The NAv1.7 blocker protoxin II reduces burn injury-induced spinal nociceptive processing
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Controlling pain in burn-injured patients poses a major clinical challenge. Recent findings suggest that reducing the activity of the voltage-gated sodium channel Nav1.7 in primary sensory neurons could provide improved pain control in burn-injured patients. Here, we report that partial thickness scalding-type burn injury on the rat paw upregulates Nav1.7 expression in primary sensory neurons 3 h following injury. The injury also induces upregulation in phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB), a marker for nociceptive activation in primary sensory neurons. The upregulation in p-CREB occurs mainly in Nav1.7-immunopositive neurons and exhibits a peak at 5 min and, following a decline at 30 min, a gradual increase from 1 h post-injury. The Nav1.7 blocker protoxin II (ProTxII) or morphine injected intraperitoneally 15 min before or after the injury significantly reduces burn injury-induced spinal upregulation in phosphorylated serine 10 in histone H3 and phosphorylated extracellular signal-regulated kinase 1/2, which are both markers for spinal nociceptive processing. Further, ProTxII significantly reduces the frequency of spontaneous excitatory post-synaptic currents in spinal dorsal horn neurons following burn injury. Together, these findings indicate that using Nav1.7 blockers should be considered to control pain in burn injury.
• Burn injury upregulates Nav1.7 expression in primary sensory neurons.
• Burn injury results in increased activity of Nav1.7-expressing primary sensory neurons.
• Inhibiting Nav1.7 by protoxin II reduces spinal nociceptive processing.
• Nav1.7 represents a potential target to reduce pain in burn injury.
KeywordsPain p-ERK1/2 Primary sensory neuron p-S10H3 Spinal cord
Burn injury is associated with moderate to severe pain that represents a significant clinical challenge . The lack of effective pain management in burn-injured patients can lead to long-term consequences including the development of anxiety, depression, post-traumatic stress disorder and chronic pain . Therefore, there is a need for the development of novel analgesic approaches to control pain in burn-injured patients.
A series of mediators produced and released during inflammation that ensues after burn injury activate a major sub-set of primary sensory neurons [1, 2]. The resulting generation and propagation of action potentials initiate nociceptive processing in the central nervous system and lead to the experience of pain. Voltage-gated Na+ channels (Nav), distinguished by their alpha sub-units [3, 4, 5], are pivotal for the generation and propagation of action potentials in neurons . Recent studies identified Nav1.7 as a putative key molecule for the development of heat hypersensitivity in burn injury [4, 7, 8]. Therefore, in the present work, we examined whether specific blockade of Nav1.7 with the selective toxin, protoxin II (ProTxII) [9, 10], is a feasible target to reduce nociceptive processing in burn injury.
Materials and methods
Animals, burn injury and treatment
We obeyed the UK Animals (Scientific Procedures) Act 1986, the guidelines of the revised National Institutes of Health Guide for the Care and Use of Laboratory Animals, Directive 2010/63/EU of the European Parliament and of the Council on the Protection of Animals Used for Scientific Purposes and the Committee for Research and Ethical Issues of IASP published in Pain, 16 (1983) 109–110 and adhered to Good Laboratory Practice and ARRIVE guidelines. Procedures were approved by veterinary services at all relevant institutions. Every effort was made to minimise the number of animals used and the potential distress. Animals were housed in climate-controlled rooms, on a 12 h light/dark cycle, with food and water ad libitum. In total, 36 male Sprague-Dawley rats (150-200 g), 5 male Wistar rats (P21), 2 wild-type (WT, ~ 22 g) mice and 2 mice lacking Nav1.7 in Nav1.8-expressing cells (Nav1.7cKO, ~ 22 g; ; both types on C57BL/6 background) were used.
