Leptin derived from adipocytes in injured peripheralnerves facilitates development of neuropathic painvia macrophage stimulationTakehiko Maedaa,1, Norikazu Kiguchia, Yuka Kobayashia, Toshihiko Ikutaa, Masanobu Ozakib, and Shiroh Kishiokaa

aDepartment of Pharmacology, Wakayama Medical University, Wakayama 641-0012, Japan; and bDepartment of Toxicology, Niigata University of Pharmacyand Applied Life Science, Niigata 950-2028, Japan

Edited by David Julius, University of California, San Francisco, CA, and approved June 12, 2009 (received for review April 1, 2009)

Nerve injury may result in neuropathic pain, characterized byallodynia and hyperalgesia. Accumulating evidence suggests theexistence of a molecular substrate for neuropathic pain producedby neurons, glia, and immune cells. Here, we show that leptin, anadipokine exclusively produced by adipocytes, is critical for thedevelopment of tactile allodynia through macrophage activation inmice with partial sciatic nerve ligation (PSL). PSL increased leptinexpression in adipocytes distributed at the epineurium of theinjured sciatic nerve (SCN). Leptin-deficient animals, ob/ob mice,showed an absence of PSL-induced tactile allodynia, which wasreversed by the administration of leptin to the injured SCN.Perineural injection of a neutralizing antibody against leptin re-produced this attenuation. Macrophages recruited to the peri-neurium of the SCN expressed the leptin receptor and phosphor-ylated signal transducer and activator of transcription 3 (pSTAT3),a transcription factor downstream of leptin. PSL also up-regulatedthe accepted mediators of neuropathic pain—namely, cyclooxy-genase-2, inducible nitric oxide synthase, and matrix metal-loprotease-9—in the injured SCN, with transcriptional activation oftheir gene promoters by pSTAT3. This up-regulation was partlyreproduced in a macrophage cell line treated with leptin. Admin-istration of peritoneal macrophages treated with leptin to theinjured SCN reversed the failure of ob/ob mice to develop PSL-induced tactile allodynia. We suggest that leptin induces recruitedmacrophages to produce pronociceptive mediators for the devel-opment of tactile allodynia. This report shows that adipocytesassociated with primary afferent neurons may be involved in thedevelopment of neuropathic pain through adipokine secretion.

adipokine � allodynia � C/EBP � fat � STAT

Neuropathic pain is elicited by a lesion or inflammation of thenervous system and is often severely debilitating and largely

resistant to treatment. The symptoms of neuropathic pain mayinclude allodynia, hyperalgesia, and spontaneous pain. A greatdeal has been reported on neuropathic pain and its possiblecauses from preclinical studies involving the development ofanimal models of pain. It has become clear that the neuroin-flammatory mechanism in the periphery nervous system (PNS)plays an important role in neuropathic pain (1). Infiltration ofinflammatory cells, as well as the activation of resident immunecells in response to nervous system damage, leads to the subse-quent production and secretion of various inflammatory medi-ators. For example, inflammatory cells, such as mast cells,neutrophils, and macrophages, produce and secrete prostanoids,nitric oxide, cytokines, chemokines, and matrix metalloproteases(2). These mediators promote neuroimmune activation and cansensitize primary afferent neurons and contribute to the devel-opment of neuropathic pain. Although the number of moleculesinvolved in neuropathic pain is increasing, their mechanisms ofaction are still unclear.

Adipose tissue distributed around internal organs, visceral fat,is well known to serve as an endocrine organ involved in obesityand metabolism. Adipocytes are also localized in the PNS (3),

but little attention has been paid to their functional importance.Adipokines secreted from adipose tissue have come to berecognized as molecular substrates contributing to obesity andrelated metabolic disorders (4). Leptin, an adipokine, is a16-kDa, nonglycosylated peptide hormone encoded by the obesegene in mice (5). Stimulation of its receptor leads to activationof the Janus kinase-signal transducer and activator of transcrip-tion (JAK-STAT) signaling pathway (6). Leptin also has a rolein the regulation of energy homeostasis (7). Recently, leptin hasemerged as a modulator of inflammation and immune responsesin the nervous system. The underlying mechanism is likely to beleptin-stimulated production of inflammatory mediators (8–10),which are also accepted molecular substrates for neuropathicpain (11–13). The present study focused on the role of leptinsecreted from adipocytes in the PNS in neuropathic pain. Here,we show that tactile allodynia induced by partial sciatic nerveligation is affected in its developmental phase by leptin possiblysecreted from adipocytes in the epineurium of the sciatic nerve(SCN). Furthermore, the leptin signaling molecules involved inthis regulation include some conventional molecular substratesfor allodynia (11–13) produced by macrophages through activa-tion of the JAK-STAT pathway.

