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shRNA mediated knockdown of Nav1.7 in rat dorsal root ganglion attenuates pain following burn injury
- Weihua Cai†1,
- Jing Cao†1,
- Xiuhua Ren1,
- Liang Qiao2,
- Xuemei Chen1,
- Ming Li1 and
- Weidong Zang1Email authorView ORCID ID profile
© Cai et al. 2016
Received: 9 November 2015
Accepted: 21 July 2016
Published: 11 August 2016
Abnormal acute pain after burn injury still torments patients severely. In this study, we investigated that one voltage gated sodium channel Nav1.7 plays a vital role in lowering heat pain threshold after burn injury, and the hypothesis that knockdown of Nav1.7 attenuates pain following burn injury.
Sixty eight adult male Sprague–Dawley rats were divided into 4 treatment groups: (1) sham, which hind paw was put on the room temperature metal plate for 15 s (2) burn model, which hind paw was put on the 85 °C metal plate for 15 s. (3) Burn injury + lentiviral vector -SCN9AsiRNA-GFP (LV- SCN9AsiRNA-GFP group, n = 18), which receive the DRG microinjection of LV- SCN9AsiRNA-GFP on the zero day. (4) Burn injury + lentiviral vector negative control (LV-NC-GFP group, n = 18), which receive the DRG microinjection of empty lentiviral vector on the zero day.
Both mechanical and heat threshold were measured from day 1 to 21. Meanwhile, expression of sodium channels Nav1.7 in injured dorsal root ganglia were measured on post-operative days 7(POD 7). Rats exhibited decreased thresholds on both mechanical allodynia and thermal withdrawl latency, accompanied by increased Nav1.7 and c-fos expression in dorsal root ganglion (DRG). And knockdown of Nav1.7 in L5DRG led to the attenuation of burn injury-induced mechanical allodynia and thermal hyperalgesia in the rats.
We provide evidence that shRNA mediated knockdown of Nav1.7 attenuates burn induced pain in rats as well as decreased the activiation of c-fos protein.
Burn injury occurs with a high prevalence and causes high morbidity . Globally, nearly 11 million people have burn pain which is severe enough to require medical attention, each year . Burn pain is a spontaneous ongoing unpleasant feeling, and induces both persistent thermal and mechanical hyperalgesia . Currently, the cure for burn-injury remains a challenge. Therefore, great effects have focused on understanding basic mechanisms of pain caused by burn injury [3, 19].
Voltage gated sodium (Nav) channels are known to play a key role in the induction of nociceptors, especially in the initial rising phase of the action potential. Nav channels are composed of a family of α-subunit (Na v 1.1–1.9) and one or more β-subunits (b1-b4) [2, 13]. Up to now, 9 subtypes of Nav channels have been identified in human, and changes in their expression may underlie hypersensitivity in pain states . Among Nav channels, Nav1.7 is encoded by the gene SCN9A . Nav1.7 has raised interest among the pain researchers, since it has been found to be related to paroxysmal extreme pain disorder , erythromelalgia , and painful neuropathy in type 2 diabetes . Recently, Nav1.7 also has been found to be essential for lowering heat pain threshold after burn injury . Therefore, Nav1.7 is considered to play a critical role in pain pathways. In addition, Nav1.7 channel availability sets the AP shape, initiation of burst firing  In this study, we aimed to investigate the role of Nav1.7 in burn injury.
In this study, we chose lentivirus vector to deliver Nav1.7 shRNA into dorsal root ganglion (DRG) neurons to knockdown SCN9A gene to investigate the role of Nav1.7 in rat model of burn injury. In addition, we examined the expression of c-fos protein because c-fos overexpression was associated with high-grade lesion [6, 16], and the c-fos has been considered as a rapidly expressed gene marker of pain  or nociceptive neuronal activation . Small hairpin RNA (shRNA) is a new tool for gene knockdown. For the expression of shRNA in the cells it is usually delivered into cells by plasmids or viral or bacterial vectors [8, 23]. Slow virus vector can not only transfect undivided neurons, but also can be integrated into the host genome, leading to the silencing of target genes for a long time and even permanently [11, 20]. Recently, the most common carrier system for the expression of shRNA is to transfect viral vector harboring the promoter of RNA pol III as well as a piece of special structure of its downstream gene into the host cell, shRNA could be cut into siRNA by Dicer enzyme in the cells [18, 22, 27].
Burn injury model
Animal experiments were approved by the Ethics committee of Henan province. Zhengzhou, Henan, China.
