Methylglyoxal modification of Na v 1.8 facilitates nociceptive neuron firing and causes hyperalgesia in diabetic neuropathy

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1 A r t i l e s Methylglyoxal modifiation of Na v 1.8 failitates noieptive neuron firing and auses hyperalgesia in diabeti neuropathy Angelika Bierhaus 1,2, Thomas Fleming 1,2, Stoyan Stoyanov 1, Andreas Leffler 2,3, Alexandru Babes 4,5, Cristian Neasu 5, Susanne K Sauer 4, Mirjam Eberhardt 4, Martina Shnölzer 6, Felix Lasitshka 7, Winfried L Neuhuber 4, Tatjana I Kihko 4, Ilze Konrade 1,8, Ralf Elvert 9, Walter Mier 1, Valdis Pirags 11, Ivan K Luki 1,12, Mihael Moros 1, Thomas Dehmer 13, Naila Rabbani 14, Paul J Thornalley 14, Diane Edelstein 15, Carla Nau 3, Josephine Forbes 16, Per M Humpert 1, Markus Shwaninger 17, Dan Ziegler 18, David M Stern 19, Mark E Cooper 16, Uwe Haberkorn 1, Mihael Brownlee 15,2, Peter W Reeh 4 & Peter P Nawroth 1 This study establishes a mehanism for metaboli hyperalgesia based on the glyolyti metabolite methylglyoxal. We found that onentrations of plasma methylglyoxal above 6 nm disriminate between diabetes-affeted individuals with pain and those without pain. Methylglyoxal depolarizes sensory neurons and indues post-translational modifiations of the voltage-gated sodium hannel Na v1.8, whih are assoiated with inreased eletrial exitability and failitated firing of noieptive neurons, whereas it promotes the slow inativation of Na v1.7. In mie, treatment with methylglyoxal redues nerve ondution veloity, failitates neuroseretion of alitonin gene-related peptide, inreases ylooxygenase-2 (COX-2) expression and evokes thermal and mehanial hyperalgesia. This hyperalgesia is refleted by inreased blood flow in brain regions that are involved in pain proessing. We also found similar hanges in streptozotoin-indued and geneti mouse models of diabetes but not in Na v1.8 knokout (Sn1 / ) mie. Several strategies that inlude a methylglyoxal savenger are effetive in reduing methylglyoxal- and diabetes-indued hyperalgesia. This previously undesribed onept of metabolially driven hyperalgesia provides a new basis for the design of therapeuti interventions for painful diabeti neuropathy. Pain and hyperalgesia are key danger signals that are evoked by physial insults, noxious hemials and inflammation. Suh danger signals are also present in patients with diabeti neuropathy despite losses in sensory and autonomi funtions in these individuals. Whereas the moleular mehanisms underlying inflammatory hyperalgesia are inreasingly being unraveled, there is urrently only a vague understanding of the mehanisms ausing neuropathi pain in metaboli diseases suh as diabetes 1. In general, hyperglyemia has been emphasized as a major risk fator in these diseases; however, normalization of gluose onentrations has shown little benefit in affeted individuals 1 3. Elevated gluose onentrations lead to inreased formation of the highly reative diarbonyl metabolite methylglyoxal 4, whih is metabolized by glyoxalase 1 (GLO1) and GLO2 to the end produt D-latate 4 7. As peripheral nerves have low GLO1 ativity 8,9, it has been reasoned that they might be partiularly vulnerable to methylglyoxal aumulation. As has been shown in the model organism Caenorhabditis elegans, the glyoxalase system is essential for neuronal integrity 1. A reent study omparing the expression of GLO1 in various inbred mouse strains showed a negative orrelation between GLO1 expression and mehanial hyperalgesia, implying that the balane between methylglyoxal and GLO1 might diretly modulate pain pereption 9. Inreased eletrial exitability seems to be the underlying ause of the generation of orthotopi or etopi impulses in experimental rat 1 Department of Mediine I and Clinial Chemistry, University Hospital Heidelberg, Heidelberg, Germany. 2 Department of Anesthesiology and Intensive Care, Hannover Medial Shool, Hannover, Germany. 3 Department of Anesthesiology, Friedrih-Alexander-University Erlangen-Nuremberg, Erlangen, Germany. 4 Institute of Physiology and Pathophysiology, Friedrih-Alexander-University Erlangen-Nuremberg, Erlangen, Germany. 5 Department of Physiology and Biophysis, Faulty of Biology, University of Buharest, Buharest, Romania. 6 Funtional Proteome Analysis B1, Core Faility Protein Analysis W12, German Caner Researh Center, Heidelberg, Germany. 7 Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany. 8 Stradins University, Riga Eastern Clinial University Hospital, Riga, Latvia. 9 Sanofi-Aventis Deutshland, Frankfurt, Germany. 1 Department of Nulear Mediine, University Hospital Heidelberg, Heidelberg, Germany. 11 University of Latvia, Pauls Stradins Clinial University Hospital, Riga, Latvia. 12 Biosistemi d.o.o., Zagreb, Croatia. 13 Zentrum für Psyhiatrie Südwürttemberg, Zwiefalten, Germany. 14 Warwik Medial Shool, Clinial Sienes Researh Institute, University of Warwik, University Hospital, Coventry, UK. 15 Albert Einstein College of Mediine, Bronx, New York, USA. 16 Baker International Institute, Heart and Institute, Melbourne, Vitoria, Australia. 17 Institute for Experimental und Clinial Pharmaology and Toxiology, University of Lübek, Lübek, Germany. 18 Institute for Clinial Diabetology, German Center at the Heinrih Heine University Leibniz Center For Researh, Düsseldorf, Germany. 19 University of Tennessee College of Mediine, Memphis, Tennessee, USA. 2 These authors ontributed equally to this work. Correspondene should be addressed to P.P.N. (peter.nawroth@med.uni-heidelberg.de). Reeived 3 November 211; aepted 22 Marh 212; published online 13 May 212; orreted after print 6 July 212; doi:1.138/nm VOLUME 18 NUMBER 6 JUNE 212 nature mediine

