RESUMEN
BACKGROUND: Mutations in CACNA1F, which encodes the Ca(v)1.4 subunit of a voltage-gated L-type calcium channel, cause X-linked incomplete congenital stationary night blindness (CSNB2), a condition of defective retinal neurotransmission which results in night blindness, reduced visual acuity, and diminished ERG b-wave. We have characterized two putative murine CSNB2 models: an engineered null-mutant, with a stop codon (G305X); and a spontaneous mutant with an ETn insertion in intron 2 of Cacna1f (nob2). METHODS: Cacna1f ( G305X ): Adults were characterized by visual function (photopic optokinetic response, OKR); gene expression (microarray) and by cell death (TUNEL) and synaptic development (TEM). Cacna1f ( nob2 ): Adults were characterized by properties of Cacna1f mRNA (cloning and sequencing) and expressed protein (immunoblotting, electrophysiology, filamin [cytoskeletal protein] binding), and OKR. RESULTS: The null mutation in Cacna1f ( G305X ) mice caused loss of cone cell ribbons, failure of OPL synaptogenesis, ERG b-wave and absence of OKR. In Cacna1f ( nob2 ) mice alternative ETn splicing produced ~90% Cacna1f mRNA having a stop codon, but ~10% mRNA encoding a complete polypeptide. Cacna1f ( nob2 ) mice had normal OKR, and alternatively-spliced complete protein had WT channel properties, but alternative ETn splicing abolished N-terminal protein binding to filamin. CONCLUSIONS: Ca(v)1.4 plays a key role in photoreceptor synaptogenesis and synaptic function in mouse retina. Cacna1f ( G305X ) is a true knockout model for human CSNB2, with prominent defects in cone and rod function. Cacna1f ( nob2 ) is an incomplete knockout model for CSNB2, because alternative splicing in an ETn element leads to some full-length Ca(v)1.4 protein, and some cones surviving to drive photopic visual responses.
Asunto(s)
Canales de Calcio/genética , Miopía/genética , Ceguera Nocturna/genética , Sustitución de Aminoácidos/genética , Animales , Canales de Calcio Tipo L , Visión de Colores/fisiología , Sensibilidad de Contraste/fisiología , Modelos Animales de Enfermedad , Electrorretinografía , Enfermedades Hereditarias del Ojo , Enfermedades Genéticas Ligadas al Cromosoma X , Humanos , Ratones , Mutación/genética , Miopía/fisiopatología , Ceguera Nocturna/fisiopatologíaRESUMEN
Congenital stationary night blindness 2A (CSNB2A) is an X-linked retinal disorder, characterized by phenotypically variable signs and symptoms of impaired vision. CSNB2A is due to mutations in CACNA1F, which codes for the pore-forming α1F subunit of a L-type voltage-gated calcium channel, Cav1.4. Mouse models of CSNB2A, used for characterizing the effects of various Cacna1f mutations, have revealed greater severity of defects than in human CSNB2A. Specifically, Cacna1f-knockout mice show an apparent lack of visual function, gradual retinal degeneration, and disruption of photoreceptor synaptic terminals. Several reports have also noted cone-specific disruptions, including axonal abnormalities, dystrophy, and cell death. We have explored further the involvement of cones in our 'G305X' mouse model of CSNB2A, which has a premature truncation, loss-of-function mutation in Cacna1f. We show that the expression of genes for several phototransduction-related cone markers is down-regulated, while that of several cellular stress- and damage-related markers is up-regulated; and that cone photoreceptor structure and photopic visual function - measured by immunohistochemistry, optokinetic response and electroretinography - deteriorate progressively with age. We also find that dystrophic cone axons establish synapse-like contacts with rod bipolar cell dendrites, which they normally do not contact in wild-type retinas - ectopically, among rod cell bodies in the outer nuclear layer. These data support a role for Cav1.4 in cone synaptic development, cell viability, and synaptic transmission of cone-dependent visual signals. Although our novel finding of cone-to-rod-bipolar cell contacts in this mouse model of a retinal channelopathy may challenge current views of the role of Cav1.4 in photopic vision, it also suggests a potential new target for restorative therapy.
