Progesterone reduces brain mitochondrial dysfunction after transient focal ischemia in male and female mice

Animals and middle cerebral occlusion

Three-month-old female and male C57BL6J mice (Janvier, le Genest-Saint-Isle, France) were housed in a temperature-controlled room on a 12-h light, 12-h dark cycle. The estrus cycle was assayed by vaginal smear examination over 10 days to check cycling regularity. The classification was proestrus, estrus, metestrus and diestrus. Females in the diestrus stage on the day of the experiment were selected.

One-hour middle cerebral occlusion (MCAO) followed by reperfusion was realized according to Frechou et al. ⁷ The criterion of exclusion was a decrease of blood flow less than 50%. On this basis, no animals were excluded. Sham-operated mice underwent the same surgical procedure except that no filament was inserted. Experimenters were blinded to the experimental conditions of mice. Mice were randomly and blindly assigned to either progesterone (8 mg/kg in oleogel), or vehicle-treated (oleogel) groups (n = 5/group). Administrations were performed intranasally immediately on reperfusion. This formulation of progesterone is efficient to rapidly deliver progesterone to blood and brain (13 ± 2.5% and 9.5 ± 0.9% of the initial dose respectively after 10 min of administration), and the reached brain levels did not vary significantly over time (2–120 min). ⁸

Ethical statement

Procedures concerning animal care and use were carried out in accordance with national guidelines (authorization 94-345 to R.G., animal facility approval 94-043-13), French ethical laws (Act 87-848 and Act 2013-11) and European Communities Council Directive (86/609/EEC); and have been approved by the ethical committee of French Ministry of Higher Education and Research C2EA-26 (project 2014_029). All experiments were performed following the ARRIVE guidelines (www.nc3rs.org.uk).

Isolation of mitochondria-enriched fraction

Mice were decapitated 6 h after MCAO (i.e. 5 h after reperfusion), and the forebrain minus the cerebellum was rapidly dissected on ice. The mitochondria-enriched fraction was immediately isolated from the freshly dissected ischemic (ipsilateral) and contralateral hemispheres. ⁶ Based on the cytochrome c oxidase (also called complex IV, CIV) activity pelleted in the mitochondrial fraction, the yield was 46% ± 7% of total mitochondria. Only 1.9 ± 0.3% of the total activity of CIV in presence of 2.5 mM laurylmaltoside was measured in the absence of this detergent, indicating that the mitochondrial outer membrane was intact. The purity of the mitochondrial fraction (75 ± 3%) was evaluated by the percentage of a cytosolic enzyme activity (the lactate dehydrogenase, LDH) in the mitochondrial pellet fraction. The purity and yield of mitochondria isolation were consistent between contralateral and ischemic hemispheres (T test, p > 0.05) and similar in all individual mouse preparations (ANOVA, p > 0.05).

Mitochondrial oxygen consumption

Mitochondrial oxygen consumption was measured polarographically in the freshly prepared mitochondria-enriched fraction. ⁶ The rate of oxygen consumption was calculated from the slope of the response of mitochondria to the successive administration of substrates and expressed as nmol O₂/min/mg protein.

Enzymatic activities

RC complexes (CI, CII, CIII and CIV), LDH, isocitrate dehydrogenase (IDH), α-ketoglutarate dehydrogenase (AKGDH), malate dehydrogenase (MDH) and pyruvate dehydrogenase complex (PDHc) activities were spectrophotometrically measured at 37℃. ⁶ All enzymatic activities were expressed as nmol/min/mg protein.

RC complexes quantity and integrity

Blue-Native polyacrylamide gel electrophoresis followed by Western-Blot analysis was performed on a part of mitochondria-enriched fraction. ⁹ Solubilized OXPHOS (5 µg) proteins were loaded on a 4–16% gradient acrylamide non-denaturing gel (Invitrogen, CA, USA). Immunoblotting was performed with monoclonal antibodies (Abcam, MA, USA) against CI (subunit GRIM19, 1:1200), CII (subunit SDHA, 1:2400), CIII (subunit UQCRC2, 1: 3000) or CIV (subunit MT-CO1, 1:2000). The secondary antibody was a peroxidase-conjugated anti-mouse immunoglobulin G (IgG) (1:4000, Sigma, MO, USA). Signals were detected using an Amersham ECL plus revelation kit (GE Healthcare, NJ, USA) with a G:BOX Chemi system (Syngene, MD, USA). Quantification was performed using ImageJ software (NIH, MD, USA).

Oxidative stress markers

Reduced glutathione (GSH) and oxidized glutathione (GSSG) concentrations were measured by reverse phase HPLC coupled with electrochemical detection. ⁶ Limits of quantification were 0.6 μM for GSH and 0.9 μM for GSSG. Activities of the mitochondrial tricarboxylic acid (TCA) cycle enzymes aconitase and fumarase were spectrophotometrically measured, and the ratio of the activities of aconitase to fumarase was calculated. ⁶ A decrease of the aconitase to fumarase activity ratio is a functional indicator of ROS production: the iron–sulfur cluster in the catalytic site of aconitase renders it very susceptible to inactivation by ROS whereas the activity of fumarase is ROS-insensitive.

Statistical analysis

For each analysis, the ratio between the ipsilateral and the contralateral hemisphere was calculated. Data were expressed as mean ± SEM and were analyzed by a commercially available program (GraphPad Prism 4.01, Graphpad Inc., CA, USA). One-way ANOVA analysis followed by Tukey post-hoc test was performed, and p values < 0.05 were considered statistically significant.

Mitochondrial oxidative stress

Ischemia reduced the GSH mitochondrial pool in males and females (respectively−23%, p < 0.05 and−24%, p < 0.05 versus Sham) and progesterone treatment restored it ( + 23%, p < 0.05 in males and + 22%, p < 0.05 in females versus MCAO) (Figure 1(a)). Mitochondrial GSSG was below the limit of quantification for all samples. Ischemia-induced oxidative damages in males and females as shown by a decrease of the aconitase to fumarase activities ratio (respectively −19%, p < 0.05 in males and −34%, p < 0.05 in females versus Sham). Progesterone did not significantly re-establish a normal ratio ( + 13%, p = 0.09 in males and + 25%, p = 0.07 in females versus MCAO) (Figure 1(b)). OPEN IN VIEWER

Mitochondrial respiration

Mitochondrial oxygen consumptions using pyruvate (nicotinamide adenine dinucleotide(NADH)-linked respiration) and succinate (flavin adenine dinucleotide (FADH₂)-linked respiration) were measured with adenosine diphosphate (ADP) (stimulation), oligomycin (ATP-synthase inhibitor) and m-chlorophenylhydrazone (m-Cl-CCP, uncoupler) (Figure 2(a)). OPEN IN VIEWER

PDHc and TCA cycle enzymes

PDHc activity, the first step of pyruvate oxidation, was not significantly modified by ischemia or by progesterone administration. No significant effects were observed for IDH, AKGDH and MDH activities, the TCA cycle enzymes involved in NADH/NAD recycling (Figure 2(d)).

RC complexes

In males, neither activity nor quantity and integrity of the CI, CII, CIII and CIV were modified by ischemia and by progesterone treatment. In females, the CII activity decreased after MCAO (−17%, p < 0.05 versus Sham), without change in CII quantity and integrity. Progesterone treatment did not significantly restore C II activity ( + 15% versus MCAO, p = 0.09) (Figure 2(e)).