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Topic:Magnetic Resonance Imaging

Magnetic Resonance Imaging (MRI) is a non-invasive diagnostic tool used in veterinary medicine to obtain detailed images of the internal structures of horses. It utilizes strong magnetic fields and radio waves to create cross-sectional images, allowing for the examination of soft tissues, bones, and joints without the need for radiation. MRI is particularly useful for diagnosing conditions in areas that are challenging to assess with other imaging modalities, such as the hoof, distal limb, and head. This page compiles peer-reviewed research studies and scholarly articles that explore the application, techniques, and advancements in MRI technology for equine diagnostics, as well as its role in improving the understanding of equine anatomy and pathology.
T2 mapping of cartilage in the equine distal interphalangeal joint with corresponding histology using 0.27 T and 3.0 T magnetic resonance imaging.
Equine veterinary journal    November 29, 2022   Volume 55, Issue 5 843-852 doi: 10.1111/evj.13900
Baker ME, Kershaw LE, Carstens A, Daniel CR, Brown H, Roberts S, Taylor SE.Low-field magnetic resonance imaging (MRI) is widely available to equine veterinarians yet is insensitive at detecting cartilage damage in the distal interphalangeal joint (DIPJ). T2 mapping is a quantitative imaging technique that can detect cartilage damage before morphological change is apparent. Objective: Validation of a T2 mapping sequence on a low-field MR system. Correlation of the mean T2 relaxation time in sections of cartilage with varying levels of pathology using low- and high-field MRI. Methods: Cross-sectional study. Methods: Eight phantoms with known (nominal) T2 values underwe...
Impact of Different Diagnostic Procedures on Diagnosis, Therapy, and Outcome in Horses with Headshaking: Recommendations for Fast-Track Advanced Diagnostic and Therapeutic Protocols.
Animals : an open access journal from MDPI    November 13, 2022   Volume 12, Issue 22 doi: 10.3390/ani12223125
Kloock T, Hellige M, Kloock A, Feige K, Niebuhr T.Most horses affected by headshaking (HS) are diagnosed with idiopathic trigeminal-mediated headshaking (i-TMHS) when no underlying disease is found. Diagnosis is made by the exclusion of differentials considering history, clinical signs, and diagnostic investigations. Therefore, in horses presented with headshaking, many diagnostic procedures and therapies are conducted. Retrospectively, the digital patient records of 240 horses with HS were analysed regarding the impact of diagnostic procedures on diagnosis, therapy, and outcome. Horses were extensively examined using a standardised protocol ...
Alterations to the synovial invaginations of the navicular bone are associated with pathology of both the navicular apparatus and distal interphalangeal joint when evaluated using high field MRI. McParland TJ, Horne CR, Robertson JB, Schnabel LV, Nelson NC.Limited information exists regarding associations between distal interphalangeal joint (DIPJ) abnormalities and synovial invagination changes in the distal sesamoid (navicular) bone. This retrospective, analytical study aimed to measure specific characteristics of the synovial invaginations of the navicular bone to determine whether any single characteristic was associated with abnormalities in the DIPJ or navicular apparatus (NA) using high field MRI and a sample of 200 horses' feet. The DIPJ and NA were graded independently by three scorers. The grades were averaged, creating a global pathol...
Palmar/plantar oblique projections improve radiographic detection of fractures of the palmar/plantar processes of the equine distal phalanx. Fairburn AJ, Sherlock CE, Mair TS.Radiography is a standard diagnostic test for horses with suspected fractures of the palmar/plantar processes (PP) of the distal phalanx, however published studies evaluating the diagnostic utility for radiography are currently lacking. The objectives of this retrospective, diagnostic case-control study were to determine the sensitivity of radiographs for the detection of PP fractures, and determine the diagnostic utility of the palmar/plantar oblique projections of the PP for the identification of PP fractures compared to standard radiographic series. The medical records of horses undergoing...
Desmopathy of the collateral ligaments of the equine distal interphalangeal joint – a prevalence in Poland between 2016-2019.
