Cdk5 and orofacial mechanical pain: Chronic orofacial pain is a significant public health concern. Patients with orofacial pain conditions often experience mechanical and thermal allodynia or hyperalgesia. Nevertheless, there are few animal models for such conditions. 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 mechanical orofacial stimulation. Using these devices, we are exploring the link between Cdk5 and orofacial pain as manifested by mechanical hypoalgesia or hyperalgesia. Is Cdk5 involved in orofacial mechanical pain sensation? To answer this fundamental question, we used a modified orofacial stimulation test (OST) on wild-type, p35-/-, and transgenic p35 (Tgp35) mice to study the effect of different mechanical stimuli on their behavior. This new behavioral testing method uses a conflict paradigm that allows animals to make a choice between receiving a reward (30% sucrose) or escaping noxious stimuli, so the animals have control over the amount of nociceptive stimulation and can modify their own behavior. Additionally, this technique provides investigator-independent testing using automatically recorded behavior of the observed animals; they incur less stress, and their behavior can be measured repeatedly in a non-biased fashion. Three different levels of painful conditions were 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). Our current findings reveal aversive behavior to mechanical stimulation with orofacial mechanical hypersensitivity in Tgp35 mice (which have increased Cdk5 activity), as evidenced by shortening of the total licking time and number of attempts the mice make to access the reward. The number of reward licking/facial contact events decreased substantially in these mice with increased mechanical pain intensity. In contrast, mice lacking p35 (with decreased Cdk5 activity) and mice lacking Cdk5 expression in sensory neurons displayed mechanical hypoalgesia. To the best of our knowledge, we are the first to report using the orofacial mechanical stimulation test in mice to demonstrate that Cdk5 plays an important role in orofacial mechanonociception. Cdk5 and orofacial inflammatory pain: Is Cdk5 involved in nociception that occurs during painful physiological conditions such as orofacial inflammation? We have previously shown that inflammation is able to induce Cdk5 activity. Currently, we are testing our mouse models that exhibit genetically altered Cdk5 activity with chemical agents that can induce inflammation in the orofacial region (carrageenan for acute inflammation and complete Freund's adjuvant to mimic chronic inflammation). We are examining if increased or decreased Cdk5 activity has an effect on pain responses following inflammation, or if there is an effect on the level of inflammation produced by these noxious chemicals. Our laboratory had previously reported that inflammation induced by carrageenan injection into the hind paws of mice increased the mRNA and protein levels of Cdk5/p35 in nociceptive neurons with a subsequent increase in Cdk5 kinase activity. To examine facial inflammatory pain, which might involve different mechanisms, we injected carrageenan into the mouse vibrissal pad. Along with increases in p35, this type of acute orofacial inflammation resulted in increased levels of inflammatory cytokines (TNF-alpha, Il-1beta, Il-6) cyclooxygenase-2, and transcription factors that are known to induce expression of Cdk5 and p35. Our results demonstrate that increased Cdk5 activity is associated with orofacial inflammatory pain. Using the same model of acute orofacial inflammation, our genetically altered Cdk5 mice demonstrated opposing phenotypes. Mice with increased Cdk5 activity exhibited enhanced expression of inflammatory markers, while Cdk5 activity-deficient mice show delayed and impaired expression of these indicators. These findings point to roles for Cdk5 not only in pain sensation, but also in the inflammatory responses that induce pain. Since TNF-alpha promotes increased p35 expression and subsequently induces increased Cdk5 kinase activity, we generated a mouse model for conditional over-expression of TNF-alpha, where Cre-mediated recombination of the transgene is needed to cause TNF-alpha overexpression (TNF-alpha glo). 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. To generate a mouse model for the study of tooth pain, we first tested in vitro a transgenic vector for conditional overexpression of TNF-alpha in a mouse odontoblast cell line (MO6-G3 cells). After thereby verifying the feasibility of recombination-activated gene expression in tissue culture, we generated TNF-alpha glo mice, a DMP1-Cre line for producing inflammation in the teeth. These mice have inflammatory infiltrates that are primarily localized only to the teeth and adjacent bone, without any major pathology in any other tissues. We were also able to demonstrate that these TNF-alpha glo/DMP1-Cre mice experience gnawing dysfunction according to dolognawmeter testing, indicating orofacial pain. We saw increased expression of inflammatory markers in the TG that innervate the tooth pulp while also seeing a modest increase in Cdk5 activity. 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 continue our efforts to collaboratively study role of TGF-beta in disease processes affecting orofacial tissues.
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