Brain Research 913 (2001) 86–89www.elsevier.com/ locate /bres

Short communication

Anti-TNF-neutralizing antibodies reduce pain-related behavior in twodifferent mouse models of painful mononeuropathy

a , a a a* ¨Claudia Sommer , Thies Lindenlaub , Philipp Teuteberg , Maria Schafers ,b aThomas Hartung , Klaus V. Toyka

a ¨ ¨ ¨Neurologische Klinik der Universitat Wurzburg, Josef-Schneider-Straße 11, 97080 Wurzburg, Germanyb ¨Biochemical Pharmacology, Universitatsstr. 10, 78464 Konstanz, Germany

Accepted 5 June 2001

Abstract

We investigated the anti-hyperalgesic effect of neutralizing antibodies (AB) to tumor necrosis factor-a (TNF) in two murine models ofneuropathy, the chronic constrictive sciatic nerve injury (CCI) which has a strong epineurial inflammatory component, and the partialsciatic nerve transection (PST), a ‘pure’ nerve injury model. In both models a single AB injection intra-operatively as well as on day 4reduced thermal hyperalgesia significantly, whereas mechanical allodynia was only reduced with intraoperative but not with delayedtreatment. 2001 Elsevier Science B.V. All rights reserved.

Theme: Sensory systems

Topic: Pain modulation: anatomy and physiology

Keywords: Hyperalgesia; Neuropathy; Cytokines; Tumor necrosis factor-a

Tumor necrosis factor-a (TNF) is a mediator of in- femoris muscle. Half of the diameter of the nerve wasflammatory and neuropathic pain. We have previously transected up to the ligature, which was then removed. Tenshown the antihyperalgesic effect of neutralizing anti- animals were unoperated untreated controls. Operated micebodies (AB) to TNF in the model of chronic constrictive were treated with 50 ml of a polyclonal sheep anti-mousesciatic nerve injury (CCI) [8,13]. Due to the presence of TNF antibody (anti-TNF-AB; purified IgG fraction, 25the constrictive ligatures, CCI combines nerve compres- mg/ml) perioperatively by application onto the exposedsion with an epineurial inflammatory lesion [2,9]. We nerve after surgery (n58 for CCI and n513 for PST) or ontherefore investigated whether anti-TNF-AB have an an- day 4 postoperatively by local epineurial injection astihyperalgesic effect also in a pure nerve lesion model, the described before [8] (n54 for CCI and n59 for PST), orpartial sciatic nerve transection (PST) [7]. with 50 ml of a polyvalent sheep IgG (10 mg/ml, Serotec,

Female C57BL/6 mice (16–21 g, n565) were used UK) as a sham treatment (n58 for CCI and n513 for(Harlan Winkelmann, Germany). In 20 mice CCI was PST) [11].performed by placing three ligatures (7 /0 prolene) around Thermal nociceptive thresholds were measured accord-one sciatic nerve [11] with a contralateral sham operation. ing to Hargreaves et al. [6] and Lindenlaub et al. [8].In 35 mice a partial sciatic transection (PST) was per- Hindpaw withdrawal latencies (means of five tests) on theformed [7]. The nerve was exposed at midthigh level and a control side were subtracted from those on the experimen-prolene 7/0 ligature was placed through the mid of the of tal side, the resulting ‘difference score’ (d.s.) giving athe nerve cranially to the branch running to the biceps measure of thermal hyperalgesia. Mechanical allodynia

was assessed with von Frey hairs using the up-and-downmethod of Dixon [3] modified for mice [11]. The 50%

*Corresponding author. Tel.: 149-931-201-2621; fax: 149-931-201-withdrawal threshold was recorded. Tissue was harvested2697.on day 8 in mice with CCI (n54) and PST (n54) treatedE-mail addresses: sommer@mail.uni-wuerzburg.de (C. Sommer),

http: / /www.uni-wuerzburg.de /neurologie / (C. Sommer). with anti-TNF-AB. A 2-mm segment taken 3 mm distally

0006-8993/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 01 )02743-3

C. Sommer et al. / Brain Research 913 (2001) 86 –89 87

to the injury site was processed for plastic embedding. sham treated with mean 0.0460.04 g on day 7), but not inToluidine blue-stained semithin sections were used to mice treated on day 4 after CCI (mean 0.0460.03, Fig.digitize the endoneurial area at 3630 with a Zeiss Ax- 1b).iophot 2 microscope (Zeiss, Germany) using a motorized Of 15 mice with PST, five each were treated with