Burn or sham injury was induced as described previously [11, 12]. Briefly, animals were anaesthetised by intraperitoneal urethane (0.02 mg/g) or isoflurane (3%, for spinal cord slice preparation) and one of the hind paws (both paws for spinal cord slices) was immersed into 60 or 37 °C water up to the knee for 2 min. Anaesthesia was maintained for up to 180 min post-injury. Following intraperitoneal sodium pentobarbital, animals were either transcardially perfused with saline then 4% paraformaldehyde or the L4–L5 dorsal root ganglia (DRGs) were dissected from both the ipsilateral and contralateral sides. ProTxII (0.1 mg/kg; Tocris) or morphine (3 mg/kg; Sigma) was injected intraperitoneally 15 min before or after the injury.
The protocol included tissue homogenisation with a pestle and mortar in ice-cold RIPA buffer (Amresco) and protease inhibitor cocktail (Sigma-Aldrich), sonication for 1 h at 4 °C, spinning for 30 min at 14,000 rpm at 4 °C and denaturing at 95 °C for 5 min. NuPAGE Novex 4–12% Bis-Tris protein gels (Invitrogen, UK) were used for separation. After transfer to PVDF membranes (Invitrogen, UK), samples were incubated in 5% non-fat milk powder (Sigma, UK) for 1 h at room temperature, then in anti-Nav1.7 and anti-β-tubulin III antibodies at 4 °C overnight followed by incubation in secondary antibodies at room temperature for 1 h and visualisation with the Luminol kit (Santa Cruz, USA; Supplementary Table 2). Membranes were examined in a G:Box (SynGene, UK) using the GeneSnap software package (Synoptics Ltd, SynGene). Analysis was done by ImageJ; Nav1.7 intensities were normalised to β-tubulin intensities in each of the 16 samples (samples from the ipsilateral and contralateral sides of 8 animals). Then, the ratio of the normalised intensities found on the ipsilateral and contralateral sides in each animal was calculated and averaged.
The L4–L5 segments of the spinal cord and the L4 and L5 DRGs were dissected, post-fixed overnight in 4% paraformaldehyde and cryoprotected in 30% sucrose. Ten-micrometre sections were cut and incubated in PBS containing 0.3% Triton-X 100 (PBST) for 10 min, then in 10% normal donkey serum (NDS) for 1 h followed by the primary antibody (Supplementary Table 1). For visualisation of the phosphorylated serine 10 in histone H3 (p-S10H3) staining, the tyramide signal amplification procedure was used . Other immunoreactions were visualised by Alexa Fluor-conjugated secondary antibodies (Supplementary Table 1). Slides were coverslipped with Vectashield (Vector Laboratories, UK) and examined with a Leica microscope attached to a Hamamatsu colour-chilled 3CCD camera.
In vitro electrophysiology
The spinal cord slices were prepared using sham-injured and burn-injured Wistar rats as described previously . One hour after the injury, 300-μm transverse slices were cut and incubated in dissection solution ((in mM) 95 NaCl, 1.8 KCl, 7 MgSO4, 0.5 CaCl2, 1.2 KH2PO4, 26 NaHCO3, 25 D-glucose and 50 sucrose) for 30 min at 35 °C, stored in a recording solution ((in mM) 127 NaCl, 1.8 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, 26 NaHCO3 and 25 D-glucose) at room temperature (21–24 °C) and allowed to recover for at least 1 h before recordings. All extracellular solutions were saturated with carbogen (95% O2, 5% CO2).
