Background:  

Burn injury (BI) occurs globally in ~11 million people per year and is the leading cause of injury in children. Despite advances in critical care and survival, the muscle wasting (MW) and neuromuscular dysfunction of BI remains unresolved. Recent studies have documented a time-dependent microgliosis (proliferated microglia), microglia activation (increased inflammatory cytokine release) together with motor neuron loss (MNL) and MW. However, the cause-and-effect relationship between the microglia changes and MNL to MW has not been established. This study tested the hypothesis that microglia-mediated cytokine release plays a pivotal role in the MNL, distant synaptic disintegration and MW after BI, and mitigation of microglia activation by α7AChR stimulation would attenuate microglia-mediated inflammatory cytokine release, MNL, synaptic (junctional) denervation/disintegration and MW changes induced by BI.

Methods:  

20~25g wild-type and α7AChR knock-out (α7KO) mice were randomly divided into sham-burn (SB) or a third degree 30% total body surface area BI group. Selective α7AChR agonist, GTS-21(10 mg/kg, bid) or saline was used after burn injury. At day 7 and 14 after the perturbations, the animals were euthanized and muscle tissues (gastrocnemius, soleus and tibialis anterior) and spinal cord (L3-4 segments) were harvested. Microglia and neurons in lumber spinal cord ventral horn section were immuno-histochemically-stained by IBA1 and Neun, respectively. And synaptophysin antibody staining was used for nerve terminal analysis, α-bungarotoxin for AChRs in muscle. Spinal inflammatory transducer-proteins (STAT3 and NF-κB) were analyzed by RT-PCR and western blots. Motor neuron apoptosis and MNL were quantitated by TUNEL and Nissl staining, respectively. The consequence of MNL on MW was quantified by weights of tibialis, soleus and gastrocnemius muscles at day 14. 

Results:

BI lead to microgliosis (5.8-fold numbers at day14) with significantly increased the expression of transcripts of pro-inflammatory protein pSTAT3 and pNF-κB, MNL in ventral horn spinal cord after burn injury combined with increased apoptosis mediated by increased caspase 3 (>10-fold). After BI, the muscle and nerve showed disintegrated and fragmented synapse with irregular edges, while the SB mice had the typical pretzel-like shape appearance with smooth boundary. Compared with SB group, the TA, SOL, and GC muscles masses were decreased 31.7%, 23.4%, and 27.5%, respectively at day 14 after BI. GTS-21 decreased microgliosis and mitigated microglia activation by decreased the expression of inflammatory markers which was not observed in A7KO. As compared to BI mice without treatment, GTS-21 rescued the disintegration of the endplate, and mitigated the partial denervation in the wild type mice, but not in the A7KO mice.  Furthermore, the TA, SOL, and GC muscles mass losses were significantly decreased to 23.3%, 18.1% and 14.7%, respectively at day 14 after BI), but not in α7KO mice.  

Conclusions:   

Our current studies demonstrate that BI-induced systemic inflammation can lead to neuro-inflammation and caspase-3 mediated apoptotic MNL. The agonist stimulation of α7AChRs ameliorates not only spinal inflammatory changes and MNL but also the synaptic disintegration and MW. These beneficial effect by GTS-21 were completely nullified in α7 AChR knockout mice, confirming the specific roles of α7 AChR stimulation and the anti-inflammatory therapeutic effects of GTS-21. GTS-21 administration in BI patients may prove useful for mitigation of MW and therefore needs further study.   

Key Words: α7acetylcholine receptors (α7AChRs), apoptosis, burn injury, caspase-3, inflammatory cytokines, inflammatory chemokines, GTS-21, microglia activation, muscle wasting, neuromuscular, synapse.