In the present study, we investigated whether Nav1.9 channel contributes to acute, subacute and chronic inflammatory pain in mice and rats using knock-out and knock-down strategies, respectively. We showed that whereas Nav1.9 is not involved in basal pain thresholds, it plays an important role in acute inflammatory pain, as well as in both mechanical and heat pain hypersensitivities produced by subacute paw and chronic joint inflammation. In the subacute inflammatory pain model, these behavioral observations were correlated with post-transcriptional modulations of Nav1.9 expression in DRG cell bodies, and with a strong upregulation of the protein in the peripheral nerve trunks. Furthermore, at a time where Nav1.9 was strongly overexpressed in DRG cell bodies, no Nav1.9 current increase could be detected, suggesting that the newly produced channels are non-functional and rather intended to be exported to nerve terminals.
The relationship between Nav1.9 and inflammatory pain has previously been pointed out by different studies, but this is the first time that one correlates behavioral, molecular and electrophysiological data on the same inflammatory models, with time-course monitoring. These previous studies also brought out some discrepancies that needed to be clarified. Firstly, heat hypersensitivity induced by intraplantar carrageenan 
or CFA 
have been reported to be either reduced 
or unaffected 
in Nav1.9-null mice. Our results show that Nav1.9 is involved in the decrease in inflammatory heat pain threshold following carrageenan injection. In the chronic monoarthritis model, Nav1.9 invalidation also weakened heat hypersensitivity phenotype, but mostly during the first week of monitoring. Those results confirm the role of Nav1.9 channel in establishing inflammatory heat pain hypersensitivity, and to a lower extent, in its maintenance.
On the other hand, it has previously been shown that Nav1.9 was not involved in inflammatory mechanical hypersensitivity following intraplantar CFA 
. However, this has never been assessed using a different inflammatory pain model, with the exception of colorectal distention following colonic TLR7 activation 
. In this last study, Nav1.9 knock-out mice presented a reduction of visceral hypersensitivity to mechanical stimulation. Furthermore, a reduction of mechanical pain hypersensitivity induced by intraplantar injections of inflammatory mediators (prostaglandin E2, bradykinin or interleukin-1β) has been observed in Nav1.9−/−
. The role played by Nav1.9 in mechanical hypersensitivity driven by inflammation is therefore far from clear and further exploration was needed. Following intraplantar carrageenan injection, our Nav1.9−/−
mice exhibited reduced mechanical hypersensitivity compared to WT littermates. This phenotype, observed with the von Frey test also used in the previously cited studies 
, was confirmed with the use of a new operator-independent dynamic weight bearing test. Moreover, our data are supported by the reduction of carrageenan-induced mechanical hypersensitivity observed in Nav1.9 knock-down rats. Finally, we observed a decrease in weight imbalance in monoarthritic knock-out mice, showing that Nav1.9 invalidation is also able to reduce chronic joint pain and the resultant posture impairment. The negative results previously published on mechanical hypersensitivity 
may be inherent to the intraplantar CFA model. Such discrepancies between inflammatory models have already been reported between carrageenan and CFA for example, considering either the efficiency of pharmacological P2X3
receptors inhibition 
, or ASIC3 gene deletion 
, on pain hypersensitivity. Although we could not generalize our conclusions to all the existing inflammatory pain models, taken together our behavioral data give numerous and strong evidences that Nav1.9 participates in lowering heat as well as mechanical pain thresholds during inflammatory states.
In the second part of this study, we assessed Nav1.9 expression in both carrageenan and monoarthritis pain models, as many different studies have reported increased, decreased or stable Nav1.9 mRNA and/or protein expression following peripheral inflammation 
. Those fragmented information were furthermore rarely correlated with behavioral data, which is an issue for the functional interpretation of possible expression variations. We therefore performed a time-course study of Nav1.9 expression allowing a correlation with the phenotype observed in animal.
A transient decrease in Nav1.9 protein level was observed in the soma of DRG neurons 8 h following intraplantar carrageenan, whereas upregulation was seen 16–24 h post-injection. These changes in somatal protein level were paralleled with an increase in cutaneous nerve trunks within the inflamed zone 24 h following carrageenan injection. These observations strongly suggest that upon inflammation Nav1.9 protein is exported to the periphery, depleting the cytoplasmic pool of channels present in the cell body of sensory neurons, before an increase in its neosynthesis. No such variation was observed in the monoarthritis model, suggesting either that the observed mechanism takes place during the subacute phase of inflammation only, or that a balance between Nav1.9 production and axonal transport has been reached within the chronic phase. As no change in Nav1.9 current properties was detected in DRG neurons innervating the inflamed paw at a time where Nav1.9 was shown to be upregulated, one can assume that a non-functional pool of channels is kept at the soma level and contributes to the increase in their axonal transport. It has been previously demonstrated that when an inflammatory soup is applied on cultured sensory neurons, the cells present a hyperexcitable behavior involving a strong upregulation of Nav1.9 current 
. Therefore, we hypothesize that the increase in peripherally located channels may contribute to fibers' excitability directly at the site of inflammation.
Nav1.9 modulation by inflammation could temporally be divided in two steps. First, we propose that a rapid modulation of Nav1.9 channels takes place in axon terminals. This idea is supported by the fact that Nav1.9 is present in nerve endings in physiological conditions 
and by the fact that in the formalin test Nav1.9 is involved in spontaneous painful behavior as soon as 10 to 15 min post-injection. Nav1.9 was also shown to be involved in mechanical hypersensitivity induced by intraplantar PGE2
within 15 min 
, and previous electrophysiological studies have revealed rapid modulation of Nav1.9 current following GPCR activation, possibly via a PKC dependent pathway 
. Secondly, an increase in axonal transport, followed by an upregulation of Nav1.9 channel expression in cell bodies, might take place within the first hours of inflammation as to sustain the excitability of nociceptive terminals.
Changes in Nav1.9 protein expression were not subsequent to modulations in mRNA level, as Nav1.9 transcript remained stable at all time-points tested. Post-transcriptional regulation of VGSCs has been reported previously. For example, Nav1.8 protein but not the corresponding mRNA has been shown to be upregulated in DRG neurons following colonic inflammation 
. The non-selective cation channel TRPV1 has also been shown to be massively and unilaterally exported to the periphery 24 h following intraplantar CFA 
, sustained by a post-transcriptional upregulation of the channel expression in DRG cell bodies. This type of regulation may occur at different levels, such as dysregulation of mRNA stability, as shown for the Nav1.7 channel 
, or modulation of its translation by RNA binding proteins, as for the ENaC channel 
Regarding to these results, Nav1.9 channel may potentially be a suitable pharmacological target for inflammatory pain care. Complete loss of function, as well as partial silencing, were both able to reduce inflammatory pain hypersensitivity, not depending on the nature of the noxious stimuli, while those treatments had no effect on pain thresholds in healthy animal. Thus, drugs inhibiting Nav1.9 should be considered as potential anti-hyperalgesics rather than analgesics, which is conceptually innovative.