Low-dose metformin treatment in the subacute phase improves the locomotor function of a mouse model of spinal cord injury

Abstract

Metformin, a first-line drug for type-2 diabetes, has been shown to improve locomotor recovery after spinal cord injury. However, there are studies reporting no beneficial effect. Recently, we found that high dose of metformin (200 mg/kg, intraperitoneal) and acute phase administration (immediately after injury) led to increased mortality and limited locomotor function recovery. Consequently, we used a lower dose (100 mg/kg, i.p.) metformin in mice, and compared the effect of immediate administration after spinal cord injury (acute phase) with that of administration at 3 days post-injury (subacute phase). Our data showed that metformin treatment starting at the subacute phase significantly improved mouse locomotor function evaluated by Basso Mouse Scale (BMS) scoring. Immunohistochemical studies also revealed significant inhibitions of microglia/macrophage activation and astrogliosis at the lesion site. Furthermore, metformin treatment at the subacute phase reduced neutrophil infiltration. These changes were in parallel with the increased survival rate of spinal neurons in animals treated with metformin. These findings suggest that low-dose metformin treatment for subacute spinal cord injury can effectively improve the functional recovery possibly through anti-inflammation and neuroprotection. This study was approved by the Institute Animal Care and Use Committee at the University of Texas Medical Branch (approval No. 1008041C) in 2010.

Keywords: inflammation; locomotor function; metformin; microglia; mortality; neuroprotection; spinal cord injury; subacute administration.

Figure 1

Effect of metformin on the survival of SCI mice. All deaths were recorded within the first 2 weeks after SCI surgery. n = 10 per group. The mortality rate between groups was analyzed by the Kaplan–Meier survival analysis, and the overall differences among groups were analyzed by the Wilcoxon test followed by Tukey-Kramer post hoc test. Control: Vehicle treated; 0 dpi or 3 dpi: the first dose of metformin given immediately or 3 days post SCI, respectively. SCI: Spinal cord injury.

Figure 2

Effect of metformin on the hind limb motor function of SCI mice. Progress of hind limb functional recovery was evaluated by BMS scoring. Data are presented as the mean ± SEM (n = 10 in each group at the start of the experiment; n = 5, 7, or 8 survived in the control, 0 dpi or 3 dpi groups, respectively, at 14 dpi and beyond). *P < 0.05, **P < 0.01, vs. control group; #P < 0.05, vs. 0 dpi group (two-way repeated measures analysis of variance followed by Tukey’s post hoc test adjustments). Control: Vehicle treated; 0 dpi or 3 dpi:, the first dose of metformin given immediately or 3 days post SCI, respectively. BMS: Basso Mouse Scale; SCI: spinal cord injury.

Figure 3

Effect of metformin on the open-field locomotor activity of SCI mice. Photobeam Activity System open-field locomotor function test was performed 4 weeks post injury. (A) The test chamber of the PAS system. (B–E) Rearing activity (B), rest time (C), average speed (D) and travel distance (E) were shown by scatter plots. Data are presented as the mean ± SEM (n = 5 in control, n = 7 in 0 dpi, n = 8 in 3 dpi). *P < 0.05, vs. control group; #P < 0.05, vs. 0 dpi group (one-way analysis of variance followed by Bonferroni post hoc test). Control: Vehicle treated; 0 dpi or 3 dpi: the first dose of metformin given immediately or 3 days post SCI, respectively. SCI: Spinal cord injury.

Figure 4

Effect of metformin on the spinal cord lesion size in SCI mice at 14 days post-injury. (A–C) Longitudinal sections of mouse spinal cords were immunostained with an antibody against GFAP (green, stained by Alexa Fluor® 488), which outlines the boundary of the lesion site. Scale bar: 500 μm. The cavity areas in the 3 dpi group and the 0 dpi group are significantly smaller than those in the control group. (D) Quantitation of the lesion area (delineated by GFAP astroglial scar). Data are presented as the mean ± SEM (n = 5). *P < 0.05, ***P < 0.001 (one-way analysis of variance followed by Bonferroni post hoc test). Control: Vehicle treated; 0 dpi or 3 dpi: the first dose of metformin given immediately or 3 days post SCI, respectively. GFAP: Glial fibrillary acidic protein; SCI: spinal cord injury.