Polish journal of veterinary sciences    July 22, 2022   Volume 25, Issue 2 311-316 doi: 10.24425/pjvs.2022.141816
Mieszkowska M, Abako J, Mieszkowski M, Zhalniarovich Y.Desmopathy of the collateral ligaments of the equine interphalangeal joint is caused by a combination of factors, including hoof shape, excessive loading and ground surface. This complex problem poses a diagnostic challenge due to the non-specificity of perineural analgesia and the limitations associated with the most popular imaging methods such as radiography and ultrasonography. The aim of this study was to retrospectively determine the prevalence of desmopathy of the collateral ligaments of the equine distal interphalangeal joint in Poland between 2016 and 2019, and to establish the freque...
Machine Learning Prediction of Collagen Fiber Orientation and Proteoglycan Content From Multiparametric Quantitative MRI in Articular Cartilage.
Journal of magnetic resonance imaging : JMRI    July 21, 2022   Volume 57, Issue 4 1056-1068 doi: 10.1002/jmri.28353
Mirmojarabian SA, Kajabi AW, Ketola JHJ, Nykänen O, Liimatainen T, Nieminen MT, Nissi MJ, Casula V.Machine learning models trained with multiparametric quantitative MRIs (qMRIs) have the potential to provide valuable information about the structural composition of articular cartilage. To study the performance and feasibility of machine learning models combined with qMRIs for noninvasive assessment of collagen fiber orientation and proteoglycan content. Retrospective, animal model. An open-source single slice MRI dataset obtained from 20 samples of 10 Shetland ponies (seven with surgically induced cartilage lesions followed by treatment and three healthy controls) yielded to 1600 data points...
Special Diagnostic Techniques in Equine Neurology (Radiography, Ultrasonography, Computed Tomography, and Magnetic Resonance Imaging).
The Veterinary clinics of North America. Equine practice    July 7, 2022   Volume 38, Issue 2 171-188 doi: 10.1016/j.cveq.2022.04.001
Garrett KS.Diagnostic imaging is often an important part of the diagnostic approach to neurologic disease. Advanced imaging techniques such as myelography, computed tomography (CT), and magnetic resonance imaging (MRI) provide more information than radiography and ultrasonography but are more limited in their availability. The clinician should be cognizant of the findings of the clinical examination when interpreting diagnostic imaging findings.
Characterization of Equine Chronic Tendon Lesions in Low- and High-Field Magnetic Resonance Imaging.
Veterinary sciences    June 15, 2022   Volume 9, Issue 6 297 doi: 10.3390/vetsci9060297
Doll CU, von Pueckler K, Offhaus J, Berner D, Burk J.In equine medicine, experience regarding MRI of chronic tendon lesions is limited, and evidence on the suitability of different sequences in 3 T high-field MRI is scarce. Therefore, macroscopically healthy and altered tendons were examined by histology and in 0.27 T low- and 3 T high-field MRI, focusing on T1-weighted (T1w) sequences to visualize chronic lesions. In high-field MRI, tendons were positioned parallel (horizontal) and perpendicular (vertical) to the magnetic field, acknowledging the possible impact of the magic angle effect. The images were evaluated qualitatively and signal inten...
Examining the Effects of the Oral Supplement Biota orientalis in the Osteochondral Fragment-Exercise Model of Osteoarthritis in the Horse.
Frontiers in veterinary science    June 1, 2022   Volume 9 858391 doi: 10.3389/fvets.2022.858391
Seabaugh KA, Barrett MF, Rao S, McIlwraith CW, Frisbie DD.Osteoarthritis (OA) is a common problem in horses. Several oral supplements have been proposed as treatments for horses with OA. The object of this study was to evaluate the use of the oil extract from the seeds of (BO) for the treatment of experimentally induced OA in horses. OA was induced in 16, 2-5 year old horses in one middle carpal joint on Day 0; the other limb underwent a sham operation. Once daily oral treatment with BO or placebo was initiated on Day 0 and continued to Day 70. All horses were exercised 5 days a week starting on Day 14 through Day 70. The horses were evaluated every...
Kinetic Analysis in Horses With Deep Digital Flexor Tendinopathy Within the Digit Diagnosed by Magnetic Resonance Imaging.
Frontiers in veterinary science    May 31, 2022   Volume 9 893026 doi: 10.3389/fvets.2022.893026
Madsen LM, Gutierrez-Nibeyro SD, Stewart MC, McCoy AM, Schaeffer DJ.To determine the stance duration and ground reaction forces (GRF) of horses with deep digital flexor (DDF) tendinopathy at the level of the foot and compare the stance duration and GRF to those of clinically sound horses. Unassigned: Prospective clinical study. Unassigned: Sixteen horses (seven horses with bilateral forelimb lameness, four horses with unilateral forelimb lameness, and five horses with no lameness). Unassigned: Analyses of kinetic variables were performed on both forelimbs from sound horses and horses diagnosed with chronic DDF tendinopathy. Stance duration and longitudinal and...