ResultsRole of Leptin in Allodynia. We examined the effect of leptindeficiency on tactile allodynia and thermal hyperalgesia inducedby PSL. PSL in lean mice elicited a significantly higher ratio ofthe withdrawal response to tactile stimuli after day 3 followingPSL than did sham-operated lean mice, indicating that tactileallodynia occurred (Fig. 1A). By contrast, ob/ob mice did notshow a significant increase in the ratio of responses during theexamined days after PSL; however, PSL did induce thermalhyperalgesia in ob/ob mice (Fig. 1B). There was no significantdifference in the ratio of tactile responses between sham-operated ob/ob and lean mice during the study period after PSL.These results suggest that tactile allodynia was not developed inob/ob mice. Consistent with these results, perineural injection ofa neutralizing antibody for leptin into the ligated SCN reducedthe magnitude of PSL-induced tactile allodynia in ICR mice(Fig. 1C). PSL induced an increase in tyrosine phosphorylationof Ob-Rb (P-Ob-R), the long isoform of the leptin receptor, inthe SCN, indicating that PSL stimulated leptin signaling (Fig.S1B). The behavioral effect of the neutralizing antibody wasassociated with the attenuation of the increase in PSL-inducedexpression of P-Ob-R (Fig. S1B). To test whether leptin replace-

Author contributions: T.M. and M.O. designed research; T.M., N.K., and T.I. performedresearch; T.M. and Y.K. analyzed data; and T.M. and S.K. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1To whom correspondence should be addressed. E-mail: tmaeda@wakayama-med.ac.jp.

This article contains supporting information online at www.pnas.org/cgi/content/full/0903524106/DCSupplemental.

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ment could prevent the failure of ob/ob mice to develop tactileallodynia, the effect of leptin injection into the SCN in mice thathad undergone PSL was examined. Daily perineural injection ofleptin (1 �g) into the ligatured SCN after PSL reversed thefailure of ob/ob mice to develop tactile allodynia on day 5 afterPSL (Fig. 1D). By contrast, treatment of ob/ob mice with leptinon days 7–11 after PSL did not affect the failure of these miceto develop PSL-induced tactile allodynia (Fig. 1E). To testwhether the effect of perineurally applied leptin involved asystemic action, leptin was administered s.c. Systemic adminis-tration at the same dose as used in the perineural injection didnot lead to tactile allodynia in ob/ob mice with PSL (Fig. S1 A).Leptin injections on either days 0–4 or days 7–11 produced nosignificant effect on tactile allodynia in lean mice (Fig. S1 C andD). These results indicate that endogenous leptin works in theearly phase of PSL-induced tactile allodynia.

PSL-Induced Up-Regulation of Leptin in Perineural Adipocytes. West-ern blotting revealed that PSL induced rapid expression of leptinprotein in the SCN, the level of which declined after day 7following PSL, with no significant change being observed insham-operated mice (Fig. 2A). PSL also increased the level ofleptin mRNA in the SCN at 3 h after PSL (Fig. 2B). However,PSL did not significantly affect the serum level of leptin at 3 hafter operation (3.11 � 0.58 ng/mL in sham and 3.97 � 0.70ng/mL in PSL, n � 7; P � 0.181). The expression of leptin mRNAis transcriptionally enhanced by CCAAT/enhancer-binding pro-tein-� (C/EBP-�), a transcription factor that is also known to bean acute-phase protein and a key regulator of adipocyte differ-entiation (14). We addressed whether PSL-induced expression ofleptin mRNA was mediated transcriptionally by C/EBP-�. AChIP assay revealed that markedly increased binding of C/EBP-�to the leptin promoter occurred in the injured SCN 3 h after PSL

