9 research outputs found

    Spatial distribution of RGCs in albino and pigmented rats.

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    <p><b>A–E</b>: magnifications from flat mounted retinas showing RGCs detected by FG tracing (<b>A</b>), Brn3a (<b>B</b>), Brn3b (<b>C</b>), Brn3c (<b>D</b>) and Brn3a+b+c immunodetection. <b>A, B</b> and <b>C, D</b> are images taken from the same retinal frame. In <b>E</b> the three Brn3 members were detected using the same fluorophore (Brn3<sup>+</sup>RGCs). <b>F–Y</b>: Representative isodensity maps showing the retinal distribution of RGCs in SD (albino) and PVG (pigmented rats). <b>F–I:</b> fluorogold traced RGCs, <b>J–M</b>: Brn3a<sup>+</sup>RGCs, <b>N–Q</b>: Brn3b<sup>+</sup>RGCs, <b>R–U</b>: Brn3c<sup>+</sup>RGCs, <b>V–Y:</b> Brn3<sup>+</sup>RGCs. For each marker and strain is shown the distribution of RGCs in one left and one right retina. Notice that, because FG and Brn3a or Brn3b and Brn3c were double detected, maps F&J, G&K, H&L, I&M, N&R, O&S, P&T and Q&U come from the same retinas. Isodensity maps are created from the data gathered after automated quantification. At the bottom of each one is shown the number of RGCs counted in the retina wherefrom the map has been generated. These maps express the RGC density according to a colour scale (bottom right in <b>I, M, Q, U</b> and <b>Y</b>) that ranges from 0 RGCs/mm<sup>2</sup> (blue) to a maximum density (red) that is 3,200 RGCs/mm<sup>2</sup> or more for FG<sup>+</sup>, Brn3a<sup>+</sup>and Brn3<sup>+</sup>RGCs; 1,800 RGCs/mm<sup>2</sup> or more for Brn3b<sup>+</sup>RGCs, and 1,600 RGCs/mm<sup>2</sup> or more for Brn3c<sup>+</sup>RGCs. The maximum density was adjusted to these numbers to allow the visualization of high and low density areas within the retina. Retinal orientation is shown in F–I: superior (S), nasal (N) temporal (T) and inferior (I). <i>Bars:</i> 20 µm (A), 1 mm (F, H).</p

    Total number of contralateral and ipsilateral RGCs in albino and pigmented rats.

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    <p>Mean number ± standard deviation (SD) of contralateral-RGCs (left retinas) and ipsilateral-RGCs (right retinas) in the albino (SD) and pigmented (PVG) rats. It is shown as well the number of ipsilateral-RGCs that express Brn3a, Brn3b or Brn3c. n = number of analyzed retinas. *The pigmented strain has significantly more ipsilateral-RGCs than the albino one (t-test p<0.001). <sup>§</sup>In the albino strain there are significantly more ipsilateral-RGCs that express Brn3a, Brn3b or Brn3c than in the pigmented one (t-test p<0.001).</p

    Total numbers and densities of Brn3 positive RGCs in albino and pigmented rats.

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    <p>In this table are shown the total numbers of the different RGC populations (FG-traced, Brn3a, Brn3b or Brn3c positive RGCs) in albino (SD) and pigmented (PVG) rats. In the right-most column is shown the total number of Brn3<sup>+</sup>RGCs counted when Brn3a, Brn3b and Brn3c were detected with the same fluorophore. As explained in methods, FG and Brn3a and Brn3b and Brn3c were detected in the same retinas. The area of each retina was measured allowing the calculation of the mean retinal density of each population. Data are shown as the mean ± standard deviation (SD). n = number of analyzed retinas.</p

    Distribution of ipsilateral RGCs in albino and pigmented rats.

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    <p><b>A, E</b> Photomontages of an albino (<b>A</b>) and a pigmented (<b>E</b>) retina showing RGCs traced from the ipsilateral colliculi (ipsilateral-RGCs). <b>B–D, F–H:</b> retinal silhouettes showing the distribution of Brn3a<sup>+</sup>ipsilateral-RGCs <b>(B, F</b>), Brn3b<sup>+</sup>ipsilateral-RGCs (<b>C, D</b>) and Brn3c<sup>+</sup>ipsilateral-RGCs (<b>D, H</b>) in albino (<b>B–D</b>) and pigmented (<b>F–H</b>) rats. At the bottom of each retina is shown its number of ipsilateral-RGCs (in brackets). <b>I:</b> Histogram showing the percentage of Brn3a, Brn3b and Brn3c positive ipsilateral-RGCs with respect to their total number. <b>J–L:</b> Magnifications from the inferotemporal quadrant of ipsilaterally-traced retinas showing ipsilateral-RGCs and Brn3a (<b>J</b>), Brn3b (<b>K</b>) and Brn3c (<b>L</b>) RGCs. Arrows point to ipsilateral-RGCs that are Brn3 positive. Retinal orientation is shown in A: superior (S), nasal (N) temporal (T) and inferior (I). <i>Bars:</i> 1 mm (A,E), 50 µm (J).</p

    Injured RGCs down-regulate Brn3a expression.

