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Second harmonic generation analysis of early Achilles tendinosis in response to in vivo mechanical loading Abraham, Thomas; Fong, Gloria; Scott, Alex Jan 26, 2011

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RESEARCH ARTICLE Open AccessSecond harmonic generation analysis ofearly Achilles tendinosis in response toin vivo mechanical loadingThomas Abraham3, Gloria Fong1,2, Alex Scott1,2*AbstractBackground: Tenocytes have been implicated in the development of tendinosis, a chronic condition commonlyseen in musculoskeletal overuse syndromes. However, the relation between abnormal tenocyte morphology andearly changes in the fibrillar collagen matrix has not been closely examined in vivo. Second harmonic generation(SHG) microscopy is a recently developed technique which allows examination of fibrillar collagen structures witha high degree of specificity and resolution. The goal of this study was to examine the potential utility of SHG andmultiphoton excitation fluorescence (MPEF) microscopy in understanding the relation between tenocytes and theirsurrounding collagenous matrix in early tendon overuse lesions.Methods: Histological preparations of tendon were prepared from adult male Sprague-Dawley rats subjected to anAchilles tendon loading protocol for 12 weeks (Rat-A-PED), or from sedentary age-matched cage controls. Secondharmonic generation and multiphoton excitation fluorescence were performed simultaneously on these tissuesections in at least three different areas.Results: SHG microscopy revealed an association between abnormal tenocyte morphology and morphologicalchanges in the fibrillar collagen matrix of mechanically loaded Achilles tendons. Collagen density and organizationwas significantly reduced in focal micro-regions of mechanically loaded tendons. These pathological changesoccurred specifically in association with altered tenocyte morphology. Normal tendons displayed a regulardistribution of fibre bundles, and the average size of these bundles as determined by Gaussian analysis was0.47 μm ± 0.02. In comparison, fibre bundle measures from tendon regions in the vicinity of abnormal tenocytescould not be quantified due to a reduction in their regularity of distribution and orientation.Conclusions: SHG microscopy allowed high resolution detection of focal tendon abnormalities affecting thefibrillar collagen matrix. With ongoing repetitive loading, these tenocyte-associated focal collagen defects couldpredispose to the progression of overuse pathology.BackgroundMultiphoton and associated microscopy methods havebeen widely used for imaging dynamic interactions incells and tissues with submicron resolution [1-3].Among these methods, the second harmonic generation(SHG) imaging method shares many of the features ofmulti photon excitation fluorescence (MPEF) micro-scopy, including identical equipment requirements andintrinsic capability of generating 3D images. Highlyordered ECM macromolecules such as the fibril-formingcollagens produce SHG signal without the need for anexogenous label. The SHG signals derived from collagenmacromolecules are strongly depend on the collagendensity, where SHG signal intensity (ISHG) scales as ISHG≈ N2 Here N is the effective number of radiating mole-cules. Likewise, isotropic macromolecules generateMPEF signal due to their endogenous fluorescence.Multiphoton excitation fluorescence intensity (IMPEF)emitted by these molecules undergoing multiphotonexcitation can be expressed as, IMPEF ≈ N. While SHGsignal intensity scales as a square of number of collagenmolecules, the MPEF intensity scales linearly with the* Correspondence: Thomas.Abraham@hli.ubc.ca1Centre for Hip Health, Vancouver Coastal Health Research Institute,University of British Columbia, Vancouver, CanadaFull list of author information is available at the end of the articleAbraham et al. BMC Musculoskeletal Disorders 2011, 12:26http://www.biomedcentral.com/1471-2474/12/26© 2011 Scott et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.number of other endogenously fluorescent molecules.Since MPEF and SHG involve different principles andcontrast mechanisms, SHG and MPEF can be capturedsimultaneously to visualize structural changes of biologi-cal macromolecules. Particularly, SHG signals originat-ing from the collagen structures can