Inflammation, Vol. 21, No. 1, 1997

AN EXPERIMENTAL STUDY OF THENEUROGENIC AND THE IMMUNOLOGICALCONTRIBUTION TO "TENNIS ELBOW" IN

RATS

EVA HAKER,1'2 ELVAR THEODORSSON,3and THOMAS LUNDEBERG,2'4

'Department of Physical- Therapy

^Physiology and PharmacologyKarolinska Institutet, Stockholm, Sweden

^Department of Clinical Chemistry, University Hospital, Linkoping, Sweden

"Department of Physical Medicine and RehabilitationKarolinska Hospital, Stockholm, Sweden

Abstract—In the present study the content of substance P (SP)-, neurokinin A(NKA)-, calcitonin gene-related peptide (CGRP)- and neuropeptide Y (NPY)-likeimmunoreactivity (-L1) was measured in rats cerebrospinal fluid (CSF), plasma andperfusates (PF) from both elbow enthesis during acute inflammation. Either substanceP, SP, (10-5 M, 0.01 ml) or human recombinant interleukin-1 a (hrll-l a, 0.01 ml)were injected into the right enthesis of the extensor carpi radial is brevis (ECRB).The left ECRB and both ECRBs of control rats, were injected with 0.01 ml saline.Samples of CSF, plasma and PF from both ECRBs were obtained at 2, 6, and 24 hfollowing injection and neuropeptide-LI was analysed by specific radioimmunoas-says. Neuropeptide-LI was compared with control values and between the treatedgroups. In both treated groups NKA- and CGRP-LI was increased in CSF and NKA-LI decreased in plasma, while CGRP- and NPY-LI were raised to a similarly sig-nificant degree in the enthesis of the ECRB. SP-LI was increased in ECRB PF incomparison with controls and NKA-LI levels were higher in the hrll-l a group bothin comparison with controls and between treated groups. In summary an unilateralinjection of either SP or hrll-1 a into the enthesis of the ECRB of the rat showed asimilar influence at 2, 6, and 24 h following injection. The most pronounced changesin neuropeptide-LI occurred in the ECRB PF of both treated groups.

INTRODUCTION

Lateral epicondylalgia or tennis elbow is a common painful condition affectingthe lateral aspect of the epicondyle (1). Age/related or degenerative changes,

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due to ischaemia in the tendon, probably predispose to the lesion (2). Todaythe etiology is believed to involve microtears at the enthesis of the extensorcarpi radialis brevis (ECRB) (3 1992). Tendon biopsies from the elbow ofpatients with tennis elbow demonstrate microscopic changes with several abnor-malities such as inflammatory granulation, blood vessel wall changes, glycos-aminoglycan infiltration and fibrocartilaginous transformation (4). Computerizedinfrared thermography and isotopic bone scanning show abnormal forearm tem-peratures and a significant unilateral cooling suggested to be a part of a soma-tosympathetic response in some patients (3). These findings suggest that thenervous system may contribute to lateral epicondylalgia. This is supported byrecent studies showing that the nervous system plays a role in rheumatoid arthri-tis in humans (5) and in experimentally induced arthritis in rats (6 1986). Neu-ropeptides released from afferent nerve fibers are suggested to be involved inneurogenic inflammation. Substance P (SP) (7), neurokinin A (NKA) (8) andcalcitonin gene-related peptide (CGRP) (9) possibly contribute to plasma extra-vasation and vasodilatation indicating that they may be part of an inflammatoryreaction. Furthermore, SP (10, 11), NKA (12) and CGRP (12-14) influenceimmune activity and may in this way also participate in the inflammatory reac-tion. It has been shown that SP (15) and interleukin (IL-1) (16), exacerbatedexperimentally induced joint inflammation. Both SP (17) and IL-1 (18) have theability to stimulate synoviocytes and release collagen degradating enzymes. Thesympathetic nervous system has also been shown to contribute to inflammatoryprocesses in rats (19) and levels of neuropeptide Y-like immunoreactivity (NPY-LI) released from efferent nerve fibers were enhanced in the synovial fluid ofrheumatoid arthritis patients (20, 21).

In human rheumatoid arthritis (22-24) and experimentally induced arthritis(25, 26) interleukin-1 (IL-1) activity is enhanced both systematically and locally,while in human osteoarthritis is it localised in the damaged joints only (27). Nostudy has to our knowledge been carried out elucidating the role of IL-1 in tenniselbow. IL-1 is responsible for specific systematic and local effects in rheumatoidarthritis and experimentally induced arthritis as well as general systematic non-specific reaction, e.g. fever, acute/phase protein synthesis, PGE production,immune system activation (28). IL-1 has been shown to be responsible forcartilage degradation in vivo (29) and in vitro (30) and bone resorption in vitro(31, 32) IL-1 inhibitory IgG has been found present in 17% of patients withrheumatoid arthritis (33) suggesting a specific antigen function.

