Genetic and Pharmacological Inhibition of p38α Improves Locomotor Recovery after Spinal Cord Injury

Abstract

One of the mitogen-activated protein kinases, p38α plays a crucial role in various inflammatory diseases and apoptosis of various types of cells. In this study, we investigated the pathophysiological roles of p38α in spinal cord injury (SCI), using a mouse model. Lateral hemisection at T9 of the SC was performed in wild type (WT) and p38α^+/- mice (p38α^-/- showed embryonic lethality). p38α^+/- mice showed a better functional recovery from SCI-associated paralyzed hindlimbs compared to WT mice at 7 days post-injury (dpi), which remained until 28 dpi (an end time point of monitoring the behavior). In histopathological analysis at 28 dpi, there was more axonal regeneration with remyelination on the caudal side of the lesion epicenter in p38α^+/- mice than in WT mice. At 7 dpi, infiltration of inflammatory cells into the lesion and expression of cytokines in the lesion were reduced in p38α^+/- mice compared with WT mice. At the same time point, the number of apoptotic oligodendrocytes in the white matter at the caudal boarder of the lesion of p38α^+/- mice was lower than that of WT mice. At 14 dpi, more neural and oligodendrocyte precursor cells in the gray matter and white matter, respectively, were observed around the lesion epicenter of p38α^+/- mice compared with the case of WT mice. At the same time point, astrocytic scar formation was less apparent in p38α^+/- than in WT mice, while compaction of inflammatory immune cells associated with the wound contraction was more apparent in p38α^+/- than in WT mice. Furthermore, we verified the effectiveness of oral administration of SB239063, a p38α inhibitor on the hindlimb locomotor recovery after SCI. These results suggest that p38α deeply contributes to the pathogenesis of SCI and that inhibition of p38α is a beneficial strategy to recovery from SCI.

Keywords: p38 mitogen-activated protein kinases; recovery of locomotor activity; spinal cord injury (SCI); tissue degeneration; tissue regeneration.

FIGURE 1

Time course of hindlimb locomotor behavior after spinal cord injury (SCI). Wild type (WT) and p38α^+/- mice with SCI were observed daily until 28 dpi, and scored based on BMS. Data are shown as mean ± SEM (n = 5). The difference between WT (filled squares) and p38α^+/- (open circles) mice was statistically significant (^∗P < 0.05) as determined by Mann–Whitney U test for unpaired values at each time point.

FIGURE 2

Histopathological changes after SCI. (A) Typical profiles of HE-stained sagittal SC sections from WT and p38α^+/- mice at 1, 2, and 4 weeks post-injury. (B) Transverse SC sections from WT and p38α^+/- mice at 4 weeks post-injury (1 mm caudal of the lesion epicenter) or with sham-operation (the corresponding site) were stained by a KB method. LFB-staining area was surrounded with the dotted lines. (C) Quantitative evaluation of (A) and (B). Left column, SCI-associated leukocyte infiltration into SC of WT (open bars) and p38α^+/- (closed bars) mice. Right column, ratio of LFB-staining area to right hemi-SC area in WT (open bars) and p38α^+/- (closed bars) mice. Data are shown as mean ± SEM (n = 5). ^∗P < 0.05 (ANOVA followed by Tukey’s test), ^#P < 0.05 (Student’s t-test for unpaired values). (D) Representative images of Texas Red-BDA-labeled axons in the white matter of SC of WT and p38α^+/- mice at 4 weeks post-injury. In the SCI group, sagittal sections of SC at 5 mm rostral and 5 mm caudal to the lesion epicenter were observed. In the sham-operated group, SC segment corresponding to the lesion epicenter (T9) of the SCI group was observed. Texas-Red BDA staining area caudal to the lesion epicenter was expressed as the percentage of the whole field. Data are shown as mean ± SEM (n = 5). ^∗P < 0.05 (Student’s t-test for unpaired values).

