Patients with diabetic-induced neuropathy experience a variety of abnormal sensations, including spontaneous pain, hyperalgesia, and hypersensitivity to innocuous stimuli (i.e., tactile allodynia). Such paresthesia is frequently resistant to treatment with antidepressants or local anesthetics.1 Local anesthetics inhibit nerve conduction by blocking voltage-gated Na+ channels and are used clinically to relieve neuropathic pain.2,3 However, the effect is transient because the concomitant production of inflammatory mediators continues to activate nociceptive neurons.4 Recent reports suggest that local anesthetics exert beneficial effects on inflammation and wound healing.5 In addition, the local anesthetic lidocaine has a protective role against tissue dysfunction and mortality in the endotoxin shock model6,7 and down-regulates lipopolysaccharide-induced production of proinflammatory molecules such as inducible nitric oxide synthase (iNOS) and monocyte chemotactic protein-1 (MCP-1) in macrophage cell lines.8–10
Accumulating evidence indicates that microglial cells, resident immune cells originally derived from myeloid cells and recruited into the spinal cord, have a critical role in the development of mechanical allodynia after peripheral nerve damage.11 Indeed, the microglial inhibitor minocycline attenuates the development of neuropathic pain but not existing hypersensitivity in a rat model of neuropathy; thus, microglial inhibition in the early phase of neuropathic pain development might be critical to prevent the exacerbation of tactile allodynia.12 Gu et al.13 demonstrated that intrathecal administration of lidocaine reversed tactile allodynia by inhibiting p38 phosphorylation in microglial cells in a chronic constriction injury model. In addition, lidocaine attenuated adenosine triphosphate–induced cytokine production in microglial cells in vitro, suggesting a mechanism for its analgesic effects.14
It has been suggested that the inhibiting microglial activation may attenuate tactile allodynia in streptozotocin (STZ)-induced diabetic neuropathy.15,16 Although lidocaine has been shown to exert antiinflammatory actions, the mechanism by which lidocaine attenuates tactile allodynia has yet to be elucidated in the STZ-induced diabetic model. Therefore, in the present study, we evaluated the effects of lidocaine, administered by continuous intraperitoneal infusion early in the course of diabetic neuropathy, on tactile allodynia in STZ-injected mice. We also assessed microglial accumulation by ionized calcium binding adaptor molecule-1 (Iba-1) immunostaining and p38 activation in the dorsal horn, gene induction of proinflammatory mediators in vivo, and chemotactic response in interferon (IFN)-γ–stimulated primary microglial cells in vitro. Our findings suggest that systemic administration of lidocaine in the early phase of neuropathic pain development produces long-lasting analgesic effects, possibly by inhibiting p38 phosphorylation in spinal microglial cells in diabetes-induced neuropathy.
Male, 8- to 10-week-old, C57BL6 mice were obtained from Charles River (Yokohama, Japan). The procedures used in this study were approved by the Animal Research Committee of Juntendo University.
Diabetes-Induced Neuropathic Pain Model
STZ (150 mg/kg; Nacalai, Kyoto, Japan) was dissolved in 0.05 M citrate buffer (pH 4.5) and intraperitoneally injected into mice. Control mice were injected with the citrate buffer only. Mice with high blood glucose levels (>300 mg/dL) at 7 days after STZ injection were used in experiments. To evaluate tactile allodynia, calibrated von Frey filaments (0.08–2.0 g) were applied to the plantar surface of the hindpaw from beneath the mesh floor.15 The 50% paw withdrawal threshold was determined using the up-down method.17,18
Administration of Lidocaine After STZ Injection
Fourteen days after STZ injection, mice were anesthetized with 2% isoflurane, and an Alzet osmotic pump (DURECT, Cupertino, CA) containing saline (vehicle) or lidocaine (2% or 10%) (Sigma-Aldrich, St. Louis, MO) was transplanted into the peritoneum. Continuous intraperitoneal infusion was performed from day 14 to day 21 at a rate of 0.5 μL/h. For mice receiving minocycline, 40 mg/kg minocycline was injected intraperitoneally once a day from day 14 to day 21.
