Progesterone treatment shows greater protection in brain vs. retina in a rat model of middle cerebral artery occlusion: Progesterone receptor levels may play an important role

Abstract

Background/objective: To determine whether inflammation increases in retina as it does in brain following middle cerebral artery occlusion (MCAO), and whether the neurosteroid progesterone, shown to have protective effects in both retina and brain after MCAO, reduces inflammation in retina as well as brain.

Methods: MCAO rats treated systemically with progesterone or vehicle were compared with shams. Protein levels of cytosolic NF-κB, nuclear NF-κB, phosphorylated NF-κB, IL-6, TNF-α, CD11b, progesterone receptor A and B, and pregnane X receptor were assessed in retinas and brains at 24 and 48 h using western blots.

Results: Following MCAO, significant increases were observed in the following inflammatory markers: pNF-κB and CD11b at 24 h in both brain and retina, nuclear NF-κB at 24 h in brain and 48 h in retina, and TNF-α at 24 h in brain.Progesterone treatment in MCAO animals significantly attenuated levels of the following markers in brain: pNF-κB, nuclear NF-κB, IL-6, TNF-α, and CD11b, with significantly increased levels of cytosolic NF-κB. Retinas from progesterone-treated animals showed significantly reduced levels of nuclear NF-κB and IL-6 and increased levels of cytosolic NF-κB, with a trend for reduction in other markers. Post-MCAO, progesterone receptors A and B were upregulated in brain and downregulated in retina.

Conclusion: Inflammatory markers increased in both brain and retina after MCAO, with greater increases observed in brain. Progesterone treatment reduced inflammation, with more dramatic reductions observed in brain than retina. This differential effect may be due to differences in the response of progesterone receptors in brain and retina after injury.

Keywords: Focal ischemia; NF-kB; inflammation; middle cerebral artery occlusion; progesterone; progesterone receptor; rat; retina; retinal ischemia.

Fig. 1

Increased NF-κB pathway activation in brain and retina following MCAO was attenuated by progesterone treatment. Phosphorylated NF-κB expression in brain (A) and retina (B). Nuclear NF-κB expression in brain (C) and retina (D). Cystosolic NF-κB expression in brain (E) and retina (F). Expression of NF-κB pathway markers as determined by western blot and quantified by densitometry. Bands were normalized to the appropriate loading control (β-Actin for cytosolic blots and Histone H3 for nuclear blots). Results expressed as means ± SD. * = p<0.03; ** = p<0.01; *** = p < 10.001.

Fig. 1

Increased NF-κB pathway activation in brain and retina following MCAO was attenuated by progesterone treatment. Phosphorylated NF-κB expression in brain (A) and retina (B). Nuclear NF-κB expression in brain (C) and retina (D). Cystosolic NF-κB expression in brain (E) and retina (F). Expression of NF-κB pathway markers as determined by western blot and quantified by densitometry. Bands were normalized to the appropriate loading control (β-Actin for cytosolic blots and Histone H3 for nuclear blots). Results expressed as means ± SD. * = p<0.03; ** = p<0.01; *** = p < 10.001.

Fig. 2

Increased cytokine levels in brain and retina following MCAO were attenuated by progesterone treatment. IL-6 expression in brain (A) and retina (B). TNF-α expression in brain (C) and retina (D). CD11b expression in brain (E) and retina (F). Expression of inflammatory markers as determined by western blot. Bands were quantified by densitometry and normalized to β-Actin loading controls. Results expressed as means ± SD. * = p < 0.03; ** = p < 0.01; *** = p < 0.001.

Fig. 2

Increased cytokine levels in brain and retina following MCAO were attenuated by progesterone treatment. IL-6 expression in brain (A) and retina (B). TNF-α expression in brain (C) and retina (D). CD11b expression in brain (E) and retina (F). Expression of inflammatory markers as determined by western blot. Bands were quantified by densitometry and normalized to β-Actin loading controls. Results expressed as means ± SD. * = p < 0.03; ** = p < 0.01; *** = p < 0.001.

Fig. 3

Progesterone receptor was upregulated in brain and downregulated in retina after MCAO. PR-A expression in brain (A) and retina (B). PR-B expression in brain (C) and retina (D). Expression of inflammatory markers as determined by western blot. Bands were quantified by densitometry and normalized to β-Actin loading controls. Results expressed as means ± SD. * = p < 0.03; ** = p < 0.01; *** = p < 0.001.

Fig. 4

PXR was upregulated in brain but downregulated in retina after MCAO. PXR expression in brain (A) and retina (B). Expression of inflammatory markers as determined by western blot. Bands were quantified by densitometry and normalized to β-Actin loading controls. Results expressed as means ± SEM. * = p < 0.03; ** = p < 0.01; *** = p < 0.001.

Fig. 5

Progesterone treatment after MCAO resulted in greater improvement on tests of behavioral function (Grip Strength and Sticky Tape Tasks) vs. retinal function (electroretinogram; ERG). Results are normalized to sham and expressed as % recovery with progesterone, means ± SD. These data were reanalyzed from behavioral data presented in Allen et al., 2015, so that we could determine whether progesterone’s effects on behavioral vs. retinal function concurred with the larger reductions in inflammatory markers observed in brain vs. retina in progesterone-treated MCAO rats (Allen et al., 2015).