4. DISCUSSION
In this study, we used the DREADDs approach as a novel tool to examine
the role of BLA astrocytes in DNP. Our results showed that inhibiting
the activation of BLA astrocytes attenuated mechanical allodynia and
pain-related negative emotions in DNP rats. Contrastively, temporary
activation of BLA astrocytes induced allodynia rather than anxious
behavior in naive rats. In addition, we found that koumine alleviates
mechanical allodynia and anxiety-like behavior in DNP rats, inhibits the
activation of BLA astrocytes and suppresses the inflammatory response.
Furthermore, sustained activation of BLA astrocytes by chemogenetic
mimics chronic pain, and koumine can alleviate its pain hypersensitivity
and anxiety-like behavior. This evidence suggests that BLA astrocytes
are involved in the regulation of pain and pain-related negative
emotions in DNP rats and may be an important target through which KM
exerted an anti-DNP effect, all of which may suggest a new target for
drug development.
In recent decades, the contribution of astrocytes to pain has received
considerable attention (Li et al., 2019). Studies have shown that in
chronic pain conditions, spinal cord astrocytes are activated, inducing
cell proliferation and hypertrophy, accompanied by functional changes
(Donnelly et al., 2020). A previous study identified a correlation
between spinal astrocyte activation and DNP (Feldman EL et al., 2019).
In terms of mechanical allodynia, Liao et al. highlighted that spinal
astrocyte activation promoted the development of mechanical allodynia in
rats with DNP (Liao et al., 2011). Additionally, numerous studies have
shown that inhibiting spinal astrocyte activation can moderate
diabetes-induced mechanical allodynia (Nakagawa and Kaneko, 2010; Dauch
et al., 2012; Zuo et al., 2015). These studies all support the notion
that astrocyte activation is an integral part of neuropathic pain
pathogenesis. Although the role of astrocytes in pain modulation is well
established, most studies have focused on astrocytic reactions in the
spinal cord, whereas a supraspinal understanding of the correlation
between astrocytes and neuropathy is still limited.
The amygdala is an important component of the limbic system and plays a
key regulatory role in pain sensation and pain emotion (Hua et al.,
2020). The BLA, as the main afferent nucleus in the amygdala, has
extensive fiber projections to many regions, such as the cerebral
cortex, thalamus and brainstem; the BLA can transmit and integrate the
emotional and cognitive components of pain from higher brain regions,
such as the cortex and thalamus, and subsequently transmits this
information to the amygdala complex, where it is combined with sensory
components of pain to capture the complete experience of pain perception
(Thompson and Neugebauer, 2017). According to previous literature, in a
state of chronic neuropathic pain, the activation of brain BLA neurons
is involved in the coding of pain, and plastic changes in pain-related
neurons are manifested as abnormally increased excitability (Kang et
al., 2021). Chemical damage to the bilateral BLA can significantly
reduce neuropathic pain-like behaviors in rats, resulting in significant
analgesic effects (Li et al., 2013). Furthermore, there is increasing
evidence that the BLA is closely associated with negative emotions (Sah,
2017) and cognitive changes (Sun et al., 2019; Zamyad et al., 2021). At
the same time, a recent study showed that amygdala astrocytes are
significantly activated during the development of neuropathic pain
(Sagalajev et al., 2018), all of which suggests a potential regulatory
role of BLA astrocytes in the pathogenesis of DNP.
To gain insight into the regulatory role of BLA astrocytes in DNP,
specific and precise regulation of BLA astrocyte activity in natural
physiological and pathological environments is needed. In this study, we
employed DREADDs approach to specifically manipulate BLA astrocytes
because, compared with traditional pharmacological methods, DREADDs have
a strong targeting selectivity, an accurate localization, a lack of
neuronal off-target effects and noninvasiveness, which makes it a
decisive technique for in vivo exploration of the physiological and
pathological effects of astrocytes (Roth, 2016; Pickering and Mazarakis,
2021). The gfaABC1D is a common astrocyte-specific promoter in studies
involving DREADDs and has been widely used to study the function of
astrocytes in vivo. However, it has also been reported that the gfaABC1D
promoter may leak into other cells (Taschenberger et al., 2017). This
result indicates that it is important to carefully investigate the
specificity of the gfaABC1D-M4-EGFP virus. Therefore, our experimental
design excluded the effects of gfaABC1D-M4-EGFP virus on neurons or
microglia, which is consistent with Griffin’s research (Griffin et al.,
2019). Our results showed that the green fluorescence of
gfaABC1D-M4-EGFP virus was specifically expressed only in BLA astrocytes
and not in neurons and other cells, indicating that the gfaABC1D
promoter has the specificity of infecting astrocytes (Supplementary
materials).
In the present study, we used a gfaABC1D-M4-EGFP virus to suppress BLA
astrocyte activity to evaluate whether inhibition of BLA astrocytes
might be beneficial for alleviating mechanical hyperalgesia. We observed
that after the verified inhibition of BLA astrocytes with CNO, the
mechanical pain threshold was significantly increased, which was similar
to the results observed by Marcello et al. after pharmacological
intervention (Marcello et al., 2013), indicating that inhibiting BLA
astrocytes can effectively alleviate mechanical allodynia in rats with
DNP. To further evaluate the effects of BLA astrocytes on mechanical
allodynia, we injected the gfaABC1D-M3-EGFP virus into the BLA of naive
rats. We observed that, after CNO was administered to activate the
virus, the mechanical pain threshold of rats was significantly reduced
and returned to baseline by the 6th hour, indicating that activation of
BLA astrocytes is necessary and sufficient to induce neuropathic
pain-like behavior. These data vigorously illustrated the function of
BLA astrocytes in the pathogenesis of DNP. In addition, our previous
studies showed that MCx astrocytes also play a role in regulating DNP
(Lu et al., 2021), suggesting that cerebral astrocytes are expected to
be a novel therapeutic target for DNP.