Asunto(s)
Canales de Calcio/metabolismo , Distrofia del Cono/metabolismo , Modelos Animales de Enfermedad , Enfermedades Hereditarias del Ojo/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/metabolismo , Miopía/metabolismo , Ceguera Nocturna/metabolismo , Sinapsis/metabolismo , Animales , Canales de Calcio/deficiencia , Canales de Calcio/genética , Canales de Calcio Tipo L , Enfermedades Hereditarias del Ojo/patología , Femenino , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Ratones , Ratones Noqueados , Mutación , Miopía/patología , Ceguera Nocturna/patologíaRESUMEN
We quantified rod- and cone-related electroretinogram (ERG) responses following subretinal injections of the human-derived retinal pigment epithelial (hRPE) cell line ARPE-19 at age P23 to prevent progressive photoreceptor loss in the Royal College of Surgeons (RCS) rat. Culture medium-injected eyes served as sham controls. At P60, in comparison with sham-injected eyes, all recordings from hRPE-injected eyes showed preserved scotopic a- and b-waves, oscillatory potentials, double-flash-derived rod b-waves and photopic cone b-waves, and flicker critical fusion frequencies and amplitudes. Although the actual preservation did not exceed 10% of a-wave and 20% of b-wave amplitude values in non-dystrophic RCS and deteriorated rapidly by P90, rod- and cone-related ERG parameters were still recordable up to P120 unlike the virtually unresponsive sham-injected eyes.
Asunto(s)
Células Fotorreceptoras de Vertebrados/fisiología , Epitelio Pigmentado Ocular/trasplante , Animales , Línea Celular , Electrorretinografía , Humanos , Inyecciones , Degeneración Macular/terapia , Modelos Animales , Ratas , Ratas Mutantes , Células Fotorreceptoras Retinianas Conos/fisiopatología , Células Fotorreceptoras Retinianas Bastones/fisiopatología , Retinitis Pigmentosa/terapia , Factores de TiempoRESUMEN
In normal rodents there is a precise topology of the retinocollicular projection, the nasotemporal and ventrodorsal axes of the retina being respectively projected onto the caudorostral and mediolateral axes of the contralateral superior colliculus (SC). We evaluated the distribution of regenerated retinal ganglion cell (RGC) axon terminals in the SC of adult hamsters in which an unbranched peripheral nerve graft was directed from the retina to the contralateral SC. Responses to visual stimulation of individual RGCs were recorded from terminal arbors of their regenerated axons in the reinnervated SC. Retinal positions of these RGCs were inferred from the locations of their visual receptive fields. At some sites in the reinnervated SC, axon terminal arbors converged from widely separated RGCs. Conversely, axon terminal arbors at widely separated sites in the SC could emanate from contiguous RGCs. To assess whether any tendency for order was superimposed on the apparent disorganization of the regenerated projection, we evaluated the relative positions of pairs of RGC terminals in the SC in relation to the relative retinal locations of the corresponding pairs of RGCs. Among the 983 pairs of RGCs able to be evaluated from nine animals studied 30-60 weeks after grafting, there was a statistically significant 3/2 tendency for the more nasally situated of two RGCs to project its terminal more caudally in the SC than that of the more temporally situated RGC. A similar tendency toward appropriate organization was not found with respect to the ventrodorsal axis of the retina and the mediolateral axis of the SC.
Asunto(s)
Axones/fisiología , Regeneración Nerviosa/fisiología , Nervios Periféricos/trasplante , Células Ganglionares de la Retina/fisiología , Colículos Superiores/fisiología , Animales , Cricetinae , Femenino , Supervivencia de Injerto/fisiología , Mesocricetus , Traumatismos del Nervio Óptico/cirugía , Estimulación Luminosa , Terminales Presinápticos/fisiología , Terminales Presinápticos/ultraestructura , Recuperación de la Función , Células Ganglionares de la Retina/citología , Colículos Superiores/citología , Trasplante Autólogo , Campos Visuales/fisiologíaRESUMEN
The pattern of axonal regeneration, specificity of reinnervation, and terminal arborization in the brainstem by axotomized retinal ganglion cell axons was studied in rats with peripheral nerve grafts linking the retina with ipsilateral regions of the brainstem, including dorsal and lateral aspects of the diencephalon and lateral aspect of the superior colliculus. Four to 13 months later, regenerated retinal projections were traced using intraocular injection of cholera toxin B subunit. In approximately one-third of the animals, regenerated retinal axons extended into the brainstem for distances of up to 6 mm. Although axons followed different patterns of ingrowth depending on their site of entry to the brainstem, within the pretectum, they innervated preferentially the nucleus of the optic tract and the olivary pretectal nucleus in which they formed two types of terminal arbors. Within the superior colliculus, axons extended laterally and formed a different terminal arbor type within the stratum griseum superficiale. In the remaining two-thirds of the animals, retinal fibers formed a neuroma-like structure at the site of entry into the brainstem, or a few fibers extended for very short distances within the neighboring neuropil. These experiments suggest that regenerated retinal axons are capable of a highly selective reinnervation pattern within adult denervated retinorecipient nuclei in which they form well defined terminal arbors that may persist for long periods of time. In addition, these studies provide the anatomical correlate for our previous functional study on the re-establishment of the pupillary light reflex in this experimental paradigm.
