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. 2017 Aug 1;34(15):2342-2352.
doi: 10.1089/neu.2016.4915. Epub 2017 Jun 14.

Compression Decreases Anatomical and Functional Recovery and Alters Inflammation after Contusive Spinal Cord Injury

Michael B Orr  1   2 , Jennifer Simkin  1   3 , William M Bailey  1 , Neha S Kadambi  4 , Anna Leigh McVicar  1 , Amy K Veldhorst  1 , John C Gensel  1
Affiliations

Compression Decreases Anatomical and Functional Recovery and Alters Inflammation after Contusive Spinal Cord Injury

Michael B Orr et al. J Neurotrauma. .

Abstract

Experimental models of spinal cord injury (SCI) typically utilize contusion or compression injuries. Clinically, however, SCI is heterogeneous and the primary injury mode may affect secondary injury progression and neuroprotective therapeutic efficacy. Specifically, immunomodulatory agents are of therapeutic interest because the activation state of SCI macrophages may facilitate pathology but also improve repair. It is unknown currently how the primary injury biomechanics affect macrophage activation. Therefore, to determine the effects of compression subsequent to spinal contusion, we examined recovery, secondary injury, and macrophage activation in C57/BL6 mice after SCI with or without a 20 sec compression at two contusion impact forces (50 and 75 kdyn). We observed that regardless of the initial impact force, compression increased tissue damage and worsened functional recovery. Interestingly, compression-dependent damage is not evident until one week after SCI. Further, compression limits functional recovery to the first two weeks post-SCI; in the absence of compression, mice receiving contusion SCI recover for four weeks. To determine whether the recovery plateau is indicative of compression-specific inflammatory responses, we examined macrophage activation with immunohistochemical markers of purportedly pathological (CD86 and macrophage receptor with collagenous structure [MARCO]) and reparative macrophages (arginase [Arg1] and CD206). We detected significant increases in macrophages expression of MARCO and decreases in macrophage Arg1 expression with compression, suggesting a biomechanical-dependent shift in SCI macrophage activation. Collectively, compression-induced alterations in tissue and functional recovery and inflammation highlight the need to consider the primary SCI biomechanics in the design and clinical implementation of immunomodulatory therapies.

Keywords: infinite horizons; inflammation; mice; microglia.

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Conflict of interest statement

No competing financial interests exist.

Figures

<b>FIG. 1.</b>
FIG. 1.
Compression impairs functional recovery from contusion spinal cord injury. Time course of locomotor recovery assessed by Basso Mouse Scale (A) and BMS subscore (B–E). (A) Over a 28-day period, groups with increased impact force score lower than groups with decreased impact force and groups with compression score lower than noncompression groups. Significance between comparable groups at each time point is detailed in Table 2 (n = 9–11; mean ± standard error of the mean [SEM]). (B–E) 28 dpi BMS subscores (n = 9–11). (B) Composite BMS subscore is significantly different between compression and noncompression groups. (C) Coordination is graded as 2 = mostly coordinated (>50%), 1 = some coordination (<50%), or 0 = no coordination. (D) Plantar stepping is graded as 1 = consistent or 0 = less than consistent. (E) Paw position is graded as 2 = parallel throughout, 1 = parallel and rotated, or 0 = rotated throughout. Groups with compression performed significantly worse than force-matched counterparts for every subscore. B–E: ****p < 0.0001, **0.01, *0.05. Mean ± SEM.
<b>FIG. 2.</b>
FIG. 2.
Compression significantly decreases anatomical recovery from contusion spinal cord injury. (A) Tissue sparing at 28 dpi is decreased by both increased force and compression at the epicenter and at regions rostral and caudal to the epicenter (n = 8–9, mean ± standard error of the mean [SEM]). (B) Tissue sparing at 3, 7, 14, and 28 dpi, reflected by eriochrome cyanine (EC) for myelin and neurofilament (NF) for axons, at the lesion epicenter is decreased by both increased impact force and compression (3 dpi n = 2–4, 7 dpi n = 3, 14 dpi n = 5, 28 dpi n = 8–9, mean ± SEM). (C) Sections, representative of the mean of each group, reflect decreased tissue sparing in groups that receive compression. EC (blue), NF (brown) Scale bar, 400 μm. ****Significance for compared groups detailed in Table 3.
<b>FIG. 3.</b>
FIG. 3.
Compression alters spinal cord injury macrophage activation at 7 dpi. (A) Three regions of interest (ROI; dorsal and left and right) within the lesion epicenter of each animal were analyzed at 7 dpi for macrophage density (total tomato lectin [TomL] positive area). The TomL + area was similar across the four injury paradigms: 50 kdyn, 75 kdyn without compression (open bars) and 50 kdyn, 75 kdyn with compression (black bars). (B) Percent of TomL+ cells positive for Arginase 1 (Arg1) across treatments. (C) Percent of TomL+ cells positive for CD206 across treatments. (D) Percent of TomL+ cells positive for CD86, and (E) percent of TomL+ cells positive for macrophage receptor with collagenous structure (MARCO) (p = 0.03 main effect of compression). B–E) n = 3–4, error bars = SEM). (F) Representative images for analysis of Tomato lectin+ (blue)/MARCO+ (green) cells across treatments. Scale bar = 50 μm.
<b>FIG. 4.</b>
FIG. 4.
Compression alters spinal cord injury macrophage activation at 14 dpi. (A) Four regions of interest within the lesion epicenter of each animal (as depicted in Fig. 3A and ventral) were analyzed at 14 dpi for macrophage density (total tomato lectin [TomL] positive area). The TomL + area was comparable across all four injuries: 50 kdyn, 75 kdyn without compression (open bars) and 50 kdyn, 75 kdyn with compression (black bars). (B) Percent of TomL+ cells positive for Arginase 1 (Arg1) across treatments (p = 0.036 main effect compression). (C) Representative images for analysis of tomato lectin + (blue)/Arg1+ (red) cells across treatments. Scale bar = 50 μm. (D) Percent of TomL+ cells positive for CD206, and (E) percent of TomL+ cells positive for CD86. n = 5, error bars = standard error of the mean.
<b>FIG. 5.</b>
FIG. 5.
Compression decreases functional recovery, anatomical recovery, and alters inflammatory macrophage profile regardless of contusion severity. Both 50 and 75 kydn spinal cord injury contusion groups were collapsed and the effect of 20s of sustained compression compared between aggregate groups. (A) Animals with compression injuries perform worse, according to the Basso Mouse Scale, at all time points than noncompression injury counterparts (n = 19–20). In addition, (B) noncompression injured animals demonstrate improved function from 14 to 28 dpi, while compression injured animals have stunted recovery after 14 dpi (n = 19–20). (C) Tissue sparing at the lesion epicenter, determined by eriochrome cyanin for myelin and neurofilament for axons, significantly decreases by 14 dpi with the addition of compression (3 dpi n = 6, 7 dpi n = 6, 14 dpi n = 10, 28 dpi n = 17). (D) Tomato lectin-labeled macrophages increase expression of the pro-inflammatory marker, macrophage receptor with collagenous structure (MARCO) (n = 6) and decrease Arginase 1 expression (n = 10). ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, data are mean ± standard error of the mean. BMS, Basso Mouse Scale.
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