be used to generatehigh resolution images of individual collagen fibre bun-dles and quantify collagen density in a given tissue.Hence, SHG provides the potential to investigate precisespatial relations between collagen organization andunderlying tenocyte biology.Tendinosis is a histological entitity characterized bycollagen remodeling and angiofibroblastic hyperplasia.Tendons which rupture often demonstrate evidence ofpre-existing tendinosis [5]. However, initiating events inthe development of tendinosis have not been closelyexamined. There is increasing recognition of the need todetect early signs of tendon injury so that appropriateprevention strategies can be implemented, as the pathol-ogy is frequently career- or activity-limiting [6]. Theimaging modalities currently in use to investigate thestructural reorganization of collagen in tendon tissuesare primarily based on conventional light microscopy.Histochemical or immunolabeling procedures are con-sidered at best semi-quantitative, since these methodsare highly sensitive to the accessibility of the epitope inantigens recognized by the antibodies. In addition,bright field or fluorescent images of immunolabeled tis-sue samples are always convolved with significantamount of background, which make the quantificationfurther problematic. Higher resolution transmissionelectron microscopy, while able to visualize collagenalterations with greater sensitivity, imposes other limita-tions including the need to dissect samples into smallerpieces which can obscure the spatial patterns in whichabnormalities occur [7,8].Research into the early pathogenesis of tendinosis hassuggested that tenocytes may initiate degradation andremodelling of the load-bearing collagen matrix inresponse to mechanical loading. This model runs coun-ter to a traditional “wear and tear” hypothesis of col-lagen microtrauma [9]. Dudhia et al. demonstrated thatin response to cyclic physiologic loading, tendonexplants which contained viable tenocytes experienced asignificant loss of mechanical integrity, whereas underthe same loading conditions, explants without viabletenocytes did not experience a loss of load-bearing func-tion [10]. Cyclic loading has also been shown to increasethe expression of matrix metalloproteinases by teno-cytes, which would support a model of tenocyte-drivenchanges of potential relevance to understanding earlypathological change [11-13]. A cross-sectional clinicalstudies identified abnormal tenocyte morphology as apotential early feature of tendinosis [14].The goal of the current study was to determinewhether SHG microscopy could be used to examine apotential association between abnormal tenocyte mor-phology and local reductions of collagen density in arelevant in vivo model of mechanical overload. Wehypothesized that collagen density and fibrillar organiza-tion would be significantly reduced in highly localizedareas associated with abnormal tenocyte morphology.MethodsAnimalsTwelve male Sprague-Dawley rats aged 4 months, weight448.7 ± 10 grams were obtained locally and housed singlyin standard cage conditions. Following a 48 hour acclima-tization period, rats were divided into controls (n = 6,standard cage care), or runners (n = 6, standard cagecare plus treadmill running), providing 12 tendons pergroup. Ethic approval for the study was obtained fromthe UBC animal care committee (A07-0274).Tendon mechanical loadingRunners were subjected to a previously publishedmechanical loading protocol (Rat-A-PED) using a dedi-cated rat treadmill to induce Achilles tendinosis (Exer3/6, Columbus Instruments, Columbus, OH) [15]. Ratswere acclimatized to the treadmill by gradually increas-ing their exposure over a 2 week period. Subsequently,the treadmill was adjusted to a 10 degree uphill grade.Following the acclimatization period, rats ran for 1 hour/ day at 1 km / hr. After 12 weeks of exercise or seden-tary cage activity, rats rested for 72 hours.Tissue sampling and processingRats were euthanized with CO2, and whole bilateralAchilles tendons were removed with the muscle stillattached, oriented longitudinally in Tissue Tek embed-ding medium (Tissue Tek, Sakura Finetek, Torrance,CA), then frozen in isopentane chilled in liquid nitro-gen. Tendons were cryosectioned onto charged glassslides at 5 micron thickness and processed for H&E(general morphology), Alcian