2 a r t i l e s and human diabeti neuropathy 11,12. Redued potassium ondutane and redued Na + /K + ATPase ativity in diabeti nerves have been proposed as possible auses of inreased exitability 12 14, but hanges in neuronal voltage-gated sodium hannels (VGSCs), whih trigger, shape and propagate ation potentials, ould also diretly explain the inreased exitability. The effet of diabetes on VGSC gene expression has yielded results with some disrepanies that annot aount for the oexistene of positive and negative symptoms in diabeti neuropathy One partiular VGSC, tetrodotoxin-resistant (TTXr) Na v1.8, is exlusively expressed in pain-signaling neurons or noieptors 18,19 ; however, Na v1.8 transription and expression are dereased in diabeti rats 15,16 with inreased TTXr sodium urrents 16. These seemingly ontraditory findings led to a hypothesis that posttranslational modifiations in Na v1.8 by methylglyoxal ould inrease either voltage sensitivity or funtional hannel availability, ausing the hyperexitability that is responsible for diabeti hyperalgesia and spontaneous pain. In this study, we show that inreased onentrations of methylglyoxal may aount for metaboli hyperalgesia. The link between hyperalgesia and methylglyoxal is provided by nonenzymati modifiations of Na v1.8, whih, in turn, modify its funtion. Our data provide evidene for a previously unidentified pathway in whih methylglyoxal diretly indues hyperalgesia and may provide new therapeuti options for the treatment of painful metaboli neuropathy. RESULTS We found that plasma methylglyoxal disriminated patients with type 2 diabetes from healthy ontrols (Fig. 1a). Assessment of pain, determined by self report in the foot and graded on a ten point numerial sale, showed that the patients with diabetes ould be subdivided into two groups: those with no pain and those with pain. Patients were onsidered to have pain when they reported more than seven points on the numeri sale. The mean pain sores for the two groups were 1.7 ± 1.2 and 7.7 ± 1. (mean ± s.e.m.) (for those with no pain and those with pain, respetively). Patients with pain also reported signifiantly a P <.1 Conentration of plasma diarbonyls (nm) 1, Deoxygluosone Glyoxal Methylglyoxal ontrol P <.1, no pain P <.1, pain b Intensity of symptom Pain, feet : no pain : pain P <.1 P =.47 P =.91 Burning Tingling COX-2:GAPDH normalized ratio ontrol more burning sensations and had a trend towards inreased dysesthesia (Fig. 1b). The groups did not differ signifiantly in their neuropathy defiit sore or in a range of metaboli variables (Supplementary Table 1). We subsequently found that diabetes-affeted individuals with pain had signifiantly higher onentrations of plasma methylglyoxal ( 6 nm) ompared to either healthy ontrols or diabetesaffeted individuals with no pain (Fig. 1a), whih is onsistent with reent observations in patients with type 1 diabetes 21. To establish whether these elevated onentrations of methylglyoxal ould affet neuronal funtion, we exposed ultured mouse dorsal root ganglion (DRG) neurons from healthy wild-type () mie to plasma from either healthy ontrols or diabetes-affeted individuals with or without pain. We determined the indution of COX-2 as an indiret readout for altered neuronal funtion. Plasma from patients with pain indued signifiantly more COX-2 transription in the neurons than plasma from ontrols or patients with no pain (Fig. 1). Treatment with trypsin of the plasma from patients with pain did not prevent COX-2 expression in the neurons. The supplementation with exogenous methylglyoxal of plasma isolated from individuals with diabetes without pain to elevate the onentrations to those observed in patients with pain ( 6 nm) resulted in further inreases in COX-2 transription in the neuron (Supplementary Fig. 1b). As the ativity of GLO1 is low in peripheral nerves and is even lower in peripheral nerves of diabeti mie 8,9 (Fig. 1d), we reasoned that neurons might be partiularly likely to aumulate methylglyoxal. Inhibition of GLO1 (ref. 22) by the ell-permeable inhibitor s-p-bromobenzylglutathione ylopentyl diester in nondiabeti mie resulted in profound thermal hyperalgesia when the methylglyoxal plasma onentrations exeeded 6 nm (Fig. 2a). mie in whih we indued diabetes with streptozotoin (STZ-indued diabeti mie) (8-week duration) showed a pronouned lateny redution in noxious heat withdrawal (Fig. 2b). Nondiabeti GLO1 knokdown (Glo1 /+ ) mie 23 showed an equally pronouned lateny redution ompared to STZ-indued diabeti mie. STZ-indution of diabetes in Glo1 /+ mie (8-week P <.1 P <.1 P <.1, no pain, pain, pain + trypsin d GLO1 ativity (mm min 1 mg 1 ) Kidney Figure 1 Methylglyoxal and pain in individuals with diabetes. (a) Plasma diarbonyl onentrations in the patients with type 2 diabetes haraterized in Supplementary Table 1 with and without pain. Data represent the mean ± s.e.m. n = 1 patients per group (omparisons were made using analysis of variane (ANOVA)). (b) Intensity of neuropathy symptoms in the studied diabetes-affeted individuals with and without pain as assessed by self report in the foot and graded on a ten point numerial sale. Data represent the mean ± s.e.m. n = 1 patients per group (unpaired, two-tailed Student s t test). () Quantifiation of COX-2 mrna expression in DRGs isolated from healthy C57BL/6 mie treated with heat-inativated plasma from healthy subjets (ontrols) or diabetes-affeted individuals with and without pain. Where indiated, plasma samples from patients with diabetes and pain were pretreated with trypsin and neutralized using Soybean inhibitor. Data represent the mean of three independent experiments performed in tripliates ± s.d. n = 3 mie per group. GAPDH, glyeraldehyde-3-phosphate dehydrogenase;, not signifiant (unpaired, two-tailed Student s t test). (d) Organ survey for GLO1 ativity in tissue extrats from healthy and STZ-indued diabeti mie. Data represent the mean ± s.d. n = 1 mie per group. Brain Liver Heart Organs Spleen Lungs Siati nerve nature mediine VOLUME 18 NUMBER 6 JUNE

