Research ArticleNEUROSCIENCE

Age- and sex-dependent effects of metformin on neural precursor cells and cognitive recovery in a model of neonatal stroke

See allHide authors and affiliations

Science Advances  11 Sep 2019:
Vol. 5, no. 9, eaax1912
DOI: 10.1126/sciadv.aax1912
  • Fig. 1 In vitro and in vivo metformin treatment leads to an age- and sex-dependent expansion of the NSC pool.

    (A and B) Fold change in the number of neurospheres from the SVZ of neonatal (P8), juvenile (P17), and adult (7 weeks) females [(A) neonate: −Met, n = 16 mice; +Met, n = 16 mice; P < 0.001; juvenile: −Met, n = 7 mice; +Met, n = 7 mice; adult: −Met, n = 15 mice; +Met, n = 15 mice; over three to seven independent experiments per age group, P < 0.05; Student’s t test] and males [(B) neonate: −Met, n = 15 mice; +Met, n = 15 mice; P < 0.001; juvenile: −Met, n = 10 mice; +Met, n = 10 mice; adult: −Met, n = 11 mice; +Met, n = 11 mice; over four to five independent experiments per age group, Student’s t test] following in vitro metformin administration (1 μM). (C and D) Fold change in the number of neurospheres from the SVZ of neonatal (P8), juvenile (P17), and adult (7 weeks) females [(C) neonate: −Met, n = 4 mice; +Met, n = 5 mice; P < 0.05; juvenile: −Met, n = 9 mice; +Met, n = 8 mice; adult: −Met, n = 10 mice; +Met, n = 8 mice; P < 0.05; over three to five independent experiments per age group, Student’s t test] and males [(D) neonate: −Met, n = 6 mice; +Met, n = 6 mice; P < 0.05; juvenile: −Met, n = 6 mice; +Met, n = 5 mice; adult: −Met, n = 7 mice; +Met, n = 5 mice; over three to four independent experiments per age group, Student’s t test] following in vivo administration with vehicle or metformin (20 or 200 mg/kg). Experiments across different ages were analyzed using a Student’s t test. *P < 0.05, ***P < 0.005, ****P < 0.001. Met, metformin.

  • Fig. 2 Estradiol in the female niche is sufficient for the metformin-induced expansion of the NSC pool.

    (A) Experimental paradigm and fold change in the number of neurospheres from the SVZ of female juvenile mice that received estradiol (5.6 mg/kg) or vehicle for 11 days and metformin (200 mg/kg) or vehicle for 7 days in vivo (Est + vehicle, n = 9 mice; Est + Met, n = 8 mice; over five independent experiments; P < 0.05; Student’s t test). (B) Experimental paradigm and fold change in the number of neurospheres from control and ovariectomized mice following administration of vehicle or metformin (200 mg/kg) for 1 week in vivo (Ov + vehicle, n = 8 mice; Ov + Est, n = 8 mice; over three independent experiments; Student’s t test). (C) Fold change in the number of neurospheres passaged in the presence or absence of 1 μM metformin in vitro (−Met, n = 7 mice; +Met, n = 7 mice; over four experiments; Student’s t test). *P < 0.05. Est, estradiol; Met, metformin; Ov, ovariectomized; n.s., not significant.

  • Fig. 3 Testosterone inhibits the metformin-induced expansion of the stem cell pool.

    (A) Experimental paradigm and fold change in the number of neurospheres from the SVZ of male juvenile mice that received estradiol (5.6 mg/kg) or vehicle for 11 days and metformin (200 mg/kg) or vehicle for 7 days in vivo (Est + vehicle, n = 5 mice; Est + Met, seven mice from three to four independent experiments, Student’s t test). (B) Experimental paradigm and fold change in the number of neurospheres from male neonatal mice that received testosterone only or testosterone and metformin for 4 days in vivo (Test + vehicle, n = 7 mice; Test + Met, n = 7 mice over seven independent experiments; Student’s t test). (C) Experimental paradigm and fold change in the number of neurospheres from control and castrated males following vehicle or metformin (200 mg/kg) administration for 1 week in vivo (Cast + vehicle, n = 9 mice; Cast + Met, n = 9 mice; over three independent experiments; P < 0.05; Student’s t test). (D) Fold change in the number of neurospheres passaged in the presence or absence of 1 μM metformin in vitro (−Met, n = 7 mice; +Met, n = 7 mice; over four experiments; P < 0.01; Student’s t test). (E) Coculture paradigm and fold change in the number of neurospheres from YFP male cells plated in an overwhelmingly female environment, in the presence or absence of 1 μM metformin, in vitro (n = 9 independent experiments, P < 0.05, Student’s t test). (F) Coculture paradigm and fold change in the number of neurospheres from YFP female cells plated in an overwhelmingly male environment in the presence or absence of 1 μM metformin in vitro (n = 8 independent experiments, P < 0.05, Student’s t test). *P < 0.05, **P < 0.01. Cast, castrated; Est, estradiol; Met, metformin; Test, testosterone.

