Background: Correction of aberrant catabolic signaling in muscle has not rectified the muscle wasting (MW) of burn injury (BI). The functional integrity of the neuromuscular apparatus is highly dependent on a healthy motor neuron, but its role in MW has received little attention after BI. In many inflammatory central nervous system (CNS) diseases (e.g., encephalitis), MW invariably occurs. BI is associated with neuroinflammation, evidenced by the activation of microglia and proinflammatory cytokine release. The source of cytokine release and its relationship to motor neuron degeneration and MW after BI is unknown. The microglia are the resident innate immune cells of the CNS and are dependent on colony-stimulating factor-1 (CSF1) for its survival. Thus, inhibition of CSF-1 will deplete microglia. In this study, we tested the hypothesis that microglia-released increased IL-1β leads to motor neuron degeneration which in turn results in MW and transient depletion of microglia normalizes IL-1β release leading to mitigation of neuronal degeneration and MW.  

Methods: A 30% body surface area BI or sham-BI (SB) was produced in C57BL/6 mice under anesthesia. Starting 24 hours after BI, the mice received the intraperitoneal (i.p) injection of either a CSF1 inhibitor, Plexxikon 5622 (PLX) 40 mg/kg or equal volume saline twice daily for 7 days. The lumbar spinal cord and hindlimb muscle samples were obtained at day 7 and 14 after BI. Immunofluorescence staining was performed on the spinal cord ventral horn using Iba1 and NeuN to identify and count microglia and ventral horn motor neuron numbers, respectively. Spinal inflammatory cytokine transcripts (TNF-α, IL-1β) were analyzed by RT-qPCR. Tibialis (TA) and gastrocnemius (GC) muscle wet weights were assessed at day 7 and 14.

Results: BI caused time-dependent significant spinal microglia activation evidenced as peak increase of IL-1β levels at day 14 compared to sham-BI (Fig. 1 a). TNF-α levels also significantly increased at day 7 and 14 after BI (Fig. 1 b) compared to SB. PLX started at day 1-postburn did not mitigate cytokines at day 7, possibly related to the latent period of onset of peak PLX effect. At day 14, the PLX treatment normalized IL-1β  levels to control levels (similar to sham-BI mice (Fig. 1a) but not TNF-α levels. There was a time-dependent increase in Iba1-positive microglia cells at day 7 and 14 in BI compared to SB, which were significantly decreased by PLX at both 7 and 14 days post-BI (Fig. 2 b). Motor neuron numbers detected by NeuN staining decreased at day 7 and day 14 after BI compared to SB (Fig.2 c). PLX treatment mitigated the BI-induced motor neuron loss both at day 7 and 14 (Fig. 2c). The tibialis (Fig. 3 a) and gastrocnemius (Fig. 3 b) muscle mass significantly decreased at day 7 and day 14 after BI compared with SB. PLX treatment did not attenuate the MW at day at day 7 paralleling the high TNF-α and IL-1β levels at that time. In contrast, when PLX decreased BI-induced IL-1β levels at day 14, the motor neuron loss and muscle mass loss were mitigated compared to untreated BI mice.

Conclusions: This study suggests that there is an interaction between BI-induced microglia numbers (microgliosis), cytokine release, motor neuron numbers and muscle changes. The parallel relationship between these variables was most obvious at day 14. Depletion of microglia with PLX mitigated microgliosis and normalized IL-1β levels together with beneficial effects on motor neuron loss and MW. As indicated earlier, PLX depleted microglia by 20 % by day 7. The increase of TNF-α and IL-1β levels at day 7 after PLX administration suggests that the peak effect of PLX possibly takes longer than 7 days. Future studies should examine the longer duration of PLX administration and its effect on microglia, cytokines, motor neuron and muscle mass. Short-term PLX5622 could be a promising approach for reversing MW caused by BI.