¨ ¨scanning table (Marzhauser, Germany) and Image Pro Plus anti-TNF-AB or with IgG at the time of surgery or with(4.0) software. A grid was overlaid with a 50-mm distance anti-TNF-AB on day 4 after surgery. Two additional micebetween intersection points where the underlying structure were controls. Mechanical allodynia was attenuated on(intact myelinated fiber, degenerating myelinated fiber, days 5–7 in mice treated with anti-TNF-AB at the time ofedema, or endoneurial cell) was identified and counted on surgery (mean 0.4560.02 g, P,0.05 versus IgG with300–400 data points per nerve. Results were expressed as mean 0.1360.01 g), but not in mice treated on day 4percentage of all data points [10]. Differences between (mean 0.1360.01 g, Fig. 1d). Twelve additional micetreatment groups were assessed with ANOVA followed by received PST, four each were treated with anti-TNF-ABLSD test (Statistica software) for morphometric and ther- either at the time of surgery or on day 4 after surgery, ormal behavioral data. Friedman’s test was used for statisti- with IgG at the time of surgery. Two mice were controls.cal analysis of mechanical thresholds with Newman– Thermal hyperalgesia was reduced on days 7 and 14 inKeul’s test for post hoc analysis. mice treated with anti-TNF-AB at the time of surgery (d.s.

Twelve mice with CCI were treated with anti-TNF-AB 22.262.1 and 22.660.2) and in those treated on day 4or with IgG at the time of surgery (n54 each) or with (d.s. 22.063.0 and 22.160.8) versus IgG-treated miceanti-TNF-AB on day 4 after surgery (n54). The mice were (d.s. 24.060.9 and 23.461.1) (Fig. 1c).behaviorally tested before surgery, on day 3 and on day 7. To assess whether the attenuation in thermal hy-Three additional mice were controls, whose thresholds did peralgesia seen with anti-TNF-AB treatment was compar-not change during the course of the experiment. Thermal able in mice with CCI and with PST tested in parallel, fourhyperalgesia was present in IgG-treated mice (d.s. mice with CCI and four with PST received anti-TNF-AB23.860.6) on day 7 and reduced in mice treated with treatment, three were controls. Treatment with TNF-AB atanti-TNF-AB at the time of surgery (d.s. 21.760.4) and the time of surgery produced the same reduction of thermalon day 4 (d.s. 21.960.6) (Fig. 1a). Mechanical allodynia difference scores in mice with CCI (d.s. betweenwas attenuated in the mice treated with anti-TNF-AB at the 21.660.3 and 22.260.8) and PST (d.s. betweentime of surgery only (mean 0.2960.11 g, P,0.05 versus 21.860.2 and 22.160.5) compared to IgG treatment (d.s.

Fig. 1. Hyperalgesia to thermal stimuli is reduced on day 7 after CCI (a) and after PST (c) when anti-TNF-antibodies (TNF-AB) are given intraoperativelyor on day 4 (TNF-AB day 4). (b) Mechanical allodynia is reduced by intraoperative AB-administration in CCI (b) and PST (d) but not by AB-treatment onday 4. Data are mean6S.E.M. *P,0.05 versus sham treatment (IgG).

88 C. Sommer et al. / Brain Research 913 (2001) 86 –89

Fig. 2. (a)Treatment with anti-TNF-AB at the time of surgery reduces thermal hyperalgesia to the same degree in mice with CCI and with PST. Data aremean6S.E.M. *P,0.05 versus sham treatment. (b) Morphometric assessment of the degree of nerve de- and regeneration at 8 days after injury. Data aremean6S.D.

between 23.660.3 and 23.860.4 in CCI and between Acknowledgements23.060.6 and 23.560.7 in PST) (Fig. 2a). Quantificationof indicators of nerve de- and regeneration on semithin We would like to thank B. Dekant and L. Biko forsections on day 8 did not show a difference in the amount technical assistance. Supported by Deutsche Forschungs-of nerve degeneration between PST and CCI (Fig. 2b). gemeinschaft SFB 353.

Our main findings are that neutralization of TNF canreduce pain-related behaviors in two neuropathy models,and that the effects on thermal hyperalgesia and on

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