Int J Clin Exp Med 2014;7(12):4877-4886 www.ijcem.com /ISSN:1940-5901/IJCEM0003574 Original Article Effect of adenoviral delivery of prodynorphin gene on experimental inflammatory pain induced by formalin in rats Xionggang Chen1, Tingting Wang2, Caizhu Lin1, Baihong Chen1 1Department of Anesthesiology, The First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, P. R. China; 2Department of Ophthalmology, The First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, P. R. China Received November 3, 2014; Accepted November 13, 2014; Epub December 15, 2014; Published December 30, 2014 Abstract: Circumstantial evidences suggest that dynorphins and their common precursor prodynorphin (PDYN) are involved in antinociception and neuroendocrine signaling. DREAM knockout mice had increased levels of PDYN and dynorphin expression, and reduced sensitivity to painful stimuli. However, some data support the notion that the up-regulation of spinal dynorphin expression is a common critical feature in neuropathic pain. It is not clear whether the production of dynorphin A can be increased when more PDYN is present. In this study we investigated the changes in pain behaviors, spinal PDYN mRNA expression and dynorphin A production on formalin-induced pain in rats receiving the pretreatment of adenoviral delivery of PDYN. Our results showed that the adenoviral transfer of PDYN gene was sufficient to reduce pain behaviors resulting from formalin injection, and the antinociceptive effect after receiving the pretreatment of adenoviral delivery of PDYN was mediated at the level of the spinal cord via KOR. Keywords: Adenoviral delivery, prodynorphin, dynorphin A, formalin, hyperalgesia, spinal dorsal horn Introduction The endogenous kappa-opioidergic system also participates in facilitation of spinal nociceptive It is currently accepted that the endogenous transmission [1, 13]. Several experimental kappa-opioidergic system plays an important models of chronic pain states show that a sig- role in the endogenous pain control system nificant upregulation of spinal dynorphin A and [1-4]. The endogenous ligands for kappa-opioid its precursor peptide, PDYN, is a common con- receptor (KOR) consist of prodynorphin (PDYN), sequence of nerve injury [14]. Previous obser- dynorphin A, dynorphin B, α-neo-endorphin and vations showed that the mechanism of signifi- big dynorphin [5], whereas PDYN is the precur- cant increase of PDYN expression in peripheral sor of dynorphins [6-8]. Actions of these pep- neuropathy was proposed to explain the para- tides are preferentially mediated through KOR dox of dynorphin actions [15]. Otherwise, a and are critical for regulation of nociceptive study with PDYN knock-out mice that up-regu- transmission [9, 10]. Otherwise, the endoge- lated spinal dynorphin was required for the nous kappa-opioidergic system also includes maintenance of persistent neuropathic pain downstream regulatory element antagonistic [16]. Recent findings implicate a direct excitato- modulator (DREAM) which inhibits transcription ry action of dynorphin A at bradykinin receptors of the PDYN gene [11, 12]. In DREAM knockout and NMDA receptors to promote hyperalgesia mice, there was increased expression of PDYN in nerve injured rats, and its upregulation may mRNA, but endogenous dynorphin increases promote nociceptive input due to injury [17-22]. might be limited to within physiological ranges All these data support the notion that the up- [6]. Therefore, the expression of endogenous regulation of spinal dynorphin expression is a PDYN, dynorphins may be under the tight con- common critical feature of expression of neuro- trol of DREAM in central nerve system. pathic pain [23]. Prodynorphin and inflammatory pain Previous study found that PDYN knockout mice LacZ-a (Microbix Biosystems, Inc.) were all almost didn’t produce dynorphin [24], indicat- digested with Hind III and EcoR I, and the ing that dynorphin cannot be produced without digested fragment s were re-collected from the PDYN. Dynorphin A is an endogenous opioid low melt agarose gel and fast ligated directly by neuropeptides derived from the PDYN gene [6, T4 DNA ligase. The reaction product was trans- 25]. However, we are not aware whether the formed into E. coli (DH5α), and the positive bac- production of dynorphin A is obviously increased terial colony was selected. The recombinant when more PDYN is present. To our knowledge, eukaryotic expression vector pDC316-PDYN the involvement of the pretreatment of PDYN in was identified by using restriction endonucle- inflammation-induced pain has not been previ- ases digestive reaction and DNA sequencing. ously investigated. Therefore in this study we The HEK 293 cells were cotransfected by the investigated the behavior change of rats receiv- shuttle plasmid of pDC316-PDYN and the skel- ing the pretreatment of adenoviral delivery of eton plasmid of pBHG which was adenoviruses PDYN, PDYN mRNA expression and dynorphin A type 5 with deletions in E1 and E3, and