Male SD rats (weight 180–220 g) were purchased from Animal Experiment Center of Henan Province, housed 4 per cage and maintained on a 12 h light/dark schedule in a temperature-controlled at 25 °C environment with available access to food and tap water. The rats were randomly divided into 2 groups (n = 16): Burn injury group: A metal plate cover floated on which water circulated from 85 °C water bath was arranged. One hind paw of each rat was held in contact with the metal plate cover for 15 s to establish the burn model. Sham group: Sham-treated animals went through the identical treatment except that the hind paw was placed on the room temperature metal plate instead of 85 °C. Burn injury was performed n the rats as described previously . Anesthesia was induced with 5 % isoflurane and maintained at 2.5 % (V/VO2 2 l.min-1) before the process and remained throughout the duration of the process. Rats were allowed to recover from anesthesia in their home cages within 10 min after operation. According to the behavioral test results, animals displayed the extreme acute pain on 7 d after operation. Therefore, animals were sacrificed on 7 d to collect DRGs.
Rats were habituated to the test apparatus for at least 30 min before test on test days.
Behavioral test was performed blindly to treatment group. Hind paw radiant heat (Hargreaves’) test: Rats were placed in plastic chambers on a glass surface maintained at 25 °C. A radiant heat source was focused on the hind paw and latency to respond was recorded in three trials per paw, separated by at least 10 min. Heat intensity was 30 % and cutoff to avoid tissue damage was 30 s. von Frey test of mechanical threshold: rats were placed in plastic chambers on a wire mesh grid and stimulated with von Frey filaments according to the up-down method described previously .
Application of lentiviral vector-mediated shRNA
Thirty six male SD rats were randomly divided into 2 groups (n = 18): Burn injury + empty lentiviral vector -SCN9AsiRNA-GFP (LV3- SCN9AsiRNA-GFP group), burn injury + lentiviral vector negative control (LV3-NC-GFP group). Recombinant Lentivirus were offered by Shanghai GenePharma Co.,Ltd. On the zero day, burn injury model rats in LV3-NC group and LV3- SCN9A group received DRG microinjection  of 4 μl empty lentiviral vector and LV3- SCN9A vector, respectively. On the day before burn injury and 4 d, 7 d, 14 d, 21 d after burn injury (Post Operation Days, POD4, POD7.POD14, and POD21), mechanical withdrawal threshold and thermal withdrawal latency were measured. Rats were scarified on 7 d after operation and DRG was collected.
Under deep anesthesia with isoflurane, the rats were perfused with normal saline followed by cooled 4 % paraformaldehyde in 1 M phosphate buffer. L4 -6 DRGs were collected at 4 d, 7 d, 14 d, and 21 d. DRG was paraffin embedded and sectioned at 20 mm. For single labeling, 3 sets of DRG sections (4–5 sections/DRG) were collected. After being dewaxed by gradient with xylene ethanol, the sections were rinsed by PBS 5 min/times. After Antigen repair, the sections were blocked with goat serum for 2 h at room temperature. Then, the sections were incubated with Nav1.7 mouse monoclonal antibody (1 mg/mL; abcam) and C-fos goat anti-mouse antibody (1:500; Beijing Zhongshan biotechnology) for 30 min at 37 °C then at 4 °C overnight. The sections were then incubated with goat anti-rabbit antibody conjugated with FITC (1:80; Shanghai Weiao biotech) or donkey anti-mouse antibody conjugated with Cy3 (1:200; Jackson ImmunoResearch) for 2 h or DAPI (1:1000; Sigma) at room temperature (RT) and covered with BSA (10 %; Shanghai Weiao biotech). All stained sections were viewed with an epifluorescence microscope (Olympus Corporation, Japan).
Western blot analysis
Total protein was extracted from L4–6 DRGs of the rats using tissue protein extraction reagent (Weiao Biotech, Shanghai, China). The proteins were loaded and separated by sodium dodcyl sulfate (SDS)- polyacrylamide gels, electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore, MA, US). The membranes were then blocked with the blocking buffer (5 % fat free dry milk with TBST) for 2 h at RT and incubated with rabbit anti-Nav1.7 (1:600, Abcam, England) at 4 °C for 2 nights. Finally, the membranes were incubated with goat anti-rabbit antibody (1:800, Zhongshan Jinqiao, China) for 2 h at RT and signal was detected with ECL detection reagents (Alphalmager proteinsimple, San Jose, USA).
