Experimental Studies On The Construction And Analgesic Effect Of N-methyl-D-aspartate Receptor 2B Subunit Vaccine

BackgroundNeuropathic pain is defined as pain initiated or caused by a primary lesion or dysfunction in the nervous system. The reasons responsible for neuropathic pain include trauma, ischaemic injury, infection/inflammation, cancer, drugs and compression etc. Patients with neuropathic pain do not respond to non-steriodal anti-inflammatory drugs and have resistance or insensitivity to opiates. The current pharmacological mainstays of clinical management are tricyclic anti-depressants and certain anticonvulsants, but these have only achieved clinical significant pain relief in less than 50% of patients and are associated with sub-optimal side effect profiles. Therefore, an important issue of pain research is to find a novel method of analgesia that is safe and effective. Peripheral nerve injury may result in a persistent pain due to abnormal activity in peripheral afferents and a sustained input to the spinal cord that leads to alterations in the activity and excitability of spinal cord neurons. These spinal changes involve central sensitization of dorsal horn neurons. Spinal N-methyl-D-aspartate (NMDA) receptor plays an important role in the facilitation and maintenance of central sensitization of spinal dorsal horn neurons. NMDA receptor antagonists such as ketamine, dextromethorphan and MK-801 have been reported to produce symptomatic relief in a number of neuropathies including postherpetic neuralgia, central pain caused by spinal cord injury and phantom limb pain. However, these agents also induce unacceptable side effects at analgesic doses including hallucinations, dysphoria and disturbances of cognitive and motor function, which exclude their widespread use.The NMDA receptor complex comprises multiple protein subunits, of which there are at least one NMDAR1 (NR1) subunit and at least one of a family of NR2 subunits (NR2A-NR2D). NR2B subunit is distributed discretely in the CNS and it is possible to support a reduced side effect profile of approach that act selectively at this site. The development of the modern vaccine technology is not only to develop vaccines aiming directly at ectogenic antigen, but also to endogenous antigen. The vaccine made from the tumour cell epitope has been proved to prevent and treat efficiently the tumour. The NMDAR1 epitope vaccine desigened by During et al provided a novel strategy for prevention and cure of stroke and epilepsy. Therefore, as an alternative approach to treat neuropathic pain, we hypothesise that a humoral autoimmune response targeting the NR2B subunit of NMDA receptor would relieve pain like behaviours produced by peripheral injury.Methods and Results1. The immunized and preemptive analgesic effect of NR2B peptide vaccine on rats with neuropathic painMethods NR2B peptide ( Sequence: TNGKHGKKINGTWNGMIGEY) is derived from C-terminal region of NMDA receptors 2B subunit. NR2B peptide is incomplete immunogens but is made fully immunogenic by coupling them to a suitable carrier protein KLH. Female SD rats were divided into three groups randomly: group PBS (vaccined with PBS), group KLH (vaccined with KLH) and group NR2B (vaccined with NR2B-KLH). Rats were immunized subcutaneouly three times at a two-weekly intervals. NR2B-KLH was emulsified in Freund's Complete Adjuvent or Freund's Incomplete Adjuvent (1:1, v/v) just before using. NR2B-specific Ig G titers in sera were determined by ELISA. By day 7 after the third immunization, surgical procedures were performed under 10% Chloral Hydrate anesthesia. After skin and muscle incision, 2 of the 3 terminal branches of the sciatic nerve ( tibial, and common peroneal nerves) with 9-0 silk suture were tightly ligated. Behavioral testing (50% paw withdraw thresholds and the duration of the withdrawal after the heat stimulation ) took place on every other day after surgery, till 7 days after surgery. CSF (10~20μl) was drawn from the cisterna magna using a 27-gauge needle on day 1 before SNI and on day 7 after SNI, respectively. NR2B-specific Ig G titers in CSF were determined by ELISA . On day 7 after the surgery, the L4~6 segment of the spinal cord was harvested. The number of c-Fos positive neurons in spinal cord dorsal horn was determined using immunohistochemistry. The expression of NR2B protein in spinal cord were determined using western blotting.The reaction of sera from rats with NR2B subuits in the spinal cord horn were tested using immunohistochemistry.Results After the second vaccination with NR2B peptide , NR2B-specific IgG were detected in six of nine rats(>0.5μg/ml). After the third vaccination with NR2B peptide, all of the rats had more than 2.2μg/ml NR2B-spicific antibody titer. Titers of NR2B-specific IgG peaked 42 days post initial immunization and persisted over the 70 days evaluated. No NR2B-specific antibody was detected in sera from rats of group PBS or KLH. On day 7 after surgery, the 50% paw withdraw thresholds of group NR2B were significantly higher than that of group PBS or KLH (P<0.05). The paw withdrawal duration to radiant heat stimulation in group NR2B were less than that of group PBS or KLH (P<0.05). The number of c-Fos positive neurons in spinal cord dorsal horn of NR2B group were less than that