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 various acknowledged experimental assays to measure the behavioral responses of mice to different kinds of painful stimuli. Currently, our lab has three behavioral testing devices to measure mechanical, thermal, and chemical related pain in the orofacial region. Mechanical pain is measured using an Orofacial Stimulation Test (OST) that is designed so that the mice must stick their snout through a drinking window that can be equipped with abrasive wires in order to get access to the reward bottle containing 30% sucrose. Using a similar device to OST called OPAD (Orofacial Pain Assessment Device), we have also examined the effects of Cdk5 on thermal sensitivity, where a mouse must make contact with two thermodes to access the reward bottle. The thermodes can be set at noxious hot or cold temperatures. Lastly, chemical aversion can be tested using a device called the lickometer to measure the consumption of water spiked with offensive chemicals, including the pungent plant compounds capsaicin and mustard oil. Using these behavioral tests, we are exploring the link between Cdk5 and orofacial pain as manifested by hypoalgesia or hyperalgesia. In our studies with genetically engineered mice, we have determined that the regulatory subunit p35 acts as a rate limiting factor affecting Cdk5 activity in pain sensing neurons, where its expression level is a key factor in regulating aversion to noxious stimuli. Overexpression of p35 in mice leads to both orofacial thermal and mechanical hyperalgesia while p35 knockouts display hypoalgesia when compared to littermate controls. Increased Cdk5 activity, however, can also be induced by binding with another regulatory subunit, p39. Developmentally, Cdk5 is important for normal neuronal migration as reduced Cdk5 activity can cause lamination patterning defects, resulting in embryonic lethality. In contrast, the p35 knockout mice are viable, even though there are still lamination patterning defects. The Cdk5 activator p39 must, therefore, play some compensatory role in activating Cdk5 to prevent embryonic lethality. Due to its importance in regulating Cdk5 activity during development, we wanted to determine if p39 also plays a role in pain sensitivity in adult mice. Our behavioral assays, however, show that, unlike p35, the Cdk5 activator p39 plays no major role in pain sensitivity, but this information has, none the less, allowed us to focus primarily on testing interfering molecules that block the interactions between Cdk5 and its activator p35 as a therapeutic means of inhibiting inflammatory induced pain hypersensitivity. Unlike senses like vision and smell, a major component of nociception, the detection of noxious stimuli, involves the activation of ion channels on the peripheral nerve ending, resulting in depolarization of the neuron and subsequent neuronal firing. During inflammation, ion channels that relay painful mechanical, thermal, or chemical stimuli can become sensitized and display altered channel activity that promotes hyperalgesia. We have shown that Cdk5 activity is induced under inflammation and, this, in turn, has effects on pain sensitivity. We are currently trying to identify substrates of Cdk5 that are involved in pain perception. Previously, we identified TRPV1, an ion channel that is activated by noxious heat and acidity, as a substrate of Cdk5. We have screened other proteins implicated in pain perception for possible interactions with Cdk5 and have newly determined that the ion channel known as P2X2a as a substrate of Ckd5. P2X2a is involved in the detection of chemical signals released upon tissue injury. Specifically, P2X2a is activated by release of adenosine triphosphate, an intracellular molecular involved in DNA synthesis and cell metabolism. We have shown that Cdk5 is able to modify the receptor P2X2a leading to a delay in use-dependent desensitization. Basically, rather than gradually desensitizing with continued activation, the P2X2a channel is more responsive to use over time than normal when modified by Cdk5. 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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