blue / fast nuclear red(stains sulphated glycosaminoglycans and cell nuclei), orPicrosirius red (enhances birefringence of collagen whenviewed under polarized light).Multiphoton and second harmonic generation microscopyThe microscope system used in our present experimentsis the same as described previously [1]. Specifically, thelaser used for SHG as well as the MPEF was a mode-locked femto-second Ti:Sapphire Tsunami (Spectra-Physics, Mountain View, CA) synchronously pumped by aMillenia Xs J (Spectra-Physics) diode-pumped solid-statelaser capable of delivering up to 10 W pumping power at532 nm. The power attenuated laser was directed to aAbraham et al. BMC Musculoskeletal Disorders 2011, 12:26http://www.biomedcentral.com/1471-2474/12/26Page 2 of 6Leica AOBS RS scan head (4000 Hz) coupled with Leicaupright microscope system (Heidelberg, Germany). Thelaser beam was focused on the specimen through Leciawater immersion objectives. The water immersion objec-tives used and their lateral resolution (Rxy) values follow:20X/0.7 NA (Rxy≈0.50 μm); 63X/1.2 NA (Rxy≈0.292 μm).Upon entering the Leica microscope system, the laserbeam was directed to the scanning mirrors, then througha 670 nm long pass dichroic mirror (RSP 670, Leica) andsubsequently focused on the specimens through the objec-tive lens. The backscattered emission from the sample wascollected through the objective lens. Leica Confocal Soft-ware TCS SP2 was used for the image acquisition. Non-descanned detectors and spectral scanning mode both inthe reflection geometry were used for capturing the MPEFimages as well as for the spectral signal characterizationsrespectively. In the non-descanned PMT detectors (R6357,Hamamatsu, Shizuoka, Japan), a 700 nm short pass filter(E700SP, Chroma Technology, USA) was used to preventthe scattered IR laser radiation from reaching the detectorand a 455 long pass dichroic beam splitter (455 DCXRU,Chroma Technology, USA) was used to separate SHG sig-nal from the MPEF signal. SHG signal in forward directionwas captured using a non-descanned detector in the trans-mission geometry equipped with a 440/20 nm band passfilter (MP 440/20, Chroma Technology) and high NA con-denser. All SHG and MPEF spectral data were generatedusing the de-scanned PMT detector (R6357, Hamamatsu)located inside the scan head where the emission signalswere delivered through the AOBS detection system withthe maximum confocal pinhole setting at 600 μm via thespectral dispersion prism. The width of the slits in front ofeach PMT could be software adjusted such that each PMTcould detect spectral regions spanning from a 5 nm band-width up to the overall spectral capacity of the system(400-800 nm). With this instrument configuration, a seriesof individual images were collected using a narrow detec-tion window with a width of ~5 nm, and each imagedetected at this specific emission wavelength band (i.e. ~ 5nm) provided a data point in the spectral graph. The gainand the offset of the PMTs were adjusted for optimizeddetection using the color gradient to avoid pixel intensitysaturation and background. Images (8 bit) acquired atslow scan speed i.e. 10 sec per 512 X 512 pixels.Statistical analysisLateral line profiles of collagen fibers were generatedusing Leica comprehensive image processing softwarefrom 2D spectrally clean SHG images. The sizes of thecollagen fibers and their size distribution were plottedby fitting the lateral line profiles to a Gaussian curveusing Origin Lab Software.A Mann-Whitney U test was used to detect differ-ences in collagen density and blood flow. Values areexpressed as means ± SE. Results were considered statis-tically significant if a was less than 0.05.ResultsSHG and MPEF images of normal rat Achilles tendonIn order to determine the microregional properties ofcollagen surrounding abnormal tenocytes in mechanicallyloaded tendon, we assessed standard histological prepara-tions using multiphoton and second harmonic generation(SHG) microscopy. Initially, normal tendon was analyzed.Representative images originating from spectrally cleanSHG signal originating from collagen (1A) and the MPEFsignal originating from endogenously fluorescent tissuecomponents (Figure 1B) are shown. A broad rangeof infrared laser excitation wavelengths (from 800 to900 nm), with a scan interval of 10 nm were employed todetect the SHG signal. The