RESUMEN
Neurons are continuously produced at different rates and locations in the teleost retina. Goldfish rods are homogeneously distributed and maintain a stable density throughout growth, whereas little is known about their postsynaptic partners. We examined the distribution and density of mixed-input ON bipolar cells (ON mBCs) in 57 goldfish of various sizes by immunolabeling their retinas with an antibody against PKCα and counting PKCα-positive neurons in wholemounts. Cell densities were correlated with morphometric data for the same animals, and the spatial resolution of the ON mBC mosaic was calculated in each case. The distribution of ON mBCs is homogeneous throughout growth. For a 10-fold change in body size (i.e., from 20 to 200 mm), the total number of ON mBCs increases 2.8 times, while retinal area expands around 10 times. As a consequence, the density of ON mBCs in large fish falls to â¼1/3 of that of small animals, and intercellular spacing doubles. The eye and the lens become around three times larger from small to large fish. This causes the retinal magnification factor (and thereby the image projected onto retina) to augment by the same amount. Because the retinal magnification factor rises more than the intercellular spacing in the same animals, the spatial resolution of the ON mBC mosaic improves from 0.8 to 1.4 cycles/degree as the body size increases from 20 to 200 mm. As ON mBCs are mostly rod-driven, our results suggest that the scotopic acuity of the goldfish may improve as the animal grows.
Asunto(s)
Carpa Dorada/anatomía & histología , Carpa Dorada/crecimiento & desarrollo , Neuronas Retinianas/citología , Animales , Tamaño Corporal , Recuento de Células , Proteínas de Peces/metabolismo , Carpa Dorada/metabolismo , Inmunohistoquímica , Tamaño de los Órganos , Proteína Quinasa C-alfa/metabolismo , Neuronas Retinianas/metabolismoRESUMEN
Most systematic studies of the avian visual system have focused on Neognathous species, leaving virtually unexplored the Palaeognathae, comprised of the flightless ratites and the South American tinamous. We investigated the visual field, the retinal topography, and the pattern of retinal and centrifugal projections in the Chilean tinamou, a small Palaeognath of the family Tinamidae. The tinamou has a panoramic visual field with a small frontal binocular overlap of 20°. The retina possesses three distinct topographic specializations: a horizontal visual streak, a dorsotemporal area, and an area centralis with a shallow fovea. The maximum ganglion cell density is 61,900/ mm(2) , comparable to Falconiformes. This would provide a maximal visual acuity of 14.0 cycles/degree, in spite of relatively small eyes. The central retinal projections generally conform to the characteristic arrangement observed in Neognathae, with well-differentiated contralateral targets and very few ipsilateral fibers. The centrifugal visual system is composed of a considerable number of multipolar centrifugal neurons, resembling the "ectopic" neurons described in Neognathae. They form a diffuse nuclear structure, which may correspond to the ancestral condition shared with other sauropsids. A notable feature is the presence of terminals in deep tectal layers 11-13. These fibers may represent either a novel retinotectal pathway or collateral branches from centrifugal neurons projecting to the retina. Both types of connections have been described in chicken embryos. Our results widen the basis for comparative studies of the vertebrate visual system, stressing the conserved character of the visual projections' pattern within the avian clade.
Asunto(s)
Aves/anatomía & histología , Aves/fisiología , Retina/anatomía & histología , Retina/fisiología , Campos Visuales/fisiología , Animales , Encéfalo/anatomía & histología , Recuento de Células , Técnicas de Trazados de Vías Neuroanatómicas , Neuronas Retinianas/citología , Neuronas Retinianas/fisiología , Vías Visuales/anatomía & histologíaRESUMEN
BACKGROUND: Acute pancreatitis is an inflammatory disease of the pancreas due to enzymatic autodigestion which can cause necrosis or multiple organ failure; its pathophysiology is not fully known yet. AIM: To evaluate the correlation between clinical and therapeutic data in patients with mild acute pancreatitis. METHODS: A retrospective study in 55 medical records of patients admitted with acute mild pancreatitis was realized to analyze the association between age, leukocytosis, serum glutamic-oxaloacetic transaminase and lactate dehydrogenase, glucose, antibiotics, time admission and Ranson´s scores. RESULTS: There was a positive association between less intensive care (strict hydration, analgesia and monitoring of vital signs), early antibiotic therapy (monotherapy), early return to diet after 48 hours and laboratory control of the serum amylase and lipase (high in the first week and decreasing after 10 days, without any prognostic value). CONCLUSIONS: Changes in the management of patients with mild acute pancreatitis, such as enteral nutrition, rational use of lower spectrum antibiotics and intensive care, have contributed significantly to the reduction of hospitalization time and mortality. .
