with 10 microm amiloride and 78.4% with 100 microm amiloride (p = 0.015). CONCLUSIONS: L-arginine increases intracellular cyclic-GMP and may give rise to retinal cells through this mechanism. In addition, amiloride in concentrations greater than 10 microm protects against L-arginine-induced retinal cell death.

The Effect of Various Factors on Variability of Retinal Nerve Fiber Layer Thickness Measurements Using Optical Coherence Tomography

PURPOSE: To evaluate the effects of various factors on the variability of retinal nerve fiber layer (RNFL) thickness measurements using the Stratus optical coherence tomography (OCT) in normal and glaucomatous eyes. METHODS: Four hundred seventy-four subjects (103 normal eyes and 371 glaucomatous eyes) were scanned to determine the RNFL thickness measurements using the Stratus OCT. Measurements were obtained twice during the same day. The standard deviation (SD) was used to compare the variability in RNFL thickness measurements of the normal subjects to that of the glaucomatous patients. Multivariate regression analysis was used to evaluate which covariates were independent predictors of SD in overall mean RNFL thickness. RESULTS: The mean SD of all RNFL thickness measurements was larger in the glaucoma group except in one sector. In the multivariate regression analysis, the average signal strength (SS) and the relative SS change (difference in SS between initial and repeat scans, divided by initial SS) were independent predictors of the SD in the RNFL thickness measurements (partial R2 = 0.018, 0.013; p = 0.016, 0.040, respectively). CONCLUSIONS: Glaucomatous eyes tend to be more variable than normal eyes in RNFL thickness measurement using the Straus OCT. The average SS and the relative SS changes appear to correlate with the variability in RNFL thickness measurement. Therefore, the results of the RNFL analysis should not be interpreted independently of these factors.

Intravenously Administered Anti-recoverin Antibody Alone Does Not Pass through the Blood-Retinal Barrier

PURPOSE: Cancer-associated retinopathy is a paraneoplastic retinal degeneration which may primarily result from auto-immune mediated apoptosis. It has been hypothesized that high titer of auto-antibodies are able to cross the blood-retinal barrier (BRB) and to enter retinal cells to activate apoptotic pathway which has been already well-established. However, it still remains to be elucidated whether auto-antibodies could cross BRB in the retina. Herein, we demonstrated that intravenously administrated anti-recoverin antibodies could not pass through BRB and not lead to retinal cell death. METHODS: Anti-recoverin antibody was intravenously injected to C57BL/6 mice, which were sacrificed 1 and 7 days to obtain eye. Vascular endothelial growth factor was intravitreally injected to induce BRB breakdown, which was confirmed by fluorescein angiography and western blotting for zonula occludens (ZO)-1, ZO-2 and occludin. To investigate the location of anti-recoverin antibody in the retina, immunofluorescein was performed. The retinal toxicity of intravenous anti-recoverin antibody was evaluated by histological examination and transferase-mediated dUTP nick-end labeling. Immunofluorescein staining for glial fibrillary acidic protein was done to address glial activation as well. RESULTS: Intravenously administrated anti-recoverin antibodies were exclusively distributed on retinal vessels which were co-localized with CD31, and led to neither increase of glial fibrillary acidic protein expression, as an indicator of retinal stress, nor apoptotic retinal cell death. Moreover, even in the condition of vascular endothelial growth factor-induced BRB breakdown, anti-recoverin antibodies could not migrate across BRB and still remained on retinal vessels without retinal cytotoxicity. CONCLUSIONS: Our results suggest that high titer of intravascular anti-recoverin antibodies could not penetrate into the retina by themselves, and BRB breakdown mediated by dysregulation of tight junction might not be sufficient to allow anti-recoverin antibodies to pass through BRB.

Functional assessment of magno, parvo and konio-cellular pathways; current state and future clinical applications

The information generated by cone photoreceptors in the retina is compressed and transferred to higher processing centers through three distinct types of ganglion cells known as magno, parvo and konio cells. These ganglion cells, which travel from the retina to the lateral geniculate nucleus [LGN] and then to the primary visual cortex, have different structural and functional characteristics, and are organized in distinct layers in the LGN and the primary visual cortex. Magno cells are large, have thick axons and usually collect input from many retinal cells. Parvo cells are smaller, with fine axons and less myelin than mango cells. Konio cells are diverse small cells with wide fields of input consisting of different cells types. The three cellular pathways also differ in function. Magno cells respond rapidly to changing stimuli, while parvo cells need time to respond. The distinct patterns of structure and function in these cells have provided an opportunity for clinical assessment of their function. Functional assessment of these cells is currently used in the field of ophthalmology where frequency-doubling technology perimetry selectively assesses the function of magno cells. Evidence has accrued that the three pathways show characteristic patterns of malfunctions in multiple sclerosis, schizophrenia, Parkinson's and Alzheimer's diseases, and several other disorders. The combination of behavioral assessment with other techniques, such as event related potentials and functional magnetic resonance imaging, seems to bear promising future clinical applications

Neurochemical phenotype and birthdating of specific cell populations in the chick retina

The chick embryo is one of the most traditional models in developing neuroscience and its visual system has been one of the most exhaustively studied. The retina has been used as a model for studying the development of the nervous system. Here, we describe the morphological features that characterize each stage of the retina development and studies of the neurogenesis period of some specific neurochemical subpopulations of retinal cells by using a combination of immunohistochemistry and autoradiography of tritiated-thymidine. It could be concluded that the proliferation period of dopaminergic, GABAergic, cholinoceptive and GABAceptive cells does not follow a common rule of the neurogenesis. In addition, some specific neurochemical cell groups can have a restrict proliferation period when compared to the total cell population.