(Fig. 2C). Adipose tissue in the epineurium of the SCN has beennoted previously, and more recently it was shown to function asan endocrine organ through the production and secretion ofadipokines, such as leptin (3). Immunohistochemistry of theSCN was performed to determine the distribution of leptin andC/EBP-�, molecules expressed specifically in adipocytes. Adi-pocytes, stained with BODIPY, were distributed in theepineurium of the SCN in sham-operated (Fig. 2K) and PSL(Fig. 2G) mice. The adipocytes in the SCN of sham-operatedmice expressed C/EBP-� and leptin peripheral to BODIPY-stained neutral lipids (Fig. 2 I and J). Increased staining for leptinwas observed in the perineural adipocytes of the ligatured SCN(Fig. 2F). These data suggest that leptin is up-regulated in theepineurial adipocytes of the injured SCN, at least in part throughtransactivation of the leptin gene by C/EBP-�.

Leptin Stimulates Macrophages Through Activation of STAT3. It hasbeen found that phosphorylation of both STAT3 and JAK2follows activation of the leptin receptor (15); therefore, to testwhether the JAK-STAT pathway is activated by leptin during thedevelopment of tactile allodynia, we evaluated the levels ofphospho-STAT3 (pSTAT3), an active form of STAT3, in theSCN of PSL mice. A rapid increase in the level of pSTAT3occurred in the SCN of PSL mice (Fig. 3A). Macrophagescontribute to pain resulting from peripheral nerve injury (16).Furthermore, leptin stimulates tyrosine phosphorylation ofJAK2 and STAT3 in macrophages (17). We examined whetherPSL stimulates the JAK-STAT pathway in macrophages re-cruited to the injured SCN. Macrophages were recruited to theperineurium of the SCN at 3 h after PSL (Fig. 3K), whereas therewere far fewer in the SCN of sham-operated mice (Fig. 3F). Thenumber of macrophages at 3 h after PSL was significantlydifferent between sham-operated (27.6 � 6.0 cells, n � 7) andPSL (64.7 � 9.3 cells, n � 7; P � 0.006 vs. sham) mice. The leptinreceptor and pSTAT3 were coexpressed in macrophages in theinjured SCN (Fig. 3H). Additionally, the possible function of theJAK-STAT pathway in tactile allodynia was assessed. A singleadministration of AG490 (40 mg/kg i.p.), a JAK2 inhibitor,prevented the development of tactile allodynia (Fig. S2 A) andinhibited the increase in the levels of phospho-JAK2 (pJAK2),an active form (Fig. S2B), and pSTAT3 (Fig. S2C) in the injuredSCN. These results indicate a significant role of JAK-STATpathway activation, downstream of leptin, in recruited macro-phages in the development of tactile allodynia.

Transcriptional Activation of Molecules Involved in Allodynia Devel-opment Through pSTAT3 in Macrophages. Many studies have re-ported key molecules related to neuropathic pain established byperipheral nerve injury. Most of these studies focused onexpression of these molecules in the spinal cord and DRG, butthere have been few reports of the molecules involved in theinjured SCN. Furthermore, the mechanisms underlying theexpression of these molecules remain to be determined. Toinvestigate which allodynia-related molecules are driven byactivation of the JAK-STAT pathway in the injured SCN, wefocused on 3 allodynia-related molecules reported previously:cyclooxygenase-2 (COX-2), inducible nitric oxide synthase(iNOS), and matrix metalloprotease-9 (MMP-9). A ChIP assaytk;2elucidated a marked increase in pSTAT3 binding on thepromoter sequence in the injured SCN from 3 h after PSL (Fig.4 A–C). Transcription levels of all 3 molecules also increasedcoincidentally with enhanced binding of pSTAT3 (Fig. 4 D–F).We used a macrophage cell line, J774A.1, to examine whethertranscription of these 3 molecules was enhanced in macrophages.In line with the findings in the injured SCN, J774A.1 cells treatedwith leptin showed a significant increase in the mRNA levels foriNOS and MMP-9 (Fig. 4 H and I). On the other hand, the levelof COX-2 mRNA was not significantly changed, although LPS