3 A r t i l e s a Plasma methylglyoxal (nm) Time (d) Lateny (s) b Lateny (s) P <.1 P <.1 Nondiabeti Glo1 /+ P <.1 Diabeti Glo1 /+ Plasma methylglyoxal (nm) Lateny (s) 1, 1, 8 6 MG (µg) Injeted Plasma MG (nm) P <.1 P <.1 P < ± ± ± ± ± 7 P <.1 P <.1 Plasma threshold onentration d Delta fore (g) 1 3 P <.1 P <.1 Figure 2 Exogenous methylglyoxal (MG) and STZ-indued diabetes indue thermal hyperalgesia. (a) Inhibition of GLO1 by the ell-permeable inhibitor S-p-bromobenzylglutathione ylopentyl diester in healthy mie. Hyperalgesia was assessed by Hot Plate assay at day before appliation of the inhibitor. Conseutive assessments of methylglyoxal plasma onentrations and thermal hyperalgesia were made at seleted time points. Data represent the mean ± s.d. n = 3 mie per time point. (b) Thermal hyperalgesia in nondiabeti and GLO1 knokdown (Glo1 /+ ) mie and diabeti mie 8 weeks after STZ-indued diabetes, as measured by Hot Plate assay. Data represent the mean ± s.e.m. n = 25 mie per group (ANOVA). () Responses to heat stimulation by Hot Plate assay in methylglyoxal-treated mie. Plasma was isolated for methylglyoxal anaylsis by HPLC. Data represent the mean ± s.d. (n = 3 mie per group) for methylglyoxal plasma onentrations and the mean ± s.e.m. (n = 1 mie per group) for hyperalgesia determination (ANOVA). (d) Mehanial hyperalgesia (tatile allodynia) in nondiabeti and Glo1 /+ mie and STZ-indued diabeti mie, as determined by Frey filament test. Data represent the mean ± s.e.m. n = 1 mie per group (ANOVA). Nondiabeti Glo1 /+ duration) had no further sensitizing effet. Systemi administration of methylglyoxal resulted in an inrease in methylglyoxal plasma onentrations and a dose-dependent thermal hyperalgesia response within 3 h of administration (Fig. 2 and Supplementary Fig. 2a ). The methylglyoxal-indued sensitization to noxious heat was obsured when the hot plate temperature applied was 55 C rather than 5 C, implying that methylglyoxal lowered the heat threshold but did not disable the noifensive response (Supplementary Fig. 2d)., GLO1 defiieny and systemi methylglyoxal administration also indued pronouned mehanial hyperalgesia (Fig. 2d and Supplementary Fig. 2e). Inreased onentrations of methylglyoxal in diabeti mie are aused, in part, by redued GLO1 transription, expression and ativity (Supplementary Fig. 3a ). We ahieved fully omparable results to those in the STZ-indued diabeti mie when we studied methylglyoxal onentrations in spontaneously diabeti BKS db/db mie 24 (Supplementary Fig. 4). The observed robust thermal and mehanial hyperalgesia in the STZ-indued diabeti mie after 8 weeks of hyperglyemia ours at a time in whih nonspeifi STZ-derived inflammatory proesses have dispersed 24 and no gross morphologial hanges in peripheral nerves are present on the ultrastrutural level (data not shown). In STZ-indued diabeti mie, we found an indution of COX-2 immunoreativity in the hindpaw epidermis (Supplementary Fig. 3d), whih is onsistent with the COX-2 induing effet of patient plasma on DRG neurons (Fig. 1 and Supplementary Fig. 1b) and with exessive prostaglandin formation in diabeti rat skin 18. Thus, non-neuronal and neuronal indution of COX-2, a marker of ellular ativation and a produer of sensitizing eiosanoids 18, may be driven by inreased onentrations of methylglyoxal. Overexpression of GLO1 in STZ-indued diabeti mie (8-week duration) by somati gene transfer resulted in an inrease in siati GLO1 ativity, dereased plasma methylglyoxal onentrations and redued thermal hyperalgesia (Fig. 3a ). This effet a b d e GLO1 ativity (mm min 1 mg 1 ).2.1 P <.1 pgl3 GLO1 Plasma methylgyoxal (nm) P <.1 1, 8 6 pgl3 GLO1 Lateny (s) 1 pgl3 P <.1 GLO1 GERP peptide 1 Gly Glu Arg Pro GEAP peptide 1 Gly Glu Ala Pro Figure 3 Overexpressing GLO1 or savenging methylglyoxal by a syntheti peptide redues thermal hyperalgesia in methylglyoxal-treated and diabeti mie. STZ-indued diabeti mie were treated f Day Day 1 Day 3 Day 6 every 2 d for 1 d with either GLO1-overexpressing vetor (GLO1) or ontrol vetor (pgl3). (a) GLO1 ativity in siati nerve tissue. Data represent the mean ± s.d. n = 5 mie per group (unpaired, two-tailed Student s t test). (b) Plasma methylglyoxal onentrations. Data represent the mean ± s.e.m. n = 1 mie per group (unpaired, two-tailed Student s t test). () Thermal hyperalgesia after 1 d of treatment with the GLO1-overexpressing vetor by intravenous injetion into the tail vein. Data represent the mean ± s.e.m. n = 1 mie per group (unpaired, two-tailed Student s t test). (d) A syntheti peptide, GERP 1 (GERP), was designed ontaining ten arginine residues per moleule to at as an effetive in vivo savenger of methylglyoxal. The peptide GEAP 1 (GEAP) served as an arginine-free ontrol. (e) mie were injeted with GERP or the ontrol peptide GEAP before P <.1 P <.1 injetion of methylglyoxal. Hyperalgesia was assessed 3 h after methylglyoxal injetion by Hot Plate assay. Data represent the mean ± s.e.m. n = 1 mie per group (ANOVA). (f) The effets of GERP and GEAP on diabetes-indued hyperalgesia in STZ-indued diabeti mie. Data represent the mean ± s.e.m. n = 1 mie per group (ANOVA). Lateny (s) +GERP +GERP +GEAP +GEAP Lateny (s) +GERP +GERP +GEAP +GEAP MG P <.1 +GERP +GERP +GEAP MG+GERP +GEAP +GERP MG+GEAP +GERP +GEAP +GEAP 928 VOLUME 18 NUMBER 6 JUNE 212 nature mediine