emission spectrum obtainedfrom the wavelength scan revealed a strong SHG signalmanifested by a narrow peak at 440 nm, which is exactlyhalf of the excitation wavelength (i.e. 880 nm). Similarinfrared laser excitation wavelength (i.e. 880 nm) wasused to generate the MPEF images. There was a strongSHG signal originating from the collagen-rich tendon-proper region. In contrast, muscle fibres generated nosignal and intramuscular connective tissue only a veryfaint signal. Likewise, paratendinous structures includingvessels and their surrounding extracellular matrix andadipose tissue, emitted virtually undetectable signals. Thisis in keeping with the known distribution of fibrillar col-lagens within these tissues [16]. Thus, when examiningmusculotendinous anatomical preparations, the SHG sig-nal was extremely specific for load-bearing collagen-richtendinous structures.Collagen fibrillar organization and tenocyte abnormalityAfter confirming the specificity of collagenous structuresvisualized with SHG in normal tendons, we next examinedthe collagen organization in sedentary and mechanicallyloaded tendons. All Achilles tendons analyzed from ani-mals subjected to the mechanical loading protocol demon-strated microregions of abnormal tenocyte morphology,typified by the appearance of tenocytes with prominentcytoplasm and rounded morphology (Figure 1D-F). Thesemorphological features were absent in normal rat Achillestendons, consistent with the original description of thisexperimental model [15]. Abnormally appearing tenocytesin tendons from rats subjected to mechanical loadingdemonstrated increased Alcian Blue staining both intra-and pericellularly (Additional file 1, Figure S1), anddecreased birefringence of Picrosirius Red stained sectionsexamined with polarized light (Figure 1H). Collagen fibresin the vicinity of abnormal tenocyte morphology displayeda reduced SHG signal level and a distinctive pericellularorganization suggestive of localized remodelling activityAbraham et al. BMC Musculoskeletal Disorders 2011, 12:26http://www.biomedcentral.com/1471-2474/12/26Page 3 of 6(Figure 1D-F, H). This abnormal appearance of fibrillarcollagen occurred in highly localized microregions sur-rounding abnormal-appearing tenocytes. These microre-gions were distributed throughout the midsubstance ofthe Achilles tendon. In contrast, normal tendon displayedregularly spaced, tightly packed fibrillar collagen, withslender spaces occupied by elongated tenocytes.Quantitative analysis of SHG signal in tendinopathySHG signals emitted from the Achilles tendon properregion in normal tendon vs tendinosis lesions were nextcharacterized by spectral analysis. The wavelength scansof both normal tendon from sedentary rats, and of ten-don surrounding abnormal tenocytes from rats subjectedto the mechanical loading protocol, showed a highlyspecific SHG signal manifested by a narrow peak at 440nm (Figure 2). The resolution of collagenous structuresvisualized with SHG could be estimated by plottingSHG signal intensity vs distance (along a digital lineplaced perpendicular to the direction of fibres). Normaltendons displayed a regular distribution of fibre bundles(Figure 2B, lower plot), and the average size of thesebundles as determined by Gaussian analysis was 0.47μm ± 0.02. In comparison, fibre bundle measures fromtendon regions in the vicinity of abnormal tenocytesdisplayed a less regular distribution and orientation,which made analysis of fibre bundle size not feasible(Figure 2B, upper plot).Figure 1 Multiphoton and SHG signals originating from tendon histological preparations. MPEF images show general tissue morphologyof tendon and associated structures, while spectrally clean SHG images specifically reveal collagen present in the same region. RepresentativeSHG (A,D), MPEF (B,E) or combined images (C,F,I) obtained from standard histological thin tissue section are shown. In normal healthy tendon(A-C), SHG demonstrates tightly bundled, longitudinally oriented collagen fibres. Tenocytes (B, C, asterisk) are inconspicuous due to their sparsecytoplasm. In early tendinosis tendon, collagen surrounding abnormal tenocytes (D-F) demonstrates a disturbed organization, and the averageSHG signal is greatly reduced (c.f. panel A). The tenocytes in tendinosis tendon demonstrate much more prominent cytoplasm and a rounded,as opposed to