IL-1 is known to exist in two molecular forms, IL-1 a and IL-1 /?. IL-1 amay be functionally active in its cell-associated form as well as in its secretedform, whereas IL-1 /3 is effective only in the secreted form (34). It has beenshown that IL-1 can regulate its own production by inducing IL-1 a and IL-1/3 gene expression in synovial fibroblasts and peripheral blood monocytes (35)and can in this way promote the inflammatory process.

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In order to evaluate the neurogenic and immunological aspects in acuteexperimentally induced tennis elbow we examined the effects of injection ofeither SP or hrll-l a into the enthesis of rats' ECRB on changes in neuropeptideswith a suggested role in the inflammatory processes.

MATERIAL AND METHODS

The study was carried out on 90 male albino Sprague-Dawley rats, weighting 250-300 g.The rats were anaesthetized with chloralhydrate (0.4 g/kg). The skin overlaying the elbow wasshaved and the elbows were flexed 90 degrees during the ECRB injections. 30 rats were givensubstance P (SP) 0.01 ml 10~5M and 30 rats 0.01 ml human recombinant interleukin-1 a (hrll-la) 0.1 mg/ml via a 27 gauge needle into the right enthesis of ECRB. Both groups also received0.01 ml of saline into the left ECRB. 30 control rats were injected with 0.05 ml saline into bothECRBs.

After periods of 2, 6 or 24 h following the injections the rats were again anaesthetized withchloralhydrate. A 2 cm long longitudinal skin incision was made bilaterally exposing the elbow. Aneedle (27-gauge size used throughout the experiments) was inserted into each ECRB from themedial aspect and another on the lateral side. The right and left ECRB were simultaneously perfusedwith saline 0.9 through the needle, using a syringe pump set at 0.2 ml/min, and the perfusate wascollected through the needle. Perfusion was carried out for 10-15 minutes and a 1.5-2 ml perfusatewas collected for each ECRB. For the collection of CSF following ECRB perfusion, rats wereplaced in a stereotaxic frame. The atlanto-occipital membrane was exposed by retracting the over-laying muscles and 80-150 jil samples of CSF were obtained through a needle connected with a1 (tl syringe via a polythylene tubing. Blood (1.5-4.5 ml) was then collected by puncture of theheart into a vacutainer tube containing heparin 143 1U with Trasylol 500 IU ml. Blood sampleswere centrifuged and the plasma removed and frozen. All samples were rapidly cooled and storedat 45°C until analysis.

Samples of the CSF, plasma and PF were extracted using a reverse-phase C18 cartridge (SepPak, Waters) and analysed using competitive radioimmunoassays (36). Radioimmunoassay of tachy-kinins other than SP, i.e. NKA was done using antiserum K 12 (37, 38), SP using antiserum SP(39), CGRP using antiserum R8 (36) and NPY using antiserum NPYI (40). The lower detectionlimit in all extracted samples was 0.1 fmol/ml for all peptide assessments.

Statistical analysis was carried out using Mann-Whitney U-test with SPSS software.

RESULTS

Cerebrospinal Fluid (CSF). SP-L1 was increased in the CSF at 2 and6 h following ECRB injection. No significant changes remained in SP-LI at 24h after treatment. At 2 h SP-LI was increased to a greater extent in the SPinjected group than in the hrll-l a group. NKA-LI was also increased in bothgroups compared with controls. Injection either of SP or hrll-l a increasedCGRP-LI at 2 and 6 h when comparing both groups to controls, but the increase

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was greater in the SP injected group. NPY-LI was increased in the hrll l-otgroup at only 6 and 24 h following injection, Table I.

Plasma. No significant changes in SP-LI were seen in plasma followingeither SP or hrll-l a injection. NKA-LI was reduced in both treated groups incomparison with controls although it remained higher in the SP injected groupthan in the hrll-l a group. In comparing the treatment groups CGRP-LI washigher in the SP group than in the hrll-l a at 2 and 6 h. NPY-LI was enhancedin the SP group at 2, 6 and 24 h compared with controls and to a larger extentcompared with the hrll-l a group at 2 h, Table I.

Perfusates (PF) of Both ECRBs. There was generally a significant increasein neuropeptide-LI in perfusates from both ECRB, following SP or hrll-l a,but with exceptions, Table I. The differences between SP and hrll-l a wereminor both in considering injected ECRB vs controls and between the twogroups, Table I.

