Fig 1.
Retinal morphometry analysis in nondiabetic (control) and diabetic (STZ) wild-type (Trpv4+/+) and Trpv4-/- mice.
(A) Representative images of hematoxylin/eosin-stained retinas and (B) corresponding quantification of retinal thickness, cell density per mm2, and nuclear eccentricity. Retinal pigment epithelium (RPE), outer segments (OS), outer nuclear layer (ONL), outer limiting membrane (OLM), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). From each of six animals per group, three equally distributed sections (300 μm x 100 μm) throughout the entire retina were analyzed. Scale bar, 100 μm. (C) Representative DWI images with enlargements corresponding to white squares in control and STZ Trpv4+/+ and Trpv4-/- mouse eyes. (D) Plots represent the thickness measurements (μm, mean ± s.e.m.; n = 8–11, N = 3) for retinas of control and STZ Trpv4+/+ (white) and Trpv4-/- (grey) mice in DWI images. *, significant differences of P < 0.05. n.s., not significant.
Fig 2.
Water diffusion, BRB permeability, nuclear distance distribution, and nuclear area in retinas of control and STZ Trpv4+/+ and Trpv4-/- mice.
(A) Representative images of ADC maps in control and STZ Trpv4+/+ and Trpv4-/- mouse eyes. ADC values between 0 and 0.6 μm2/s are displayed in color to enhance contrast, as the retina shows markedly reduced diffusion as compared to the vitreous. (B) Summary of ADC values in three separate groups of control and STZ Trpv4+/+ and Trpv4-/- mice. (C) Evaluation of the Evans blue dye content in retinas from control and STZ Trpv4+/+ and Trpv4-/- mice. For B and C, values are mean ± s.e.m. (n = 9–12 per group). (D) Representative fields (90 μm x 50 μm), randomly chosen in hematoxylin/eosin-stained retina images from control and STZ Trpv4+/+ and Trpv4-/- mice, to illustrate how the distances between the center of the nucleus and all immediate neighbouring nuclei have been measured; summary of the distribution of mean internuclear distance evaluated in 3 fields per groups. Red line corresponds to data distribution. Scale bar, 50 μm. (E) Summary of the mean nuclear area in the ONL, INL, and GCL of control and STZ Trpv4+/+ and Trpv4-/- mice (n = 3, N = 6). *, significant differences of P < 0.05; n.s., not significant.
Fig 3.
Retinal thickness assessment in control and STZ mice that received a single intravitreal injection of TRPV4 antagonist GSK2193874 on the left eye and vehicle (PBS) on the right eye, 24 h before the end of the 4-week STZ treatment.
(A) Representative images of non-diffusion weighted images and enlargement corresponding to white boxes. Signal (S) is shown normalized with respect to the median signal intensity of the entire volume (Smedian). Approximate locations of vitreous, retina, and choroid-RPE complex are indicated in vertical black and gray lines. (B) Representative DWI showing the retina as a hyperintense band. (C) Summary of retinal thickness measurements from control and STZ mice treated with vehicle (white) and GSK2193874 (grey) in DWI images (n = 10–12, N = 4). *, significant differences of P < 0.05; n.s., not significant. (D) Corresponding plot of retinal-segment analysis (μm, mean ± s.e.m.). Vehicle-treated STZ retinas were significantly thinner than vehicle-treated control ones. GSK2193874-treated STZ retinas had similar thickness than vehicle-treated ones, except in the nasal central part of the retina (10–30 °, red box).
Fig 4.
Water diffusion in control and STZ mice that received a single intravitreal injection of TRPV4 antagonist GSK2193874 on the left eye and vehicle (PBS) on the right eye, 24 hours before the end of the 4-week STZ treatment.
(A) Representative images of ADC maps. (B) Summary of ADC and (C) corresponding retinal segment analysis in four separate groups. Error bars correspond to s.e.m. *, significant difference (P < 0.05); n.s., not significant. In C, vehicle-treated STZ retinas had a significantly higher ADC than that of vehicle-treated control retinas. The ADC of GSK2193874-treated STZ retinas was not different from that of vehicle-treated retinas and GSK2193874-treated control retinas.