Figure 5

Effect of metformin on the neuron and astrocytes in the spinal cord of SCI mice. GFAP (green, stained by Alexa Fluor® 488) and NeuN (red, stained by Alexa Fluor® 568) double-immunostained spinal cord tissue (longitudinally sectioned) around the lesion site at 14 dpi. (A–L) Merged images of the control, 0 dpi and 3 dpi groups (A, E, I; 4× original magnification, scale bar: 500 μm). GFAP (B, F, J) immunostaining and (C, G, K) NeuN immunostaining (10× original magnification, scale bars: 300 μm). Merged images (D, H, L; 40× original magnification, scale bar: 100 μm). In the 0 dpi and 3 dpi groups, more neurons were observed around the lesion site with a reduced number of astrocytes. In A, E and I, large yellow square insets are enlarged in B–C, F–G and J–K, respectively; whereas small yellow insets enlarged in D, H and L, respectively. The yellow arrows indicate neurons. (M, N) The number of neurons in 40× fields. (M) and GFAP immunopositivity (N). Data are presented as the mean ± SEM (n = 5) *P < 0.05, **P < 0.01 (one-way analysis of variance followed by Bonferroni post hoc test). Control: Vehicle treated; 0 dpi or 3 dpi: the first dose of metformin given immediately or 3 days post SCI, respectively. GFAP: Glial fibrillary acidic protein; NeuN: neuronal nuclear antigen; SCI: spinal cord injury.

Figure 6

Effect of metformin on the microglia/macrophage activation in the spinal cord of SCI mice. Iba-1 (red, stained by Alexa Fluor® 568) immunostained (longitudinally sectioned) spinal cord tissue around the lesion site at 14 dpi. Iba-1 immunofluorescence intensity in the 3 dpi group was significantly lower than that in the control group (A, D, G). Merged images (4× original magnification, scale bar: 500 μm). The large yellow square insets are enlarged in B, E and H showing Iba-1 staining (10× original magnification, scale bar: 300 μm). The small insets are enlarged in C, F and I (40× original magnification, scale bar: 100 μm). The yellow arrows indicate microglia/macrophage. (J) Quantitation of Iba-1 immunoreactivity. Data are presented as the mean ± SEM (n = 4). *P < 0.05, vs. control group (one-way analysis of variance followed by with Bonferroni post hoc test). Control: Vehicle treated; 0 dpi or 3 dpi: the first dose of metformin given immediately or 3 days post-SCI, respectively. Iba-1: Ionized calcium-binding adapter molecule 1; SCI: spinal cord injury.

Figure 7

Effect of metformin on the neutrophil infiltration in the spinal cord longitudinal sections around the lesion site of SCI mice. Gr-1 (green, stained by Alexa Fluor® 488) immunostained spinal cord longitudinal sections around the lesion site at 14 dpi. (A, D, J) Merged images (4× original magnification, scale bar: 100 μm). The large yellow square insets are enlarged in B, E and H (10× original magnification, scale bar: 100 μm), and the small insets enlarged in C, F and I (40× original magnification, scale bar: 100 μm). In the 3 dpi group, the number of neutrophils was significantly reduced compared to the control group. Blue: Nuclear counterstain with DAPI. (J) Number of neutrophils. Data are presented as the mean ± SEM (n = 4) *P < 0.05, vs. control group (one-way analysis of variance followed by Bonferroni post hoc test). Control: Vehicle treated; 0 dpi or 3 dpi: the first dose of metformin given immediately or 3 days post-SCI, respectively. DAPI: 4′,6-Diamidino-2-phenylindole; Gr-1: monoclonal anti-Ly-6G/Ly-6C; SCI: spinal cord injury.