The Analgesic Effect Of APA Microcapsulized NIH3T3/rPENK Subarachnoid Transplantation On Neuropathic Pain In Rats

Background and ObjectiveNeuropathic pain was resulted from nerve injury or dysfunction. It is a painful nerve disease. Up to now, traditional analgetics can not treat neupathie pain efficiently. As a sort of endogenous opioid peptide,Enkephalin eases pain mainly through restraining the transmission of pain message. Upon transfection into a packaging cell line , the retroviru vector can integrate the enkephalin gene and stably express. Therefore, in order to treat neuropathic pain, we can transplant the cells with enkephalin-gene into the spinal subarachnoid space of patients. But potential host effect and serious immunological reaction are inevitable in transplanting different kinds. In order to prevent immunological reaction, NIH3T3/rPENK were embedded in APA (alginate-polylysine-alginate) microcapsule to reach the goal of immunological isolation. Then APA-NIH3T3/rPENK were transplanted into the rat's spinal subarachnoid space to observe its effects on the suppression of neuropathic pain. The concentration of the enkephalin in the rats cerebral spinal fluid and the intensity of NMDAR2B proteins in the dorsal horn of spinal cord were examined. The mechanism of analgesia and immunologically isolated effect were explored, this would offer the experimental foundation for the clinical application of the technique of transplanting APA-NIH3T3/rPENK into spinal subarachnoid space.Methods1.Observation of the microcapsule cells and detectation of the secretion of enkephalin:The NIH3T3 cells transfected with rat enkephalin gene were microcapsuled using a high-voltage electrostaic system. Then, the cells were maintained in DMEM with 10% fetal bovine serum(FBS), at 37°C in an atmosphere containing 5% CO₂. The growth of the cells were observed and the microcapsule's penetrating effect to enkephalin was checked every other day.2.Detectation of the thermal threshold of neuropathic pain rats20 adult male SD rats were randomly divided into two groups(n=10):CCI rats and sham-operated rats.Right sciatic nerve was ligated in CCI group,but it was only exposed in sham-operated group.The thermal threshold of left and right hindpaws were measured once every other day from the fifth day of post-operation to 42th day.3.Observation of the analgesic effect of transplanted NIH3T3/rPENK and APA- NIH3T3/rPENK30 adult male SD rats were randomly divided into three groups (n=10):APA, NIH3T3/rPENK and APA- NIH3T3/rPENK group. 40ul suspension including about three hundred empty APA microcapsules (group APA), 40ul suspension including about 2×10⁶ NIH3T3/rPENK (group NIH3T3/rPENK) and 40ul suspension including about 2×10⁶ APA microcapsulized NIH3T3/rPENK (group APA-NIH3T3/rPENK) were injected into the spinal subarachnoid space of CCI rats on 12th day after CCI operation respectively.Measurement of thermal threshold: The thermal threshold of the operated hindpaws were measured from the 2^th day post-transplantation, every day initially, every other day 5 days later, until the 21^th day post-transplantation.Antagonist test: The effect of intraperitoneal naloxone (0.08mg/kg) on the analgesic effect was tested on the 11^th day post-transplantation in all groups.Artificial cerebral spinal fluid (ACSF) perfusion: ACSF was perfused into the spinal subarachnoid space of CCI rats using transfusion-pump. 1ml of CSF was Collected at 0°C,then it was destroyed with boiled water,after centrifugation,the supernate was stored at -20°C. The concentration of enkephalin was measured with radioimmunoassay.Expression of NMDAR2B gene: The intensity of NMDAR2B protein was determined by immunohistochemical technique on the 21th day of post-transplantation in three groups.Results1.Observation of the microcapsule cells:Cells began to proliferate and mass along with time.Cloning cell dumpling appeared on the 12^th day. Detectation of the secretion of enkephalin: The concentration of enkephalin was 12.9±1.6pg/ml on 3rd day of culture. It increased