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Uphill treadmill running does not induce histopathological changes in the rat Achilles tendon Dirks, Rachel C; Richard, Jeffrey S; Fearon, Angela M; Scott, Alexander; Koch, Lauren G; Britton, Steven L; Warden, Stuart J Mar 11, 2013

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RESEARCH ARTICLE Open AccessUphill treadmill running does not inducehistopathological changes in the ratAchilles tendonRachel C Dirks1,2, Jeffrey S Richard1,3, Angela M Fearon4,5, Alexander Scott4,5, Lauren G Koch6,Steven L Britton6 and Stuart J Warden1,2,3*AbstractBackground: The purpose of this study was to investigate whether uphill treadmill running in rats createdhistopathological changes within the Achilles tendon consistent with Achilles tendinosis in humans.Methods: Twenty-six mature rats selectively bred for high-capacity running were divided into run and cage controlgroups. Run group rats ran on a treadmill at a 15° incline for a maximum duration of 1 hr/d, 5 d/wk for 9 weeks atincreasing speeds, while rats in the cage control group maintained normal cage activity. After 9 weeks, Achillestendons were harvested for histological processing and semi-quantitative histopathological analysis.Results: There were no significant group differences within each of the individual histopathological categoriesassessed (all p≥ 0.16) or for total histopathological score (p = 0.14).Conclusions: Uphill treadmill running in rats selectively bred for high-capacity running did not generate Achillestendon changes consistent with the histopathological presentation of Achilles tendinosis in humans.Keywords: Animal model, Tendinitis, Tendinopathy, Tendinosis, OveruseBackgroundThe Achilles tendon connects the muscles of the calf(gastrocnemius and soleus) to the calcaneus, and is thelargest and strongest tendon in the body. Functioning totransmit the muscle contractile forces necessary for hu-man stance and locomotion, the Achilles tendon mustbe able to withstand large-magnitude tensile loads. Al-though structurally designed to withstand these loads,injuries of the Achilles tendon thought to result from re-petitive loading are common [1-5]. The most common ofthese injuries is termed Achilles tendinopathy (tendo– =tendon, –pathy = disease).Achilles tendinopathy refers to a clinical conditioncharacterized by activity-related Achilles tendon pain asso-ciated with focal tendon tenderness and intratendinous im-aging changes. The underlying pathology has historicallybeen thought to be one of inflammation and the conditionhas traditionally been labeled as ‘Achilles tendinitis’. How-ever, histopathological studies have consistently shown thepathology underlying tendinopathy to be one of progres-sive tendon degeneration (tendinosis) rather than inflam-mation (tendinitis) [6-8]. Thus, the use of the termtendinosis is preferred when describing the pathology asso-ciated with Achilles tendinopathy.Despite consistent identification of the pathologyunderlying Achilles tendinopathy, little is known aboutthe pathological process/es taking place within the ten-don. This limited knowledge has restricted treatmentoptions, with clinical management presently being moreof an art than a science. In order to address this void, asuitable animal model of Achilles tendinosis is required.As knowledge regarding human Achilles tendinosis iscurrently centered around its histopathological features,a suitable animal model is one in which the histopatho-logical features of the injured animal Achilles tendonreplicate those observed in the human condition.* Correspondence: stwarden@iupui.edu1Center for Translational Musculoskeletal Research, School of Health andRehabilitation Sciences, Indiana University, Indianapolis, IN, USA2Department of Anatomy and Cell Biology, Indiana University School ofMedicine, Indianapolis, IN, USAFull list of author information is available at the end of the article© 2013 Dirks et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Dirks et al. BMC Musculoskeletal Disorders 2013, 14:90http://www.biomedcentral.com/1471-2474/14/90Treadmill running represents a potential means of re-petitively loading tendons in rats to induce histopatho-logical changes. Although established for the generationof supraspinatus tendinosis [9], treadmill running hashad variable success in developing tendinosis-like changesin rat Achilles tendons [10-15]. Soslowsky and colleagues[13] used the same downhill (10º