Asunto(s)
Regeneración Nerviosa/fisiología , Núcleo Olivar/citología , Nervio Peroneo/trasplante , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/fisiología , Factores de Edad , Animales , Axones/fisiología , Neoplasias del Tronco Encefálico , Toxina del Cólera , Femenino , Neuroma , Neuronas/fisiología , Neuronas/trasplante , Neuronas/ultraestructura , Núcleo Olivar/patología , Nervio Peroneo/patología , Ratas , Ratas Sprague-Dawley , Colículos Superiores/citología , Vías Visuales/patologíaRESUMEN
It has been shown that photoreceptor degeneration can be limited in experimental animals by transplantation of fresh RPE to the subretinal space. There is also evidence that retinal cell transplants can be used to reconstruct retinal circuitry in dystrophic animals. Here we describe and review recent developments that highlight the necessary steps that should be taken prior to embarking on clinical trials in humans.
Asunto(s)
Trasplante de Células/métodos , Epitelio Pigmentado Ocular/citología , Retina/citología , Degeneración Retiniana/cirugía , Animales , Supervivencia Celular , HumanosRESUMEN
The heterozygous P23H transgenic rat is a model of autosomal dominant retinitis pigmentosa, in which a mutation in the rhodopsin gene leads to a rapid loss of rods and a more protracted loss of cones. It has been suggested that rods play an essential role in preserving cones. We tested this hypothesis by examining whether higher levels of dysfunctional rhodopsin in rats homozygous for the P23H mutation would result in exacerbated cone dysfunction when compared with heterozygous P23H rats. Electroretinogram (ERG) responses were recorded from P21 to P250 in Sprague-Dawley (SD) and homozygous P23H rats. Both scotopic and photopic intensity response ERGs were severely depressed already at P21 when compared with age-matched SD rats. Furthermore, flicker amplitudes and critical fusion frequencies were also lower in P23H compared with SD rats at P21. Scotopic and photopic intensity responses as well as flicker amplitude and critical fusion frequencies declined rapidly up to P60, reaching a steady state that was maintained up to P200. We conclude that in rats homozygous for P23H rhodopsin mutations, the severe loss of rod function already seen by P21 is accompanied by substantial cone functional loss at that age. While rod-related responses are more severely affected than cone-related responses at all ages, their actual rate of decline with age is surprisingly similar. Both undergo a biphasic temporal pattern of decline: very rapid (P21-P60) followed by very slow (P60-P200) deterioration in response parameters, implying a tight link between rod and cone functional deterioration.
Asunto(s)
Mutación/fisiología , Células Fotorreceptoras Retinianas Conos/fisiología , Rodopsina/genética , Envejecimiento/fisiología , Animales , Animales Modificados Genéticamente , Electrorretinografía , Fusión de Flicker , Homocigoto , Masculino , Ratas , Ratas Sprague-Dawley , Retina/crecimiento & desarrollo , Degeneración Retiniana/fisiopatología , Células Fotorreceptoras Retinianas Bastones/fisiología , Rodopsina/fisiologíaRESUMEN
Subretinal injections of human retinal pigment epithelial (RPE) cells early in the course of retinal degeneration in Royal College of Surgeons (RCS) rats can rescue photoreceptors. Fourteen injected animals were studied using a double flash electroretinogram (ERG): 10 were examined longitudinally and four terminally with immunohistochemistry. The proportion of cone contribution to the ERG b-wave rather than the absolute size of isolated cone response proved to be a reliable indicator of function over time and a predictor of the proportion of cones identified anatomically in the area of optimal photoreceptor rescue.
