Synovial Membrane Microarthroscopy of the Equine Midcarpal

Joint

ALBERTO SERENA, DMV, MS, MRCVS, R. REID HANSON, DVM, Diplomate ACVS & ACVECC, andSTEVEN A. KINCAID, DVM, PhD

Objective—To evaluate the value of microarthroscopy in the equine midcarpal joint using the vitalstains methylene blue, trypan blue, neutral red, and Janus green B to observe components of thesynovial lamina propria, vascular architecture, and synoviocytes.Study design—Experimental.Animals—Ten horses.Methods—Microarthroscopy of left and right midcarpal joints was performed with and withoutvital staining of the synovium. Four vital stains (methylene blue, trypan blue, neutral red, and Janusgreen B) were evaluated, with each stain used in 5 joints. Synovial biopsy specimens were collectedfrom the dorsomedial and dorsolateral aspects of the joint.Results—All dyes were biocompatible. At �60 without vital staining, synovial surface topography,vascular network, and translucency were observed. Intra-articular vital dyes improved evaluation ofsynovial surface topography. At �150 with vital staining, individual synoviocytes were clearlyidentified with all dyes, except neutral red. Although methylene blue provided the best in vivomicroscopic differentiation of the structure of the intima, trypan blue had superior retention inconventionally processed synovial biopsies.Conclusions—Methylene blue, trypan blue, neutral red, and Janus green B stains can be used safelyfor microarthroscopy. Good visualization of cells and vascular network can be obtained by mi-croarthroscopy, and microarthroscopic evaluation of the synovium compares favorably with con-ventional histologic evaluation of biopsy specimens.Clinical relevance—Microarthroscopy may be beneficial in both research and clinical diagnosis ofequine articular diseases.r Copyright 2005 by The American College of Veterinary Surgeons

Key words: horse, joint pathology, synovial membrane, microarthroscopy, vital stains.

INTRODUCTION

EQUINE MUSCULOSKELETAL injuries, in par-ticular those affecting synovial joints, are the most

common reasons for poor performance and wastage.1

Even in non-racing horses, lameness associated with jointdisease is the most common cause of early retirement.2

The synovial membrane has a central role in the patho-genesis of inflammatory joint diseases of the horse.3

The synovial membrane is composed of an intimamade of 1–4 layers of synoviocytes and a subintima orlamina propria. In addition to blood vessels, lymphaticsand nerves, the subintima is composed of connectivetissue types (areolar, fibrous, adipose) that determine

Study supported by the Birmingham Racing Association, Birmingham, AL.

Presented at the American College of Veterinary Surgeons Forum, Denver, CO, October 2004, and at the Veterinary Orthopedic

Society Conference, Snowmass, CO, March 2005.

Address reprint requests to Alberto Serena, DMV, J.T. Vaughan Large Animal Teaching Hospital, College of Veterinary Medicine,

Auburn University, 1500 Wire Road, Auburn, AL 36849. E-mail: serenal@auburn.edu.

Submitted December 2004; Accepted February 2005

From the Departments of Clinical Sciences, and Anatomy, Physiology, and Pharmacology, College of Veterinary Medicine, Auburn

University, Auburn, AL.

r Copyright 2005 by The American College of Veterinary Surgeons

0161-3499/04

doi:10.1111/j.1532-950X.2005.00048.x

310

Veterinary Surgery

34:310–317, 2005

synovial classification. Areolar type synovial membranehas a thick intima and abundant blood vessels, numerousfolds and villi, and responds to inflammation primarilyby an increase in the number of blood vessels.4 Fibroustype synovium usually serves as a gliding surface of thejoint and contains minimal numbers of synoviocytes andvessels whereas adipose type synovium represents a tran-sitional area and has various surface folds.4 Areolar typesynovium is more metabolically active than the fibrousand adipose types.

Synoviocytes are a functionally heterogeneous popu-lation of cells.5,6 There are 3 types of synoviocytes withinthe synovial intima of the horse: type A synoviocytes arephagocytic cells, type B synoviocytes have fibroblastic andsecretory properties, and type C synoviocytes represent atransitional form between types A and B synoviocytes.5

Synovial membrane evaluation is typically performedby microscopy and arthroscopy. Optical and electronmicroscopy require small, chemically preserved samplesof synovial membrane and yield detailed information onmorphology and metabolic status of synoviocytes, andvillus vasculature. These methods use isolated tissue sam-ples and thus only provide limited regional informationthat may not necessarily be representative of an articulardisease process.

