Neuroprotective Effect of Tauroursodeoxycholic Acid on N-Methyl-D-Aspartate-Induced Retinal Ganglion Cell Degeneration

Retinal ganglion cell degeneration underlies the pathophysiology of diseases affecting the retina and optic nerve. Several studies have previously evidenced the anti-apoptotic properties of the bile constituent, tauroursodeoxycholic acid, in diverse models of photoreceptor degeneration. The aim of this study was to investigate the effects of systemic administration of tauroursodeoxycholic acid on N-methyl-D-aspartate (NMDA)-induced damage in the rat retina using a functional and morphological approach. Tauroursodeoxycholic acid was administered intraperitoneally before and after intravitreal injection of NMDA. Three days after insult, full-field electroretinograms showed reductions in the amplitudes of the positive and negative-scotopic threshold responses, scotopic a- and b-waves and oscillatory potentials. Quantitative morphological evaluation of whole-mount retinas demonstrated a reduction in the density of retinal ganglion cells. Systemic administration of tauroursodeoxycholic acid attenuated the functional impairment induced by NMDA, which correlated with a higher retinal ganglion cell density. Our findings sustain the efficacy of tauroursodeoxycholic acid administration in vivo, suggesting it would be a good candidate for the pharmacological treatment of degenerative diseases coursing with retinal ganglion cell loss.This work was supported by project grants from Spanish Ministerio de Economía y Competitividad-FEDER (http://www.mineco.gob.es) #BFU2012‐36845, Instituto de Salud Carlos III RETICS (http://www.oftared.com) #RD12/0034/0010 and Organización Nacional de Ciegos Españoles (http://www.once.es) to NC; Ministerio de Ciencia e Innovación #JCI‐2009‐05224 to VGV; Universidad de Alicante (http://www.ua.es) #2010-48536273 to GE; Instituto de Salud Carlos III (http://www.isciii.es) #PI13/02098 and RETICS #RD12/0034/0006 to PdV; and FUNDALUCE

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

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 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

Effect of NMDA and TUDCA on the ERG OPs.

<p>(A) Representative examples of filtered OP traces from scotopic ERGs recorded before (thin traces) and after (bold traces) NMDA-induced retinal damage, in vehicle- (upper graph) or TUDCA-treated (lower graph) rats, in response to a 1 cd·s/m² stimulus (arrow). (B) Amplitude (mean ± SEM) of maximum OPs before retinal damage (pre-lesion, n = 11), and after NMDA-induced lesion in rats either treated with vehicle (NMDA, n = 5) or TUDCA (NMDA+TUDCA, n = 6). Asterisks indicate statistical significance (ANOVA, Bonferroni’s test) for pre-lesion <i>vs</i>. NMDA, and NMDA <i>vs</i>. NMDA+TUDCA groups, **P<0.01. No significant differences were found when comparing pre-lesion <i>vs</i>. NMDA+TUDCA groups.</p

Quantitative analysis of RGC survival after NMDA-induced damage in the presence or absence of TUDCA.

<p>(A) Percentage (mean ± SD) of surviving RGCs with respect to control (untreated, 100%), in NMDA-injected retinas of animals treated with vehicle or TUDCA. *P<0.05; Student’s t-test. (B) RGC density was determined in 16 regions of interest at 4 different eccentricities along the dorsal-ventral and nasal-temporal axes of the retina, as represented schematically in the picture, and the data were plotted as an average (mean ± SD) of the 4 values corresponding to each eccentricity. (C) RGC density (mean ± SD) in each of the 8 regions of interest along the dorsal-ventral axis of the retina. (D) RGC density (mean ± SD) in each of the 8 regions of interest along the nasal-temporal axis of the retina. Asterisks in (B-D) indicate statistical significance *P<0.05, **P<0.01 and ***P<0.001; two-way ANOVA. In all cases (A-D) RGC density was determined in a total of 6 rats per experimental group (n = 6).</p