Muscle/bone atrophy occurs in the early phase after CCI
In neurogenic inflammation after nerve injury, M1 macrophage infiltration into peripheral nerves has been reported to be important in pain amplification [9,10,11,12,13,14,15,16,17]. We investigated the influence of M1 macrophages on muscle/bone atrophy after nerve injury in mice. The numerical values obtained in this study are shown in Additional file 1.
One week after CCI in C57BL/6 J mice, biceps femoris and gastrocnemius muscle weights were significantly lower in the CCI group than in the control group (P = 0.001 and P < 0.001, respectively; Fig. 2a).
Total hindlimb bone density was measured by CT performed weekly for 5 weeks (Fig. 2b). Total femoral and tibial bone densities decreased significantly at 5 weeks after CCI (P = 0.005 and P < 0.001, respectively). Multiple comparisons with 0 weeks revealed that total femoral bone density decreased significantly at 2 and 3 weeks after CCI (P = 0.037 and P = 0.035, respectively); total tibial bone density decreased significantly at 2, 3, and 5 weeks after CCI (P = 0.025, P = 0.016, and P < 0.001, respectively). However, the control group showed no significant difference in total femoral and tibial bone densities at 5 weeks after CCI (P = 0.808 and P = 0.480, respectively).
M1 macrophages infiltrate muscles/bones after CCI
We investigated the molecular mechanism underlying muscle/bone atrophy after CCI. We focused on neurogenic inflammation involvement, particularly of M1 macrophages, which has recently attracted attention because of neuropathic pain [12,13,14,15,16,17].
Inflammatory cells and osteoclasts in the femur were analyzed by immunohistochemical analysis. One week after CCI in C57BL/6 J mice, TRAP staining of the right femur revealed that osteoclast numbers (Fig. 2c) were significantly higher in the CCI group than in the control group (P = 0.043).
The nerve/muscle cell dynamics in LysM Cre tandem TOMATO mice were observed with intravital microscopy 1 week after CCI. Infiltration into lysosome-producing cells around the sciatic nerves was observed in the CCI group (Fig. 2 da); such infiltration was mild in the control group (Fig. 2db). To observe inflammatory responses in the acute phase, the hindlimbs of CAG-EGFP mice were observed immediately after CCI. In the CCI group, macrophage infiltration from blood vessels to muscles was observed 2 h after CCI (Fig. 2dc); this was not observed in the control group (Fig. 2dd).
M1 macrophage infiltration into the hindlimbs observed by intravital microscopy was quantified by flow cytometry performed 1 week after CCI (Fig. 2e). In the biceps femoris and gastrocnemius muscles, M1 macrophage numbers (Ly6C+/F4/80+ and CD301−/CD11b+) were significantly higher in the CCI group than in the control group (P < 0.001 and P = 0.049, respectively).
TNFα, IL-1β, CCL2, and CCR2 are involved in M1 macrophage infiltration into muscles/bones after CCI
In neuropathic pain, inflammatory cytokines are involved in neurogenic inflammation [9,10,11]. We thought that inflammatory cytokines were also involved in M1 macrophage infiltration and muscle/bone atrophy after CCI. To investigate cytokine changes in biceps femoris and gastrocnemius muscles after CCI, RT-PCR was performed 1 week after CCI on C57BL/6 J mice. mRNA expression levels are expressed relative to those of the control group (Fig. 2f). Concerning the biceps femoris and gastrocnemius muscles, relative mRNA expression was significantly higher in the CCI group than in the control group for TNFα, IL-1β, CCL2, and CCR2 (P < 0.001 for all).
M1 macrophage infiltration into muscles/bones is suppressed by clodronate liposomes
The second aim of this study was to clarify whether muscle/bone atrophy can be avoided by suppressing M1 macrophages. We depleted M1 macrophages using clodronate liposomes after CCI.
One week after CCI, compared with the untreated group, the macrophage depletion group showed significantly lower numbers of osteoclasts on immunohistochemical analysis (P = 0.049; Fig. 3a) and of M1 macrophages on flow cytometry in the biceps femoris and gastrocnemius muscles (P < 0.001 and P < 0.001, respectively; Fig. 3b).
