Tissue damage-induced axon injury-associated responses in sensory neurons – requirements, prevention, and potential role in persistent post-surgical pain

Pain resulting from tissue damage, including surgical incision, is often only partially responsive to standard treatments focusing on inflammation, suggesting additional mechanisms are involved. Tissue damage leads to expression in dorsal root ganglion (DRG) sensory neurons of genes associated with axonal injury and regeneration, most notably activating transcription factor 3 (ATF3) and GAP-43. ATF3 expression is associated with sensitization of cellular physiology and enhanced amplitude/duration of a nociceptive reflex. It is unclear how tissue damage leads to these changes in the sensory neurons, but it could include direct damage to the tissue-innervating axons and inflammation-associated retrograde biochemical signalling. Using the CTM reflex to map innervation fields, we examined the necessity and sufficiency of incision, inflammation, and axonal conduction for induction of ATF3 in response to skin incision. Incision outside the innervation field, but close enough to induce inflammation inside the innervation field, was not sufficient to induce ATF3 expression in the field-innervating DRG. Incision inside the innervation field led to strong expression of ATF3. Anti-inflammatory treatments did not prevent this induction of ATF3. In rodent models of repeated injury - a major etiological factor for chronic pain - ATF3 expression was synergistically-increased and the threshold for paw-withdrawal to mechanical stimulation was significantly decreased for an extended duration. Together, these results suggest that actual damage to axons innervating the skin is both necessary and sufficient for induction of ATF3, expression of which appears additionally increased by repeated injury. Further, pre-treatment of the nerves innervating the incised skin with bupivacaine, a local anesthetic commonly used to reduce surgical pain, did not prevent induction of ATF3, indicating that conduction of action potentials is not necessary for induction of ATF3. We also determined that closure of incision with surgical glue significantly reduced incision-induced expression of GAP-43. Intriguingly, treatment with polyethylene glycol (PEG), known to enhance membrane integrity after injury among other effects, reduced incision-associated ATF3 expression and electrophysiological changes. These results suggest that pain resulting from tissue damage may arise from a mix of ATF3-/axonal-damage-associated mechanism as well as ATF3-independent inflammation-related mechanisms and therefore require a mix of approaches to achieve more complete control.