For statistical analysis, GraphPad Prism software was used. Behavioral data and immune fluorescence intensity were analyzed by either the Student’s t-test to compare 2 groups or ANOVA followed by planned comparisons of multiple groups. In both cases, when significant main effects were observed, P < 0.05 was considered to be statistically significant.
Nociceptive behaviors after burn injury
After burn injury, SD rats displayed increased sensitivity to heat and mechanical stimuli. To elucidate basic mechanism of burn injury-induced sensitization, we first established a model of focal second-degree burn in rats.
Protein expression change after burn injury
Nociceptive behaviors after treatment
Protein expression change after treatment
Burn injury arouses worldwide attention due to the large number of the patients and the abnormal acute pain which torments the patients in duration of therapy. Burn injury causes the release of various inflammatory mediators, as well as the Nav1.7 and c-fos proteins which are related to local and systemic inflammation or neurons activation. In this study, we established a rat model of focal second-degree burn injury to simulate the burn condition, for the investigation of mechanisms underlying burn-related pain. Using this model, we performed behavioral experiments and also examined the expression of sodium channel isoforms in the peripheral nervous system. We found that Nav1.7 is associated with burn-induced hypersensitivity to heat stimuli. Next we injected lentiviral vector-mediated shRNA into DRG microinjection to knockdown SCN9A gene to understand the role of Nav1.7 in burn injury induced pain.
Recently, several Nav1.7 inhibitor has been utilized, for instance,benzazepinones could block Nav1.7 sodium channels, and methadone could be a local anaesthetic-like inhibitor of Nav1.7 sodium channels . However, the application of these inhibitors has several concerns, including the specificity and side effects such as neurotoxicity. Therefore, in our present study, we chose the lentiviral vector-mediated shRNA to specifically knockdown Nav1.7 to elucidate the role of Nav1.7 in burn injury pain.
To our knowledge, there are various ways to deliver drugs and biological to DRG, including epidural injection (Long term catheterization), intrathecal injection , direct injection of the ganglion (DRG microinjection), and injection of peripheral tissue or the peripheral nerve like sciatic nerve. Considering that Nav1.7 is mostly expressed in DRG, we chose the DRG microinjection method to deliver lentiviral vector shRNA.
Previous study reported that the expression of Nav1.7 was increased in DRG neurons of burn-injury rats, thus we hypothesized that inhibiting Nav1.7 expression could prevent or relieve pain after burn injury, and alleviate the lesion in the burn injury-related neurons by reducing the expression of c-fos protein. In this study, we injected lent virus vector Nav1.7 shRNA to the DRG of the burn-injury rats at the day we perform the burn injury. Our data showed that the MWT of the left hind leg of burn-injury rat model had a significant rise at 4 d, 7 d and 21 d, exhibiting an apparent resistance to damage effect. We chose the 7 d after burn injury as the typical time point for protein expression analysis by Western Blot, because it’s the time point that the ipsilateral withdrawal thresholds and latencies in the shRNA groups were both significantly increased.
In summary, our data showed that shRNA mediated knockdown of Nav1.7 in L5DRG led to the attenuation of burn injury-induced mechanical allodynia and thermal hyperalgesia as well as the decrease of the activation of c-fos protein.in the rat hind paw. Therefore, Nav1.7 is a potential target to treat burn injury pain, and lentivirus mediated Nav1.7 shRNA have potential clinical application for gene therapy of burn injury pain.
DRG, dorsal root ganglion; LV, lentiviral vector; NC, negative control; POD, post-operative days; PWL, paw withdrawal latency; PWT, 50 % paw withdrawal threshold; RT, room temperature
We gratefully acknowledge the National Natural Science Foundation Committee of China and Zhengzhou University for their financial support.
This work was supported by independent innovation project of Zhengzhou University, Zhengzhou University, Zhengzhou, Henan, China, and grant from the National Natural Science Foundation of China (No. 81471144;81200866), China, and Special scientific research fund for young teachers of Zhengzhou University, Zhengzhou, Henan, China
Availability of data and materials
All the data and materials will be available upon publication of the study results.
JC and WDZ contributions to the conception and design. WHC involved in drafting the manuscript. WHC, LQ performed operations and carried out the molecular studies. XHR, XMC and ML collected and analyzed the data. All authors read and approved the final manuscript.
The authors declared that they have no competing interest.
Consent for publication
Ethics approval and consent to participate
Animal experiments were approved by the Ethics committee of Henan province. Zhengzhou, Henan, China.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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