Whole-cell patch-clamp recordings (at 21–24 °C) were performed from the superficial dorsal horn neurons clamped at − 70 mV in the presence of 10 μM bicuculline and 5 μM strychnine in the bath solution as described previously . The intracellular pipette solution contained (in mM) 125 gluconic acid lactone, 15 CsCl, 10 EGTA, 10 HEPES, 1 CaCl2, 2 Mg2ATP and 0.5 NaGTP and was adjusted to pH 7.2 with CsOH. An Axopatch 1D (Axon Instruments, USA) amplifier, a Digidata 1440A digitizer (Molecular Devices, USA) and the pCLAMP 10.5 software package were used for recordings. Low-pass filter (2 kHz), 10-kHz sampling rate and 80% series resistance compensation were used. Spontaneous excitatory post-synaptic currents (sEPSCs) with an amplitude of 5 pA or greater (at least twice of the noise) were included in the frequency and amplitude analyses. Basal activity recording was followed by recoding in the presence of ProTxII (10 nM in 0.1% BSA, Tocris) for 5 min. In the end of the recording protocol, capsaicin (200 nM) was applied to find whether the neuron received nociceptive input.
Data were analysed as previously reported . In brief, power calculations were used to estimate sufficient sample size using an online-based software (http://homepage.stat.uiowa.edu/~rlenth/Power/) and statistical analyses were carried out using the SPSS program (IBM SPSS statistics 22.0 for Windows). Data from immunostaining were analysed using a generalised linear model (GzLM), with significance assessed with the Wald chi-squared test. For Western blotting, independent t tests were used. In all cases, differences were regarded significant at p < 0.05. The statistical significance of ProTxII on sEPSCs was tested using paired t test and t test with Bonferroni correction for multiple comparisons. The sEPSC frequency after ProTxII application was also normalised against the pre-application control value. Data are expressed as mean ± standard error of mean; n refers to the number of biological repetitions.
In our pilot experiments, we tested several anti-Nav1.7 antibodies and immunolabelling procedures on sections cut from rat L4 and L5 DRGs. While all the antibodies provided similar staining pattern, the antibody supplied by Millipore (Supplementary Table 1) and the procedure described above produced the highest signal-to-noise ratio, which we found suitable for quantitative analysis.
In rat L4–L5 DRGs, a significant proportion of neurons exhibited immunostaining (Fig. 1c). No immunopositive neurons were visible when the primary antibody was replaced by normal serum (data not shown) or exhausted with the immunising peptide (Supplementary Fig. 1a). In the positive control, an already characterised antibody (anti-TRPV1 antibody; ) showed the characteristic immunopositivity (Supplementary Fig. 1b).
Nav1.7 immunopositivity was found in about 25% of the neurons in naive rat DRGs (25.69 ± 6.03%, n = 4). The size distribution confirmed that Nav1.7 is expressed predominantly in small- and medium-size neurons (Fig. 1d). The average area of the Nav1.7-immunopositive neurons was significantly smaller than that of the Nav1.7-immunonegative cells (positive 406.14 ± 33.64 μm2, n = 4; negative 572.10 ± 33.33 μm2, n = 4; p = 0.0128, GzLM).
Burn injury upregulates Nav1.7 expression in primary sensory neurons
Quantification of Nav1.7-immunopositive neurons at various time points after the injury confirmed the upregulation of Nav1.7 expression in ipsilateral DRG at 3 h post-injury (naive 25.69 ± 6.03%, 3 h 55.81 ± 14.11%, n = 4 for each; p = 0.05, GzLM; Fig. 2c). However, no increase in the proportion of Nav1.7-immunopositive DRG neurons in contralateral DRGs at any time point following burn injury (not shown) or at either side following a sham injury was found (naive 25.08 ± 2.97%, n = 4, p = 0.693; sham ipsilateral 25.72 ± 9.81%, n = 4, p = 0.797; sham contralateral 27.67 ± 5.97%, n = 4, p = 0.857; 5 min 22.24 ± 2.57%, n = 4, p = 0.747; 30 min 40.05 ± 9.55%, n = 4, p = 0.185; 1 h 22.32 ± 8.28%, n = 4, p = 0.753; 3 h 18.96 ± 9.18%, n = 4, p = 0.532; GzLM).
