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Osteoarthritis and Cartilage xxx (2016) 1e8

Relationship between structural pathology and pain behaviour in amodel of osteoarthritis (OA)

L.N. Nwosu y z *, P.I. Mapp y z, V. Chapman y x, D.A. Walsh y zy Arthritis Research UK Pain Centre, Clinical Sciences Building, Nottingham, NG5 1PB, UKz School of Medicine, University of Nottingham, UKx School of Life Sciences, University of Nottingham, UK

a r t i c l e i n f o

Article history:Received 17 November 2015Accepted 17 June 2016

Keywords:Pain behaviourMonosodium iodoacetateSynovitisKnee OAStructural pathology

* Address correspondence and reprint requests to: LUK Pain Centre, Department of Academic RheumatoloCity Hospital, Clinical Sciences Building, Hucknall RoaTel: 44-115-840-4732.

E-mail address: (L.N. N 2016 The Authors. Published by Elsevie(

Please cite this article in press as: Nwosu LN(OA), Osteoarthritis and Cartilage (2016), ht

s u m m a r y

Objectives: To address the hypothesis that different types of established osteoarthritis (OA) pain be-haviours have associations with different aspects of articular pathology, we investigated the relationshipbetween structural knee joint pathology and pain behaviour following injection of a low vs a high dose ofmonosodium iodoacetate (MIA) in the rat.Methods: Rats received a single intra-articular injection of 0.1 mg or 1 mg MIA or saline (control). Painbehaviour (hind limb weight bearing asymmetry (WB) and hindpaw withdrawal threshold (PWT) topunctate stimulation) was assessed. Cartilage and synoviumwere examined by macroscopic visualisationof articular surfaces and histopathology.Results: Both doses of MIA lowered PWTs, 1 mg MIA also resulted in WB asymmetry. Both doses wereassociated with cartilage macroscopic appearance, proteoglycan loss, abnormal chondrocytemorphology, increased numbers of vessels crossing the osteochondral junction, synovitis and macro-phage infiltration into the synovium. PWTs were more strongly associated with chondrocytemorphology, synovitis and macrophage infiltration than with loss of cartilage surface integrity.Conclusions: Both pain behaviours were associated with OA structural severity and synovitis. Differencesin pain phenotype following low vs higher dose of MIA were identified despite similar structural pa-thology. OA structural pathology as traditionally measured only partially explains the MIA-induced painphenotype. 2016 The Authors. Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International.

This is an open access article under the CC BY license (


Osteoarthritis (OA) is a chronic debilitating disease affectingaround 8.8million people in the UK1. Pain is the commonest clinicalsymptom that leads OA sufferers to seek medical care. OA paincontributes to loss of joint function, disability and reduced qualityof life in the ageing population2, and is an important unmet clinicalneed. Our incomplete knowledge of the sources of OA pain, and therelationship between pain phenotypes and pathology hindersprogress in the identification of better targets for treatments whichimprove OA pain.

.N. Nwosu, Arthritis Researchgy, University of Nottingham,d, Nottingham, NG5 1PB, UK.


r Ltd on behalf of Osteoarthritis Re

, et al., Relationship betweentp://

The classification of radiographic OA depends on the presence ofosteophytes and joint space narrowing (JSN). Radiographic evi-dence of OA is associated with pain3,4, but this association is oftenonly weak5,6. Some patients report pain despite minimal radio-graphic changes7, whilst others with abnormal knee radiographsreport no pain8. Magnetic resonance imaging (MRI) has revealedassociations of bone marrow lesions or synovitis with pain9e11

although their contributions as direct sources of OA pain remainuncertain5. Central sensitization can moderate the link betweenjoint damage and pain, through complex pain-amplifying neuro-plastic alterations to the central nervous system12.

Preclinical models of knee OA have potential to extend under-standing of OA pain mechanisms, and the contributions of specificstructural features to OA pain. Intra-articular injection of theglycolysis inhibitor monosodium iodoacetate (MIA) into the rattibiofemoral joint produces cartilage and subchondral bone pa-thology that are similar to those seen in human OA knees4,5. Intra-articular MIA injection also leads to pain-related behaviours

search Society International. This is an open access article under the CC BY license

structural pathology and pain behaviour in a model of osteoarthritis2016.06.012

L.N. Nwosu et al. / Osteoarthritis and Cartilage xxx (2016) 1e82

(weight bearing (WB) asymmetry and reduced hindpaw with-drawal thresholds (PWTs) to punctate stimulation)6,7 that resemblepain on WB and more widespread reduced pain thresholds13

observed in human OA. Lowered PWTs implicates a contributionof central sensitisation14, whereas WB asymmetry likely reflects acombination of peripheral and central sensitisation.