Asunto(s)
Electrorretinografía , Epitelio Pigmentado Ocular/fisiología , Células Fotorreceptoras Retinianas Conos/fisiología , Degeneración Retiniana/fisiopatología , Animales , Humanos , Epitelio Pigmentado Ocular/trasplante , Ratas , Degeneración Retiniana/cirugíaRESUMEN
A primary neuronal culture was prepared from the ventral mesencephalon, centered on the A8, A9 and A10 dopaminergic nuclei of the embryonic day 14 rat, and studied from 12 h to 28 days. At 12 h after plating, and before cell death ensued, 95% of the cells stained positive for neuron specific enolase; 20% for tyrosine hydroxylase; 5% for vimentin and < 0.1% for glial fibrillary acidic protein. In the presence of the mitotic inhibitor cytosine arabinoside (2.0 microM), neuronal growth and survival were surprisingly normal up to the ninth day in culture, but deteriorated rapidly thereafter. In the absence of a mitotic inhibitor, and in the presence of proliferating but non-confluent glia, the tyrosine hydroxylase positive neurons that survived to the 10th day, had retracted neurites and a rounded soma, suggesting an inhibition of cell development. Those tyrosine hydroxylase positive neurons that survived this adverse phase of development tended to produce elaborate neuritic profiles after the 11th day, coincident with confluence of the astrocyte monolayer at the 12th day. By the 21st day in culture, and persisting up to the 28th day, 60% (61 +/- 10, n = 20) of the surviving neurons stained positive for tyrosine hydroxylase. When plated on an established, ventral mesencephalic monolayer of astrocytes, at the seventh day in culture, neuritic growth and branching of the tyrosine hydroxylase positive neurons were greater, compared with similar neurons grown on poly-D-lysine, and the signs of arrested development (retraction of neurites and rounded soma) seen at the 10th day after plating on poly-D-lysine, were not observed. We conclude that in the primary culture studied, and under the experimental conditions used, the survival of dopaminergic neurons was independent of glia during the first nine days, and critically dependent on glia thereafter. The resurgence of growth of dopaminergic neurons after 10 days in vitro, and their subsequent selective survival in culture, suggest that confluent type-1 astrocytes produce factors that act selectively on the dopaminergic neuronal phenotype. The successful identification of these dopaminergic-specific, neurotrophic factors could lead to an increased understanding of the etiology of Parkinson's disease, and suggest new directions for therapeutic intervention.
Asunto(s)
Astrocitos/fisiología , Dopamina , Mesencéfalo/citología , Neuronas/citología , Animales , Biomarcadores , Supervivencia Celular , Células Cultivadas , Medios de Cultivo/farmacología , Medios de Cultivo Condicionados/farmacología , Medio de Cultivo Libre de Suero/farmacología , Sustancias de Crecimiento/fisiología , Mesencéfalo/embriología , Proteínas del Tejido Nervioso/análisis , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Tirosina 3-Monooxigenasa/análisisRESUMEN
We have used the P23H line 1 homozygous albino rat to study how progressive photoreceptor degeneration affects rod and cone relay pathways. We examined P23H retinas at different stages of degeneration by confocal microscopy of immunostained sections and electroretinogram (ERG) recordings. By 21 days of age in the P23H rat retina, there is already substantial loss of rods and reduction in rod bipolar dendrites along with reduction of metabotropic glutamate receptor 6 (mGluR6) and rod-associated bassoon staining. The cone pathway is relatively unaffected. By 150 days, when rods are absent from much of the retina, some rod bipolars remain and dendrites of rod and cone bipolar cells form synaptic complexes associated with cones and horizontal cell processes. These complexes include foci of mGluR6 and bassoon staining; they develop further by 270 days of age. Over the course of degeneration, beginning at 21 days, bipolar axon terminals atrophy and the inner retina undergoes further changes including a reduced and disorganized AII amacrine cell population and thinning of the inner plexiform layer. Electroretinogram (ERG) results at 23 days show reductions in a-wave amplitude, in rod and cone-associated b-waves (using a double flash paradigm) and in the amplitude of oscillatory potentials (OPs). By 38 days, rod scotopic a-wave responses and OPs are lost. B-wave amplitudes decline until 150 days, at which point they are purely cone-driven and remain stable up to 250 days. The results show that during the course of photoreceptor loss in the P23H rat, there are progressive degenerative changes, particularly in the rod relay pathway, and these are reflected in the changing ERG response patterns. Later reactive changes involving condensation of cone terminals and neurotransmitter receptors associated with rod and cone bipolar dendrites and with horizontal cell processes suggest that at this stage, there are likely to be complex changes in the relay of sensory information through the retina.
