Pharmacological activation of AMPK inhibits incision-evoked mechanical hypersensitivity and the development of hyperalgesic priming in mice

Highlights

• A novel resveratrol-based topical cream alleviates acute postoperative pain and hyperalgesic priming.

• Combination treatments of resveratrol and metformin have additive effects on acute sensitivity and hyperalgesic priming.

• AMPK activators do not negatively influence wound healing.

Abstract

New therapeutics to manage post-surgical pain are needed to mitigate the liabilities of opioid and other analgesics. Our previous work shows that key modulators of excitability in peripheral nociceptors, such as extracellular signal-regulated kinases (ERK) are inhibited by activation of adenosine monophosphate activated protein kinase (AMPK). We hypothesized that AMPK activation would attenuate acute incision-evoked mechanical hypersensitivity and the development of hyperalgesic priming caused by surgery in mice. Here we have used a variety of administration routes and combinations of AMPK activators to test this hypothesis. Topical administration of a resveratrol-based cream inhibited acute mechanical hypersensitivity evoked by incision and blocked the development of hyperalgesic priming. We also observed that systemic administration of metformin dose-dependently inhibited incision-evoked mechanical hypersensitivity and hyperalgesic priming. Interestingly, low doses of systemic metformin and local resveratrol that had no acute effect were able to mitigate development of hyperalgesic priming. Combined treatment with doses of systemic metformin and local resveratrol that were not effective on their own enhanced the acute efficacy of the individual AMPK activators for post-surgical mechanical pain alleviation and blocked the development of hyperalgesic priming. Finally, we used dorsal root ganglion (DRG) neurons in culture to show that resveratrol and metformin given in combination shift the concentration–response curve for AMPK activation to the left and increase the magnitude of AMPK activation. Therefore, we find that topical administration is an effective treatment route of administration and combining systemic and local treatments led to anti-nociceptive efficacy in acute mechanical hypersensitivity at doses that were not effective alone. Collectively our work demonstrates a specific effect of AMPK activators on post-surgical pain and points to novel therapeutic opportunities with potential immediate impact in the clinical setting.

Introduction

One out of four chronic pain patients suffer from persistent pain because of prior surgery (Crombie et al., 1998) and up to 50% of patients who undergo common surgical procedures develop chronic pain (Kehlet et al., 2006, Johansen et al., 2012). Surgery remains a major cause of persistent pain despite the fact that analgesics for the treatment of acute post-surgical pain are widely available. This suggests that treatments targeting the molecular pathology to prevent or block the transition to chronic post-surgical pain are needed. New ways to interfere with acute post-surgical pain are also needed given the growing negative impact of opioids on society (Skolnick and Volkow, 2016, Volkow and Collins, 2017). While there have been advances in our understanding of signaling pathways and mediators that play an important role in driving post-surgical pain (Velnar et al., 2009, Ling et al., 2017), treatment approaches that target these pathways or mediators are currently not clinically utilized.

In animal models of post-surgical pain, there is an increase in interleukin 6 (IL-6) and nerve growth factor (NGF) levels in serum and skin around the incision site, which signal through the ERK and mechanistic target of rapamycin (mTOR) pathways inducing nociceptive sensitization (Matsuda et al., 1998, Sato and Ohshima, 2000, Banik et al., 2005, Bryan et al., 2005). We have shown that both of these pathways are negatively regulated by adenosine monophosphate (AMP)-activated protein kinase (AMPK) in nociceptors (Melemedjian et al., 2010, Tillu et al., 2012b, Mejia et al., 2016). AMPK is a ubiquitous energy sensing kinase, activated by an increase in intracellular AMP/ATP ratio during energy deprivation. The anti-diabetic drug metformin activates AMPK indirectly via inhibition of mitochondrial complex I (Owen et al., 2000, Shaw et al., 2005) or via inhibition of AMP deaminase (Ouyang et al., 2011). Both of these mechanisms lead to increased AMP levels in cells which allosterically activates AMPK (Hardie, 2015). Resveratrol activates AMPK through multiple mechanisms, but its mechanism of action in neurons appears to be distinct. In neuronal-like cell lines, and cortical and DRG neurons resveratrol does not alter AMP/ATP cellular ratios but activates AMPK via a mechanism that requires the upstream kinase liver kinase B1 (LKB1) (Dasgupta and Milbrandt, 2007), which phosphorylates the α subunit of AMPK to increase kinase activity (Shaw et al., 2005). We have demonstrated that the AMPK activators, metformin, A769662, and R419 inhibited translation regulation signaling pathways and nascent protein synthesis in DRG neurons resulting in a resolution of neuropathic allodynia induced by peripheral nerve injury, in the case of metformin and A769662 (Melemedjian et al., 2011), or incision-evoked mechanical pain in the case of R419 (Mejia et al., 2016). Additionally, we demonstrated that resveratrol profoundly inhibited ERK and mTOR signaling in sensory neurons in a time- and dose-dependent fashion and local injection of resveratrol around the surgery site attenuated surgery-induced mechanical hypersensitivity in a model of post-surgical pain (Tillu et al., 2012b).

The aim of the present study was to further establish AMPK activation as a mechanism for the prevention of post-surgical persistent pain states and to introduce novel therapeutics and therapeutic strategies that employ this mechanism of action for use in humans. To do this, we utilized multiple AMPK activators and administration routes, including topical and local resveratrol, as well as systemic metformin. Our experimental endpoint was incision-induced mechanical hypersensitivity and hyperalgesic priming in mice where we observed efficacy for the attenuation of acute mechanical hypersensitivity and prevention of development of hyperalgesic priming.