Mice were deeply anesthetized with sodium pentobarbital (100 mg/kg intraperitoneally) and perfused transcardially with saline. The L3-6 segments of the spinal cord were removed, fixed in 4% paraformaldehyde overnight at 4°C, and placed in a 30% sucrose solution for 24 hours at 4°C. Transverse spinal cord sections (30 μm thick) were incubated overnight with rabbit monoclonal antiphospho-p38 antibody (1:100; Cell Signaling Technology, Danvers, MA) at 4°C, and then incubated with Alexa Fluor 546-labeled antirabbit immunoglobulin G (1:500; Invitrogen, Carlsbad, CA). After blocking with nonimmune rabbit immunoglobulin G, the sections were incubated with rabbit polyclonal anti–Iba-1 antibody (Wako, Osaka, Japan), which was prelabeled with Alexa Fluor 488 according to the manufacturer's instructions (Invitrogen). Fluorescent images were obtained using the LSM510 imaging system (Carl Zeiss, Aalen, Germany). The number of Iba-1+ cells was counted using KS-400 imaging software (Carl Zeiss) as described previously.15
Quantitative Polymerase Chain Reaction
Total RNA was extracted using Sepazol reagent (Nacalai). The synthesis of first strand cDNA was performed using the High Capacity RNA-to-cDNA (Applied Biosystems, Carlsbad, CA) according to manufacturer's instructions. Quantitative polymerase chain reaction (PCR) was performed with the ABI Prism 7200 Sequence Detection System using SYBR Green PCR Master Mix (Applied Biosystems), and target gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase. Primers used for quantitative PCR were chemokine (C-C motif) receptor 2 (CCR2): forward: 5′-CTCAGTTCATCCACGGCATAC-3′; reverse: 5′-GACAAGGCTCACCATCATCG-3′; glyceraldehyde 3-phosphate dehydrogenase: forward: 5′-TGAAGCAGGCATCTGAGGG-3′; reverse: 5′-CGAAGGTGGAAGAGTGGGAG-3′; iNOS: forward: 5′-CCAAGCCCTCACCTACTTCC-3′; reverse: 5′-CTCTGAGGGCTGACACAAGG-3′; interleukin (IL)-1β: forward: 5′-GCTTCAGGCAGGCAGTATC-3′; reverse: 5′-AGGATGGGCTCTTCTTCAAAG-3′.
Isolation of Primary Microglial Cells
Microglial cells were prepared from the spinal cord of newborn C57BL/6 mice (postnatal days 1–3) as described previously.19 Briefly, cells were kept at 37°C in a 5% CO2 and the cell culture medium was changed every 3 to 4 days. After 14 days, microglial cells were separated from the underlying astrocyte monolayer by gentle agitation using their differential adhesive properties. Microglial cells were then cultured in Dulbecco's modified Eagle medium/ nutrient mixture F-12 with 10% fetal bovine serum. Cultures routinely consisted of approximately 98% microglial cells as determined by staining with Iba-1 (Wako).
Primary microglial cells were cultured for 48 hours with different concentrations of lidocaine in the presence or absence of 10 ng/mL IFN-γ (PeproTech, Rocky Hill, NJ). Cells were then incubated with 5 mg/mL MTT (Sigma) dissolved in phosphate-buffered saline for 2 hours at 37°C. The cell culture medium was removed and 30 μL dimethyl sulfoxide was added to each well. The absorbance (570 nm) of aliquots taken from each well was determined using an enzyme-linked immunosorbent assay plate reader.
Cells were incubated with 10 ng/mL IFN-γ and/or 5 μM lidocaine for 24 hours before chemotaxis was evaluated; lidocaine was added 30 minutes before stimulation by IFN-γ. The chemotaxis assay was performed in a 24-well chemotaxis plate with 8-μm pore inserts (BD Biosciences, San Jose, CA) as described previously.20 Briefly, 10 ng/mL MCP-1 (PeproTech) was diluted in Dulbecco's modified Eagle medium with 0.1% bovine serum albumin and placed in the bottom wells. Cell suspension (2 × 106 cells) was placed on the top wells of the chamber. After a 2-hour incubation at 37°C with 5% CO2, cells in the bottom wells were counted at 20× magnification by light microscopy.
Values are presented as mean ± SEM. Differences among groups were analyzed using 1-way analysis of variance with Bonferroni analysis. P values <0.05 were considered to be statistically significant.
