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Molecular Roles of Cdk5 in Neuronal Functions and Pain Signaling

Cdk5 在神经元功能和疼痛信号传导中的分子作用

基本信息

批准号：
9155517
负责人：
Ashok Kulkarni
金额：
$ 122.79万
依托单位：
NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9155517
关键词：
Acute Adult Affect Afferent Neurons Alzheimer&apos s Disease Animal Model Animals Attention Deficit Disorder Behavior Blood flow Burning Pain Carrageenan Cell Cycle Cell Line Cells Chemical Agents Chemicals Chronic Complex Conflict (Psychology)Cornea Cyclin-Dependent Kinase 5 Dental Pulp Devices Diagnosis Disease Elderly Esthesia Etiology Event Exhibits Face Freund&apos s Adjuvant Functional disorder Gene Expression Genetic Recombination Genetically Engineered Mouse Goals Huntington Disease Hyperalgesia Hypersensitivity Inflammation Inflammatory Inflammatory Infiltrate Inflammatory Response Injection of therapeutic agent Investigation Laboratories Link Malignant Neoplasms Malignant neoplasm of prostate Measures Mechanical Stimulation Mechanics Mediating Medicine Messenger RNA Methods Mitotic Modeling Molecular Mus Neurodegenerative Disorders Neurons Nociception Odontoblasts Oral mucous membrane structure Orofacial Pain Pain Pain intensity Parkinson Disease Pathology Pathway interactions Patients Phenotype Phosphotransferases Physiological Play Population Process Proline Proteins Public Health Pulpitis Reporting Research Research Personnel Rewards Role Salivary Glands Sensory Signal Transduction Sjogren&apos s Syndrome Spinal Spinal Ganglia Stimulus Stress Structure Structure of mucous membrane of nose Structure of trigeminal ganglion Sucrose System Techniques Testing Thalamic structure Time Tissues Tooth structure Transforming Growth Factor beta Transgenes Transgenic Organisms Tumor Necrosis Factor-alpha Woman allodynia base behavior test bone chronic pain cyclooxygenase 2 cytokine experience human TNF protein in vitro testing inflammatory marker inflammatory pain malignant endocrine gland neoplasm men mouse model nerve supply neurochemistry neurodevelopment neurophysiology neuropsychiatry nitinol novel orofacial overexpression painful neuropathy peripheral pain programs response tissue culture transcription factor transmission process vector

项目摘要

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.

Cdk5与口面机械性疼痛：慢性口面疼痛是一个重要的公共卫生问题。患有口面部疼痛的患者通常会经历机械和热异常性疼痛或痛觉过敏。然而，很少有动物模型来模拟这种情况。为了研究口面痛觉过敏并确定Cdk5活性是否参与其中，我们使用了特殊的装置来量化小鼠对疼痛的机械口面刺激的反应。使用这些装置，我们正在探索Cdk5与机械性痛觉减退或痛觉过敏表现的口面部疼痛之间的联系。