O embrião de galinha é um dos mais tradicionais modelosde estudos da neurociência do desenvolvimento e seu sistema visual tem sido um dos mais exaustivamente estudado. Aretina tem sido utilizada como modelo para estudar o desenvolvimento do sistema nervoso. Aqui, nós descrevemos as características morfológicas que caracterizam cada estádio da retina em desenvolvimento e os estudos do período de neurogênese de algumas subpopulações de células neuroquímicamente específicas da retina usando uma combinação de imunohistoquímica e autoradiografia de timidina-tritiada. Conclui-se que o período de proliferação das células dopaminérgicas, GABAérgicas, colinoceptivas e GABAceptivas não segue uma regra comum. Além disso, alguns grupos celulares neuroquimicamente distintos podem ter um período de proliferaçãomais restrito quando comparado ao da população total destas células.

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.

Shank 2 expression coincides with neuronal differentiation in the developing retina

The retinal activity for vision requires a precise synaptic connectivity. Shank proteins at postsynaptic sites of excitatory synapses play roles in signal transmission into the postsynaptic neuron. However, the correlation of Shank 2 expression with neuronal differentiation in the developing retina remains to be elucidated regardless of previous evidences of Shank 2 expression in retina. Herein, we demonstrated that with progression of development, Shank 2 is initially detected in the inner plexiform layer at P2, and then intensively detected in inner plexiform layer, outer plexiform layer, and ganglion cell layer at P14, which was closely colocalized to the neurofilament expression. Shank 2 was, however, not colocalized with glial fibrillary acidic protein. Shank 2 expression was increased in the differentiated retinoblastoma cells, which was mediated by ERK 1/2 activation. Moreover, Shank 2 expression was colocalized with neurofilament at the dendritic region of cells. In conclusion, our data suggests that Shank 2 is expressed in the neurons of the developing retina and could play a critical role in the neuronal differentiation of the developing retina.

Retinal incorporation and differentiation of mesenchymal stem cells intravitreally injected in the injured retina of rats / Incorporação e diferenciação retiniana de células tronco mesenquimais intravítreas em ratos

PURPOSE: To evaluate the pattern of retinal integration and differentiation of mesenchymal stem cells (MSCs) injected into the vitreous cavity of rat eyes with retinal injury. METHODS: Adult rat retinas were submitted to laser damage followed by transplantation of DAPI-labeled BM-MSCs grafts. To assess the integration and differentiation of BM-MSCs in laser-injured retina, host retinas were evaluated 2.4 and 8 weeks after injury/transplantation. RESULTS: Our results demonstrated that the grafted cells survived in the retina for at least 8 weeks and almost all BM-MSCs migrated and incorporated into the neural retina, specifically in the outer nuclear layer (ONL), inner nuclear layer (INL) and ganglion cell layer (GCL) while a subset of grafted cells were found in the subretinal space posttransplantation. At 8 weeks immunohistochemical analysis with several retinal specific markers revealed that the majority of the grafted cells expressed rhodopsin, a rod photoreceptor marker, followed by parvalbumin, a marker for bipolar and amacrine cells. A few subsets of cells were able to express a glial marker, glial fibrillary acidic protein. However, grafted cells failed to express pan-cytokeratin, a retinal pigment epithelium marker. CONCLUSIONS: These results suggest the potential of BM-MSCs to differentiate into retinal neurons. Taken together, these findings might be clinically relevant for future mesenchymal stem cell therapy studies concerning retinal degeneration repair.

OBJETIVO: Avaliar o padrão de integração e diferenciação retiniana de células tronco mesenquimais (CTM) injetadas na cavidade vítrea de ratos portadores de lesões retinianas. MÉTODOS: Ratos Wistar adultos foram submetidos a múltiplas lesões retinianas utilizando-se YAG laser e injeção intravítrea de células tronco mesenquimais. A fim de se avaliar a integração e diferenciação retiniana, o tecido retiniano lesado pelo YAG laser / tratado pelas células tronco, foi avaliado 2, 4 e 8 semanas após a lesão. RESULTADOS: As células injetadas na cavidade vítrea sobreviveram na retina por pelo menos 8 semanas e quase todas células tronco mesenquimais migraram e incorporaram-se na retina neural, especificamente nas camadas nucleares externa e interna e camada de células ganglionares. Uma pequena quantidade de células foi encontrada no espaço sub-retiniano. A análise imuno-histoquímica de 8 semanas mostrou que a maioria das células injetadas expressou rodopsina (marcador para fotorreceptores), parvalbumina (marcador para células bipolares e amácrinas), GFAP (marcador de células gliais). As células injetadas não expressaram a pancitoqueratina, que é a marcadora de células do epitélio pigmentar da retina. CONCLUSÕES: Ocorre aparente diferenciação e incorporação de células tronco mesenquimais na retina de ratos após injeção intravitrea destas células.