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Fig. 1. Leptin was involved in the development of tactile allodynia. (A andB) Time course of tactile allodynia and thermal hyperalgesia. *, P � 0.05; **,P � 0.01; ***, P � 0.001 vs. sham in lean mice. #, P � 0.05; ##, P � 0.01; ###, P �0.001 vs. sham-operated ob/ob mice. (C) Leptin-neutralizing antibody (Nab; 1ng) or normal goat IgG (10 ng) as a control was injected into the perineural SCNof ICR mice every other day from day 0 (immediately after PSL) to day 4 afterPSL. A behavioral test was conducted on day 5 after PSL. ***, P � 0.001. (D andE) Recombinant mouse leptin (lep; 1 �g) or vehicle (veh) was perineurallyadministered to the SCN with surgery in ob/ob mice, once daily in the earlyphase [from day 0 (immediately after PSL) to day 4 after PSL] or in the latephase (from day 7 to day 11 after PSL). A behavioral test was conducted on day5 after PSL in the early-phase group, or on day 12 after PSL in the late-phasegroup (n � 8). ***, P � 0.001. ns, not significant.

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treatment induced a marked increase in the level of this mRNA(Fig. 4G).

Leptin-Stimulated Macrophages Facilitate Allodynia Development.We addressed whether leptin-stimulated macrophages in theinjured SCN may be responsible for the development of tactileallodynia. Peritoneal macrophages (PMs) from naive ob/ob miceshowed pSTAT3 expression (Fig. 5A) when treated with leptin(1 �g/mL for 3 h). PMs were distributed in the epineural SCNin PSL (Fig. 5C) and sham-operated (Fig. 5D) ob/ob mice 1 dayafter they were percutaneously administered to the perineuralSCN, which was performed immediately after PSL. These resultssuggest the possibility that leptin treatment evoked JAK-STATpathway activation in PMs, and that injected PMs are functionalin the SCN. Administration of leptin-treated PMs evoked tactileallodynia in ob/ob mice subjected to PSL, but administration ofvehicle-treated PMs did not have a significant effect in thesemice (Fig. 5E). The ratio of withdrawal responses was notsignificantly different between sham-operated mice injectedwith leptin-treated or vehicle-treated PMs.

DiscussionWe provide evidence that leptin, possibly derived from adipo-cytes in the epineurium of the SCN, contributes to the devel-opment of tactile allodynia through the production of themolecular substrates for neuropathic pain by stimulation of theJAK-STAT pathway in macrophages, as supported by the fol-lowing results: (i) ob/ob mice, which are leptin-deficient, failedto show PSL-induced tactile allodynia; (ii) PSL enhanced tran-scription of the leptin gene in the injured SCN; (iii) PSL resultedin recruitment of pSTAT3-positive macrophages to theepineurium of the SCN; (iv) PSL induced expression of mRNAsfor MMP-9, COX-2, and iNOS, molecules known to underlieallodynia development, through pSTAT3 transactivation, in theinjured SCN; (v) MMP-9 and iNOS mRNA levels were alsoincreased in a macrophage cell line treated with leptin; and (vi)

Administration of leptin-treated PMs reversed the failure ofob/ob mice to show PSL-induced tactile allodynia.

C/EBP-� has been identified as a transcription factor thatbinds to the leptin promoter, working through a consensus

A B C

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Fig. 2. Expression of leptin was increased in the adipo-cytes in the epineurium of the injured SCN. (A) Time courseof leptin expression in the SCN. Leptin expression wascorrected based on the level of �-tubulin. (B) RT-PCR forleptin mRNA in the SCN 3 h after PSL. Leptin expression wascorrected based on the level of GAPDH. (C) ChIP analysis ofbinding of C/EBP-� to the leptin promoter. Immunoprecipi-tation (IP) of complexes of C/EBP-� binding and the genepromoter was corrected for using the gene without IP(input) (n � 4). *, P � 0.05; **, P � 0.01; ***, P � 0.001 vs.sham-operated mice. (D–K) Sections of the SCN from sham-operated (H–K) and PSL (D–G) mice at 3 h of PSL werestained with BODIPY (G and K), which binds neutral lipids inadipocytes, and were immunostained for C/EBP-� (E and I)and leptin (F and J). (D and H) Merged images. A controlsection of the SCN of a PSL mouse at 3 h, for which normalIgG was substituted for the primary antibody tested,showed no immunostaining for leptin or C/EBP-� in theligatured SCN (Fig. S3 Upper). (Scale bars: 100 �m.)

