Patomorfologinis žirgų varlės kaulo pažeidimų vertinimas Pathomorphology of equine navicular bone lesions

LITHUANIAN UNIVERSITY OF HEALTH SCIENCES

VETERINARY ACADEMY

Faculty of Veterinary Medicine

Sandra Eriksson

Patomorfologinis žirgų varlės kaulo pažeidimų

vertinimas

Pathomorphology of equine navicular bone

lesions

MASTER THESIS

of Integrated Studies of Veterinary Medicine

Supervisor: Dr. Jūratė Sabeckienė

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THE WORK WAS DONE IN THE DEPARTMENT OF VETERINARY PATHOBIOLOGY CONFIRMATION OF THE INDEPENDENCE OF DONE WORK

"PATHOMORPHOLOGY OF EQUINE NAVICULAR BONE” I confirm that the presented Master Theses

1. has been done by me; Sandra Eriksson

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 Sandra Eriksson

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

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

2017-12-15 Sandra Eriksson

CONCLUSION OF THE SUPERVISOR REGARDING DEFENCE OF THE MASTER THESES

2017-12-15 Jūratė Sabeckienė

THE MASTER THESES HAVE BEEN APPROVED IN THE DEPARTMENT OF VETREINARY PATHOBIOLOGY

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

Reviewers of the Master Theses 1)

2)

(name, surname) (signatures)

Evaluation of defence commission of the Master Thesesis (date of approbation) (name, surname of the defence

com

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CONTENT LIST

1.1 Functional anatomy of the navicular bone ... 12

1.2 Gross pathology of the navicular bone ... 13

1.2.1. Fibrocartilage alterations ... 13

1.2.2. Parasagittal plane splits ... 13

1.2.3. Dorso-palmar fibrocartilage erosion ... 14

1.2.4. Yellow discoloration ... 15

1.2.5. Adhesions ... 15

1.3. Histopathology of the navicular bone ... 16

1.3.1. Fibrillation of the fibrocartilage ... 16

1.3.2. Chondrocyte cluster formation and loss of cellular density ... 17

1.3.3. Necrosis of fibrocartilage and subchondral bone ... 17

1.4. Hoof functional anatomy ... 18

1.5. Hoof external pathologies ... 19

1.5.1. Hoof conformations effect on the navicular bone ... 19

1.5.2. High heels ... 20

1.5.3. Underrun heels ... 21

1.5.4. Long toe ... 21

1.5.5. Long toe-underrun heel ... 22

1.5.6. Frog pathologies ... 22

1.5.7. Laminitic rings ... 23

1.5.8. Contracted heels ... 24

1.6. Individual parameters affecting the pathologies of the navicular bone ... 25

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1.6.2. Age related changes in the navicular bone ... 25

2. RESEARCH METHODS AND MATERIAL ... 26

2.1. Sampling population ... 26

2.2. Pathomorphological lesions of navicular bone ... 27

2.3. Collection of samples for histopathological investigation ... 27

2.4. Histopathological preparation and staining ... 28

2.5. External hoof pathologies ... 28

2.6. Hoof measurements ... 29

3. RESEARCH RESULTS ... 30

3.1. Sagittal ridge area examination ... 30

3.2. Gross pathology of navicular bone ... 31

3.2.1. Graphical representation of the identified gross pathological findings ... 31

3.2.1.1. Gross pathology of navicular bone seen in all the hooves ... 31

3.2.1.2. Gross pathology of navicular bone in each age groups ... 31

3.2.1.3. Gross pathology of navicular bone in each weight groups ... 32

3.2.1.4. External hoof conformations effect on gross pathology of the NB ... 33

3.2.1.4.1. Long toe-underrun heel and the effect on gross pathologies of the NB 33 3.2.1.4.2. Contracted heels and their effect on gross pathologies of the NB ... 35

3.2.1.4.3. Frog damage and the effect on gross pathologies of the NB ... 37

3.2.1.4.4. Laminitic rings and the effect on gross pathologies of the NB ... 38

3.2.2. Photographic documentation of the pathomorphological findings in the NB ... 39

3.2.2.1. Fibrocartilage thinning ... 39

3.2.2.2. Parasagittal plane splits ... 40

3.2.2.3. Fibrocartilage erosion ... 40

3.2.2.4. Yellow discoloration ... 41

3.2.2.5. Adhesions ... 42

3.3. Histopathology of the navicular bone ... 43

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3.3.1.1. Histopathology of navicular bone seen in all the hooves ... 43

3.3.1.2. Histopathology of the navicular bone in each age group ... 44

3.3.1.3. Histopathology of the navicular bone in each weight group ... 44

3.3.1.4. Fibrocartilage alteration table ... 45

3.3.1.4.1. Fibrocartilage alteration pictures ... 45

3.3.1.4.2. Fibrocartilage erosions ... 46

3.3.1.4.3. Cartilage necrosis ... 47

3.3.1.5. Tables describing the chondrocyte clusters distribution ... 47

3.3.1.5.1. Chondrocyte clusters in the hooves analysed in both age groups ... 47

3.3.1.5.2. Chondrocyte clusters distribution pictures ... 48

3.3.2. Photographical representation of histopathological findings ... 49

3.3.2.1. Loss of cellular density ... 49

3.3.2.2. Subchondral bone necrosis ... 49

3.3.2.3. Cartilage bulging ... 50

4. DISCUSSON OF RESULTS ... 51

CONCLUSION ... 56

SUGGESTIONS AND RECOMMENDATION ... 57

LIST OF LITTERATURE ... 58

ANNEXES ... 60

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SUMMARY

Pathomorphology of equine navicular bone lesions

Sandra Eriksson

Master Thesis

The purpose of this study was to investigate the possible correlation between the external hoof conformations of the horse and the presence of navicular bone pathologies and to identify the most common gross and histopathological lesions. The horses included into the study were grouped according to age and weight with the purpose of differentiating the actual pathological changes from age-related changes in the navicular bone, and to see the possible correlation between weight and the presence of lesions in the navicular bone.

Both front hooves from 60 horses were used in the study and these were collected immediately after slaughtering. The external analysis of the hoof conformations was done photographically and through specific linear and angular measurements. The hooves were then subjected to sagittal plane splitting and the gross internal pathologies of the hoof were evaluated. The external hoof pathologies were compared with the internal gross lesions identified. 47 samples of the navicular bone were used for the histopathological analysis, these were collected from the central portion of the sagittal ridge as it is the most common region for development of pathological alterations.

The results showed that 92% of the hooves had gross pathological changes of the navicular bone. Thinning of the dorso-palmar fibrocartilage was the most frequently occurring gross pathological lesions occurring in 82% (n=49) of the hooves. Other identified lesions included parasagittal splitting, erosions of the dorsal fibrocartilage and yellow discoloration, each of them being present in 58% of the hooves. The rarest finding was adhesions seen in 8% (n=5) of the hooves.

The histopathological evaluation showed that 70% of the hooves suffered from various types histopathological alterations. The most common histopathological finding was fibrocartilage alterations, other common histopathological findings include: chondrocyte cluster present in 55% (n=26) of the hooves and loss of cellular density, 38% (n=18).

