Synaptic contacts in OPL.

<p>Quantification of the number of photorreceptor synaptic ribbons at the OPL along retinal sections in control and safranal-administered P23H animals (n = 8 and n = 6, respectively in). *** <i>P</i><0.001; Student's <i>t</i>-test.</p

Horizontal cells and their synaptic connectivity.

<p>Vertical sections of retinas from wild-type rats (Sprague Dawley, SD) (A, D, G) and P23H animals treated with vehicle (B, E, H) or safranal (C, F, I). (A–C) Horizontal cells labeled with antibodies against calbindin. Note that the number of horizontal cell terminals in safranal-treated rats was higher than in vehicle-treated animals. (D–F) Labeling of photorreceptor synaptic ribbons with antibodies against bassoon (red). (G–I) Double immunolabeling for calbindin and bassoon showing a larger number of synaptic contacts (arrows) between photoreceptor and horizontal cells in safranal-treated rats (H) than observed in the control rats (I). All images were collected from the central area of the retina, close to the optic nerve. ONL: outer nuclear layer, OPL: outer plexiform layer, INL inner nuclear layer. Scale bar: 20 μm.</p

Photoreceptor cell bodies number.

<p>Representative retinal section stained with TO-PRO (blue), γ-transducin (green) and recoverin (red) from a wild-type animal (Sprague Dawley, SD, A), a P23H rat treated with vehicle (B) and a P23H rat treated with safranal (C). All images were collected from the central area of the retina, close to the optic nerve. Note that the number of photoreceptor rows in the vehicle-treated P23H rat is low (B), as compared to those present in the retina of the safranal-treated P23H animal (C). ONL: outer nuclear layer, INL: inner nuclear layer, IPL: inner plexiform layer. Scale bar: 20 μm.</p

Retinal capillary network.

<p>(A–B) Whole-mount retinas from P23H rats treated with vehicle (A) or safranal (B), stained with NAPDH diaphorase. Note that the retinal capillary network was more extensive, with more capillary loops in safranal-treated animals. (C) Measurements of the relative capillary density (capillary area/retinal area) showing higher values in safranal-treated rats than in control animals (n = 6 and n = 3, respectively). ** <i>P</i><0.01; Student's <i>t</i>-test.</p

Retinal function in control and safranal-treated P23H rats.

<p>(A–B) Example of scotopic (A) and photopic (B) ERG traces from a P120 rat treated with vehicle (left) or safranal (right). Units on the left of panels represent the luminance of the flashes in log cd·s/m². (C–D) Stimulus intensity curves for mixed scotopic a-waves (C) and b-waves (D) from rats administered safranal (squares) or vehicle (circles). (E–F) Intensity response of photopic a-waves (E) and b-waves (F).</p

Primary antibodies.

<p>Primary antibodies.</p

ON rod bipolar cells and their synaptic connectivity.

<p>Immunolabeling of retinal vertical sections from wild-type rats (Sprague Dawley, SD) (A, D, G) and P23H animals treated with vehicle (B, E, H) or safranal (C, F, I). Nuclei stained with TO-PRO (blue). (A–C) Staining of retinal ON rod bipolar cells with PKC (green). Note that cell bodies and dendrites were preserved by safranal. (D–F) Labeling of photorreceptor synaptic ribbons with antibodies against bassoon (red). (G–I) Double immunolabeling for PKC and bassoon, showing the preservation by safranal of synaptic contacts (arrows) between photoreceptors and bipolar cells. All images were collected from the central area of the retina, close to the optic nerve. ONL: outer nuclear layer, OPL: outer plexiform layer, INL inner nuclear layer. Scale bar: 20 μm.</p

Immunohistochemical analysis of RGCs after NMDA-induced damage in the presence or absence of TUDCA.

<p>Confocal images of whole-mounted retinas labelled with the RGC markers Brn3a (green) and RBPMS (red). A representative image of the dorsal area of the retina is shown for (A) untreated, (B) NMDA and (C) NMDA+TUDCA experimental groups. High magnification images (D-I) correspond to central (D-F) and peripheral (G-I) areas of the retina for the three experimental groups. Scale bar 1 mm (A-C), 50 μm (D-I).</p

Effect of NMDA and TUDCA on the ERG intensity-response functions.

<p>The graph represents mixed scotopic ERG amplitude (mean ± SEM) versus stimulus intensity previous to retinal damage (pre-lesion, circles, n = 11), and after NMDA-induced lesion in rats either treated with vehicle (NMDA, squares, n = 5) or TUDCA (NMDA+TUDCA, triangles, n = 6). Scotopic pSTRs, nSTRs, a-waves and b-waves recorded after retinal insult in TUDCA-treated rats reached higher values than those obtained in vehicle-administered animals. Asterisks indicate statistical significance (ANOVA, Bonferroni’s test), *P<0.05, **P<0.01.</p

Effect of NMDA and TUDCA on the rat full-field ERG.

<p>(A) Experimental timeline indicating the days of intraperitoneal injection with TUDCA or vehicle, ERGs and intravitreal delivery of NMDA. (B) Representative scotopic ERG waveforms performed before (thin traces) and after (bold traces) NMDA-induced retinal lesion in rats treated with TUDCA or vehicle. Units on the left indicate input flash intensities in log cd·s/m².</p