In this study, we have shown for the first time that repetitive epidural administration of the local anesthetic ropivacaine suppressed both activated microglia and astrocytes concurrently with alleviation of thermal hyperalgesia in a rat model of neuropathic pain. Furthermore, upregulated NGF in the injured DRG was involved in the suppression of activated microglia, but not astrocytes, and may contribute to the prolonged analgesic effect of ropivacaine.
In recent years, microglia have been increasingly receiving much attention due to their potential as therapeutic targets for intractable pain. Many compounds that modify microglial function successfully alleviate neuropathic pain [15, 16, 18]. Consistently, the present study showed that ropivacaine suppressed activated microglia and relieved neuropathic pain via the upregulation of NGF expression in the DRG, suggesting NGF-dependent microglial inhibition by ropivacaine. Thus, NGF upregulated in the DRG by ropivacaine may act on microglia through transport along the DRG axons and be released into the spinal cord. Under some conditions, NGF receptors, TrkA and p75 neurotrophin receptors, are reported to be expressed in microglia and to be involved in their function [34–37]. Alternatively, NGF may diminish the injury-induced expression of activators for microglia in DRG neurons. It is well known that NGF is a key neurotrophic factor for maintaining the function of a subpopulation of DRG neurons  and restores damaged functions of the primary afferents in a wide range of disorders [22, 23, 39–41]. Upon nerve injury, DRG neurons begin to release CCL2 in the dorsal spinal cord, leading to microglial activation [11, 42, 43]. On the contrary, interleukin (IL)-10, an anti-inflammatory cytokine, is also increased after nerve injury . IL-10 suppresses the p38 MAPK activation and tumor necrosis factor-α expression in microglia activated by lipopolysaccharide . Therefore, NGF may modulate the expression of injury-induced cytokines in the DRG neurons, and thereby indirectly prevent the activation of microglia.
The ropivacaine-induced analgesia was abolished by NGF blockade, whereas NGF by itself, even at quite a high dose, could not relieve neuropathic pain. While it is well established that NGF has a hyperalgesic action in the periphery , the roles of NGF in nociceptive modulation in the spinal cord are still controversial. It was reported that intrathecal administration of NGF induces thermal hyperalgesia in intact rats . On the contrary, other reports have indicated that NGF may be rather beneficial for the treatment of neuropathic pain when administered intrathecally [20, 21, 26]. Although the reason for the discrepancy in the results is not yet understood, the present study implies that ropivacaine could be a feasible lead compound for local upregulation of NGF in the injured DRG. Although we cannot speculate at present that all local anesthetics have an NGF-upregulation effect similar to ropivacaine, further studies using other drugs seem to be warranted because other local anesthetics, including butamben , bupivacaine [3, 48] and lidocaine [4, 5, 7], have similar long-term analgesic effects to ropivacaine. In the present study, ropivacaine dose-dependently reduced the pain-related behavior. At all concentrations of ropivacaine examined, the rats showed transient motor paralysis after administration of ropivacaine. This dose-dependency could be interpreted as an analgesic effect of ropivacaine that is related to voltage-gated sodium channel blockade. However, the experiment examining the prolonged effect of ropivacaine showed that the analgesic effect of 0.2% ropivacaine continued for at least 2 days after the recovery from the hyperalgesia that was established at day 10 by the repeated daily injections. Therefore, the prolonged effect of ropivacaine in the present study seems to be beyond the transient voltage-gated sodium channel blockade. In addition to voltage-gated sodium channel blockade, local anesthetics are known to affect many different molecules including G protein-coupled receptors and immune cells . Therefore, molecular targets other than voltage-gated sodium channels may contribute to the NGF upregulation and prolonged analgesic effect, although the channel blockade may initiate these processes.
Astrocytes have also been shown to play an important role in neuropathic pain [11, 13], although much less is known about the molecular basis. Upon activation, astrocytes increase their synthesis of inflammatory factors similar to microglia [11, 12], and compounds that inhibit activated astrocytes have been shown to attenuate neuropathic pain [12, 17]. Consistent with these findings, activated astrocytes were also inhibited by ropivacaine treatment. However, the ropivacaine-induced suppression of activated astrocytes was not prevented by blockade of NGF action. Therefore, the inhibitory effect of ropivacaine on astrocyte activation seems to be NGF-independent, in clear contrast to the effect on microglia. It still remains to be elucidated whether the inhibition of astrocyte activation is required for the analgesic effect of ropivacaine.