M1 macrophage depletion suppressed muscle/bone atrophy after CCI
Muscle weight and total bone density changes were compared between the two groups to investigate their effects on muscle/bone atrophy.
In the macrophage depletion group, biceps femoris and gastrocnemius muscle weights were significantly higher 1 week after CCI than in the untreated group (P < 0.001 and P < 0.001, respectively; Fig. 3c), whereas there were no significant differences in total femoral and tibial bone densities at 2 weeks after CCI (P = 0.794 and P = 0.531, respectively; Fig. 3d).
M1 macrophage infiltration into muscle/bone after nerve injury was significantly lower in dexamethasone, pregabalin, and loxoprofen groups than in the untreated group
To clarify whether M1 macrophage infiltration and muscle/bone atrophy can be avoided by clinically usable drugs, we used dexamethasone and loxoprofen as anti-inflammatory drugs (mainly for peripheral neuropathic pain) and pregabalin, neurotropin, and amitriptyline (the latter two for central pain) as neuropathic pain drugs.
One week after CCI, immunohistochemical analysis revealed that osteoclast numbers were significantly lower in the drug groups (P = 0.003). According to multiple comparisons with the untreated group, osteoclast numbers were significantly lower in the dexamethasone, pregabalin, and loxoprofen groups (P = 0.005, P = 0.038, and P = 0.042, respectively). There were no significant differences in osteoclast numbers between the neurotropin and amitriptyline groups and the untreated group (P > 0.99 and P > 0.99, respectively; Fig. 4a).
Flow cytometry of the biceps femoris muscles 1 week after CCI showed that M1 macrophage numbers were significantly lower in the drug administration groups (P = 0.002). On multiple comparisons with the untreated group, it was found that M1 macrophage numbers were significantly lower in the dexamethasone, pregabalin, and loxoprofen groups (P < 0.001, P = 0.043, and P = 0.034, respectively). There were no significant differences in M1 macrophage numbers between the neurotropin and amitriptyline groups and the untreated group (P > 0.99 and P > 0.99, respectively). In the gastrocnemius muscles 1 week after CCI, M1 macrophage numbers were significantly lower in the drug administration groups (P = 0.033). According to multiple comparisons with the untreated group, there were no significant differences in M1 macrophage numbers in any group (dexamethasone, P = 0.134; pregabalin, P > 0.99; loxoprofen, P > 0.99; neurotropin, P > 0.99; and amitriptyline, P = 0.096; Fig. 4b).
Muscle weight and total bone density after nerve injury were significantly higher in dexamethasone, pregabalin, and loxoprofen groups than in the untreated group
Muscle weight and total bone density changes in the drug administration groups were compared with those in the untreated group to investigate their effects on muscle/bone atrophy. These examinations were conducted only for the dexamethasone, pregabalin, and loxoprofen groups, which showed a significant difference in M1 macrophage infiltration into muscles/bones.
One week after CCI, muscle weight was significantly higher in the drug administration groups (biceps femoris, P = 0.001 and gastrocnemius, P = 0.002). According to multiple comparisons with the untreated group, biceps femoral muscle weights were significantly higher in the dexamethasone, pregabalin, and loxoprofen groups (P < 0.001, P = 0.036, and P = 0.030, respectively) and gastrocnemius muscle weights were significantly higher in the dexamethasone and pregabalin groups (P < 0.001 and P = 0.046, respectively). However, there were no significant differences with the loxoprofen group (P = 0.085; Fig. 4c).
Regarding the femur, there were no significant differences in total bone density in the dexamethasone group at 3 weeks after administration (P = 0.176). Total bone density significantly decreased in the pregabalin and loxoprofen groups [pregabalin, P = 0.002, multiple comparisons with 0 week, significant difference at 2 (P = 0.011) and 3 (P = 0.002) weeks and loxoprofen, P = 0.001, multiple comparisons with 0 week, significant difference at 2 (P = 0.006) and 3 (P < 0.001) weeks]. Regarding the tibia, there were no significant differences in total bone density in all groups at 3 weeks after drug administration (dexamethasone, P = 0.789; pregabalin, P = 0.648; loxoprofen, P = 0.092; Fig. 4d).