Neuropathic pain can result from nerve damage caused by cancer, infection, autoimmune disease, and trauma.
Pain and loss of function are intimately associated with the response of the nervous system against neural damage. It has become clear that neuro-immune communication in the peripheral nervous system has an important role in the development of neuropathic pain. 1 Infiltration of immune cells in response to nerve damage leads to the subsequent production and secretion of proinflammatory mediators such as nitric oxide, chemokines, cytokines, and matrix metalloproteases (MMPs), which are involved in inflammation and tissue remodeling associated with nerve injury. 2 3 – 5
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of the nuclear hormone receptor superfamily; there are 3 isoforms (PPARα, PPARβ, and PPARγ).
In the pain pathway, PPARγ is expressed in the dorsal root ganglion, the dorsal horn of the spinal cord, adipocytes around the sciatic nerve (SCN), and macrophages. 6 7 – One study reported that intrathecal injection of the blood-brain barrier (BBB)-impermeant PPARγ agonist rosiglitazone produced a rapid antiallodynia effect through spinal PPARγ activation. 9 Similarly, oral administration of rosiglitazone attenuated hyperalgesia in tibial and sural nerve transection-induced neuropathy and decreased oxidative stress and calcium levels. 7 A BBB-permeant PPARγ agonist, pioglitazone, also attenuated tactile allodynia when oral administration was initiated immediately after partial sciatic nerve ligation (PSNL). 10 These reports suggest that PPARγ activation suppresses neuropathic pain development. However, primary PPARγ targets have not been identified. 8
Macrophages derived from circulating monocytes are activated primarily in the early phase of inflammation after peripheral nerve injury.
4 , PPARγ signaling has been implicated in tissue protection through the phenotypic switch of monocyte-lineage cells. 11 It has been demonstrated that PPARγ signaling promotes neuroprotection and neurological improvement after cerebral ischemia and spinal cord injury by modulating inflammatory gene expression in macrophage/microglia. 12 13 , Therefore, we evaluated whether rosiglitazone could produce analgesic effects in a PSNL model, focusing on determining the optimal treatment time and the role of PPARγ signaling in peripheral macrophages. 14 15 METHODS
Eight- to ten-week-old male C57BL6 mice were obtained from Charles River (Japan). The procedures used in this study were approved by the Animal Research Committee of Juntendo University.
Mice were deeply anesthetized with inhalation of 2% isoflurane (Abbott, Japan). The SCN of the lateral hindlimb was exposed and half of the nerve was tightly ligated with 8-0 silk suture thread as described previously.
On the contralateral side, sham operation was performed. Briefly, the SCN of the lateral hindlimb was exposed, but not subjected to ligation. Rosiglitazone (Cayman Chemical, Ann Arbor, MI) was dissolved in dimethyl sulfoxide (DMSO) and then diluted in phosphate-buffered saline (PBS; pH7.2) (1:3 solution). Rosiglitazone (0.5 mg/mL) was intraperitoneally injected into the mice with 3 mg/kg or 10 mg/kg. For local rosiglitazone treatment after PSNL, 5 μg (0.25 mg/mL, 20 μL) or 10 μg (0.5 mg/mL, 20 μL) rosiglitazone was injected at the ligation site. 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. 16 The 50% paw withdrawal threshold was determined using the up-down method. 17 18 , 19 Immunohistochemistry
