LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES

VETERINARY ACADEMY

Faculty of Veterinary Medicine

MASTER THESIS

of Integrated Studies of Veterinary Medicine

PATHOMORPHOLOGICAL EVALUATION OF DEEP DIGITAL

FLEXOR TENDON LESIONS IN HORSES

Viktoria Eklund

Head of the work: Dr. Jūratė Sabeckienė

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2 THE WORK WAS DONE IN THE DEPARTMENT OF VETERINARY PATHOBIOLOGY

CONFIRMATION OF THE INDEPENDENCE OF DONE WORK

I confirm that the presented Master Thesis “PATHOMORPHOLOGICAL EVALUATION OF DEEP DIGITAL FLEXOR TENDON LESIONS IN HORSES”

1. has been done by me;

2. has not been used in any other Lithuanian or foreign university;

3. I have not used any other sources not indicated in the work and I present the complete list of the used literature.

2017-12-15 Viktoria Eklund

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CONFIRMATION ABOUT RESPONSIBILITY FOR CORRECTNESS OF THE ENGLISH LANGUAGE IN THE DONE WORK

I confirm the correctness of the English language in the done work.

2017-12-15 Viktoria Eklund

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CONCLUSION OF THE SUPERVISOR REGARDING DEFENSE OF THE MASTER THESIS

2017-12-15 Jūratė Sabeckienė

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THE MASTER THESIS HAVE BEEN APPROVED IN THE DEPARTMENT/CLINIC

(date of approbation) (name, surname of the manager of department/clinic)

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Reviewers of the Master Thesis

1) Prof. dr. Albina Aniulienė, Department of Veterinary Pathobiology

2) Assist. Indrė Stasiulevičiūtė, Large Animal Clinic

(name, surname) (signatures)

Evaluation of defense commission of the Master Thesis:

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SUMMARY

PATHOMORPHOLOGICAL EVALUATION OF DEEP DIGITAL FLEXOR TENDON LESIONS IN HORSES

Viktoria Eklund Master Thesis

The objective of this work was to investigate the occurrence and frequency of gross and microscopic pathomorphological lesions of the deep digital flexor tendon (DDFT) of horses at the level of the navicular bone, and their correlation with the horses’ age, weight and hoof conformation.

For this purpose, 71 cadaveric front hooves from 41 horses slaughtered for reasons other than this study were collected. Signalment and case history other than age and weight were not considered. The age ranged from 4 to 22 years and weight ranged from 430 to 865 kg.

The hooves were measured, photographed and dissected for further histological sampling of the DDFT and associated structures. External and internal gross pathologies of the DDFT (in the mid-sagittal view and in the specimen obtained for the histological slide preparation) were recorded at the same time as photography. For the purpose of histopathological specimen preparation, samples of 10-15 mm thickness were taken from the distal DDFT at the level of the sagittal ridge on the flexor surface of the NB of each hoof.

The DDFT dorsal border lesions (found in 87% of the horse population) were thermal damage (87%), traumatic fibrillation (56%), adhesion formation (8%) and parasagittal plane splits (3%). The histopathological investigation of core lesions (found in 86% of the horse population) revealed degeneration/core necrosis (83%), fibroplasia/fibrocartilaginous metaplasia (83%), fibrillar disorganization (86%), increased cellularity (39%), thickening of interfascicular septa (47%) and other lesions (10%). The significant external gross lesions encountered were long toe (48% of the horse population), high heel (35%), underrun heel (21%) and contracted heel (62%). These results indicate a high incidence of various pathomorphological lesions in nearly 90% of the investigated feet.

Neither a correlation between age and occurrence of pathomorphological lesions of the distal DDFT, nor a correlation between weight and lesions, were found in this study. Similarly, no strong correlation between hoof conformation and distal DDFT lesions was found. This means that the occurrence of pathomorphological lesions encountered in both this study and preceding research is not primarily related to either the age or weight of the individual, or the hoof conformation. It is

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6 more likely the result of individual predisposition further enhanced by nonphysiological biomechanical elements such as type of athletic discipline and hoof maintenance (shoeing).

Key words: Equine, Hooves, Deep digital flexor tendon, Tendon pathology, Pathomorphology, Tendon histology, Hoof angles, Navicular disease, Tendonitis

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SANTRAUKA

ŽIRGŲ GILIOSIOS PIRŠTŲ LENKIAMOSIOS SAUSGYSLĖS PATOMORFOLOGINIŲ PAŽEIDIMŲ VERTINIMAS

Viktoria Eklund Master Thesis

Šio darbo tikslas buvo nustatyti dažniausiai pasitaikančius išorinius ir mikroskopinius žirgų giliosios pirštų lenkiamosios sausgyslės (GPLS) patomorfologinius pakitimus, randamus varlės kaulo srityje, bei jų koreliaciją su žirgų amžiumi, svoriu ir kanopų forma.

Šiam tikslui buvo atrinkta 71 priekinių kojų kanopa iš 41 žirgų, atvežtų į skerdyklą dėl kitų priežasčių nei šis tyrimas. Tyrimui buvo naudojama informacija tik apie žirgų amžių ir svorį. Amžius svyravo nuo 4 iki 22 metų, o svoris nuo 430 kg iki 865 kg.

Kanopos buvo matuojamos, fotografuojamos ir preparuojamos, ruošiant tolimesniems GPLS ir šalia esančių struktūrų tyrimams. Buvo vertinamos kiekvienos kojos išorinės ir vidinės GPLS patologijos. Histopatologiniam tyrimui, mėginiai buvo imami ties distaliniu varlės kaulo paviršiumi, pjaustant sausgyslę 10-15 mm storio sagitaliais pjūviais.

GPLS dorsalinio paviršiaus pažeidimai (rasti 87% tirtų žirgų) buvo: terminis pažeidimas (87%), trauminė fibriliacija (56%), sąaugų formavimasis (8%) ir parasagitalinės plokštumos plyšiai (3%). Histopatologinio tyrimo metu nustatyti sausgyslės pažeidimai (rasti 86% tirtų žirgų) buvo: sausgyslės degeneracija/nekrozė (83%), fibroplasia/fibrokremzlinė metaplazija (83%), fibriliarinė deorganizacija (86%), padidėjęs ląstelių tankis (39%), tarpskaidulinės pertvaros sustorėjimas (47%) ir kiti pažeidimai (10%). Svarbiausi išoriniai kanopų formos pakitimai buvo: ilgas kanopos priekis (rastas 48% tirtų žirgų), aukšti užpenčiai (35%), ilgi palindę užpenčiai (21%) ir susiaurėję užpenčiai (62%). Šie rezultatai rodo, kad daugiau kaip 90% tirtų kanopų turėjo daug įvairių patomorfologinių formos pakitimų.

Koreliacijos nebuvo nei tarp amžiaus ir patomorfologinių GPLS pakitimų, nei tarp svorio ir pakitimų. Taip pat nebuvo didelės koreliacijos tarp kanopos formos ir GPLS pakitimų. Tai reiškia, kad tyrimo metu nustatyti pakitimai nėra tiesiogiai susiję nei su individo amžiumi, nei su svoriu, nei su kanopų forma. Panašu, kad šie pakitimai labiau susiję su individualiu gyvūno polinkiu tam tikrai patologijai, kurį sustipriną tokie nefiziologiniai biomechaniniai faktoriai, kaip sportinė disciplina ir kanopų priežiūra (kaustymas).

