A hypothermia mimetic molecule (zr17-2) reduces ganglion cell death, gliosis, and electroretinogram distortion in male rats subjected to perinatal asphyxia

Abstract

Introduction: Perinatal asphyxia (PA) represents a major problem in perinatology and may cause visual losses, including blindness. We, and others, have shown that hypothermia prevents retinal symptoms associated to PA. In the present work, we evaluate whether a hypothermia mimetic small molecule, zr17-2, has similar effects in the context of PA. Methods: Four experimental groups were studied in male rats: Naturally born rats as controls (CTL), naturally born rats injected s.c. with 50 µL of 330 nmols/L zr17-2 (ZR), animals that were exposed to PA for 20 min at 37°C (PA), and rats that were exposed to PA and injected with zr17-2 (PA-ZR). Forty-five days after treatment, animals were subjected to electroretinography. In addition, morphological techniques (TUNEL, H&E, multiple immunofluorescence) were applied to the retinas. Results: A reduction in the amplitude of the a- and b-wave and oscillatory potentials (OP) of the electroretinogram (ERG) was detected in PA animals. Treatment with zr17-2 resulted in a significant amelioration of these parameters (p < 0.01). In PA animals, a large number of apoptotic cells was found in the GCL. This number was significantly reduced by treatment with the small molecule (p < 0.0001). In a similar way, the thickness of the inner retina and the intensity of GFAP immunoreactivity (gliosis) increased in PA retinas (p < 0.0001). These parameters were corrected by the administration of zr17-2 (p < 0.0001). Furthermore, injection of the small molecule in the absence of PA did not modify the ERG nor the morphological parameters studied, suggesting a lack of toxicity. Discussion: In conclusion, our results indicate that a single s.c. injection of zr17-2 in asphyctic neonates may provide a novel and efficacious method to prevent the visual sequelae of PA.

Keywords: apoptosis; cold-shock proteins; electroretinogram; gliosis; hypothermia; hypothermia mimetics; perinatal asphyxia.

FIGURE 1

Schematic drawing of the experimental procedure. Normally delivered pups were assigned to the control groups. Perinatal asphyxia (PA) was generated by introducing the uterine horns in a water bath at 37°C for 20 min. Half of the animals were injected with zr17-2 and the other half with vehicle to generate the four experimental groups.

FIGURE 2

Small molecule zr17-2 prevents changes in the electroretinogram induced by perinatal asphyxia. (A) Representative electroretinograms of 45 day-old animals (n = 9 per group) subjected to PA with and without small molecule injection. The red line corresponds to the right eye whereas the blue line is the recording of the left eye. (B) Amplitude of the a-wave in the four experimental groups. (C) Amplitude of the b-wave in the four experimental groups. In both cases, perinatal asphyxia (PA) induced a significant decrease in the a- and b-wave compared to control (CTL), whereas the small molecule partially prevented it. Each bar represents the mean ± SEM of 9 animals. Two way ANOVA. Asterisks indicate significant differences. *:p < 0.05; **:p < 0.01; ***:p < 0.001; ****:p < 0.0001.

FIGURE 3

Small molecule zr17-2 prevents changes in the oscillatory potentials induced by perinatal asphyxia. (A) Representative oscillatory potentials (OP) of the electroretinograms of 45 day-old animals (n = 9 per group) subjected to PA with and without small molecule injection. The red line corresponds to the right eye whereas the blue line is the recording of the left eye. (B) Sum of amplitudes of the OP in the four experimental groups. Perinatal asphyxia (PA) induced a significant decrease in the OPs compared to control (CTL), whereas the small molecule prevented it. Each bar represents the mean ± SEM of 9 animals. Two way ANOVA. Asterisks indicate significant differences. **:p < 0.01; ****:p < 0.0001.

FIGURE 4

Apoptosis induced by perinatal asphyxia is prevented by treatment with the small molecule. Representative images of apoptotic cells (red arrows) localized in the ganglion cell layer as labeled by the TUNEL assay in the four experimental groups (A). Scale bar: 40 µm. Graphical representation of TUNEL-positive cell number (B). The PA group showed a significant increment in the number of TUNEL positive cells compared to the CTL group. The small molecule (PA-ZR) significantly prevented apoptosis induction. Two way ANOVA. Each bar represents the mean ± SEM of 5 animals. Asterisks indicate significant differences. ****:p < 0.0001.

FIGURE 5

Inner retina thickening is prevented by the small molecule. Representative histological images of the retina of animals of the 4 experimental groups, taken 45 days after birth, and stained with hematoxylin-eosin (A). Three layers of the retina are labeled in the pictures for reference: outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL). A black vertical bar demarcates the inner retina (IR). Scale bar = 50 μm. Quantification of the IR thickness is shown as a histogram (B). Bars represent the mean ± SEM of all samples (n = 5 animals per group, 4 measurements per animal). Asterisks represent statistically significant differences. ****p < 0.0001. Statistical test: Two way ANOVA followed by Holm-Sidak post-hoc test.

FIGURE 6

The small molecule prevents gliosis. Representative confocal microscopy images, labeled with an antibody against GFAP (red), of the retina of animals of the 4 experimental groups taken 45 days after birth. Nuclei were counterstained with DAPI (blue) (A). Three layers of the retina are labeled in the pictures for reference: outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL). Size bar = 20 μm. Graphical representation of the intensity of GFAP staining in the inner retina (B). Two way ANOVA. Each bar represents the mean ± SEM of 5 animals. Asterisks indicate significant differences. ****:p < 0.0001.