4 a r t i l e s was independent of the pathway involving the advaned glyation endproduts (AGEs) and their reeptor (the AGE-RAGE pathway), as systemi administration of arboxymethyl-lysine (8 nm, 3 h) did not indue thermal hyperalgesia (data not shown). Further, injetion of methylglyoxal (5 µg, 3 h) into RAGE knokout (Ager / ) mie aused thermal hyperalgesia that was indistinguishable from that observed in the STZ-indued diabeti mie (data not shown). In ontrast, treatment with either aminoguanidine, a savenger of free methylglyoxal, or alagebrium (ALT-711), a ompound that breaks AGE-derived rosslinks in proteins, inhibited methylglyoxal-indued hyperalgesia in methylglyoxal-treated mie (Supplementary Fig. 5), implying that de novo post-translational modifiations of membrane proteins are responsible for the inreased sensitivity and exitability of primary sensory neurons in these mie. To savenge exessive onentrations of methylglyoxal in vivo, we designed an arginine-rih peptide (GERP 1 ) and a ontrol peptide devoid of arginine (GEAP 1 ) (Fig. 3d). Systemi administration of GERP 1 redued thermal hyperalgesia in both methylglyoxal-treated mie (Fig. 3e) and STZ-indued diabeti mie (Fig. 3f), with a half-life of approximately 8 h, whereas systemi administration of GEAP 1 had no effet in either of these groups. To identify a possible protein target of post-translational modifiations by methylglyoxal, we determined the expression of VGSCs in DRGs. Transient reeptor potential V1 (TRPV1) and COX-2 expression served as positive ontrols for DRG expression 25,26. Expression of Na v1.7 and Na v1.9 did not differ between the DRGs from nondiabeti and STZ-indued diabeti mie, whereas Na v1.8 expression was slightly lower, but not signifiiantly lower, in the STZ-indued diabeti mie ompared to the nondiabeti mie 15,16 (Fig. 4a). Methylglyoxal-indued modifiations of arginine residue(s) within the DIII-DIV linker of Na v1.8 (omprising its inativation gate) might alter the gating properties of Na v1.8. Immunopreipitations of DRGs isolated from rats treated with methylglyoxal, healthy mie treated with methylglyoxal, STZ-indued diabeti mie (8-week duration) and healthy Glo1 /+ mie showed that modifiations of Na v1.8 by methylglyoxal were inreased in the presene of a GAPDH normalized ratio d Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] mie Diabeti mie P <.1 Na v 1.7 Na v 1.8 Na v 1.9 TRPV1 COX Minimally modified (1:5 molar ratio) Maximally modified (1:1 molar ratio) b 25 kda 64 kda 5 kda 36 kda Inativation Gate from murine Na v 1.8 (1,42 1,47) (1) (1) (1) (1) Unmodified peptide: [M+H]+631 (2) Modified peptide: [M+H]+685[+54]=1 Arginine modified by MG-H1 (2) (2) m/z Na v 1.8 Na v 1.7 TRPV1 Con MG Con MG Con MG 25 kda 25 kda e GADPH normalized ratio kda 5 kda 36 kda +sirna P <.1 62% P <.1 Na v 1.7 Na v kda 5 kda 36 kda 5% P >.5 TRPV1 46% f sirna Na v 1.8 sirna Negative ontrol Figure 4 Methylglyoxal requires Na v 1.8 to indue hyperexitability and thermal hyperalgesia. (a) Quantifiation by real-time PCR of mrna expression of the voltage-gated sodium hannels Na v 1.7, Na v 1.8 and Na v 1.9, TRPV1 and COX-2 in the DRGs of healthy and STZ-indued diabeti mie. Data represent the mean ± s.e.m. n = 1 mie per group (unpaired, two-tailed Student s t test). (b) Methylglyoxal-modifiation of Na v 1.8, Na v 1.7 and TRPV1 in vivo from ontrol (Con) and methylglyoxal-treated (MG) Wistar rats. The experiment was repeated three times with idential results, and one representative western blot is shown. () Methylglyoxal-modifiation of Na v 1.8 in vivo from ontrol healthy (Con), methylglyoxal-treated (MG), STZ-indued diabeti (Diab) and DRGs Na v1.8 Mouse Con MG Con Diab Con Glo1 /+ Con Diab 25 kda 25 kda 25 kda 25 kda Lateny (s) g Methylglyoxal Na v 1.7 Na v 1.8 Na v 1.9 TRPV P <.1 h Sn1 +/+ Sn1 / 7 P < MG P <.1 P <.1 healthy Glo1 /+ (Glo1 /+ ) mie. For eah group, DRGs were pooled from at least ten mie. Human siati nerve material was obtained from biopsies from patients with (Diab) or without diabetes (Con). The experiment was repeated three times with idential results, and one representative western blot is shown. (d) Methylglyoxal diretly binds to the arginine residue in the Na v 1.8 ativation gate. The DIII-DIV linker of murine Na v 1.8 was synthesized and inubated without (ontrol) or with methylglyoxal in an either 1:5 (minimally modified) or 1:1 (maximally modified) molar ratio 35. A single peak with [M+H] + 6,31 (peak 1) was observed under ontrol onditions. Under both minimal and maximal onditions, a modified peptide was observed with a mass differene of 54 Da (peak 2; 6,85 Da versus 6,31 Da), whih orresponds to the modifiation of the arginine residue at position 43 of the peptide by methylglyoxal to form the hydroimidazolone MG-H1. (e) Effets of in vivo sirna treatment on the expression of Na v 1.7, Na v 1.8 and TRPV1 in healthy DRGs, as determined by real-time PCR. Data represent the mean ± s.d. n = 3 mie per group (unpaired, two-tailed Student s t test). (f) Effets of in vivo sirna treatment for Na v 1.8 in healthy DRGs, as determined by immunoytohemistry. The experiment was repeated three times with idential results, and one representative staining is shown. Sale bars, 5 µm. (g) Effet of in vivo sirna treatment for Na v 1.7, Na v 1.8 and Na v 1.9 on methylglyoxal-indued thermal hyperalgesia in mie. Data represent the mean ± s.e.m. n = 1 mie per group (unpaired, two-tailed Student s t test). (h) Effet of exogenous methylglyoxal on hyperalgesia in healthy mie, littermates of the Na v 1.8 knokout mie (Sn1a +/+ ) and Na v 1.8 knokout mie (Sn1a / ). Data represent the mean ± s.d. n = 5 or 6 mie per group (unpaired, two-tailed Student s t test). Lateny (s) Human nature mediine VOLUME 18 NUMBER 6 JUNE