Raktiniai žodžiai: žirgai, kanopos, gilioji pirštų lenkiamoji sausgyslė, sausgyslės patologijos, patomorfologija, sausgyslių histologija, kanopų kampai, varlės kaulo liga, sausgyslių uždegimas.

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ABBREVIATIONS

DC – Digital cushion

DDF – Deep digital flexor muscle DDFT – Deep digital flexor tendon DIPJ – Distal interphalangeal joint DP – Distal phalanx

DWA – Dorsal hoof wall angle ECM – Extracellular matrix GRF – Ground reaction force

HAVS – Hand-arm vibration syndrome HLA – Heel angle

H&E – Hematoxylin and eosin

LUHS – Lithuanian University of Health Sciences MRI – Magnetic resonance imaging

NB – Navicular bone ND – Navicular disease NS – Navicular syndrome

PA – Palmar angle of the distal phalanx PPS – Parasagittal plane splits

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INTRODUCTION

The equine limb is a good example of an exquisite mechanical construction, designed to withstand excessive forces and a heavy load with consistent changes in the surrounding environment. In addition to the natural forces acting upon the equine leg and more precisely – the hoof, the domestication of the horses with concomitant changes in environment, nutrition, and behavioral patterns and the invention of applying metal shoes to the hooves, rather than breeding for soundness, have led to the general increase of hoof pathologies [1,2].

The distal phalanx is the foundation of the equine hoof and with all its surrounding structures it forms a strong and elastic construction, that can carry the horse over long distances and a clear majority of terrain with endurance and comfort [1]. There are no muscles in the equine digit; instead the locomotion of the hooves is governed exclusively by the extensor- and flexor tendons inserting on the dorsal and palmar aspect of the hooves, thus enabling extension and flexion of the digit, and to some extent the weight-bearing properties of the limb [1,3].

The horses’ hoof conformation and function is an everlasting hot topic in the equine science, whether the discussion revolves around to shoe or not to shoe; optimal dietary choices; or the ‘natural’ angles and measures of the hooves to function optimally and thus minimizing the risk of injury [1,4]. Furthermore, there has been a marked increase in horses presented with lameness associated with pain in the caudal foot, a condition commonly referred to as the Navicular syndrome (NS), and damage to the navicular bone (NB) and the deep digital flexor tendon also known as the Navicular Disease (ND). For this reason, the work with demystifying the etiology and the sequence of events in the course of these conditions has in the past few decades further intensified [1].

Before the introduction of magnetic resonance imaging (MRI) in equine medicine to diagnose soft tissue injuries, horses that presented with caudal foot pain were commonly diagnosed with NS/ND. Now it is well known that caudal foot pain can be related to any of the internal structures and true ND is therefore not as often encountered anymore [1,5]. Nevertheless, studies have shown that there is a substantial amount of horses having pathomorphological changes in the NB and deep digital flexor tendon (DDFT) regardless of them being lame or not [6]. Hence, in order to better understand the pathogenesis of lesions of the DDFT, further investigations of the type and frequency of lesions with concurrent estimation of the correlation to various factors such as age, diet, environment and athletic performance could be useful.

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10 The objective of this study was to describe the most common pathomorphological lesions of the distal DDFT at the level of the navicular bone in horses; their frequency; and correlation with the horses’ age, weight and hoof conformation.

The tasks of the work:

1. To determine the pathomorphological lesions of the distal DDFT at the level of the navicular bone in horses.

2. To determine frequency of the pathomorphological lesions of the distal DDFT

3. To determine the correlation between distal DDFT lesions and the horses’ age and weight. 4. To determine the correlation between hoof conformation and distal DDFT lesions.

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1. LITERATURE REVIEW

1.1. The deep digital flexor tendon

1.1.1. Functional anatomy

Functional anatomy studies the relation between bodily structures and their functions [7], since both areas are individually unique but at the same time closely intertwined. It is therefore difficult to study one subject without learning the other to get a complete understanding of the reasoning behind the magnificent architecture of the equine locomotor apparatus.

The deep digital flexor muscle (DDF) of the front limb of the horse runs along the caudal aspect of the entire forearm deep to the superficial digital flexor muscle and flexor muscles of the carpus, and is composed of three heads: the humeral head; the radial head; and the ulnar head, originating from the conjunctions with the humeral medial epicondyle, the radius and ulna [3,8]. The muscle compartments merge just proximal to the carpus to form a common thick tendon unit of the deep digital flexor muscle – the deep digital flexor tendon (DDFT), which continues the DDF all the way down to its insertion points at the distal end of the middle phalanx and the flexor surface of the distal phalanx (Fig. 1) [1,3,8]. As a matter of fact, the tendon section of the entire

muscle-Fig. 1: Anatomy of the horse hoof, sagittal view. Abbreviations: MP – Middle phalanx, DP – distal phalanx, NB – navicular bone, DC – digital cushion, DDFT – deep digital flexor tendon. The image in the upper right corner visualizes the dorsal surface of the DDFT, which is pale yellow/offwhite and smooth. (Source: Viktoria Eklund)

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12 tendon unit is much greater than the muscle compartment, due to the biomechanical feature of the equine limb, where the ability of the flexor tendons to prevent motion rather than create it is far more important than pure muscle strength [9]. Nevertheless, the main function of the DDFT is flexion of the distal phalanx (DP) and thereby the whole foot [8]. Furthermore, the DDFT inserts along the entire brim of the palmar aspect of the DP, which creates a strong connection through its entheses with the DP. This is a necessity for the stabilizing and motoric function of the muscle-tendon unit, thus providing support and flexion of the distal part of the limb. One unique feature possessed by the horse is the presence of the so-called inferior check ligament that attaches the DDFT to the palmar carpal ligament of the equine limb [8]. Because of this tough fibrous band, the rotation of the fetlock and coffin joints exerted by the body weight of the horse during impact can be prevented with minimal muscle action, thus significantly reducing the load on the DDFT [9].

Despite the fact that the tendon structure possesses an impressive tensile strength and resistance to wear, there are still areas of concern along the length of the flexor tendons where bony protuberances of the skeletal pillar of the equine limb put excessive stress on the tendon during locomotion, for example on the caudal aspect of the carpus and the metacarpophalangeal (fetlock) joint [3,8]. In order to prevent injury, the tendons are surrounded by protective synovial sheaths at these locations, thus allowing frictionless movement over the joint surfaces [8].

