In the present study, we showed that: 1. SNL induced persistent mechanical allodynia with NMDAR phosphorylation and astrocytic activation in spinal dorsal horn. 2. Intrathecal application of NMDAR antagonist ketamine alleviated mechanical allodynia with decreased NMDAR phosphorylation in a quick but short response, whereas astrocytic cytotoxin LAA relieved mechanical allodynia with attenuated astrocytic activation in a late but persistent manner. 3. Combining ketamine with LAA suppressed neuropathic pain in a quick and stable way, whereas, NMDAR phosphorylation and astrocytic activation were both much more suppressed than those of either single drug administration. Taken together, combination of NMDAR antagonist and astrocytic inhibitor exhibited some additive and complementary analgesic effects on SNL-induced neuropathic pain.
Effects of ketamine on neuropathic pain
Clinical study shows that intrathecal ketamine is useful for treating neuropathic pain [
27] at a sub-anesthetic dosage due to the sedation and other side effects when it is applied at high dose [
7]. In the present study, intrathecal ketamine attenuates SNL-induced mechanical allodynia in a dose dependant manner. Spinal NMDAR plays an important role in the development of central sensitization and neuropathic pain via the induction of long-term potentiation (LTP) in dorsal horn nociceptive synaptic transmission. As a functional subunit of NMDAR, NR1 phosphorylation is significantly increased in the ipsilateral spinal cord after nerve injury and coincides with mechanical allodynia [
28]. Previous study suggests that ketamine combined with methamphetamine could down-regulate NR1 receptor phosphorylation in rats (phosphorylation site: serine 897) [
29]. Moreover, it is reported that NR1 subunit knockout mice are more resistant to ketamine than control wide type mice, indicating that NMDAR NR1 subunit contributes to mediation of ketamine anesthesia or analgesia [
30]. The detailed mechanisms underlying ketamine-induced dephosphorylation of NR1 are still unclear. Ketamine could block Ca
2+ influx through the NMDA receptor, and then the lowered intracellular Ca
2+ decreases the activity of protein kinase C (PKC) and some other intracellular signals [
31,
32].Inhibition of PKC activity could block capsicin-induced phosphorylation of NR1 [
33]. Additionally, PKA activation mediates NR1 phosphorylation in SNL-induced neuropathic pain [
28]. Therefore, it is possible that ketamine blocks NMDA receptor and inhibits intracellular PKA, PKC or signals activity, and then decreases NR1 phosphorylation.
In the present study, intrathecal ketamine alleviated mechanical allodynia mainly via suppressing the function of NR1. Therefore, blocking NMDA receptor may be the primary anti-allodynic mechanism of ketamine observed in the present study.
Effects of the astrocytic specific inhibitor on neuropathic pain
Spinal microglia and astrocytes are both important for the development of neuropathic pain. In SNL-induced neuropathic pain, studies showed that activation of ERK and p38 only presented in spinal microglia at the initiation of neuropathic pain [
23,
34]. Zhuang
et al
[
23] also observed that there was a sequential activation of neurons, microglia and astrocytes following SNL. These results support a role for microglia in the early establishment of SNL-induced neuropathic pain and predict the role for astrocytes in the maintenance of neuropathic pain. Therefore, astrocytes are one of the important cell types for maintenance of SNL-induced neuropathic pain, which is very important for the chronic process of neuropathic pain. Cumulating evidence has shown a critical role for activated astrocytes in nerve injury-induced neuropathic pain [
35]. Ultrastructural evidence suggests that cell degeneration and death are confined to astrocytes after the injection of LAA into the striatum [
36]. Consistent with a previous study [
21], we demonstrated that LAA relieved late phase mechanical allodynia in a dose dependent manner. In the present experiment, we injected LAA and observed that SNL-induced mechanical allodynia was suppressed and GFAP expression was down regulated. These results indicated that inhibition of astrocytic activation may be a useful way to conquer neuropathic pain, which may be a complementary option to modern neuronal based therapeutics.