Asunto(s)
Vías Nerviosas/patología , Vías Nerviosas/fisiopatología , Células Fotorreceptoras Retinianas Conos/patología , Degeneración Retiniana/patología , Degeneración Retiniana/fisiopatología , Células Fotorreceptoras Retinianas Bastones/patología , Células Amacrinas/metabolismo , Células Amacrinas/patología , Células Amacrinas/ultraestructura , Animales , Animales Modificados Genéticamente , Dendritas/metabolismo , Dendritas/patología , Dendritas/ultraestructura , Modelos Animales de Enfermedad , Electrorretinografía , Inmunohistoquímica , Potenciales de la Membrana/genética , Microscopía Confocal , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Proteínas del Tejido Nervioso/metabolismo , Vías Nerviosas/metabolismo , Terminales Presinápticos/metabolismo , Terminales Presinápticos/patología , Terminales Presinápticos/ultraestructura , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Conos/ultraestructura , Degeneración Retiniana/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/ultraestructura , Visión Ocular/genéticaRESUMEN
The Royal College of Surgeons rat has a retinal pigment epithelial cell defect which causes a progressive loss of rods occurring primarily over the first few months of life. We have studied the consequences of this degenerative process on visual sensitivity across the visual field. Sensitivities were determined in the superior colliculus for unit responses recorded from 22 days up to one year of age from sites encompassing the whole visual field representation. Following visual sensitivity assessment, retinae were examined anatomically at the light and electron microscopic level. At 22 days of age, sensitivities in dystrophic rats were comparable to those of non-dystrophics at any age (40+/-1 and 41+/-1dB, respectively), despite the fact that signs of degenerative events were clear at the electron microscopic level, including presence of pyknotic photoreceptor nuclei, disorganised outer segments and accumulation of debris. However, loss in sensitivity was first detected only at 28-36 days of age (27+/-4dB). From then on, sensitivities progressively decreased to reach a plateau by 180-240 days (4+/-2dB). Starting around 90 days and onward, there was a positive gradient of sensitivities from temporal to nasal field. Drops in visual sensitivity were parallelled by several changes in visual response properties, including prolonged latency, inconsistent responsiveness, appearance of bursting spontaneous activity and activation of units by stimuli presented outside their classical receptive fields. The measure of visual sensitivities by recording visual responses at specific sites in the superior colliculus provides a reliable point-to-point assessment of retinal function comparable to visual perimetry testing in humans. This experimental approach provides the background for answering questions arising during the development of potential experimental therapies for retinal degeneration using animal models like the Royal College of Surgeons rat.
Asunto(s)
Degeneración Retiniana/patología , Colículos Superiores/fisiopatología , Visión Ocular , Percepción Visual , Animales , Mapeo Encefálico , Microscopía Electrónica , Estimulación Luminosa , Células Fotorreceptoras de Vertebrados , Ratas , Tiempo de Reacción , Retina/patología , Degeneración Retiniana/fisiopatología , Campos VisualesRESUMEN
The dystrophic RCS rat undergoes progressive photoreceptor degeneration due to a primary defect in retinal pigment epithelial (RPE) cells. This has a major impact on central visual responsiveness. Here we have examined how functional deterioration is contained by subretinal transplantation of immortalized human RPE cells. Transplantation was done at three to four weeks of age prior to significant photoreceptor loss and recipients were kept on cyclosporin. At six months of age, sensitivity maps and multi-unit response properties were obtained across the visual field by recording at 76 equidistant sites encompassing the whole superior colliculus.A significant degree of functional protection, both in terms of area of responsive retina and response characteristics was observed following RPE transplantation. At best, the sensitivity, latency of onset, and response rise time were all maintained within normal ranges and this was achieved with no more than half of the normal complement of photoreceptors. Although partial, the degree of anatomical preservation (both in terms of outer nuclear layer thickness and area of rescue) correlated well with the level of preserved visual sensitivities. Sham injections also resulted in rescue, though the area of preservation was strictly confined to the needle injury site and the response properties were significantly worse than with RPE injections. This study shows that central physiological responsiveness and correlated retinal morphology can be preserved in an animal model of retinal disease by implantation of an immortalized cell line. The use of retinal sensitivity measurements provides a background for assessing higher visual functions in these animals and a direct comparison for human perimetry measures.
