Table 1.
Primary antibodies.
Fig 1.
(A) Scanning electron microscopy picture of TUDCA-loaded microspheres. Insets: particle size distribution. (B) Cumulative in vitro release of TUDCA (μg/mg MSs) over 28 days from TUDCA-loaded PLGA MSs in PBS (pH 7.4).
Fig 2.
Electroretinographic responses in vehicle- and TUDCA-PLGA-MSs-treated eyes.
Representative scotopic ERG traces from postnatal days P30 (A), P60 (B), P90 (C) and P120 (D) P23H rats treated with unloaded PLGA microspheres (left, control) or microspheres containing TUDCA (right, TUDCA). Units to the left of the panels indicate the flash luminance in log cd·s/m2. Note that ERG responses reached higher values in eyes treated with TUDCA-PLGA MSs, as compared to vehicle-treated eyes.
Fig 3.
Effect of TUDCA-loaded PLGA microspheres on the ERG stimulus-response functions.
Stimulus-response curves for mixed scotopic a-waves (A, C, E, G) and b-waves (B, D, F, H) from P30 (A, B), P60 (C, D), P90 (E, F) and P120 (G, H) P23H rats treated with TUDCA-PLGA MSs (squares) or vehicle (circles). ERG a- and b-waves obtained in P23H rats treated with TUDCA-PLGA MSs reached higher values than those recorded in vehicle-treated animals. *p < 0.05; ANOVA, Bonferroni’s test. n = 17 for P30, P60, P90 and n = 12 for P120.
Fig 4.
Number of rows of photoreceptor nuclei in eyes treated with vehicle or TUDCA-PLGA MSs.
(A-D) Retinal sections from P23H rats at P90 (A, B) and P120 (C, D), administered unloaded PLGA MSs (A, C) or TUDCA-loaded PLGA MSs (B, D), counterstained with hematoxylin and eosin. (E, F) Mean number of photoreceptor rows along central sections of the retina in vehicle- or TUDCA-PLGA-MSs-treated animals (n = 5 and n = 9, respectively). *p < 0.05, **p < 0.01; ANOVA, Bonferroni’s test. ONL: outer nuclear layer, INL: inner nuclear layer, OPL: outer plexiform layer, IPL: inner plexiform layer, GCL: ganglion cell layer. Scale bar, 100 μm.
Fig 5.
Photoreceptor morphology in vehicle- and TUDCA-PLGA-MSs-treated eyes.
Triple immunolabeling for cone arrestin (in green), VGLUT1 (in blue) and CtBP2 (in red) of retinal vertical sections from a P120 normal rat (Sprague Dawley, SD) (A) and P23H rats at P90 (B, C) and P120 (D, E), treated with unloaded PLGA MSs (B, D) or TUDCA-loaded PLGA MSs (C, E). Note that the typical cone pedicles (in green) containing synaptic vesicles (in blue) surrounding synaptic ribbons (in red) were less deteriorated in TUDCA-PLGA-MSs-treated P23H rats than in vehicle-treated animals. Scale bar, 10 μm.
Fig 6.
ON-rod bipolar cells and their synaptic contacts in vehicle- and TUDCA-PLGA-MSs-treated eyes.
Examples of retinal vertical sections from a P120 normal rat (Sprague Dawley, SD) (A) and P23H rats at P90 (C, D) and P120 (E, F), treated with unloaded PLGA MSs (C, E) or TUDCA-loaded PLGA MSs (D, F). (B) Higher magnification of panel A. Retinal ON rod bipolar cells were stained with the α isoform of protein kinase C (α-PKC) (in green), and synaptic ribbons in photoreceptors were labeled with Bassoon (in red). Nuclei were stained with TO-PRO (in blue). Note that the density of Bassoon-positive puncta (arrows) associated with dendritic tips of bipolar cells was higher in TUDCA-PLGA-MSs-treated than in vehicle-treated P23H rat retinas. ONL: outer nuclear layer, INL: inner nuclear layer, OPL: outer plexiform layer, IPL: inner plexiform layer, GCL: ganglion cell layer. Scale bar, 20 μm.
Fig 7.
Horizontal cells and their synaptic contacts in vehicle- and TUDCA-PLGA-MSs-treated eyes.
Retinal vertical sections from a P120 normal rat (Sprague Dawley, SD) (A, B) and P23H rats at P90 (C, D) and P120 (E, F), treated with unloaded PLGA MSs (C, E) or TUDCA-loaded PLGA MSs (D, F). Horizontal cells were stained for calbindin (in green), synaptic ribbons were labeled using anti-Bassoon antibodies (in red) and synaptic vesicles in the photoreceptor were stained using antibodies against VGLUT1 (in blue). Note that pairings between photoreceptor axon terminals and horizontal cell dendrites were more numerous in TUDCA-PLGA-MSs-treated retinas, as compared to those observed in vehicle-treated retinas. ONL: outer nuclear layer, INL: inner nuclear layer, OPL: outer plexiform layer. Scale bar, 20 μm.