| BackgroundPrimary osteosarcoma and cancer bone metastasis are mostly end-stage patients,one of the main symptoms is pain.This kind of pain is also called bone cancer pain(bone cancer pain,BCP).The quality of life of patients with BCP is seriously affected,and it is also a great mental pressure and economic burden for patients’ families and society.BCP is the most complex and unique mechanism in all kinds of chronic pain.It has two characteristics:neuropathic pain caused by tumor cell invasion and inflammatory pain caused by local inflammatory mediators.Clinicians often use opioids to alleviate BCP,but side effects such as opioid addiction and obstinate constipation limit their wider use.It is of great significance to find a reliable and less side effect method to alleviate BCP.For non-cancerous chronic pain,neuroelectric stimulation has been used to treat chronic pain.Peripheral electrical stimulation of intractable chronic pain,characterized by neuropathic pain,is sensitive to some extent.The mechanism of peripheral nerve stimulation(peripheral nerve stimulation,PNS)on chronic pain is very complicated and has not been fully explained.It is possible that the analgesic effect is played by acting on peripheral nerve fibers A and C at the spinal level,at the same time reducing the release of inflammatory factors such as substance P norepinephrine(5-HT),GABA)and multiple regulation of pain.Different parameters of stimulation conditions,the effect of pain treatment is also different.Therefore,it is of great significance to select appropriate electrical stimulation parameters.Like c-fos and Zif268,Arc(activity-regulated cytoskeleton-associated protein,Arc),also known as arg3.1,belongs to a family containing many types of early-response genes.One characteristic of Arc is that it can be induced by high frequency(>100 Hz)peripheral electrical nerve stimulation.Nother feature is that synaptic plasticity plays a crucial role,while peripheral nerve stimulation can induce Arc expression in hippocampal gyrus and neocortex.In this study,on the one hand,in the pain model of bone cancer in rats,the best peripheral nerve stimulation parameters were selected by using the method of pain behavior(thermal pain behavior and mechanical pain behavior).On the other hand,the relationship between the expression of Arc and GluR1 protein and peripheral nerve stimulation in spinal cord was observed.Further more,intrathecal injection of Arc shRNA is used to clarify the mechanism of Arc in the treatment of bone cancer pain by peripheral nerve electrical stimulation.Part I:Construction and evaluation of rat tibial cancer pain modelObjectiveTo construct and evaluate the rat bone cancer pain model by means of weight change,heat pain and mechanical pain behavior,tibial imaging and tibial histology.Methods Female Sprague-Dawley rats weighing 160-180g were injected with Walker 256 tumor cells in the right tibia of mice(female SD neonatal rats weighing 60-80 g)and Walker256 breast cancer sarcoma cells were inoculated with Walker 256 tumor cells for 1 day,The effects of heat withdrawal latency(TWL)and mechanical withdrawal threshold(MWT)were observed at 3d,5d,7d,9d,11d,13d,15d and 21d.On the 14th day after tumor cells,the destruction of the right tibial bone and the histomorphology(HE staining)were examined by radiographic imaging.The growth of the tumor was observed under the microscope.Results 1.The weight gain rate of the rats on the 13th day after Tumor cell implantation(TCI)was significantly slower,and the rate of slow growth was time-dependent.2.The TWL and MWT values were significantly decreased in the fifth day after TCI,and decreased with the passage of time.3.On the 14th day after TC,X-ray showed that there were obvious osteoporosis in the upper and lower tibia of the operation.At the same time,the bone cortex had obvious defects,the structure of the bone was unclear and the degree of bone destruction was significantly increased.Histopathological examination showed that the bone marrow cavity filled with nuclear large deep staining of tumor cells,tumor cells were active growth status,both sides of the cortical bone and bone marrow were significant damage.Summary TCI rats were sensitive to heat pain and mechanical pain,severe tibial bone destruction and active tumor cells in the bone marrow cavity.The rat tibial cancer pain model was established successfully.PartⅡ:Effects of different parameters on peripheral nerve stimulation on bone cancer painObjective To observe the effect of peripheral nerve electrical stimulation on bone pain in different parameters(including frequency,intensity and duration of action)by pain behavior,and to screen out the best parameters for the treatment of bone cancer pain.Methods(10Hz,0.3mA,0.6mA)were used to measure the frequency,intensity and stimulation time of the stimuli,including the frequency of stimulation(120Hz),medium(60Hz)and low(10Hz)(10 