Dissertation
Dissertation > Medicine, health > Medical research methods > Experimental medicine, medical experiments > Medical Laboratory Animal Science

Expression and Activity of Cdk5/p35 in Rat Model of Trigeminal Neuropathic Pain

Author LiWei
Tutor CaiXiaoHui
School Southern Medical University,
Course Clinical Stomatology
Keywords Trigeminal neuralgia Chronic constriction injury Cdk5 p35
CLC R-332
Type Master's thesis
Year 2011
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Background cycle-dependent protein kinase 5 (cyclin-dependent kinase 5, Cdk5) is one of the Cdks family members, belonging to the serine / threonine kinase family, because Cdk5 has named homologous sequences with other members of the Cdk, p35 its main activator. Cdk5/p35 originally from bovine brain separation and refining come. Cdk5 expression is increased with the development of the brain and reach the highest, p35 expression was born from the rat to the expression of the second week of cdk5 expression in the adult rat are the highest expression of p35 in the adult rat brain was meaningful reduction. Various stages of brain development in the rat p35 expression and cdk5 activity is closely related to, but not associated with the expression of the Cdk5. This prompted the Cdk5 the activity depends on p35 expression, p35 expression regulates Cdk5 activity. Studies have shown that mice lacking p35 expression, and make cdk5 activity affected, another activator p39 upregulation emergency, can not completely replace the function of p35. p35 and p39 are defective in mice, Cdk5 kinase activity, with the Cdk5 deficient mice, indicating that at least in the the mouse development process, whether there cdk5 other adjustment factor, P35 cdk5 activity regulation is not completely other molecular protein substitute. Moreover, neural cell cultures of Cdk5 and p35 overexpression induced neurite outgrowth. 2006, scholars unless the mouse model (p35-/ - mice) and p35 overexpression model the (Tgp35 mice), control WT mice with wild-type p35 gene knock, found that Cdk5 activity was significantly decreased p35-/ - mice, the pain response to thermal stimulation was significantly delayed; pain Allergies of Cdk5 levels significantly upregulated Tgp35 mice to thermal stimulation. Cdk5/p35 been shown to be involved in the regulation of the nervous system pain signal passed Cdk5/p35 overexpression can increase the number of synaptic structure and postsynaptic activity greatly improve. That of Cdk5 activator p35 overexpression can cause increase of synaptic structure and allergies and the pain reaction, and also regulate the presynaptic expression of protein material after the area, thereby affecting neurotransmitter release and receptor binding. Trigeminal neuralgia (Trigeminal neuralgia, TN) is the trigeminal nerve distribution area the paroxysmal electrical shock like severe pain, which lasted a few seconds to a few minutes, intermission asymptomatic. The pain can be caused by any oral or facial stimulus. More common in the elderly, mostly unilateral. So far, TN on pathogenesis remain unclear, difficult to establish the exact animal model. Domestic and foreign scholars generally agreed that the surrounding factors is the major causative factor in TN decision most scholars agree that the vascular compression doctrine surrounding factors, Therefore, most of the animal models by ligation of the infraorbital nerve simulation vascular compression and in infraorbital nerve dominate at the surface of the skin to mechanical stimulation, analog trigeminal neuralgia trigger points. Located on the outside of the medulla oblongata and trigeminal spinal tract of the inside of the spinal trigeminal nucleus (Spinal trigeminal caudal subnucleus, Vc), five from outside to inside. Spinal trigeminal nucleus is The noxious stimuli maxillofacial organizations want the central conduction portal, caudal subnucleus and adjacent areas feel somatosensory primary hub convey maxillofacial noxious stimuli information. Therefore, we have adopted the the Vc organization Cdk5/p35 to detect the expression and activity. Above background, the experiments were prepared by three and neuropathic pain rat model and to detect changes in the group of trigeminal neuralgia rat model of Cdk5 and p35 expression and activity. Purpose we assume the Cdk5/p35 exist in neurons in the rat TN model Vc, elaborated no Cdk5/p35 the model of how to play the role, and trigeminal sensory afferent nerve the bulbar under section Vc neurons, so we consider the infraorbital nerve chronic constriction injury (chronic constriction injury on the infraorbital branch of the trigeminal nerve, the ION-CCI) rats as experimental model in TN, inspection ION-CCI rats Vc Organization the expression in Cdk5/p35 and Cdk5 activity lay the experimental foundation for the study of the correlation of Cdk5/p35 TN pathogenesis. 3 Materials and Methods 3.1 Experimental Animal grouping the same batch of SD male rats, weighing 180g, 36 were randomly divided into six groups, divided into normal group, sham operation group, ION-CCI 1d group, ION-CCI 3d group, ION the-CCI 7d group, ION-CCI 14d group. Rats by Southern Medical the Zhujiang Hospital animals Laboratories conventional solid diet feeding, with free access to water, humidity 40% -50%. 3.2 infraorbital nerve ligation normal group do not need any treatment, sham group only exposed infraorbital nerve suture wounds, the rest of the surgery group exposed side of the infraorbital nerve ligation, but to keep the nerves microvascular circulation, suture wounds. 3.3 infraorbital nerve specimens and Vc tissue preparation called to take the body weight of rats, respectively, according to the grouping flag chloral hydrate anesthesia after intraperitoneal injection of 350mg/kg body weight, take the the ligation District infraorbital nerve HE weil's staining. Paxinos and Watson stereotaxic atlas of the rat brain given good Vc coordinate position cut the rat Vc organization block, were placed in the corresponding mark good test tube, placed at -80 ℃ cryopreservation. 3.4 Vc tissue antigen protein extraction removed by tag vitro tissue blocks, into the homogenizer, add the cell lysis buffer, the homogenate was placed on ice to conduct a thorough homogenate, the homogenate was transferred to centrifugation the tube, at 4 ℃, 12000rpm centrifuged 5min, extracting the supernatant is mounted on the already marked centrifuge tube, and placed in the -20 ℃ refrigerator. Western blot, 3.8 Cdk5 immunoprecipitation and activity analysis 3.9 3.5 Vc organizations supernatant determination of the protein content of 3.6 Cdk5 3.7 p35 detected by Western blotting experiments on rats in each group OD values ??of Cdk5 and p35 and Cdk5 activity using SPSS13.0 statistical software for statistical analysis, the use of single-factor analysis of variance (one-way ANOVA), the first test of homogeneity of variance, such as the homogeneity of variance using LSD test for multiple comparisons; If heterogeneity of variance, Dunnett's multiple comparison. 4 Results (1) ION-CCI was the form of time-dependent induction of p35 in rat nerve injury side Vc organization raised; (2) of Cdk5 expression among the experimental group showed no significant difference, Cdk5 activity and p35 expression have consistency, and to p35 upregulation curve showed an increasing trend. Cdk5 activity and p35 expression consistent and showed an increasing trend, ION-CCI 14 days Vc organization Cdk5 activity (114 Kcpm), Vc organizations, ION-CCI one day in the Cdk5 activity (68 Kcpm) 2 times, among the groups were significant differences (P lt; 0.01) 5, Conclusion (1) experimental rats Vc organization, association of Cdk5 activity Cdk5 protein expression; (2) experimental group the rat Vc organization Cdk5 activity and p35 expression with consistency.

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