Analgesic Effects Of Intrathecal Injection Of PcDNA3.1(+)-hPPE Recombinant Plasmid On Neuropathic Pain In Rats

ObjectiveTreatment of neuropathic pain is extremely difficult, current analgesic agents may be limited with regard to their analgesic effects or side effects. By targeting a specific receptor or other specific protein targets, gene therapy approach to the treatment of neuropathic pain may provide great analgesic efficacy without the limitations associated with current pharmacotherapy.Enkephalin(Enk),as a type of endogenous opioid peptides,has been tested to have analgesic effect by numerous investigations using animal models. So the possibility of using Enk gene therapy as a potential novel treatment for neuropathic pain is taken into consideration. The success of gene therapy rests on the development of a vector that can selectively and efficiently deliver a gene to the target cells. Advantages of non-viral vectors include their nonimmunogenicity and feasibility to be produced on a large scale. Theoretically, non-viral gene transfer systems using naked plasmid DNA has the characteristics of safety,reliablility and simplicity,up to now, plasmid DNA has been the predominant non-viral vectors utilized in research.To find more convenient, effective, economic and safe antinocicepive method, the the recombinant plasmid pcDNA3.1(+)-hPPE was constructed using gene recombination technology and transfected into the subarachnoid space of CCI model rats via direct intrathecal injection and the antinocicepive effects were evaluated in CCI model rats. Then,to investigating the feasibility of delivering exogenous genes into the subarachnoid space using naked plasmid DNA, the recombinant plasmid pcDNA3.1(+)-EGFP was constructed and was transfected into the subarachnoid space of normal rats via direct intrathecal injection.Methods1. The expression of pcDNA3.1(+)-EGFP in normal rat. We investigated the feasibility of delivering exogenous genes into the subarachnoid space using naked plasmid DNA. 20 Spague-Dawley male rats weighing 220-250g were randomly distributed into two groups:control group (n =6) and experimental group(n =48) which was further divided into 8 subgroups (24-hour subgroup, 1-week subgroup, 2-week subgroup, 3-week subgroup,4-week subgroup, 5-week subgroup, 6-week subgroup and 7-week subgroup). The rats of experimental group were injected into the spinal subarachnoid with 30μg/100μl of the pcDNA3.1(+)-Enhanced green fluorescence protein(EGFP) recombinant plasmid.2.Results of the behavioral tests. The chronic constriction injury of the sciatic nerve (Bennett model; CCI model): 20 Spague-Dawley male rats weighing 220-250g were randomly distributed into two groups (n =10,for each group), ten CCI and ten sham-operated groups. In CCI group, right sciatic nerves were separated in the midthigh region and loosely tied four ligatures( 6-0, silk thread) so that they touched but barely constricted the nerve; In the sham-operated rats, the sciatic nerves were separated but not ligated. In all rats, the contralateral sides were not disturbed. The paw withdrawal thermal latency (PWTL) was assessed once daily for 3 days before the CCI operation and the PWTL were detected every other day from day 5 to 6 weeks after the operation in both group.3. To evaluate the efficacy of the pcDNA3.1(+)-hPPE recombinant plasmid on neuropathic pain after intrathecal administration,20 CCI model rats were randomly distributed into two groups (n =10), pcDNA3.1(+)-hPPE group and pcDNA3.1(+) group. In the pcDNA3.1(+)-hPPE group,the pcDNA3.1(+)-hPPE recombinant plasmid was intrathecally injected in a dose of 30μg/100μl;in the control group, the eukaryotic expression vector pcDNA3.1(+) was intrathecally injected in a dose of 30μg/100μl. PWTL of two groups was assessed from day 2 to day 18 after intrathecal administration. Naloxone (0.08mg/kg) was intraperitoneally injected in both groups at days 8 after intrathecal injection, PWTL was measured before and from 10-60 minutes after intraperitoneal Naloxone injection at interval of 10 minutes. CSF samples were obtained for the determination of the concentration of Leucine enkephalin using radio-immunological assay, and the changes in NR2B protein expression in the spinal cord was detected by immunohistochemical techniques in both groups at 18 days intrathecal injection.Results1 .To determine the sites of cellular uptake and expression of the transfected gene, autofluorescence in nerve tissue,heart,liver,spleen,lung,kidney were detected at different time points (At 24 hours ,weeks 1, 2, 3, 4, 5, 6 and 7 following intrathecal injection). At 24 hours following the pcDNA3.1(+)-EGFP recombinant plasmid injection a clear EGFP expression mainly in meninges and the lumbar dorsal roots was detected. No EGFP was found in the other tissues. The most intensive autofluorescence was seen between 7 and 14 days after the pcDNA3.1(+)-EGFP recombinant plasmid injection and the intensity was decreased after 4 weeks. In control group ( without any treatment), no EGFP activity was noted at either time point.2. In CCI group,the animals tended to avoid weight-bearing on the affected foot, both at rest and while walking. The foot was often held in an everted position with the toes plantar-flexed. Sham-operated rats had normal posture and gait. Behavioural testing to detect signs of thermal hyperalgesia was carried out on all CCI and sham-operated rats,responses to thermal stimuli were tested with a Plantar Analgesia Instrument(Ugo-Basile, Italy).The right PTWL were significantly lower than the left PTWL in CCI group from 7 to 33 days after the operation (P < 0:01). There were no significant differences in withdrawal latencies between the paws left and right in the sham-operated rats at any of the time tested (P>0.05) . 3.Then; were no significant differences between withdrawal latencies of the two hindpaws in the pcDNA3.1(+) group after intrathecal injection at any of the time tested (P < 0.05); The right PTWL were significantly higher than the left PTWL in the pcDNA3.1(+)-hPPE group from 2 to 18 days after intrathecal injection (P > 0:05).The attenuation of the thermal hyperalgesia induced by CCI in rats can be blocked by naloxone:.The concentration of the leucine enkephalin in CSF of the pcDNA3.1 (+)-hPPE group significantly higher than those of the leucine enkephalin in CSF of the pcDNA3.1(+) group, 149.52±17.83vs.45.55±12.58 pg/ml, t= 15.581, P=0.001<0.01. The expression of NR2B protein in the spinal cord was minimal in normal group ;in the pcDNA3.1(+) group, the expression of NR2B protein has significantly increased, in the pcDNA3.1(+)-hPPE group, the expression of NR2B protein was significantly decreased in the spinal cord.Conclusions1. Naked DNA can be transfected into meninges and perilemma through the intrathecal injection and exogenous gene can be expressed.2. The pcDNA3.1(+)-hPPE recombinant plasmid transfected into the subarachnoid space of CCI model rats via intrathecal injection could significantly attenuate thermal hyperalgesia.