min,20 min,and 40 min).The seventh day(7 days of bone cancer pain model rats had significant painful behavior changes)Bone cancer pain in the sciatic nerve of rats by heat pain and mechanical pain.And other indicators to observe the different combinations of different electrical stimulation of bone pain.Results The effect of peripheral nerve stimulation at 60Hz and 120Hz on the thermal hyperalgesia and mechanical pain of rats with bone cancer pain was significantly inhibited under the condition of stimulating intensity and time of action.At the same time,the inhibition of 120Hz group was stronger than 60Hz,but no significant difference,10Hz frequency of bone pain in rats with no significant effect on pain behavior.The peripheral nerve electrical stimulation of 0.3mA and 0.6mA intensity had a significant effect on the thermal hyperalgesia and mechanical pain of rats with bone cancer pain under the condition of stimulating frequency and time of action,and the mitigation effect was not significant.The 0.1 mA intensity of electrical stimulation had no significant effect on pain behavior in rats with bone cancer pain.Under the condition of stimulating intensity and time of action,the peripheral nerve stimulation at 10min,20min and 40min had obvious inhibitory effect on the thermal and mechanical pain of rats with bone cancer pain,and the inhibition of 20min and 40min.The effect was significantly stronger than lOmin,and 20min,40min between the inhibition of no significant difference.Summary 60Hz,0.3mA and 20min are the best parameters for the treatment of bone cancer pain in three parameters,frequency,intensity and time of action.Part Ⅲ:The role of spinal cord Arc-G1uR1 in relieving bone cancer pain by peripheral electrical stimulationObjective To observe the effect of Arc and GluRl in the treatment of bone cancer pain by peripheral nerve stimulation.Methods The effects of peripheral nerve stimulation at different frequencies on the expression of Arc,GluR1 and pNR2B protein in the spinal cord of normal rats and bone cancer rats were detected by immunoblotting and immunohistochemistry.In order to further clarify the effect of Arc and GluRl on the treatment of bone cancer pain by peripheral nerve stimulation,lentiviral particles carrying Arc shRNA were injected intrathecally one day before electrical stimulation.Real-time PCR and Western blot were used to detect the expression of Arc mRNA and protein in spinal cord And the effects of intrathecal injection of Arc shRNA on the analgesia of bone pain and the expression of Arc and GluRl in spinal cord were observed by heat pain and mechanical pain behavior method and protein immunoblotting method.Results 1.The expression of Arc protein in the spinal cord of normal rats was significantly increased at 60Hz and 120Hz,and the expression level of GluRl protein was significantly decreased in the central region of Ⅰ-Ⅱ layer of spinal dorsal horn.At the same time,between 60Hz and 120Hz,There was no significant difference in the expression of GluR1.The electrical stimulation at 10Hz had no significant effect on the expression of Arc and GluRl.The expression of pNR2B protein did not change significantly during the whole electrical stimulation.2.The expression of Arc protein was further induced by electrical stimulation at 60Hz and 120Hz in rats with bone cancer pain,and the expression of GluRl was significantly decreased,and the expression of pNR2B was not changed significantly.3.After intrathecal injection of Arc shRNA,the expression of Arc mRNA and protein in the spinal cord of rats with bone cancer was significantly decreased.Intrathecal injection of Arc shRNA,60Hz peripheral nerve stimulation to alleviate the role of bone cancer pain was offset,while inhibiting the electrical stimulation induced Arc expression,GluRl protein expression increased.Summary Peripheral nerve stimulation can induce the expression of Arc protein in the spinal cord of normal rats and bone cancer rats,and inhibit the expression of GluRl.Intrathecal injection of Arc shRNA can inhibit the analgesic effect of peripheral nerve stimulation on bone cancer pain Spinal cord expression and GluRl expression increased.ConclusionI:TCI rats were sensitive to heat pain and mechanical pain,severe tibial bone destruction and active tumor cells in the bone marrow cavity.The rat tibial cancer pain model was established successfully.Ⅱ:60Hz,0.3mA and 20min are the best parameters for the treatment of bone cancer pain in three parameters,frequency,intensity and time of action.Ⅲ:Peripheral nerve stimulation can induce the expression of Arc protein in the spinal cord of normal rats and bone cancer rats,and inhibit the expression of GluR1.Intrathecal injection of Arc shRNA can inhibit the analgesic effect of peripheral nerve stimulation on bone cancer pain Spinal cord expression and GluR1 expression increased. |
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