RACIONAL: Pancreatite aguda consiste de doença inflamatória do pâncreas por autodigestão enzimática que pode ocasionar necrose ou mesmo falência múltipla de órgãos e de fisiopatologia ainda não totalmente conhecida. OBJETIVO: Avaliar as correlações existentes entre dados clínicos e terapêuticos em pacientes com pancreatite aguda leve. MÉTODOS: Foi realizado estudo retrospectivo em 55 prontuários de pacientes internados por pancreatite aguda leve para análise de associação entre idade, leucocitose, dosagem sérica de transaminase glutâmico-oxalacética e de desidrogenase lática, glicemia, antibioticoterapia, tempo de internação e escores de Ranson. RESULTADOS: Houve associação positiva entre cuidados intensivos menores (hidratação rigorosa, analgesia e monitorização de sinais vitais), antibioticoterapia precoce (monoterapia), retorno precoce da dieta após 48 horas e controle laboratorial dos níveis séricos de amilase e lipase (elevados na primeira semana e decrescentes após 10 dias, porém sem valor prognóstico). CONCLUSÕES: Mudanças no manejo de pacientes com pancreatite aguda leve, tais como nutrição enteral, uso racional de antibióticos de menor espectro e cuidados intensivos têm contribuído significativamente para a redução do tempo de internação e mortalidade. .
Asunto(s)
Animales , Masculino , Ratas , /antagonistas & inhibidores , /metabolismo , /metabolismo , N-Metilaspartato/farmacología , Péptidos/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Neuronas Retinianas/fisiología , Muerte Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Necrosis , Fenantrenos/farmacología , Ratas Sprague-Dawley , Neuronas Retinianas/citología , Neuronas Retinianas/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Polylaminin (polyLM) is a flat biomimetic polymer of laminin capable of promoting axonal growth both in vitro and in vivo. It is assembled in a cell-free system when laminin 111 is incubated in acidic pH, whereas incubation in neutral buffer leads to the formation of bulky and irregular polymers (LM). In the present work, we compared the behaviors of cells isolated from the P1 rat retina on polyLM and LM. PolyLM induced cellular spreading and the outgrowth of neurites in contact with the substrate, whereas LM led to the formation of large clusters of cells, with neurites growing only inward. After 24 hr in culture, the number of cells on polyLM increased threefold, and this increase was inhibited by 60% in the presence of the PKA inhibitor H89 and by 41% in the presence of the PKC inhibitor chelerythrine chloride, whereas both inhibitors abolished neuritogenesis. Neither the cell number nor the outgrowth of neurites was affected by the ERK1/2 inhibitor PD98059 on polyLM. On the other hand, PD98059 was able to reduce the cell number on LM, whereas the other inhibitors were not. Immunostaining of P1 retina with an antilaminin antibody revealed that the protein was expressed not only at its inner surface but also within the neuroblast layer in close contact with individual cells. Our results indicate that, when provided in its active polymerized form, laminin can influence both neuritogenesis and proliferation of retinal cells.
Asunto(s)
Laminina/metabolismo , Neuritas/metabolismo , Polímeros/metabolismo , Neuronas Retinianas/metabolismo , Animales , Recuento de Células , Células Cultivadas , Ratas , Neuronas Retinianas/citologíaRESUMEN
In this study, we describe a simple and reliable method to study neuroprotective effects in living and organized neural tissue. This method, which was based on retinal explants for in vivo focal lesions, was conceived as a collection of modular procedures, which can be customized for particular demands. With this model, it is possible to combine immunohistochemistry with image data analysis to track the two- or three-dimensional redistribution of proteins as a time/space function of primary cell loss. At the same time, it is possible to finely control the exposure of the tissue to specific drugs and molecules. In order to illustrate the use of the proposed method, we tested the effects of two different nanotube compounds on retinal explant viability. Transcriptome analyses can be separately performed in the lesion focus and penumbra with laser capture microdissection followed by polymerase chain reaction analyses. In addition, other common experimental drawbacks, such as high individual variance, are eliminated. With intraocular injections, treatments can be verified in vivo, with one eye serving as the experimental tissue and the other serving as the control tissue. In summary, we describe a flexible and easy method, which can be useful in combination with a broad variety of recently developed neuroprotective strategies, to study neurodegeneration.