Using conventional arthroscopy, gross inflammationof the equine synovium typified by synovial hyperemiaand hypertrophy can be characterized. These relativelynon-specific signs of inflammation are commonly ob-served with intra-articular fractures, septic arthritis, anddegenerative joint disease and, to a minor extent, withosteochondritis dissecans (OCD).3 Depending on jointanatomy, conventional arthroscopy permits evaluation ofmost of the synovial membrane in vivo, but at magni-fications � � 10 so the synovial microarchitecture can-not be evaluated and only subjective evaluation ofinflammatory status is possible.

To improve resolution of in vivo alterations an art-hroscope with magnification similar to that of a lightmicroscope (up to � 150) has been used in humans.7

Arthroscopic observation of the joint at these magnifica-tions permits excellent histologic examination of thesynovium in vivo. Thus, microarthroscopy has potentialvalue for both research on, and clinical diagnosis of,equine joint diseases, by bridging the gap that existsbetween conventional arthroscopy and histologic evalua-tion. In humans, microarthroscopy allows precise mor-phologic differentiation among types of joint disease,highlighting alterations of the synovial membrane typicalof osteoarthritis (OA), post-traumatic synovitis, and rheu-matoid arthritis.8

Microarthroscopic diagnostic criteria used in humanmedicine are synovial membrane translucency, characterof the vascular network, synoviocyte number and mor-

phology.7–9 Normal human synovium is translucent andcontains tortuous vessels and star-shaped cells (type Asynoviocytes). In OA, the synovium is translucent/opaque,blood vessels are straight and hypertrophied, with evidentneovascularization, and cells are numerous and round inshape (type B synoviocytes).10 Rheumatoid arthritis ischaracterized by villous hyperplasia, the synovial mem-brane is opaque because of congestion and edema, thevascular plexus is irregular with no clear outline, andsynoviocytes are numerous and round (type B).

A sterile 1% solution of methylene blue is typicallyused for human microarthroscopy.7 Other vital stainshave not been evaluated after intra-articular administra-tion; however, differing affinities for cells and intracellu-lar organelles could make real time identification of cellspossible during microarthroscopy. Dyes that can be usedare either intravital dyes composed of particles that areengulfed by phagocytic cells or supravital dyes that dif-fuse into the cell cytoplasm to localize in vacuoles ororganelles.11 Trypan blue (1% solution) is an intravitaldye used to localize phagocytic cells and supravital dyesinclude methylene blue, Janus green B, and neutral red.Methylene blue stains the nucleus, Janus green B localizesin mitochondria, and neutral red accumulates in lyso-somes.11 Thus using dye properties11 evaluation of equinesynovial membrane to determine synoviocyte distribution(type A and B) and functional/metabolic status of resi-dent cells may be possible.

Precise morphologic characterization of normal equinesynovial membrane in vivo using microarthroscopic tech-niques may contribute to the understanding of the syno-vium’s role in joint homeostasis. Microarthroscopicstudies might allow characterization of morphologicchanges that occur in vivo during pathologic processes,thus the morphologic response of the synovium to a par-ticular intra-articular or systemic treatment could bedocumented by in vivo observation.12

Use of microarthroscopic techniques for diagnosis ofequine joint disease remains undefined.3 Our goals wereto evaluate microarthroscopy in horses and to provide aprecise microarthroscopic morphologic characterizationof normal equine synovium. An additional goal was toevaluate the ability of intra-articularly administered vitalstains to aid in identification of cell types.

MATERIALS AND METHODS

Ten healthy adult horses (4360 kg) of mixed breed andsex, with no history or signs of carpal joint disease were stud-ied. All horses had complete physical and lameness examina-tions performed to determine health status, both midcarpaljoints were radiographed, and synovial fluid was collected forevaluation of cell count and total protein concentration.Criteria for inclusion in the study included an absence of

311SERENA, HANSON, AND KINCAID

lameness at a trot on a hard surface, both in a straight line andin a circle, and at a trot after carpal flexion (60 seconds), noradiographic abnormalities, and normal synovial fluid analysis(total leukocytes o500 cells/mL, total protein o2.5 g/dL).