FIGURE 3

Inflammatory profiles 1 week after SCI. (A) Typical profile of CD3⁺CD45⁺ cells in the lesion epicenter of the two genotypes at 1 week post-injury. Asterisks indicate double-positive cells. (B) Quantitative evaluation of (A). CD3⁺ cells and CD45⁺ cells in the injured site within 500 μm rostrocaudal of the lesion epicenter increased in both WT (open bars) and p38α^+/- (closed bars) mice at 1 week post-injury, the number of which was significantly lower in p38α^+/- mice than WT mice. Data are shown as mean ± SEM (n = 5). ^∗P < 0.05 (ANOVA followed by Tukey’s test). (C) SCI-induced changes in expression of cytokines in the SC between WT and p38α^+/- mice. Collected SC protein sample from five mice of each group (WT-sham, WT-SCI, p38α^+/--sham or p38α^+/--SCI) were subjected to protein array for 308 molecules. Three independent experiments were conducted (15 mice in each group). Using a densitometer, each signal was normalized to the positive internal controls included in the array membrane (P1-a), and expressed as induction ratio of the sham-operated value. Among 61 molecules (≥2, induction ratio in WT group), 15 molecules showed a significant difference in their induction ratio between the two genotypes. Data are shown as mean ± SEM (n = 3). The difference between WT (open squares) and p38α^+/- (filled squares) mice was statistically significant (^∗P < 0.05) as determined by Student’s t-test for unpaired values.

FIGURE 4

Changes in numbers of apoptotic oligodendrocytes in the white matter 1 week after SCI. (A) Typical profile of cleaved caspase-3⁺CNPase⁺ cells in the white matter at the caudal boarder of the lesion of the two genotypes at 1 week post-injury. Asterisks indicate double-positive cells. (B) Cleaved caspase-3⁺CNPase⁺ cells in the injured site within 500 μm rostrocaudal of the lesion epicenter increased in both WT (open bars) and p38α^+/- (closed bars) mice at 1 week post-injury, the number of which was significantly lower in p38α^+/- mice than WT mice. Data are shown as mean ± SEM (n = 4–5). ^∗P < 0.05 (ANOVA followed by Tukey’s test).

FIGURE 5

Astrocytic scar formation and neuroinflammation-associated cells at 2 weeks post-injury. (A,E) Representative images of sagittal sections showing GFAP⁺ reactive astrocytes and Iba1⁺ cells at 2 weeks post-injury. (B,C,F,G) Higher magnification images from the boxed area, b, c, f and g in (A) and (E). Iba1⁺ cells were compacted to the lesion center between rostrocaudal reactive astrocytes, which was more apparent in the SC of p38α^+/- mice than that of WT mice. (D,H) Higher magnification images of GFAP⁺ reactive astrocytes from the boxed area, d (in A) and h (in E). GFAP⁺ reactive astrocytes at 1 mm caudal to the lesion epicenter decreased in p38α^+/- mice compared with WT mice.

FIGURE 6

Change in numbers of NG2⁺ cells in the white matter at 2 weeks after SCI. (A) Typical profile of NG2⁺ cells in the lesion epicenter of the two genotypes at 2 weeks post-injury. (B) NG2⁺ cells in the injured site within 500 μm rostrocaudal of the lesion epicenter in the SCI group were increased in the two genotypes at 1 week post-injury, the number of which was significantly higher in p38α^+/- mice (closed bars) than WT mice (open bars). Data are shown as mean ± SEM (n = 4–5). ^∗P < 0.05 (ANOVA followed by Tukey’s test).

FIGURE 7

Effect of SB239063 on hindlimb locomotor behavior after SCI. WT mice with SCI received oral administration of vehicles (filled squares) and SB239063 (10 mg/kg per day, filled circles) at 1, 2, and 3 dpi. Functional recovery from SCI was better in mice with SB239063 treatment than those with vehicle administration. Data are shown as mean ± SEM (n = 6). ^∗P < 0.05, a significant difference between the two groups by Mann–Whitney U test for unpaired values at each time point.