Identifying specific aspects of joint pathology that contribute todifferent OA pain phenotypes might help identify pain phenotype-specific peripheral treatment targets. We hypothesised thatdifferent types of established OA pain behaviours may have asso-ciations with different aspects of articular pathology. To addressthis question, we have used two doses of MIA that result in twodifferent pain profiles in the rat and then identified associationsbetween WB asymmetry and lowered PWTs and a range ofmacroscopic and histopathological changes in the knee. A sec-ondary question addressed is whether there is delayed progressionof structural pathology in the lower dose MIA model.

Materials and methods


Studies used male SpragueeDawley rats (Charles River, Kent,UK) (n 64) weighing 250e300 g at time of intra-articular injec-tion. Studies were conducted in accordance with UK Home Officeregulations and followed the guidelines of the International Asso-ciation for the Study of Pain. Rats were housed in groups of four percage under standard conditions with a 12 h light/dark cycle, withunlimited access to food and water. Rats, anaesthetised with iso-flurane (2% in O2), received a single intra-articular injection of MIA(0.1 mg/50 ml or 1 mg/50 ml, based on previous studies14e16) insterile 0.9% normal saline through the infrapatellar ligament of oneknee. In two separate experiments (experiment 1 24 rats,experiment 2 40 rats), rats were randomly assigned to theexperimental (MIA) and control groups (saline) and results fromboth studies combined. The groups included 1 mg MIA; n 18,0.1 mgMIA; n 18 and saline-injected rats; n 8 stopped at day 20and 0.1 mg MIA; n 10 and saline-injected rats; n 10, stopped atday 42 (Supplementary Fig. 1). Rats were killed by an overdose ofcarbon dioxide and tissues harvested at 20 days (1 mg; n 18 and0.1 mg MIA; n 18) or 42 days (0.1 mg MIA; n 10) post-injection.Previous studies indicated that OA pathology and pain behaviourwere fully developed by 20 days after intra-articular injection of1 mg MIA17. All outcome measurements were carried out by anexperimenter blinded to intra-articular injections.

Behavioural measurements of OA pain

Pain behaviours were measured as withdrawal thresholds (g) topunctuate stimulation of the hind paw18 and as hind limb weight-bearing asymmetry. Weight-bearing asymmetry was assessed asdifference between hind limbs as a percentage of total weightborne through both hind limbs19. Measurements were obtainedimmediately prior to intra-articular injection (day 0) and at regularintervals from day 3e20 (1 mg or 0.1 mgMIA) or day 42 (saline and0.1 mg MIA).

Joint pathology

Synovia with patellae from both knees were harvested at sac-rifice and immediately embedded in optimal cutting temperature(OCT) and snap frozen over melting isopentane. Tibiofemoral jointswere then isolated and dissected to assess the severity of damage tothe chondral surfaces. Macroscopic lesions were graded using themethod of Guingamp15; grade 0 normal appearance, 1 slight

Please cite this article in press as: Nwosu LN, et al., Relationship between(OA), Osteoarthritis and Cartilage (2016),

yellowish discolouration of the chondral surface, 2 little cartilageerosion in load bearing areas, 3 large erosions extending down tothe subchondral bone and 4 large erosions with large areas ofsubchondral bone exposure. Five chondral compartments of theknee: femoral groove, medial and lateral femoral condyles andmedial and lateral tibia plateaus were scored then summated togive a maximum possible score of 20.

Following macroscopic scoring, joints were fixed in neutralbuffered formalin for 48 h, and then decalcified in 10% formic acid-formalin for 7 days at room temperature, split into anterior andposterior blocks, and embedded in paraffin. Six frontal sections perrat (three anterior and three posterior) were stained with haema-toxylin and eosin (H&E) and corresponding consecutive sections forSafranin-O-Fast green20. Chondropathy and chondrocytemorphology were scored on H&E sections whereas proteoglycancontent of the cartilagewas scored on Safranin-O Fast green stainedsections. Chondropathy was scored using the Janusz method aspreviously describ