Mice were deeply anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal injection) and perfused transcardially with saline. On the PSNL side, the SCN within 0.5 cm of the ligation site was removed. Tissues were fixed in 4% paraformaldehyde overnight at 4°C, and placed in 30% sucrose solution for 24 hours at 4°C. Sections (30 μm thick) were incubated overnight with primary antibody to F4/80 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C and then incubated for 1 hour at room temperature with the secondary antibody labeled with Alexa Fluor 488 (1:500; Invitrogen, Carlsbad, CA) followed by nuclear staining with 4′-6-diamidino-2-phenylindole (DAPI). Fluorescent images were obtained using LSM510 imaging systems (Carl Zeiss, Aalen, Germany). The number of F4/80
+ DAPI + cells with clearly visible cell bodies in the SCN was manually counted using KS-400 software (Carl Zeiss) as described previously. 20 , 21 Polymerase Chain Reaction Analysis
Total RNA of peritoneal macrophages and the SCN within 0.5 cm of the ligation site was extracted with Sepazol reagent (Nacalai, Japan). First strand cDNA was synthesized using the High Capacity RNA-to-cDNA (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions. Primers used for reverse transcription-polymerase chain reaction (RT-PCR) were as follows: glyceraldehyde-3-phosphate dehydrogenase (G3PDH): forward: 5′-CAGAGCAGCGTCCCTGTGAAT-3′, reverse: 5′-GGCTTACAGTGAACCACCTT-3′; chemokine (C-C motif) receptor 2 (CCR2): forward: 5′-GGTCATGATCCCTATGTGG-3′, reverse: 5′-CTGGGCACCTGATTTAAAGG-3′; inducible nitric oxide synthase (iNOS): forward: 5′-AGCCTGTGAGACCTTTGATG-3′, reverse: 5′-CACACAGTTTGGTGTGGTGT-3′; cyclooxygenase-2 (COX-2): forward: 5′-CACAACAGAGTGTGCGACAT-3′, reverse: 5′-GTGAGTCCATGTTCCAGGAG-3′; CD11b: forward: 5′-CAGATCAACAATGTGACCGTATGG-3′, reverse: 5′-CATCATGTCCTTGTACTGCCGC-3′; MMP-9: forward: 5′-GTCTTCCTGGGCAAGCAGTA-3′, reverse: 5′-GGCTTAGAGCCACGACCATAC-3′; PPARγ: forward: 5′-GGAAGACCACTCGCATTCCTT-3′, reverse: 5′-TCGCACTTTGGTATTCTTGGAG-3′.
Quantitative PCR was performed on an ABI Prism 7200 Sequence Detection System (Applied Biosystems) using SYBR Green Mix (Applied Biosystems) according to the manufacturer's instructions. Target gene expression was normalized to G3PDH. The primers used for quantitative PCR were CCR2: forward: 5′-CTCAGTTCATCCACGGCATAC-3′, reverse: 5′-GACAAGGCTCACCATCATCG-3′; G3PDH: forward: 5′-TGAAGCAGGCATCTGAGGG-3′, reverse: 5′-CGAAGGTGGAAGAGTGGGAG-3′.
Isolation of Peritoneal Macrophages
Mice were intraperitoneally injected with 3 mL of 3% thioglycollate (Sigma, St Louis, MO). After 3 days, peritoneal macrophages were collected by peritoneal lavage with 8 mL of cold PBS. Cells were incubated overnight in 24-well tissue culture plates with RPMI 1640 cell culture medium. Nonadherent cells were removed with PBS by repeated washing.
Cell Viability Assay
Peritoneal macrophages were incubated with 5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma) dissolved in PBS for 2 hours at 37°C. The culture medium was removed and 30 μL DMSO was added to each well. Aliquots were obtained from each well and absorbance at 570 nm was read with an enzyme-linked immunosorbent assay plate reader.
Transplantation of Peritoneal Macrophages into the PSNL Site
Peritoneal macrophages were incubated with 50 μM rosiglitazone for 72 hours and fluorescently labeled with PKH67 (Sigma) according to the manufacturer's instructions. Rosiglitazone-treated macrophages (1 × 10
5 cells in 10 μL PBS) were injected perineurally to the SCN immediately after PSNL. Chemotaxis Assay
The chemotaxis assay was performed in a 24-well chemotaxis plate with 8-μm pore inserts (BD Biosciences, San Jose, CA) as described previously.
