Expression And Activity Of Cdk5/p35 In Rat Model Of Trigeminal Neuropathic Pain

1. BackgroundCyclin-dependent kinase 5 (Cdk5) is a member of the family of Cdks, it belongs to serine/threonine kinase family, Cdk5 named for its sequence homology with the other cdk family. p35 is the major activator of cdk5. It was first extracted and purified from mammalian brain cell. cdk5 expression gradually with brain development and became most prominent in the adult brain, On the other hand, the expression of p35 was most prominent in newborn to 2 week of age and significantly decreased in the adult rat brain. The activity of cdk5 correlates with the expression of p35 but not with the expression of cdk5 in brain development. These data suggest that the activity of cdk5 may depend on the expression of p35, p35 regulates the activity of cdk5. Research has shown, p35 deficient mice severely affects the activity of cdk5, p39 compensatory increase also can not completely replace the function of p35. p35 and p39 are deficient mice, cdk5 has not kinase activity. Regardless of whether there are other regulatory factors cdk5, at least during the development of the mice, these both regulatory factors impact on the cdk5 can not be replaced by other molecules. In addition, the nerve cell cultures have shown that the overexpression of cdk5/p35 induced the growth of neuritis. In 2006, some scholars researched on the p35 gene knockout model p35-/- mice and p35 over-expression mouse models Tgp35 mice, using the wild WT mice as a control. The experiment found that the pain response of thermal stimulation of p35-/- mice was significantly delayed for its Cdk5 activity was significantly decreased. However, heat stimulation of the pain of Tgp35 mice was allergic reaction, since its cdk5 levels were significantly upward, cdk5/p35 had been proved to participate to regulate the pain signaling in the nervous system. The overexpression of cdk5/p35 can cause significant increase in synaptic structure and significantly improve the activity of the post-synaptic. It shows that overexpression of p35 activator of Cdk5 can cause the increase in synaptic structure and pain of allergic reaction, and it also regulate the protein substances of presynaptic and postsynaptic areas, thereby affects neurotransmitter release and receptor binding.Trigeminal neuralgia is happening paroxysmal and click to like severe pain in the trigeminal nerve distribution area, it lasts seconds to minutes. There are asymptomatic in interval.Pain may be due to any oral or facial stimulation. It commons in elderly and mostly is unilateral. So far, the pathogenesis of TN remains unclear, the exact model is difficult to establish. Domestic and foreign experts believe that surround factor is the main pathogenic factor of TN. Most experts agree that the vascular compression theory in the surround factors is the main pathogenic factor. Therefore, most animal models simulated by ligation of the infraorbital nerve vascular compression, and stimulate the surface of the skin by dominating the infraorbital nerve to simulate of the trigger points of trigeminal neuralgia.Spinal trigeminal nucleus caudalis subnucleus (Vc) is a portal that nociceptive information transmits from peripheral to the central. Three is a variety of terminal of neuropeptide-positive fibers which conduct pain in Vc. Most of them derived from the trigeminal ganglion neurons, and formed of synaptic connections with a large number of cell bodies and dendrites that distributed in the superficial layers of Vc neurons. So we test expression and activity of the cdk5/p35 in Vc tissue.Comprehensive the background of the study, we tested the expression of p35/Cdk5 in a rat model of trigeminal neuropathic pain and the activity of cdk5/p35.2. ObjectiveWe assume Cdk5/p35 are present in the neurons of trigeminal nerve spinal tract rat model of trigeminal caudal subnucleus (Vc), there is no reported cdk5/p35 how to play in a rat model of trigeminal neuralgia pain, and the sensory nerves of trigeminal nerve afferent the lower medulle oblongata of the Vc neurous. So we consider making the ION-CCI rat as experimental study model of the trigemninal neuropathic pain, and test the expression and activity of cdk5/p35.3. Materials and methods3.1 Animal groups36 SD male rats that were weighing 180 gram were divided into six groups according to normal group, sham group, ION-CCI-1, ION-CCI-3, ION-CCI-7, ION-CCI-14. All rats feed by a regular solid diet, free access to water, humidity 40%-50% in the animal laboratory of zhujiang hospital.3.2 The ligation of infraorbital nerveThe normal group without any treatment, the sham group just exposed the infraorbital nerve, then suture the wound, the other operation groups exposed one side of infraorbital nerve and ligate it. But it must maintain the neural microcirculation, then suture the wound.3.3 Preparation of infraorbital nerve and Vc tissueWeigh the rats according to the groups, Narcotize the rats by intraperitoneal injection of chloral hydrate 3ml/kg. Taking the nerve tissue of infraorbital nerve ligation area to make the HE and WEIL stain, then observed. Cutting the rats'Vc tissue according to Paxinos and Watson rat brain stereotaxic map. Put the tissues into the test tubes then store in the -80℃fridge3.4 The extraction of antigen protein of Vc tissueTaking out the tissue from the marked test tubes, then put them into homogenizer, after added the cell lysis solution and placed the homogenizer on ice and thoroughly homogenized, make the liquid of after homogenated into centrifuge tubes. At 4℃and 12000rpm, pick up the supernatant to the marked test tubes and put inside the-20℃fridge after centrifuged 5 minutes.3.5 Test the protein content of supernatant of Vc tissue3.6 Test the western blotting of cdk5 according to the measured protein content 3.7 Test the western blotting of p35 according to the measured protein content3.8 Test the immunoprecipitation and enzyme activity of cdk5 according to the measured protein content3.9 we will get the OD of cdk5 and p35 and the activity of cdk5 value in these groups of experiment, then using SPSS13.0 to analysis. One-way ANOVA test these data. First we test Homogeneity of variances. If P>0.05, using LSD test to compare between groups. If p<0.05,using Welch test to compare between groups.4. Results(1) Western blotting showed ION-CCI induced a time-dependent upregulation of p35 primarily within the ipsilateral superficial laminae of Vc revealed(2) In contrast the expression of Cdk5 was constant during day 1-14 in Vc after ION-CCI. Cdk5 activity on day 14 in Vc after ION-CCI (114 Kcpm) was snout 2 time the height of that on day 1 in Vc after ION-CCI (68 Kcpm). There was significant difference between each group(P<0.01)5. Conclusion(1) There was no significant relationship between the expression and activity of Cdk5 in these groups of experiment in the rats'Vc tissue(2) Expression of the activity of cdk5 is consistent with the expression of p35.