Arthroscopy

Horses were anesthetized, positioned in dorsal recumbency,and both carpi aseptically prepared for surgery. To avoidinflammation and morphologic changes of the synovium,synovial fluid samples were collected immediately before art-hroscope insertion. An 18-cm-long, 4-mm-diameter, 301 for-ward viewing, contact microarthroscope (Karl Storz ‘‘Andrea-Dias’’, Karl Storz Veterinary Endoscopy, Goleta, CA; Fig 1)was used. A lateral arthroscopic portal was used, halfway be-tween the extensor carpi radialis tendon and the commondigital extensor tendon, and midway between the proximaland distal rows of carpal bones with the joint flexed at 901.13

The microarthroscope provided magnification up to� 150. A hand piece on the telescope was progressivelyturned in a clockwise direction to increase magnification andin a counterclockwise direction to return to a panoramic viewof the joint cavity. The tip of the scope was maintained inclose contact with the synovium at higher magnification andthis was difficult, but was facilitated by stopping fluid ingress.Panoramic observation of the joint cavity was initially per-formed without vital staining. Increased magnification wasused to observe microscopic details of the synovium (e.g.,erythrocytes); however, identification of most cells (synovio-cytes) required vital staining. Microarthroscopic images weredigitally recorded and stored (Pro 2 DVD, Stryker Endos-copy, San Jose, CA).

After examination of the synovial surface and synovial bi-opsy, skin portals were closed with non-absorbable suture inan interrupted vertical mattress pattern. Horses were recov-ered from anesthesia and observed daily for 15 days for jointeffusion and for lameness at the walk. In addition, from days5–15, carpi were flexed daily for 1minute, and horses weretrotted on a hard surface both in a straight line and in circle.Both carpi were bandaged for 5 days. Horses were adminis-tered phenylbutazone (4.4mg/kg orally once daily for 3 days)after surgery. Skin sutures were removed after 12 days.

Vital Staining

Filtered (0.22mm filter, Millex-GV, Millipore, Carrigtwo-hill, Ireland) 1% sterile solutions of methylene blue (Eastam

Kodak Co., Rochester, NY), trypan blue, neutral red, andJanus green B (Sigma-Aldrich Co., St. Louis, MO) were used.Vital stains were obtained as a pure powder and diluted to 1%in an isotonic solution. Each stain (5mL/joint) was injectedinto 5 different joints through an 18-g needle inserted intra-articularly medial to the extensor carpi radialis tendon. Afterinjection the joint was repeatedly flexed and extended slightlyto allow stain diffusion and homogeneous contact with theentire synovium. Five minutes later the joint was thoroughlyrinsed through the arthroscopic cannula with sterile Normo-sol-R solution (Abbott Laboratories, Abbott Park, IL) andinspection of the joint at increasing magnification was per-formed systematically from medial to lateral. To obtain acontact view, the tip of the scope was gently pushed againstthe area of the synovium to be evaluated, and then magni-fication increased.

Synovial Biopsy

Synovial membrane was collected from the dorsolateraland dorsomedial aspects of the synovium for histologic eval-uation. Four specimens were collected from each joint using a6 � 12mm Ferris-Smith rongeur (Scanlan, Saint Paul, MN).Two specimens were collected from the lateral and 2 from themedial aspect of the joint. Histologic evaluation of synoviumspecimens was used to substantiate, compare, and contrastmicroscopic structure with microarthroscopic images to de-termine what morphologic features could be observed in vivo.

Specimen Preparation

Two different techniques were used to determine the bestmethod for histologic evaluation of vitally stained synovialbiopsies. Specimens were fixed in buffered 10% formalin andin 2-methylbutane cooled by liquid nitrogen. Formalin-fixedspecimens were dehydrated in graded alcohol and cleared inmethyl salicylate.14 A single-edged razor blade was used to cutthin sections from the tissue specimens biopsies that weremounted in a synthetic resin on microscopic glass slides forviewing. Sections (15mm) were cut from frozen specimens us-ing a cryostat then mounted on microscopic glass slide. Ad-ditional specimens from 1 horse were prefixed in ammoniummolybdate before primary fixation in buffered 10% formalinto stabilize methylene blue staining of the tissues.15

RESULTS

Technically, microarthroscopy was comparable toconventional arthroscopy, the only difference being thatthe tip of the scope must be in contact with the synoviumat higher magnification (contact view). Mild subcutane-ous edema of the left carpus from fluid extravasation in 2horses resolved within 3–4 days. No other adverse effectsassociated with microarthroscopy occurred.