Briefly, 10 ng/mL monocyte chemotactic protein (MCP)-1 (PeproTech) in Dulbecco's modified Eagle medium with 0.1% bovine serum albumin was placed in the bottom wells, and cell suspension (2 × 10 22 6 cells) was placed on the top wells of the chamber. After a 2-hour incubation at 37°C with 5% CO 2, the cells in the bottom wells were counted at 20× magnification by light microscopy. Statistical Analysis
Values are presented as mean ± SEM. Differences among groups were analyzed using 1-way analysis of variance with Bonferroni test.
P values <0.05 were considered to be significant. RESULTS
Systemic Administration of Rosiglitazone in the Early Phase of Neuropathic Pain Development Ameliorates Tactile Allodynia
To determine the optimal timing of rosiglitazone treatment after the PSNL model, mice received rosiglitazone by intraperitoneal injection once daily in the early phase of neuropathic pain development (postoperative days [PODs] 1–3) (
Fig. 1A), the late phase of neuropathic pain development (PODs 5–7) ( Fig. 1B), or after neuropathic pain was established (PODs 26–28) ( Fig. 1C). We found that rosiglitazone significantly reduced tactile allodynia when administered in the early phase (PODs 1–3) and late phase (PODs 5–7), but had little effect after neuropathic pain developed (PODs 26–28) ( Fig. 1C). Rosiglitazone had long-lasting analgesic effects (PODs 5–21) when injected in the early phase, which corresponds with the initiation of macrophage infiltration 4 , ( 11 Fig. 1A). Rosiglitazone injected during the late phase (PODs 5–7) produced analgesic effects on POD 8, but the withdrawal threshold was reduced to that of the vehicle control on POD 14 ( Fig. 1B). These data indicate that rosiglitazone attenuates neuropathic pain development triggered by peripheral macrophages at the inflamed site when administered soon after nerve damage occurs. Once macrophages are activated and recruited to the injured site, the effects of peripheral PPARγ signaling may be limited to transient analgesia. In addition, repeated administration of rosiglitazone began to produce analgesic effects on POD 5 when injected on PODs1 to 3 and on POD 8 when injected on PODs 5 to 7, indicating a delayed onset of action. Figure 1:
Systemic administration of rosiglitazone in the early postoperative period attenuated the development of tactile allodynia. Rosiglitazone was intraperitoneally injected once daily on postoperative days (PODs) 1 to 3 (A), PODs 5 to 7 (B), or PODs 26 to 28 (C) after partial sciatic nerve ligation (PSNL). *
P < 0.05. Each point represents the mean ≥ SEM ( n = 5–9). Rosiglitazone Inhibits Both Macrophage Infiltration and Induction of Proinflammatory Mediators
To determine whether macrophage infiltration around the PSNL site is decreased or delayed in rosiglitazone-treated mice, we analyzed tissues harvested from the PSNL site of mice treated with DMSO vehicle control or 10 mg/kg rosiglitazone on PODs 1 to 3 by immunostaining against the macrophage marker F4/80. High macrophage infiltration was observed at the PSNL site of control mice (PODs 1–7), whereas rosiglitazone-treated mice exhibited lower macrophage recruitment (
Fig. 2A). Few F4/80 + cells were observed on the PSNL side on POD 14 or on the sham-operated side throughout the experimental period ( Fig. 2B). Infiltration of F4/80 + colabeled with DAPI (F4/80 +DAPI +) on the PSNL site was significantly lower in rosiglitazone-treated mice (2410 ± 1000 cells/mm 2) than in control mice (5179 ± 1595 cells/mm 2) on POD 7 ( Fig. 2C). Macrophage infiltration to the PSNL site was also evaluated by analyzing the expression of CD11b, a monocyte-specific cell surface marker ( Fig. 3). RT-PCR results show that CD11b expression was higher on the PSNL side than the sham side, reaching peak mRNA levels on POD 3 in control mice. However, upregulation of CD11b was attenuated in the rosiglitazone-treated mice. Similarly, levels of the proinflammatory mediators MMP-9, iNOS, and COX-2 were markedly higher in control mice than in rosiglitazone-treated mice on PODs 1 to 7, indicating that rosiglitazone administered in the early phase inhibits both macrophage infiltration and induction of proinflammatory mediators. Figure 2:
Local infiltration of macrophages was decreased at the partial sciatic nerve ligation (PSNL) site of mice treated with 10 mg/kg rosiglitazone on postoperative days (PODs) 1 to 3. A, Time course of macrophage infiltration at the PSNL site. B, Absence of macrophages in the sham-operated site on POD 7. Green = F4/80; blue = 4′-6-diamidino-2-phenylindole (DAPI)-stained nuclei. Scale bar, 100 μm. C, Quantification of F4/80
+DAPI + cells on POD 7. * P < 0.05. Each column represents the mean ± SEM ( n = 6–7). Figure 3:
Rosiglitazone down-regulated gene induction of proinflammatory molecules underlying allodynia development. Gene induction of CD11b, matrix metalloprotease (MMP)-9, inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX)-2 after treatment with 10 mg/kg rosiglitazone on postoperative days (PODs) 1 to 3 as determined by reverse transcription-polymerase chain reaction. Four mice in each group were evaluated at each time point after partial sciatic nerve ligation (PSNL). Representative results are shown. G3PDH = glyceraldehyde-3-phosphate dehydrogenase; V = vehicle; R = 10 mg/kg rosiglitazone (rosi).
Local Injection of Rosiglitazone Was Sufficient to Attenuate Tactile Allodynia
A single intrathecal injection of rosiglitazone has been shown to rapidly attenuate hyperalgesia.
To determine whether the peripheral action of rosiglitazone in the early phase is sufficient to ameliorate tactile allodynia, rosiglitazone (5 or 10 μg) was locally injected to the ligation site immediately after PSNL (PODs 0–2). The direct injection of 10 μg rosiglitazone to the PSNL site significantly attenuated tactile allodynia up to POD 7 ( 7 Fig. 4). Figure 4:
Local injection of rosiglitazone attenuated tactile allodynia. Rosiglitazone (5 or 10 μg) was directly injected to the partial sciatic nerve ligation (PSNL) site once daily on postoperative days 0 to 2. Each point represents the mean ± SEM (
n = 5–6). Rosiglitazone Ameliorates Tactile Allodynia by a Macrophage-Mediated Mechanism
To determine whether the effects of rosiglitazone on macrophages are sufficient to ameliorate tactile allodynia, rosiglitazone-treated peritoneal macrophages prelabeled with PKH67 were transplanted to the PSNL site. We found that transplanted peritoneal macrophages remained localized at the PSNL site on POD 7 (
Fig. 5A), and their viability was not affected by incubation with rosiglitazone in vitro ( Fig. 5B). Transplantation of rosiglitazone-treated peritoneal macrophages significantly reduced tactile allodynia at the PSNL site on POD 3, whereas vehicle treatment resulted in the progression of tactile allodynia ( Fig. 5C). Figure 5:
Local transplantation of rosiglitazone-treated peritoneal macrophages (PM) to the partial sciatic nerve ligation (PSNL) site attenuated the development of tactile allodynia. Peritoneal macrophages pretreated with 50 μM rosiglitazone for 3 days ex vivo were transplanted into the ligation site. Engraftment of peritoneal macrophages prelabeled with PKH67 was assessed on postoperative day (POD) 7. A, Green = PKH67; blue = 4′-6-diamidino-2-phenylindole (DAPI)-stained nuclei; scale bar, 50 μm. B, Cell viability was evaluated by MTT assay (
n = 3 for each). C, Tactile allodynia was improved by transplantation of rosiglitazone-treated peritoneal macrophages to the PSNL site. * P < 0.05. Each point represents the mean ± SEM ( n = 6–7). Rosiglitazone Has Antiinflammatory Effects in Peritoneal Macrophages
Next, the direct effects of rosiglitazone on macrophages were evaluated in peritoneal macrophages isolated from mice. Cells were stimulated with 10 ng/mL interferon (IFN)-γ, which acts as a proinflammatory regulator of peripheral macrophages and induces tactile allodynia.