decline) treadmill runningprogram (17 m/min, 1 hr/d, 5 d/wk for up to 16 weeks)as they used to induce supraspinatus tendinosis in an un-successful attempt to induce mechanical and geometricchanges within the rat Achilles tendon. A possible expla-nation for the lack of an effect may be that downhill run-ning in quadrupeds results in a forward shift of the centerof mass [16] resulting in increased forelimb loading (andelevated subacromial compression) combined with a rela-tive decrease in hindlimb loading.Glazebrook et al. [11] replicated the same runningprogram as Soslowsky and colleagues [13], but furtheredthe work by running rats uphill (10º incline) rather thandownhill for up to 12 weeks. Uphill running requires thecalf muscles (and other antigravity muscles) to contractconcentrically to raise the center of mass with each step.The net result may be increased Achilles tendon loadingas the increased muscle forces are transmitted to theskeleton. Glazebrook et al. [11] showed uphill runningresulted in histological changes consistent with humanAchilles tendinosis, including a reduction in collagenorganization and an increase in tenocyte number [11].However, the latter observations were not replicated byHeinemeier et al. [12] who completed a comprehensivestudy using the same uphill running program, but withthe modification increased running speed (20 m/min).The aim of this study was to build upon these previousstudies and investigate whether uphill treadmill runningat a higher (15º) incline and speed (up to 30 m/min) cre-ates histopathological changes within the rat Achillestendon consistent with Achilles tendinosis in humans.Specifically, we assessed the effect of uphill treadmill run-ning on Achilles tendon calcification, adipocytes, synoviumattached to the tendon, collagen arrangement, tenocytemorphology, cellularity, and vascularization in rats selec-tively bred for high-capacity running (HCR).MethodsEthics statementAll procedures were performed following a priori approvalfrom the Indiana University Institutional Animal Care andUse Committee (Animal Welfare Assurance #A4091-01).AnimalsTwenty-six male HCR rats (age = 24.8 ± 3.2 wk; weight =374.0 ± 30.3 g) were acquired from the University ofMichigan (Ann Arbor, MI) and acclimated for 2 weeks.HCR rats have been artificially selected for aerobic capacityfrom a founder population of genetically heterogeneous N:NIH rats [17]. Animals in the current study were from the26th generation of HCR rats, with this strain of rat beingused due to their known ability to run long distances. Allanimals were maintained under standard conditions andprovided ad libitum access to food and water.Treadmill runningAnimals were randomly divided into two groups: cagecontrol (n = 11) and running (n = 15). Rats in the cagecontrol group maintained normal cage activity through-out the duration of the study. Rats in the running groupran 5 d/wk for 9 weeks on a treadmill at a 15° incline.Rats were acclimated to the treadmill initially startingwith 5 minutes at 10 m/min. The duration and speed ofrunning were gradually increased throughout weeks 1and 2 until the rats were running for 60 minutes at25 m/min (Table 1). The duration was kept constant forthe remainder of the study while speed was progressivelyincreased up to 30 m/min by the final week of running.HistologyAnimals were euthanized after 9 weeks of the running regi-men, and one of their Achilles tendons was harvested andfixed in 10% neutral buffered formalin for 48 hours beforebeing transferred to 70% ethanol. Tendons were embeddedin paraffin, and 6 μm thick midsubstance sagittal sectionsTable 1 Running protocol used for the running group ofratsDuration (min) Speed (m/min)Week 1 Day 1 5 10Day 2 5 10Day 3 5 10-15Day 4 5 10-25Day 5 30 15-25Week 2 Day 6 45 15-25Day 7 50 15-25Day 8 55 15-25Day 9 60 15-25Day 10 60 15-25Week 3 60 15-25Week 4 60 20-25Week 5 60 20-25Week 6 60 20-25Week 7 60 20-27.5Week 8 60 20-27.5Week 9 60 20-30Hyphenated values in the speed column represent the gradual increase inspeed at the beginning of each running session. Running began at the lowerspeed and was increased by 1 m/min each minute until the higher speed wasachieved. Rats ran at this higher speed for the remainder of the duration.Dirks et al. BMC Musculoskeletal Disorders 2013, 14:90 Page 2 of 5http://www.biomedcentral.com/1471-2474/14/90were cut using a microtome and stained with hematoxylinand eosin. Sections were viewed on a Zeiss Axiophot