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Fig. 3. PSL-induced stimulation of intracellular signaling downstream of theleptin receptor in macrophages. (A) Time course of STAT3 activation. ThepSTAT3 expression was corrected based on the level of STAT3 expression (n �4). *, P � 0.05; ***, P � 0.001 vs. sham-operated mice. (B–K) The sections of SCNfrom sham-operated (B–F) and PSL (G–K) mice were immunostained forpSTAT3 (B, D, G, I), leptin receptor (E and J), and F4/80, a marker for macro-phages (F and K). (C–F, H–K) High-power magnification of the outlined areasin B and G, respectively. (C and H) The colocalization of pSTAT3, leptin receptorand F4/80 in sham-operated and PSL mice, respectively. A control section, forwhich normal IgG was substituted for the primary antibody used, showed noimmunostaining for pSTAT3 or leptin receptor in the injured SCN (Fig. S3Lower). (Scale bars: C–F and H–K, 10 �m; B and G, 150 �m.)

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C/EBP-binding site in the proximal leptin promoter (18, 19); itplays a critical role in the transcription of acute-phase proteins(14). Transactivation of the leptin promoter by C/EBP-� wasrapidly increased (Fig. 2C). Although it remains unclear hownerve damage induces leptin up-regulation through the trans-activation of C/EBP-�, the prompt induction of transcriptionindicates that leptin may work in the induction phase of tactileallodynia. This hypothesis was robustly confirmed by the resultthat leptin administration immediately after PSL was moreeffective in reversing the absence of tactile allodynia in ob/obmice (Fig. 1D) than administration commencing 7 days after PSL(Fig. 1E). By day 7 after PSL, leptin expression returned to thebasal level (Fig. 2A), whereas binding of C/EBP-� to the leptinpromoter remained elevated until day 14 after PSL (Fig. 2C). Theresults of the present study cannot explain this different temporalprofile, but the discrepancy might be resolved by further studies onthe time course of the transcriptional activity of a potential repres-sor of leptin (20) in adipocytes in the injured SCN.

The present evidence indicates that adipocytes in theepineurium of the SCN are an important source of leptin. First,

LPS and cytokines, which are possible inducers of neuropathicpain, reportedly both induce leptin expression (21). Second,increased levels of leptin protein were observed in adipocytes inthe epineurium of the SCN 3 h after PSL (Fig. 2F), when thelevel of leptin expression in the SCN was significantly increased,as revealed by Western blot analysis and RT-PCR (Fig. 2 A andB). Secreted leptin may diffuse into the perineurium and act onmacrophages recruited by cytokines and chemokines (1) and/orleptin itself (22). In addition, leptin might be delivered to theendoneurium and involved in allodynia development: althoughthe perineurium forms a diffusion barrier between theepineurium and the endoneurium, these 2 compartments arenonetheless connected by the vascular network of the peripheralnerve (23). Third, at 3 h after PSL, we observed no significantdifference in the serum levels of leptin between sham-operated andPSL mice. This result suggests that leptin locally up-regulated in theinjured SCN contributes more to the development of tactile allo-dynia than circulating leptin: the significance of its role is less insystemically distributed adipocytes.

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Fig. 4. PSL up-regulates molecules underlying allodynia development in macrophages through pSTAT3. (A–C) ChIP analysis of pSTAT3 binding on the promotersequences of COX-2 (A), iNOS (B), and MMP-9 (C) in the SCN (n � 5 in A and B; n � 4 in C). (D–F) Time course of mRNA expression for COX-2 (D), iNOS (E), andMMP-9 (F) in the SCN (n � 4). Sham and PSL are abbreviated as S and P, respectively. White and black columns indicate sham and PSL, respectively. (G–I) Effectof leptin on the mRNA expression of COX-2 (G), iNOS (H), and MMP-9 (I) in J774A.1 cells, a macrophage cell line. The cell line was cultured in serum-free culturemedium including leptin (16 �g/mL) or LPS (0.1 �g/mL) for the indicated number of hours. The cell line was cultured in drug-free medium for 24 h as a controlgroup, indicated by C (n � 6–9). *, P � 0.05; **, P � 0.01; ***, P � 0.001 vs. sham treatment (A–F) or control (G–I).