to 197.0±38.1pg/ml on 11th day of culture. With cells in the capsule gradually expanding, extracapsular secretion of the enkephalin increased obviously.2.The rule of thermal threshold of the neuropathic pain rats:5 days after CCI,the thermal threshold of ligated side was lower than the contralateral side,but it was not significant(P>0.05);7 days after CCI ,the thermal threshold of the ligated side was significantly lower than the contralateral side(P<0.05);7～33 days after CCI ,the difference of ligated side and contralateral side was of statistical significance(P<0.05 or P<0.01);5 weeks after CCI,the difference of the two sides was indistinctive(P > 0.05).In sham-operation group, the difference was not significant between right and left hind-paw(P>0.05).3. Measurement of thermal threshold after transplantation: Transplants of APA didn't show evident change. The thermal threshold of NIH3T3/rPENK group on 3～21 days of post-transplantation and APA-NIH3T3/rPENK group on 5～21 days of post-transplantation were evidently higher than APA group ( P＜0.01 or P＜0.05) .Compared with APA-NIH3T3/rPENK group, the thermal threshold of NIH3T3/rPENK group was evidently lower two weeks later (P<0.05).It demonstrated that transplantation of NIH3T3/rPENK into the spinal subarachnoid space of CCI rats could attenuate neuropathic pain, and this effect was weakened as time going.APA microcapsule offered immune isolation so that it could prolong the time of analgesia and made stronger analgesia after two weeks.4. Antagonist test: 10 days after transplantation, opioid peptide receptor antagonist naloxone was injected into the abdominal cavity of 3 groups rats. The difference of the thermal threshold between pre-injection and post-injection was not distinct in APA group (P>0.05) .But compared with pre-injection,the thermal threshold of post-injection was significantly lower in NIH3T3/rPENK and APA-NIH3T3/rPENK groups (P<0.01) . The analgesic effects were reversed by intraperitoneal naloxone.5.Concentration of enkephalin in cerebral spinal fluid perfusion fluid: 21 days after transplantation, the concentration of the leucine-enkephalin in APA group was lower than that in NIH3T3/rPENK group and APA-NIH3T3/rPENK group (P<0.01). The concentration in NIH3T3/rPENK group was lower than that of APA-NIH3T3/rPENK group(P<0.01).The above results demonstrated that NIH3T3/rPENK implanted into the rat's spinal Subarachnoid Space could increase the concentrations of the leucine-enkephalin and APA microcapsuled NIH3T3/rPENK prolonged elevation of time.6. Expression of NMDAR2B gene: 21 days after transplantation, NMDAR2B positive protein integral optical density in spinal dorsal horn of ligation side in APA group was significantly higher than that in NIH3T3/rPENK, APA-NIH3T3/YPENK and the control (P<0.01) . Meanwhile , NMDAR2B positive protein integral optical density in spinal dorsal horn in NIH3T3/rPENK group was obviously higher than that in APA-NIH3T3/rPENK and the control (P<0.05).The difference was not statistically significant between APA-NlH3T3/rPENK and the control. It showed that the transplantation of NIH3T3/rPENK could inhibit the expression of NMDAR2B gene of the spinal dorsal horn induced by neuropathic pain and a better result could be achieved after being covered by APA microcapsule.ConclusionNIH3T3/rPENK or APA-NIH3T3/rPENK implanted into the spinal subarachnoid space of rats could significantly reduce the painful behavior in neuropathic pain. It had close relationship with the graft's continous release of enkephalin and its action on the opioid receptor. NIH3T3/rPENK or APA-NIH3T3/rPENK implanted into the rat's spinal subarachnoid space could significantly inhibit the NR2B protein expression in the spinal dorsal horn.It indicated that the graft might exert analgesic effect by inhibiting expression of NR2B protein. Compared with the transplantation of NIH3T3/RPENK, the transplantation of APA-NIH3T3/rPENK could obviously improve analgesic effects. It showed that APA microcapsule had good effects on immune isolation.