spindle-shaped, morphology (E). Picrosirius red-stained tendon from tendinosis tendon (G, brightfield; H, polarized light) is shownas a comparison. Tendinosis regions demonstrate a loss of birefringence (H). In comparison, SHG signal reveals the presence of abnormalpericellular fibrillar collagen structures not visualized with polarized light. Scale bars represent 25 μm for A-H, and 10 μm for I.Abraham et al. BMC Musculoskeletal Disorders 2011, 12:26http://www.biomedcentral.com/1471-2474/12/26Page 4 of 6We then applied a noise removal filter to SHG images todefine the boundary between foreground and background,and the lower threshold in the histogram was set to 10%of the highest pixel intensity value. The total SHG signalintensity values thus generated were normalized by thecropped collagen area (μm2) to yield a mean collagen den-sity value. The collagen density from micro-regions corre-sponding to abnormal tenocytes in rats subjected to theoveruse protocol was 729 ± 90.3 AU, compared to thevalue of 1135 ± 60.1 in normal tendon (p < 0.05).DiscussionSecond harmonic generation (SHG) resulted in high reso-lution, spectrally clean signals originating from fibrillarcollagen. The collagen matrix surrounding abnormal teno-cytes demonstrated an abnormal organization and areduced density compared to sedentary cage controls.This reduction in SHG signal occurred specifically in asso-ciation with the appearance of abnormal tencoytes in thetendons subjected to mechanical loading. These resultsprovide support to an emerging model of tendinosiswhere degradation and/or remodelling of the load-bearingcollagen matrix in response to exercise is mediated bythe local activity of tenocytes, rather than being due to apassive accumulation of fatigue damage [10,14].Glazebrook et al. recently reported increased collagenstaining using this overuse tendinopathy model basedon assessment of H&E preparations [15]. However, weare confident that the reduced SHG signal observed inthe current study accurately reflects a loss of collagendensity in the tendinosis samples. Indeed, H&E evalua-tion of tendinosis samples may be misleading as tendi-nosis lesions can be highly eosinophilic, but we havefound these eosinophilic regions to generate very weakor absent SHG signals indicating that they may com-prise provisional matrix with a higher component ofnon-collagenous elements. We are therefore able here torefine and further validate the description of earlypathology which occurs in this model.In the current study, SHG was used to estimate thecollagen fibre bundle diameter with submicron resolu-tion in normal tendons, but not in tendinosis samples.The longitudinal alignment of collagen fibres wasreduced in the tendinosis tendons, in association with areduction of SHG signal intensity. The technique ofmeasuring fibre bundle diameter relied on the fibresdemonstrating an orderly, longitudinal orientation,which was disrupted in areas of tendinosis.ConclusionsSecond harmonic generation analysis of mechanicallyloaded tendon provides the ability to examine collagenorganization in standard histological preparations, pro-viding insight into mechanisms of early load-inducedtendon changes. Tenocyte responses to mechanical load-ing in vivo are accompanied by highly localized foci ofreduced collagen density which may predispose to theprogression of overuse tendinopathy.Additional materialAdditional file 1: Figure 1S Glycosaminoglycan distribution inmechanically loaded and sedentary tendon. Alcian blue staining intendinosis (A) and normal healthy (B) tendon. Tendinosis tendondemonstrates increased sulphated glycosaminoglycan intra- and peri-Figure 2 Analysis of SHG signal intensity in rat Achilles tendonfrom collagen surrounding normal or abnormal tenocytes.(A) Three representative spectral scans from a single normal (solidline) and a single tendinosis tendon (dashed line) are shown. TheSHG signal arising from the tendon proper region peakedconsistently at 440 nm as expected. (B) When SHG signal intensitywas plotted as a function of position along a line drawnperpendicular to the collagen fibre bundles, the fibre bundle sizecould be estimated with sub-micron resolution in normal tendon,but not in tendinosis tendons.Abraham et al. BMC Musculoskeletal Disorders 2011, 12:26http://www.biomedcentral.com/1471-2474/12/26Page 5 of 6cellularly, particularly in chains of abnormal-appearing tenocytes. Mastcells also stain blue with this technique in the paratendon regions (notshown). 