Activated Microglia Mediate the Motor Neuron-, Synaptic Denervation- and Muscle Wasting-Changes in Burn Injured Mice

Background

Muscle wasting (MW) of burn injury (BI) remains unresolved. Microglia-mediated inflammatory cytokine/chemokine release, motor neuron loss (MNL) and MW is observed after BI but connection of the central changes to synaptic-denervation and MW is unelucidated. Stimulation of microglia α7acetylcholine receptors (α7AChRs), a Chrna7gene-product, exhibits anti-inflammatory properties (decreased cytokine/chemokines). Hypothesis tested was that exploitation of the microglia α7AChR anti-inflammatory properties mitigates cytokine inflammatory responses, MNL, synaptic-denervation and MW of BI.

Methods

Wild-type or α7AChR knockout (A7KO) mice received 30% body BI or Sham BI (SB) under anaesthesia with and without selective α7AChR agonist, GTS-21. Lumbar spinal cord tissue and hindlimb muscles were harvested. Immunohistochemistry, TUNEL assay for apoptosis and/or Nissl staining were used to examine microglia (Iba1 staining), MNL (NeuN staining) and synapse morphology (synaptophysin for nerve and α-bungarotoxin for muscle α7AChR). Spinal cytokine/chemokine transcripts, inflammatory transducer-protein expression and tibialis, soleus and gastrocnemius muscle weights were measured.

Results

BI to Wild-type mice caused significant microgliosis (5.8-fold increase, p < 0.001) and upregulated TNF-α, IL-1β, CXCL2, MCP-1 transcripts, and inflammatory transducer-protein (STAT3 and NF-κB, p < 0.01) expression together with increased transcripts of iNOS (p < 0.01) and CD86 (p < 0.01) at day 14 reflective of inflammatory M1 microglia phenotype. Significant apoptosis, MNL (32.2% reduction, p < 0.05), increased spinal caspase-3 expression (> 1100-fold, p < 0.05) and synaptic denervation were observed with BI. The tibialis muscle endplates (synapse) of SB had a smooth pretzel shaped appearance with good apposition of presynaptic nerve to postsynaptic muscle. In BI mice, the normal pretzel-like appearance was lost, and the endplates were fragmented with less nerve to muscle apposition. Tibialis, soleus, and gastrocnemius mass were decreased 31.7% (p < 0.01), 23.4% (p < 0.01) and 27.5% (p < 0.01) relative to SB. The A7KO mice with SB showed significant MNL loss (61.5% reduction, p < 0.05), which was aggravated with BI, accompanied by significantly higher expression of STAT3 and Nf-kB (p < 0.05). GTS-21 ameliorated the spinal expression of above enumerated cytokines/chemokines, inflammatory transducer-proteins (p < 0.05) together with mitigated MNL (p < 0.05), synaptic denervation (p < 0.05) and decreased MW of tibialis (25%), gastrocnemius (15%) and soleus (20%) relative to untreated wild type BI mice (p < 0.01). GTS-21 beneficial effects were absent in the A7KO mice.

Conclusions

Microglia-mediated inflammatory responses play pivotal role in MNL as decrease of inflammatory responses improved MNL; α7AChR stimulation also mitigated synaptic denervation and MW changes of BI. α7AChRs have a role in spinal homeostasis even in uninjured state.