Lidocaine Ameliorates Tactile Allodynia in STZ-Induced Diabetic Mice
Mice injected with 150 mg/kg STZ exhibited a marked increase in blood glucose levels on day 7 (STZ-vehicle: 513 ± 110; STZ-2% lidocaine: 445 ± 110; STZ-10% lidocaine: 499 ± 105; and STZ-minocycline: 376 ± 36 mg/dL). Hyperglycemia persisted through the entire experimental period (Fig. 1A), but mean body weight on day 21 was similar to body weight at day 0 (Fig. 1B).
In all treatment groups, the paw withdrawal threshold decreased gradually from day 7, reaching a minimum approximately on day 14 (STZ-vehicle: 0.11 ± 0.09; STZ-2% lidocaine: 0.24 ± 0.36; STZ-10% lidocaine: 0.13 ± 0.17; and STZ-minocycline: 0.22 ± 0.40 g) (Fig. 2). However, the withdrawal threshold was significantly increased in mice that received 2% or 10% lidocaine by continuous infusion, reaching a maximum level 7 days after lidocaine treatment was initiated (STZ-vehicle: 0.047 ± 0.045; STZ-2% lidocaine: 0.88 ± 0.64; STZ-10% lidocaine: 1.39 ± 0.46; and STZ-minocycline: 1.13 ± 0.41 g; P < 0.001). The analgesic effects lasted until day 28 in mice that received 10% lidocaine.
The analgesic effect of 10% lidocaine was comparable to that of the microglial inhibitor minocycline, injected intraperitoneally daily from day 14 to day 21. Mice receiving minocycline treatment exhibited similar changes in blood glucose levels and body weight to other groups (Figs. 1 and 2). In contrast, lidocaine infused from day 41 to day 47 after STZ injection did not attenuate tactile allodynia up to day 61 (data not shown).
Lidocaine Inhibits Microglial Infiltration and p38 Activation in the Dorsal Horn of Diabetic Mice
To demonstrate the effects of lidocaine on microglial activation in the dorsal horn, we immunostained L3-6 spinal cord sections taken on day 21 after the STZ injection from mice treated with a 7-day continuous infusion of vehicle or lidocaine. Compared with control mice, vehicle-treated diabetic mice showed a 1.9-fold increase in the number of Iba-1–positive cells in the dorsal horn, but this increase was inhibited by lidocaine (control: 270 ± 106; STZ-vehicle: 513 ± 75; STZ-10% lidocaine: 289 ± 61 cells/mm2; P < 0.001) (Fig. 3, A and B). However, morphological changes or increase in Iba-1 immunoreactivity in microglial cells were not observed. Increased phosphorylation of mitogen-activated protein kinase family member p38 in Iba-1+ microglial cells was observed 21 days after STZ injection in STZ-vehicle–treated mice, but 10% lidocaine inhibited p38 phosphorylation in Iba-1+ microglial cells (Fig. 3C). The level of CCR2, the receptor for MCP-1, also increased 3.3-fold in STZ-vehicle–treated mice, but this up-regulation was significantly attenuated by 10% lidocaine (control: 1.0 ± 0.10; STZ-vehicle: 3.15 ± 0.22; STZ-10% lidocaine: 2.17 ± 0.0.27; … . ”--P = 0.033) (Fig. 3D).
Lidocaine Inhibits IFN-γ–Induced Activation of Primary Microglial Cells In Vitro
Next, we evaluated whether lidocaine directly affects monocyte lineage cells with in vitro experiments using primary microglial cells isolated from mice. It has been demonstrated that IFN-γ signaling is a key component in the molecular machinery through which resting microglial cells transform to the activated state in neuropathic pain.21,22 We first assessed the effect of lidocaine on cell viability with the MTT assay. Although high concentrations of lidocaine induce apoptosis by activating caspase-3 through the mitochondrial pathway in a lymphoid cell line,23,24 cell viability was not affected by 0.5 to 50 μM lidocaine in our study (Fig. 4A). Next, we investigated whether lidocaine has an effect on microglial chemotactic response to MCP-1. The IFN-γ–induced increase in migration was inhibited by pretreatment with 5 μM lidocaine (control: 67 ± 4; IFN-γ: 111 ± 7; lidocaine: 65 ± 2; IFN-γ + lidocaine: 81 ± 3 cells; P < 0.001) (Fig. 4B). Activated microglial cells produce proinflammatory mediators such as iNOS and IL-1β, which promote the infiltration of immune cells in the dorsal horn.25,26 Lidocaine has been reported to inhibit nitric oxide production in the activated murine macrophage cell line8 and IL-1β in microglial cells.14 Consistent with the previous reports, IFN-γ–induced gene induction of iNOS and IL-1β was inhibited by pretreatment with 5 μM lidocaine (Fig. 4C).