20× objective lens.AcknowledgementsThe study was funded by the University of British Columbia operating funds.Infrastructure support was provided by the Centre for Hip Health andMobility and the James Hogg Centre for Cardiovascular Research. TA wassupported by Providence Healthcare Society. We thank Ingrid Barta forhistology services, and the Jack Bell Centre animal care staff.Author details1Centre for Hip Health, Vancouver Coastal Health Research Institute,University of British Columbia, Vancouver, Canada. 2Department of PhysicalTherapy, University of British Columbia, Vancouver, Canada. 3James HoggResearch Centre-St Paul’s Hospital, Vancouver, Canada.Authors’ contributionsAS and TA conceived of the study. AS supervised the tendon mechanicalloading protocol and carried out the imaging and histological examination.GF assisted with imaging analysis. TA and AS conducted the image analysis.TA designed and oversaw the SHG and MPEF microscopy. All authors readand approved of the final manuscript.Authors’ informationAlex Scott is a physical therapist engaged in full time research and teaching.Gloria Fong is a Research Assistant with a BSc in Medical and LaboratorySciences. Thomas Abraham is a specialist in optical physics engaged in fulltime research.Competing interestsThe authors declare that they have no competing interests.Received: 23 August 2010 Accepted: 26 January 2011Published: 26 January 2011References1. Abraham T, Carthy J, McManus B: Collagen matrix remodeling in3-dimensional cellular space resolved using second harmonic generationand multiphoton excitation fluorescence. J Struct Biol 2010, 169:36-44.2. Strupler M, Pena AM, Hernest M, Tharaux PL, Martin JL, Beaurepaire E,Schanne-Klein MC: Second harmonic imaging and scoring of collagen infibrotic tissues. Opt Express 2007, 15:4054-4065.3. Zipfel WR, Williams RM, Christie R, Nikitin AY, Hyman BT, Webb WW: Livetissue intrinsic emission microscopy using multiphoton-excited nativefluorescence and second harmonic generation. Proc Natl Acad Sci USA2003, 100:7075-7080.4. 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Woo SL, Tkach LV: The Cellular and Matrix Response of Ligaments andTendons to Mechanical Injury. In Sports Induced Inflammation. Edited by:Leadbetter W, Buckwalter J, Gordon SE, Parkridge, IL. American Academy ofOrthopaedic Surgeons; 1990:189-203.10. Dudhia J, Scott CM, Draper ER, Heinegard D, Pitsillides AA, Smith RK: Agingenhances a mechanically-induced reduction in tendon strength by anactive process involving matrix metalloproteinase activity. Aging Cell2007, 6:547-556.11. Yang G, Im HJ, Wang JH: Repetitive mechanical stretching modulatesIL-1beta induced COX-2, MMP-1 expression, and PGE2 production inhuman patellar tendon fibroblasts. Gene 2005, 363:166-172.12. Archambault J, Tsuzaki M, Herzog W, Banes AJ: Stretch and interleukin-1beta induce matrix metalloproteinases in rabbit tendon cells in vitro.J Orthop Res 2002, 20:36-39.13. Thornton GM, Shao X, Chung M, Sciore P, Boorman RS, Hart DA, Lo IK:Changes in mechanical loading lead to tendon-specific alterations inMMP and TIMP expression: Influence of stress-deprivation andintermittent cyclic hydrostatic compression on rat supraspinatus andAchilles tendons. Br J Sports Med 2010, 44:698-703.14. Cook JL, Feller JA, Bonar SF, Khan KM: Abnormal tenocyte morphology ismore prevalent than collagen disruption in asymptomatic athletes’patellar tendons. J Orthop Res 2004, 22:334-338.15. Glazebrook MA, Wright JR Jr, Langman M, Stanish WD, Lee JM: Histologicalanalysis of achilles tendons in an overuse rat model. J Orthop Res 2008,26:840-846.16. Josza L, Kannus P: Human Tendons. Champaign, Illinois: Human Kinetics;1997.Pre-publication historyThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/12/26/prepubdoi:10.1186/1471-2474-12-26Cite this article as: Abraham et al.: Second harmonic generation analysisof early Achilles tendinosis in response to in vivo mechanical loading.BMC Musculoskeletal Disorders 2011 12:26.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/submitAbraham et al. BMC Musculoskeletal Disorders 2011, 12:26http://www.biomedcentral.com/1471-2474/12/26Page 6 of 6

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