in PBS or KLH group (P<0.05). NR2B-specific IgG of NR2B group was positive on day 7 after SNI. There is a significant reduction for NR2B protein level in the lumbar spinal cord in group NR2B compared with that in group PBS or KLH (P< 0.01). Sera from rats immunized with NR2B-KLH react with NR2B protein in spinal horn.2. Evaluation of NR2B peptide as subunit vaccines against neuropathic pain being existed previously Methods After skin and muscle incision, 2 of the 3 terminal branches of the sciatic nerve (tibial, and common peroneal nerves) with 9-0 silk suture were tightly ligated. Behavioral testing (50% paw withdraw thresholds and the duration of the withdrawal after the heat stimulation ) took place on every day after surgery, till 7days after surgery. Female SD rats developed hyperalgesia were divided into three groups randomly: PBS,KLH and NR2B. Rats were immunized with PBS,KLH or NR2B, respectively, three times subcutaneously at a two-weekly intervals, in combination with Complete Freund's adjuvant (CFA) or Incomplete Freund's adjuvant (ICFA).By day 14 after the third immunization, 50% paw withdraw thresholds and the paw withdraw duration to radiant heat stimulation were assessed and then sera were harvested for detection of titers of NR2B antibody. The L4~6 segment of the spinal cord was harvested. The astrocytic activation was determined using immunohistochemistry with antibodies of GFAP (an astrocyte marker). The expressive level of NR2B protein in spinal cord horn were detected using western bloting.Results The titers of NR2B antibody in NR2B group was 6.94±2.04μg/ml on day 14 after the third immunization and there was no detection of NR2B antibody in PBS or KLH groups. All rats have developed a marked hyperalgesia 7 days after SNI, however, three times immunization later, mechanical hypersensitivity and heat hyperalgesia in NR2B groups were relieved, compared with PBS or KLH groups. The expression of GFAP indicated that astrocytic in group NR2B became less intense responses in the ipsilateralⅠ~Ⅳlemanie of dorsal horn than control group(PBS or KLH). Western blotting analysis showed that NR2B peptide vaccine decreased the expressive level of NR2B protein in spinal cord horn after the third immunization.3. Construction of recombinant adenovirus analgesic vaccine encoding NR2B geneMethods NR2B gene was obtained by digesting the plasmids pcDNA3.1-NR2B with EcoRⅠand BamHⅠand was cloned into the adenovirus shuttle vector pDC515. Then the vector was transferred into 293 cell and the expression of NR2B gene in 293 cell was identified by RT-PCR and western blotting. The shuttle plasmid pDC515-NR2B and the genomic plasmid pBHGfrt(del)E1,3FLP were cotransfected into the package 293 cells using lipofectamine TM 2000. The contransfected 293 cells were observed persistently for the production of CPE and the viral plaques. The viral plaques were picked and were put into infecting the passage cell 293. The recombinant Ad5/ NR2B were identified by PCR,RT-PCR and western blotting. The amplification and purification of the recombinant Ad5/NR2B were made and the titer and purity of the virus were assayedResults First, transgene NR2B was cloned into the small adenovirus shuttle vector pDC515. The presence and oritention of the insert were confirmed using restriction endonuclease analysis and then the plasmid was termed as pDC515-NR2B. Second, pDC515-NR2B are cotransfected with an Ad genomic plasmid pBHGfrt△E1,3FLP into E1 complementing cell line 293 cells. The recombination between the two contransfected plsmids was mediated by frt site specific recombinase, FLP. The formation of viral plaques begins with the infection of one cell by one virus followed by multiple cycles of complete infection when released viruses infect surrounding cells. By day 15 following contransfection, well-defined plaques can be observed under the microscope . In order to build up working stocks of virus from plaque isolates before extensive experimentation, the presence and oritention of transgene NR2B in the recombinant adenovirus vector were confirmed using NR2B-specific PCR. The viral stocks of rAd5/NR2B were generated by the transient transfection of 293 cells and purified using CsCl ultracentrifugation gradients. The final titer was 5×1011 (Plaque Forming Unit, PFU)/ml and the purity is 100% using identification of HPLC methods.4. rAd5/NR2B vaccine induces protective immunity against neuropathic painMethods The rAd5/NR2B (1×108 PFU) was administered via an orogastric tube into the stomachs of a group of rats (n=16), with a control group (n=16) receiving a similar dose of a recombinant Ad5 virus expressing EGFP or PBS, and followed by a booster immunization with the same dose two weeks after the initial immunization. A week later, expression of NR2B gene in the intestine M cells was assessed by RT-PCR. NR2B-specific Ig G titers in sera were determined by ELISA at a two-weekly intervals. Two weeks after the boost immunization, SNI model was established through the ligation of tibial nerve and peroneal nerve. A week after the SNI procedure, the number of c-Fos positive neurons in the spinal dorasl horn were assessed using immunohistochemistry. 