lightmicroscope and tendon damage assessed using a modifiedBonar histopathology scale [18-20]. The entire slide wasassessed for the presence of adipocytes, synovial lining, andcalcification. These three characteristics were graded either0 (not present) or 1 (present). Following this assessment,the most pathological region of the tendon was determinedbased upon collagen arrangement under polarized light. Inthis region, tendons were graded for collagen arrangement(one field of view at 100x), tenocyte morphology (fourfields of view at 200x), cellularity (one field of view at100x), and vascularity (up to 10 fields of view at 400x). Agreater number of fields were viewed for characteristics re-quiring assessment at higher magnification in an effort tograde a similar total area of tissue for each outcome. Thecharacteristics were graded between 0 (normal) and 3(maximum pathology). The sum of all 7 categories gave acompletely normal tendon a score of 0 and a tendon withmaximum damage a score of 15. Samples were randomizedand graded by two independent blind examiners (R.C.D.and A.M.F.). Discrepancies in scoring were resolved by dis-cussion, with a third examiner (A.S.) being consulted whenconsensus could not be reached.StatisticsStatistical analyses were performed using the StatisticalPackage for Social Sciences (SPSS 19.0; IBM) software,with tests being two-tailed with a level of significance setat 0.05. Kolmogrov-Smirnov and Levene tests were usedto test for the presence of a normal distribution andhomogeneity of variance, respectively. Mann–WhitneyU tests were used to compare histopathological scoresbetween groups because the data did not meet the as-sumptions required for parametric statistics.ResultsAnimals in the running group ran on the treadmill anaverage of 51.2 ± 7.5 km during the study (Figure 1).Tendons from the cage control and running groups weresimilar on gross histological appearance (Figure 2). Therewas 70-80% agreement in initial grade between examinersfor each histological characteristic. There were no groupdifferences within each of the individual histopathologicalcategories assessed (all p > 0.16) or for total histopatho-logical score (p = 0.14) (Table 2).DiscussionHistopathological evaluation of the tendon specimensfailed to differentiate between the control rats and therunning rats. These data suggest that uphill treadmillrunning in rodents may not be a suitable animal modelfor the study of human Achilles tendinosis. Achillestendinosis in humans is characterized by tissue degen-eration with a failed reparative response [6-8]. Thesechanges are identified histologically as collagen fiberdisorganization, hypercellularity with atypical tenocyteproliferation and morphology, and neovascularization[7,21,22]. We did not observe changes in these or other0102030405060700 1 2 3 4 5 6 7 8 9Average cumulative running distance (km)WeeksFigure 1 Cumulative distance ran on the treadmill by rats inthe run group. Error bars represent standard deviation.Figure 2 Representative photomicrographs of the Achillestendon in the A) run group and B) control group. Note theuniform appearance of tightly packed, well-aligned collagen fibrilswith interspersed, spindle-shaped tenocytes aligned parallel to thefibrils in the tendons from both groups (Stain = Hematoxylin &Eosin; Magnification = 500x).Table 2 Differences (mean ± SD) in individualhistopathological categories and total histopathologicalscore in Achilles tendons from cage control and run groupsHistopathological characteristic Cage control Run p-valueAdipocytes 0.09 ± 0.30 0 ± 0 0.69Synovial lining 0.64 ± 0.50 0.57 ± 0.51 0.74Calcification 0 ± 0 0 ± 0 1.00Collagen arrangement 1.91 ± 0.83 1.70 ± 0.70 0.38Tenocyte morphology 0.91 ± 0.70 0.78 ± 0.60 0.66Cellularity 1.37 ± 0.50 1.00 ± 0.60 0.16Vascularity 0.91 ± 0.83 0.78 ± 0.95 0.61Total score 5.82 ± 1.94 4.83 ± 1.83 0.14Dirks et al. BMC Musculoskeletal Disorders 2013, 14:90 Page 3 of 5http://www.biomedcentral.com/1471-2474/14/90individual histopathological categories for human Achillestendinosis in the current animal study.Uphill treadmill running in rats has had variable suc-cess in producing tendinosis-like changes in the Achillestendon, with some investigators reporting preliminarytendinosis-like changes [10,11,15] whereas others reportingno evident pathology [12]. The current study used a run-ning program (15° incline with speeds of up to 30 m/min)seemingly more intense than these previous studies, butwas