Asunto(s)
Trasplante de Tejido Encefálico/métodos , Degeneración Macular/terapia , Epitelio Pigmentado Ocular/trasplante , Recuperación de la Función/fisiología , Retinitis Pigmentosa/terapia , Colículos Superiores/fisiología , Percepción Visual/fisiología , Potenciales de Acción/genética , Animales , Línea Celular Transformada , Modelos Animales de Enfermedad , Humanos , Degeneración Macular/genética , Degeneración Macular/fisiopatología , Degeneración Nerviosa/genética , Degeneración Nerviosa/fisiopatología , Degeneración Nerviosa/terapia , Estimulación Luminosa , Células Fotorreceptoras/patología , Células Fotorreceptoras/fisiopatología , Epitelio Pigmentado Ocular/fisiopatología , Ratas , Ratas Mutantes , Tiempo de Reacción/fisiología , Células Ganglionares de la Retina/patología , Células Ganglionares de la Retina/fisiología , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/fisiopatología , Factores de Tiempo , Resultado del Tratamiento , Vías Visuales/patología , Vías Visuales/fisiopatologíaRESUMEN
PURPOSE: To examine whether congenic Schwann cells grafted into the subretinal space of dystrophic Royal College of Surgeons (RCS) rats can prevent photoreceptor loss and maintain visual function. METHODS: Purified neonatal Schwann cells derived from congenic rats were grafted into the subretinal space of 3- to 4-week-old dystrophic RCS rats. Graft placement was confirmed using Schwann cells labeled in vitro with the fluorescent dye Hoechst 33342 or in grafted eyes processed for electron microscopy (48-hour to 1-month survival). At longer intervals, up to 9 months after surgery, animals were examined for photoreceptor survival; preservation of a visual reflex, head-tracking to moving stripes; and preservation of visual receptive fields associated with the region of graft placement. RESULTS: One week after the graft was performed, Schwann cells had integrated into the subretinal space with little evidence of a reactive response. When screened for head-tracking to moving stripes, Schwann cell-grafted animals performed better than sham-treated or control dystrophic animals. Threshold sensitivity measurements and visual field assessment made by recording from the superior colliculus also showed a significant level of preserved function compared with control animals. Functional rescue was correlated with photoreceptor survival and could be observed for at least 9 months after grafting. CONCLUSIONS: Schwann cells injected into the subretinal space limit functional deterioration and prolong photoreceptor survival. It is suggested that they act by local release of growth factors that either support photoreceptors directly and/or stimulate phagocytosis in RPE cells.
Asunto(s)
Retina/cirugía , Degeneración Retiniana/fisiopatología , Degeneración Retiniana/cirugía , Células de Schwann/trasplante , Animales , Conducta Animal/fisiología , Bencimidazoles , Trasplante de Células , Electrofisiología , Colorantes Fluorescentes , Actividad Motora/fisiología , Células Fotorreceptoras de Vertebrados/fisiología , Ratas , Ratas Mutantes , Retina/patología , Retina/fisiopatología , Degeneración Retiniana/patología , Degeneración Retiniana/prevención & control , Nervio Ciático/citología , Colículos Superiores/fisiología , Visión Ocular/fisiología , Campos Visuales/fisiologíaRESUMEN
In the ventral mesencephalon of the E14 rat fetus, 90% of the dopaminergic, tyrosine hydroxylase positive (TH+) cells are localized in 1.0 mm3 of tissue. This same ventral mesencephalic region also contains 90% of the dopamine content of the E14 ventral brainstem (2.2 +/- 0.3 nmol/mg protein). When cells were prepared for culturing from this localized area, and plated at a density of 2.5 x 10(5) cells/cm2, 17-21% of the cells were TH+, at 4 and 12 h, and at 1, 5, 7 and 10 days after plating. The percentage of TH+ cells was also 17-21% when examined at 4 h, 12 h or 5 days after plating at densities ranging from 7.8 x 10(3) to 2.5 x 10(5) cells/cm2. However, cell survival at a density of less than 6.2 x 10(4) cells/cm2 was poor after 5 days in culture. Based on the degree of neurite elongation and complexity, cell maturation appeared to be complete at 5 days in culture (DIV5), and appeared to be maintained at this level up to DIV10. By DIV14, neurite retraction was evident, and the cells were more rounded. These signs may indicate the inception of senescence in the cultures. A benztropine-sensitive, concentration-dependent dopamine uptake mechanism was demonstrated in the cultures at DIV7, and DA could be released from preloaded cells using 50 mM K+. Five morphological subtypes of TH+ cells were identified in the cultures. This primary culture of the ventral mesencephalic, dopaminergic area, with a high percentage of TH+ cells, is suitable for use in acute biochemical and cellular studies, between DIV 5 and DIV10.