The results further proved that both age and weight had an effect on the development of navicular bone pathologies. Gross pathological and histopathological lesions were present in an overall higher percentage in horses >10 years of age and horses weighing over 565kg. The most significant age correlation was seen for cartilage thinning, present in 91 % (n=29) of the horses >10 years old and 72%, (n=20) in the horses ≤ 10 years old. For the weight groups, gross pathological lesions were 2-4 percentage points (pp.) higher in horses > 565kg in all categories cases except for yellow discoloration. When evaluating the histopathological lesions in the two weight groups the

7 most significant difference between them was the loss of cellular density, 44% in horses > 565kg and 32 % in horses weighing < 565kg. The p values however were more than 0,05 and demonstrated the results regarding weights effect on the prevalence of gross and histopathological lesions were not statistically relevant for the common horse population.

Presence of external hoof pathologies was proven to be correlated with the development of navicular bone lesions as the hooves having poor hoof conformation and external hoof pathologies had a higher risk of developing various gross internal lesions. Further reviewing of the results did however prove that the development of navicular bone pathologies does not solely depend on poor hoof conformation but may be affected by other parameters as well.

Key words: Horses, navicular bone, external hoof conformation, navicular bone lesions,

fibrocartilage alterations, fibrocartilage erosion, thinning of navicular bone cartilage, chondrocyte clusters.

SANTRAUKA

Patomorfologinis žirgų varlės kaulo pažeidimų vertinimas

Sandra Eriksson

Master Thesis

Šio tyrimo tikslas buvo nustatyti galima koreliaciją tarp išorinės žirgų kanopų sandaros ir varlės kaulo patologijų, įvertinant dažniausiai pasitaikančius makroskopinius ir histopatologinius varlės kaulo pažeidimus. Vertinant, nuo amžiaus priklausomus, varlės kaulo pažeidimus, tirti žirgai buvo suskirstyti pagal amžių ir svorį. Taip pat šios grupės buvo naudotos ieškant koreliacijos tarp žirgų svorio ir varlės kaulo pažeidimų.

Tyrimui naudotos abi priekinės 60 žirgų kanopos, surinktos skerdimo metu. Išorinių kanopos patikimų analizė atlikta kanopas fotografuojant ir naudojant specialius linijinius bei kampų matavimus. Vėliau kanopos buvo perpjautos per sagitalinę liniją ir nustatyti vidiniai kanopų struktūrų pažeidimai, kurie buvo lyginami su išoriniais kanopų sandaros pakitimais. Patologiniam histologiniam tyrimui buvo naudojami 47 varlės kaulo mėginiai, paimti iš centrinio sagitalinio varlės kaulo krašto, kuriame dažniausiai nustatomos patologijos.

Tyrimo metu 92% kanopų varlės kauluose nustatyti makroskopiniai patologiniai pažeidimai. Dažniausiai nustatytas makroskopinis pakitimas buvo dorso-palmarinės dalies varlės kaulo kremzlės suplonėjimas 82 % (n=49), kiti pakitimai buvo: parasagitaliniai plyšiai, erozijos ir geltonoji pigmentacija (po 58%). Rečiausias nustatytas pakitimas – sąaugos, rastos 8% (n=5) kanopų varlės kaulų.

8 Histopatologinio tyrimo metu patologijos buvo rastos 70% kanopų. Mažesnis mikroskopinių pažeidimų skaičius, lyginant su makroskopiniai pažeidimais susidaro dėl to, kad geltonoji pigmentacija, rasta makroskopinio tyrimo metu, nebuvo matoma patologiniuose histologiniuose preparatuose. Dažniausiai rasta patologinė histologinė patologija buvo varlės kaulo skaidulinės kremzlės pažeidimai, kuriems priklausė kremzlės fibriliazija, erozijos ir nekrozė. Kiti rasti pakitimai buvo: chondrocitų telkiniai 55% ir ląstelingumo sumažėjimas 38%.

Tolimesni tyrimai parodė, kad amžius ir svoris turi įtakos pažeidimų formavimuisi, nes didesnis kiekis pažeidimų buvo nustatyti vyresniems nei 10 metų ir sunkesniems nei 565 kg žirgams. Stipriausia koreliacija buvo matoma tarp pagrindinės pasitaikančios patologijos, kremzlės suplonėjimo ir amžiaus: 91% (n=29) pažeistų kanopų vyresniems nei 10 metų žirgams ir 72% (n=20) pažeistų kanopų jaunesniems nei 10 metai žirgams. Lyginant svorio grupes, visose patologijų grupėse, išskyrūs geltonąją pigmentaciją, makroskopinių patologijų rasta 2-4% daugiau sunkesniems nei 565 kg žirgams. Lyginant dvi svorio grupes ir histopatologinius pakitimus, nustatytas didžiausias skirtumas matomas ląstelingumo sumažėjime: 44% pirmoje grupėje (> 565kg) ir 32% antroje grupėje (< 565kg). Kitų žirgų svorio ir varlės kaulo makroskopinių ir mikroskopinių pažeidimų koreliacija, tirtų žirgų populiacijoje, nebuvo statistiškai reikšminga.

Išoriniai kanopos sandaros pažeidimai koreliavo su varlės kaulo patologijų vystymusi, nes fiziologiškai neteisinga kanopų forma ir išorinės kanopų sandaros patologijos didina įvairių varlės kaulo pažeidimų riziką. Tolimesni tyrimai parodė, kad varlės kaulo patologijos kyla dėl daugelio faktorių ir kanopos išorinės sandaros pokyčiai yra tik vienas iš šių faktorių.

Raktiniai žodžiai: žirgai, varlės kaulas, išorinė kanopų sandara, varlės kaulo pažeidimai,

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ABBREVIATIONS

PP – Proximal phalanx DP – Distal phalanx DC – Digital cushion

DDFT – Deep digital flexion tendon NB – Navicular bone

ND – Navicular disease

PDA – Podotrochlear apparatus DIPJ – Distal inter phalangeal joint HE – Hematoxylin and eosin ECM – Extracellular matrix

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INTRODUCTION

Navicular disease, a commonly used term in the field of equine medicine and one that despite its name is not dependent on a pathology of the navicular bone itself (1). The term has various definitions which are often the cause of confusion among publishers in the field of veterinary medicine. For the purpose of this study navicular disease is defined as degenerative changes affecting the navicular bone compromising the mechanical function of the bone and surrounding tissues (2). Navicular disease is a complex of disease processes responsible for 1/3 of cases of chronic forelimb lameness in horses in North America (3) and it is well known that it often, although not exclusively, involves the navicular bone. The pain and dysfunction resulting from the degenerative processes occurring in the navicular bone and its supportive structures makes it one of the most important diseases in the field of equine veterinary medicine (4).

Gross pathologies affecting the navicular bone have been described in several studies (3,4,5,6) and the most commonly mentioned ones are yellow discoloration, dorso-palmar cartilage thinning and navicular bone necrosis (5,6,7,8). When it comes to histological investigations authors have been able to identify some common histopathological changes in the bone including: fibrocartilage lesions, subchondral bone necrosis, fibrillations of dorso palmar fibrocartilage, loss of cellular density and chondrocyte alterations (5,8). Despite extensive research conducted on the histopathological alterations in the NB (1,5,8) there is limited information regarding the normal histological appearance of the navicular bone and the descriptions in literatures are often limited or non-existent (9,10,11,12,13).