A Descriptive Study of the Carpal Joint of Healthy Donkeys Using Ultrasonography, Computed Tomography, and Magnetic Resonance Imaging.
Veterinary sciences    May 23, 2022   Volume 9, Issue 5 249 doi: 10.3390/vetsci9050249
Salem M, El-Shafaey ES, Farag AMM, El-Khodery S, Al Mohamad Z, Abass M.This study was conducted to establish a detailed anatomic reference for the carpal joint of apparently healthy donkeys using ultrasonography (US), computed tomographic (CT), and magnetic resonance imaging (MRI). Ten orthopedically sound adult donkeys were used for US examination of the carpal joint in each forelimb. Additionally, the carpi of ten donkey cadavers were subjected to CT and MRI examinations. The carpal joint was divided into four zones to simplify examination. US assessment of the carpal joint included transverse and longitudinal sonograms. CT was performed using three planes: axi...
When radiography and ultrasonography are not enough: the use of computed tomography and magnetic resonance imaging for equine lameness cases.
Journal of the American Veterinary Medical Association    May 11, 2022   Volume 260, Issue 10 1113-1123 doi: 10.2460/javma.22.03.0136
Garrett KS.MRI and CT have enhanced our diagnostic abilities for equine lameness beyond what is available using radiography and ultrasonography. This has allowed veterinarians to better prognosticate and treat lameness conditions, improving patient outcomes. This article discusses the basic principles behind MRI and CT, their advantages and disadvantages, the different types of equipment available for clinical use in horses, the typical diagnostic workup prior to pursuing advanced imaging, and common regions where MRI and CT are used clinically. The companion Currents in One Health by Spriet, AJVR, July ...
The existence of intertransverse joints in young warmblood foals.
Journal of the American Veterinary Medical Association    April 27, 2022   Volume 260, Issue 10 1206-1210 doi: 10.2460/javma.22.02.0051
Spoormakers TJP, Bergmann W, Veraa S, van Weeren PR, Brommer H.To verify the existence of intertransverse joints (ITJs) in young foals. 11 warmblood foals. Postmortem examination of the lumbar area in foals < 200 days old using CT, MRI, dissection, and histomorphology. Data were analyzed with descriptive statistics. Age of foals varied between 1 and 200 days (median, 11 days). Ten foals had 6 lumbar (L) vertebrae, and 1 foal had 5. All 11 foals, irrespective of age, had ITJs between the first sacral and last lumbar vertebrae and between the last and second-to-last lumbar vertebrae. In 6 foals (all with 6 L vertebrae), ITJs also existed between the four...
Leveraging MRI characterization of longitudinal tears of the deep digital flexor tendon in horses using machine learning. ELKhamary AN, Keenihan EK, Schnabel LV, Redding WR, Schumacher J.While MRI is the modality of choice for the diagnosis of longitudinal tears (LTs) of the deep digital flexor tendon (DDFT) of horses, differentiating between various grades of tears based on imaging characteristics is challenging due to overlapping imaging features. In this retrospective, exploratory, diagnostic accuracy study, a machine learning (ML) scheme was applied to link quantitative features and qualitative descriptors to leverage MRI characteristics of different grades of tearing of the DDFT of horses. A qualitative MRI characteristic scheme, combining tendon morphologic features, alt...
Multi-modality imaging of aggressive submural neoplasia of the hoof in two horses.
Australian veterinary journal    April 5, 2022   Volume 100, Issue 7 336-341 doi: 10.1111/avj.13161
Nagel H, Lang H, Sole Guitart A, Lean N, Allavena R, Sprohnle-Barrera C, Young A.This case series presents a 20-year-old Quarter horse gelding and a 10-year-old Thoroughbred gelding with a histologic diagnosis of aggressive submural neoplasia, including an anaplastic sarcoma and a squamous cell carcinoma respectively. The current case series describes these neoplasias with radiography, computed tomography, magnetic resonance imaging and histopathology with immunohistochemistry. The purpose of this case series is to identify the clinical pattern of chronic reoccurring lameness in these cases and highlight the appearance of expansile osteolytic pathology of the distal phalan...