5 A r t i l e s a b CGRP (pg ml 1 ) 16 P <.5 14 Saline 12 MG 1 P < (C57BL/6) (C57BL/6) Sn1 / KCI (6 mm) Heat (45 C) 35 P < MG NCV (m s 1 ) Weeks NCV (m s 1 ) Weeks P <.1 + MG + MG + GERP + GERP Total brain slie density (optial density/mm 2 ) P <.1 P <.1 P <.1 P <.1 MG GERP + + Positive ontrols + formalin C57BL/6 Negative ontrol + MG Sn1 / Glo1 /+ Figure 5 Methylglyoxal auses a asade of neurohemial hanges and ativates brain regions involved in pain proessing. (a) Effet of systemi methylglyoxal treatment on KCl- and heat-indued CGRP release from isolated hindpaw skin of healthy and Na v 1.8 knokout (Sn1a / ) mie after systemi methylglyoxal pretreatment. Data represent the mean ± s.e.m (two-tailed Student s t test). (b) Maximal dorsal tail NCV determined in healthy mie untreated (ontrol) or pretreated with methylglyoxal (n = 7 mie in eah group) and in healthy (ontrol) and STZ-indued diabeti mie (diabetes) (n = 12 mie in eah group). Data represent the mean ± s.d. (unpaired, two-tailed Student s t test). () CBF hanges show inreased brain ativity. Sample olorized oronal setions showing differenes in brain ativation after heat stimulation; inreased CBF is indiated in red. Eah image is taken from a single brain setion in the individual mie. Data represent the mean ± s.d., n = 4 mie per group (ANOVA), and one representative brain setion is shown for eah ondition. Sale bars, 5 mm. methylglyoxal, diabetes and the defiit in GLO1 (Fig. 4b). Further, in siati nerve tissue isolated from patients with or without diabetes who had amputations resulting from peripheral artery disease, we observed that the patients with diabetes had substantially more modifiations of Na v1.8 by methylglyoxal than the patients without diabetes (Fig. 4). Inubation of a peptide, omprising the inativation gate of Na v1.8, with methylglyoxal followed by a mass spetrometry analysis onfirmed that methylglyoxal binds to the arginine residue within the Na v1.8 inativation gate sequene (Fig. 4d). When we inubated plasma from diabetes-affeted individuals with the peptide, peptide mapping onfirmed methylglyoxal binding to the arginine residue in the Na v1.8 inativation gate (Supplementary Fig. 6). Systemi treatment of mie with sirna targeting Na v1.7, Na v1.8 or TRPV1 resulted in a signifiant inhibition of the respetive mrna (Fig. 4e,f). The Na v1.8 knokdown was paralleled a by loss of methylglyoxal-indued thermal hyperalgesia (Fig. 4g and Supplementary Fig. 7a). In addition, the Na v1.8 knokout mie were not only proteted from methylglyoxal-indued hyperalgesia but atually showed hypoalgesia; we observed wild-type littermates of the NA V 1.8 knokout mie (Sn1a + / + ) to have a similar methylglyoxal-indued hyperalgesia to the C57BL/6 mie used throughout the study (Fig. 4h and Supplementary Fig. 7b). We also found that in vivo pretreatment with methylglyoxal of healthy or Na v1.8 knokout mie failitated the potassiumindued and noxious-heat indued release of the proinflammatory neuropeptide alitonin gene-related peptide (CGRP) from utaneous noieptors in vitro (Fig. 5a), indiating inreased responsiveness to both nonspeifi and heat-transdued membrane depolarization of the skin nerve endings as a result of the pretreatment with methylglyoxal. Sensitization to heat, but not the heat response itself, was absent in skin flaps from the methylglyoxal-pretreated Na v1.8 knokout mie. Methylglyoxal pretreatment and diabetes both signifiantly redued the maximal tail nerve ondution veloity (NCV) (Fig. 5b) despite the absene of overt morphologial hanges (data not shown), a prelinial signature of the imminent large-fiber neuropathy. We next determined the erebral blood flow (CBF) hanges in the pain-proessing regions of the brain 27 in mie we stimulated with laser-evoked noxious heat. We visualized the blood flow distribution in these mie using pseudoolored frontal brain setions showing the resting and ativated brain regions (Fig. 5). We found inreased heat responses in healthy mie pretreated with methylglyoxal and in STZ-indued diabeti mie ompared to healthy mie, whereas the methylglyoxal-savenging peptide GERP 1 showed an antihyperalgesi effet in the healthy methylglyoxal-pretreated and STZ-indued diabeti mie. Na v1.8 knokout mie did not have an enhaned heat response after methylglyoxal pretreatment. Formalin injetion in healthy mie, to indue pain without radiant heat, or healthy Glo1 /+ mie plus heat stimulation showed an enhaned erebral blood flow omparable to that in methylglyoxalpretreated or STZ-indued diabeti mie. Notably, the areas of inreased blood flow in all mouse brains studied (bregma, 1.72 to 2.41) orrespond to the brain areas shown to be ativated in rats (bregma, 2.3) with diabetes-indued neuropathi pain 28. We used urrent-lamp reordings from ultured DRG neurons to haraterize the diret effets of exogenous methylglyoxal on sensory neuron exitability (Fig. 6). Small- and medium-sized neurons from healthy and Na v1.8 knokout mie did not differ in terms of resting membrane potential, urrent threshold and voltage threshold 29. In the neurons, exposure to 1 µm methylglyoxal for 3 h resulted in a signifiant depolarizing shift by 8.2 mv in the resting membrane potential, a redued urrent threshold for the ativation of ation potentials and a redution in the depolarization required