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    <p>Plot depicting the distribution of RGCs according to their expression level of Brn3a. The number of RGCs (ordinate axis) is plotted against the intensity of their Brn3a signal in control, IONC and IONT injured retinas at 3 and 5 days post-lesion.</p

    RGC nuclear sizes and expression of Brn3 factors.

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    <p><b>A</b>: Histogram showing the percentages of Brn3a, Brn3b or Brn3c positive nuclei that are small, medium, large or very large. <b>B</b>: Histogram showing for each Brn3 combination the percentage of small, medium, large and very large nuclei (each Brn3 combination was considered 100%). <b>C</b>: Brn3a immunodetection showing the different sizes of RGC nuclei. An example of each size range is shown (S: small, M: medium, L: large, VL: very large).</p

    Brn3b and Brn3c expression is rapidly lost after optic nerve injury.

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    <p><b>A-P</b>: Magnifications from SD flat mounted retinas analyzed 3 and 5 days after intraorbital nerve crush (<b>A–H</b>) and intraorbital nerve transection (<b>J–P</b>). As internal control, Brn3a was double immunodetected with Brn3b (<b>A–B, E–F, I–J, M–N)</b> or with Brn3c (<b>C–D, G–H, K–L, O–P</b>). In these images is observed that Brn3b and Brn3c expression decreases already 3 days after both injuries. This low signal impeded the automated quantification of Brn3b and Brn3c positive RGCs. Brn3a<sup>+</sup>RGCs were counted in these retinas and isodensity maps showing the distribution of the surviving RGCs were created (<b>Q–T</b>, at the bottom of each map is shown the number of Brn3a<sup>+</sup>RGCs counted in the retina wherefrom the map has been generated). These maps illustrate that IONT induces a quicker loss of RGCs than IONC, as evidenced by the higher densities observed in <b>Q</b> and <b>S</b> than in <b>R</b> and <b>T</b>. Colour scale (T bottom right) is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049830#pone-0049830-g004" target="_blank">figure 4M</a>. Four (IONC 5d) or five (rest of the groups) retinas were analyzed per marker. <i>Bars:</i> 50 µm (J), 1 mm (Q).</p

    Co-expression of Brn3 transcription factors in albino and pigmented rats. A–O:

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    <p>Magnifications from SD flat mounted retinas in which FG and Brn3a (<b>A–C</b>); Brn3a detected with two different antibodies -goat and mouse anti-Brn3a-(<b>D–F</b>); Brn3a and b (<b>G–H</b>), Brn3a and c (<b>J–L</b>) and Brn3b and c (<b>M–O</b>) have been double detected. In images like these taken from SD (albino) and PVG (pigmented) rat retinas the percentage of co-localization of each marker (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049830#pone-0049830-g002" target="_blank">figure 2</a>) was calculated. <b>P–R:</b> cross-sections from SD rat retinas, in which Brn3a (<b>P</b>), Brn3b (<b>Q</b>) and Brn3c (<b>R</b>) have been detected to show that these proteins are only expressed in the ganglion cell layer. In these images, all nuclei have been counterstained with DAPI. Arrows point to those RGCs that only express one of the Brn3s. <i>Bar</i>: 50 µm (C,R).</p

    Brn3 co-expression in albino and pigmented rat retinas.

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    <p><b>A:</b> Stacked-bar graph showing the percentage of Brn3 co-expression in both rat strains. In each bar is shown the number of cells counted. First and second bars: percentage of Brn3b<sup>+</sup>RGCs that express or not Brn3a. Third and fourth bars: percentage of Brn3c<sup>+</sup>RGCs that express or not Brn3a. Fifth and sixth bars: percentage of Brn3b<sup>+</sup>RGCs that express or not Brn3c. Seventh and eighth bars: percentage of Brn3c<sup>+</sup>RGCs that express or not Brn3b. Percentage was calculated considering 100% the total number of cells per marker (see results for further explanation). <b>B</b>: Inference of the total number of RGCs that express one, two or the three Brn3 members in the albino strain. These numbers were calculated based on the co-expression percentages and on the total number of RGCs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049830#pone-0049830-t001" target="_blank">table 1</a> and results). The final population for each marker combination is shown in the grey squares.</p
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