Progesterone exerts neuroprotective effects and improves long-term neurologic outcome after intracerebral hemorrhage in middle-aged mice

Abstract

In this study, we examined the effect of progesterone on histopathologic and functional outcomes of intracerebral hemorrhage (ICH) in 10- to 12-month-old mice. Progesterone or vehicle was administered by intraperitoneal injection 1 hour after collagenase-induced ICH and then by subcutaneous injections at 6, 24, and 48 hours. Oxidative and nitrosative stress were assayed at 12 hours post-ICH. Injury markers were examined on day 1, and lesion was examined on day 3. Neurologic deficits were examined for 28 days. Progesterone posttreatment reduced lesion volume, brain swelling, edema, and cell degeneration and improved long-term neurologic function. These protective effects were associated with reductions in protein carbonyl formation, protein nitrosylation, and matrix metalloproteinase-9 activity and attenuated cellular and molecular inflammatory responses. Progesterone also reduced vascular endothelial growth factor expression, increased neuronal-specific Na(+)/K(+) ATPase ɑ3 subunit expression, and reduced protein kinase C-dependent Na(+)/K(+) ATPase phosphorylation. Furthermore, progesterone reduced glial scar thickness, myelin loss, brain atrophy, and residual injury volume on day 28 after ICH. With multiple brain targets, progesterone warrants further investigation for its potential use in ICH therapy.

Keywords: Inflammatory response; Intracerebral hemorrhage; Neurologic function; Neuroprotective effects; Progesterone.

Fig. 1

Progesterone (PROG) decreases brain lesion volume and edema on day 3 after collagenase-induced ICH. (A) Representative images of Luxol fast blue/Cresyl Violet-stained brain sections on day 3 after collagenase-induced ICH. The area of the lesion is indicated by a lack of staining; scale bar = 2 mm. (B) Brain lesion volume was measured on Luxol fast blue/Cresyl Violet-stained brain sections. Quantification analysis revealed that brain lesion volume was smaller in the progesterone-treated group than in the vehicle-treated group 3 days after ICH (n=10–11 mice/group, *p<0.05). (C) Progesterone post-treatment reduced brain swelling after collagenase-induced ICH compared with that of the vehicle-treated group (n=10–11 mice/group, *p<0.05). (D) Progesterone post-treatment reduced brain water content in the ipsilateral striatum after collagenase-induced ICH compared with that of the vehicle-treated group (n=6 mice/group, *p<0.05). All values are means ± SD. Ipsi, ipsilateral; Contra, contralateral.

Fig. 2

Progesterone (PROG) post-treatment ameliorates neurologic deficits after collagenase-induced ICH. (A) Progesterone post-treatment improved the neurologic function of mice on days 7, 14, 21, and 28 after ICH compared with that of the vehicle-treated group (n=12 mice/group, *p<0.05). (B) Neurologic deficit scores for each of the individual tests on days 1, 3, 7, 14, 21, and 28 after ICH (n=12 mice/group, all p>0.05). Values are means ± SD.

Fig. 3

Progesterone (PROG) decreases oxidative stress and MMP-9 activity after collagenase-induced ICH. Immunoblotting analysis showed that progesterone decreased protein carbonyl (A) and 3-nitrotyrosine (B) immunoreactivity on multiple protein bands at 12 hours after ICH. (C) Representative gel showing relative activity of MMP-2 and MMP-9 on day 1 after ICH in brain lysates from sham and ICH mice post-treated with vehicle or progesterone. Bar graphs show the quantitative analysis of nitrotyrosine (A, 14 to 191 kDa), carbonyls (B, 29 to 98 kDa), and MMP-2 and MMP-9 activity (C) from each group (n=5 mice for each treatment group, and n=3 mice for sham group). All data are shown as mean ± SD. *p<0.05 vs. ICH+vehicle.

Fig. 4

Progesterone (PROG) reduces microglial/macrophage and astrocytic activation, neutrophil infiltration, and Fluoro-Jade B (FJB)⁺ cell number after collagenase-induced ICH. (A–C) Immunostaining for Iba1 (A), myeloperoxidase (MPO; B), and glial fibrillary acidic protein (GFAP; C) in the perihematomal region on day 3 after collagenase-induced ICH. Inset represents higher magnification of MPO-positive neutrophil. Scale bar = 50 μm. (D) FJB histological staining of degenerating cells in sections collected 3 days after collagenase injection. Inset represents higher magnification of FJB-positive cell. Scale bar = 25 μm. (E–G) Bar graphs show quantification analysis of activated microglia/macrophages and astrocytes, infiltrating neutrophils, and FJB-positive cells (n=6 mice/group, *p<0.01 or 0.05 versus vehicle-treated group). Values are means ± SD.

Fig. 5

Progesterone (PROG) decreases HMGB-1, IL-1β, and VEGF expression on day 1 after collagenase-induced ICH. (A) Representative Western blot showing relative protein expression of COX-1, COX-2, HMGB-1, IL-1β, and VEGF in brain lysates from sham and ICH rats post-treated with vehicle or progesterone. β-Actin was used as a loading control (n=5 mice for each treatment group, and n=3 for sham group). (B–D) Bar graphs show the quantitative analysis of COX-1, COX-2, HMGB-1, IL-1β, and VEGF expression from each group (n=5 mice for each treatment group, and n=3 mice for sham group, ^#p<0.05 or *p<0.01 versus vehicle-treated group). Values are means ± SD.

Fig. 6

Progesterone (PROG) increases protein expression of Na⁺/K⁺ ATPase (NKA) α3, but decreases protein expression of NKA pSer-23 on day 1 after collagenase-induced ICH. (A and B) Representative Western blots showing relative protein expression of NKA α1 and NKA α3 (A), and NKA pSer23 and NKA pSer943 (B) in brain lysates from sham-operated and ICH rats post-treated with vehicle or progesterone. β-Actin was used as a loading control. (C and D) Bar graphs show the quantitative analysis of NKA α1, NKA α3, NKA pSer23, and NKA pSer943 expression from each group (n=5 mice for each treatment group, and n=3 mice for sham group, *p<0.01 versus vehicle-treated group). Values are means ± SD.

Fig. 7

Progesterone (PROG) post-treatment decreases glial scar thickness (astrogliosis), myelin loss, brain atrophy, and lesion volume on day 28 after collagenase-induced ICH. (A and B) Immunostaining for glial fibrillary acidic protein in the perihematomal region on day 28 after collagenase-induced ICH; scale bar = 50 μm. (C) Quantitative analysis of glial scar thickness (n=12 mice/group, *p<0.01 vs. ICH + vehicle). (D) Immunostaining with Luxol fast blue in the perihematomal region on day 28 after collagenase-induced ICH; scale bar = 50 μm. (E) Quantitative analysis of white matter damage (n=12 mice/group, *p<0.01 vs. ICH + vehicle). (F and G) Representative images of Luxol fast blue/Cresyl Violet-stained brain sections on day 28 after collagenase-induced ICH; scale bar = 2 mm. (H and I) Bar graphs show the quantitative analysis of brain atrophy (H) and lesion volume (I) from each group (n=12 mice/group). Quantitative data are shown as mean ± SD. *p<0.01 vs. ICH + vehicle.