Drugs combination
The clinical concept of balanced or associative manner proposes to use a combination of analgesics and other treatments to provide better pain relief and minimized side effects [
8‐
10]. However, all of them only concerned about the neuronal participation in the neuropathic pain states. Spinal astrocytes play important roles in the development of SNL-induced neuropathic pain. Therefore, combining ketamine with the astrocytic inhibitor may provide a potential strategy for treating neuropathic pain.
In the present study, we observed that combination of ketamine and LAA showed additive and complementary effects of anti-allodynia. The analgesic effect of coadministration appeared earlier than that of intrathecal ketamine alone. Moreover, the peak effect of coadministration was much stronger than that of individual ketamine or LAA injection. Furthermore, the combination of drug administration exerted more powerful inhibition on NR1 phosphorylation and astrocytic activation. Our results suggest that combination of NMDAR antagonist ketamine and astrocytic cytotoxin LAA is an effective way to relieve SNL-induced mechanical allodynia. Then we further search for the underlying mechanisms of this effect.
Intrathecal ketamine enhanced the analgesic effect of LAA on tactile allodynia and potentiated the inhibitory effect of LAA on astrocytic activation. As observed in our previous study, inhibiting astrocytic activation is probably a novel analgesic mechanism of ketamine [
37]. Some opioid receptors (μ, κ and δ subtypes) and toll like receptors (TLRs) are expressed on astrocytes. It is shown that ketamine can function on opioid receptors [
38,
39] and TLRs [
40‐
42] and thus inhibiting astrocytic activation. Besides, nitric oxide (NO) released following NMDAR activation could induce astrocytic activation [
43]. Consequently, blocking NMDAR-NO pathway probably prevents astrocytic activation. All of these evidence supported our results that intrathecal ketamine facilitated the effects of astrocytic inhibitor LAA both on anti-allodynia and GFAP down-regulation.
On the other hand, our study observed that astrocytic toxin LAA could facilitate the effects of ketamine on neuropathic pain relief and reduction of pNR1 level. Studies indicated that some "gliotransmitters", proinflammatory cytokine and chemokine released from activated astrocytes could facilitate neuronal activity [
20,
44‐
46]. Therefore, blocking astrocytic activation might dephosphorylate NR1. Recent studies show that glutamate exocytose from astrocytes enhanced synaptic strength at excitatory synapses [
45,
47]. This effect is mainly mediated by neuronal NMDAR. Therefore, inhibiting astrocytic activation (e.g. with LAA in our study) could down-regulate activation of NMDAR via preventing glutamate release from astrocytes. In addition, emerging literature implicates a role for glia-cytokines-neurons in persistent pain [
12]. Nerve injury induces astrocytic activation and release of some proinflammatory cytokines. It is confirmed that pain-induced up-regulation of IL-1β is selectively localized to astrocytes, while IL-1 receptor (IL-1R) is exclusively expressed on neurons [
12,
48]. Furthermore, it is showed that IL-1R and NR1 are co-localized in spinal neurons [
48]. Applying IL-1R antagonist and glial inhibitor, attenuates NMDAR phosphorylation [
48]. In vitro application of IL-1β induces NR1 phosphorylation, which is blocked by an IL-1R antagonist [
12]. These results further provide evidence for our study that LAA could decrease pNR1, and thus enhances the role of ketamine.
LAA is a kind of toxin and cannot be used as a clinical drug although it can produce significant analgesic effects. However, in the present study, LAA is used to confirm the active participation of astrocytes in neuropathic pain. More importantly, we want to provide a potential strategy on the treatment of neuropathic pain. It is expected that in the future, some analgesic treatments targeting astrocytes with limited side effects will be developed.
In conclusion, the results of the present study show that ketamine and LAA individually present anti-allodynic effects in neuropathic pain, whereas their coadministration exhibits additive and complementary effects on mechanical allodynia. This combination of these two drugs provided a quick, stable and enhanced effect, with less side effects of ketamine in current treatment. Although LAA is now just used in experimental study but not clinical research or therapy, our results provide a new potential strategy for treating clinical neuropathic pain.