Fig 1. Karl Storz ‘‘Andrea-Dias’’ microarthroscope. The

hand piece is turned in clockwise direction to increase the

magnification.

312 MICROARTHROSCOPY OF EQUINE MIDCARPAL JOINT

Intraarticular Observations

Synovial villi were numerous on the dorsal aspectwhereas the proximal and distal cul-de-sac and the mostlateral and most medial aspects of the midcarpal synoviumwere smooth. At � 60, the vascular pattern within thevilli, and erythrocyte motion were easily appreciated (Fig2). Some villi had a spiral arrangement of blood vesselswhereas in others vessels had a straighter configuration.

Intraarticular Observations After Vital Staining

Synovial staining with vital dye was necessary to eval-uate microscopic structure at high magnification. At� 150, synoviocytes, numerous in some regions andsparse in others, were identified (Fig 3). Some synovio-cytes were spherical whereas others were star-shapedcells. A consistent finding with all vital stains was thatthorough rinsing of the articular cavity with sterileNormosol-R solution 5 minutes after dye injection wasnecessary to visualize components of the synovium.Without lavage, the synovium was over stained, whichproduced a very dark background that prevented goodvisualization of the synovium. Substantial differences inidentification of synovial membrane components duringmicroarthroscopy was documented for each vital staintested.

Methylene Blue. A supravital dye that stains the nu-cleus, methylene blue provided the best localization ofcells (Fig 3), highlighted the vascular network, and thetranslucency or opacity of the matrix. At � 150, round-and stellate-shaped synoviocytes were differentiated.

Trypan Blue. An intravital dye that localized inphagocytic cells, trypan blue permitted identification ofsynoviocytes and appreciation of synoviocyte shape wasgood. Background matrix and blood vessels could be seenbut were not well defined (Fig 4).

Neutral Red. Cells, cell shape, blood vessels, andbackground matrix were all poorly differentiated withthis supravital dye that localizes in lysosomes. Synovialmembrane constituents could not be satisfactorily recog-nized because of a diffuse, intense red staining of thesynovium.

Janus Green B. Synoviocytes were identified and cellshape (round versus star-shaped cells) was appreciatedusing this supravital stain that localizes in mitochondria.

Fig 2. Microarthroscopic view of equine synovium stained

with methylene blue (�60). The vascular loop at the tip of a

single villus is visible.

Fig 3. Microarthroscopic view of equine synovium stained

with methylene blue (�150) depicting visible synoviocytes.

Fig 4. Microarthroscopic view of equine synovium stained

with trypan blue (�100).

313SERENA, HANSON, AND KINCAID

Blood vessels were not well highlighted and backgroundmatrix could not be seen (Fig 5).

Histologic Observations of Vital Dye Stained

Formalin-Fixed Specimens

Methylene Blue. Eluted during processing of bothformalin-fixed specimens and ammonium molybdate/for-malin fixed specimens. Synoviocytes and blood vesselscould be identified but different synovial componentslacked definition (Fig 6).

Trypan Blue. Cell identification was considered verysatisfactory. Trypan blue had the best retention in for-malin-fixed specimens and provided the best three-di-

mensional visualization of cells in the synovial membrane(Fig 7). Matrix background and blood vessels identifica-tion, however, was considered poor.

Neutral Red. Excellent identification of synoviocyteswas possible, matrix background was clear, and bloodvessels could be identified (Fig 8).

Janus Green B. Elution of dye occurred and cell dif-ferentiation was not possible.

Histologic Observations from Vital Dye Stained

Frozen Sections

Sections of frozen specimens retained all stains exceptJanus green B. Histologic evaluation of frozen sections ofsynovial membrane was considered satisfactory, exceptfor specimens stained with Janus green B, but not supe-rior to evaluation of formalin-fixed specimens.

Good correlation existed between cell and vascularnetwork characterization observed by microarthroscopyand histologic evaluation of both formalin fixed and fro-zen biopsy specimens.