Complex central nervous system malformations in a Dutch Warmblood foal.
Journal of veterinary internal medicine    April 1, 2022   Volume 36, Issue 3 1173-1178 doi: 10.1111/jvim.16407
Louie EW, Streeter R, Story M, Scrivani PV, Barrett M, Mullen KR.A neonatal Dutch Warmblood colt was evaluated for inability to stand, incoordination and intention tremor. Despite partial improvement in clinical signs during the first 4 days of hospitalization, neurological deficits remained. Magnetic resonance imaging identified a unilateral infratentorial arachnoid cyst-like lesion with ipsilateral compression and displacement of the cerebellar hemisphere, absent corpus collosum, polymicrogyria, suspect leukoencephalopathy, and noncompressive occipitoatlantal malformation. Improvement in clinical signs during the first 6 months of life suggests that ho...
Validation of ultrasonography for measurement of cartilage thickness in the equine carpus. Andersen C, Griffin JF, Jacobsen S, Østergaard S, Walters M, Mori Y, Lindegaard C.Articular cartilage thinning is an important hallmark of osteoarthritis (OA), and ultrasonography (US) is a clinically accessible tool potentially suitable for repeated evaluation. The aim of the present prospective methods comparison study was to validate US as a tool for measuring cartilage thickness in the carpus of the horse. Eight Standardbred trotters underwent US examination with 9 and 15 MHz linear transducers. Six anatomical locations in the radiocarpal joint (RCJ) and middle carpal joint (MCJ) were examined. The same joints were assessed by ultrahigh field (9.4 Tesla) magnetic reson...
Ex vivo comparison of 3 Tesla magnetic resonance imaging and multidetector computed tomography arthrography to identify artificial soft tissue lesions in equine stifles.
Veterinary surgery : VS    March 15, 2022   Volume 51, Issue 4 648-657 doi: 10.1111/vsu.13798
Aßmann AD, Ohlerth S, Suárez Sánchez-Andráde J, Torgerson PR, Bischofberger AS.To determine the diagnostic performance of computed tomographic arthrography (CTA) and 3 Tesla magnetic resonance imaging (MRI) for detecting artificial meniscal, meniscotibial ligament (MTL) lesions and cruciate ligament (CL) lesions in horses. Methods: Ex vivo controlled laboratory study. Methods: Nineteen stifles from adult horses. Methods: Stablike defects (n = 84) (16 mm long, 10 mm deep) were created in the menisci (n = 35), CLs (n = 24), and MTLs (n = 25) via arthroscopy prior to MRI and CTA (80 mL contrast at 85 mg/mL per joint). Two radiologists, unaware of the lesions...
Three-dimensional printed surgical guides for keratoma removal in horses using computed tomography or magnetic resonance imaging-based segmentation.
Veterinary surgery : VS    February 28, 2022   Volume 51 Suppl 1 O43-O52 doi: 10.1111/vsu.13786
Biedrzycki AH, Morton AJ, Perez-Jimenez EE, Elane GL, Roe HA, Trolinger-Meadows KD.To report the technique, surgical approach, and postoperative features in horses treated via a 3-dimensional (3D) printed guide-assisted keratoma resection created using computed tomography (CT) or magnetic resonance imaging (MRI)-based segmentation. Methods: Five client-owned horses. Methods: Short case series. Methods: Horses were placed under general anesthesia for imaging (CT and MRI) and underwent a second anesthesia for surgery. Two horses had guides created from CT-based imaging, 3 horses had guides created from MRI. Various sized nonarbored hole saws were used to create accurate and pr...
Magnetic resonance imaging of the normal equine pituitary gland. Hobbs KJ, Porter E, Wait C, Dark M, MacKay RJ.Magnetic resonance imaging is increasingly available for evaluation of horses with suspected pituitary adenomas; however, published studies describing the MRI features of normal equine pituitary glands are currently lacking. This retrospective, descriptive, anatomic study describes the MRI appearance and size of presumed normal equine pituitary glands and assesses potential correlations between pituitary size, brain size, and patient weight. These findings serve as a reference for MRI evaluation of the equine pituitary gland. Inclusion criteria consisted of adult horses (2-15 years old), over ...