21 , Pretreatment of cells with 50 μM rosiglitazone markedly inhibited IFN-γ–induced gene induction of COX-2 and iNOS ( 23 Fig. 6A). The expression of C-C motif receptor, CCR2, was also down-regulated by rosiglitazone ( Fig. 6, A and B). Quantitative PCR revealed that the 3.3-fold increase in CCR2 expression in IFN-γ–stimulated cells was suppressed by rosiglitazone. The increased chemotactic activity to MCP-1 induced by IFN-γ was also dose-dependently attenuated by rosiglitazone ( Fig. 6C). Figure 6:
Rosiglitazone (ROSI) suppressed gene induction of proinflammatory mediators and chemotaxis after stimulation with interferon (IFN)-γ in peritoneal macrophages. A, Expression of cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), and chemokine (C-C motif) receptor 2 (CCR2), and peroxisome proliferator-activated receptor (PPAR)-γ was evaluated by reverse transcription-polymerase chain reaction (RT-PCR). B, CCR2 expression was evaluated by quantitative PCR. Cells were stimulated with 10 ng/mL IFN-γ with or without 50 μM rosiglitazone in A and B. C, The effect of rosiglitazone on chemotaxis to monocyte chemotactic protein-1 promoted by IFN-γ was evaluated. Experiments were performed in triplicate. *
P < 0.05. Each column represents the mean ± SEM ( n = 3 for each). G3PDH = glyceraldehyde-3-phosphate dehydrogenase. DISCUSSION
PPARγ ligands can ameliorate the inflammatory response by switching macrophage polarization to be tissue protective, suggesting a therapeutic potential of PPARγ agonists in the treatment of neuronal damage.
24 , It has been reported that the macrophage/microglia lineage is activated at the early phase corresponding to the onset of neuropathic pain, whereas activation of glial cells including spinal astrocytes contributes to the progression of neuropathic pain in the later phase. 25 Macrophage infiltration to the injured nerve reaches a peak 2 to 4 days after PSNL. 5 11 , We demonstrated that rosiglitazone-activated PPARγ signaling attenuated tactile allodynia throughout the experimental period when repeatedly administered in the early phase but not in the late phase of neuropathic pain development ( 26 Fig. 1), suggesting that the exacerbation of acute inflammation triggered by macrophages is the critical phase of pain development. Moreover, both local injection of rosiglitazone ( Fig. 4) and transplantation of rosiglitazone-treated peritoneal macrophages to the injured site ( Fig. 5) produced significant analgesic effects. These results suggest that the primary target of rosiglitazone might be local macrophages. However, we could not completely exclude the possibility that rosiglitazone acts on glial cells, fibroblasts, and/or adipocytes localized at the injured site or targets primary neurons directly.
Peripheral sensitization results from the accumulation of endogenous factors released from activated nociceptors or non-neuronal cells that reside within or infiltrate to the injured area.
Vasodilation induced by downstream products of iNOS or COX-2 is consistent with the invasion of circulating immune cells so that a dense cellular infiltrate composed primarily of macrophages forms at the lesion site. Activation of resident macrophages rushing to the lesion site and invasion of additional monocytes from the peripheral blood are orchestrated by chemokine signaling. Accordingly, mice lacking CCR2 show a marked attenuation of monocyte recruitment in response to various inflammatory stimuli in the central nervous system. 27 28 , Moreover, development of mechanical allodynia was completely abrogated in CCR2-deficient mice. 29 These reports may suggest that regulating macrophages in the initial phase could be crucial to controlling neuropathic pain. Reduced sensitization to mechanical stimuli in the rosiglitazone-treated mice and CCR2-deficient mice could be explained by fewer macrophages accumulating at the PSNL sites. In this study, rosiglitazone inhibited IFN-γ–induced gene expression of CCR2 in peritoneal macrophages as reported previously ( 11 Fig. 6B), and reduced the chemotactic response to MCP-1 ( 30 Fig. 6C). Because both systemic and local administration of rosiglitazone were effective to attenuate tactile allodynia, we could not determine whether rosiglitazone alters the migration of circulating monocytes or local proliferation of resident macrophages ( Fig. 4). However, in vitro data support the idea that rosiglitazone directly regulates macrophage infiltration to the inflamed sites.