Immunohistochemical characterization of the chick marginal retina

The retina is a highly differentiated tissue with a complex layered structure that has been extensively characterized. However, most of the previous studies focused on the histology of the central retina while little is known about the cellular composition, organization and function of the marginal retina. Recent research has identified a subpopulation of multipotential progenitor cells in the marginal regions of the retina, closest to the ciliary body ("ciliary marginal zone"). These cells are capable of differentiation in response to an appropriate stimulus. Thus, it is possible that the structure and composition of the marginal retina are distinct from those of the central retina to accommodate the potential addition of newly formed neurons. To characterize the cellular profile of the chick marginal retina, we labeled it immunohistochemically for markers whose staining pattern is well established in the central retina: calbindin, calretinin, protein kinase C, and choline acetyltransferase. Calbindin was present at very low levels in the marginal retina putative photoreceptor layer. Calretinin-positive horizontal cells were also sparse close to the ciliary marginal zone. The bipolar cells in the marginal outer plexiform layer were positive for anti-protein kinase C antibodies, but the density of labeling was also decreased in relation to the central retina. In contrast, the marginal starburst cholinergic amacrine cell pattern was very similar to the central retina. From these data we conclude that the structure of the marginal retina is significantly different from that of the central retina. In particular, the expression of late retina markers in the marginal retina decreased in comparison to the central retina.

Stem cells: potential source for retinal repair and regeneration

Stem cells have been studied in several fields of Medicine, and their applications are not too far from the clinical practice. Retinal impairment by neuronal death has been considered incurable due to the limited regenerative capacity of the central nervous system. The capacity of stem cells to regenerate tissues, as well as their plasticity makes them a potential source for retinal repair. The stem cells are a great promise for the therapy of inherited retinal disorders and retinal-neuronal degenerative diseases, such as retinitis pigmentosa and allied retinal dystrophies, which can result in blindness. Because of the accessibility, expansibility, and multipotentiality mesenchymal stem cells are expected to be useful for clinical applications, especially in regenerative medicine and tissue engineering. Mesenchymal stem cells are clonogenic, nonhematopoietic stem cells present in the bone marrow. Given the appropriate microenvironment, they could differentiate into cardiomyocytes or even into cells of nonmesodermal derivation including hepatocytes and neurons. So far, the results of a few studies are consistent with the belief that cell-based therapies using mesenchymal stem cells may be effective when it comes to retinal damaged tissue repair.

Células-tronco têm sido estudadas em várias áreas da Medicina e suas aplicações brevemente deverão estar incorporadas à prática clínica. O dano retiniano pela morte neuronal é considerado incurável devido a pobre capacidade regenerativa do sistema nervoso central. A capacidade das células-tronco em regenerar tecidos, assim como sua plasticidade, faz que estas sejam uma fonte potencial de células para a regeneração retiniana. Células-tronco são muito promissoras para o tratamento das distrofias retinianas, como a retinose pigmentar e outras doenças neurodegenerativas, que podem evoluir para cegueira. As células-tronco mesenquimais são o tipo mais provável de células-tronco a serem utilizadas na prática clínica devido a sua fácil acessibilidade e multipotencialidade de diferenciação em vários tecidos. As células-tronco mesenquimais são células clonogênicas, não-hematopoiéticas, localizadas na medula óssea. Desde que seja proporcionado um microambiente apropriado, estas células podem se diferenciar em cardiomiócitos e até mesmo em células de origem não-mesodérmica, como hepatócitos e neurônios. Até o presente momento, os resultados dos estudos iniciais são animadores em relação ao uso de células-tronco mesenquimais e uso eficaz destas no reparo de tecidos retinianos lesados.

In vitro culture of bone marrow mesenchymal stem cells in rats and differentiation into retinal neural-like cells / 华中科技大学学报（医学）（英德文版）

In order to study the in vitro culture and expansion of bone marrow mesenchymal stem cells in rats (rMSCs) and the possibility of rMSCs differentiation into retinal neural cells, the bone marrow-derived cells in SD rats were isolated and cultured in vitro. The retinal neural cells in SD rats were cultured and the supernatants were collected to prepare conditioned medium. The cultured rMSCs were induced to differentiate by two steps. Immunofluorescence method and anti-nestin, anti-NeuN, anti-GFAP and anti-Thy1.1 antibodies were used to identify the cells derived from the rMSCs. The results showed that the in vitro cultured rMSCs grew well and expanded quickly. After induction with two conditioned media, rMSCs was induced to differentiate into neural progenitor cells, then into retinal neural-like cells which were positive for nestin, NeuN, GFAP and Thy1.1 detected by fluorescence method. The findings suggested that rMSCs could be culture and expanded in vitro, and induced to differentiate into retinal neural-like cells.