DISCUSSION

Using a microarthroscopic technique in the equinemidcarpal joint we were able to identify synovial vasc-ular architecture, background matrix, and synoviocytes.These morphologic observations were substantially en-hanced by the use of intra-articular administration of vi-tal dyes, particularly methylene blue, which alloweddistinct observation of synoviocytes morphology, to thepoint to be able to distinguish round-shaped versus star-shaped cells.

Fig 5. Microarthroscopic view of equine synovium stained with

Janus green B (�80).

Fig 6. Light microscopic image of equine synovium vitally

stained with methylene blue and fixed in formalin (�40). Be-cause of stain elution during fixation, there is no clear dis-

crimination of cellular detail.

Fig 7. Light microscopic image of equine synovium vitally

stained with trypan blue and fixed in formalin (�40). Syno-viocytes composing the intima are readily identified.

314 MICROARTHROSCOPY OF EQUINE MIDCARPAL JOINT

Microarthroscopy is used diagnostically especially inhuman rheumatology. Diagnostic criteria used are tissuetranslucency, structure and appearance of the vascularnetwork, and number and morphology of synoviocytes.8

Our observations in the equine midcarpal joint supportuse of these criteria for evaluation of equine synovialmembrane. Microarthroscopic identification of humansynoviocytes has been based on shape extrapolated fromultrastructural studies.16 Star-shaped cells were identifiedas type A synoviocytes and round cells as type B syno-viocytes. Interestingly, ultrastructural studies of equinecarpal joints identified type A synoviocytes as spherical inshape, and type B synoviocytes as star-shaped because ofcytoplasmic processes that extend radially.17 The discrep-ancy between humans and horses in morphological clas-sification is speculative, but may be because of differencesin joint capsule and synovium anatomy, and biomechan-ical demands. The currently accepted classification sys-tem accounts for some overlap in function between the 2principal cell types.3 Synoviocyte morphology and pro-posed function might not be strictly correlated, as sug-gested by the description of an intermediate cell type(synoviocytes type C).4

Equine synovial membrane reportedly has nonspecifichistopathologic changes when inflammed.3 However, thesynovial membrane has a critical role during joint inflam-mation. Synoviocytes release IL-1 and other cytokinesthat stimulate the synthesis of collagenase, gelatinase, ca-seinase, and PGE2 from monolayers of articular carti-lage.18 Microarthroscopy may provide additional real timeinformation about the equine synovium, by observing invivo synovial morphology of normal and diseased joints.

To evaluate cells within the synovium, microarthros-copy requires vital staining of the synovial membrane.Vital dyes are used regularly in human ophthalmic sur-gery but infrequently in joints with methylene blue beingmost commonly used. We had hoped that by use of dif-ferent vital stains we might detect different synovial cellpopulations, or different metabolic states within specificcell types, because of differing stain chemical propertiesand affinities. Despite different chemical characteristics ofthe 4 stains used, selective staining of synoviocytes didnot occur. Differentiation between the types of synovio-cytes could only be based on their morphologic appear-ance (round or star-shaped) during microarthroscopy.

Vital staining of synovial tissues observed with themicroarthroscope in our study was similar to that of intoto and en bloc techniques used for conventional histo-logic observation.14 These simplistic techniques allowidentification of individual cells and groups of cells andpermit observation of the 3-dimensional relationshipsand orientation of cells and tissues that are not possibleusing other imaging techniques.14 For these methods tobe efficacious, dyes must readily penetrate tissues, haveselective staining specificity, and for vital staining theymust be biocompatible. Importantly, dyes must be re-tained in tissues and be able to withstand intra-articularlavage, or dehydration and clearing techniques used forconventional histologic processing. Methylene blue, neu-tral red, and Janus green B are cationic dyes that bond toanionic molecules. A characteristic of cationic dyes istheir susceptibility to elution from cells and tissues byalcohol during dehydration19 but resistance to washingwith water during tissue processing or saline during mi-croarthroscopy. Thus, the intense intra-articular stainingof synovial tissues by methylene blue, Janus green B, andneutral red and their elution during histologic processingcan be explained by their ionic nature. Unfortunately,neither shortening times for fixation/dehydration/clearingnor pre-fixation in ammonium molybdate15 preventedloss of staining from our biopsy specimens. Because try-pan blue is an anionic dye, it has good tissue retention invivo and in biopsy specimens. Like Frizziero et al,7–10 wefound that methylene blue was an effective vital dye forsynovium and provided excellent intra-articular stainingof cells and tissues.