Several studies have been done on the gross pathological and histopathological appearance of the navicular bone (1,2,5,6,7,14), despite this the amount of information on how the hoof appeared prior to the pathological sampling and evaluation is limited in these studies (5,7,8,14). Therefore, the aetiological factors causing the pathological alterations in the NB are still under investigation (8).

It is well known that hoof conformation and hoof angles have a tremendous impact on the forces acting on the navicular bone (4,15,13,16) the relationship between this and the actual pathological findings however is poorly understood and remains open to speculation. Therefore, one of the main purposes of this study was to investigate the possible correlation between the external hoof conformations of the horse and the presence of navicular bone pathologies.

Individual parameters of the horse may also affect the development of NB pathologies thus the horses used in this study were grouped according to age to be able to identify which pathological changes in the navicular bone were caused by a normal age-related phenomenon. Horses were also divided into groups based on weight to see if there was any possible correlation between weight and extent of damage in the bone which has been suggested in other studies (12,17).

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The aim of the work: To evaluate pathomorphological lesions of navicular bone in horses. Objectives:

1. To determine the most frequently occurring lesions of navicular bone in horses.

2. To compare lesions with the horse’s age and weight.

3. To determine the possible dependence between hoof conformation and navicular bone lesions.

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1. REVIEW OF LITERATURE

1.1 Functional anatomy of the navicular bone

The portion of the lower distal forelimb extending from the fetlock (metacarpophalangeal joint) all the way to the hoof is the main source of weight bearing in the horse (12,13). The three most important bones in this region (with regard to the load applied to the navicular bone) is the proximal, middle and distal phalanges with the last two holding the largest significance to the hoof functional anatomy (11,10). The navicular bone is a small flattened bone located ventrally to the articulate surface of proximal and distal phalanx (Fig. 1). The bone is anchored to the surrounding bones by the ligaments forming the suspensory apparatus including: the proximal sesamoidean suspensory ligament which anchors the bone to the middle phalanx cranially and the distal sesamoidean impair ligament which attaches the navicular bone to the coffin bone caudally (13,16,11,10). The suspensory apparatus forms a central part of the podotrochlear apparatus (includes the impair ligament, collateral sesamoidean ligament, navicular bursa, and DDFT) and together with the navicular bone they act as a pulley redirecting the forces exerted by the DDFT during motion (12).

The DDFT is the main flexor tendon of the distal forelimb, it extends along the ventral portion of the navicular bone (Fig. 1), running all the way to its insertion on the distal end of middle phalanx and a portion of the distal phalanx as well (12,16,11) . Any factors adjusting the position and flexion of the DDFT will in turn affect the forces acting on the NB (1,16,18).

Between the NB and the DDFT lies a small fluid pocket known as the navicular bursa which acts as a cushion against the high forces these structures subjects one another to (18).

Fig. 1. Picture of a sagittal section of the hoof displaying the middle Fig.2 The DDFT and NB after the

and distal phalanges, navicular bone, deep digital flexion tendon sampling. Note the shiny, white, smooth the digital cushion. (Sandra Eriksson and Viktoria Eklund). surface of the bone. (Sandra Eriksson).

DDFT

TT

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1.2 Gross pathology of the navicular bone

In young and healthy horses, the fibrocartilaginous structure of the navicular bone is smooth and shiny (Fig. 2). Any form of thinning of the fibrocartilage with or without roughening along its surface is classified as a pathological alteration, often seen in horses above 5 years of age (7). Thinning of the cartilage appears as a darkening of the surface of the dorso-palmar cartilage of the NB and it is principally a generalized lesion, but it may also be localized over a certain area of the bone. A partial fibrocartilage loss over the articulate surface of the NB is a frequently identified pathology in ND affected horses and it is classified as one of the earliest signs the disease (1). Despite the high occurrence of these degenerative changes in clinically affected horses, studies have failed to determine the significance of low grate articulate changes in the development of ND as they have been identified in both sound and navicular disease affected horses (1). A sever loss of fibrocartilage surface (>50% loss of surface area) is a rare finding and this type of pathology is only seen in severe advanced stages of the disease (8).

Although fibrocartilage alterations are common pathological findings on post-mortem examinations of horses it is hard to detect in vivo since these changes are often missed on a standard x-ray (1). This leads to many such clinical cases going undiagnosed until they become severe enough to cause bone alteration in form of full-thickness erosions, which leads to a late stage diagnosis and poorer prognosis for the horse.

1.2.2. Parasagittal plane splits

A frequently identified pathology during the gross pathological examination of the NB are bands of fibrocartilage damage running along the length of the bone (8). These alterations are referred to as parasagittal splits. They are visible as “stripes” running in a longitudinal direction over the surface to the DDFT and occasionally the corresponding surface of the NB (1). In some cases, the bands extend over entire length of the bone, but they may also be restricted to a localized area, most commonly the medial or lateral portion of the bone. Minor fibrillations have been described as a pathological finding in both lame and sound horses (1) while deep sagittal plane splits have only been identified in lame horses (1). These changes are commonly described as an early sign of pathological changes in the bone (1). The damage to the NB ensures when parasagittal plane splits of the DDFT creates sharp edges, these are capable of causing fibrocartilage damage and eventually ulceration of the NB with the lesion extending into the spongiosa portion of the bone (1).

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1.2.3. Dorso-palmar fibrocartilage erosion

The most commonly described pathological evidence of navicular disease is erosive and ulcerative damage to the dorsal surface of navicular bone and the corresponding surface of DDFT (3). Erosions often occurs as a sequalae to fibrillation of the dorso-palmar fibrocartilage of the NB, fibrillation is believed to be a result of increased pressure exerted on the NB by the DDFT. An increase in pressure causes the friction between these two structures to intensify durian motion, eventually leading to cartilage damage and leakage of synovia. This in turn leads to erosion which may eventually reach all the way into the subchondral bone resulting in necrosis (5).

Depending on the severity, erosion may occur with or without subchondral bone changes (3,8,9). The damage occurs principally in the distal half of the NB, especially around the central sagittal ridge area (3,9,11) and it is often identified on the gross examination of the surface of the NB. The lesions are commonly visible as red-brown, slightly rough areas on the dorso-palmar surface of the NB (Fig. 3), in more severe cases they present as small depressions on the surface of the bone (Fig. 3). When progressing further, the lesions appear as severe erosions approaching ulcerations and they are often seen in the sagittal sections of the hooves as “holes” at the dorso-palmar surface of the navicular bone (9) . Eventually these pathologies develop into deep ulcerative lesions extending into the flexor cortex reaching the subchondral bone (9). These changes are often seen clearly on the gross pathological examination of the bone, however in early stages they might only be detectable during histological examination (9).

Fig. 3. Yellow discoloration of DDFT and NB and severe

erosion (red arrows) on the surface of the bone, a shadowed area revealing a thinning in the density of the cartilage (area between the green lines). (Sandra Eriksson and Viktoria Eklund)

Although dorso-palmar fibrocartilage erosions of the NB are common post-mortem findings in horses the significance and nature of these lesions are poorly understood. (7). It has been proposed that such changes can be either primary or secondary (3) and there are two main theories proposed for their aetiologies. The first theory suggests that these lesions appear due to vascular occlusion of

DDFT

15 the blood supply to the bone leading to ischemia and necrosis among other secondary bone changes. The second theory proposes that erosions of the NB occurs secondary to biomechanical alterations of the hoof (3,5,13,20) which increasing the forces acting on the NB. The majority of experimental studies support the biomechanical theory which is the most widely accepted one today (17,1,8).

1.2.4. Yellow discoloration

Yellow discoloration of the articulate surface of navicular bone and the corresponding surface of the DDFT is a common pathological finding when examining the NB (1,8). Certain authors describe this as a normal age-related finding (3) while others consider it as an early stage in NB degeneration (1). Yellow coloration is a general term and such changes may also be referred to as thermal lesions. The exact reason for the appearance of this yellow colour is not clearly understood and therefore still under investigation. One article found that these changes were seen in healthy adult horses but not in immature horses (individuals <5 years of age), this would support the theory of this pathology simply being an age-related occurrence (8) . Another author however managed to identify the presence of yellow discoloration in all types of horses, those with ND, normal adult horses without ND and one immature healthy horse (≤ 4 years old) (1). Thus, the possibility of yellow discoloration being not only an age-related change but also an acceleration of the normal age-related changes occurring in the NB cannot be ruled out completely.

In articles where horses having yellow discoloration on the surface of the dorso-palmar navicular cartilage were examined, all of them showed some degree of thinning and loss of cellularity in this area (8). The location where this colour change appears also seems to be extending over areas containing fibrocartilage lesions or adjacent to such lesion (Fig. 3). In the cases where a full-thickness erosion of the fibrocartilage was present the yellow discoloration was only present on the area surrounding the lesion (8) (1), indicating it may only be possible for it to appear on the cartilage surface and not on the surface of the bone itself. There are also cases where a yellow coloration of the fibrocartilage occurred without any visible lesions (8). In these cases, the changes appeared centred on the sagittal ridge (8). If the reason for their development is the compressive forces exerted on the NB this would be the most logical place for their appearance since it is the area of the bone subjected to the highest pressure (8).

1.2.5. Adhesions

Injury to the navicular bone commonly occurs as a result of the high loading forces of the DDFT increasing the pressure in the navicular bone area (3), thus these structures are often damaged concurrently and any form of primary navicular bone lesions are rare. Adhesions consists of bundles

16 of tendon fibres that has been torn from the DDFT and formed an attachment to the palmar surface of the NB. These adhesions are typically seen either dorsal to an DDFT lesion (1), cantered on the sagittal ridge or medio-lateral to it.

It has been proposed that fibrillation of the DDFT predisposes the bone to adhesion formation between the DDFT and an eroded surface of the NB (3). Whether the fibrillation of the DDFT occurs primary or secondary to the erosion of the NB cartilage has not been determined and the question remains open to debate (1,7). In a previous study of NB pathologies (8) there were cases where adhesions appeared in samples of NB with a slightly denuded but intact dorso-palmar fibrocartilage (8) and in the same study adhesions occurred in samples of NB which had a of full thickness fibrocartilage loss extending into the subchondral bone surface (1). This suggests that cartilage erosion and subchondral bone involvement is not necessary for development of adhesion since NB with an intact but slightly denuded fibrocartilage and intact subchondral bone may be affected as well. Among all the current studies on NB pathology, adhesions have never been reported as an occurrence between a healthy NB and DDFT, instead it is described as a severe lesion occurring in horses suffering from end-stage ND (1,3,14)

1.3. Histopathology of the navicular bone

Some of the first microscopical degenerative changes appearing in the cartilage of the navicular bone of ND affected horses are: reduction of cartilage thickness, roughening of the surface of the cartilage and occasionally fibrillation (7,5,20). Fibrillation is a very common finding on histopathological examination and as it progresses it may extend into several layers of the fibrocartilage eventually reaching the subchondral bone causing necrosis and other lesions in the internal osseous structures (1,8). Regardless of its degree fibrillation is classified as a degenerative change of the NB occurring in the surface of the fibrocartilage with small portions of the cartilage breaking apart and the normal smooth surface becoming disrupted (Fig. 41).

The lesion may be mild, moderate, or severe (Fig.41-43) depending on the stage of pathological process (22). It can occur along the entire surface of the NB or it may be localized to a certain portion of the bone. The reason for their occurrence is the same as for cartilage erosion and aetiologies includes: increased loading of the NB, more intense friction between DDFT and NB and increases pressure in the NB area.

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1.3.2. Chondrocyte cluster formation and loss of cellular density

Clusters of chondrocytes are called isogenic groups (18) and they are a common finding in hyaline cartilage which is generally present at bone forming sites, this is not surprising as these clusters are a result of dividing chondrocytes (18). The number of chondrocytes within a single cluster is variable but they rarely exceed >5 chondrocytes (8,14). Chondrocyte clusters may be located anywhere within the cartilage (Fig. 11) but they are most commonly seen at the edge of a zone of cartilage thinning (8). Their presence in a normal fibrocartilage should be modest and they should be surrounded with a population of normal chondrocytes constituting the majority of the cell population.

Reduced cellularity and an increased number of chondrocyte clusters within the dorso-palmar fibrocartilage of the NB are signs of degeneration of the fibrocartilage (9). As the degree of fibrocartilage thinning increases there is a concurrent increase in the loss of cellular density of the cartilage and the number of chondrocyte clusters present. A normal cartilage has a uniform distribution of chondrocytes within the cartilage, loss of cellular density means that there is loss function since there are not enough cells present to maintain the proper function of the cartilage.

Fig. 4. Occasionally chondrocyte gathers in huge

cluster formations (red arrow). Giemsa staining, (Sandra Eriksson and Viktoria Eklund).

1.3.3. Necrosis of fibrocartilage and subchondral bone

Severe erosions and ulcerations of the dorso-palmar fibrocartilage of NB have been described in multiple studies (1,8,14) while necrotic findings of the fibrocartilage and subchondral portion of the bone are rarely encountered. (7). Subchondral bone necrosis has been known to occur as a result of deep focal erosion of the fibrocartilage progressing until it reaches the spongiosa thereby exposing the underlying bone (1). Without the protective covering of the cartilage all the forces acting on the navicular area will be directly absorbed by the exposed bone portion resulting in a progression of the lesions. A study involving horses diagnosed with severe ND showed that a significant percentage of them had damages extending into the subchondral bone (1).

18 Bone loss and damage to the internal osseous structures are considered to be signs of advanced ND (8). Histological characteristics of such lesions includes osteonecrosis and fibroplasia (8) and loss of the normal cellular arrangement within the bone. Although subchondral bone lesions are generally seen dorsal to extensive fibrocartilage damage there are some instances where a full thickness erosion of NB occurred without any other fibrocartilage lesion surrounding this area (8).

1.4. Hoof functional anatomy

The hoof is one of the central parts in the horse’s locomotor apparatus, this small structure has to be capable of countering the biomechanical forces excreted upon it by the horse’s massive body size. Each structure of the hoof has its own specialized function; the caudal structures of the hoof is used for vertical support, countering concussion forces and dissipating energy while the cranial portion of the hoof is designed for protection and propulsion of the horse during movement.

There are 5 main important structural components in the hoof, these includes: the hoof wall, the sole, the frog, digital cushion and the bars (11) (Fig. 5). The hoof wall is the outer supportive cover of the hoof and it has three main layers. The outermost layer is the stratum externum consisting of hardened insensitive epidermis, it is a thin-walled, waxy covering waterproofing the hoof against external moisture and dirt. The middle layer is the stratum medium which makes up the main portion of the hoof wall, it forms the division between the external insensitive portion of the hoof and the internal sensitive layers (13). The innermost layer is known as the stratum lamellatum, it is made up of several hundred cornified sheets known as “lamellae” and it forms the main sensitive portion of the hoof (13,10). The hoof wall serves as the protector of the hoof, shielding the bone from injury by caused by external forces.

Fig. 5. Picture of the main anatomical structure of the hoof. (Sandra Eriksson and Viktoria Eklund).

FROG SOLE BAR HOOF WALL WHITE LINE HEEL BULBS

19 The sole is the ventral covering of the hoof (Fig. 5), it is the portion which interacts with the ground surface and protects the internal structures from damage (such as rough surfaces and sharp objects) during landing and propulsion phase (13). The sole fills the area between the hoof wall and the frog (10), together with the hoof wall the sole shares some of the weight bearing of the horse.

The junction between the frog and the wall is known as the white line, it is the visible portion of stratum internum (Fig. 5) (10). The frog and digital cushion are the main shock absorbing structures in the equine foot (Fig. 5). The frog is a wedge-shaped structure on the solar surface of the hoof, longitudinally along its centre runs a groove known as the frog sulcus (10). By projecting upward, the frog becomes increasingly narrow until it ends in a sharp point, at the distal end it increases in thickness until it reaches the heel bulbs (Fig. 5). The bars are located at the turning point in the back of the hoof and they end in two pointed projections proximal to the heel bulbs (21) (Fig. 5). The bars are essential for structural integrity, support, and movement of the hoof (21).

1.5. Hoof external pathologies

Forces applied on the navicular region is influenced by three main components: weight, hoof conformation and usage of the horse (17). Several studies have revealed a breed predisposition for developing navicular bone pathologies, typically affected breeds includes male quarter horses and thoroughbred geldings (17,5,22,1,12), while ponies, Arabian and Frieser horses are rarely affected by the condition. The conclusions drawn from this is that there the hereditary hoof conformation among these breeds causes higher loading forces on the navicular bone thus predisposing it to injury (1,2).

Quarter horses are muscular, compact horses with relatively small hooves causing high pressure to be distributed over a small surface area, thoroughbreds on the contrary have a tendency to develops long toe and collapsed heels which in several studies has proven to increase the loading forces of the DDFT on the navicular bone (16,18). This information indicates that foot conformation, heel angles and heel height can influence the development of navicular bone pathologies (15,16,18,22).

To identify horses having various external hoof pathologies such as underrun heels and high heels angular measurements can be used. Recent publications refer to the ideal angulation of the dorsal hoof wall as the angle by which the dorsal hoof wall is in alignment or parallel to the hoof pastern axis, seen as a line draw through the three-distal phalanges (15,18,19). Two main angle measurements are used to identify horses with high and low hoof angles: the toe angle, which is the angle between dorsal hoof wall and horizontal ground and the second being heel angle, the angle between ventral hoof wall and horizontal ground (Fig. 6).

20

Fig. 6. Displaying the measurement of heel angle, S and

toe angle, Q.(Sandra Eriksson and Viktoria Eklund)

For the purpose of this study the normal toe angle was determined to range between 45-500_C,

(average 42,50_{C) for the front hooves (23) and 49-60}0_{C, (average 55} 0_{C) for the hind hooves (12). A}

normal healthy hoof should have no more than an 8-degree difference between the toe angle and the heel angle, therefore a heel angle >8 degrees were termed as high. All these ranges are described as the standard norm for a healthy horses’ hooves according current western farrier therapies (12,19,24). These measurements were used together with other important hoof characteristic to aid in identifying hooves with high heels and underrun heels or low heels.

1.5.2. High heels

A high heel has several definitions and various angles can be used to identify a true high heel hoof conformation, for the purpose of the study high heel was defined as one in which the hoof had a difference between the toe angle and heel angle of more than 8degrees. High heels will subsequently give the hooves a high dorsal hoof wall angle which turn which increases the tendency of landing toe-first at initial ground contact (12). If the horse has a tendency for a toe-first landing, there is a greater concussion to the navicular bone and suspensory apparatus since the shock absorptive structures are located in the caudal potion of the hoof (12). The caudal portion of the hoof reduces concussion through the viscoelastic properties of the digital cushion, frog and venous plexus (13).

A small alteration in hoof angle of only 10_{C away from its normal position will result in a}

four-fold increase in the peak forces acting on the NB (1). It has further been proven that an alteration in the toe and heel angle away from its position of alignment with the hoof pastern axis is associated with changes in angulation in the DIPJ and an increase in forces acting on the NB (15,22). The degree by which this increased loading force influences the pathology of the navicular bone in practice has

S Q

21 not yet been proven, although one study has shown a week correlation between alteration of the toe angle and navicular bone pathologies (22).

1.5.3. Underrun heels

Underrun heels occur when the angle of the heel is at least 5 degrees lower than the angle of the toe (18). Sound horses with normal heel angles land with the hoof level to the ground and, while horses with extremely low hoof angles however have been proven to have an increased frequency of heel-first contact (12). A correlation between damage navicular bone and a shift in weight bearing to the heel region of the hoof have been described in literatures (12). A normal hoof angle of 54 0_C

ensures that 43 % of the weight is supported by the heels, if the hoof angle is lowered to 39 0_{C 75%}

of the weight is supported by the heels (12). The result is abnormal caudal weight bearing eventual leading to crushing and collapse of the heels due to overload (13).

Underrun heels occur as a result of improper loading of the heels and poor development of hoof structures (13). This condition is often seen in combination with a long toe conformation, a long and flared toe typically end up pulling the heel more forward away from its normal position (13,18) eventually leading to an underrun heel formation. In such cases when the hoof is both overgrown and underrun the heel tubules receives almost no support at all causing them to collapse and become increasing parallel to the ground (13). As previously mentioned the heel is the main supportive element for energy dissipation and shock absorption and if the weight is no longer efficiently supported by the heels these functions are reduced as well.

1.5.4. Long toe

A deviation in the hoof wall growth pattern indicates an unbalance in the hoof leading to an uneven hoof development (12). Practically all hooves included into this study suffered from “farrier absence syndrome” meaning they had not been trimmed with regular intervals and some of them may never have been trimmed at all. Unlike their wild counterparts travelling miles each day in a variety of different terrains our domestic horses spend a great deal of their lives in soft pastures or locked inside stables, hence there is a need of a constant care and trimming of their hooves (13).

One of the main problematics with an overgrown toe besides the uneven balance of the hoof is that the hoof wall will assume a majority, if not all of the weight support as the frog, sole and digital cushion may only be partially in contact with the ground. A such peripherally loaded foot will eventually lead to alterations of the internal hoof structures and result in deterioration and forward shift of the supportive soft tissue structures within the caudal part of the hoof (13).

22

1.5.5. Long toe-underrun heel

One of the most commonly encountered hoof problem when dealing with poor hoof conformation is the long toe-low heel syndrome, it has been reported in 77% case of horses displaying a certain degree of chronic heel pain (17,13). Long toe-low heel condition is defined as a <5-degree difference between heel and toe angle of the foot, a definition chosen for this study due to its usage in similar studies of the NB pathologies (12). A low heel angles as seen in collapsed heels coupled with a long toe assuming majority of the weight bearing have been mentioned as a cause of alterations in the biomechanics of the hoof predisposing horses to development of NB pathologies (22).

Horses with a long-toe, low-heel conformation will have an altered area of ground contact, instead of occurring beneath the caudo-palmar portion of the foot which contains all the supportive and shock absorbing soft tissue structures, the area of ground contact is shifted forward and occurs under the palmar process of distal phalanx (13). There are limited weight bearing structures and no significant supportive tissue in this portion of the foot (11). This is the reason why such long horses with long toe-low-heel conformations are likely to develop various hoof pathologies including sole bruising, wall cracks and flares. Limited caudal weight bearing also contributes to the deterioration of the DDFT and NB cartilage as a result of high frequency waves generated during locomotion being distributed directly to these structures instead of the shock absorption area in the heels (13,1,12).

1.5.6. Frog pathologies

The frog is essential to the balance, blood flow and energy dissipation within the hoof. When the hoof is properly balanced the frog and sole are in contact with the ground surface allowing more micro vessels within the hoof cartilages to open. The blood flow through these channels are essential for nutrition supply to the hoof and for energy dissipation. A severe reduction in frog diameter (≤2/3 of original length remaining) reduces the dynamic movement of the frog and thus its ability to ensure the proper movement of lateral cartilage of the hoof and the vessel dilation within the soft tissue structures (13,17). The second outcome of a compromised frog structure is that the biomechanical stresses which were supposed to be absorbed and dissipated by the frog and associated soft tissues are shifted to the more rigid structures including: coffin bone, hoof wall, DDFT and NB (13). These structures may not be robust enough to support such forces (13), they become compresses and start rubbing against one another resulting in friction leading to thermal damage and erosion of the NB.

Frog pathologies are known to be associated with horses suffering from pain originating in the navicular area (13). The pain is typically a secondary response attributed to the lack of weight bearing in posterior portion of the foot and the increased loading forces acting on the NB and the PDA (12). The frog damages investigated in this study includes: frog atrophy, degeneration and necrosis (3).

23

1.5.7. Laminitic rings

Laminitis is defined as failure to maintain the connection between the dermal and epidermal lamellae resulting in a displacement of the distal phalanx within the hoof capsule (23). The hoof wall is largely an insensitive structure, but the inner laminar corium (lamellae) is extremely sensitive and when the blood flow to this area is impaired it causes the inflammation and swelling in the soft tissue structures. The lamella serves an important role in keeping the coffin bone in place and as it swells it loses its hold on the bone which can ultimately lead to ventral displacement of the bone a condition also known as “sinkers” (Fig.8) (25). In some cases, the bone may not only sink but also rotate away from the dorsal hoof wall which leads to a condition known as “coffin bone rotation”. (23).

Laminitis may present in several ways, but the end result is similar and ranges from mild to severe pain and lameness typically involving both front limbs (23) or occasionally the back limbs. The horse displays a reluctance to bear weight on the cranial portion of the foot and subsequently they will adapt an altered stance and gait, this may in turn affect the soft tissue structures of the navicular apparatus resulting in abnormal loading and development of pathologies in the bone.

The main external sign of laminitis is an unequally spaced growth pattern appearing as ring formations across the dorsal hoof wall (Fig. 7). The rings arise when the hoof wall growth in the heel region becomes more rapid compared to the toe region (healthy hooves grow faster in the toe region compared to the heel region) (26). Eventually, the hoof curls upward as it grows creating a ring-shaped pattern on the wall surface (Fig. 7) (26). These rings also result in disruption of the normal wall angles since each ring changes the direction of the dorsal hoof wall. In this study all such ring formations are classified as lamellar ring pathologies. By studying the sagittal section of the hoof, lamellar separation is clearly visible as a widening of the with line (Fig. 8). In all cases where the distance between the phalanx and toe wall was greater than 18-19 mm this was classified as a lamellar separation, as set out in the criteria by current therapies when treating equine lameness (12).

Fig. 7. Severe laminitic ring formation with rings Fig. 8. Severe case of “sinkers” with coffin bone almost

curving over the surface of the hoof wall. penetrating the sole (red arrow). Severe stretching of white (Sandra Eriksson and Viktoria Eklund line (blue arrow). (Sandra Eriksson and Viktoria Eklund)

24

1.5.8. Contracted heels

An improper development of the internal hoof structures leading to a toe-first change in landing pattern is the main reason for development of contracted heels (24). It is a condition that is hard to define but the general appearance of such a hoof is one with a narrow tall frog apex with a base that seems to flow into the heel bulb and with bars that starts moving inward forming sharp points and exaggerating the central frog sulcus (13) (Fig. 9).

The condition may appear at a very young age in horses not subjected to enough heel-first movement in various terrain, it may also develop later in life due to any condition that discourages the horse from using the caudal portion of the hoof (13). Neglect, excessive hoof wall growth, improper shoeing and chronic injury to the heel resulting in pain are just a few examples of a situation limiting the horses heel-ground interaction. A contracted heel is not able to provide an efficient shock absorption or an even distribution of the weight and loading forces (13,12) thus it will cause an increased tension in the navicular bone area. The condition may initially be mild and reversible but as it progresses it can result in a severe alteration of the overall shape of the hoof (Fig. 10).

Fig. 9. A mild contracted heel with the bars Fig. 10. Severe contracted heels. The hoof has

starting to point inwards towards the hoof started to assume a “bell-shaped” appearance. (arrows). (Sandra Eriksson and Viktoria Eklund). (Sandra Eriksson and Viktoria Eklund)

25

1.6. Individual parameters affecting the pathologies of the navicular bone

Important factors influencing the forces acting on the navicular region includes weight, hoof conformation and usage of the horse. There is literature describing the relationship between weight of the horse and the loading of the NB (12). A study also showed that the peak compression forces that the DDFT exerts on the navicular bone was 0,77 times the body weight of the horse when measured at slow trot (1,2). This compression occurred at 70% of the stance duration in the stride phase (12,17). Small hoof conformation in combination with a muscular body (as seen in quarter horses also seems to predispose horses to the development of NB pathologies (12,17).

1.6.2. Age related changes in the navicular bone

Despite the findings of severe degenerative NB lesions occurring in very young horses (7,14) some authors have referred to the degenerative changes identified in the NB as age-related changes not necessarily contributing to pain and lameness of the horse (6) (3). Some author even claims that there is no correlation between age and the grade of histological abnormalities in NB structures (1,20) . Other publications however (1) proved that although age-related changes are seen in the NB as a constant ongoing natural degenerative process similar to those seen in the joints several hoof conformations and pathologies can lead to an earlier development of such lesions causing them to occur at a very young age. In such situations these NB alterations should be classified as primary pathologies not contributed sole to old age of the horse (3).

26

2. RESEARCH METHODS AND MATERIAL

The study was conducted on hooves taken from left and right distal thoracic limb of 40 horses selected from a slaughterhouse in Lithuania between the years 2016-2017. All limbs were dissected at the slaughterhouses by disarticulating the metacarpophalangeal joint leaving only the hoof. The hooves were transported immediately after collection from the slaughterhouse to the LSMU Veterinary Academy, Department of Veterinary Pathobiology and they were prepared for gross examination the same day.

20 hooves taken from 10 horses were also used in the study to evaluate the articulate surface of the distal interphalangeal joint and determine which part of the bone was subjected to most damaged, this section was then chosen for sampling for the histopathological analysis. The evaluation was done by performing a parasagittal plane split of the hoof and disarticulation of the distal interphalangeal joint, then grossly evaluating the surface of the navicular bone and DDFT. The central portion of the sagittal ridge was proven to be the area with the most sever pathological changes including yellow discoloration, adhesions, cartilage erosion and parasagittal splitting. All the samples collected for the gross pathological and histopathological analysis were therefor collected from this site.

2.1. Sampling population

60 hooves taken from the remaining 30 horses were used in the study for the gross pathological and histopathological analysis. The horses chosen for this study were either of Zemaitukai pure breed or a cross-breeds of Lithuanian draft horses. The horses varied in ages from 4 to 21 years old (average 12 years). They were grouped according age intervals to identify which pathological findings had an age correlation and which findings were unrelated to age. Navicular bone pathologies are most common in horses above 10 years of age (3,13,4) and that is why horses were grouped into age group according horses ≤10 years old (Group 1 n=14) and horses >10 years (Group 2 n=16), this gave an almost equal number in each group, which was another reason for the age interval selection.

The slaughter weight of each horse was also recorded, and it varied from 465kg-865kg (average 591kg). The weight has influence on the biomechanics of the hoof (6,) and therefore we wanted to compare the possible relationship between two different weight groups of horses and see if there was any correlation between the horses’ weight and the development of navicular bone pathologies. For the purpose of having and equal sample population the horses were grouped into horses between 465kg-565kg (Group 1 n=15), (average 531kg) and horses between 570-865kg (Group 1 n=15), (average 650kg).

Detailed information about each horse can be seen in table 1, containing the case detail about the horses in each group and the number of hooves examined during gross pathological and

27 histopathological examination. The reason for the reduced number of samples used for histopathological examination (n=47) compared to the total number of hooves collected (n=60) were that a few samples were destroyed during processing. There were also a small number of samples that were of too poor quality to be able to perform an accurate evaluation of their pathologies thus they were excluded from the histopathological examination as well.

Groups Number of horses

n=30

Number of hooves used in pathological

examinations n=60

Number of hooves used in histological

examination n=47 Age group (years)

Group 1 4-10 14 28 26 Group 2 >10 16 32 21 Weight groups (kg) Group 1 465-565 15 30 22 Group 2 >565 15 30 25 Summary (number) Total 30 60 47

Table 1. Detail about the distribution of horses in each group and the number of hooves examined during gross

pathological and histopathological examination.

2.2. Pathomorphological lesions of navicular bone

The first part of the gross pathological analysis involved evaluation of the sagittal section of the hooves. Sagittal plane splitting of the hoof was performed using mechanical saw dividing the hooves in two equal parts, the side with the least processing artefacts was chosen for photographic documentation. The sagittal section was evaluated for any gross hoof pathologies and abnormalities such as navicular bone lesion, cartilage erosion, adhesions and yellow discoloration. All data collected were documented in excel tables and they were used for graphical representations of the pathologies within each group and to calculate the statistical data. The p value for each data was calculated using chi-square test.

2.3. Collection of samples for histopathological investigation

A second sagittal plane splitting was done parallel to the first one leaving a 1-1,5 cm sample slice containing all of the structure in the sagittal plane from the coffin bone to the distal portion of the DDFT. From this structure the navicular bone was carefully extracted by dissecting the DDFT at the proximal end of the bone and the distal sesamoidean impair ligament at the distal end of the NB.

28 The final sample included the navicular bone section together with approximately 1 cm tendon at the proximal end and 2mm piece of the digital cushion below the ventral portion of the bone.

2.4. Histopathological preparation and staining

Each of the samples taken for histopathological analysis were numerated with the individual horse’s number and left/right hoof markings and then placed in a sealed bottle of 10% formalin buffered solution. After formalin preparation the samples were placed in decalcification solution for several weeks before being extracted for histological preparation. The navicular bone was isolated and cut longitudinally along the cross-section area into thin, 1-2,5mm slices and placed inside the plastic cassettes used for the fixation procedure. Samples were fixated in automatic machine. The embedding was done manually using liquid paraffin for embedding and then cooling on ice-plate.

Histological sections were prepared using microtone and the samples were cut in 4-6µm sizes depending on degree of decalcification to ensure minimal tearing of the samples. For each navicular bone sample at least 2 slides were prepared. Staining was done using giemsa staining and H&E. Different staining was selected for their different properties, giemsa stain enables a clearer more distinct view of the cartilage as it takes on a distinct blue colour while H&E staining gives a better overview of the individual cell population. All slides were evaluated under microscope and all samples having signs of fibrocartilage alterations, cartilage erosion, chondrocyte clusters, loss of cellular density and cartilage bulging were recorded and the summarized in an excel table for further graphical analysis and statistical data calculations.

All the fibrocartilage alterations of the NB identified in the histological section were classified into three subgroups according severity of the lesions (see Table 2). This was done in order to identify how many hooves suffered from each degree of fibrocartilage alterations. The percentage of hooves having these types of damages were summarised and the results were compared between the two age groups. The number of hooves having evidence of chondrocyte clusters were also divided into three subgroups (see Table 2). There was a high variability in the orientation of chondrocytes within the cartilage layers of each sample and thus the hooves having presence of chondrocyte clusters were divided into three subgroups according to which layers these clusters occupied.

2.5. External hoof pathologies

The first step after taking the samples from the slaughterhouse was preparing them for hoof conformation and external pathologies evaluation. All of the hooves were cleaned and photographed using a Samsung NX20 model camera, and all photographs were in RAW set 400 format to enable visualization of even the smallest structures. The pictures were taken against a white background and

29 each of the hooves were marked with an individual number as well as left and right. They were placed at a pre-determined distance away from the camera, this ensured the reliability of any measurements and comparisons that were done using the photographic images alone. Photographs were taken of 5 different angles: solar surface, ventro-dorsal projection, latero-medial projection, cranio-caudal and caudo-cranial projection. During the photographic documentation the number of horse suffering from frog pathologies, laminitic rings, contracted heels, underrun heels and long toe were summarized.

2.6. Hoof measurements

Measurements of dorso-palmar hoof angle (toe angle) and heel angle was done (see annex 1). Theses angles were included into the study as they have a great significance on the biomechanical load acting on the navicular bone (9, 10, 11, 12) and due to the limitation of time preventing a full analysis of several different hoof angles. The results of the hoof angle measurements helped identify presence of hoof conformational abnormalities such as high heel, underrun heel and long toe-low heel hoof conformation. The distances of the sole, frog length, distance between the heel triangles and hoof wall among other parameters were measured (see annex 1). These measurements were done as they were used as criteria’s for determining the presence of certain external hoof pathologies such as contracted heel, laminitic rings and frog pathologies.

Table 2. Demonstration of the division of navicular bone lesions into subcategories.

Na vuc il ar bone Gross pathologies Fibrocartilage thinning Parasagital plane splits Fibrocartilage erosion Yellow discoloration Adesions Histopathologies Fibrocartilage alteration Fibrocartilage fibrillation Fibrocartilage erosion Fibrocartilage necrosis Subchondral bone necrosis Cartilage bulging Chondrocyte clusters Peripheral clusters Clusters in all layers Deeper layer clusters Loss of cellular density

30

3. RESEARCH RESULTS

3.1. Sagittal ridge area examination

All of the hooves taken from the 10 horses used in this study to evaluate the articulate surface of the distal interphalangeal joint had damage in the sagittal ridge area of the NB and not a single hoof had a completely normal articulate surface of the metacarpophalangeal joint. The hoof with least amount of damage had an intact sagittal ridge area only showing mild yellow discoloration unilaterally on both sides of the sagittal ridge (Fig.11). Half of the hooves (n=5) had damage located solely on the central sagittal ridge portion of the cartilage (Fig. 13) and the second half of the hooves (n=5) had damage on the entire surface sagittal ridge of the NB (Fig. 12). Thus, the central portion of the sagittal ridge was the only location that was constantly affected by some degree of cartilage damage and some of the most severe lesions were seen in this area as well.

Fig. 11. sagittal ridge area with the surface is still intact Fig. 12. Severe damage extends over the entire sagittal

and there is no erosion or loss of cartilage surface. The ridge area with erosion and yellow discoloration of

only noticeable finding is a mild yellow discoloration the NB and DDFT. (Sandra Eriksson & Viktoria Eklund.) the NB area surrounding the sagittal ridge and surface

of the. DDFT. (Sandra Eriksson & Viktoria Eklund).

Fig. 13. Demonstrating a large well-demarcated single,

oval Erosive lesion of the NB situated in the middle of the sagittal ridge. (Sandra Eriksson & Viktoria Eklund). NB DDFT NB DDFT NB DDFT NB DDFT

31

3.2. Gross pathology of navicular bone

60 hooves taken from 30 horses were used in this study for examination of gross pathologies. The findings within two age groups were analysed separately and then compared with each other, the same procedure was done for the two weight groups. Several hooves had more than one pathology present and therefore in the graphs the summary of the percentage of the lesions in each category exceeds 100%. There was no correlation between the right and left hooves, pathologies could vary widely between them and due to the time limitation in this study their differences were not compared.

3.2.1.1. Gross pathology of navicular bone seen in all the hooves

The most common gross pathological findings were thinning of the cartilage 82 % (n=49) (Fig. 14). Adhesions was the rarest finding among all the hoof samples and it was only present in 8 % (n=5) of the hooves (Fig. 14). Only 8 % (n=5) were healthy and had no identifiable pathological lesions (Fig. 14) All the hooves that were identified as healthy, having no sign of any gross pathological findings, came from different horses. This means that in total out of 60 hooves only 5 of them were normal, however none of the horses had two normal hooves.

The P value for yellow discoloration, adhesion and thinning of the cartilage was 0,38, 0,75 and 0,06 respectively thus the results were not statistically significant. The p value for parasagittal splitting and palmar cartilage erosion were both 0,023 and thus significant meaning similar findings is to be expected if taken a random sample from the common horse population.

Fig. 14. Comparison of the most common pathomorphological lesions in all the hooves (n=60). (Sandra eriksson).

3.2.1.2. Gross pathology of navicular bone in each age groups

Only 5 of the 60 hooves used in this study were healthy meaning they had no signs of any gross pathological lesions, 11% of them (n=3) belonged to age group 1 and 6% (n=2) belonged to age group

8% 58% 58% 58% 8% 82% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Healthy Parasaggital splitting Palmar cartilage Erosion Yellow discoloration Adhesions Thinning of cartilage

32 2. A comparison between the types of gross pathologies identified within each age group revealed an overall higher percentage of lesions in hooves of age group 2 (n=32) compared to group 1 (n=28) (Fig. 16). This applied to all pathological findings except yellow discoloration which was 11pp. more common in group 1 compared to group 2. Cartilage thinning was the dominant pathological finding in both groups, 91 % (n=29) of the hooves in group 2 and 71% (n=20) of the hooves in group one (Fig. 16). The largest percentage point (pp.) difference was seen in parasagittal splitting and palmar cartilage erosion both being 29pp. higher in group 2 (72%) compared to group 1 (43%) (Fig. 16).

The p value was calculated to see if the age really had an effect in development of gross pathological lesions and the results showed that only parasagittal splitting and palmar cartilage erosion had p values of approximately 0,02 and thus proving the significance of the results. The other yellow discoloration, adhesion and cartilage thinning had p values of 0,38, 0,75 and 0,055 respectively and thus they were not statistically significant.

Fig. 16. The Graph shows a comparison between distribution of the gross pathological lesions identified in age group 1

(n=28) and age group 2 (n=32). (Sandra Eriksson).

3.2.1.3. Gross pathology of navicular bone in each weight groups

Only 5 of the 60 hooves used in this study were healthy meaning they had no signs of any gross pathological lesions, 7% of them (n=3) belonged to weight group 1 and 10% (n=3) belonged to weight group 2. When comparing the types of pathological lesions identified in each weight group it showed that all of the cartilage lesions were slightly more prevalent in weight group 2, while adhesions and yellow discoloration were more common in weight group 1 (Fig 18). There was also a slightly higher percentage point, 10pp. (n=3) of healthy hooves in group 1, 10% (n=3) compared to the 7 % (n=2) of normal hooves in group 2 (Fig.18).

11% 43% 43% 64% 7% 71% 6% 72% 72% 53% 9% 91% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Healthy Parasaggital splitting Palmar cartilage Erosion Yellow discoloration Adhesions Thinning of cartilage Group 1 Group 2

33 For the weight groups all of them had p values above 0,05 indicating no statistical significance of the results. P values ranged from the lowest value 0,39 for yellow discoloration to 0,66 for parasagittal splitting and palmar cartilage erosion which had the highest value.

Fig. 18. Comparison between gross lesions in weight group 1 (n=30) and weight group 2 (n=30). (Sandra Eriksson).

3.2.1.4. External hoof conformations effect on gross pathology of the NB

All 60 hooves were evaluated for several external hoof pathologies and then compared to the internal gross pathological finding identified in each hoof. The external hoof conformations evaluated in this this study included: long toe-low heel conformation, underrun heel, laminitic rings and frog damage. Since there could be more than one internal gross pathology and external hoof pathology in each hoof the summary of the percentage of the lesions in each category exceeded 100%. Horses which did not have the specific external hoof conformation evaluated in each graph were termed normal, although they might still have other hoof pathologies besides the ones being investigated.

The p value for the internal lesions present for each individual hoof having the investigated external hoof pathology was calculated to see if they increased the risk of developing certain internal gross pathologies when having a typical external gross lesion. The p value would also prove if the results from this study would be statistically relevant in which case it would reflect the trend in the general horse population.

3.2.1.4.1. Long toe-underrun heel and the effect on gross pathologies of the NB

Long toe and underrun heels were most commonly present together (n=22) while other hooves either had underrun heels alone (n= 14) or in rare cases only long toe (n=8). Horses not having underrun heels or long toe were termed normal (n=16) regardless of any other external hoof pathologies present. A comparison of these two hoof conformations was made with regard to the percentage of identified gross pathological lesions in both groups.

7% 57% 57% 63% 10% 80% 10% 60% 60% 53% 7% 83% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Healthy Parasaggital splitting Palmar cartilage Erosion Yellow discoloration Adhesions Thinning of cartilage Group 1 Group 2