1. Aim and background
We previously showed that hiPSCs-derived Müller cells behaved as primary human Müller cells to high glucose and high lipid exposure. Palmitic Acid (PA), the most elevated circulating free fatty acid in humans, was demonstrated to induce a strong Müller glial cell activation , resulting in a significantly increased expression of pro-inflammatory factors, such as ATF3 and IL8, as well as of pro-angiogenic factors such as VEGF and ANGPTL4.
Several in vitro studies have demonstrated the stimulating effect of free fatty acids on either Müller glial cells (Capozzi ME et al., 2018) or on endothelial cells (Othman A et al., 2013;
Capozzi ME et al., 2016). However, only few evidence exits regarding their effect on the retina in vivo (Naveh-Floman N et al., 1984). We thus aimed to investigated the effect of PA in vivo.
2. Preliminary results
a) Intravitreal injection of palmitic acid
To confirm the reaction of activation of Müller glial cells to high lipid exposure, we studied the effect of intravitreal injection of palmitic acid (PA) in C57Bl6/J male mice. The first experiment aimed to analyze Müller glial cell reaction after one intravitreal injection of PA compared with one intravitreal injection of PBS. Mice were sacrificed 24 hours and 96 hours after the intravitreal injection. The relative expression of GFAP and VEGF in the entire retina was determined by quantitative RT-PCR.
No significant increase in GFAP expression indicative of a glial reaction in Müller glial cells, as well as no increase in VEGF expression were detected (Figure 31).
Figure 31 : Relative expression of GFAP and VEGF in the entire retina of C57Bl6/J mice after one intravitreal injection of either palmitic acid or PBS.
A) The mean value of GFAP expression in the retina was of 0.7616 and 0.3735 at 24h and 96h respectively after PBS intravitreal injection; and was of 0.6836 and 0.8235 at 24h and 96h respectively after PA intravitreal injection (p = 0.8519 at 24h and 0.1240 at 96h).
B) The mean value of VEGF expression in the retina was of 0.9837 and 0.4258 at 24h and 96h respectively after PBS intravitreal injection; and was of 1.001 and 0.5775 at 24h and 96h respectively after PA intravitreal injection (p = 0.9695 at 24h and 0.2862 at 96h).
All results were normalized with expression of S26 and expressed as mean ± SEM (n = 3 in each group)
In a second experiment, we performed three intravitreal injections of PA in C57Bl6/J male mice, at baseline, 48 hours and 7 days to increase PA intravitreal level. Mice were sacrificed 24 hours after the third intravitreal injection. The relative expression of GFAP and VEGF in the entire retina was determined by quantitative RT-PCR.
Similarly to the first experiment, ,no significant increase in GFAP expression or VEGF expression in Müller glial cells were detected.
b) Sub-retinal injection of palmitic acid
Given the property of the internal limiting membrane, we hypothesized that intravitreal injection of PA failed to activate Müller glial cells because it was not able to enter the retina and reach the glial cells. In human, fatty acids may enter the retina via the blood circulation, in case of blood-retinal barrier rupture. We then conducted an experiment to analyze glial cell
reaction after sub-retinal injection of PA. The relative expression of VEGF, ANGPTL4 and IL6 in the entire retina was determined by quantitative RT-PCR.
A non-significant increase in VEGF, ANGPTL4 and IL6 relative expression was detected 24 hours after one sub-retinal injection of PA (Figure 32).
Figure 32 : Relative expression of VEGF, ANGPTL4 and IL6 in the entire retina of C57Bl6/J mice 24 hours after one sub-retinal injection of either palmitic acid or PBS (control).
A) The mean value of VEGF expression in the retina was of 0.8463 and 0.9733 after PBS and PA subretinal injection respectively (p = 0.3614).
B) The mean value of ANGPTL4 expression in the retina was of 0.5738 and 1.038 after PBS and PA subretinal injection respectively (p = 0.1661).
C) The mean value of IL6 expression in the retina was of 0.07444 and 0.3271 after PBS and PA subretinal injection respectively (p = 0.2330).
All results were normalized with expression of S26 and expressed as mean ± SEM (n = 6 in the control group and n = 8 in the PA-treated group)
c) Total retinal explants exposed to palmitic acid
We also analyzed the reaction of total retinal explants cultured in PA medium. As previously described, total retinal explants were obtained by entire retina dissection from C57/Bl6 mice and were incubated in either low glucose fatty acid-free medium (DMEM with 5 mM glucose plus BSA 0,83 %, plus ethanol 0,5 % and 1.8g mannitol for osmotic control); or in low glucose medium with PA (DMEM with 5 mM glucose plus BSA-bound PA 0.5 mM and 1.8g mannitol for osmotic control) for 48 hours.
A significant increase of ANGPTL4 and IL6 relative expression was detected in the entire retinal after a 48-hours-PA exposure (Figure 33).
Figure 33 : Relative expression of ANGPTL4 and IL6 in total retinal explants obtained from C57Bl6/J mice and cultured for 24 hours in either low glucose fatty acid-free medium palmitic acid (control) or in low glucose medium with palmitic acid (Palm).
A) The mean value of ANGPTL4 expression in the retina was of 3.522 in control explants versus 7.198 in PA-treated explants (p = 0.0301).
B) The mean value of IL6 expression in the retina was of 0.2562 in control explants versus 0.6770 in PA-treated explants (p = 0.0041). All results were normalized with expression of S26 and expressed as mean ± SEM (n = 6 in each group)
3. Perspectives
In these preliminary results, we showed that palmitic acid did not exert any significant pro-inflammatory or pro-angiogenic effect on retinal Müller glial cells, when injected intravitreally or sub-retinally. These results might be explained by the only slight contact between PA and the Müller glial cells using these injection routes in non-diabetic wild-type mice, having preserved inner limiting membrane and preserved blood-retinal barrier.
It may be of interest to study the effect of sub-retinal injections of PA in an animal model of blood-barrier rupture or in newborns animals. The potential pro-angiogenic effect of PA on Müller glial cells could also be studied in vitro in oxygen-induced retinopathy model.
III. Histopathologic study of hyperreflective fluid in post-mortem