Mitogen-activated protein kinase-activated protein kinase 2 (MK2) contributes to secondary damage after spinal cord injury

Abstract

The inflammatory response contributes importantly to secondary tissue damage and functional deficits after spinal cord injury (SCI). In this work, we identified mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAPK2 or MK2), a downstream substrate of p38 MAPK, as a potential target using microarray analysis of contused spinal cord tissue taken at the peak of the inflammatory response. There was increased expression and phosphorylation of MK2 after SCI, with phospho-MK2 expressed in microglia/macrophages, neurons and astrocytes. We examined the role of MK2 in spinal cord contusion injury using MK2(-/-) mice. These results show that locomotor recovery was significantly improved in MK2(-/-) mice, compared with wild-type controls. MK2(-/-) mice showed reduced neuron and myelin loss, and increased sparing of serotonergic fibers in the ventral horn caudal to the injury site. We also found differential expression of matrix metalloproteinase-2 and 9 in MK2(-/-) and wild-type mice after SCI. Significant reduction was also seen in the expression of proinflammatory cytokines and protein nitrosylation in the injured spinal cord of MK2(-/-) mice. Our previous work has shown that macrophages lacking MK2 have an anti-inflammatory phenotype. We now show that there is no difference in the number of macrophages in the injured spinal cord between the two mouse strains and little if any difference in their phagocytic capacity, suggesting that macrophages lacking MK2 have a beneficial phenotype. These findings suggest that a lack of MK2 can reduce tissue damage after SCI and improve locomotor recovery. MK2 may therefore be a useful target to treat acute SCI.

Figure 1.

A, Western blots for total and pMK2 in naive (N) spinal cord and at 1, 3, 7, 14 and 28 d after SCI show increasing levels of total and pMK2 protein beginning 3 d after injury, reaching a peak by day 7. B, Quantification of pMK2 from Western blots. The values show fold increase relative β-actin (*p < 0.05; n = 4; see supplemental Fig. 7, available at www.jneurosci.org as supplemental material for comparison with total MK2). C–J, Representative micrographs of immunofluorescence labeling of spinal cord sections 12 h (C, F) and 7 d (D, G, I, J) after SCI labeled for pMK2, cell type-specific antibodies, and DAPI staining (merged images). C, D, Double labeling with anti-pMK2 and anti-NeuN for neurons at 500 μm caudal to epicenter. F, G, Double labeling for anti-pMK2 and anti-GFAP for astrocytes at 500 μm caudal to epicenter. I, J, Double labeling for pMK2 and anti-Mac-1 for macrophages at epicenter of injury (I) and microglia (J) at 500 μm caudal to epicenter. Arrows point to pMK2-immunopositive cells and insets in panels show higher-magnification images of pMK2⁺ cells from the boxed areas. Note that pMK2 labeling is localized primarily to the nuclei of macrophages/microglia, which are double-labeled with Mac-1, a cell surface label. As a result, the two labels are not superimposed but the staining nevertheless shows colabeling of the same cell. Quantitative analysis of pMK2⁺ neurons (E) and astrocytes (H) at 12 h and 7 d after injury. n = 6 per time point. Scale bar, 100 μm.

Figure 2.

A, MK2^−/− mice show significant improvement in locomotor recovery, assessed using BMS analysis, compared with wild-type mice starting as early as 5 d after injury and improving even further during the 28 d duration of the study. B, The BMS subscores, which measure finer aspects of locomotor control, also show marked improvement in MK2^−/− mice between days 10 and 28 after SCI. *p < 0.05; n = 10–11 per group.

Figure 3.

A, Quantification of ventral horn neuron survival at various distances rostral and caudal to the injury epicenter (0) reveals significantly greater neuronal survival in MK2^−/− mice, relative to wild-type animals, taken 28 d after SCI. B, C, Representative micrographs showing sparing of ventral horn neurons in wild-type (B) and MK2^−/− (C) mice in tissue sections stained with cresyl violet at 500 μm caudal to the injury epicenter; arrows point to surviving neurons. D, Quantification of myelin sparing in the dorsal column in serial tissue sections stained with Luxol Fast Blue rostral and caudal to the lesion epicenter. Note the significantly greater spared myelin in MK2^−/− mice relative to wild-type animals. E, F, Representative micrographs showing LFB staining of tissue sections from wild-type (E) and MK2^−/− (F) mice. G, Optical density measurements of serotonergic (5-HT) immunoreactivity indicate that MK2^−/− mice show an almost twofold increase in 5-HT innervation of the ventral horn 1 mm caudal to the lesion epicenter compared with wild-type animals. H, I, Representative micrographs showing 5-HT immunoreactivity in the ventral horn of the spinal cord from wild-type (H) and MK2^−/− (I) mice. *p < 0.05; n = 9–11 per group; Scale bar, 200 μm.

Figure 4.

A, B, Densitometric values of Western blots for total Hsp25 (A) and phosphorylated Hsp25 (B) in spinal cord tissue 7 d after SCI, each normalized to β-actin. Note the significant reduction in pHsp25 levels in MK2^−/− mice [KO (knockout)] compared with wild-type mice (WT), but no change in total Hsp25 levels between the two strains (n = 3 per group). C, Western blots showing pHSP25 and total HSP25. D, Quantification of 3-nitrotyrosine (3-NT) levels in the lateral funiculus 7 d after SCI, measured by optical density reading of tissue sections, reveals significantly reduced protein nitration in MK2-null mice relative to wild-type animals (n = 10 per group). Similar results were also seen in the dorsal column. E, Representative examples of gelatin zymography showing reduced MMP-9 and increased MMP-2 in spinal cord tissue of MK2^−/− (KO) and WT mice at 7 d after SCI (n = 4 per group). *p < 0.05.

Figure 5.

MK2^−/− macrophages retain their ability to phagocytose myelin. MK2 knock-out (KO) and wild-type (WT) macrophage cell lines were incubated with or without fluorescent DiI-labeled myelin in vitro for 30 min and analyzed by FACS to measure myelin phagocytosis. A, FACS data show a 25% reduction in the number of MK2^−/− macrophages that phagocytose myelin compared with wild-type macrophages. B, For the 75% of cells that phagocytose myelin, there is no difference in the mean fluorescence intensity of the cells, indicating that these MK2^−/− macrophages are able to phagocytose myelin as effectively as wild-type macrophages. *p < 0.05; n = 3 per group.