The vascularization of the flexor tendons originate from the main arteries and veins in the front limbs which branches out to supply every tissue, thus running along the entire length of the legs. However, due to the main purpose of the tendon to be pressure resistant and resilient, the space for vasculature is scarce in order to prevent debilitation of the tissue. As a result the vessels are of minimal diameter and sparse amount, and the delivery of nutrients and oxygen occurs mainly in one of three ways: by the intrinsic systems at the myotendinous and osteotendinous entheses of the tendon; via the extrinsic pathways through the paratenon; or via the synovial sheaths surrounding the tendon [10,11,12]. As a matter of fact, this is the reason why the tendons are naturally white in color as opposed to the bright red and well vascularized muscles [12]. Consequently the healing properties of tendons are slow and poor because of the inability of inflammatory cells and healing components to infiltrate the tissue. Furthermore, tendons have a low metabolic rate and thus oxygen consumption, and a well-developed anaerobic energy generation [11] which enables them to carry a load and uphold tension for prolonged periods of time, without being subjected to oxygen deprivation and subsequent necrosis. However the low metabolic rate further compromises the healing of the tendon after injury [11]. Another key point regarding the vascularization of the DDFT at the level of the NB is the presence of persistent compressional forces exerted upon both the tendon and the bone, which consequently compromises the blood supply even further [11]. This is important from a clinical perspective as the poor vascularization at these sites further predisposes

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13 the tendon to degeneration and, in the case of the DDFT at the level of NB, commonly sagittal/parasagittal splits [12].

The innervation of the limbs of the horse consists of nerves that generally follow the same route as the blood vessels, thus forming neurovascular bundles. The innervation of the DDFT in the front limb is maintained by the main branches of the median and ulnar nerves [3] originating from the brachial plexus. However, most of the nerve fibers terminate on the surface of the tendon, and few enter the main body [11].

At the level of the navicular bone, the distal DDFT is dorsally covered by the navicular bursa; a sac-like fluid-filled structure which promotes the stress-free sliding of the DDFT over the palmar surface of the NB by reducing the friction and wear through lubrication as the horse moves. The DDFT has a constant angle of insertion upon the DP during both stance and movement, and this is made possible by the NB serving as a pulley to redirect the hauling forces of the DDF, which is imperative as the angle of the insertion upon the DP changes markedly as the bones move [1,9]. Hence, without a pulley in this area, the DDFT would have to suffer severe tear and subsequent destruction. Furthermore, the dorsal surface of the DDFT is here covered by a delicate layer of hyaline cartilage which gives the DDFT at this level a slightly different appearance histologically (for a more detailed description, see section 1.1.2. Histology) compared to the rest of the length of the tendon. The main function of the cartilage is to provide increased strength and resilience to the DDFT, as well as offering a smooth gliding surface as the constant movement over the NB puts excessive stress on the tendon [13].

The distal DDFT is on its palmar surface closely related to the digital cushion (DC) of the equine hoof via a fibrous sheath, a wedge-shaped mass which is primarily composed of collagen, fat and vasculature and plays a significant role in the shock absorption, stability and blood circulation of the foot [1,14]. In the healthy, well developed hoof there is also a substantial amount of supportive fibrocartilage that furthermore improves the supportive function of the DC as well as encouraging the development of the vasculature with subsequent enhanced circulation of the palmar foot of the horse [1].

Furthermore, the DDFT and the DC are both flanked laterally by- and connected to the ungual cartilages whose true function has not yet been fully established, but seems to be connected with the stability and shock absorption of the digit [1].

The DDFT plays a key role in the passive Stay-Apparatus of the horse, which enables quadrupeds to maintain a standing position for extended periods of time with minimal muscular effort due to the passive action of the non-tiring tendons [3]. Furthermore, the DDFT supports the interosseous suspensory apparatus of the fetlock joint by providing an elastic tendinous mechanism to the movement of the limb, thus yielding a smooth, anti-concussive transition in the step.

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1.1.2. Histology

The DDFT has the same main histological assembly as most tendinous structures with a sparse amount of cells but an abundant extracellular matrix (ECM) composed primarily of delicate bundles of collagen fibers (collagen accounts for roughly 60-85% of the tissue dry weight [15,16]), mainly type I (approximately 95% [15,16]), arranged in parallel rows surrounding the tenocytes from which they are originally synthesized (Fig. 2) [10,17]. Along with the collagen fibers, the proteoglycans, glycoproteins and the elastin constitute a significant part of the ECM [10]. The collagen bundles are assembled into subfascicles surrounded by a subtle endotenon composed of a woven mesh of areolar connective tissue interlaced with thin elastic fibers, and these primary bundles are subsequently arranged alongside each other into fascicles to comprise the entire tendon encased by a tough outer sheath – the epitenon. The elastic fibers of the endotenon tends to pull the collagen bundles into a crinkled wavy formation termed ‘crimp’ [18] when the tendon is in an unloaded state, compared to the straight formation of the parallel rows of collagen fibers in a loaded state of the tendon. The crimp length and angle can be measured microscopically, and has been shown to decrease in the time period between birth and two years of age [18]. Lateral cohesion between the collagen fibers and fibrils is one of the features of the crimp, thus preventing slippage and allowing the tendon to resist high uniaxial loads during activity. Hence, the elastic fibers promote the elasticity of the tendon as they recoil when the load of the tendon is removed and further prevent strain injuries during the loaded phase. The epitenon consists of a tightly woven network of dense irregular connective tissue that holds all the fascicles together to form the tendon that is externally surrounded by loose, fatty areolar connective tissue, the paratenon, which holds mucinous fluid. Since the tendon slides continuously against surrounding tissues, the need for a friction free movement is evident, which is provided by the paratenon [11].

The tenocytes found in the tendon tissue are of two different morphological types according to their metabolic profile. Type 1 tenocytes is the spindle-shaped, metabolically less active cell type with an elongated nucleus, compared to type 2 tenocytes or tenoblasts that are highly metabolically active and thus of a rounded form, containing numerous cytoplasmic organelles and an ovoid nucleus [11,19]. Type 1 tenocytes represent the end-stage of the maturation process of the type 2 tenocytes, and are thus mainly found in mature, healthy tissues whereas type 2 tenocytes is the predominant cell type of immature tissue or tissue subjected to regenerative activity due to damage [11,19]. Altogether, both types of tenocytes comprise roughly 90-95% of the cellular compartment of normal tendon tissue, whereas the remaining 5-10% consists of chondrocytes, synovial cells and vascular cells [20]. However, at the cellular level the distal DDFT differs from the rest of the tendon where it passes over the sesamoid bones, as it here assumes a structure that more resembles the conformation of fibrocartilage, with numerous chondrocytes spread evenly throughout the tissue

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15 between the parallel rows of collagen (Fig. 3) [18]. Thus, the cellular distribution does not comply with the standard tendon structure.

Fig. 2: Photograph illustrating the characteristic ‘crimp’ pattern. Abbreviations: TC – Tenocytes, CF – Collagen fibers. White areas in the sample are processing artefacts. (Source: Viktoria Eklund)

Fig. 3: Photograph illustrating the dorsal border of the DDFT covered with hyaline cartilage (HC), and fibrochondrocytes (FCC) interspersed in between the collagen fibers. White areas in the sample are processing artefacts. (Source: Viktoria Eklund)

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1.1.3. Biomechanical significance

During normal locomotion the equine limb is put under a remarkable amount of stress, predominantly during the weight-bearing stance phase of the stride when the limb is in contact with the ground. The force that is exerted upon the limb by the ground during the loaded phase is defined as the Ground Reaction Force (GRF), and due to the synchronized efforts of all the individual components of the limb this force is dissipated throughout the leg as it moves proximally. Hence, a pathological state of any of the tissues may influence the dissipation and transmission of the GRF up the limb and thus further aggravate the condition by redistributing the load unevenly [13]. When the horse is lame it will instinctively put less weight on the affected part of the limb in order to minimize the pain that subsequently results in a decreased GRF on this limb, which can be objectively measured [21].