  • Fig. 4 Metformin has sex-dependent effects on proliferation but enhances neurogenesis in both females and males.

    (A) Experimental paradigm. (B) Average number of BrdU+ cells in the SVZ of adult females treated with vehicle or Met (200 mg/kg) for 1 week (Vehicle, n = 4 mice; Met, n = 4 mice; P < 0.05; Student’s t test). (C) BrdU+ cells (arrows) in the SVZ of vehicle- or Met-treated female mice. (D) Average number of BrdU+ cells in the SVZ of adult males treated with vehicle or Met (200 mg/kg) for 1 week (Vehicle, n = 3 mice; Met, n = 3 mice; Student’s t test). (E) BrdU+ cells (arrows) in the SVZ of vehicle- or Met-treated male mice. (F) Experimental paradigm. (G) Average number of Ki67 + Dcx + cells in the SVZ of female mice that received Met (200 mg/kg) for 1 week in vivo (Vehicle, n = 5 mice; Met, n = 5 mice; P < 0.05; Student’s t test). (H) Ki67+Dcx+ cells (arrowheads) in the SVZ of vehicle- or Met-treated female mice. (I) Average number of Ki67 + Dcx + cells in the SVZ of male mice that received Met (200 mg/kg) for 1 week in vivo (Vehicle, n = 4 mice; Met, n = 3 mice; P < 0.05; Student’s t test). (J) Ki67+Dcx+ cells (arrowheads) in the SVZ of vehicle- or Met-treated male mice. *P < 0.05. Scale bars, 50 μm. CC, corpus callosum; Dcx, doublecortin; LV, lateral ventricle; Met, metformin; Veh, vehicle.

  • Fig. 5 Long-term metformin treatment leads to cognitive improvements in females, but not males, following early postnatal H-I injury.

    (A) Experimental paradigm. (B) Trial times in the puzzle box task for female mice comparing naïve (uninjured), H-I injured, and H-I+ metformin-treated cohorts (Naïve, n = 11 mice; H-I, n = 13 mice; H-I + Met, n = 15 mice; *P < 0.05, **P < 0.01, F8,288 = 31.73) repeated-measures two-way ANOVA). (C) Trial times in the puzzle box task for male mice comparing naïve (uninjured), H-I injured, and H-I + metformin-treated cohorts, which resulted in a trial effect (Naïve, n = 8 mice; H-I, n = 11 mice; H-I + Met, n = 13 mice; *P < 0.05, **P < 0.01, F8,232 = 32.93 repeated-measures two-way ANOVA). *P < 0.05, **P < 0.01. Met, metformin.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaax1912/DC1

    Fig. S1. Metformin at 1 μM increases the number of neurospheres in an age- and sex-dependent manner.

    Fig. S2. Metformin has no effect on the number of neurospheres from the adult male SVZ regardless of metformin dose or duration of treatment.

    Fig. S3. The absolute number of neurospheres is the same in male and female C57 mice at each age examined.

    Fig. S4. Lack of sex-dependent effects of estradiol and testosterone on primary and secondary neurospheres from the SVZ of adult female and male mice.

    Fig. S5. Testosterone and metformin eliminate the effect of metformin on NPCs in the female SVZ.

    Fig. S6. Motor impairments do not account for the deficits in task acquisition in females and males.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Metformin at 1 μM increases the number of neurospheres in an age- and sex-dependent manner.
    • Fig. S2. Metformin has no effect on the number of neurospheres from the adult male SVZ regardless of metformin dose or duration of treatment.
    • Fig. S3. The absolute number of neurospheres is the same in male and female C57 mice at each age examined.
    • Fig. S4. Lack of sex-dependent effects of estradiol and testosterone on primary and secondary neurospheres from the SVZ of adult female and male mice.
    • Fig. S5. Testosterone and metformin eliminate the effect of metformin on NPCs in the female SVZ.
    • Fig. S6. Motor impairments do not account for the deficits in task acquisition in females and males.

    Download PDF

    Files in this Data Supplement:

Navigate This Article