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Studies of rodents with genetic abnormalities of leptin orleptin receptors have revealed deficits in macrophage phagocy-tosis and the expression of proinflammatory cytokines (24).Macrophages in the PNS contribute to pain resulting fromperipheral nerve injury (16). Thus, our hypothesis was thatmacrophages are target cells for leptin in the perineural SCN ofPSL mice. This hypothesis is supported by the following findings.First, macrophages expressing leptin receptors and pSTAT3, aprimary leptin receptor signaling molecule, were seen in theinjured SCN 3 h after PSL (Fig. 3H), when the expression levelof pSTAT3 was maximal in the injured SCN (Fig. 3A). Second,treatment with leptin led to the production of molecules respon-sible for allodynia development in a macrophage cell line, witha peak level at 3 h after PSL (Fig. 4 H and I). The molecularsubstrates for allodynia development are beginning to be unrav-eled by accumulated evidence; among these, we focused onmolecules with pSTAT3-binding consensus sequences in thepromoter or enhancer, including MMP-9 (11), iNOS (12), andCOX-2 (13). These were all up-regulated via pSTAT3 in theinjured SCN 3 h after PSL (Fig. 4 A–F). The expression levels ofmRNAs for MMP-9 and iNOS but not COX-2 were enhanced inthe macrophage cell line after leptin treatment (Fig. 4 G–I). Thereason why the change in COX-2 expression in the macrophagecell line was different from that in the SCN was not elucidatedby the present study. A possible explanation is that in the earlyphase after peripheral nerve injury, the increase in COX-2expression is noted in Schwann cells rather than in macrophages(25). Another is that COX-2 expression is increased by synergywith IFN-� treatment in the cell line (26). Further studies areneeded to clarify the mechanism underlying the PSL-inducedincrease in COX-2 expression in the SCN.

We applied in vitro leptin-treated PMs to the SCN of ob/obmice that had received PSL to determine the critical role ofleptin-stimulated macrophages in the development of tactileallodynia. Administration of leptin-treated PMs reversed thefailure of ob/ob mice to develop PSL-induced tactile allodynia(Fig. 5E). These results strongly suggest that the distribution ofleptin-stimulated macrophages in the injured SCN is critical forthe development of PSL-induced tactile allodynia. The effects ofleptin-treated PMs are consistent with the effect of leptinreplacement on the absence of tactile allodynia in ob/ob mice(Fig. 1D). Moreover, these results raise the possibility thatmacrophages are the key target of leptin during the development of

tactile allodynia. On the other hand, the administration of leptin-treated PMs did not induce tactile allodynia in sham-operated ob/obmice (Fig. 5E), which was also consistent with the effect ofperineural injection of leptin (Fig. 1D). These results suggest thatleptin-stimulated macrophages play a critical role in the early phaseof PSL-induced tactile allodynia but not in the late phase.

The behavioral data presented here suggest that leptin ismore important in mediating tactile allodynia than thermalhyperalgesia. Different pain modalities have distinct mecha-nisms (27). Some studies have reported that tactile allodyniaand thermal hyperalgesia are not both always elicited byperipheral nerve injuries. IL-6 knockout mice show an absenceof tactile allodynia, but normal thermal hyperalgesia in thesemice can be induced by nerve injury (28). IL-6 stimulates theglycoprotein 130 (gp130)-JAK-STAT pathway, as does leptin(6); thus, the development of specific modalities of abnormalpain might depend on the type of intracellular signaling beingrecruited. This possibility needs to be studied in the future.

