- Short report
- Open Access
Activation of Erk in the anterior cingulate cortex during the induction and expression of chronic pain
© Wei and Zhuo; licensee BioMed Central Ltd. 2008
- Received: 12 June 2008
- Accepted: 23 July 2008
- Published: 23 July 2008
The extracellular signal-regulated kinase (Erk) activity contributes to synaptic plasticity, a key mechanism for learning, memory and chronic pain. Although the anterior cingulate cortex (ACC) has been reported as an important cortical region for neuronal mechanisms underlying the induction and expression of chronic pain, it has yet to be investigated whether or not Erk activity in the ACC may be affected by peripheral injury or in chronic pain state. In the present study, we use adult rat animal models of inflammatory and neuropathic pain and demonstrate that Erk signaling pathway in the ACC is potently activated after peripheral tissue or nerve injury. Furthermore, we demonstrate that mechanical allodynia significantly activated Erk activity at synaptic sites at two weeks after the injury. We propose a synaptic model for explaining the roles of Erk activity during different phases of chronic pain. Our findings suggest that cortical activation of Erk may contribute to both induction and expression of chronic pain.
- Chronic Pain
- Anterior Cingulate Cortex
- Mechanical Allodynia
- Formalin Injection
- Synaptic Potentiation
Recent studies have consistently indicated that Erk signaling cascade plays an important role in activity-dependent plasticity in the central nervous system (CNS) and may contribute to the molecular mechanisms underlying learning, memory and persistent pain. Previous studies have found that tissue and nerve injury transiently activates Erk pathway in the spinal dorsal horn neurons, and the activation of Erk is required for the central sensitization during the development of hyperalgesia and allodynia [1–5]. In supraspinal structures, it has been reported that activation of Erk in the amygdala is induced by peripheral injury , and the increased Erk activity in this region is acquired for behavioral sensitization to mechanical stimulation after injury . The ACC has been found to be an important site for cortical regulation of nociception and persistent pain after amputation [8–11]. Long-term potentiation (LTP) in ACC neurons is the likely synaptic model for persistent pain [12–16]. Recent studies using pharmacological inhibitors has showed that the activity of Erk contributes to synaptic potentiation caused by LTP induction protocols , and that such inhibitors are relatively selective and do not affect basic synaptic transmission. However, little is known about the possible involvement of Erk in the ACC after tissue or nerve injury in adult animals.
Phantom pain is chronic pain occurring after losing or amputation of a part of limb or organ. Although the animal model for studying central mechanisms of phantom pain is rare, investigations of changes in central synapses in the ACC after amputation in animals may reveal molecular mechanism for plastic changes that are caused by amputation. We have previously demonstrated that amputation of a single digit resulted in a loss of synaptic cortical depression [20, 9, 11] in brain slice preparations, and caused LTP of synaptic responses in the ACC to peripheral sensory stimulation or local synaptic stimulation [11, 21], demonstrating that central plasticity takes place in the synapses of ACC neurons after the amputation. Because the Erk activity is shown to be required for cingulate LTP , we decided to test if tissue and nerve injury after the amputation would activate the Erk pathway in the ACC neurons. As shown in Fig. 1B, we found that larger number of the layer II pyramidal-like neurons in the ACC expressed P-Erk immunoreactivity at two weeks after single digit amputation. Activation of Erk activity in the ACC is bilateral. Interestingly, the P-Erk expression in these neurons was present in cytoplasm of both cell body and dendrites (Fig. 1B). There was some P-Erk in neurons of the deeper layers, suggesting the possible interaction between neurons in different layers of the ACC. This data further indicates that there is a prolonged activation of Erk in the layer II neurons after amputation.