Asunto(s)
Proteínas del Ojo/genética , Fármacos Neuroprotectores/farmacología , Retina/citología , Neuronas Retinianas/citología , Técnicas de Cultivo de Tejidos , Animales , Aptámeros de Nucleótidos/farmacología , Aptámeros de Péptidos/farmacología , Pollos , Proteínas del Ojo/antagonistas & inhibidores , Proteínas del Ojo/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Inyecciones Intraoculares , Masculino , Imagen Molecular , Nanotubos , ARN Interferente Pequeño/genética , Ratas , Retina/efectos de los fármacos , Retina/lesiones , Retina/metabolismo , Neuronas Retinianas/efectos de los fármacos , Neuronas Retinianas/metabolismo , Análisis de la Célula IndividualRESUMEN
Analyzing cell morphology is crucial in the fields of cell biology and neuroscience. One of the main methods for evaluating cell morphology is by using intracellular fluorescent markers, including various commercially available dyes and genetically encoded fluorescent proteins. These markers can be used as free radical sources in photooxidation reactions, which in the presence of diaminobenzidine (DAB) forms an opaque and electron-dense precipitate that remains localized within the cellular and organelle membranes. This method confers many methodological advantages for the investigator, including absence of photo-bleaching, high visual contrast and the possibility of correlating optical imaging with electron microscopy. However, current photooxidation techniques require the continuous use of fluorescent or confocal microscopes, which wastes valuable mercury lamp lifetime and limits the conversion process to a few cells at a time. We developed a low cost optical apparatus for performing photooxidation reactions and propose a new procedure that solves these methodological restrictions. Our "photooxidizer" consists of a high power light emitting diode (LED) associated with a custom aluminum and acrylic case and a microchip-controlled current source. We demonstrate the efficacy of our method by converting intracellular DiI in samples of developing rat neocortex and post-mortem human retina. DiI crystals were inserted in the tissue and allowed to diffuse for 20 days. The samples were then processed with the new photooxidation technique and analyzed under optical microscopy. The results show that our protocols can unveil the fine morphology of neurons in detail. Cellular structures such as axons, dendrites and spine-like appendages were well defined. In addition to its low cost, simplicity and reliability, our method precludes the use of microscope lamps for photooxidation and allows the processing of many labeled cells simultaneously in relatively large tissue samples with high efficacy.
Asunto(s)
Fluorescencia , Luz , Iluminación/métodos , Oxidación-Reducción , Animales , Biomarcadores , Humanos , Iluminación/instrumentación , Microscopía Fluorescente , Ratas , Neuronas Retinianas/citología , Neuronas Retinianas/metabolismo , Coloración y Etiquetado/métodosRESUMEN
Recently, many efforts have been made to develop N-methyl-D-aspartic acid receptor antagonists for treating different pathological conditions such as thrombo-embolic stroke, traumatic head injury, Huntington's, Parkinson's, and Alzheimer's diseases). However, as side-effects limit the use of most antagonists, new drugs are still required. In this work, we performed a (quantitative) structure-activity relationship analysis of 17 phenyl-amidine derivatives (1a-1q), reported as N-methyl-D-aspartic acid receptor antagonists, and used this data to rationally design the triazolyl-amidines. The best (quantitative) structure-activity relationship model constructed by multiple linear regression analysis presented high data fitting (R = 0.914) was able to explain 83.6% of the biological data variance (R(2) = 0.836), presented a satisfactory internal predictive ability (Q(2) = 0.609) and contained the descriptors (E(HOMO), Ovality and cLogP). Our assays confirmed that glutamate promotes an extensive cell death in avian neurons (77%) and 2a and 2b protected the neurons from the glutamate effect (from 77% to 27% and 45%, respectively). The results of neurotoxicity and cytotoxicity on Vero cells suggested the favorable profile of 2a and 2b. Also, the molecular modeling used to predict the activity, the interaction with the receptor and the pharmacokinetic and toxicity of the triazolyl-amidines pointed them as a promising class for further exploration as N-methyl-D-aspartic acid receptor antagonists.
