Chronic orofacial pain is a significant public health concern. Patients with orofacial pain conditions often experience both mechanical and thermal allodynia or hyperalgesia. In order to study orofacial hyperalgesia and determine whether Cdk5 activity is involved, we have utilized special devices to quantify the responses of mice to painful orofacial stimulation. Using these devices, we are exploring the link between Cdk5 and orofacial pain as manifested by hypoalgesia or hyperalgesia. In our studies, we used mice lacking the Cdk5 activator p35 (p35-/- with about an 85% reduction in Cdk5 activty), mice null for Cdk5 in nociceptive neurons (deletion of Cdk5 activity only in nociceptive neurons), or mice transgenically expressing an additional copy of p35 (Tgp35 with an overall 50% increase in Cdk5 activity). To examine if Cdk5 activity is involved in orofacial mechanical pain sensation, we tested our mice using a modified orofacial stimulation test (OST). The OST employs a conflict paradigm that allows animals to make a choice between receiving a reward or escaping painful stimuli. In the case of OST, mice can only get to a solution of 30% sucrose (a reward) if they can endure putting their snout through a series of abrasive wires. Three different degrees of noxious mechanical stimulation are achieved by interfering with their access to a reward (30% sucrose) using plates with different numbers of Nitinol wires (pain level 1: 6+6 wires, level 2: 9+9 wires, and level 3: 13+13 wires). OST testing, therefore, allows for testing of mechanical pain sensitivity. Our findings revealed that increased Cdk5 activity in mice (Tgp35) leads to orofacial mechanical hypersensitivity, as evidenced by shortened total licking time and a decreased number of attempts to access the reward. In contrast, mice lacking p35 (with decreased Cdk5 activity) and mice lacking Cdk5 expression in sensory neurons displayed mechanical hypoalgesia. Using a similar device to OST called OPAD (Orofacial Pain Assessment Device), we have also examined the effects of Cdk5 on thermal sensitivity, particularly towards noxious heat. With OPAD, mice must put their snout between two thermodes to obtain a 30% sucrose reward. We tested the responses to noxious heat by setting the thermodes at 45C. Again, the mice with increased Cdk5 activity displayed increased aversive behavior (decreased licking) to a noxious orofacial stimulus, this time using hot temperature. In contrast, mice with decreased Cdk5 activity displayed significantly increased number of licks compared to control animals. The perception of noxious heat is, in part, relayed via the pain-transducing ion channel TRPV1 (Transient Receptor Potential Vanilloid 1). TRPV1 is additionally a substrate of Cdk5. To examine the interactions between Cdk5 and TRPV1 further in vivo, we examined the level of oral aversion to the TRPV1 agonist capsaicin in our mice. Capsaicin causes a burning sensation in the mouth when consumed, so we used a device known as the lickometer to measure the number of times mice lick water spiked with capsaicin. Mice with increased Cdk5 activity displayed increased aversion to capsaicin expressed by decreased number of licks compared to control animals. In contrast, mice with decreased Cdk5 activity showed decreased aversion to capsaicin consumption. Our results indicate that Cdk5 activity modulates TRPV1 channel activity in vivo and that increased TRPV1 sensitivity is a result of direct modification of TRPV1 by Cdk5. We wanted to develop mouse models mimicking conditions of orofacial inflammation and subsequent hyperalgesia in order to better understand the neuronal pathways involved in pain perception. We have animal models of inflammatory hyperalgesia through injections of either carrageenan or CFA, but we also wanted to complement these studies using genetic approaches to induce orofacial pain in mice. To develop an animal model of orofacial pain, we decided to generate a transgenic mouse that allows for conditional overexpression of the proinflammatory cytokine tumor necrosis factor- (TNF-alpha) as a means of causing a type of inflammation without injection of a microbial trigger or foreign immunostimulatory chemical. In the resulting TNF-alpha-glo mice, overexpression of TNF-alpha requires recombination of the transgene by the enzyme Cre (Cyclization Recombinase). These mice have been used to conditionally overexpress TNF-alpha in the teeth (to mimic pulpitis), in the sensory neurons (as a model of neuropathic pain), and in the salivary gland (to produce an inflammation similar to Sjogren's Syndrome). Most mouse models of tooth inflammation, for example, involve exposure of the tooth pulp followed by administration of lipopolysaccharide (LPS) to induce an inflammatory immune response, but we used the TNF-alpha-glo mice to develop a non-invasive means to research dental pain. TNF-alpha overexpression is restricted to cells that express dentin matrix protein 1 (DMP1), which primarily occurs in odontoblasts, but also ameloblasts, cementoblasts, and osteocytes as well. This genetically engineered mouse model, designated DMP1/TNF-glo, shows inflammation in the teeth that resembled pulpitis, including inflammatory infiltrates in the tooth pulp and vasodilation of the blood vessels. TNF-alpha expression was localized around the odontoblast layer that, in turn, caused active TNF-alpha cell signaling in the tooth pulp and recruitment of lymphocytes and macrophages. Orofacial pain resulting from tooth inflammation was validated using the dolognawmeter, a device that measures gnawing function. DMP1/TNF-glo mice took longer than the controls to chew through the hard ethylene vinyl acetate (EVA) resin dowel, which suggests masticatory dysfunction (a behavioral index of orofacial pain). Along with this tooth pain model, we also wanted to see if we could replicate in mice conditions of neuronal inflammation where TNF-alpha expression is induced after neuronal injury and neuropathic pain. We were able to use the TNF-alpha-glo mice to conditionally overexpress TNF-alpha in pain sensing neurons (SNS/TNF-alpha-glo mice). We were then able to show that increased levels of TNF-alpha in the TG induces both increased expression of the Cdk5 activator p35 and elevated Cdk5 activity. These mice also have increased orofacial aversion to noxious heat (45C) as tested by OPAD where SNS/TNF-alpha-glo mice show less licking events as compared to the controls. In summary, having shown that Cdk5 modulates orofacial mechanical pain, our current research is focused on further confirming these findings with additional Cdk5 mouse models. Furthermore, we will vigorously pursue molecular investigations into identifying novel Cdk5 substrates involved in pain signaling. Additionally, we will continue our efforts to collaboratively study role of TGF-beta in disease processes affecting orofacial tissues.
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