In addition to spontaneous pain and hyperalgesia, allodynia is the most common feature of pathological pain and is a painful response to a usually innocuous stimulus. Touch-evoked allodynia occurs often in patients with phantom pain after amputation. Most of previous studies focus on the activation of Erk at early time points after the injury, there is few studies for the possible involvement of Erk activity during allodynic stimulation. Therefore, we applied non-noxious mechanical stimuli (brushing) on amputated hindpaw in rats to identify if Erk signaling in the ACC may be activated. To our surprise, there was significantly enhanced activation of Erk in the ACC neurons after brushing the amputated hindpaw. In addition to the increased number of P-Erk labeled neurons in the layer II, we also observed that there was stronger expression of P-Erk in the dendrites distributed in the layer I (Fig. 1C), as compared with that in rats with amputation alone. Most of P-Erk labeled pyramidal neurons in the layer II exhibited strong immunoreactivity in the main and distal apical dendrites, which branched upward to the superficial layer I of the ACC (see Fig. 1C). We also observed that there were many P-Erk labeled granular-like neurons in the deeper layers of the ACC. Again, we found similar activation pattern at bilateral sides of the ACC. By contrast, non-noxious brushing alone in normal rats did not cause any detectable Erk activation in the ACC. The enhanced expression of P-Erk in ACC neurons and their dendrites after brushing normal skin of amputated hindpaw suggests that Erk activity are recruited at distal synapses in ACC neurons. It may contribute to local synaptic plasticity and/or neuronal modulation during allodynia after amputation.
Although we cannot distinguish the contribution of Erk activities to pain perception, pain-related emotional responses or pain-related memory in the present study, we believe that such increased Erk activity may contribute to long-term plastic changes in the ACC caused by the injury or amputation. In fact, in brain slices, we demonstrate that Erk activity is required for synaptic potentiation in the ACC excitatory synapses . One major function of Erk may contribute to cortical plasticity during the expression of behavioral allodynia, and serve as one of key signaling protein kinase in chronic pain .
Adult male Sprague Dawley rats weighing 250–300 gram were used. Animals were kept in cages (2 animals per cage) at an ambient temperature of 20–25°C under a 12 hr light/dark cycle and had free access to food and water. We adhered to the ethical guidelines for investigation of experimental pain in conscious animals .
Animal models of chronic pain
For inflammatory pain model, fifty microliters of a 5% formalin solution (dissolved in saline) were injected into the plantar surface of the left hindpaw. For rat model of amputation, under brief anesthesia with halothane, the third digit of the left hindpaw was amputated. In some cases, at 2 week after the amputation, a brush stimulus (with number 4 camel's hair artist's brush) was applied for 3 min by stroking vertically at the dorsal part of amputated hindpaw. At 15 min, 45 min, 90 min and 120 min after formalin injection, 2 week after the amputation, or 15 min after brush stimulus, rats were deeply anesthetized with halothane and perfused transcardially with 100 ml of saline, followed by 500 ml of cold 0.1 M phosphate buffer containing 4% paraformaldehyde. The brain was removed, post-fixed for 4 hr, and then cryoprotected. Coronal sections (25 μm) through the ACC were cut using a cryostat. Sham groups without formalin injection or amputation were performed as controls. Sections from sham and experimental animals were processed simultaneously for immunostaining.
Immunostaining was performed using free-floating sections . Briefly, the ACC sections were first treated with 0.75% Triton X-100 and 1% H2O2 in PBS for 1 hr, and then processed for 30 min in 3% normal goat serum, followed by incubation with anti-phospho-p44/42 Erk (Thr202/Tyr204) monoclonal antibody (diluted 1:500; Cell Signaling, Beverly, MA) overnight at room temperature. Secondary reactions with biotinylated goat anti-mouse immunoglobulin (1:400; Vector Laboratories, Burlingame, CA) for 1 h were followed by avidin-biotin-peroxidase complexes (1:100; Vector Laboratories) for 1 h. A nickel-intensified diaminobenzidine with glucose oxidase was used as the final chromogen. Sections were washed several times, mounted on gelatinized slides, dehydrated through a series of ethanol solutions, cleared in xylene, and covered with glass coverslips. Controls, performed by replacing primary antibody with 1% NGS in the protocol, exhibited no staining.
This work was supported by grants from the EJLB-CIHR Michael Smith Chair in Neurosciences and Mental Health, Canada Research Chair, CIHR84256, CIHR66975 and NIH NINDS NS42722 to Dr. Min Zhuo.
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