Spinal expression of autophagic markers following intraplantar formalin injection

Autophagy is a physiological intracellular mechanism contributing to protein and organelles degradation, cellular remodelling and survival. An imbalance on the fine-tuning of this process can impact basal functions leading to cellular dysfunction and this has recently been implicated in several human diseases, including neurodegeneration and cancer.Recent data from our group have shown autophagy impairment in the spinal cord following spinal nerve ligation and suggested a potential role for this degradative pathway in this experimental model of neuropathic pain (Berliocchi et al, 2012). Aim of this study was to investigate whether spinal expression of autophagic markers can be altered also in models of inflammatory pain.Inflammatory pain was induced by a single intraplantar injection of 5% buffered formalin solution (20ul) in the left hindpaw of male C57BL/6 mice (22-25g). Pain-related behaviour was assessed by scoring licking/biting of the injected paw in 5min bins, for 50 minutes. The expression of the main autophagic markers beclin-1, LC3 and p62 was investigated in spinal cord lysates by western blot analysis.Formalin injection into the hind paw induced a biphasic pain response characterised by a first phase of 10 minutes in which licking/biting of the paw was high, followed by a transient decline in these behaviours and a subsequent second phase of pain response lasting about 30 minutes.A decrease in beclin-1 and LC3 expression was observed 4 days following formalin injection in the L4-L5 portion of the spinal cord ipsilateral to the injured paw in comparison to the contralateral side. The two distinct phases of the test may be used to address different aspects of nociception since the first phase seems to be due to direct activation of primary afferent sensory neurons, whereas the second phase is dependent on peripheral inflammation and changes in central processing. The analysis of the autophagic markers beclin-1 and LC3 did not reveal major changes at early time points, but a decreased spinal expression 4 days after formalin injection. Our data indicate that autophagy is modulated in the spinal cord following intraplantar injection of formalin and prompt further studies for the characterization and the understating of these changes.