| BACKGROUND AND OBJECTIVEChronic pain is the most common and most intractable disease.For those patients withadvanced cancer, pain is often the most painful experience in the final stage of their lives.Studies have shown that about two-thirds of patients with advanced cancer will experienceosseous metastasis, which are considered to be the most common cause of cancer pain.It’sone of the primary causes why the survival of terminal cancer patients is affected thatcontinuous infiltration of tumor cell, normal sclerotin instruction is destroyed results inserious pain response when the sick bone is under mild pressure. Effective analgesia hasbecome in deadly need. Intravenous or oral opioids for pain relief are the best ways, butbring a lot of side effects, such as excessive sedation, respiratory depression, constipation,addiction and etc. Patient-controlled analgesia technology brings the gospel to patientswith cancer pain, but there are also some problems including hard to maintaining,increasing the chances of infection, higher costs and other issues. Finding more convenient,effective, lasting and economic, safe analgesia becomes a subject of public concern. Inrecent years, gene therapy of chronic pain and using exogenous vector to mediateendogenous protein’s effect on peripheral and central neurons directly or indirectly havehigher purity than chemicals or protein preparation, less side effects, low toleranceincidence, and can take advantage of the specificity and long-term of vectors expression tosolve clinical problems posed by long-term medication.In previous studies，recombinant adenovirus expressing beta-endorphin (Ad-NEP)treatment of the rat model with CCI pain has a positive meaning. Recombinantadenovirus expressing-endorphin (Ad-NEP) treatment of bone cancer pain has not beenreported. This study was to construct adenovirus carrying the nerve growth factor leader peptide-endorphin, which is inoculated via the lateral ventricle, transfects hostependymal cells, long-term and efficient expresses human endogenous-endorphin,elevates-endorphin levels in cerebrospinal fluid, effects on the spinal segment μ-opioidreceptors(MOR). Through cerebrospinal fluid circulation, inhibits c-fos and c-junexpression in neurons by G protein-mediated signaling pathways, thereby inhibits uploadof nociceptive signals, enhances inhibitory modulation system, changes neuronal plasticity,and increases the pain thresholdwhich. The present study may try a new way for long-termrelief of chronic pain patients and provide experimental basis in adenoviral transfectionefficiency and safety in the central nervous system for further research.METHOD1. Create rat model of bone cancer pain and explore the feasibility assessmentThe rats were divided into tumor group, sham group, and normal group. Walker256ratbreast cancer cells rats was vaccinated in the weanling rats’ abdominal cavity, rat asciteswere collected after7days, separated and extracted. Then plant this ascites mixture into theright side tibia of the tumor group rats. Sham group was injected a mixture of inactivatedascites mixture, normal group not treated. On the same day,2,4,6,8,10,12,14,16,18,20,22days after inoculation surgery, in the same time every day, to three groups, randomly anddouble-blind assess the use of spontaneous walking limb, measure thermal stimulationinduced pain behavior and mechanical stimulation induced pain behavior as well. On the22thday after inoculation surgery, the rats, after anesthesia, accepted a CT scan of theirlower limbs. CT scanning conditions:80KV,50MA, matrix512×512, thickness0.7mm.Observe whether the right tibia sclerotin changes.2. The effect on β-endorphin in cerebrospinal fluid after intracerebroventricularinjection of Ad-NEP with recombinant adenovirus expressing β-endorphinThe original titer of Ad-NEP is5×1011PFU. It was diluted of4different titers (5×1010PFU,5×109PFU,5×108PFU, and5×107PFU) for standby. Experimental SD rats were divided into six groups. Among them, Group A, Group B, Group C, group D andgroup E correspond to5×107PFU,5×108PFU,5×109PFU,5×1010PFU,5×1011PFUfive different titers of Ad-NEP, besides, group F is physiological saline control. Fivedifferent titer groups of Ad-NEP were injected into the lateral ventricle of correspondingnormal healthy rats. Rats CSF from different groups were collected after4days and theconcentration of-EP was determined. Meanwhile, F group was injected with saline at thecorresponding time. Adopt statistical analysis to-EP concentration in rat cerebrospinalfluid of different concentration groups and normal control group, correlation analysis to thelogarithm of viral titers and CSF-EP concentration values, presume suitable viral titer forlateral ventricle injection and discuss the correlation of viral titer and expression.3. The effects on bone cancer pain after intracerebroventricular injection of Ad-NEP.Prepare a titer of5×109PFU Ad-NEP according to the second part of the experimentresults. Use walker256rat breast cancer cells to create bone cancer pain model in ratsaccording to the second part of the experiment results. The rats were randomly divided intoAd-NEP group (group A), control group (group B) and normal group (group C). The firsttwo groups copied rat model of bone cancer pain according to the first part of theexperiment method and were injected5×109PFU of Ad-NEP into lateral ventricles of ratson the10thday after inoculation, while the control group was injected with the samevolume of saline. Normal rats received no treatment. On the same day,2,4,6,8,10,12,14,16,18,20,22days after inoculation surgery, in the same time every day,to three groups, randomly and double-blind measure weight, evaluate spontaneous walkinglimb use, measure thermal stimulation induced pain behavior