to reah the voltage threshold (Fig. 6a,b). The relative derease in the urrent threshold in the neurons was larger (44%) than the derease in voltage threshold (3%), whih may indiate that the membrane resistane was inreased by exposure to methylglyoxal. The rise time of the ation potential was longer in methylglyoxal-treated (1.7 ms ±.1 ms (mean ± s.e.m)) than in ontrol untreated neurons (1.3 ms ±.8 ms, P <.1), refleting the lowered voltage threshold as a result of methylglyoxal treatment. Exposure of the neurons to a lower onentration of methylglyoxal (1 µm), orresponding to the plasma onentrations found in patients with diabetes and pain, for an extended period of time (12 14 h) also rendered their resting membrane potentials more depolarized (Fig. 6; 4.3 mv, P <.1), however, their urrent and voltage thresholds were only redued insignifiantly. Additional pretreatment of these neurons with aminoguanidine, whih inhibits protein modifiations by methylglyoxal, was able to prevent the depolarizing effet of methylglyoxal, as well as the redution of eletrial thresholds (Fig. 6). Pretreatment of Na v1.8 knokout neurons with methylglyoxal also resulted in a signifiant depolarizing shift by 5.2 mv of the resting membrane potential but did not indue a derease in the urrent or voltage thresholds (Fig. 6b). Methylglyoxal pretreatment did not signifiantly hange the amplitude or duration of either the 93 VOLUME 18 NUMBER 6 JUNE 212 nature mediine

6 a r t i l e s a Voltage (mv) Current (na) Voltage (mv) Current (na) Time (ms) µm methylglyoxal Time (ms) 5 b RMP (mv) RMP (mv) 1 µm MG P <.1 P < Sn1 / P <.1 P <.1 1 µm MG + 1 µm MG 1 µm AG I threshold (pa) 16 1 µm MG 8 I threshold (pa) P < µm MG V threshold (mv) P <.1 1 µm MG + 1 µm 1 µm MG 1 µm AG MG 1 µm MG V threshold (mv) 1 µm MG Sn1 / Sn1 / µm MG + 1 µm AG 1 µm MG d Na v µm MG f g h i Normalized urrent mv 1 µm MG 5 ms 2 ms 3 na DRG TTXr P <.1 Normalized urrent Normalized urrent Na v µm MG 14 mv 1 P <.1 DRG TTXs mv 5 ms 5 ms e Na v mv 5 ms 2 ms 5 na Na v µm MG 14 1 Normalized urrent mv 14 mv mv 3 s 5 ms 3 s.8 1 ms 1 ms P <.1 Na v µm MG.4.2 Na v µm MG Normalized urrent +5 mv 5 ms 5 ms +5 mv +5 mv Normalized urrent Figure 6 Effets of methylglyoxal on ation potential generation and Na + urrents in sensory neurons. (a) Examples of ation potentials (upper traes) evoked by urrent pulses (lower traes) in neurons from mie stimulated with methylglyoxal. (b) The effet of methylglyoxal on the resting membrane potential, urrent threshold for pulses (I threshold ) and depolarization required to reah the threshold ( V threshold ) in and Na v 1.8 knokout (Sn1a / ) neurons. Data represent the mean ± s.e.m. n 13 neurons in eah group. () The effets of methylglyoxal on the resting membrane potential, I threshold and V threshold in neurons in the presene and absene of aminoguanidine (AG). Data represent the mean ± s.e.m. n 13 neurons per group. (d) A typial urrent trae of reombinant rat Na v 1.8 in ND7/23 ells ativated by depolarizing steps (see insert) in the presene of TTX and voltage dependene of the steady-state fast inativation of Na v 1.8 in ND7/23 ells (n = 12/12 (ontrol/methylglyoxal treatment)). (e) A typial urrent trae of reombinant rat Na v 1.7 expressed in HEK293T ells, ativated as shown in insert, but without any addition of TTX, showing the voltage dependene of the steady-state fast inativation of Na v 1.7 (n = 13/14)., not signifiant. (f) The voltage dependene of the steady-state fast inativation of TTXr Na + urrents reorded in DRG neurons (n = 14/15). (g) The voltage dependene of the steady-state fast inativation of TTXs urrents reorded in Na v 1.8 knokout DRG neurons (n =11/11). (h) The voltage dependene of the steady-state slow inativation of heterologously expressed Na v 1.8 (n = 12/1). (i) The voltage dependene of the steady-state slow inativation of heterologously expressed Na v 1.7 expressed in HEK293T ells (n = 1/9)., not signifiant. All datasets shown in this figure were analyzed by two-tailed Student s unpaired t test ation potential or the afterhyperpolarization in or Na v1.8 knokout neurons (data not shown). These data suggest that biophysial hanges in Na v1.8 funtion are responsible for the methylglyoxal-indued hyperexitability of sensory neurons but not for their depolarization. We further assessed the effets of methylglyoxal pretreatment (3 h) using voltage lamp reordings on reombinant rat Na v1.8 and Na v1.7 in ND7/23 and HEK293T ells, respetively (Fig. 6d,e and Supplementary Fig. 8). We found methylglyoxal to have no effet on the voltage dependene of ativation (data not shown). However, the midpoint of the steady-state fast inativation of Na v1.8 was signifiantly shifted toward more depolarized potentials (from V.5 = 58 mv ±.5 mv (mean ± s.e.m.) to V.5 = 48.7 mv ±.9 mv, n = 12), an effet that signifies inreased hannel availability at voltages orresponding to the ation potential threshold (Fig. 6d). We did not find this effet on fast inativation when we applied methylglyoxal only briefly (3 min) before beginning the test protool (data not shown). This is ompatible with the time required for posttranslational protein modifiations to our and exludes rapid, nonspeifi membrane effets. Methylglyoxal did not shift the steady-state fast inativation of Na v1.7 (Fig. 6e). To reprodue the differential effets of methylglyoxal on the steady-state fast inativation of Na v1.8 and Na v1.7 (Fig. 6d,e and Supplementary Fig. 8), we performed idential experiments on native TTXr and tetrodotoxin-sensitive (TTXs) Na + urrents, whih are the exeutive ation potential generators in noieptive DRG neurons 26. nature mediine VOLUME 18 NUMBER 6 JUNE