In summary, this report demonstrates that leptin in the PNSis a novel mediator of tactile allodynia induced by peripheralnerve injury. Our results established that adipocytes in theepineurium of SCN may have a crucial role in synthesizing andsecreting leptin into the perineurium and endoneurium. Thepresent study shows that blocking locally elevated signaling ofleptin constitutes a therapeutic approach for the treatment ofchronic pain. It is significant to bear in mind that the patho-physiology associated with the overexpression and hypersecre-tion of leptin—namely, obesity and diabetes mellitus—mightinduce subjects to have increased susceptibility to neuropathicpain: a higher prevalence of peripheral neuropathy has beenreported in subjects with obesity and impaired glucose tolerance(29). Obese patients with non-insulin-dependent diabetes mel-litus have shown poor results in clinical neuropathy tests (30).These reports suggest that body mass control, which could lowerthe levels of leptin in adipocytes, may be a therapeutic approachto reduce diabetic neuropathy.

Materials and MethodsSubjects. The procedures used in these studies were approved by the AnimalResearch Committee of Wakayama Medical University in accordance withJapanese Government Animal Protection and Management Law, JapaneseGovernment Notification on Feeding and Safekeeping of Animals, and TheGuidelines for Animal Experiments in Wakayama Medical University (approvalnos. 271 and 322). For the behavioral tests and PM samples, 5-week-old male,leptin-deficient, obese ob/ob mice (B6.V-Lepob/J) and their lean littermates (12weeks old) were obtained from Charles River. We found that the body weight inob/ob mice at 5 weeks of age was similar to that in lean mice at 12 weeks of age(29.5 � 1.5 g in lean mice and 30.1 � 2.5 g in ob/ob mice, n � 8; P � 0.875). MaleICR mice (5 weeks old, 27.1 � 2.2 g in body weight, n � 8) were used for otherexperiments. Mice were subjected to PSL according to the method described byMaeda et al. (31). Behavioral studies were conducted by using the modifiedprocedures described by Maeda et al. (31), as shown in the SI Methods.

Drug Administration. AG490 (Sigma–Aldrich) at 40 mg/kg was administered i.p.to ICR mice 30 min before PSL. For perineural injection, mice were injectedpercutaneously at the SCN under brief anesthesia with ether to study the roleof leptin in allodynia. A single injection of 10 �L of recombinant mouse leptin (1�g) or a neutralizing antibody against mouse leptin (1 ng; R & D Systems) wasgiventomouseSCNimmediatelyafterPSLand3hbeforebehavioral testing,oncedaily. Vehicle (PBS) or normal goat IgG (10 ng; R & D Systems) was given to thecontrol groups for leptin treatment or the neutralizing antibody, respectively.

Western Blotting, Immunohistochemistry, ELISA, RT-PCR, and ChIP. These meth-ods were performed according to the modified methods described by Maedaet al. (31). Brief methods are described in the SI Methods.

Macrophage Cell Line in Culture. The murine macrophage J774A.1 cell line(JCRB9108; Health Science Research Resources Bank, Osaka, Japan) was incu-bated in serum-free medium including vehicle (PBS), LPS (0.1 �g/mL), or leptin (16�g/mL).

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Fig. 5. Administration of leptin-treated macrophages reverses the failure ofallodynia development in ob/ob mice. PMs from ob/ob mice were cultured for3 h in culture medium, including Hoechst 33342 (for nuclear staining) andleptin (1 �g/mL) or vehicle (PBS). (A and B) Some cultured PMs were immu-nostained for pSTAT3 and F4/80. Cultured PMs were washed with drug-freePBS and then perineurally injected into the SCN of a different ob/ob mouseimmediately after PSL and on days 2 and 4 after PSL. (C and D) The SCNsisolated from PM-administered sham-operated and PSL mice were sec-tioned on day 1 after PSL. Arrowheads, double-stained (A) or nuclear-stained (C and D) cells. (Scale bars: A and B, 10 �m; C and D, 100 �m.) (E)Tactile allodynia was evaluated on day 5 of PSL (n � 7– 8). **, P � 0.01. ns,not significant.

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PM Injection. PMs were obtained by using a modification of the techniquedescribed by de Jonge et al. (32) and were injected as shown in theSI Methods.

ACKNOWLEDGMENTS. This research was supported by Grant-in-Aid for Sci-entific Research 18613014 and Grant-in-Aid for Exploratory Research19659404 from the Japan Society for the Promotion of Science.

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