50% paw withdraw thresholds of mechanical stimulation were assessed using the up-down paradigm every other day after SNI and withdrawal duration of heat response were also assessed at the same timepoint. Serial spinal sections from rats were stained with sera from rats treated with PBS , rAd5/EGFP or rAd5/NR2B. CSF (20~50μl) was drawn from the cisterna magna using a 27-gauge needle on day 1 before SNI, on day 7 after SNI and on day 42 after SNI, respectively. Six weeks after SNI, the expression of NR2B gene in the lumbar spinal cord were assessed using western blotting.Results Following the primary immunization and the booster immunization, NR2B-spicific IgG in sera were 5.5.±1.9μg/ml and 13.4±3.2μg/ml,respectively. In contrast, NR2B-spicific IgG were not detected in sera from the control groups (na?ve and rAd5/EGFP).By day 7 after the primary vaccination with rAd5/NR2B, the expression of NR2B protein were detected in the intestine of rats immunized with rAd5/NR2B. By day 7 after surgery, the 50% paw withdraw thresholds of group rAd5/NR2B were significantly higher than control groups (na?ve and rAd5/EGFP) (P<0.05). The paw withdrawal duration to radiant heat stimulation in grouprAd5/NR2B were less than the control groups (na?ve and rAd5/EGFP) (P<0.05). The significant difference of 50% paw withdraw thresholds and paw withdrawal duration to radiant heat stimulation among the three groups remains till 42 days after the surgery. NR2B-spicific IgG in CSF was only detected on day 7 and 42 post the initial immunization in rAd5/NR2B group. And only sera from rats immunized with rAd5/NR2B reacted with NR2B protein in the lumbar spinal cord dorsal horn. Western botting analysis showed that immunization with rAd5/NR2B decreased the expression of NR2B protein in the spinal dorsal horn. 5. rAd5/NR2B vaccine induces protective immunity against the affective component relating to neuropathic painMethods SD rats without preference one compartment to the others before conditioning were selected and were divided into three groups randomly: na?ve, rAd5/EGFP and rAd5/NR2B. The rAd5/NR2B (1×108 PFU) was administered via an orogastric tube into the stomach of a group of rats, with a control group receiving a similar dose of a recombinant Ad5 virus expressing EGFP or PBS. Two weeks later, re-immunization were performed by the same dose of vaccine. Titers of NR2B antibody in sera were determined using ELISA at week 2 following the booost immunization and at the same time, SNI model was established through the ligation of tibial nerve and peroneal nerve. From the day 7 after SNI on, conditioned place avoidance (CPA) test were carried out. CSF (20μl) was drawn from the lateral ventricle using a 27-gauge needle on day 1 before SNI and on the day of SNI-CPA completion ,respectively. Anti-NR2B antibody titer in CSF were detected using ELISA and CSF was screened against ACC membrane extract by immunoblot analysis. The NR2B protein expression in ACC were assessed using western blotting and immunohistochemistry.Results Two weeks following the booster immunization, titers of NR2B antibody in rAd5/NR2B groups was up to 13.5±2.3μg/ml. CPA scores in rAd5/NR2B group was significantly less than that in na?ve or rAd5/EGFP group(P<0.05). ( rAd5/NR2B group: 138.1±29.2S, na?ve group: 198.1±49.5S,rAd5/EGFP group: 236.8±40.2S). By day 13 after SNI, NR2B antibody in CSF of rAd5/NR2B group could be detected and react with NR2B protein of ACC membrane extracts. The expressive level of NR2B protein in ACC of rAd5/NR2B-i mmunized rats was decreased significantly compared with rats in na?ve or rAd5/EGFP group.6. Statistical AnalysisAll of the analyses were performed by SPSS 12.0 software package. Measurement data were presented as means±SD. The differences between multiple individual means were analyzed by one-way ANOVA. Categorical data were presented as rate. The differences between multiple individual rates were analyzed by Chi-square test. P < 0.05 was considered statistically significant in all tests.ConclusionNR2B peptide vaccine could induce the humoral immunological response to produce NR2B-specific antibody in sera. NR2B-specific antibody could react with NR2B subunit in spinal dorsal horn to reduce the NR2B protein expression and then have a pre-emptive analgesic effect on neuropathic pain. As for neuropathic pain existed previously, NR2B peptide vaccine could also relieve hyperalgesia. NR2B gene was inserted into the adenovirus shuttle vector pDC515 and then pDC515-NR2B was constructed successfully. The vaccine rAd5/NR2B was constructed successfully using cotransfection with pDC515-NR2B and pBHGfrt(del)E1,3FLP, which induced the higher titer of NR2B-specific antibody than NR2B peptide vaccine. The oral immunization with rAd5/NR2B could not only produce the analgesic effect against pain-related sensory but also alleviate the pain-related affective response.SummaryThe present study firstly demonstrated that NR2B peptide vaccine not only induced the humoral immunological response but also NR2B-spicific antibody could react with NR2B protein in the spinal dorsal horn to fight against neuropathic pain. In order to improve the immunological effect, rAd5/NR2B vaccine could construct.The present study made a research about the analgesic effect of rAd5/NR2B vaccine on both sensory and affective component of neuropathic pain, providing a novel direction for the treatment of neuropathic pain.