Trypan blue staining provided the best visualization ofcells and tissues in dehydrated and cleared biopsy spec-imens. Staining of nuclei was unexpected because trypanblue is an anionic dye. One possible explanation may havebeen the tissue pH. With neutral pH, the amine group ofanionic dyes is capable of bonding to anionic molecules,such as nucleic acids of cells;19 thus it is possible that thephysiologic environment of normal equine synovium fa-cilitated such bonds resulting in nuclear staining. We hadanticipated that trypan blue particles would be phagocyti-

Fig 8. Light microscopic image of equine synovium vitally

stained with neutral red and fixed in formalin (�40).Synoviocytes, as well as blood vessels, are identified precisely.

315SERENA, HANSON, AND KINCAID

zed by type A-synoviocytes, which would facilitate theirintra-articular and in vitro identification. However, thisdid not occur which may reflect either the relatively shortstaining time required for microarthroscopy so thatphagocytosis did not occur or that preparation of thedye did not result in particle formation.

Ultrastructural analysis of synoviocyte phagocyticproperties has shown that 10 minutes after intra-articu-lar injection, colloidal gold particles were located in thelamina propria of synovium but not within synovio-cytes.20 However, a similar study by Luckenbill and Co-hen21 indicated that some carbon particles were identifiedin avian synoviocytes at 15 minutes after intra-articularinjection. Thus in our equine joints, the short exposure ofsynoviocytes to trypan blue may have been insufficienttime for phagocytosis to occur, yet trypan blue readilyentered synoviocytes judged by the nuclear staining. She-ehan and Hrapchak11 suggested that intravital dyesshould have the capacity to flocculate to a particulatesize that could be phagocytized by cells. Dyes we used,including trypan blue, were filtered with a 0.22mm filter(Millex-GV, Millipore, Carrigtwohill Co., Cork, Ireland)to minimize the potential for foreign body reaction orinfection; however, filtration may have also minimizedflocculation of dye molecules and prevented or delayedphagocytosis. In addition to filtration, dyes were steri-lized (autoclave) before use. In retrospect, filtration couldhave been avoided, at least for trypan blue, to obtainlarger particle sizes that could flocculate and be phago-cytized by synoviocytes. This may have allowed selectivetype A synoviocytes (phagocytic cells) to be stained bytrypan blue.

Supravital staining of synovial tissues presented anunusual histologic challenge compared with other organsbecause of the physiologic environment of joints. Janusgreen B and neutral red readily penetrated the synoviumand obscured cellular detail. Staining of mitochondria byJanus green B may have occurred but was not visible.Another consideration is that Janus green B will onlystain mitochondria if oxygen is present in cells21 and asoxygen content decreases so does Janus green B stainintensity. We did not evaluate oxygen tension in thesynovium but joint lavage may have adversely affectedoxygen tension. Elution of methylene blue from speci-mens processed for histology limited the value of biopsyspecimens and was more pronounced for formalin fixedthan frozen specimens. Neutral red was considered inap-propriate for microarthroscopic synovium evaluation be-cause cell identification was not possible. Interestingly,neutral red provided excellent synoviocyte and bloodvessel identification for histologic evaluation of synovialbiopsies.

Although histologic evaluation of frozen section offersa possible clinical advantage over intraoperative imaging,

this method lacks the definition achieved with histologicevaluation of formalin fixed specimens. Overall, histo-logic evaluation of formalin fixed specimens was con-sidered superior to frozen section for evaluation of vitallystained specimens.

Thus we found that methylene blue was the best stainof those evaluated for microarthroscopic evaluation ofequine synovium. Trypan blue and Janus green B stain-ing also permitted precise identification of synoviocytesduring microarthroscopy, but the vascular network andmatrix were not as well visualized as with methylene blue.Seemingly, microarthroscopy may be useful to documentin vivo morphologic changes in equine synovium at mag-nifications up to �150 and thus facilitate targeted biopsyfor more specific synovial membrane evaluation. The ex-cellent anatomic detail of normal equine synovium weobserved suggests that microarthroscopy is very likely tobe valuable for evaluating equine joint disease in both aclinical and an experimental setting. However, furtherstudies are needed to determine the experimental andclinical value of the technique.

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