Browsing by Subject "Automated quantification"

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    Axotomy-induced retinal ganglion cell death in adult mice: quantitative and topographic time course analyses
    (Elsevier, 2011-02-24) Galindo Romero, Caridad; Avilés Trigueros, Marcelino; Jiménez López, Manuel; Valiente Soriano, Francisco Javier; Salinas Navarro, Manuel Ángel; Nadal-Nicolás, Francisco Manuel; Villegas Pérez, Maria Paz; Vidal Sanz, Manuel; Agudo Barriuso, Marta; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Anatomía Humana y Psicobiología; Facultades de la UMU::Facultad de Medicina
    The fate of retinal ganglion cells after optic nerve injury has been thoroughly described in rat, but not in mice, despite the fact that this species is amply used as a model to study different experimental paradigms that affect retinal ganglion cell population. Here we have analyzed, quantitatively and topographically, the course of mice retinal ganglion cells loss induced by intraorbital nerve transection. To do this, we have doubly identified retinal ganglion cells in all retinas by tracing them from their main retinorecipient area, the superior colliculi, and by their expression of BRN3A (product of Pou4f1 gene). In rat, this transcription factor is expressed by a majority of retinal ganglion cells; however in mice it is not known how many out of the whole population of these neurons express it. Thus, in this work we have assessed, as well, the total population of BRN3A positive retinal ganglion cells. These were automatically quantified in all whole-mounted retinas using a newly developed routine. In control retinas, tracedretinal ganglion cells were automatically quantified, using the previously reported method (SalinasNavarro et al., 2009b). After optic nerve injury, though, traced-retinal ganglion cells had to be manually quantified by retinal sampling and their total population was afterwards inferred. In naïve whole-mounts, the mean ( standard deviation) total number of traced-retinal ganglion cells was 40,437 ( 3196) andofBRN3Apositive ones was 34,697( 1821). Retinal ganglion cell loss was first significant for both markers 5 days post-axotomy and by day 21, the last time point analyzed, only 15% or 12% of traced or BRN3A positive retinal ganglion cells respectively, survived. Isodensity maps showed that, in control retinas, BRN3A and traced-retinal ganglion cells were distributed similarly, being densest in the dorsal retina along the naso-temporal axis. After axotomy the progressive loss of BRN3A positive retinal ganglion cells was diffuse and affected the entire retina. In conclusion, this is the first study assessing the values, in terms of total number and density, of the retinal ganglion cells surviving axotomy from 2 till 21 days post-lesion. Besides, we have demonstrated that BRN3A is expressed by 85.6% of the total retinal ganglion cell population, and because BRN3A positive retinal ganglion cells show the same spatial distribution and temporal course of degeneration than traced ones, BRN3A is a reliable marker to identify, quantify and assess, ex-vivo, retinal ganglion cell loss in this species.
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    Inherited photoreceptor degeneration causes the death of melanopsin-positive retinal ganglion cells and increases their coexpression of Brn3a
    (Association for Research in Vision and Ophthalmology, 2015-07-20) García Ayuso, Diego; Di Pierdomenico, Johnny; Esquiva, Gema; Nadal-Nicolás, Francisco Manuel; Pinilla, Isabel; Cuenca, Nicolás; Vidal Sanz, Manuel; Agudo Barriuso, Marta; Villegas Pérez, Maria Paz; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Facultades de la UMU::Facultad de Medicina
    Purpose: To study the population of intrinsically photosensitive retinal ganglion cells (melanopsin-expressing RGCs, m+RGCs) in P23H-1 rats, a rat model of inherited photoreceptor degeneration. Methods: At postnatal (P) times P30, P365, and P540, retinas from P23H dystrophic rats (line 1, rapid degeneration; and line 3, slow degeneration) and Sprague Dawley (SD) rats (control) were dissected as whole-mounts and immunodetected for melanopsin and/or Brn3a. The dendritic arborization of m+RGCs and the numbers of Brn3a+RGCs and m+RGCs were quantified and their retinal distribution and coexpression analyzed. Results: In SD rats, aging did not affect the population of Brn3a+RGCs or m+RGCs or the percentage that showed coexpression (0.27%). Young P23H-1 rats had a significantly lower number of Brn3a+RGCs and showed a further decline with age. The population of m+RGCs in young P23H-1 rats was similar to that found in SD rats and decreased by 22.6% and 28.2% at P365 and P540, respectively, similarly to the decrease of the Brn3a+RGCs. At these ages the m+RGCs showed a decrease of their dendritic arborization parameters, which was similar in both the P23H-1 and P23H-3 lines. The percentage of coexpression of Brn3a was, however, already significantly higher at P30 (3.31%) and increased significantly with age (10.65% at P540). Conclusions: Inherited photoreceptor degeneration was followed by secondary loss of Brn3a+RGCs and m+RGCs. Surviving m+RGCs showed decreased dendritic arborization parameters and increased coexpression of Brn3a and melanopsin, phenotypic and molecular changes that may represent an effort to resist degeneration and/or preferential survival of m+RGCs capable of synthesizing Brn3a.
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    Number and spatial distribution of intrinsically photosensitive retinal ganglion cells in the adult albino rat
    (Elsevier, 2013-01-04) Galindo Romero, Caridad; Jiménez López, Manuel; García Ayuso, Diego; Salinas Navarro, Manuel Ángel; Nadal-Nicolás, Francisco Manuel; Agudo Barriuso, Marta; Villegas Pérez, Maria Paz; Avilés Trigueros, Marcelino; Vidal Sanz, Manuel; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Facultades de la UMU::Facultad de Medicina
    Intrinsically photosensitive retinal ganglion cells (ipRGCs) respond directly to light and are responsible of the synchronization of the circadian rhythm with the photic stimulus and for the pupillary light reflex. To quantify the total population of rat-ipRGCs and to assess their spatial distribution we have developed an automated routine and used neighbour maps. Moreover, in all analysed retinas we have studied the general population of RGCs - identified by their Brn3a expression - and the population of ipRGCs - identified by melanopsin immunodetection - thus allowing the co-analysis of their topography. Our results show that the total mean number ± standard deviation of ipRGCs in the albino rat is 2047 ± 309. Their distribution in the retina seems to be complementary to that of Brn3a(+)RGCs, being denser in the periphery, especially in the superior retina where their highest densities are found in the temporal quadrant, above the visual streak. In addition, by tracing the retinas from both superior colliculi, we have also determined that 90.62% of the ipRGC project to these central targets.