p-CREB is a marker for neuronal activation by burn injury
Phosphorylated CREB (p-CREB) is a common downstream effector of various pathways implicated in regulating transcriptional changes associated with use-dependent increase in the activity and excitability (sensitisation) of primary sensory neurons by noxious stimuli . Hence, we assessed whether p-CREB identifies activated primary sensory neurons in our burn injury model.
p-CREB is expressed in Nav1.7-expressing neurons
Double immunostaining revealed that while in the naive rats, about 25% of the very few cells with p-CREB-immunopositive nuclei exhibited Nav1.7 immunopositivity, 5 min after the burn injury, ~ 90% of the cells with p-CREB-immunopositive nuclei also exhibited immunopositivity for Nav1.7 (87.5 ± 7.98%, n = 4; Fig. 3c, f, g). The co-expression pattern was very similar at 3 h post-injury (p = 0.983; Fig. 3h).
Very few Nav1.7-immunopositive neurons showed p-CREB immunopositivity in naive animals (13.3 ± 3.33%, n = 4). The co-expression pattern increased to around 80% 5 min after the burn injury (77.58 ± 10.16%, n = 4), and this proportion remained similar at 3 h post-injury (63.42 ± 10.31%, n = 4; p = 0.758).
Together, these data support recent reports on the pivotal role of Nav1.7 in the development of burn injury-associated pain . Those reports also showed that Nav1.7 is particularly important in the development of burn injury-associated heat hyperalgesia . We have shown most recently that burn injury induces a rapid and sustained upregulation of p-S10H3 in a sub-population of spinal dorsal horn neurons . Our findings also indicate that p-S10H3 can be used as a marker for nociceptive activation of spinal cord neurons involved in the development of inflammatory heat hyperalgesia . Therefore, next, we assessed the effect of blocking Nav1.7 on p-S10H3 expression in the spinal dorsal horn.
Nav1.7 blockade partially reduces burn injury-induced nociceptive activation in spinal cord neurons
Morphine injection 15 min before the injury completely prevented the upregulation of p-S10H3 expression by burn injury (morphine-before 1.66 ± 0.88, n = 3, p = 0.646, GzLM; Fig. 4a, b, e, g). Morphine, injected 15 min after the induction of the burn injury, significantly reduced the number of neurons exhibiting p-S10H3-immunopositive nuclei (morphine-after 11.33 ± 1.20, n = 3, p < 0.001, GzLM; Fig. 4a, b, f, g). The number of activated neurons found in the morphine-after group was not significantly different from that found in naive animals. Both ProTxII and morphine had similar effects on the expression of phosphorylated ERK (p-ERK) 1/2 in the spinal cord (Supplementary Fig. 4).
ProTxII reduces sEPSC frequency following burn injury
sEPSC frequency exhibited a robust and significant increase following burn injury (3.1 ± 0.6 Hz; Fig. 5a, b, p = 0.002). ProTxII significantly decreased the sESPC frequency to 2.1 ± 0.5 Hz (66.2 ± 8.1% of the control value; Fig. 5a, b). The average sEPSC amplitude was − 17.8 ± 3.4 pA, and ProTxII did not change that (− 17.1 ± 3.6 pA). All neurons responded to capsaicin (31.5 ± 7.1 Hz; n = 10; p = 0.003). The capsaicin response was not different in the sham and injured groups.
Similar to previous reports, we found that a significant proportion of primary sensory neurons express Nav1.7 [5, 17]. While we did not test whether Nav1.7 is functional in the Nav1.7-expressing primary sensory neurons, previous findings that a significant proportion of primary sensory neurons do express such currents [4, 5, 14, 17, 19] indicate that at least a proportion of the Nav1.7-immunopositive neurons express functional Nav1.7 channels.
Both Western blotting and immunostaining revealed that Nav1.7 expression is increased in DRGs by 3 h post-injury. Similar upregulation has been reported in other peripheral inflammatory models [20, 21]. Increased density of Nav1.7-mediated currents in primary sensory neurons following burn injury was also reported recently . The increased Nav1.7 expression and the increased density of Nav1.7-mediated currents, 3 h and 2 days after the injury, respectively, support the view that Nav1.7 significantly contributes in enhancing nociceptive signalling of primary sensory neurons during the entire course of burn injury .