Asunto(s)
Encéfalo/embriología , Neuronas/enzimología , Tirosina 3-Monooxigenasa/análisis , Animales , Encéfalo/citología , Encéfalo/enzimología , Supervivencia Celular , Células Cultivadas , Técnicas de Cultivo/métodos , Embrión de Mamíferos , Femenino , Neuritas/fisiología , Neuritas/ultraestructura , Neuronas/citología , Embarazo , RatasRESUMEN
Neonatal rats, when spinalized on the fourteenth postnatal day, showed minimal recovery of function in their hindlimbs. Bridging the cut spinal cord with E16 fetal spinal cord tissue did not improve functional recovery. Bridging, plus treatment with GM1 ganglioside, caused a significant (p less than 0.05) improvement in function, versus the bridged animals treated with saline. The E16 spinal cord transplants survived poorly, or not at all. Contact of the hindlimbs with a surface is necessary to elicit function. Regrowth of descending fibers into the caudal region of the cord is probably not involved in functional recovery. It is suggested that functional recovery is mediated by hindlimb proprioceptive afferents, which activate the lumbosacral motor central pattern generator.
Asunto(s)
Gangliósido G(M1)/farmacología , Miembro Posterior/inervación , Actividad Motora , Regeneración Nerviosa , Médula Espinal/fisiología , Análisis de Varianza , Animales , Animales Recién Nacidos , Trasplante de Tejido Fetal/fisiología , Músculos/inervación , Ratas , Médula Espinal/trasplanteRESUMEN
Although the RCS rat is widely used as a model of progressive photoreceptor loss, it is unclear how the relative rod and cone functions change with age. Rod and cone b-waves were isolated using a double flash ERG paradigm. In contrast to cones, rods never reached normal functional maturity levels, and the ERG b-wave changed from being predominantly rod-driven to being purely cone-driven by age 74 days, at which point, b-waves were progressively replaced by negative STR-like (scotopic threshold response) waves that persisted up to age 180 days. A double flash commonly abolished this wave and unveiled a b-wave.
Asunto(s)
Adaptación a la Oscuridad , Células Fotorreceptoras Retinianas Conos/fisiopatología , Degeneración Retiniana/fisiopatología , Células Fotorreceptoras Retinianas Bastones/fisiopatología , Envejecimiento/fisiología , Animales , Progresión de la Enfermedad , Electrorretinografía , Estimulación Luminosa/métodos , Ratas , Ratas MutantesRESUMEN
Dark-adapted full field electroretinogram (ERG) and visual receptive field thresholds (recorded from the superior colliculus) were correlated in a model of retinal degeneration, the Royal College of Surgeons rat. In both untreated and retinal pigment epithelium cell transplanted rats, optimal correlation was between b-wave amplitude and preserved visual field area with thresholds under a defined level. The work shows that the magnitude of the b-wave can be used to predict the computed area and degree of visual field preservation recorded in the central nervous system. These observations validate using ERG to assess residual visual function and the effect of transplantation.
Asunto(s)
Epitelio Pigmentado Ocular/trasplante , Degeneración Retiniana/etiología , Animales , Adaptación a la Oscuridad/fisiología , Electrorretinografía/métodos , Modelos Animales , Ratas , Degeneración Retiniana/cirugía , Retinitis Pigmentosa/etiología , Campos Visuales/fisiologíaRESUMEN
Royal College of Surgeon (RCS) rats undergo retinal degeneration due to the inability of retinal pigment epithelial (RPE) cells to phagocytose shed outer segments. We explored the effect of introducing Schwann cells to the subretinal space of RCS rats (before the onset of retinal degeneration), by relying on electroretinogram (ERG) recordings and correlative retinal morphology. Scotopic ERGs recorded from cell-injected eyes showed preserved amplitudes of mixed a-wave b-wave, rod b-waves, and cone b-waves over controls (sham-injected eyes); photopic b-wave amplitudes and critical flicker fusion were also improved. Normal retinal morphology was found in areas of retinas that had received cell injections. Since Schwann cells have no phagocytic properties, their therapeutic effect is best explained through a paracrine mechanism (secretion of factors that ensure photoreceptor survival).
