Involvement of Cav3.2 channels in the
development of inflammation and related mechanical hypersensitivity:
genetic evidence
Taking into consideration that no pharmacological tool is exclusively
specific to Cav3.2 channels, we investigated the
contribution of these channels in inflammation and related
allodynia/hyperalgesia using mice constitutively deleted of the CACNA1H
gene (Cav3.2 KO). In the carrageenan model,
Cav3.2 KO mice did not develop allodynia (Figure
2A left ) nor hyperalgesia (Figure 2B left ) compared to their
control wild-type (WT) littermates. Edema development was observed in
both WT and Cav3.2 KO mice but to a significantly lesser
extent in the latter (Figure 2C left ). In addition, the
pro-inflammatory cytokine IL-6 level in the edema was significantly
lower in Cav3.2 KO mice (Figure 2D left ). In
the CFA model, similar results were obtained: a lack of mechanical
nociceptive behavior in Cav3.2 KO mice (Figures
2A right and 2B right ) and a significant decrease in both edema size
(Figure 2C right ) and IL-6 level in the edema (Figure
2D right ) compared to those in WT animals. Thus, these findings
underscore the importance of Cav3.2 subtype channels in
inflammation and related allodynia/hyperalgesia.
Functional location of
Cav3.2 channels involved in inflammatory pain-like
symptoms
Next, we gained further insights into the role of Cav3.2
channels in inflammatory pain by investigating the impact of their
inhibition at different locations. We assessed the effect of the
deletion of Cav3.2 channels located in a population of
primary sensory neurons in mice, named C-LTMRs. These fibers express
both the sodium channel Nav1.8 and the calcium channel
Cav3.2 (François et al., 2015) and have been shown to
contribute to inflammatory pathological pain (Seal et al., 2009; Delfini
et al., 2013; François et al., 2015; Reynders et al., 2015; Urien et
al., 2017; Bohic et al., 2020). We therefore used a genetic mouse model
in which the expression of Cav3.2 channels is
conditionally knocked out in C-LTMRs, by crossing
Cav3.2GFP-flox KI with
Nav1.8cre KI mice as previously
described (François et al., 2015). In this mouse model
(Cav3.2Nav1.8 cKO), a significant
decrease in allodynia and hyperalgesia in the von Frey test was observed
after carrageenan (Figure 3A ) or CFA (Figures 3B )
injections compared to control
Cav3.2GFP-flox KI littermates. These
results evidenced that the Cav3.2 channels located on
C-LTMRs are required for the development of inflammatory
allodynia/hyperalgesia. This involvement of C-LTMRs-located
Cav3.2 channels could arise from their projection in the
spinal cord (pre-synaptic Cav3.2 channels) or their
origin in peripheral tissue (Cav3.2 channels on primary
afferent fibers). Therefore, the contribution of both locations was
studied. First, peripheral Cav3.2 channels involvement
was evaluated using an intraperitoneal injection of ABT-639 (10, 30 and
100 mg/kg), a peripherally restricted T-type calcium channels inhibitor.
After such treatment, we observed a robust analgesic effect on allodynia
and hyperalgesia at the two highest doses tested (30 and 100 mg/kg) in
both carrageenan (Figure 4A ) and CFA (Figure 4B )
models. This result was confirmed by performing an intraplantar
injection of ABT-639 (2.5 µg / mouse in 20 µl) in the CFA model
(Figure 4C ). Second, an intrathecal injection of ABT-639 (10 µg
/ mouse in 5 µl) was performed in the CFA model to determine the
contribution of Cav3.2 channels in primary afferent
spinal terminals to allodynia/hyperalgesia. The injection partially
reduced the responses to the von Frey test (Figure 4D ). Thus,
both pharmacological and genetic tools identified the involvement in
inflammatory pain of Cav3.2 located in primary afferent
neurons at both the peripheral and spinal terminals.