Burn injury induced a biphasic upregulation in the expression of p-CREB, a marker for neurons activated by various painful peripheral pathologies, including inflammation of various origins in primary sensory neurons [18, 22]. The increase at 5 min could be due to the activation of neurons by the excessive heat and/or molecules released from the degenerated cells. The increase at 180 min could be due to the activation of neurons by inflammatory mediators . Importantly, we found a high degree of co-expression between Nav1.7 and p-CREB after burn injury indicating that Nav1.7-expressing neurons are activated by this injury.
We analysed p-ERK 1/2 and p-S10H3 expressions to find the effect of morphine and ProTxII on spinal nociceptive processing. While p-ERK1/2 is well-established, p-S10H3 is a novel marker for nociceptive activation of spinal dorsal horn neurons [11, 12, 23]. As confirmed in the present study, burn injury induces sustained upregulation in both p-ERK1/2 and p-S10H3 expressions in the spinal dorsal horn [11, 12].
Both morphine and ProTxII, which respectively activates the μ-opioid receptors (MOR; ) and inhibits Nav1.7 , significantly reduced the burn injury-induced upregulation of both p-ERK1/2 and p-S10H3 expressions. While the finding that morphine reduces spinal nociceptive processing is in full agreement with a large body of previous findings [25, 26], the effect of ProTxII appears to be in contrast to previous reports that intravenous or intrathecal ProTxII injection does not reduce pain-related behaviour . Although due to animal welfare considerations, we did not assess pain-related behaviour, the similar magnitude of inhibitory effects by morphine and ProTxII administration following the injury suggests that similar to morphine , ProTxII is also highly likely to produce an analgesic effect.
The lack of effect by ProTxII on pain-related behaviour was attributed to the inability of the toxin to access Nav1.7 in intact peripheral nerves and to pass the blood-brain barrier [10, 28]. However, a recent finding has demonstrated that ProTxII can access Nav1.7 in the spinal cord following intrathecal delivery as well as the peripheral nerve after perineural application . Our data suggest that in addition to those, ProTxII may also reach Nav1.7 following intraperitoneal injection. While we did not assess the site of action, based on previous findings, we propose that the ProTxII-produced inhibitory effect on spinal nociceptive processing could be due to ProTxII-induced inhibition of Nav1.7 expressed on free nerve endings at the injured tissues as well as the central terminals of Nav1.7-expressing primary sensory neurons.
The significantly larger effect of morphine than of ProTxII on the upregulation of both p-S10H3 and p-ERK1/2 when applied before the injury could be due to the differing respective access to MOR and Nav1.7 of morphine and ProTxII in various parts of primary sensory neurons in naive conditions [25, 26].
The effect of morphine or ProTxII applied 15 min after the injury that models the time course of burn-injured patients receiving analgesics for the first time shows that both drugs are able to induce a significant downregulation in the expression of both markers, hence reducing spinal nociceptive processing. Interestingly, ProTxII produces a greater downregulation in p-S10H3 than p-ERK1/2 expression, which could be due to both Nav1.7 and p-S10H3 being involved in the development of heat hypersensitivity [4, 7, 8, 12], whereas p-ERK1/2 is involved in the development of both thermal and mechanical hypersensitivities . Nevertheless, the downregulation of p-ERK1/2 and p-S10H3 by morphine or ProTxII 15 min after the injury indicates that ongoing activity of MOR- and/or Nav1.7-expressing primary sensory neurons is needed for the nociceptive activation of the spinal dorsal horn neurons in burn injury.