Asunto(s)
Amidinas/química , Fármacos Neuroprotectores/química , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Triazoles/química , Amidinas/farmacología , Animales , Muerte Celular , Chlorocebus aethiops , Ácido Glutámico/toxicidad , Simulación del Acoplamiento Molecular , Fármacos Neuroprotectores/farmacología , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Neuronas Retinianas/citología , Neuronas Retinianas/efectos de los fármacos , Relación Estructura-Actividad , Triazoles/farmacología , Células VeroRESUMEN
The tyrant flycatchers represent a monophyletic radiation of predominantly insectivorous passerine birds that exhibit a plethora of stereotyped prey capture techniques. However, little is known about their retinal organization. Using retinal wholemounts, we estimated the total number and topography of neurons in the ganglion cell layer in the generalist yellow-bellied elaenia (Elaenia flavogaster) and the up-hover-gleaner mouse-colored tyrannulet (Phaeomyias murina) with the optical fractionator method. The mean estimated total number of neurons in the ganglion cell layer was 4,152,416 +/- 189,310 in E. flavogaster and 2,965,132 +/- 354,359 in P. murina. Topographic maps of isocounting lines revealed a similar distribution for both species: a central fovea and a temporal area surrounded by a poorly defined horizontal streak. In addition, both species had increased numbers of giant ganglion cells in the dorsotemporal retina forming an area giganto cellularis. In E. flavogaster, these giant ganglion cells were also distributed across the nasal and ventral retinal peripheries, which is in agreement with the generalist habits of this species. However, in P. murina these cells were rarely seen along the nasal and ventral peripheries, possibly reflecting a lesser need to perceive movement because this species captures stationary insects resting on foliage. Thus, we suggest that the retinas of the tyrant flycatchers in the present study show a general common pattern of neuron distribution in the ganglion cell layer irrespective of their foraging habits. We also suggest that the distribution of giant ganglion cells is indicative of the visual requirements of the species.
Asunto(s)
Conducta Apetitiva , Células Ganglionares de la Retina/citología , Neuronas Retinianas/citología , Pájaros Cantores/anatomía & histología , Animales , Recuento de Células , Conducta Exploratoria , FotomicrografíaRESUMEN
Oxidative damage is involved in triggering neuronal death in several retinal neurodegenerative diseases. The recent finding of stem cells in the retina suggests that both preventing neuronal death and replacing lost neurons might be useful strategies for treating these diseases. We have previously shown that oxidative stress induces apoptosis in cultured retinal neurons. We now investigated the response of Müller cells, proposed as retina stem cells, to this damage. Treatment of glial cell cultures prepared from rat retinas with the oxidant paraquat (PQ) did not induce glial cell apoptosis. Instead, PQ promoted their rapid dedifferentiation and proliferation. PQ decreased expression of a marker of differentiated glial cells, simultaneously increasing the expression of smooth muscle actin, shown to increase with glial dedifferentiation, the levels of cell-cycle markers, and the number of glial cells in the cultures. In addition, glial cells protected neurons in coculture from apoptosis induced by PQ and H(2)O(2). In pure neuronal cultures, PQ induced apoptosis of photoreceptors and amacrine neurons, simultaneously decreasing the percentage of neurons preserving mitochondrial membrane potential; coculturing neurons with glial cells completely prevented PQ-induced apoptosis and preserved mitochondrial potential in both neuronal types. These results demonstrate that oxidative damage activated different responses in Müller glial cells; they rapidly dedifferentiated and enhanced their proliferation, concurrently preventing neuronal apoptosis. Glial cells might not only preserve neuronal survival but also activate their cell cycle in order to provide a pool of new progenitor cells that might eventually be manipulated to preserve retinal functionality.