the production on Formalin-induced pain. We recombinant plasmid of Ad5-PDYN was ob- hypothesized that rats receiving the pretreat- tained. The expression of the transfected genes ment of adenoviral delivery of PDYN by the tail was evaluated by PCR and immunocytochemi- vein would show a modulatory antinociceptive cal staining. Virus was purified by double CsCl action and dynorphin A down-regulation in the centrifugation and subsequently dialyzed as spinal cord. described previously [29, 30]. Final yields as assessed by plaque assays on 293 cells were 12 Materials and methods approximately 1 × 10 plaque forming units (pfu)/ml [31]. Experimental animals Recombinant adenoviral injection Adult male SPF grade Sprague-Dawley rats Group Ad5 received respectively Ad5, with 1 × weighing between 250 and 300 g were housed 1011 pfu in 100 µl of PBS by the tail vein in day at a constant ambient temperature of 24°C ± 1 and day 7; group Ad5-PDYN received respec- 1°C, humidity of 40%-70% under a 12 h light/ tively Ad5-PDYN, with 1 × 1011 pfu in 100 µl of dark cycle and given food and water ad libitum. PBS by the tail vein in day 1 and day 7, while The rats were individually housed in plastic group C were injected with normal saline as cages with wood-chip bedding for at least 1 day control. Infusions of the recombinant adenovi- before surgery. All experimental procedures ruses into the tail vein of rats anesthetized with were approved by the Institutional Animal Care pentobarbital sodium (50 mg/kg, i.p.) were per- and Use Committee of First Affiliated Hospital formed as reported previously [32]. of Fujian Medical University and were in accor- dance with the guidelines for the use of labora- Experiment 1: The SD rats were randomly divid- tory animals [26]. All efforts were made to mini- ed into three groups (n = 10): group C, group mize animal suffering and to reduce the num- Ad5 and group Ad5-PDYN to assess whether ber of animals used. adenoviral delivery of prodynorphin inhibits experimental inflammatory pain induced by for- Recombinant adenoviral vectors malin in rats. Experiment 2: The SD rats were randomly divid- Construction of the recombinant adenoviruses ed into group Ad5 (n = 5) and group Ad5-PDYN was determined as described previously [27]. (n = 10) to determine whether the possible role Briefly, PDYN gene (GenBank accession num- of nor-binaltorphimine (nor-BNI) after receiving ber. NM 019374) primer sets (Sence: the pretreatment of adenoviral delivery of 5’-GACATGGCGTGGTCCAGGCTGATG-3’; Antisen- PDYN, and nor-BNI was dissolved in artificial ce: 5’-GTCTCAAACATCTAAATCT TCAGAATAGG- cerebrospinal fluid (aCSF) [33]. Rats in the con- TATTGGG-3’) were designed via PDYN cDNA trol group received aCSF infusions. We used was cloned by PCR technique, and then insert- the method of intrathecal injection to infuse ed into the cloning vector pUC57. The recon- Nor-BNI or aCSF [34]. Intrathecal injection (i.t.) structed plasmid with PDYN gene was detected was given in a volume of 5 µl through an inter- by the electrophoresis and the sequence analy- vertebral space at the level of the 5th or 6th sis [28]. Plasmid pUC57-PDYN and pDC316- lumbar vertebra using a microsyringe. 4878 Int J Clin Exp Med 2014;7(12):4877-4886 Prodynorphin and inflammatory pain Formalin-induced nociceptive behaviors and se: 5’-CACCCGCGAGTACAACCTTC-3’; antisense: pain intensity scoring 5’-CCCATACCCACCATCACACC-3’) were designed and synthesized by Invitrogen. PCR was per- On day 6 after second virus injection, the rats formed after reverse transcription at 42°C for were placed in a transparent glass trough and 60 min and initial denaturation at 95°C for 10 were allowed to get habituated for about 30 min. The temperature cycles (Roche, Bran- min. Then formalin (50 µl, 5%) was injected sub- chburg, NJ) were 94°C/30 s (denaturing), cutaneously (s.c.) into the large lateral footpad 58°C/30 s (annealing), and 72°C/30 s (exten- on the plantar surface of the right hind paw by sion). A total of 32 cycles and a final 5-min 100 µl microsyringe. Each rat was measured extension at 72°C were conducted. The PCR 12 times and Formalin-induced nociceptive amplified fragments, 239 bp for PDYN and 207 behaviors of rats were observed by trained indi- bp for β-actin, were separated on 3% ethidium viduals who were blinded to experimental con- bromide stained agarose gel (Invitrogen, ditions, and continued for the next 60 min [35]. Carlsbad, CA). The PCR gel image was captured Pain intensity scoring (PIS) of nociceptive be- and analyzed by a gel documentation system havior was recorded per 5 min by assigning (BIO-RAD, USA). The positive PCR bands were weights to the following categories: 0 = the purified and sequenced, and the resulting injected paw is no abnormalities; 1 = no or little sequences were identical to the targeted cDNA weight is placed on the injected paw (weight = sequences. Each experiment was run three 1); 2 = the injected paw is elevated and not in times and each sample was run in triplicate.