Hypoxia and the expression of HIF-1alpha and HIF-2alpha in the retina of streptozotocin-injected mice and rats

Abstract

Decreases in retinal blood flow in diabetics could render the retina hypoxic. In mouse and rat models of diabetes, a decrease in retinal blood flow occurs early, within 3-4 weeks of the induction of hyperglycemia, although information is scarce on whether this early decrease in flow induces hypoxia. The purpose of the current study was to determine whether hypoxia-inducible factor (HIF) levels increase following 4 and/or 12 weeks of hyperglycemia in streptozotocin (STZ)-injected mouse (C57BL/6) and rat (Wistar) retinas. Additionally, retinal tissue hypoxia was measured with pimonidazole following 12 weeks of hyperglycemia. These aims were accomplished via immunostaining of cross-sections from enucleated eyes. In mice, staining for HIF-1alpha and HIF-2alpha showed a contrasting pattern, with HIF-1alpha higher in the inner retina than outer, but HIF-2alpha higher in the outer retina than inner. However, in rats, staining for both HIF-1alpha and HIF-2alpha was more intense in the inner retina. The HIF-1alpha staining intensities and patterns were similar between diabetic animals and their non-diabetic counterparts following 4 and 12 weeks of hyperglycemia. The same was true for HIF-2alpha except for a trend toward an increase following 12 weeks of hyperglycemia in mice. Pimonidazole staining showed significant decreases throughout all layers of the central retina and most layers of the peripheral retina of rats (but not mice), following 12 weeks of hyperglycemia. In summary, despite early decreases in flow in rats and mice, retinal HIF-1alpha and HIF-2alpha were not found to be increased, and the extent of hypoxia may even decrease after 12 weeks of hyperglycemia in rats.

Figure 1

HIF-1α fluorescent intensity values in frozen cross-sections of the retina of mice (panel A) and rats (panel B). N= 7 control mice, 7 STZ mice, 8 control rats, and 11 STZ rats, with the STZ injected 4 weeks prior to collection of the eye. * p<0.05 between the indicated groups.

Figure 2

Representative cross-sectional images of HIF-1α staining from the central retina of control and diabetic rats. Panels A and D show HIF-1α staining, panels B and E show the DAPI counterstain, and panels C and F show the merged HIF-1α/DAPI overlay. The magnified section from panel A is labeled for the ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), and photoreceptor layer (PR).

Figure 3

HIF-1α fluorescent intensity values in frozen cross-sections of the retina of mice (panel A) and rats (panel B). N= 8 control mice, 6 STZ mice, 8 control rats, and 9 STZ rats, with the STZ injected 12 weeks prior to collection of the eye.

Figure 4

HIF-2α fluorescent intensity values in frozen cross-sections of the retina of mice (panel A) and rats (panel B). N= 7 control mice, 7 STZ mice, 8 control rats, and 9 STZ rats, with the STZ injected 4 weeks prior to collection of the eye.

Figure 5

HIF-2α fluorescent intensity values in frozen cross-sections of the retina of mice (panel A) and rats (panel B). N= 8 control mice, 6 STZ mice, 7 control rats, and 8 STZ rats, with the STZ injected 12 weeks prior to collection of the eye. * p<0.05 between the indicated groups.

Figure 6

Representative cross-sectional images of HIF-2α staining from the peripheral retina of control and diabetic mice. Panels A and D show HIF-2α staining, panels B and E show the DAPI counterstain, and panels C and F show the merged HIF-2α/DAPI overlay.

Figure 7

Anti-pimonidazole (hypoxia) fluorescent intensity values in frozen cross-sections of the retina of mice (panel A) and rats (panel B). N= 8 control mice, 6 STZ mice, 7 control rats, and 8 STZ rats, with the STZ injected 12 weeks prior to collection of the eye. * p<0.05, ** p<0.01, and *** p<0.001 between indicated groups.

Figure 8

Representative cross-sectional images of anti-pimonidazole (hypoxia) staining from the central retina of control and diabetic rats. Panels A and D show anti-pimonidazole staining, panels B and E show the DAPI counterstain, and panels C and F show the merged anti-pimonidazole/DAPI overlay.