A hypothermia mimetic molecule (zr17-2) reduces ganglion cell death and electroretinogram distortion in a rat model of intraorbital optic nerve crush (IONC)

Abstract

Introduction: Ocular and periocular traumatisms may result in loss of vision. Our previous work showed that therapeutic hypothermia prevents retinal damage caused by traumatic neuropathy. We also generated and characterized small molecules that elicit the beneficial effects of hypothermia at normal body temperature. Here we investigate whether one of these mimetic molecules, zr17-2, is able to preserve the function of eyes exposed to trauma. Methods: Intraorbital optic nerve crush (IONC) or sham manipulation was applied to Sprague-Dawley rats. One hour after surgery, 5.0 µl of 330 nmol/L zr17-2 or PBS, as vehicle, were injected in the vitreum of treated animals. Electroretinograms were performed 21 days after surgery and a- and b-wave amplitude, as well as oscillatory potentials (OP), were calculated. Some animals were sacrificed 6 days after surgery for TUNEL analysis. All animal experiments were approved by the local ethics board. Results: Our previous studies showed that zr17-2 does not cross the blood-ocular barrier, thus preventing systemic treatment. Here we show that intravitreal injection of zr17-2 results in a very significant prevention of retinal damage, providing preclinical support for its pharmacological use in ocular conditions. As previously reported, IONC resulted in a drastic reduction in the amplitude of the b-wave (p < 0.0001) and OPs (p < 0.05), a large decrease in the number of RGCs (p < 0.0001), and a large increase in the number of apoptotic cells in the GCL and the INL (p < 0.0001). Interestingly, injection of zr17-2 largely prevented all these parameters, in a very similar pattern to that elicited by therapeutic hypothermia. The small molecule was also able to reduce oxidative stress-induced retinal cell death in vitro. Discussion: In summary, we have shown that intravitreal injection of the hypothermia mimetic, zr17-2, significantly reduces the morphological and electrophysiological consequences of ocular traumatism and may represent a new treatment option for this cause of visual loss.

Keywords: apoptosis; electroretinogram; hypothermia mimetics; intravitreal injection; therapeutic hypothermia; traumatic optic neuropathy.

FIGURE 1

Schematic drawing of the different treatments generating the four experimental groups of eyes used in this study: i) Control eyes (CTL) sham operated and injected with PBS, ii) Traumatism control (intraorbital optic nerve crush, IONC) and injected with PBS, iii) Control ZR (CTL ZR) sham operated and injected with zr17-2, iv) Treated traumatism IONC operated and injected with zr17-2 (IONC ZR). The experimental procedure timeline is indicated, as well as the small molecule structure.

FIGURE 2

Electroretinograms and quantification of the a- and b-waves on the four experimental groups. Representative electroretinograms of vehicle- (A) and zr17-2-treated (B) animals subjected to IONC (red lines) or sham-operated (blue lines). The a- (C) and b-waves (D) were quantified. Bars represent the mean ± SD of all animals (n = 9 per experimental group). Asterisks represent statistically significant differences (ANOVA). *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

FIGURE 3

Electroretinograms and quantification of oscillatory potentials on the four experimental groups. Representative electroretinograms of vehicle- (A) and zr17-2-treated (B) animals subjected to IONC (red lines) or sham-operated (blue lines). Oscillatory potentials of the four groups were quantified (C). Bars represent the mean ± SD of all animals (n = 9 per experimental group). Asterisks represent statistically significant differences (ANOVA). *: p < 0.05; ****: p < 0.0001.

FIGURE 4

Anatomical structure of the retinas on the four experimental groups. Representative sections of the retina of treated animals stained with hematoxylin and eosin (A). The nuclear layers of the retina are indicated: outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL). Size bar = 50 µm. Quantification of the number of ganglion cells per 1,000 µm (B). Bars represent the mean ± SD of all animals (n = 21 fields per experimental group). Asterisks represent statistically significant differences (ANOVA). ****: p < 0.0001.

FIGURE 5

TUNEL positive cells on the four experimental groups. Representative images of retinas from animals in the four experimental groups taken 6 days after surgery (A). The nuclear layers of the retina are indicated: outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL). TUNEL positive cells (arrows) were found mainly in the GCL and the INL. Size bar = 20 μm. Quantification of the results for the GCL (B) and the INL (C) is shown as histograms. Bars represent the mean ± SD of all animals (n = 18 fields per experimental group). Asterisks represent statistically significant differences (ANOVA). ****: p < 0.0001.

FIGURE 6

R28 cells responses to Al (mal)₃ and zr17-2. First, a toxicity assay was performed to determine the IC₅₀ of Al (mal)₃ on R28 (A). Then, cells were exposed to 200 µM Al (mal)₃ and increasing concentrations of zr17-2. Aluminum maltolate drastically reduced cell number whereas zr17-2 significantly prevented cell death in a dose-dependent fashion (B). Bars represent the mean ± SD of all wells (n = 8 per experimental condition). Asterisks represent statistically significant differences with the Al (mal)₃ treatment (second bar) (ANOVA). *: p < 0.05; **: p < 0.01.