unable to find histopathological evidence of Achillestendinosis. These findings support those of Heinemeieret al. [12] who found that an uphill running program hadno effect on the histological appearance and actually im-proved some mechanical properties of the rat Achilles ten-don. These cumulative data suggest that uphill treadmillrunning in isolation in rats is unable to induce the samehistopathological changes as observed in human Achillestendinosis. Ng et al. [14] recently described a unique bi-pedal running model wherein rats ran at 17 m/min on atreadmill at a 20° decline, but with the animals in an up-right posture. This model resulted in a decrease in Achillestendon mechanical properties as well as histologicalchanges associated with human Achilles tendinosis; how-ever, the model has yet to be replicated.One of the strengths of our study was the age of theanimals which were older than those used in previousstudies. The use of older animals may facilitate the de-velopment of tendon pathology if it occurs. Anotherstrength of our study was the use of animals selectivelybred for aerobic capacity. The use of HCR rat enabledus to run our animals at a greater incline and at a fasterpace than in previous studies, with the intent that theseparameters would potentiate the generation of Achillestendon degeneration. However, this study strength mayalso be a weakness as the selective breeding of our ani-mals for aerobic capacity may also have led to the devel-opment of a tendon phenotype that enhanced tendonresistance to degeneration. Similarly, our study was lim-ited by its relatively small number of animals and limitedoutcome measures. We do not believe increasing oursample size would have altered the study conclusions asthe total histopathological score in treadmill ran rats inthe current study were actually about 25% better than incage controls. Similarly, we do not believe includingadditional outcome measures are indicated at this stageas histological tendon changes are considered the car-dinal sign of the human condition we were attemptingto replicate. A final limitation of the current study wasthe relatively prolonged treadmill acclimation periodwhich lasted 2 weeks and subsequent relatively shortperiod (7 weeks) of running at full speed and duration.These factors may have potentiated tendon adaptationto running and/or limited the ability to produce detect-able pathology.ConclusionsIn summary, we were unable to identify histopathologicalchanges in the Achilles tendon of rats that ran uphill on atreadmill. These cumulative data suggest that uphill run-ning in isolation in rats is unable to induce the same histo-pathological changes as observed in human Achillestendinosis.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsRCD and SJW conceived the project; LGK and SLB provided animals; RCD,JSR and SJW performed treadmill running; RCD, AMF and AS performedhistological assessments; RCD wrote the first draft of the manuscript. Allauthors participated in the trial design, provided feedback on drafts of themanuscript, and read and approved the final manuscript.Author details1Center for Translational Musculoskeletal Research, School of Health andRehabilitation Sciences, Indiana University, Indianapolis, IN, USA. 2Departmentof Anatomy and Cell Biology, Indiana University School of Medicine,Indianapolis, IN, USA. 3Department of Physical Therapy, School of Health andRehabilitation Sciences, Indiana University, 1140 W. Michigan St., CF-326,Indianapolis, IN 46202, USA. 4Centre for Hip Health and Mobility, VancouverCoastal Health and Research Institute, Vancouver, BC, Canada. 5Departmentof Physical Therapy, University of British Columbia, Vancouver, BC, Canada.6Department of Anesthesiology, University of Michigan Medical School, AnnArbor, MI, USA.Received: 29 December 2012 Accepted: 6 March 2013Published: 11 March 2013References1. Kannus P: Etiology and pathophysiology of chronic tendon disorders insports. 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Acta Orthop Scand 1997,68:170–175.doi:10.1186/1471-2474-14-90Cite this article as: Dirks et al.: Uphill treadmill running does not inducehistopathological changes in the rat Achilles tendon. BMCMusculoskeletal Disorders 2013 14:90.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitDirks et al. BMC Musculoskeletal Disorders 2013, 14:90 Page 5 of 5http://www.biomedcentral.com/1471-2474/14/90


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