Several reports demonstrated that the activation of PPARγ signaling promotes tissue repair by reducing the production of proinflammatory mediators and in macrophages.
30 – Induction of iNOS, COX-2, and MMP-9 was attenuated at the PSNL site of rosiglitazone-treated mice from PODs 1 to 7 ( 32 Fig. 3). In vitro, IFN-γ–induced gene induction of COX-2 and iNOS was inhibited by rosiglitazone treatment of peritoneal macrophages. Proinflammatory mediators contribute to axonal damage, but they also modulate stimulus-induced neuronal activity and the early onset of neuropathic pain. Our results indicate that rosiglitazone may also attenuate tissue disruption by proinflammatory mediators and promote tissue repair through macrophage-mediated mechanisms. Stimulation of macrophages leads to the activation of nuclear factor-κB. 33 SUMOylation of PPARγ by rosiglitazone promotes transrepression of nuclear factor-κB target genes such as iNOS in macrophages. 34 Rosiglitazone has a delayed onset of action after repeated injection ( 31 Fig. 1), which may be attributable to the phenotypic switch of macrophages to an antiinflammatory phenotype or the activation of PPARγ-dependent pathways to balance inflammatory signaling pathways in macrophages. Alternatively, the analgesic effects of PPARγ signaling in macrophages may ultimately be performed by different types of cells. Further analysis is required to clarify the molecular mechanisms underlying PPARγ-mediated analgesia.
In a study by Churi et al.,
the effect of a single intraperitoneal injection of rosiglitazone on POD 7 was evaluated for 240 minutes in a spared nerve injury model; the treatment did not decrease hyperalgesia. In the present study, the effects of a single intraperitoneal or local injection of rosiglitazone immediately after PSNL were assessed for 3 days; similarly, we found that this single injection did not attenuate tactile allodynia (data not shown). These data suggest that a single injection of rosiglitazone does not reverse neuropathic pain development, supporting the idea that repeated administration in the early phase is required for the onset of rosiglitazone. 7
Daily oral administration (PODs 0–6) of a different type of PPARγ agonist, pioglitazone, significantly reduced tactile allodynia from POD 5 to 14.
However, pioglitazone is permeable to the BBB, and the primary site of action was not identified. 8 However, taken together with our data, both of the PPARγ agonists required repeated administration to inhibit neuropathic pain development. Although PPARγ is expressed in dorsal horn neurons, BBB-impermeant rosiglitazone is likely to exert an analgesic effect primarily outside the central nervous system. These data indicate that early blockade of the initial immune response by activating PPARγ in the peripheral macrophages may be sufficient to reverse neuropathic pain development. 15
In summary, we demonstrated that rosiglitazone administration in the early phase of neuropathic pain development markedly reduced tactile allodynia by modulating macrophage infiltration to the SCN injury. We therefore propose PPARγ regulation of the macrophage-mediated inflammatory response as a novel therapeutic target for treating neuropathic pain development.
Name: Yoshika Takahashi, MD.
Contribution: This author performed experiments.
Name: Maiko Hasegawa-Moriyama, MD, PhD.
Contribution: This author performed experiments, contributed to study design, and wrote the manuscript.
Name: Takashi Sakurai, MD, PhD.
Contribution: This author contributed to study design and wrote the manuscript.
Name: Eiichi Inada, MD, PhD. Contribution: This author contributed to study design. REFERENCES
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