During the stance phase the horse moves through four subsequent stages starting with the initial ground contact followed by the impact; loading/unloading; and break-over phases [1]. The manner of the initial ground contact is classified as heel-first; flat-footed; or toe-first and is important because it influences the subsequent movement pattern of the hoof in the following phases, thus affecting the amount of concussion exerted upon the limb during impact. A flat-footed or slightly heel-first landing is the one deemed to generate the smoothest transition into the stance phase [9,22,23], thus creating least concussion. Moreover, the DDF aids in positioning the hoof and promote a minimally concussive landing during this phase. In a horse with chronically ill-balanced feet and a distinct heel-first or toe-first landing, the amount of concussion during the impact phase increases, causing vigorous vibrations and subsequent friction damage to the DDFT [1]. Whereas the DDFT only exerts passive support of the distal limbs during the loading phase, it plays a key role during break-over as it solely flexes the distal phalanx. Moreover, it is during this phase that the highest amount of tension is exerted on the DDFT as its purpose to flex the distal interphalangeal joint (DIPJ) is resisted by the action of the GRF working in the opposite direction [1]. It has been shown that the pressure applied upon the NB and bursa by the DDFT is at its highest right before the initiation of break-over.

Furthermore, several authors [1,9] point out the importance of the ability of the hoof to “cut” into the ground and thus allowing full movement of the fetlock joint without inducing excessive dorsiflexion and subsequently excessive strain on the DDFT during break-over. In order to cut into the earth and thus reduce the resistance of the ground to the

Fig. 4: a. Normal movement and dorsiflexion of fetlock when hoof cuts into ground. b. Excessive dorsiflexion of the fetlock when hoof cannot cut into the substrate [9].

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17 motion of the hoof, the hoof should have a rather sharp angle in the toe region. With this in mind it is rather clear that when the horse is shod, the sharpness of the toe is diminished due to the flat surface of the shoe and the hoof will consequently not dig into the ground as efficiently. Furthermore, a compact and impervious surface will provide the same difficulty and thus the same excessive strain on the distal limb (Fig. 4) [9].

1.2. Pathologies of the distal deep digital flexor tendon

Generally speaking, for optimal function it is imperative for a tendon to maintain its structure and cellular organization. Consequently, when disruption of the normal structural arrangement of the tendon occurs, weakening and subsequent damage follows [24]. However, as the tendon structure is poorly vascularized, it is generally accepted that tendon injury does not involve a classic inflammatory pathway with infiltration of leukocytes, but rather a local reaction where the resident cells release inflammatory substances [11,24]. Ultimately, chronic degenerative changes and subsequent injuries in response to an accumulation of micro-damage and an altered cell/matrix reaction [24] are the commonly encountered pathologies when examining the distal DDFT at the level of the NB. The lesions can be categorized as dorsal border lesions or core lesions according to their location and nature [25]. The most common dorsal border lesions are thermal damage, traumatic fibrillation, adhesions and parasagittal plane splits (PPS) [25]; and the most common core lesions are degeneration/core necrosis, fibrillar disorganization, fibroplasia/fibrocartilaginous metaplasia, increased cellularity and thickening of the interfascicular septae (Fig. 5).

Fig. 5: Categorization of the DDFT lesions.

DD

FT

le

sions

Dorsal border lesions

Thermal damage

Traumatic fibrillation

Adhesions

Parasagittal plane splits

Core lesions

Degeneration/necrosis Fibrillar disorganization

Fibroplasia/fibricartilaginous metaplasia Increased cellularity

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1.2.1. Dorsal border lesions

Thermal damage: As the DDFT slides back and forth over the palmar/plantar surface of the NB friction takes place, thus producing heat and subsequent thermal damage. Due to the fact that tendons are poorly vascularized, this heat is dissipated relatively slowly which allows for higher temperatures to build up within the tendon. Furthermore, with an unbalanced hoof where distinct heel-first or toe-first landings occur, the excessive vibrations caused by the third order acceleration from the improper landing create even more friction and heat between the DDFT and the NB [26,27]. Thermal damage is grossly seen as a yellow discoloration of the dorsal surface of the DDFT at the level of the NB (Fig. 6).

Traumatic fibrillation of the dorsal border of the DDFT is a common occurrence, and has been seen in both sound and lame horses at post mortem investigations [5] As cells of the synovia in the navicular bursa migrate to sites of injury, dorsal fibrillation of the DDFT may predispose to adhesion formation between the DDFT and the palmar aspect of the NB [5]. Fibrillation is characterized by an irregular dorsal surface of the DDFT, with torn fibers commonly curling up in the navicular bursa [28] (Fig. 7).

Adhesions occur at the site of injury to the distal DDFT that creates disadvantageous interconnections between the DDFT and the NB, thus interfering with the natural sliding movement of the DDFT over the palmar surface of the NB. This phenomenon occurs as a result of cell migration from within the navicular bursa to the site of injury where they infiltrate the lesion and create subsequent adhesion formation [29] (Fig. 8-9).

Parasagittal plane splits (PPS) may affect the entire thickness of the tendon and characteristically run parallel to the longitudinal fiber arrangement of the tendon. It has been stated that PPS of the DDFT most commonly occur at the level of the navicular bone [25]. Splits can affect only the superficial layer (20% of DDFT thickness), or extend further through the entire thickness of the tendon (Fig. 10). The PPS are characteristically flanked by chondrones along the entire rim [28].

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19 Fig. 6: Thermal damage on dorsal surface of the DDFT

(black arrow). (Source: Viktoria Eklund)

Fig. 8: Adhesion. (Source: Viktoria Eklund) Fig. 9: Adhesion between the DDFT and NB. Sagittal view. (Source: Viktoria Eklund)

Fig. 7: Traumatic fibrillation on the dorsal surface of the DDFT (transparent arrows) and fibrocartilage erosion on the surface of the NB (black arrow). (Source: Viktoria Eklund)

Fig. 10: Dorsal view of the DDFT. Full thickness parasagittal plane split. (Source: Deep digital flexor tendinopathy in the foot. M. C. Schramme, 2011 [28])

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1.2.2. Core lesions

Degeneration/core necrosis: It has been demonstrated that degenerative alterations in tendon tissue is characterized by an increase in the collagen (mainly type III collagen) deposition and turnover, resulting in deposits of collagen bundles in areas of injury (Fig. 11-12). This increase in collagen production may be the result of thermal damage as hyperthermia is suspected to change the biochemical environment of the tenocytes [30]. Studies have shown that tenocytes are sensitive to temperatures above 45ᵒC where, as a response to the heat, they produce an increased amount of pro-inflammatory cytokines, thus subsequently supporting the matrix degradation of the tendon [27]. Over an extended period of time, chronic tendon degeneration weakens the tendon and predisposes it to subsequent injuries [29]. There are various kinds of degeneration, such as lipoid degeneration, mucoid degeneration and so on [31]. Mucoid degeneration is seen as an increase in the production and turnover of ECM in the area of injury (in comparison to lipid degeneration where the normal tendon structure is replaced by fatty tissue) where the formerly linear pattern of collagen fibers has been disrupted. A commonly encountered pathology in areas of mucoid degeneration is focal calcification, as the normal cells that prevent mineralization have been destroyed [12].