DISCUSSION

In the present experiment an ECRB injection of hrll-l a increased SP-,NKA-, CGRP- and NPY-LI in the PF from both ECRBs. It has previously beenshown that SP (41), NKA (8) and CGRP (8, 9) participate in neurogenic inflam-mation (23, 42), and increased concentrations of these neuropeptide-LI havebeen shown in the PF from both ECRBs at 2, 6 and 24 h following unilateralinjection of either Freund Adjuvans or carrageenan (1 or 43). A bilateral increasein the concentrations of these neuropeptide-LI during different models of acutemonoarthritis has recently been presented (44, 45). It has recently been proposedthat the central events in the spinal cord dorsal horn are sufficient and necessaryfor the initiation of neurogenic inflammation and the resultant pain and hyper-algesia (46). IL-1 increases SP content in cultured sympathetic ganglia (47) andit has been reported that endopeptidase 24.11 which is responsible for SP andNKA metabolism (48) inactivates IL-1 (49). Our findings concerning SP-LI inPF following hrlL-la are in line with results (50) when using lower doses thanthose presently used (59 ^g). An intraarticular injection of hrll-l a (100 mg)into the rabbit knee joint enhanced SP-LI in the joint fluid, but no SP-LI wasfound following injection of 1 /*g. However, dose differences in IL-la mayresult in different mechanisms of action, and species differences might also haveinfluenced the outcomes (51).

SP and NKA have been shown to induce the release of IL-1 from humanmonocytes (52) and enhance 11-1 activity in a mouse macrophage cell lineP388D1 (53). Injection of SP into an ECRB enhanced all neuropeptide-LI inthe PF of both ECRB. The bilateral changes of neuropeptide-LI in ECRB PF

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were not always detected, particularly those in NKA-LI. The lack of contralat-eral release might be related to a specific effect of injected SP on NKA-LI releasein ECRB. SP has been shown to have an excitatory action with regard to sensoryneurons (42) indicating that the unilateral release of NKA-LI could be a directeffect of SP.

However, the different reactions of SP- and NKA-LI to SP injection remainunclear. SP and NKA have been shown to be synthesized from the same pre-cursor-gene (54) but separate and differing reactions of these peptides cannot beexcluded. In our laboratory during capsaicin perfusion of the rat knee joint,pretreated with adenosine, NKA-LI increased significantly but SP-LI did not(55).

A problem in the analysis of SP-LI following SP injections is whether theactivity noted represents the exogenously injected peptide or an endogenouslyreleased amount. The elimination of SP in plasma has been found to take about10 min (56). The time profile for elimination in ECRB PF is unascertained butreasonably short, and injected SP would not be present at 2 h in the ECRB. Itis interesting to note similar time related increases of SP-LI, NKA-LI and CGRP-LI following injection. The three peptides are found to be co-localized in sensoryneurons (54, 57) and the present results indicate a simultaneous release followingstimulation with one of them (SP or hrll-l a). A local inflammatory processcan therefore, through the presence of one stimulatory agent, release severalpeptides which can interact increasing inflammation progressively through sep-arate mechanisms. The intraarticular injection of either hrll-l a or SP enhancedneuropeptide-LI in CSF as early as 2 or 6 h, but only NKA-LI remained increasedat 24 h following injection. The release of NKA in the spinal cord has beenshown to be more widespread than that of SP-LI during experimentally inducedacute arthritis in cats (58, 59). The greater enhancement of NKA- and CGRP-LI in the CSF of the SP group compared to that of IL-1 a at 2 and 6 h can berelated to the greater excitatory effect of SP directly on sensory neurons. Therelease of NKA- and CGRP-LI but not SP-LI in CSF, and all three peptides inPF, indicates the possibility of a difference in the release of peptidergic neuronsperipherally vs centrally. Activation of separate populations of neurons can beanother explanation. NKA-LI decreased in the plasma of both treated groups.This could be related to enzyme systems circulating in plasma (48), althoughthe reason for its decrease in plasma while increased in CSF and joint PF remainsunclear, and further experiments are needed to explore this phenomena.

Coderre et al. (60) demonstrated the role of the sympathetic nervous systemin experimentally induced arthritis. In the present study NPY-LI was mainlyelevated in the PF of both injected groups, Table I. NPY-LI was also increasedin plasma at 2, 6 and 24 h in the SP group and in CSF at 6 h in the hrll-l agroup, Table I. Such local increases might indicate a possible role for NPY intennis elbow. It has been demonstrated that NPY can modulate sensory neuron

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function and SP release dorsal root ganglion cells in rats (61). On the otherhand, NPY has been reported to have vasoconstrictor properties (62). However,neither SP nor hrll-l a injected into the ECRB were able to influence NPY-LIcontent to any greater degree.

Taken together the results of the present study show that the nervous andimmune system contributes to experimentally induced "tennis elbow" in therat.

Acknowledgments—The present study was supported by grants from Anna Greta Craafordsfoundation, Karolinska Institutet foundation, King Gustav Vth 80-year anniversay fund, ProfessorNanna Svartz foundation, the Swedish Medical Research Council, the Swedish Society againstRheumatism, Magnus Bergvalls foundation and Clas Groschinskys memorial fund.

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