Asunto(s)
Células Fotorreceptoras/patología , Degeneración Retiniana/terapia , Células de Schwann/trasplante , Animales , Biomarcadores/análisis , Supervivencia Celular , Electrorretinografía , Fusión de Flicker , Inmunohistoquímica , Microscopía Confocal , Estimulación Luminosa , Ratas , Ratas Mutantes , Células Fotorreceptoras Retinianas Conos/patología , Degeneración Retiniana/patología , Degeneración Retiniana/fisiopatología , Epitelio Pigmentado de la Retina/patología , Células Fotorreceptoras Retinianas Bastones/patologíaRESUMEN
During development, gradients of EphA receptors (nasal(low)-temporal(high)) and their ligands ephrin-As (rostral(low)-caudal(high)) are involved in establishing topography between retinal ganglion cells (RGCs) and the superior colliculus (SC). EphA5-expressing RGC axons are repulsed by ephrin-A2-expressing SC neurones. In adult rats RGCs maintain graded EphA5 expression but ephrin-A2 expression is down-regulated in the SC to a weak gradient. At 1 month after optic nerve transection, EphA5 expression is reduced in the few remaining RGCs and is no longer graded; by contrast, SC ephrin-A2 is up-regulated to a rostral(low)-caudal(high) gradient. Here we examined expression in adult rat 1 month after bridging the retina and SC with a peripheral nerve graft, a procedure that enhances RGC survival and permits RGC axon regeneration. Double labelling with cell markers revealed preservation of a nasal(low)-temporal(high) EphA5 gradient in RGCs and establishment of a rostral(low)-caudal(high) ephrin-A2 gradient within neurones of the SC. The results suggest a potential for guidance cues to restore the topography of RGC axons in the SC. However, high ephrin-A2 levels were also found in astrocytes surrounding the peripheral nerve graft insertion site. The repulsive ephrin-A2 environment offers at least a partial explanation for the observation that only a limited number of RGC axons can exit the graft to enter target central nervous system tissue.
Asunto(s)
Efrina-A2/genética , Efrina-A5/genética , Regeneración Nerviosa/fisiología , Nervio Óptico/fisiología , Nervio Ciático/trasplante , Animales , Axones/fisiología , Axotomía , Supervivencia Celular/fisiología , Efrina-A2/metabolismo , Efrina-A5/metabolismo , Femenino , Expresión Génica/fisiología , Nervio Óptico/citología , Ratas , Ratas Endogámicas , Células Ganglionares de la Retina/fisiología , Células Ganglionares de la Retina/ultraestructura , Nervio Ciático/citología , Colículos Superiores/citología , Colículos Superiores/fisiología , Vías VisualesRESUMEN
The Royal College of Surgeons (RCS) rat has a primary defect in retinal pigment epithelial cells that leads to the progressive loss of photoreceptors and central visual responsiveness. While most rods are lost by 90 days of age (P90), cones degenerate more slowly, and can be detected anatomically up to 2 years of age, despite massive neuronal death and retinal remodelling. To examine how this progressive degenerative process impacts on cone function, we recorded the electroretingram to white light flashes (1.37 log cd s m(-2)) presented at frequencies ranging from 3 to 50 Hz, under light adapted conditions (29.8 cd m(-2)). Pigmented dystrophic and congenic non-dystrophic RCS rats aged from 18 to 300 days were studied. In all responsive animals at all ages, maximal amplitudes were obtained at 3 Hz. In both non-dystrophic and dystrophic rats, there was an increase from P18 to P21 in response amplitude and critical fusion frequency. After P21, these two parameters declined progressively with age in dystrophic rats. Other changes included prolongation in latency, which was first detected prior to the initiation of amplitude reduction. While phase shifts were also detected in dystrophic RCS rats, they appeared at later degenerative stages. The latest age at which responses could be elicited in dystrophic rats was at P200, with positive waves being replaced by negative deflections. The effect of increments in the intensity of background illumination was tested at P50 in both groups. This caused a diminution in flicker response amplitude and critical fusion frequencies in non-dystrophics, while in dystrophic animals, response amplitudes were reduced only at low frequencies and critical fusion frequencies were unaltered. In conclusion, although dystrophic RCS rats undergo a progressive decline in cone function with age, the flicker responsiveness at P21 is comparable to that of non-dystrophic congenic rats, suggesting normal developmental maturation of the cone system in this animal model of retinal degeneration. Flicker responses can be recorded up to P200, at which point the retina has undergone severe regressive and reactive changes in its connectivity patterns. The fact that responses at this age consist of solely negative deflections might be a reflection of the highly pathological state of the retina.