Fibroplasia/fibrocartilaginous metaplasia/fibrillar disorganization: Migration of fibroblasts and/or chondrocytes to the site of injury typically occurs in the healing process of the wound, thus depositing ECM in the attempt to mend the lesion, however in a disorganized manner compared to the healthy undamaged tendon where the collagen bundles are arranged parallel to each other [29] (Fig. 12-13). These disordered collagen/fibrocartilage deposits have not the tensile strength and elasticity of the healthy tendon and thus make the tendon weaker in its entirety.

Thickening of interfascicular septae (Fig. 11) can easily be detected on histological specimen as the normally thin, negligible strands are filled up with proteoglycan accumulation and occluded, hyalinised blood vessels, thus creating a web of homogen thick strands [28].

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21 Fig. 11: Degeneration/core necrosis (transparent arrows) and thickening of interfascicular septa

(black arrow). (Source: Viktoria Eklund)

Fig. 12: Degeneration and fibroplasia (black arrow); fibrillar disorganization (asterisks). (Source: Viktoria Eklund)

Fig. 13: Fibroplasia/fibrocartilaginous metaplasia and fibrillar disorganization. (Source: Viktoria Eklund)

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1.3. The influence of hoof conformation on the deep digital flexor tendon

The conformation of the equine hoof has been shown to strongly relate to the soundness of the hoof and thus the entire horse. Whether it concerns the alignment of the phalanges, the angles of the hooves or the size and distribution of the various anatomical parts, they all play a significant role in the overall health of the horse as proper alignment, weight distribution and shock absorption will provide the animal with a healthier life [1]. It is important to realize that the conformation of the hoof is affected by many factors where genetics, diet and environment play major roles, as well as the fact that the internal structures are continually changing and adapting to concomitant changes in the surroundings. It is also apparent that all equine hooves follow a basic pattern of conformation that can be assumed to be the optimum, even though there is a high variety in the conformation between individuals [32].

Due to the high amount of factors affecting the conformation of the equine foot, it is usually difficult to pinpoint one single etiology of an occurring pathology which furthermore seldom comes alone [1]. Mostly the clinical condition is a result of the convergence of many factors which further complicates the work with determining the course of treatment for each individual case. Nevertheless, there are many concrete factors that can be objectively measured to aid in the determination of the status of the individual hoof. The front and rear hoof angles, and the frog:sole ratio are measurements that have a direct impact on the health of the distal DDFT and, when they are not within what is considered to be the normal range, may have a detrimental effect with subsequent pathological lesions of the DDFT [33,34].

1.3.1. Hoof angles

The anterior/posterior hoof angles of a particular foot are the composition of the length and height of the toe and the heels of that hoof. Thus, a long toe and a low heel will subsequently give a narrower toe angle whereas a shorter toe and higher heel give a wider toe angle.

Moreover, there is a variety in the established optimal hoof angles as well when comparing several authors [35,36], and an increasing number of researchers on the subject have discovered significant variations between feral horses’ feet in completely diverse environments where the horses’ hooves are exposed to dissimilar substrates [1,4,35,34,33]. To put it differently, the studies have shown that the hooves are highly affected by the environment, and no single hoof shape is optimal for all horses. Nevertheless two of the hoof parameters, namely the dorsal hoof wall angle (DWA) and the palmar angle of the distal phalanx (PA), varied little between the horse populations which suggests that these might have useful prescriptive value in the determination of the health status of the hoof [1,33]. Another angle of potential value is the heel angle (HLA), which is useful when evaluated together with the DWA [2].

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23 The dorsal hoof wall angle (Fig. 14) marks the caudal angle formed by the intersection of two lines drawn from the lateral view: along the dorsal hoof wall and along the ground surface [33]. The range of mean DWA between five different feral horse populations in one study [33] varied between 53,3 ± 2,6ᵒ and 56,8 ± 4,4ᵒ. Another author [4] found the DWA to vary between 48ᵒ - 62ᵒ in the front feet of a population of wild mustangs with the mean DWA 54ᵒ, and a third author [35] found the DWA to be smallest in horses roaming on hard substrate (51ᵒ - 57ᵒ) whereas the horses living on soft substrate had a DWA of 57ᵒ - 68ᵒ. Obviously it is difficult to find one clear answer, as has been shown throughout history [37].

The heel angle (Fig. 14) is together with the DWA an indicator of the relationship between the cranial and caudal portion of the hoof, as these two parameters should normally closely resemble each other to confirm that the heels are placed far enough back to support the internal structures of the caudal foot [2]. Thus, a HLA markedly narrower than the DWA with concurrent rearrangement of the horn tubules of the heel region in a more caudocranial fashion, could indicate that the heel is underrun (see 1.4.3. Underrun heel for further information) [2].

A mistake commonly made is to assume that the angle of the DP is the same as the angle of the dorsal wall of the hoof [38]. However, this is only true in correctly trimmed and balanced, healthy hooves, as in reality a significant portion of the equine population exhibits some degree of deviation between the angle of the DP and the DWA. Hence, trimming the hooves and aligning hoof pastern axis according to the DWA without preceding confirmation of the actual position of the DP, may result in DP misplacement and predisposition to injury [38]. In other words, a correction of the hoof pastern axis in relation to the DWA might actually cause a broken forward/back pastern axis.

1.3.2. Frog:sole ratio

The frog:sole ratio is an important indicator of the proportions of the hoof, as the bottom of the foot is the only true guide to be used when assessing the condition, as well as for trimming an optimal shape for that individual foot [1,38]. Moreover, the ideal frog:sole ratio will allow the hoof to maintain an optimal weight distribution where adequate ground contact of the frog and sole will mediate proper engagement of the sensory apparatus in the caudal foot as well as promoting vascular perfusion of the internal structures of the hoof, thus allowing for sufficient energy dissipation [1]. The frog:sole ratio is calculated by dividing the distance from the base of the frog to the apex of the frog with the distance from the apex of the frog to the tip of the toe, and should be roughly 2:1 (Fig. 15) [39].

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24 Fig. 14: Dorsal hoof wall angle (DWA) and heel

angle (HA). (Source: Viktoria Eklund)

Fig. 15: Frog:sole ratio. (Source: Viktoria Eklund)

Fig. 17: High heel. Red markings indicate the heel height over the live sole plane, yellow line demarcates the level of the frog. (Source: Viktoria Eklund)

Fig. 18: Underrun heel. Horny tubules of the heels are ‘dragged’ cranially because of the long toe, thus assuming a more horizontal pattern (indicated by the red lines). (Source: Viktoria Eklund)

Fig. 16: Markedly long toe. (Source: Viktoria Eklund)

Fig. 19: Contracted heel (white asterisks). Notice how the heels and the hoof wall are pivots medially and pinch the frog and the heel bulbs. (Source: Viktoria Eklund)

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25

1.4. Pathological conformation of the hoof and its impact on the deep digital flexor

tendon

There are numerous conformational abnormalities affecting the equine hoof, however, only those posing a potentially harmful influence on the morphology and function of the DDFT will be discussed here. Pathological conformation occurs when there is a distortion of the optimal foot parameters previously discussed (see section 1.3. The influence of hoof conformation on the deep digital flexor tendon). Furthermore, it is important to understand that there are currently not a generally accepted single set of standards for the healthy versus pathological conformation of the hoof, but rather some disagreement on this matter [1,4,37], thus making it an important topic for future research and further clarification on the subject.