7 A r t i l e s We onduted the experiments in ultured DRG neurons of mie in the presene of TTX to monitor the effets on Na v1.8 and of Na v1.8 knokouts to monitor the effets on Na v1.7 and other TTXs subunits. We found a small but signifiant methylglyoxal-indued shift in the midpoint (from V.5 = 41 mv ±.2 mv to V.5 = 36 mv ±.2 mv, n = 15 (ontrol) or 14 (methylglyoxal treatment)) of the steady-state fast inativation for the TTXr urrents (Fig. 6f) but not for the TTXs urrents (Fig. 6g). Thus, methylglyoxal inreased the known relative resistane of the TTXr ation potential generator against fast inativation, whih may have ontributed to the inreased exitability. Using the same heterologous expression systems as desribed above, we then studied slow steady-state inativation using reombinant VGSC.Whereas this voltage-dependent funtion of Na v1.8 was not influened by pretreatment with methylglyoxal (Fig. 6h), we did observe a signifiant shift toward more hyperpolarized potentials (from V.5 = 53 mv ±.8 mv to V.5 = 68 mv ±.6 mv, n = 1 (methylglyoxal treatment) or 9 (ontrol)) with Na v1.7 as a result of methylglyoxal pretreatment (Fig. 6i), reduing the availability of the Na v1.7 hannels at the resting membrane potential by half. As Na v1.7 is known to be ruial for ation potential triggering in noieptive neurons, the slow inativation of these ion hannels ould aount for the methylglyoxal-indued hypoalgesia observed in the Na v1.8 knokout mie (Fig. 4g). The ontrary effets of methylglyoxal on Na v1.8 and Na v1.7 funtions (sensitization and inativation, respetively) argues against an indiret ation of methylglyoxal, for example, by induing COX-2 expression, whih might enhane prostaglandin formation 3. Although this and other inflammatory mediators inrease the voltage sensitivity of Na v1.8, they are not known to enhane the slow inativation of Na v1.7 (refs. 19,25). DISCUSSION Our data support a new mehanisti onept of painful peripheral neuropathy in diabetes. Post-translational modifiation of the noieptor-speifi sodium hannel Na v1.8 by methylglyoxal is assoiated with enhaned sensory neuron exitability and hyperalgesia, whereas methylglyoxal slows myelinated nerve ondution and drives Na v1.7 into slow inativation. As rystallographi and site-direted mutagenesis studies on these VGSCs are not yet available, the exat moleular link between methylglyoxal and hanges in hannel funtion is unknown. Other modifiations seondary to treatment with methylglyoxal annot be exluded as being responsible for the funtional hanges observed, suh as the depolarizing effet. However, the biohemial and funtional data presented here support the hypothesis that methylglyoxal-dependent modifiation(s) in Na v1.8 have a role in diabetes-assoiated hyperalgesia that is independent of degenerative or regenerative hanges in the nerve. The onentration of plasma methylglyoxal is inreased in patients with diabetes as a result of inreased formation of methylglyoxal from exessive glyolysis 4 and dereased degradation and detoxifiation by the glyoxalase system. Other pathologial states resulting in inreased onentrations of methylglyoxal are uremia and ishemiareperfusion. Although symptoms of painful diabeti neuropathy arise spontaneously, suh as burning and prikling sensations in the feet, tatile allodynia is a lassial sign of this ondition, and burning pain an be provoked by warming the patients feet 31. Thus, the thermal and mehanial hypersensitivity in diabeti, healthy methylglyoxalinjeted or GLO1-defiient mie an be onsidered to reflet, at least in part, the human symptoms. To the best of our knowledge, methylglyoxal is the first endogenously produed small moleule (as opposed to ytokines, some of whih indue systemi hyperalgesia) that indues thermal and mehanial hyperalgesia in healthy mie by aute systemi administration. Systemi appliation of methylglyoxal, however, gave rise to some ambiguity as to the target of its post-translational modifiation. CGRP release experiments, however, onfirmed the indution of peripheral hypersensitivity by methylglyoxal and the dependene of this indution on Na v1.8 expression. In diabeti neuropathy, the axonal transport of neuropeptide-ontaining dense ore vesiles is impeded, whih leads to redued basal release from the skin nerve endings. However, stimulated neuroseretion is failitated in diabeti neuropathy, suggesting an inreased sensitivity, exitability or both 32. These findings in the skin do not exlude additional effets of methylglyoxal on the nonpeptidergi subpopulation of noieptors or on spinal terminals where further sensitization of synapti transmission ould our 33. The altered funtion of Na v1.8 (redued inativation, as well as inreased whole-ell exitability) is onsistent with methylglyoxal binding to TTXr sodium hannels and with the lak of methylglyoxalindued hyperalgesia in Na v1.8 knokout mie and sirna-treated healthy mie. The prominent methylglyoxal-indued neuronal depolarization and the onomitant inrease in membrane resistane both inrease the hane for sensory funtions, for example, heat-indued generator potentials or aidental spontaneous depolarization, to reah the relatively high voltage threshold of Na v1.8 and generate ation potentials 34. Notably, methylglyoxal also auses a major slow inativation of Na v1.7, an essential VGSC and threshold hannel in noieptive neurons. This inativation of the TTXs sodium hannel would be aggravated in vivo by the methylglyoxal-indued depolarization, and only unmyelinated nerve fibers, whih have a high abundane of the inativation-resistant Na v1.8 in their