| Bone cancer pain, which impair patients’ physical and psychological function, is amalignant disease-related pain state dominated by a battery of symptoms including tonicbackground pain at rest, spontaneous pain at rest and movement-evoked pain. While thebackground pain at rest could be handled by opioid analgesia now, the other twocomponents are still difficult to deal with. Most late-stage individuals are too frail tobenefit from current therapeutic regimens, such as gold standard therapy-palliativeradiotherapy. Therefore, it is very important to clarify the pathophysiologic mechanismsof bone cancer pain.Research numbe r one Expression of Nav1.8and Nav1.9on the ipsilateral DRGin a rat model of bone cancer painObjective In attempt to understand the underlying mechanisms of cancer-induced bonepain, we established a rat model of bone cancer pain and investigated the presence oftwo tetrodotoxin-resistant sodium channels, Nav1.8(SNS/PN3) and Nav1.9(SNS2/NaN), on the ipsilateral dorsal root ganglia (DRG) neurons in this bone cancerpain animal model.Methods Fifty-seven female Sprague-Dawley rats were randomized into three groups:Sham operation group (Sham, n=17), cancer-bearing animals killed after7days (C7,n=17) and cancer-bearing animals killed after14days group (C14, n=18). Afterinoculation of5μL1×104/μL walker256tumor cells (for C7and C14groups) or5μLsaline solution vehicle (for sham group) into the left tibia medullary cavity ofSprague–Dawley rats, mechanical pain paw withdrawal threshold (PWT) and thermalpain PWT were measured with Von Frey filaments and a CO2laser heat stimulator.Series of tests including body weight, bone radiology, bone histology were carried outat the final time point of each group to make sure the animal model is successfullyestablished. The ipsilateral lumbar4-5DRG were dissociated7(C7group) or14(sham and C14groups) days after the establishment of bone caner pain model, respectively.Real-time RT-PCR, Western bolt and Immunofluorescence were applied to determinethe mRNA and protein expression of Nav1.8and Nav1.9in ipsilateral lumbar4-5DRG.Results During the obvervation, there was an increase of body weight in sham groupand a decrease of body weight in two cancer pain groups from post-operative day5today7. Mechanical and thermal pain PWT in cancer group displayed a profounddecrease from post-operation day7to the end of the experiment, while sham-operatedrats did not show significant responses to any stimulation within the subthresholdintensity. Erosion of the bone and infiltration of the tumor cells could be detected byradiological and histological assay. Compared to Sham group, the relative mRNAexpression of Nav1.8and Nav1.9exhibited a significant up-regulation in C14group(8.9times and9times, respectively, P<0.01) but not C7group (1.5times and2.4times,respectively, P>0.05). Western blot and Immunofluorescence revealed an apparentincrease of Nav1.8(P<0.05) and Nav1.9(P<0.05) protein in C14group compared withSham group.Conclusion We successfully established an animal model of metastatic carcinoma ofbone. The decrease of mechanical and thermal pain PWT occurred almost concurrentlywith the up-regulation of mRNA and protein levels of Nav1.8and Nav1.9suggested thepotential involvement of the two TTXr sodium channels in the development andmaintenance of bone cancer pain. Research number two Improve ment of technology for dissociating rats’ dorsalroot ganglion cells used for patch clampObjective To Improve the previous technology for dissociating rats’ dorsal rootganglion（DRG）cells being used for whole-cell patch-clamp studies.Methods The rats’ DRG were obtained with microsurgical techniques, and then theDRG cells were acutely dissociated and acquired with optimized culture methodincluding dual enzyme digestion and dual serum incubation. After a short-time culture, the DRG neurons were observed under inverted microscope and those with normalmorphologic character were selected for the further studies. The basicelectrophysiological membrane characteristics and sodium channel currents wererecorded by Whole-cell patch-clamp technique before and after the administration ofTTX, respectively.Results The various size cultured DRG cells, which were acutely dissociated andacquired with optimized method, were morphologically intact with a round or ovalshape and smooth clear cell membrane. The resting membrane potential was in a rangeof55.22±4.39mV. Some slowly inactivated tetrodotoxin-resistant sodium currents wererecorded after the administration of tetrodotoxin. The highest giga-seal successrate(73.8%) with whole-cell patch-clamp was obtained in mid-diameter DRG neurons,which was significantly higher than those gotten from the small and large diameterneurons (P<0.05).Conclusion The optimized simple protocol, including dual enzyme digestion and dualserum incubation, can acquire morphologically intact rats’ DRG neurons in a highseparation efficiency. The dissociated mid-diameter DRG neurons under these methodsare suitable for patch clamp research. |
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