1.4.1. Long toe

As the domestic horses are not able to naturally abrade their hooves in the same fashion as the feral horses due to metal shoes preventing wear, and lack of consistent movement when confined in stalls or small paddocks, the hooves may grow excessive material, thus potentially assuming a distorted shape with the dorsal hoof wall migrating forward. This conformational abnormality of having a longer than normal toe will inevitably promote a toe-first landing and delay the break-over time for that particular hoof, thus promoting excessive strain on the internal structures of the foot and especially the DDFT [1,40]. Subsequently the long toe will redistribute the area of ground contact during impact further cranially, thus inhibiting the natural activation of the caudal foot with diminishing of proprioception and energy dissipation [1] which will lead the forces from the impact to be distributed to other structures such as the DDFT instead, thus increasing the probability of developing degenerative changes.

A long toe may be confirmed by assessing the frog:sole ratio (Fig. 16), thus finding that the sole length is too long compared to the frog length (normal ratio is 2:1) [1,39].

1.4.2. High heel/Low heel

There is a rough guideline available [1] which remarks the extremes in both directions considering high/low heels. Heel height is evaluated in relation to the callused sole plane, where a heel height >20 mm over the sole plane is esteemed as a high heel (Fig. 17), and a heel height below the sole plane is graded as low.

1.4.3. Underrun heel

A rather arbitrary definition has been made where the heel was determined underrun when the HLA was more than 5ᵒ smaller than the DWA [2]. Currently the general opinion is moving towards

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26 a more holistic approach where the condition of the entire hoof is evaluated together with the state of the heels [1]. Hence, if the heels per definition of the hoof angles are underrun, but the structures of the hoof – the bars, the hoof wall or the angle of the sole – are healthy, then the heel is not considered underrun. As a matter of fact, studies have shown that it is quite common in sound horses to have an obvious discrepancy between the front and rear hoof angles [1].

However, in a situation with an underrun heel, the cause can commonly be traced back to a long, flared toe causing complete capsule rotation as it stretches forward, thus leaving the horny tubules in the heel region susceptible to bending and/or crushing [1]. Hence, instead of the heels expanding laterally during impact, they are bent and/or crushed underneath the hoof wall with subsequent distortion of the energy dissipation (Fig. 18).

1.4.4. Contracted heel

Contracted heels are commonly a consequence of poor internal structure development and toe-first landings for whatever reason. The energy dissipation through the caudal foot does not generally function well with contracted heels. All cases of contraction carry distinct signs such as a narrow frog where the heels seem to ‘pinch’ the frog and heel bulbs together (Fig. 19); distinct caudal foot sensitivity; thin and underdeveloped lateral cartilages and DC.

1.4.5. Third order acceleration of the distal phalanx

Vibrations occur even during the normal movement of perfectly balanced feet with a proper landing, as the energy from the impact causes some amount of oscillation. However, the equine limb is designed to redistribute this small amount of vibrations without causing substantial damage to the tissues) [23]. As has already been discussed, conformational abnormalities such as a long toe/high heel/low heel will yield an improper landing with subsequent rotation of the DP, so-called third order acceleration [26], thus instigating excessive vibrations in the hoof. There are currently not many studies available regarding the extent of the damage of chronic excessive vibrations in equine limbs, however in human medicine there has been numerous studies undertaken to investigate the consequences of long-term use of vibrating machines on human limbs, the so-called hand-arm vibration syndrome (HAVS) [41]. Chronic damage affecting mainly the vascular, musculoskeletal and neurological systems has been demonstrated, and the probability of similar adverse effects resulting from the amplified oscillations occurring in the equine hoof during misplacement on the ground cannot be excluded [41].

As these vibrations exerted by the rotation of the distal interphalangeal joint (DIPJ) at the instant of ground contact travels up the limb, the DDFT is affected as well, thus moving rapidly across the palmar surface of the NB causing friction with subsequent thermal damage and tendon degeneration [23]. This chronic condition with degenerative damage at the interface between DDFT

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27 and NB is known as the Navicular Disease (ND). As genuine ND is the cumulative effect of friction damage over an extended period of time rather than a single event, it is truly rarely encountered in younger individuals, however quite uncommon in older horses as well compared to how often it is diagnosed [9].

1.5. The influence of horse´s age on the deep digital flexor tendon

Age related changes can be seen in the tendinous structure at the histological level, typically as an increase in vascularization and a change in the distribution and amount of the components of the ECM (i.e. lowering of the collagen content) [10]. However, studies have been undertaken where no correlation between age and histological findings could be found, regardless of whether the studied population of horses were clinically healthy or presented with hoof pathology [42]. In other words, the histological degenerative abnormalities encountered has been suggested to be the result of individual predisposition further enhanced by nonphysiological biomechanical elements such as type of athletic discipline and hoof maintenance (shoeing) [5].

In conclusion the deep digital flexor tendon is a dense, fibrous structure closely resembling fibrocartilage at the level of the navicular bone, which main function is to initiate break-over and flexion of the distal phalanx. The stress exerted upon the DDFT during locomotion in combination with conformational deformities such as a long toe, high heel, underrun heel and/or contracted heel may have an adverse effect on the morphology of the tendon, thus resulting in the development of pathologies, both on the dorsal surface of the tendon and in the deeper layers. Specifically, lesions such as thermal damage, traumatic fibrillation, adhesion formation and parasagittal plane splits are commonly encountered on the dorsal surface of the DDFT at the level of the NB, with core lesions of the same area mainly comprising degeneration and necrosis, thickening of interfascicular septae, fibrillar disorganization and fibroplasia/fibrocartilaginous metaplasia.

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28

2. RESEARCH METHODS AND MATERIAL

This research work was carried out at the Veterinary Academy Pathology Center of the Lithuanian University of Health Sciences (LUHS) in Kaunas, Lithuania. The sampling procedure and preparation of histological specimen were undertaken between October 2016 and June 2017.

For this study, 71 cadaveric front limbs of 41 horses of a Lithuanian cold-blood and Žemaitukai mixed breed subjected to slaughter for reasons other than this study, were sectioned at the level of the metacarpophalangeal (fetlock) joint, labeled at the hoof wall and brought to the LUHS Pathology Center for further investigation. The horses were selected from a slaughterhouse in Lithuania, based on age and weight. The dissection and collection of the hooves took place immediately after euthanasia. The age ranged from 4 to 22 years (mean ± s.d. 12, 9 ± 5, 5) and weight ranged from 430 to 865 kg (mean ± s.d. 582 ± 85).

At the LUHS Pathology Center the feet were measured, photographed and dissected for further histological sampling of the deep digital flexor tendon (DDFT) and associated structures. Additionally, the macroscopic changes of the DDFT encountered during these procedures were duly recorded. Photos of each hoof were taken from the front, lateral side, rear, solar and mid-sagittal view (example of photographic recording of the hooves can be seen in Annex 2) for the possibility of detecting any occurring external abnormalities of the feet. External and internal gross pathologies of the DDFT (in the mid-sagittal view and in the specimen obtained for the histological slide preparation) were recorded at the same time as photography. The lateral photograph was used for the measurement of the DWA and HA, and the solar view was used for the measurement of the frog:sole ratio as it has been shown that digital photography is a more accurate method than using various measuring devices on the hoof [43].