and mechanical stimulationinduced pain behavior as well. In addition, define the intracerebroventricular injection dateis0day, then in the2,4,8,12days after the lateral ventricle injection of Ad-NEP, extractrats cerebrospinal fluid of three experimental groups after behavior assessment anddetermine the-EP concentrations of CSF samples with Radial Immunoassay(RIA). Onthe22thday after vaccination, that is, on the12thday after the injection of the lateralventricle, Ad-NEP group of rats accept derived brain tissue after behavioral measurement and cerebrospinal fluid extraction. Then, the biopsy specimens were drawn, and staining.Result1. In the tumor group two animals’ unforeseeable death occurred in7thday after surgery.From the second day after surgery, rats in the sham group drink and eat normally, and nodeaths occurred. In the first week after surgery, the weight in the tumor group and the othertwo groups had no significant difference, in the second week, the weight increasing oftumor group was slower than the other two groups, in the third week, the gap was evenmore pronounced. From the6thday after surgery,2rats in the tumor group15rats score3points. On the18thday after surgery, all rats in the tumor group are1point in spontaneouswalk score. From the2ndday after surgery, rats in the sham group and normal group werenormal gait and the score were all4points. As for thermal stimulation in rats induced painbehavior assessment, through testing, we found that there was no significant difference ofthermal hyperalgesia incubation period between groups. As for mechanical stimulationevoked pain behavior assessments, tests found in the early period when walking painbehavior score was higher, rats in the tumor group had no significant change betweenvaccination side and contralateral hind limb, but when obviously not enough weight onipsilateral hindfoot, it appears2-3days of mechanical pain hypersensitivity. The same rats,in the early post-vaccination, were measured a stable similar pain threshold to thefoundation values. At hyperalgesia, pain threshold measured in the same conditions and theformer one had significant difference (P <0.01). In addition, in the period when themechanical stimulation induced hyperalgesia, pain threshold of the ipsilateral andcontralateral side were also statistically significant different (P <0.05). To radiologicfindings in rats, on the22thday after vaccination, in the two rats of tumor group, CT scanfound that tumor did not attack the right side tibia, then anatomy found that tumorerosion develop until subcutaneous tissue out of right tibia. For other rats in the tumorgroup, CT scan had seen that on the superior of the right side tibia, metaphysis cortex thinningz, insect-like changed and light transmission increased. Sham group and thenormal group were not found tumor erosion.2. Animals in group E all died one after another after intracerebroventricular injection. Forthe remaining four experimental groups, the data between the groups were statisticallysignificant differences (P <0.01). Groups A and B had no statistically significant differencewith control group(group F)(P>0.05); Groups C and D were significantly different fromthe control group(group F)(P <0.01).3. The weight of inoculated rats in each group were no significant difference within a week,but from the second week, group A and group B had slower weight gain than in group C, tothe third week, the gap is more obvious, after intracerebroventricular injection we foundthat weight gain in the group A and B was significantly slower than the normal group, andweight gain between the two groups had no significant difference (P>0.05). When ratswere assessed limb use when spontaneous walking, after the10thday when havingintracerebroventricular injection of ad-NEP, on the12thday, pain behavior scores in groupA declined significantly lower than group B (P <0.05). As for mechanical stimulationevoked pain behavior assessment, we found that from the12thday after vaccination, inother words, from the2ndday after intracerebroventricular injection, rats pain threshold ofvaccination side and contralateral hind limb in group A had a significant increase comparedto group B(P <0.01). This significant difference disappeared until20days after theinoculation procedure in the vaccination side, while18days in the contralateral limb.Radioimmunoassay determination results of-EP concentrations in cerebrospinal fluidspecimens showed that from the4thday after intracerebroventricular injection of5×109PFU titers of Ad-NEP,-EP concentration largely increase in CSF in group A. On the4thday after intracerebroventricular injection, brain tissue immunofluorescencephotographs of rats in the Ad-NEP group showed that after intracerebroventricularinjection, lateral ventricle surrounding dense distribution of Ad-NEP expression product-EP was found in the rats in groupA. Conclusion1. Walker256rat breast cancer cells can build a successful model of bone cancer pain inrats, and is easy to copy.2. Recombinant adenovirus Ad-NEP with volume of30ml,5×109or5×1010PFU titer issuitable virus condition for lateral ventricle injection. This dosage may induce highconcentration of (F groups)-EP in animal cerebrospinal fluid, and also will not cause massof mortality of rats in the experiment.3. Intracerebroventricular injection of adenovirus Ad-NEP with the volume of30ml, thetiter of5×109PFU on bone cancer pain model constructed by Walker256rat breast cancercan effectively reduce hyperalgesia in rats with bone cancer pain, improve the painthreshold of the mechanical stimulation, greatly increasing the concentration of-endorphin in the brain, thereby improving the survival effect of rats with bone cancer pain. |
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