Radiographic changes of the proximal third metatarsal bone do not predict presence or severity of proximal suspensory desmopathy in a predominately Quarter Horse population.
Equine veterinary journal    February 11, 2022   Volume 55, Issue 1 24-32 doi: 10.1111/evj.13562
Hinkle FE, Selberg KT, Frisbie DD, Barrett MF.Comparison of radiography to magnetic resonance imaging (MRI) can help objectively assess the value and limitations of radiographs in orthopaedic disease processes. The tarsus and proximal suspensory origin of the metatarsus are commonly imaged regions for lameness. The knowledge gained by comparison between imaging modalities will aid in improving accuracy of radiographic interpretation. Objective: To compare the radiographic and MRI findings of the proximal third metatarsal bone (MTIII) and proximal suspensory ligament using MRI as the gold standard. Methods: Retrospective observational stud...
Measurement accuracy of foot conformation parameters on low-field magnetic resonance images in horses.
Journal of equine veterinary science    February 7, 2022   Volume 112 103894 doi: 10.1016/j.jevs.2022.103894
Bolt DM, Carrier ME, Sheridan KS, Manso-Diaz G, Berner D.Foot imbalance and malalignment of the digits are common in horses. Angle and distance measurements performed on bones, joints and hoof wall on radiographs provide essential guidance for precise corrective trimming and shoeing. This study aimed to investigate, if selected standing low-field magnetic resonance (MR) images can be used to accurately measure dorsopalmar and lateromedial foot conformation parameters in comparison to the radiographic gold standard. Images of 100 horses referred for standing low-field MR examination were selected. Foot conformation angles and distances were measured ...
Advances in the Diagnosis of Equine Respiratory Diseases: A Review of Novel Imaging and Functional Techniques.
Animals : an open access journal from MDPI    February 4, 2022   Volume 12, Issue 3 doi: 10.3390/ani12030381
Kozłowska N, Wierzbicka M, Jasiński T, Domino M.The horse, as a flight animal with a survival strategy involving rapid escape from predators, is a natural-born athlete with enormous functional plasticity of the respiratory system. Any respiratory dysfunction can cause a decline in ventilation and gas exchange. Therefore, respiratory diseases often lead to exercise intolerance and poor performance. This is one of the most frequent problems encountered by equine internists. Routine techniques used to evaluate respiratory tract diseases include clinical examination, endoscopic examination, radiographic and ultrasonographic imaging, cytological...
Osteochondral regeneration of the femoral medial condyle by using a scaffold-free 3D construct of synovial membrane-derived mesenchymal stem cells in horses.
BMC veterinary research    January 22, 2022   Volume 18, Issue 1 53 doi: 10.1186/s12917-021-03126-y
Murata D, Ishikawa S, Sunaga T, Saito Y, Sogawa T, Nakayama K, Hobo S, Hatazoe T.Medical interventions for subchondral bone cysts in horses have been extensively studied. This study investigated the regeneration of articular cartilage and subchondral bone with scaffold-free three-dimensional (3D) constructs of equine synovial membrane-derived mesenchymal stem cells (SM-MSCs) isolated from three ponies and expanded until over 1.0 × 10 cells at passage 2 (P2). Results: SM-MSCs were strongly positive for CD11a/CD18, CD44, and major histocompatibility complex (MHC) class I; moderately positive for CD90, CD105, and MHC class II; and negative for CD34 and CD45 on flow cytom...
Suspensory ligament size does not change after plantar fasciotomy and neurectomy of the deep branch of the lateral plantar nerve by ultrasonographic assessment.
Veterinary surgery : VS    December 31, 2021   Volume 51, Issue 2 259-269 doi: 10.1111/vsu.13757
Scharf A, de Solis CN, Sampson SN, Glass K, Watts AE.To determine the short-term effect of plantar fasciotomy and neurectomy (PFN) of the deep branch of the lateral plantar nerve on the proximal suspensory ligament (PSL) cross-sectional area (CSA) in horses with hindlimb proximal suspensory desmopathy (PSD). Methods: Analytical, observational, cohort study. Methods: Twenty-one horses. Methods: Records of horses with chronic PSD treated by PFN were included if a preoperative ultrasonographic examination was available and at least one postoperative ultrasonographic examination. One masked observer measured the ultrasonographic cross-sectional area...
