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.