endings, are expeted to show inreased exitability. In fat, the partial blok of Na v1.7 by methylglyoxal may explain why Na v1.8 knokout mie show hypoalgesia in response to methylglyoxal, as hardly any ation potential generator would be left in the noieptors under these onditions. Similarly, postganglioni neurons of the autonomi nervous system, whih are often affeted by diabeti neuropathy and express Na v1.7 but not Na v1.8 (refs. 33,34), would fail under the ondition of aumulating methylglyoxal. These opposing effets of methylglyoxal on TTXs ompared to TTXr VGSCs ould aount for the oexistene of positive and negative linial symptoms, suh as burning feet and diabeti gastroparesis. Support for the general impairment of TTXs sodium hannels by methylglyoxal omes from the marked slowing of NCV after systemi methylglyoxal administration. Noieptive neurons that express Na v1.8 in their peripheral terminals gain one more profit from the modifying ations of methylglyoxal, as Na v1.8 is a faster repriming ation potential generator than Na v1.7 (ref. 18). Thus, the disharge rate in response to a given depolarization of the affeted neuron an rise when the trigger funtion for disharge passes from the inativated Na v1.7 to the failitated Na v1.8. Modifiation of Na v1.8 by methylglyoxal seems to be the root of primary hyperalgesia in animals, as well as the ause of pain in patients with diabetes. Although short-term diabetes in mie is not omparable to years of diabetes in humans, our findings suggest that methylglyoxal is a valid therapeuti target for the treatment of painful diabeti neuropathy, a medial ondition for whih few effetive therapeuti options are available. Methods Methods and any assoiated referenes are available in the online version of the paper. 932 VOLUME 18 NUMBER 6 JUNE 212 nature mediine

8 a r t i l e s Note: Supplementary information is available in the online version of the paper. Aknowledgments The authors thank S. Kaymak for immunohistohemistry, A. Buhl and K. Leotta for assistane in the erebral blood flow experiments, L. Werner for support with bregma gradings and X. Du and A. Erhardt for the daily are of the mie. A. Bierhaus, J.F., M.E.C., M.B. and P.P.N. were supported by a Centre grant from the Juvenile Researh Foundation (JDRF). This work was also supported in part by grants from the Deutshe Forshungsgemeinshaft (BI-1281/3-1 to A. Bierhaus; LU728/3-1 and NA-35/3-2 in KFO 13 to P.W.R., C. Nau and A.L.), the US National Institutes of Health (2R56DK to M.B.), the European Foundation for the Study of (EFSD/Lilly-Programme, to A. Bierhaus), the German Assoiation (DDG, Christian-Hagedorn-Award to A. Bierhaus), the Manfred-Lautenshläger-Stiftung for (LSD, to P.P.N.), the Dietmar- Hopp-Stiftung (to A. Bierhaus, P.M.H. and P.P.N.) and the Network Aging Researh (NAR, to A. Bierhaus). A. Babes and C.N. were supported by grant PN2 164/7 from the Romanian Researh Counil (CNCSIS). A. Babes also reeived support from the Alexander von Humboldt Foundation. AUTHOR CONTRIBUTIO A. Bierhaus planned, performed and supervised all experiments, was responsible for the data interpretation and wrote the manusript. T.F. and S.S. performed most of the mouse experiments, and T.F. also did the biohemial analytis and ell ulture experiments. A.L. and C. Nau performed the voltage lamp studies and were involved in data interpretation. A. Babes and C. Neasu performed the urrent lamp studies and were involved in data interpretation. S.K.S., M.E. and T.I.K. performed CGRP-release experiments and single-fiber reordings in the skin-nerve preparation and were involved in data interpretation. M. Shnölzer, N.R. and P.J.T. were involved in the diarbonyl analytis and data interpretation. F.L. provided biopsies of human siati nerves. W.L.N. performed eletron mirosopy. R.E. and I.K.L. performed the nerve ondution veloity experiments and the measurements of tatile allodynia. W.M., M. Shwaninger and U.H. supervised the erebral blood flow measurements and were involved in data interpretation. T.D. was involved in the isolation and haraterization of the DRG. D.E. generated the Glo1 /+ mie. J.F. and M.E.C. provided ALT-711 and were involved in data interpretation. I.K., V.P. and M.M. performed the linial studies. P.M.H. supervised the linial studies and was involved in data interpretation. D.M.S. was involved in data interpretation and the writing of the manusript. D.Z. provided the human skin biopsies and was involved in data interpretation. M.B. provided the Glo1 /+ mie and was involved in data interpretation and the writing of the manusript. P.W.R. supervised the eletrophysiologial part of the projet and wrote the manusript. P.P.N. supervised the projet and wrote the manusript. Senior oauthorship is shared by P.W.R. and P.P.N. COMPETING FINANCIAL INTERESTS The authors delare no ompeting finanial interests. Published online at Reprints and permissions information is available online at reprints/index.html. 1. Tavee, J. & Zhou, L. Small fiber neuropathy: a burning problem. Cleve. Clin. J. Med. 76, (9). 2. Nawroth, P.P., Rudofsky, G. & Humpert, P.M. Have we understood diabetes? New tasks for diagnosis and therapy. Exp. Clin. Endorinol. 118, 1 3 (21). 3. Calutt, N.A. Potential mehanisms of neuropathi pain in diabetes. Int. Rev. Neurobiol. 5, 2528 (2). 4. Thornalley, P.J. Diarbonyl intermediates in the maillard reation. Ann. NY Aad. Si. 143, (5). 5. Brownlee, M. Biohemistry and moleular ell biology of diabeti ompliations. Nature 414, 8132 (1). 6. Karahalias, N., Babaei-Jadidi, R., Ahmed, N. & Thornalley, P.J. 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