Betulinic Acid Protects from Ischemia-Reperfusion Injury in the Mouse Retina

Abstract

Ischemia/reperfusion (I/R) events are involved in the pathophysiology of numerous ocular diseases. The purpose of this study was to test the hypothesis that betulinic acid protects from I/R injury in the mouse retina. Ocular ischemia was induced in mice by increasing intraocular pressure (IOP) to 110 mm Hg for 45 min, while the fellow eye served as a control. One group of mice received betulinic acid (50 mg/kg/day p.o. once daily) and the other group received the vehicle solution only. Eight days after the I/R event, the animals were killed and the retinal wholemounts and optic nerve cross-sections were prepared and stained with cresyl blue or toluidine blue, respectively, to count cells in the ganglion cell layer (GCL) of the retina and axons in the optic nerve. Retinal arteriole responses were measured in isolated retinas by video microscopy. The levels of reactive oxygen species (ROS) were assessed in retinal cryosections and redox gene expression was determined in isolated retinas by quantitative PCR. I/R markedly reduced cell number in the GCL and axon number in the optic nerve of the vehicle-treated mice. In contrast, only a negligible reduction in cell and axon number was observed following I/R in the betulinic acid-treated mice. Endothelial function was markedly reduced and ROS levels were increased in retinal arterioles of vehicle-exposed eyes following I/R, whereas betulinic acid partially prevented vascular endothelial dysfunction and ROS formation. Moreover, betulinic acid boosted mRNA expression for the antioxidant enzymes SOD3 and HO-1 following I/R. Our data provide evidence that betulinic acid protects from I/R injury in the mouse retina. Improvement of vascular endothelial function and the reduction in ROS levels appear to contribute to the neuroprotective effect.

Keywords: arterioles; betulinic acid; ischemia-reperfusion injury; reactive oxygen species; retina.

Figure 1

Total cell number in the ganglion cell layer (GCL) of the retina and axon number in the optic nerve. (A–D) Representative pictures of cells in the GCL stained with cresyl blue. Scale bar = 30 µm. (E) I/R markedly reduced the total cell number in the GCL in vehicle-treated mice but not in betulinic acid (BA)-treated mice (*** p < 0.001; * p < 0.05; n = 8 per group). (F–I) Representative pictures of optic nerve axons stained with toluidine blue. Scale bar = 30 µm. (J) I/R reduced axon number in the optic nerve in vehicle-treated mice but not in BA-treated mice (** p < 0.01; * p < 0.05; n = 8 per group).

Figure 2

Responses of retinal arterioles to vasoactive substances. (A) The thromboxane mimetic, U46619, elicited concentration-dependent vasoconstriction in retinal arterioles that was similar in all groups. (B) Likewise, responses to the endothelium-independent vasodilator, sodium nitroprusside (SNP), did not differ between the four groups. (C) In contrast, retinal arterioles from mice subjected to I/R displayed blunted endothelium-dependent vasodilator responses to acetylcholine, which were partially improved by treatment with BA. Values are expressed as the mean ± SE (* p < 0.05, I/R + vehicle versus vehicle; # p < 0.05, I/R + vehicle versus BA; + p < 0.05, I/R + vehicle versus I/R + BA; $ p < 0.05, I/R + BA versus vehicle; & p < 0.05, I/R + BA versus BA; n = 8 per group).

Figure 3

Dihydroethidium (DHE) staining in retinal cross-sections. (A–D) Representative pictures of retinal cross-sections from each group. Scale bar = 50 µm. (E–J) DHE staining intensity was markedly increased in retinal blood vessels from I/R- and vehicle-treated eyes (E). In none of the retinal layers, marked differences in DHE staining intensity were observed among groups (F–J) (GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; *** p < 0.001; * p < 0.05; n = 8 per group).

Figure 4

Messenger RNA expression of the hypoxic genes, HIF-1α and VEGF-A (A), the prooxidant genes (NOX1, NOX2, NOX4) (B), the antioxidant genes (SOD1, SOD2, SOD3, CAT, GPX1, HO-1) (C) and of the NOS genes (eNOS, nNOS, iNOS) (D) in the eyes treated with vehicle only, I/R + vehicle, betulinic acid (BA) only and I/R + BA. Notably, BA did not prevent upregulation of NOX2 expression induced by I/R. However, mRNA expression of the antioxidant redox genes, SOD3 and HO-1, was markedly increased in mice exposed to I/R and BA. Data are presented as the mean ± SE (** p  <  0.01; * p  <  0.05; n  =  8 per group).