Imaging and Gross Pathological Appearance of Changes in the Parasagittal Grooves of Thoroughbred Racehorses.
Animals : an open access journal from MDPI    November 24, 2021   Volume 11, Issue 12 3366 doi: 10.3390/ani11123366
Johnston GCA, Ahern BJ, Palmieri C, Young AC.(1) Background: Parasagittal groove (PSG) changes are often present on advanced imaging of racing Thoroughbred fetlocks and have been suggested to indicate increased fracture risk. Currently, there is limited evidence differentiating the imaging appearance of prodromal changes in horses at risk of fracture from horses with normal adaptive modelling in response to galloping. This study aims to investigate imaging and gross PSG findings in racing Thoroughbreds and the comparative utility of different imaging modalities to detect PSG changes. (2) Methods: Cadaver limbs were collected from twenty ...
Retinal detachment secondary to vitreoretinopathy in two closely related warmblood horses.
Open veterinary journal    November 19, 2021   Volume 11, Issue 4 672-679 doi: 10.5455/OVJ.2021.v11.i4.18
Vercruysse E, Naranjo C, Sauvage A, Vandersmissen M, Grauwels M, Monclin S.To describe the clinical, diagnostic imaging, and histopathological findings of two visually impaired closely related horses and to identify a possible cause. Methods: Two warmblood horses, with a common dam and sire, were presented to the ophthalmology department of Liège for investigation of impaired vision. Information collected included physical and ophthalmic examination findings, results of ocular ultrasound, electroretinogram, magnetic resonance imaging (MRI), and histopathology. Ophthalmic examination, ocular ultrasound and MRI revealed a complete retinal detachment (RD) in the left e...
Deep Digital Flexor Tendon Injury at the Level of the Proximal Phalanx in Frontlimbs With Tendon Sheath Distension Characterized by Standing Low-Field Magnetic Resonance Imaging in Horses: 13 Cases (2015-2021).
Frontiers in veterinary science    November 18, 2021   Volume 8 734729 doi: 10.3389/fvets.2021.734729
van Veggel ECS, Selberg KT, van der Velde-Hoogelander B, Vanderperren K, Cokelaere SM, Bergman HJ. To describe the MRI findings for 13 horses with deep digital flexor tendon (DDFT) injury at the proximal phalanx where the tendon goes from ovoid to bilobed in frontlimbs with tendon sheath distension. In addition, the prognosis of this lesion was assessed. Retrospective case series. Thirteen client-owned horses. Medical records were reviewed, and data were collected regarding signalment, history, MRI findings, and outcomes of horses. Findings of MRI were recorded and whether the case was confirmed with tenoscopy. A diagnosis of DDFT injury at the junction between ovoid and bilobed portio...
Other Clinical Problems of the Equine Foot.
The Veterinary clinics of North America. Equine practice    November 17, 2021   Volume 37, Issue 3 695-721 doi: 10.1016/j.cveq.2021.08.005
Fürst AE, Lischer CJ.Many disorders affect the equine foot, and many hoof problems have multiple predisposing causes. Surgery may be necessary after conservative management has failed. Diseases of the hoof capsule may seem simple, but their effect on performance can be long-lasting and healing is often prolonged. Diagnosis of problems within the hoof capsule is enhanced with the use of computed tomography and MRI. The prognosis of fractures has improved with strategic placement of lag screws across fracture planes using aiming devices and advanced intraoperative imaging techniques. Collaboration between the clinic...
The Frequency of Communication Between the Synovial Compartments of the Equine Temporomandibular Joint: A Contrast-Enhanced Computed Tomographic Assessment.
Frontiers in veterinary science    October 25, 2021   Volume 8 753983 doi: 10.3389/fvets.2021.753983
Pimentel KL, Carmalt JL. There is conflicting evidence regarding communication between the two compartments of the equine temporomandibular joint (TMJ). Understanding the inter-relationship between TMJ compartments is critical for diagnostic and clinical management purposes. To determine the frequency of communication between the discotemporal joint (DTJ) and the discomandibular joint (DMJ) of the equine TMJ in horses free of overt disease. A randomized, blinded, controlled cadaveric study. Equine cadaver heads ( = 20), with no reported history of potential TMJ disease, were collected and stored frozen until use. ...