A local anesthetic, ropivacaine, suppresses activated microglia via a nerve growth factor-dependent mechanism and astrocytes via a nerve growth factor-independent mechanism in neuropathic pain

Neuropathic pain is an extremely severe chronic pain caused by damage to the nervous system itself. In clinical practice, this pain syndrome remains a major issue because of the limited and variable effectiveness of existing analgesics [1]. Such poor effectiveness can be partly attributed to insufficient understanding of the analgesic mechanisms of existing drugs such as opioids, anticonvulsants and antidepressants. Systemic and epidural applications of local anesthetics relieve neuropathic pain in some cases, but the underlying mechanisms remain unclear because the effects occur at lower doses than the effective doses for blocking voltage-gated sodium channels, the primary targets of local anesthetics [2]. Furthermore, the analgesic effects of drugs persist for longer durations than those predicted on the basis of their pharmacokinetics [3‐5]. It has been reported that local anesthetics affect other ion channels and G protein-coupled receptors [2], and these effects are suggested to partially contribute to the analgesia observed with local anesthetics [2, 6]. Local anesthetics also inhibit the phosphorylation of p38 mitogen-activated protein kinase in the spinal microglia in animal models of neuropathic pain [7, 8]. However, the glial participation in the analgesic effects of local anesthetics remains speculative.

Microglia and astrocytes are the dominant glial cells in the central nervous system and play critical roles in neuroinflammation and neuronal plasticity via active communication with neurons [9, 10]. In response to peripheral nerve injury, these glial cells become activated and release a variety of proinflammatory mediators such as cytokines and chemokines in the dorsal spinal cord [11‐14]. These proinflammatory mediators act on nociceptive neurons, resulting in augmentation of nociceptive signal transmission or central sensitization. In line with these findings, it has been reported that intrathecal administration of compounds that suppress the activation of glial cells or antagonists of proinflammatory mediators alleviates neuropathic pain [11‐13, 15‐18]. Therefore, modulating glial cell function appears to represent a promising therapeutic strategy for neuropathic pain.

Nerve growth factor (NGF) is a founding member of the neurotrophic factor family and is well known to be involved in nociceptor function [19]. In the periphery, NGF is released in response to inflammation and subsequently acts on its high-affinity receptor, TrkA, expressed on a subset of nociceptive dorsal root ganglion (DRG) neurons, resulting in hyperalgesia [19]. On the other hand, NGF has a beneficial impact on neuropathic pain when administered intrathecally [20, 21]. NGF promotes functional regeneration of damaged DRG neurons [22] and improves neuropathy in streptozotocin-induced diabetic rats [23]. NGF ameliorates the increased expressions of c-jun and ATF3 in DRG neurons caused by nerve injury, suggesting its involvement in the protection of neurons [24, 25]. It has also been reported that NGF suppresses activated astrocytes in association with pain relief [26]. Thus, the roles of NGF in neuropathy are considerably complicated.

Ropivacaine was developed as an alternative to bupivacaine, which has more severe toxicity, and at present it is widely used as an epidural anesthetic at concentrations of 0.2-1% in clinical practice [27]. Ropivacaine shows increased cardiovascular safety and a shorter elimination half-life compared with bupivacaine [27‐29]. Recently, we reported that the content of NGF was upregulated in the injured DRG after repetitive epidural administration of 0.2% ropivacaine [30]. This finding implies that NGF produced endogenously upon ropivacaine treatment plays a role in the process of pain reduction. Therefore, in the present study, we further investigated the involvement of NGF in the analgesic effect of ropivacaine in a rat model of neuropathic pain with a focus on the spinal glial cells.

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 [38] 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 [44]. IL-10 suppresses the p38 MAPK activation and tumor necrosis factor-α expression in microglia activated by lipopolysaccharide [45]. 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 [19], 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 [46]. 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 [47], 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 [2]. 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.

The present study has shown that ropivacaine suppressed activated microglia and astrocytes in the spinal dorsal horn in a neuropathic pain state, concomitantly with the alleviation of pain. The suppression of activated microglia and the analgesic effect of ropivacaine were mediated by NGF signaling. These results suggest that epidural local anesthetics, including ropivacaine, may represent a new approach for glial cell inhibition and, therefore, therapeutic strategies for neuropathic pain.