The link between chronic neuropathic pain and negative moods (anxiety,
depression and fear) has proven to be increasingly significant since the
link is bidirectional, and both act as risk factors for each other
(Baliki and Apkarian, 2015). In this study, we observed an obvious
anxiety-like behavior in DNP rats, which was specifically manifested as
reduced exploration intention and activity ability. Previous studies
have shown that chronic pain in rats does not reduce total distance
traveled in open field (De Gregorio et al., 2019). However, we found
that compared to the control group, the total distance of activity in
the open field was significantly reduced in DNP rats, which may be due
to chronic pain following hind foot pain and a reluctance to exercise in
the open area, thereby reducing the harmful stimulation of the post
foot. In addition, we found that the DREADD specific inhibition of the
activation of BLA astrocytes in DNP rats could effectively alleviate the
anxiety-like behavior of DNP rats, which was similar to Xiao et al ’s
findings by using optogenetic technology (Xiao et al., 2020).
Interestingly, we found that the transient activation of BLA astrocytes
by DREADD did not significantly affect the behavior of rats in the open
field and elevated cross maze, which may be due to a single injection of
CNO activation of BLA astrocytes in naive rats caused by transient pain
is not enough to make rats produce negative emotions in the short term.
Increased attention has recently focused on the examination of the
analgesic potential of phytoconstituents, and finding active monomers
from medicinal plants plays an important role in the development of new
drugs (Almeida et al., 2001). Gelsemium, a perennial evergreen
entangling vine plant from the family Loganiaceae, contains KM, the most
dominant alkaloid in G. elegans Benth, alters variety of biological
functions and has great value in the context of new drug development
(Zhang and Wang, 2015). Previous studies have shown that KM has marked
antinociception in inflammatory and neuropathic pain without inducing
antinociceptive tolerance (Xu et al., 2012; Xiong et al., 2017). In
addition, studies have shown that KM has anti-anxiety properties without
inducing adverse neurological effects (Liu et al., 2013; Chen et al.,
2017). In line with these findings, we discovered that KM can
significantly alleviate DNP and effectively mitigate anxiety-like
behavior in DNP rats, suggesting that KM is expected to become a
candidate new drug for the treatment of DNP.
DREADDs are chemogenetic tools widely used to remotely control cellular
signaling, neuronal activity, and behavior and have emerged as powerful
tools with great potential for drug discovery and development (Lee et
al., 2014). In this study, we combined DREADD approach with in vivo
pharmacology to parse the role of BLA astrocyte in chronic pain,
demonstrating that BLA astrocytes are an important target for KM in
exerting anti-DNP effects. DREADD were used to continuously activate BLA
astrocytes in naive rats to mimic animals in a chronic neuropathic pain
model; these rats were then treated with KM to observe the antagonistic
effect of KM on the DREADD-induced effects. Similar to the report by Sun
et al. (Sun et al., 2020), our data showed that the phenotype of chronic
neuropathic pain animals can be simulated by activation of BLA
astrocytes in naive animals by CNO for 7 consecutive days, which was
specifically manifested in the continuous mechanical pain sensitivity.
Unexpectedly, we found that repeated administration of CNO to
continuously activate BLA astrocytes also induced anxiety-like behaviors
in naive rats, in contrast to the results of short-term activation of
BLA astrocytes with a single injection of CNO. The simultaneous
administration of KM, however, gradually reversed the chronic
neuropathic pain and improved anxiety-like behavior induced by
continuous activation of BLA astrocytes. By combining the chemogenetic
approach with in vivo pharmacological manipulations, it was further
confirmed that BLA astrocytes may be a potential target for DNP
treatment.
Astroglial activation is associated with increases in proinflammatory
cytokines (Sommer et al., 2018). An increasing number of studies have
shown that the proinflammatory cytokines released by activated
astrocytes play a crucial regulatory role in neuropathic pain
sensitization (Linnerbauer et al., 2020). Many experiments have
confirmed that the proinflammatory cytokines TNF-α and IL-1β are
involved in the regulation of neuropathic pain (Hung et al., 2017). In
addition, in chronic pain conditions, activated astrocytes express a
large number of chemokines including MCP-1 and CXCL1, which act on CCR2
and CXCR2 in spinal cord neurons (Zhang et al., 2017). Activated CCR2
and CXCR2 induce the activation of extracellular signal-regulated
kinases, which rapidly phosphorylate NMDA receptors, increase excitatory
synaptic transmission, and promote central sensitization after nerve
injury (Moraes et al., 2020). We observed that the STZ-induced DNP model
rats had sustained mechanical hyperalgesia and massive activation of
astrocytes in the BLA, accompanied by TNF-α, IL-1β and chemokines CXCL1
and MCP-1 in the BLA. Importantly, we also confirmed that KM can reduce
the expression of TNF-α, IL-1β, CXCL1 and MCP-1, which is consistent
with previous findings. In general, these results demonstrate that BLA
astrocytes not only are transmission intermediaries for neuropathic pain
but also actively participate in the production of neuropathic pain, and
KM can improve the mechanical hyperalgesia and anxiety-like behaviors of
DNP rats by inhibiting the activation of BLA astrocytes.
In conclusion, our current findings highlight for the first time that
activation of BLA astrocytes plays an important role in the pathogenesis
of DNP, which may be a target for the treatment of DNP. The anti-DNP
effect of KM may be related to inhibiting the activation of BLA
astrocytes, and reveal KM as a potential candidate for treating pain and
pain-related anxiety-like behaviors.