Our electrophysiological recordings confirm the significant role of Nav1.7 in spinal nociceptive processing following burn injury. Burn injury significantly increased sEPSC frequency which was significantly reduced by ProTxII. The differential effect of ProTxII in naive slices and slices prepared after burn injury could be due to Nav1.7 playing a minor role, whereas it gains a much more prominent role in generating spontaneous activity of nociceptive primary sensory neurons in naive condition and after burn injury, respectively. Alternatively, ProTxII, due to neuroinflammatory processes in the spinal cord, may have access to Nav1.7 on the central terminals of the primary sensory neuron after burn injury.
Morphine or other opioids used currently to control pain in burn-injured patients induce a series of undesirable effects [30, 31]. Based on the similar effects of morphine and ProTxII on p-ERK1/2 and p-S10H3 expressions by the spinal dorsal horn neurons and the role of p-ERK1/2 and p-S10H3 in the development of persistent pain associated with peripheral pathologies [11, 12, 32, 33], we propose that blocking Nav1.7 could reduce pain, particularly heat hyperalgesia [4, 7, 8], in burn injury with a potency equivalent to that produced by morphine.
Previous attempts to recapitulate the profound analgesic phenotype of Nav1.7−/− mice or loss of function human mutations [5, 19] by pharmacological agents produced disappointing results . However, a recent report has shown that a ProTxII-based designer peptide acting on Nav1.7 is able to reproduce the analgesic phenotype observed in mice lacking Nav1.7−/− or humans having a loss of function Nav1.7 mutations . Therefore, based on the expression pattern of Nav1.7 [5, 14, 17, 19], it is likely that blocking Nav1.7, particularly in a cell-specific manner, could produce a significant analgesic effect with significantly less undesirable effects than opioids.
This work was supported by the Chelsea and Westminster Health Charity, KTIA_NAP_13-2-2014-0005, and GACR 15-11138S, MSMT LQ1604, LH15279, CZ.1.05/1.1.00/02.0109, GAUK138215 and RVO 67985823 grants.
Compliance with ethical standards
We obeyed the UK Animals (Scientific Procedures) Act 1986, the guidelines of the revised National Institutes of Health Guide for the Care and Use of Laboratory Animals, Directive 2010/63/EU of the European Parliament and of the Council on the Protection of Animals Used for Scientific Purposes and the Committee for Research and Ethical Issues of IASP published in Pain, 16 (1983) 109–110 and adhered to Good Laboratory Practice and ARRIVE guidelines. Procedures were approved by veterinary services at all relevant institutions.
Conflict of interest
The authors declare that they have no conflict of interest.
- 12.Torres-Perez JV, Santha P, Varga A, Szucs P, Sousa-Valente J, Gaal B, Sivado M, Andreou AP, Beattie S, Nagy B et al (2017) Phosphorylated histone 3 at serine 10 identifies activated spinal neurons and contributes to the development of tissue injury-associated pain. Sci Rep 7:41221. https://doi.org/10.1038/srep41221 CrossRefPubMedPubMedCentralGoogle Scholar
- 17.Toledo-Aral JJ, Moss BL, He ZJ, Koszowski AG, Whisenand T, Levinson SR, Wolf JJ, Silos-Santiago I, Halegoua S, Mandel G (1997) Identification of PN1, a predominant voltage-dependent sodium channel expressed principally in peripheral neurons. Proc Natl Acad Sci U S A 94:1527–1532CrossRefPubMedPubMedCentralGoogle Scholar
- 22.Li MY, Lai FJ, Hsu LJ, Lo CP, Cheng CL, Lin SR, Lee MH, Chang JY, Subhan D, Tsai MS et al (2009) Dramatic co-activation of WWOX/WOX1 with CREB and NF-kappaB in delayed loss of small dorsal root ganglion neurons upon sciatic nerve transection in rats. PLoS One 4:e7820. https://doi.org/10.1371/journal.pone.0007820 CrossRefPubMedPubMedCentralGoogle Scholar
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