(E) Light touch around the non-inflamed paw induces pERK expression in the superficial dorsal horn. post-CFA day 2 reversed CFA-induced bilateral mechanical allodynia but not warmth hyperalgesia. Furthermore, L–AA, the glial inhibitor fluorocitrate, and a peptide inhibitor of c-Jun N-terminal Kinase (JNK) all reduced light touch-evoked ERK activation ipsilateral to touch. Collectively, these data suggest that (a) ERK can be activated in superficial dorsal horn neurons by low threshold mechanical activation under pathological condition and KIAA0288 (b) ERK activation by light touch is associated with mechanical allodynia and requires an astrocyte network. Keywords:ERK, Total Freunds adjuvant, Mechanical allodynia, Astrocytes, JNK The extracellular signal-regulated kinase (ERK, including ERK1 and ERK2) is usually a member of mitogen-activated protein kinase (MAPK) family. Early studies indicated a critical role of ERK in regulating mitosis, proliferation, differentiation, and survival TLK117 of mammalian cells during development (Widmannet al.1999). Recently, it has been exhibited that ERK also plays an important role in neuronal plasticity after peripheral inflammation and nerve injury (Ji and Woolf 2001;Jiet al.2003;Jiet al.2009). Normally, ERK activation (phosphorylation) in the spinal cord is only induced by high-threshold noxious stimuli and this activation is specifically localized in dorsal horn neurons of the ipsilateral medial superficial spinal cord TLK117 where main nociceptive afferents from your hindpaw terminate (Jiet al.1999). Of note ERK activation can be suppressed by analgesic compounds (Karimet al.2001;Ji TLK117 and Strichartz 2004;Kawasakiet al.2006). Spinal inhibition of ERK activation by a MEK inhibitor has also been shown to inhibit inflammatory pain hypersensitivity (Jiet al.2009). Thus, pERK (phosphorylated ERK) expression is regarded as a marker for the sensitization of dorsal horn neurons (central sensitization) following prolonged nociceptive activity (Gao and Ji 2009). Cruz et al. have shown that movement of normal joint does not induce ERK activation in the spinal cord but movement of the inflamed joint in monoarthritic rats elicits a noticeable TLK117 ERK activation in dorsal horn neurons (Cruzet al.2005). Gentle touch also induces pERK expression in dorsal horn neurons after peripheral nerve injury (Wanget al.2004;Haoet al.2005). These data suggest innocuous stimulation is also capable of evoking ERK activation under pathological conditions. Tissue injury or inflammation induces pain hypersensitivity, characterized by both mechanical allodynia (pain in response to normally innocuous stimuli) and warmth hyperalgesia (enhanced pain in response to noxious stimuli). Especially, mechanical allodynia is observed not only in the injured region but also in adjacent non-injured regions and even in contralateral side of the body (Koltzenburget al.1999;Woolf and Salter 2000;Milliganet al.2003;Baron 2009;Gaoet al.2010). Unilateral injection of total Freunds adjuvant (CFA) has been shown to induce mechanical allodynia in both the ipsilateral and contralateral TLK117 paws (Bertorelliet al.1999;Nagakuraet al.2003;Raghavendraet al.2004;Ambalavanaret al.2006). Inflammation also produces bilateral increases in the expression of cyclooxygenase-2 (COX2) (Samadet al.2001), phosphorylation of the transcription factor cAMP response element binding protein (CREB) (Ji and Rupp 1997;Messersmithet al.1998) and c-Jun N-terminal kinase (JNK) in the spinal cord (Gaoet al.2010). Particularly, JNK is activated in spinal astrocytes after nerve injury and CFA inflammation (Zhuanget al.2006;Gaoet al.2010). Intrathecal administration of JNK inhibitor (Gaoet al.2010) or astrocyte function inhibitor (Milliganet al.2003) can prevent and reverse inflammation-induced mechanical allodynia bilaterally. However, ERK activation by low threshold mechanical stimulation under inflammation has not been well characterized and the role of astrocyte network in this activation is also unknown. == Materials and methods == == Animals == Male adult Sprague Dawley rats (220260 g) were used under Harvard Medical School Animal Care institutional guidelines. Peripheral inflammatory pain was induced by an s.c. injection of CFA (Sigma, St. Louis, MO) (100 l) in the plantar surface of the left hind paws under a brief anesthesia with sevofluorane. On day 2 after CFA injection, the animals were anesthetized with isoflurane, light touch stimuli were applied manually by a cotton tip to the ventral surface of the hindpaw, once every 5 sec for 5 min. This touch stimulus did not elicit withdrawal response in normal animals. == Drug administration == For intrathecal injection, spinal cord puncture was made under brief sevofluorane anesthesia with a 27 gauge needle between the L5 and L6 level to deliver the reagents (20 l) to the CSF (Zhuanget al.2006). Immediately after the needle access into subarachnoid space (change in resistance), a brisk tail flick could be observed. The peptide inhibitor of JNK, D-JNKI-1 was kindly provided by Dr. Christopher Bonny, University of Lausanne, Switzerland (Borselloet al.2003). L–aminoadipate (L–AA) and fluorocitrate were purchased from Sigma. The L–AA and D-JNKI-1 were dissolved in 0.01 M PBS. The fluorocitrate was dissolved initially in 2 M HCl (0.1 mol/L) and then diluted in 0.01 M PBS to attain a final concentration of 0.1 nmol/l (pH 6.0). The vehicle control for fluorocitrate.