Hyperglycemia induced by STZ causes the conversion of dorsal horn microglial cells from resting to activated phenotype, which has a crucial role in the development of diabetes-induced tactile allodynia.15,16 In the present study, we demonstrated that the continuous infusion of lidocaine in the early phase but not late phase attenuates STZ-induced tactile allodynia, possibly by inhibiting p38 activation of spinal microglial cells.
Microarray expression profiles in neuropathic pain models revealed that the most highly up-regulated transcripts in the dorsal horn are immune related, and are expressed only by spinal microglial cells.27 Raghavendra et al.12 reported that inhibiting microglial activation with minocycline attenuated the development of tactile allodynia but did not alter existing mechanical allodynia. This finding is consistent with our result that continuous administration of lidocaine attenuated STZ-induced tactile allodynia only when delivered in the early phase; the time course was similar to that of repeated injections of minocycline (Fig. 2). These results suggest that lidocaine inhibits tactile allodynia primarily through a direct action on microglial cells.
Previously, it was reported that the intrathecal administration of lidocaine reverses the activation of p38 signaling in dorsal horn microglial cells in a chronic constriction injury model.13 In addition, lidocaine inhibits adenosine triphosphate–dependent induction of proinflammatory cytokines such as IL-1β through the p38 pathway.14 In primary microglial cells, the induction of iNOS and IL-1β by IFN-γ was down-regulated by lidocaine pretreatment, supporting the idea that lidocaine may attenuate cytokine production through the p38 pathway in spinal microglial cells in STZ-induced diabetes. Racz et al.21 reported that IFN-γ released by activated astrocytes and neurons promotes microglial activation by enhancing iNOS and CCR2 activity. In the present study, we demonstrated that lidocaine suppressed microglial accumulation in the dorsal horn of STZ-treated mice, and reduced the chemotactic response to MCP-1 in primary microglial cells. CCR2-deficient mice exhibit a reduced neuropathic pain response and fewer phospho-p38–positive microglial cells in the dorsal horn,28 indicating that signaling from MCP-1 to CCR2 and the activation of the downstream signaling molecule p38 in microglial cells may be critical for the development of tactile allodynia. Gao et al.29 reported that MCP-1 induces central sensitization by activating CCR2 expressed by neurons, indicating that MCP-1 may act both on microglial cells and neurons. However, lidocaine blocked neuropathic pain development, which showed a similar degree and time course of allodynia reversal to that of the microglial inhibitor minocycline (Fig. 2). Our data support the idea that lidocaine primarily alleviates STZ-induced tactile allodynia by inhibiting CCR2 signaling in microglial cells, followed by decreased microglial accumulation in the dorsal horn.
The dose of lidocaine delivered to mice by continuous infusion in the present study is nearly equivalent to the clinical dose of lidocaine administered by IV infusion or by the 5% lidocaine patch used in patients.3,6,30,31 Local injection of lidocaine produces only temporal effects possibly by blocking nerve transmission,32 and is often associated with neurotoxicity.33 However, the use of a 5% lidocaine patch relieves neuropathic symptoms without serious adverse effects.31 These previous reports suggest that continuous systemic administration of low-dose lidocaine may have long-lasting analgesic effects with negligible side effects.
In conclusion, we found that continuous infusion of lidocaine inhibited microglial activation in the dorsal horn of STZ-treated diabetic mice. Administration of lidocaine early in the course of diabetes-induced neuropathy may be an effective therapeutic approach to prevent the development of tactile allodynia.
Name: Naoko Suzuki, MD.
Contribution: This author performed experiments and wrote the manuscript.
Name: Maiko Hasegawa-Moriyama, MD, PhD.
Contribution: This author performed experiments, contributed to study, and wrote the manuscript.
Name: Yoshika Takahashi, MD.
Contribution: This author performed experiments.
Name: Yuji Kamikubo, PhD.
Contribution: This author contributed to study.
Name: Takashi Sakurai, MD, PhD.
Contribution: This author contributed to study and wrote the manuscript.
Name: Eiichi Inada, MD, PhD.
Contribution: This author contributed to study.
This manuscript was handled by: Quinn H. Hogan, MD.
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