Horses were divided into two age groups: younger horses ≤12 years old (Group 1, n=21); and older horses >12 years old (Group 2, n=20) for evaluation of the correlation between age and obtained common pathomorphological lesions of the DDFT (see Table 1). As there

were no youngsters (<4 years old) in the horse population for this study a classical arrangement of three groups could not be used. Instead, the two groups were determined by using the mean age as a divider, as well as taking into account that degenerative lesions of the DDFT commonly occur in older horses from 8-10 years of age [44]. No control group of healthy hooves was used since all of the hooves obtained for this study carried internal damage.

Table 1: Summary of the horse population characteristics. a) Gross external and internal lesions, b) Histopathology

Mean ± s. d. Number of hooves Group 1 8,2 ± 2,5 38a, 34b Group 2 17,8 ± 3 33a, 25b Category 1 524 ± 34 36a, 31b Category 2 650 ± 71 35a, 29b

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29 For the correlation between weight and dorsal border lesions, the hooves (n=71) were divided into two categories: Weight ≤ 565 kg (Category 1, n=36); and weight >565 kg (Category 2, n=35) (see Table 1). For the correlation between weight and core lesions, the horses were divided into two categories: Weight ≤565 kg (Category 1, n=31); and weight >565 kg (Category 2, n=29). For the correlation between conformational abnormalities and multiple dorsal border lesions, the number of lesions was divided into four classes: Class 0 = 0 lesions; class 1 = 1 lesion; class 2 = 2 lesions; class 3 = 3 lesions present. For the correlation between conformational abnormalities and multiple core lesions, the number of lesions was divided into four classes: Class 0 = 0 lesions; class 1 = 3 lesions; class 2 = 4 lesions; class 3 = 5 lesions present.

2.1. Pathomorphology of the deep digital flexor tendon

2.1.1. Dorsal border lesions

The pathomorphological lesions of the DDFT were divided into dorsal border lesions and core lesions. The dorsal border lesions comprised DDFT pathologies visible to the naked eye such as thermal damage, traumatic fibrillation, adhesion formation and parasagittal plane splits.

2.1.2. Core lesions

For the purpose of histopathological specimen preparation, samples of 10-15 mm thickness were taken from the distal DDFT at the level of the sagittal ridge on the flexor surface of the NB of each hoof, by making one parasagittal cut on either side of the mid-sagittal plane of the hoof. The mid-sagittal area of the DDFT and NB was chosen because of the convenience of being able to obtain samples from the same area of all hooves by cutting along the center line of the hoof (Fig. 1). The samples were fixed in 10% neutral buffered formalin and later embedded in paraffin wax prior to being sectioned at 6 μm, using a rotary microtome. Sections were stained routinely with Giemsa and H&E stain. The specimens were investigated along with concomitant photography of the encountered pathologies for further evaluation.

2.2. External lesions of the hoof

Measurements of the dorsal wall angle, heel angle and frog:sole ratio were taken. Example of the measurement table used can be seen in Annex 1.

2.2.1. The external lesions

The external lesions comprised conformational abnormalities of the hoof such as long toe, high heel, underrun heel and contracted heel. Long toe was evaluated using the frog:sole ratio, where a ratio of <1,8 was considered as long toe [1,39]. Heel height was determined in relation to

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30 the callused sole plane, where a heel height >20 mm over the sole plane was rated as a high heel, and a heel height below the sole plane was rated as low. The reference range from J. Jackson’s work [4] for the DWA was used, with a DWA of 48ᵒ - 62ᵒ being normal. Underrun heels were deemed present if the HLA was more than 10ᵒ smaller than the DWA [1,2], and there were any signs of crushing/bending of the tubular horn of the heel. Contracted heels were evaluated with regards to the overall hoof shape and the transition between the frog and the heel bulbs [1].

2.3. Statistical data

The program Microsoft® Excel2010 was used to organize all data about the hooves and perform the statistical analysis with a significance level set at P<0.05.

The horse population was confirmed to be normally distributed by the combination of a high correlation coefficient for DWA (0,984) and for the frog:sole ratio (0,995); and a linear scatter plot for each of the two parameters (Fig. 20).

For the investigation of the correlation between age/weight and pathomorphological lesions of the DDFT, a hypothesis test for the difference of two proportions was used. For the investigation of the correlation between hoof conformation and pathomorphological lesions of the DDFT, a two-tailed Fischer’s exact test was used in cases of comparison between single/combined hoof conformation and single lesion, which is recommended since it always calculates the exact p-value, while the chi-square only gives an approximation to the p-value. Hooves were then grouped into ‘normal’ vs ‘abnormal’ hoof conformation with two possible outcomes: ‘with lesion’ or ‘without lesion’.

In comparison between single/combined hoof conformation and multiple lesions, a Fischer’s exact probability test 2x4 was used.

-3 -2 -1 0 1 2 3 0 50 100 Z-score Angle Normality DWA -3 -2 -1 0 1 2 3 0 1 2 3 Z-score Frog:sole ratio Normality frog:sole ratio

Fig. 20: Linear scatter plots confirming the normal distribution for DWA and frog:sole ratio in the horse population included in this study.

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31

3. RESEARCH RESULTS

3.1. Dorsal border lesions

3.1.1. Summary

The summary of all detected dorsal border lesions during the entire study can be seen in Fig. 21.

Only six hooves (8%) had adhesion formation and two hooves (3%) had parasagittal plane splits, whereas more than half of the population had traumatic fibrillation (56%) and thermal damage (87%). One of the horses had adhesion formation on both front legs, whereas the other horses with adhesions only had it on one front leg. All horses with adhesions were ≥ 10 years of age.

3.1.2. Pathomorphology

On the gross view, thermal damage can be seen as a yellow discoloration on the dorsal surface of the deep digital flexor tendon, with nuances ranging from pale yellow to dark brown (Fig. 22). Histologically, degenerative changes along with fibrillar disorganization, thickening of interfascicular septae and fibroplasia/fibrocartilaginous metaplasia are frequently seen.

Traumatic fibrillation appears as an irregular dorsal surface of the DDFT on the gross view, with torn fibers commonly curling up in the navicular bursa (Fig. 23). In the majority of the samples, traumatic fibrillation occurs together with thermal damage. On the histological view, traumatic fibrillation is seen as collagen fibrils separated from the tendon, containing scattered chondrocytes. Commonly, these tendons carry degenerative changes, fibrillar disorganization and

0% 10% 20% 30% 40% 50% 60% 70% 80% 90%

Thermal damage Traumatic

fibrillation

Adhesion formation Parasagittal plane splits 87%

56%

8%

3% Fig. 21: Summary of all dorsal border lesions of the investigated horse population.

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32 fibroplasia/fibrocartilaginous metaplasia with an increase in cellularity and amount of ECM due to the attempts of the tissue to heal.

Adhesions are grossly viewed as fibrous interconnections between the dorsal surface of the DDFT and the palmar surface of the navicular bone (Fig. 24). Blood and red/purple discoloration are frequently observed due to the inflammation and healing activity. Thermal damage and traumatic fibrillation are commonly the precursors of adhesions, as surface injuries to the NB and tendon activates the migration of cells within the navicular bursa to the site of injury. On the sagittal view, an invasion of tendinous tissue into the cortex and medulla of the NB can be clearly seen as an indentation in the bony cortex filled with collagen. Histologically, adhesions have a unique appearance with massive fibroplasia consisting of large deposits of ECM and rounded cells infiltrating the bony tissue (Fig. 25). The tendon structure is severely disorganized and the smooth hyaline cartilage layer covering the dorsal surface is disrupted by the outgrowth of fibrous tissue reaching deep into the NB (Fig. 24). Cells constitute a relatively large portion of the fibrous tissue that extends into the bone. Entrapment of blood can be seen along the edge of the infiltrating tissue (Fig. 26), and the cartilage covering the bone is completely destroyed at the site of injury.

Where only one of the two front hooves had adhesions, there was generally little/no difference in the conformation between the hooves, and the other hoof without adhesions had severe traumatic fibrillation and thermal damage in the majority of the cases.

Fig. 23: Photograph above illustrating traumatic fibrillation and thermal damage of DDFT (asterisk). Note the simultaneous destruction of cartilage on the surface of the navicular bone.

Fig. 22: Photograph above illustrating thermal damage seen as orange/brown discoloration on the dorsal surface of the DDFT.

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33 Fig. 24: Photograph to the left: Histological section of the DDFT illustrating an adhesion formation (asterisks). Note the invasion of collagen tissue in the navicular bone and the complete destruction of the osseous cortex. White areas of the sample are processing artefacts. Giemsa stain. Magnification x 5. Photograph to the right: Sagittal section of DDFT, gross view of an adhesion between the DDFT and NB (black arrow).

Fig. 25: Sagittal section of the DDFT showing fibrous infiltration of the navicular bone (black arrow). The edge of the bone is marked by black arrowheads, and the navicular bursa is marked by asterisks. Giemsa stain. Magnification x 40.

Fig. 26: Sagittal section of the DDFT showing the margin between the navicular bone (NB) and the infiltrative tendon tissue (DDFT). Entrapment of blood along the margin is marked by black arrowheads. Giemsa stain. Magnification x 40.

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34 The parasagittal plane splits found could only be seen on the histopathological examination where the superficial hyaline cartilage was torn and a line of disrupted tissue was stretching from the surface of the dorsal border of the DDFT deeper into the tendon towards the core (Fig. 29). The line was surrounded by an increase in cellularity and fibrocartilaginous metaplasia along the entire margin of the lesion.

3.1.3. Statistics

There was no statistically significant correlations between age (Group 1, n=38; Group 2, n=33) and dorsal border lesions (P-value= 0,0592) (Fig. 27). There was no statistically significant correlation between weight (Category 1, n=36; Category 2, n=35) and dorsal border lesions (All p-values >0,05) (Fig. 27). There was no difference between left and right hoof regarding the amount and/or type of dorsal border lesions (All p-values >0,05) (Fig. 27).

3.2. Core lesions

3.2.1. Summary

For the purpose of the histopathological investigation, specimens from 59 hooves were obtained. The summary of all detected core lesions during the entire study can be seen in Fig. 28. More than four out of five hooves carried signs of degeneration/core necrosis (83%), fibroplasia/fibrocartilaginous metaplasia (83%) and fibrillar disorganization (86%), while increased cellularity (39%) and thickening of interfascicular septae (47%) were detected in less than half of

43% 43% 82% 94% 89% 86% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 1 2 Side (left/right) Age (Group 1/Group 2)

Weight (Category 1 /Category 2)

Fig. 27: Graph of the correlations between dorsal border lesions and the different attributes age,

weight and side (left/right hoof). Number 1 on the horizontal axis represents group 1 (age), category 1

(weight) and left hoof (side); number 2 represents group 2 (age), category 2 (weight) and right hoof (side).

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35 the hooves. Additionally, of all the hooves with degenerative changes, six hooves were found to have degeneration with nodular calcinosis (10%).

3.2.2. Pathomorphology

Degeneration/core necrosis can be seen as well demarcated foci in the tendon tissue where the normal tendon structure has been replaced by disorganized deposits of collagen bundles, mainly consisting of type III collagen (Fig. 30). When stained with Giemsa stain, the necrotic foci appear darker than the surrounding normal tissue, and homogenous in nature. Only mucoid degeneration with increased deposits of extracellular matrix was found in this study, thus no samples had lipid degeneration. The calcified centers of the lesions are well demarcated and ovoid with a granular appearance, and stains pink with Giemsa stain (Fig. 31).

Histopathologically, fibroplasia/fibrocartilaginous metaplasia is seen as an area with ECM deposited in a disorganized manner compared to the undamaged tendon. There is commonly an increased production of collagen and/or cartilaginous ground substance depending on the location of the lesion, and an increase in rounded cells of both tendinous and cartilaginous origin are usually present (Fig. 29-30). In the centers of the lesions there may be loss of structural components and cells due to core necrosis, with a more homogenous appearance resembling degenerative changes.

Fibrillar disorganization commonly occurs in association with degeneration and fibroplasia/fibrocartilaginous metaplasia, and is the result of the disruption of the linear pattern of the tendinous collagen fibers. The fibers run in all directions instead of parallel to each other (Fig. 30).

Increased cellularity implies increased activity of metabolically active cells in the area of injury or stress (Fig. 29-30), and thickening of interfascicular septae are commonly early signs of

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 83% 83% 86% 39% 47% 10% Fig. 28: Summary of all core lesions of the investigated horse population.

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36 increased stress to the tendinous tissue. The normally thin, negligible strands are filled up with proteoglycan accumulation and occluded, hyalinised blood vessels, thus creating a web of homogen thick strands (Fig. 32). The widened septae stain dark purple with Giemsa stain and acquire a cloudy, homogenous appearance.

Fig. 30: Sagittal section of the DDFT showing a core lesion (transparent arrow). There are core necrosis and loss of normal collagen tissue in the center of the lesion along with fibroplasia/fibrocartilaginous metaplasia (small blue spots are fibrochondrocytes); fibrillar disorganization in the periphery; and processing artefacts (black arrows). Giemsa stain. Magnification x 40.

Fig. 29: Parasagittal plane split (black arrowheads) and fibrocartilaginous metaplasia with increased cellularity (asterisk). Sagittal section of the DDFT. White areas of the sample are processing artefacts. Giemsa stain. Magnification x 40.

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37

3.2.3. Statistics

Nearly 90% of the population had some kind of pathomorphological lesion, with thermal damage being the most common one (Fig. 33). All hooves that did have dorsal border lesions had thermal damage. In a similar fashion did all hooves that had core lesions have fibrillar disorganization.

Fig. 31: Sagittal section of the DDFT showing mucoid degeneration with nodular calcinosis (white arrows). White areas of the sample are processing artefacts. Giemsa stain. Magnification x 40.

Fig. 32: Sagittal section of the DDFT showing prominent thickening of interfascicular septa (arrows). Giemsa stain. Magnification x 40.

LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY