ESTONIAN UNIVERSITY OF LIFE SCIENCES

Institute of Veterinary Medicine and Animal Sciences

Kia Pekkonen

SYNOVIAL SERUM AMYLOID A (SAA) AS A DIAGNOSTIC

MARKER FOR EQUINE JOINT DISEASE

SÜNOOVIA SEERUMI AMÜLOID A (SAA) KUI VÕIMALIK

DIAGNOSTILINE MARKER HOBUSE LIIGESEHAIGUSTE

KORRAL

Final Thesis

Curriculum in Veterinary Medicine

Supervisor: Professor Toomas Orro

Tartu 2020

Estonian University of Life Sciences Kreutzwaldi 1, 51014, Tartu Estonia

Abstract of Final Thesis

Author: Kia Pekkonen Curriculum: Veterinary Medicine

Title: Synovial serum amyloid A (SAA) as a diagnostic marker for equine joint disease

Pages: 51 Figures: 3 Tables: 4 Appendixes: 2

Chair: Chair of Clinical Veterinary Medicine Field of research and (CERC S) code: 3. Health, 3.2. Veterinary Medicine B750 Veterinary medicine, surgery, physiology, pathology, clinical studies Supervisor: Professor Toomas Orro Place and year: Tartu 2020 Joint disease of different aseptic origins, is common problem in horses. An acute phase protein, serum amyloid A (SAA), is produced by chondrocytes and it is good candidate to be diagnostic marker for equine joint disease. In this study 11 synovial samples were collected from joints with different joint diseases and 8 samples from non-pathologic joints. Samples were from stifle (n = 10), tarsal (n = 6) and fetlock (n = 3) joints. Four different pathologies were identified: osteoarthritis (OA), osteochondrosis (OC), meniscal tear and one fetlock with chronic suspensory ligament injury. This sample was included into OA group for data analysis. Samples were analyzed with commercial ELISA kit with detection limit 0.1 mg/l. Samples from OA group (n = 5) and meniscal tear group (n = 3) had the highest concentrations of SAA in pathologic joint groups with ranges from 0.1-0.60 mg/l and 0.36-0.61 mg/l, respectively. Concentrations were lower in the OC group (0.20-0.28 mg/l). No-pathologies group (n = 4) had even lower synovial SAA concentrations (0.1-0.22 mg/l). Four synovial samples from two horses were omitted from controls and were included to a separate group due to possible systemic inflammatory response (0.4-3.24 mg/l). There were no statistically significant differences between groups’ synovial SAA concentrations, except between no-pathologies group and horses with possible systemic inflammation (p < 0.05). SAA concentrations were higher in all samples with joint pathology (n = 11) when compared to no-pathologies group (n = 4; p = 0.037). Horses with higher synovial SAA concentrations tended to have higher lameness scores, and higher synovial SAA concentrations were found in samples (n = 9) from lame horses compared to samples from sound ones (n = 6; p = 0.034). As a conclusion, synovial SAA could potentially be used as a marker for equine joint disease, but in future large-scale studies are needed to evaluate synovial SAA as diagnostic marker. Keywords: Equine joint diseases, osteoarthritis, synovia, serum amyloid A

Eesti Maaülikool Kreutzwaldi 1, 51014, Tartu

Lõputöö lühikokkuvõte

Autor: Kia Pekkonen Õppekava: Veterinaarmeditsiin

Pealkiri: Sünoovia seerumi amüloid A (SAA) kui võimalik diagnostiline marker hobuse

liigesehaiguste korral

Lehekülgi: 51 Jooniseid: 3 Tabeleid: 4 Lisasid: 2

Õppetool: Kliinilise veterinaarmeditsiini õppetool ETIS-e teadusvaldkond ja CERC S-i kood: 3. Terviseuuringud, 3.2 veterinaarmeditsiin B750 Veterinaarmeditsiin, kirurgia, füsioloogia, patoloogia, kliinilised uuringud Juhendaja: Professor Toomas Orro Kaitsmiskoht ja -aasta: Tartu 2020 Erinevate aseptiliste põhjustega liigesehaigused on hobustel väga levinud. Ägeda järgu valku, seerumi amüloid A-d (SAA) toodetakse kondrotsüütide poolt ja SAA-d on potentsiaalselt võimalik kasutada kui diagnostilist markerit liigesehaiguste korral. Antud uuringus koguti liigesehaigusega hobuste liigestest 11 sünoviaalproovi ja mittepatoloogilistest liigestest 8 proovi. Proove oli põlve- (n = 10), kanna- (n = 6) ja randmeliigesest (n = 3). Diagnoositi 4-ja erinevat patoloogiat: osteoartriit (OA), osteokondroos (osteochondrosis; OC), meniski rebend ja üks proov oli kroonilise tugisidemete kahjustusega randmeliigesest. Viimati nimetatud proov liideti andmete analüüsiks OA rühmaga. Liigeseproovide SAA sisaldus määrati ELISA testiga mille avastamispiir oli 0.1 mg/l. OA rühma (n = 5) ja meniski rebendi rühma (n = 3) sünoovia SAA kontsentratsioonid olid kõrgemad, vastavalt 0,1-0,60 mg/l ja 0,36-0,61 mg/l. OC rühmas olid madalamad tulemused (0,20-0,28 mg/l). Kahe hobuse 4 liigeseproovi eemaldati kontrollproovide rühmast võimaliku süsteemse põletikuvastuse tõttu ja nende proovide tulemusi käsitleti eraldi rühmana (0.4-3.24 mg/l). Ilma liigese patoloogiata rühmas (n = 4) olid madalaimad sünoovia SAA kontsentratsioonid (0,1-0,22 mg/l). Nende rühmade SAA kontsentratsioonid ei erinenud omavahel oluliselt, välja arvatud ilma patoloogiata liigeseproovid ja võimaliku süsteemse põletikuvastusega hobuste proovid (p < 0.05). Kõigi patoloogiatega liigeseproovide (n = 11) SAA oli kõrgem võrreldes tervete liigeste proovidega (n = 4; p = 0.037). Tugevama lonkeastmega hobuste proovides oli ka kõrgem SAA tase ning lonkavate hobuste liigeseproovide (n = 9) SAA sisaldus oli kõrgem kui mittelonkavate hobuste liigeseproovides (n = 6; p = 0.034). Kokkuvõtteks võib ütelda, et sünoovia SAA-d saab potentsiaalselt kasutada hobuste liigesehaiguste diagnostikas, kuid selle kinnitamiseks on vaja laiahaardelisemaid uuringuid. Märksõnad: Hobuste liigesehaigused, osteoartriit, sünoovia, seerumi amüloid A

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TABLE OF CONTENTS

ABBREVIATIONS ................................................................................................................................... 6

INTRODUCTION ..................................................................................................................................... 8

1. LITERATURE REVIEW ..................................................................................................................... 9

1.1. Acute phase response ..................................................................................................................... 9 1.1.1. Acute phase response................................................................................................................ 9 1.1.2. Cytokines ................................................................................................................................. 9 1.1.3. Systemic effect of cytokines ................................................................................................... 10 1.1.4. Effects of nitric oxide ............................................................................................................. 10 1.1.5. Effect of APR in liver ............................................................................................................. 10

1.2. Acute phase proteins .................................................................................................................... 11 1.2.1. Classification of APP.............................................................................................................. 11 1.2.2. APP of the equine species ....................................................................................................... 12 1.2.3. SAA in equines ...................................................................................................................... 12 1.2.4. SAA as a molecule ................................................................................................................. 13 1.2.5. Specific functions of SAA ...................................................................................................... 13

1.3. Osteoarthritis ............................................................................................................................... 14 1.3.1. Physical function of joints ...................................................................................................... 14 1.3.2. Characterization of OA ........................................................................................................... 14 1.3.3. Approaches to the disease ....................................................................................................... 15

1.3.3.1. Abnormal loading of normal joint ............................................................................... 15 1.3.3.2. Normal loading of abnormal joint ........................................................................................ 15

1.3.4. Characteristics of articular cartilage ........................................................................................ 16

1.3.4.1. Organization of cartilage............................................................................................. 16 1.3.5. Pathogenesis of the OA .......................................................................................................... 17 1.3.6. Molecular pathways of OA ..................................................................................................... 17 1.3.7. Matrix degradation ................................................................................................................. 19

1.3.7.1. Matrix metalloproteinases ........................................................................................... 19

1.3.7.2. Collagen destruction ................................................................................................... 21

1.3.7.3. Role of disintegrin and metalloproteinase with thrombospondin motifs ........................ 21

1.3.8. Clinical diagnosis of OA in equines ........................................................................................ 21 1.4. Conclusions of literature review .................................................................................................. 22

2. AIM OF THE STUDY ........................................................................................................................ 23

3. MATERIALS AND METHODS ......................................................................................................... 24

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3.1. Animals and samples ................................................................................................................... 24 3.1.1. Inclusion criteria for study group ............................................................................................ 24 3.1.2. Diagnosing joint pathologies................................................................................................... 24 3.1.3. Inclusion criteria for control group .......................................................................................... 26 3.1.4. Synovial sampling and storage ................................................................................................ 26

3.2. Description of study population ................................................................................................... 26 3.2.1. Description of control group ................................................................................................... 28

3.3. Laboratory analysis ..................................................................................................................... 28 3.4. Statistical analysis ........................................................................................................................ 29

4. RESULTS ............................................................................................................................................ 30

5. DISCUSSION ...................................................................................................................................... 34

CONCLUSIONS ..................................................................................................................................... 40

ÜLDKOKKUVÕTE ................................................................................................................................ 41

ACKNOWLEDGEMENTS .................................................................................................................... 43

REFERENCES ....................................................................................................................................... 44

APPENDIXES ......................................................................................................................................... 50

Appendix 1. Data-survey used to collect anamnesis of the animals included in the study population ...... 51 Appendix 2. Non-exclusive license for depositing the final thesis and opening it for the public and the

supervisor’s confirmation for allowing the thesis for the defense ........................................................... 52

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ABBREVIATIONS

ADAMTS Disintegrin and metalloproteinase with

thrombospondin motifs

AGE Advanced glycation end product

AGP Alpha 1-acid glycoprotein

APP Acute phase protein

APR Acute phase reaction

COX-2 Cyclo-oxygenase 2

CP Ceruloplasmin

DAMP Damage-associated molecular patterns

ECM Extra-cellular matrix

ELISA Enzyme-linked immunosorbent assay

FIB Fibrinogen

HDL High density lipoproteins

HP Haptoglobin

HPA-axis hypothalamic-pituitary-adrenal

IL-1b Interleukin 1beta

IL-6 Interleukin six

iNOS Inducible nitric oxide synthase

L-score Lameness score

LPS Lipopolysaccharides

MMP Matrix metalloproteinases

MMP-1, -8, -13 Collagenases

MMP-14, -15, -16, -17, -24, -25 Membrane type matrix metalloproeinases

MMP-2, -9 Gelatinases

MMP-3, -10, -11 Stromelysins

MP Metalloproteinases

MRI Magnetic resonance imaging

NF-κB Nuclear factor-Kappa B

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NLRP3 Nucleotide-binding oligomerization domain,

leucine rich repeat and pyrin domain containing;

NO Nitric oxide

OA Osteoarthritis

OC Osteochondrosis

OCD Osteochondrosis dissecans

RAGE Advanced glycated end receptors

SAA Serum amyloid A

TLR Toll-Like receptor

TNF -α Tumor necrosing factor alpha

TRF Transferrin

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INTRODUCTION

Equine species are made for movement. Horses are stretched to jump higher, run faster and

perform better in dressage. But it does not come without a cost: while movement is the

essence of equine sports, practitioners face the challenge of lameness and joint disease on a

daily basis. Inflammatory joint diseases are a broad group of diseases that all culminate

towards common endpoint: osteoarthritis (OA). OA is described as irreversible destruction

of articular cartilage and modulation of the underlying bony structures due to inflammation

in the synovial space. To better understand, the process must be viewed in molecular bases.

The body has two mechanisms to respond to integrity challenges: Innate and acquired

immunity. Inflammation is mainly driven by the innate mechanism, better called the acute

phase response (APR) (Gruys et al., 2005). Physiological changes in APR are associated

with cytokine production and acute phase protein (APP) synthesis in the liver and locally in

the joint. Rather intensive research is done over the APR and OA in human medicine (Xia

et al., 2014; Sofat et al., 2019) and equine medicine (Jacobsen et al. 2016; McIlwraith et al.,

2018) alike. Equine OA treatment is usually restricted to pain control and anti-inflammatory

treatment like intra-articular corticosteroids (McIlwraith et al.,2012). Disease progress is

usually very advanced before diagnosis. Subjective diagnostic tactics, like lameness scoring

can make disease progress observations inconsistent. By good diagnostic markers, like

synovial APP, the diagnosis could be made more promptly, intervention more efficiently

and disease progress observed more objectively.

Serum amyloid A (SAA) is a major APP in horses. SAA is well investigated in both equine

and human OA. SAA is strongly suspected to intermediate in the pathogenesis of OA in the

synovial compartment, because SAA is produced extrahepatically in the joint by

synoviocytes and has collagen cleaving properties. Typical ranges of SAA in the equine joint

are not known yet, but SAA has been shown to increase in septic joint infections with 100-

1000 fold compared to the detection limit of robust assays. I hypothesize that if SAA is

detected with a more optimized assay, a significant difference between aseptically inflamed

joint and non-pathologic joint could be found.

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1. LITERATURE REVIEW

1.1. Acute phase response

1.1.1. Acute phase response

All domestic animals face immunological challenges constantly. Not depending on the

species, gender or age, the first reaction to immunological stress is an activation of the non-

specific innate immunity, proceeded by the specific immune reactions. The systemic

inflammatory component of innate immunity alias APR is well described systemic

phenomena of the organism to local or systemic deviation from homeostasis, invoked by

trauma, infection, stress, neoplasia or inflammation (Gruys et al., 2005; Cray et al., 2009;

Belgrave et al., 2013). Initiating factors create several reactions by the local area or tissue

itself. Pro-inflammatory cytokines are released whenever the integrity of the cells is

compromised, and affect neural signs to initiate local response (Murata et al., 2004).

Vascular system and inflammatory cells are activated, and the production of more cytokines

and other inflammatory mediators are in turn elicited. This is followed by diffusing of

cytokines into the extracellular matrix and eventually increased concentrations in circulation

(Gruys et al., 2005).

1.1.2. Cytokines

Cytokine is an umbrella term for a large group of proteins, which have a specific effect on

communication and cell interactions. Cytokines involved in APR are termed pro-

inflammatory cytokines and are mainly produced by activated macrophages (Zhang and An,

2007). The main pro-inflammatory cytokines are tumor necrosis factor-alpha (TNF-α),

interleukin-1beta (IL-1 β), and interleukin-6 (IL-6) (Gruys et al., 2005; Zhang and An,

2007). IL-1β is also produced in fibroblasts and endothelial cells during cell injury (Zhang

and An, 2007), which shows that APR can also have more local effects.

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1.1.3. Systemic effect of cytokines

Systemic effects of the cytokines are multiple in the body. Trough receptor activation in

target cells, cytokines activate the hypothalamic-pituitary-adrenal (HPA-) axis, reduce

growth hormone secretion, cause negative nitrogen balance and catabolism of muscle, elicit

clinical changes like fever and anorexia (Gruys et al., 2005). Activation of the HPA-axis

also increases glucocorticoid levels in circulation (Murata et al., 2004; Gruys et al., 2005),

which is partly seen in clinical signs. Murata et al. (2004) noted that increased glucocorticoid

production has also cytokine downregulating action. Other reported direct changes induced

by cytokines that can be measured in the laboratory include lower plasma cholesterol,

decreased leukocyte count, activation of the complement system and blood coagulation

system (Gruys et al., 2005).

1.1.4. Effects of nitric oxide

The third component inducing APR is nitric oxide (NO) production in cells. NO has been

known to be involved in the inflammation from the 1980s when Stuehr and Marletta (1985)

reported NO production in mice macrophages in response to bacterial lipopolysaccharides

(LPS). In addition to bacterial compounds, NO reacts also to cytokines like TNF-α, hypoxia

and deviations of calcium homeostasis. NO has an important role in modulation of APR;

infection control, regulatory role in vascular responses, cascades and transcription factors,

leucocyte migration, proliferation, apoptosis and in turn cytokine production (Korhonen et

al., 2005).

1.1.5. Effect of APR in liver

Pro-inflammatory cytokines, glucocorticoids and nitric oxide trigger and modulate the

systemic effect of APR (Murata et al., 2004; Gruys et al., 2005). Most of the actions are

based on rapid plasma composition change facilitated by the liver (Murata et al., 2004; Gruys

et al., 2005). Especially protein production is changed by APR, and thus there are proteins

named acute phase proteins (APP). The effect of APR may be enhancing the production of

APP or hindering it down. By this categorization, APPs are either positive, meaning their

plasma concentration rises or negative when plasma concentration decreases, respectively

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(Rodney et al., 2014). In the hepatic APR, pro-inflammatory cytokines play a major role:

especially IL-6 activates the hepatic receptors for APP production (Gruys et al., 2005). To

maintain homeostasis, the body also causes itself to down-regulate the APR. The main down

regulator action is rapid clearance of APP in the hepatocytes, anti-inflammatory cytokine

secretion, especially IL-10 secretion by Kupffer cells and gene activation pathways co-

activation at receptor binding (Gruys et al., 2005). IL-6 hepatic activation is dependent on

glucocorticoid concentrations, but on the other hand the glucocorticoids down-regulate

cytokine production by macrophages and monocytes (Murata et al., 2004; Gruys et al.,

2005). APR is a result of all these very complex interactions and all aim to repair the

homeostasis.

1.2. Acute phase proteins

1.2.1. Classification of APP

As mentioned earlier, APP are differentiated to positive and negative APP by APR influence

on the plasma concentration. Although APR is inter-species phenomena, APP reactions

differ from species to species (Cray et al., 2009; Rodney et al., 2013). Still, some general

analogies are found: Albumin, major plasma protein, drops during APR (Petersen et al.,

2004) and is negative APP. Other downregulated APP of mammals include transferrin

(TRF), transthyretin, retinol-binding protein 4 and corticosteroid-binding globulin (Schrödl

et al., 2016). The positive APP are further subdivided into major, mediate and minor classes.

Major APP are characterized to have low plasma concentrations in healthy animals, but

increase several hundred folds in hours after APR stimulus occurs and drop promptly with

recovery (Murata et al., 2004; Cray et al., 2009). Major positive APP in most species

including equines is serum amyloid A (SAA). Other equine APP moderate and minor for

equine species are alpha 1-acid glycoprotein (AGP), ceruloplasmin (CP), fibrinogen (FIB),

and haptoglobin (HP).

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1.2.2. APP of the equine species

HP is considered an intermediate APP in horses. The physiological role is suggested to be

binding hemoglobin, to be bacteriostatic, stimulate angiogenesis and immunomodulation

(Eaton et al., 1982; Delanghe et al., 1998). In horses, HP increase in serum has been studied

in viral infections and following surgery (Kent and Goodal, 1991). HP has also observed

during non-septic arthritis (Hulten et al., 2010) and saw a rise over baseline at 24h after

induction of disease. Fibrinogen is used as a general indicator for inflammation in horses,

and the measurement is easy even in less furnished laboratory (Borges et al., 2007). Lately

it has suggested to be a good tool in prognosis, like determine delayed complications post-

surgery (De Czar et al., 2019). The physiological role of fibrinogen in APR is noted, since

universally in all inflamed or injured tissues will eventually find fibrin deposits. Fibrinogen

is a large part of the reparative pathway that aims to contain the inflamed area, stop bleeding

and prevent microbial infection (Luyendyk et al., 2019). Fibrinogen is the most used clinical

APP in equine medicine, but it has drawbacks like high concentrations in healthy patients,

peak values are 2-4 times of normal concentration and show a slow decline during recovery

(Allen and Kold, 1988).

1.2.3. SAA in equines

SAA as major APP in horses (Nunokawa et al., 1993; Hultén et al., 1999; Hultén et al.,

2010; Cray, 2012; De Cozar et al., 2019; Witkowska-Piłaszewicz et al., 2019). Typical

concentrations of normal clinical status range from 0 to 20 mg/l (Nunokawa et al., 1993,

Hultén et al., 1999). Plasma concentrations of SAA has reported to increase the 100-1000

times normal range when APR is inflicted (Jacobsen and Andersen, 2010). The level of

plasma concentration is related to the size of damage affected on the tissues (Satué et al.,

2013; De Cozar et al., 2019) and can show clinically marked increase within six hours from

induction of APR (Nunokawa et al., 1993). Although a major part of SAA is produced in

the liver like other APP (Hultén et al., 1999; Gruys et al., 2005), it has been demonstrated

that it is also produced extrahepatically in variety of human tissues (Urieli-Shoval et al.,

1998). Extrahepatic production has been also proven in uterus, adipose tissue and mammary

gland of cattle (Berg et al., 2011). SAA isoforms are also secreted into colostrum with high

concentrations in equine and bovine species (McDonald et al., 2001; Orro et al., 2008).

Christoffersen et al. (2010) demonstrated that equine endometrium associated with

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Escherichia coli infection also produces extrahepatic SAA isoforms. Moreover, SAA has

been detected in synovial fluid of horses (Jacobsen et al., 2006a), and is produced locally in

the inflamed equine joint by articular chondrocytes and synoviocytes (Jacobsen et al., 2016).

1.2.4. SAA as a molecule

Multiple SAA genes and isoforms have been described (Berg et al., 2011). SAA is

heterogeneous apolipoprotein expressed in usually three different circulating acute-phase

isoforms (Hultén et al., 1999; Witkowska-Piłaszewicz et al., 2019). In horses, SAA-1 and

SAA-2 isotopes are usually produced in the liver, and secreted into the bloodstream with

APR activation (Berg et al., 2011; Satué et al., 2013). SAA-3 is found in hepatocytes as well

as multiple different tissues during APR (Satué et al., 2013), like the endothelial lining in

lungs and gastrointestinal tract, colostrum and inflamed synovial fluid of horses (Berg et al.,

2011), and SAA-3 is proven also to originate from extrahepatic tissues, like chondrocytes

and synoviocytes (Jacobsen et al., 2016). SAA-1 and SAA-2 are sometimes grouped as acute

phase (A-)SAA (Berg et al., 2011). SAA is found in blood bonded in high-density

lipoproteins (HDL). In humans and mice, fourth isotope SAA-4 is identified, which is

reflected as “constitutive”, since it is part of HDL of healthy individuals and interference

with inflammatory process minimal or negligible (Urieli-Shoval et al., 1998).

1.2.5. Specific functions of SAA

Functions of SAA are still to be discovered. Urieli-Shoval et al. (1998) suggest that SAA

has a key role in innate immune response against invading microorganisms, since it is

largely found from epithelia on lungs, mammary lining and gastrointestinal tract, or is part

of maintenance of the tissues. During APR, SAA has been linked to T-cell proliferation

and adhesion, platelet aggregation, prostaglandin secretion, immune cell migration,

neutrophil activities and metalloproteinase activation (Badolato et al., 2000). SAA is also

linked to pathogenesis of OA as driving agent, because of its ability to cleave collagen and

induction of metalloproteinase function in joint (Connolly et al., 2012).

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1.3. Osteoarthritis

1.3.1. Physical function of joints

Joints are essential parts of any organism with bones, since joints make movement possible.

Joints or articulations consist of bones, cartilages and capsule. Bones are joint together with

muscles, ligaments and tendons, which attribute to the overall health of joints. The joint

capsule is filled with synovial fluid, which lessens friction between the two cartilages.

Synovial fluid is produced by synoviocytes, which lines the capsule. Cartilage does not

contain vessels, which is why it is dependent on synovial components to nourish and take

care of waste material. Synovia is not only found from joint, but also within tendon sheets,

invagination’s of tendons and other compartments communicating with synovial spaces of

joint, where it functions as in joints to nourish, lubricate and manage waste. Equine joints

are susceptible to traumas due to lack of physical protection, in the sense that only protection

over joints is usually a thin layer of skin. Also, horses are fairly large and active animals,

and the sporting nature of the use of the animal predisposes joint to very large physical

forces.

1.3.2. Characterization of OA

OA is a progressive degenerative aseptic joint disease with loss of articular cartilage due to

limited ability to self-repairment. The disease is multifactorial and events leading to hall-

mark changes can be initiated in any component of the articulation (Ghasem et al., 2018).

OA is affecting 60% of horses (US Department of Agriculture, 2000). Typical picture

includes progressive loss of articular cartilage, change of the cartilage matrix, remodeling of

chondral bone and growth of bony material within the joint space (Bar-Or et al., 2015).

Metacarpophalangeal joint is most commonly affected with spontaneous OA due to its close-

fitting articular surfaces and tendency to produce linear erosions (McIlwraith et al., 2012).

Fetlocks are under intense use, which is why racehorses tend to produce there OA lesions

(Santschi, 2008). Carpal joints and lately also knee joints have observed to be affected more

frequently with OA (McIlwraith et al., 2012).

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1.3.3. Approaches to the disease

Classically there has been two approaches to OA development.

1.3.3.1. Abnormal loading of normal joint

The first option is abnormal loading of normal joint, which is typical path with high-

performance sport horses (McIlwraith at al., 2012). In racehorses, repetitive micro injuries

accumulate and compromise the integrity of joints and bones (Santschi, 2008). Typical site

for a racehorse is fetlocks and carpal joints, since the range of motion and impaction forces

are high – cyclic trauma will create microfractures, necrosis and remodeling of the

subchondral bone (Santschi, 2008; Lacourt et al., 2012). Sclerosis of subchondral bone is

suggested to cause secondary damage to the cartilage via decreased shock absorption

capacity (McIlwraith et al., 2012). Because of the physical destruction of cells, synovia will

demonstrate damage-associated molecular patterns (DAMP) and cause local APR in the

form of acute synovitis and capsulitis, which contribute to the cartilage degradation

(McIlwraith et al., 2012; Rosenberg et al., 2017). Another route to induce the inflammation

chain on the normal joint is single high force impact to any joint component, but the

incidence has a much lower rate than accumulation trauma (Santschi, 2008; McIlwraith et

al., 2012), but even though the event of the trauma is low, a high percentage of patients with

trauma will have OA afterward. Ligamentous tears and fractures can cause instability in the

joint, which in turn causes abnormal stress to the cartilage (McIlwraith et al., 2012).

1.3.3.2. Normal loading of abnormal joint

The second option is the normal loading of abnormal cartilage. Osteochondrosis (OC) is an

interspecies disease syndrome of juvenile, growing animals, including equines (Bourebaba

et al., 2019). Disease is characterized as a failure of endochondral ossification, and abnormal

chondrocyte differentiation and is one of the most common primary causes of degenerative

joint diseases in domestic species (Bourebaba et al., 2019). The consequence of OC type

cartilage is to create osteochondrosis dissecans (OCD). OCD can manifest as fissures and

focal loss of articular cartilage and even cause loose fragments to enter joint space

(Bourebaba et al., 2019). Tarso-crural joints are typical places for OC lesions that are created

within the first year of life (Bourebaba et al., 2019). From the activation of the lesion till

manifestation as arthritis might take several years, when adequate physical challenge is

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presented on the joint (Bourebaba et al., 2019). The five main lesions associated with OC

are subchondral fractures, subchondral cysts, wear lines/chondromalacia, osteochondrosis

with synovitis and cartilage flap (Bourebaba et al., 2019).

1.3.4. Characteristics of articular cartilage

Articular cartilage is a framework of fibrillar collagen entrapping proteoglycans (Goldring

and Goldring, 2010). Normal composition includes tissue fluid, proteoglycans and collagen

(Xia et al., 2014). Dominating fraction of the three is fluid, and fluid content in the wet mass

is up to 80%. Only cell type found in articular cartilage is articular chondrocyte, which

produces the matrix holding up the cartilage (Goldring and Goldring, 2010; Xia et al., 2014).

The major type of collagen is type II, which comprises over 80% of all collagen types

(Steinert et al., 2007). Minor collagen types found in the matrix are IX, XI which are

embedded into negatively charged proteoglycans. Collagen VI is usually found

pericellularly over chondrocytes (Steinert et al., 2007). Homeostasis of chondrocytes is

possible due to high fluid content and so can be nourished without immediate blood supply

to the cartilage (Xia et al., 2014). All supply comes from synovium, which must first diffuse

into the synovial fluid and further to cartilage. Only coordinated lysis and synthesis of

extracellular matrix (ECM) by chondrocytes can maintain the cartilage composition and

turnover. All factors affecting this equilibrium usually cause chondrocytes to favor

catabolism (Steinert et al., 2007).

1.3.4.1. Organization of cartilage

Articular cartilage is not uniform homogenous mass, but it organizes into distinct regions

from superficial to deep where the chondrocytes and ECM differ morphologically and

functionally. For example, chondrocytes in the superficial zone produce lubricin, which is

in the key role of boundary lubrication (Goldring and Goldring, 2010). Most of the

chondrocytes habituate the deeper zones and are accustomed to hypoxic stress and hypoxia

is in role with articular anabolism (Goldring and Goldring, 2010). Overall, chondrocytes

have low regeneration capacity and low metabolic activity in normal joints (Xia et al., 2014).

With aging, the chondrocytes come less resilient and due to lack of efficient waste

management, the ECM will have more advanced glycation end products (AGEs) hindering

chondrocyte survival and anabolic capacity (Goldring and Goldring, 2010).

17

1.3.5. Pathogenesis of the OA

During the early phases of OA cartilage is still intact, but the composed layers of the

chondrocytes start to differ (Goldring and Goldring, 2010, Xia et al., 2014). Due to irritation,

chondrocytes exhibit a slight proliferative phase attempting self-repair. The most commonly

observed pattern is chondrocyte clustering due to cell proliferation and conjoint change in

ECM configuration (Goldring and Goldring, 2010, Xia et al., 2014). After progress, there

starts to be evidence of increased catabolic activity. As the ECM starts to disrupt, cartilage

integrity is compromised (Xia et al., 2014). Due to poor ability to regenerate, local

inflammation and loss of homeostasis chondrocytes will first start to catabolize the ECM

and then go through apoptosis or necrosis due to hypoxic stress (Steinert et al., 2007), which

will eventually cause the cartilage to be lost. In the early phases, fibrillation and fibrillary

cartilage can be found in the area under stress (Kamm et al., 2010). Because the

chondrocytes do not migrate into damaged areas, lesions found on cartilage are usually very

patchy (Steinert et al., 2007; Lacour et al., 2012). Depending on the duration and

intervention, investigated equine joints affected with OA show different depths of

histological damage on the cartilage from barely visible cartilage differentiation to full-

thickness lesions and articular calcification (Lacour et al., 2012). After the cartilage is gone,

the joint space narrows and the subchondral bones cause friction, further mechanical

destruction and pain.

1.3.6. Molecular pathways of OA

Although synovium is not usually used in the clinical diagnosis, synovium composition is

the first compartment to be affected. When cell damage is apparent, for example due to

mechanical trauma, DAMP are released into the synovium (Rosenberg et al., 2017).

Breakdown products of ECM are OA specific DAMP, for instance fibronectin and

hyaluronan. DAMP trigger the innate immunity, aka induce APR. DAMP bind to toll-like

receptors (TLR), advanced glycated end receptors (RAGE), nucleotide-binding

oligomerization domain, leucine-rich repeat and pyrin domain-containing (NLRP3)

inflammasome on macrophages and chondrocytes, and upregulate cytokines which activate

the inflammation regulator nuclear factor-kappa B (NF-κB) (Kalaitzoglou et al., 2017;

Rosenberg et al., 2017). DAMP receptor binding is more precisely indicated in table 1. The

main cytokines in OA are IL-1" and TNF-α (Kamm et al., 2010; Jacobsen et al., 2016). In

18

the OA cartilage, receptors of IL-1" are multiplied around the OA lesions (Kamm et al.,

2010) compared to the less affected area on the same joint. As the chemical degradation

continues, more DAMP are released, fueling the chain of inflammation.

Table 1. Damage-associated molecular patterns (DAMP) in osteoarthritis

Damage-associated molecular patterns (DAMP) Pattern recognition receptors (PRR)

Extracellular matrix (ECM): biglycan, fibronectin, low molecular weight hyaluronic acid, tenacin C

TLR-4

Plasma: α1 microglobulin, α2 macroglobulin, fibrinogen, gc-globulin

TLR-4

Intracellular: HMGB1, S100 family of proteins TLR-2, TLR-4, RAGE

Crystal: calcium phosphate, calcium pyrophosphate dihydrate, uric acid

NLRP3

HMGB-1, high-mobility group box 1; LMW, low molecular weight; NLRP, nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing; TLR, toll-like receptor. Adapted by author from Sokolove and Lepus (2013).

Jacobsen et al. (2016) showed that an increase in IL-1β and TNF-α increase the production

of SAA in the chondrocytes. Similarly, to DAMP, SAA is the major ligand of TLR, thus

resulting in similar activation of the inflammatory chain of NF-κB (Ebert et al., 2015). NF-

κB is important because it increases synovial fluid concentration of catabolic substances,

like inducible nitric oxide synthase (iNOS), cyclo-oxygenase-2 (COX-2), chemokines,

aggrecanases and matrix metalloproteinases (MMP) (Kalaitzoglou et al., 2017). SAA also

can act as an autocrine molecule to induce cytokine expression for example induce IL-6, IL-

8 and prostaglandin E2 (Kamm et al., 2010; Ebert et al., 2015). Prostaglandin causes the

typical signs of inflammation, like swelling, hotness and pain. SAA is also part of the

sclerotic changes in the subchondral bone via similar activation routes as in the cartilage and

induces cartilage calcification (Ebert et al., 2015). Before it was thought that SAA was only

a messenger in the chain of the inflammation, but now it seems that it is more of a driving

force in the OA pathology; over other routes of the pro-inflammatory actions, SAA induces

19

the production TNF-α in chondrocytes and synoviocytes (Connolly et al., 2012; Ebert et al.,

2015). TNF-α is not constantly expressed in the joint, but only when it is firstly induced or

inflammation is more acutely progressing (Kamm et al., 2010). The model for the molecular

path to matrix degradation is presented in figure 1.

1.3.7. Matrix degradation

There are four different class of enzymes which indicate catabolic changes in the joint:

cysteine-, aspartate-, serine dependent proteases and metalloproteinases (MPs). Cysteine-

and aspartate dependent proteases are mainly functioning in the intracellular matrix and

optimal pH is low. Serine dependent protease, like MPs, work extracellularly and in neutral

pH (Burrage et al., 2006). All four groups are present in OA joint, but main workers of

matrix breakdown are MPs (Burrage et al., 2006; Ghasemi et al., 2018). Different MPs are

in the key role of matrix detraction (Ghasemi et al., 2018), because the group enzymes can

catabolize the breakdown of all ECM components (Burrage et al., 2006). The active

metalloproteinases can be further subdivided into matrix metalloproteinases (MMP) and “a

disintegrin and metalloproteinase with thrombospondin motifs” (ADAMTS) (Burrage et al.,

2006; Steinert et al., 2007).

1.3.7.1. Matrix metalloproteinases

MMP are made by synoviocytes and chondrocytes and secreted to synovia via inflammatory

induction (Ghasemi et al., 2018). MMP are divided into five categories: Collagenases

(MMP-1, -8, -13), gelatinases (MMP-2, -9), stromelysins (MMP-3, -10, -11), membrane-

type MMP (MMP-14, -15, -16, -17, -24, -25) and diverse subgroup containing the rest MMP

with variable tasks (MMP-7, -11, -12, -20, and MMP-23). In OA, MMP are not inhibited by

the natural inhibiting molecule, and there is left “extra” MMP in synovia (Bar-Or et al.,

2015).

20

Figure 1. Diagram showing simplified acute phase response (APR) in joint with osteoarthritis (OA). Abbreviations: IL1 - interleukin-1; TNFα - tumor necrosis factor-α; SAA - serum amyloid A; MMP - Matrix metalloproteinases; DAMP - Damage-associated molecular pattern. Adapted by author from Kamm et al. (2010), McIlwraith at al. (2012) and Ebert et al. (2015).

21

1.3.7.2. Collagen destruction

Collagen cleaving is almost exclusively mediated by MMP (Burrage et al., 2006). The major

part of the ECM of joint cartilage is made out of collagen II, which is a fibrillar triple helix

molecule. Collagenases, mainly MMP-13 make initial cleavage of the triple helix fibril in

between Gly775 and Leu776, which makes the molecule denature and lose the coiled

structure. Each collagenase can cleave the collagen triple helix, although there is some

substrate preference. Collagenases can also cleave collagen types I and III, which means the

ligaments supporting joint could be degraded by MMP as well. After collagen II is cleaved,

gelatinases further degrade the collagen/gelatin (Burrage et al., 2006). Collagen type IV is

mainly broken down by gelatinases, which comprise the basement layer of the chondrocytes

(Kvist et al., 2008).

1.3.7.3. Role of disintegrin and metalloproteinase with thrombospondin motifs

ADAMTS are a group of extracellular enzymes, which function include cleaving collagen

propeptides, inhibiting angiogenesis, and degrading cartilage proteoglycans (Porter et al.,

2005). Especially the ADAMTS-4 and -5, also known as aggrecanase-1 and -2, have been

linked into the pathogenesis of OA and TNF-alpha and IL-1beta upregulate the aggrecanases

(Porter et al.,2005; Kamm et al., 2010). It has noted that in the equine OA that when TNF-

alpha is increased, also ADAMTS-5 is highly increased in synovia (Kamm et al., 2010).

ADAMTS have role in collagen processing as procollagen N-protease (Porter et al., 2005).

1.3.8. Clinical diagnosis of OA in equines

Clinical diagnostics in equine OA start with lameness detection and grading. Typically,

assessment of lameness is varying, synovial effusion is present and there is reaction to

flexion. First-line imaging, usually radiology, reveals osteophytosis, sclerosis of

subchondral bone, occasionally cyst-like lesions and lysis in the subchondral bone, and ends-

stage joint also narrowing of the joint space (McIlwraith et al., 2012). Cartilage can be

observed with magnetic resonance imaging (MRI), which can deliver more precise

information early into the disease and provide an excellent tool for assessing joint (Smith et

al., 2016), but is rarely used due to economical restrictions. Diagnosis is usually based on

hall-mark findings of the disease.

22

In the case of OC and OCD, radiological findings are usually a way to diagnose. If possible,

computed tomography may be used to give more precise information, for example to locate

joint mice before arthroscopic removal (Lim et al., 2017).

As mentioned before, OA does not cause changes in the cartilage or the bone immediately,

but the chemical composition of synovia is usually changed drastically. Synovial sampling

is rarely used to support diagnosis, unlike in equine septic arthritis. Research to find a good

marker for realistic prognosis and progression of the disease is a hot topic in both equine and

human medicine (McIlwraith et al., 2018; Sofat et al., 2019).

1.4. Conclusions of literature review

Equine aseptic joint diseases are a large group of conditions with heterogeneous origins with

all the same result: OA. OA is driven by low-grade inflammatory response in the affected

joint and cause progressive, irreversible destruction of the joint. APR is part of the innate

immunity and is responsible for the molecular changes in the synovial fluid as well as the

systemic circulation. APR is communicating with cytokines and APP, which work as

messengers and activate extracellular matrix degradation factor within the synovial

compartments. Investigation of APP concentration in the OA joint might reveal new

unpursued clinical applications like early diagnostic markers and effective interventions.

Especially SAA has proven to be very central to the pathogenesis of OA and thus be a

potential marker protein of the future OA patients. More research is required to set standards

for SAA to further implicate it to clinical use.

23

2. AIM OF THE STUDY

Aim of this study was to investigate SAA concentrations synovial samples from horse joints

with different articular pathologies and compare these results to the synovial samples from

joints without pathologies. Additionally, synovial SAA concentrations were compared

between lame and sound horses.

24

3. MATERIALS AND METHODS

3.1. Animals and samples

The animals of the present study were client-owned and presented to the equine clinic in

Western-Finland and municipal practice in Northern-Finland. The samples were collected

both from orthopedic patients and euthanized patients in the period of 3 years starting from

autumn 2016 lasting to autumn of 2019. Horses with lameness of aseptic joint disease origin

and who were scheduled to have articular surgery or puncture were selected to the study

group. Control group included patients euthanized for other reasons than orthopedic

problems, or clinically healthy joints sampled on orthopedic patients. A total of 21 synovial

fluid samples were collected. The collection and data surveys (Appendix 1) was done by

attending veterinarian. Attendance to study was on a volunteer basis and all samples were

collected with owners’ consent.

3.1.1. Inclusion criteria for study group

Horses which needed the intervention of aseptic joint disease were enrolled to study group.

Aseptic joint disease pathology should be based on anamnesis, palpation, lameness

examination, local blocks and imaging. All horses included in the study group should be

spontaneously lame or reacted positively to the flexion test.

3.1.2. Diagnosing joint pathologies

When diagnosing orthopedic problems, all started with the evaluation of the horse standing

afar. Then the horse was clinically examined and joints palpated. Typical signs of

inflammation, like heat, redness, swelling and pain was detected by palpation. Then patients’

gait was evaluated.

25

Aseptic joint diseases all have a common factor of causing pain and loss of normal function

due to it. Usually the level and origin of pain are evaluated by assessing patients’ gait

deviation from normal. Because every patient and veterinarian are individual, standardized

lameness scoring is used to reduce the subjectivity of the evaluator. The lameness scale used

in this study is presented in table 2.

Table 2. Description of used scale for lameness (L-score)

L-score Description

0 Lameness not perceptible under any circumstances

1 Lameness is difficult to observe and is not consistently apparent, regardless of

circumstances

2 Lameness is difficult to observe at a walk or when trotting in a straight line but

consistently apparent under certain circumstances (e.g. weight-carrying,

circling, inclines, hard surface, etc.)

3 Lameness is consistently observable at a trot under all circumstances

4 Lameness is obvious at a walk

5 Lameness produces minimal weight bearing in motion and/or at rest or a

complete inability to move

If the hypothesized location of pain was not clear or otherwise not readily apparent

pathologies wanted to be found, a flexion test was performed on the joints of interest to get

a marked reaction on the pathologic one. When there was a clear perception of location, this

hypothesis was to be tested by performing diagnostic nerve and joint blocks. When the site

of pain was clear, imaging was used to define the reason for lameness. Arthritis and

osteochondrotic lesions were diagnosed with radiographic findings.

Meniscal tears are hard to diagnose without physically opening the joint. These cases were

identified with radiographic changes and specialized ultrasound examination was performed

after lameness examination and positive local nerve blocks. Enrolled horses were scheduled

for surgical removal of torn material and thus diagnosis was always confirmed.

26

3.1.3. Inclusion criteria for control group

Patients that were to be euthanized without systemic, severe infectious or inflammatory

disease can donate control synovial sample from joint deemed clinically normal. The initial

control group included three euthanized horses. Two of the horses were euthanized due to

eye disease, and the third was euthanized due to severe meniscal tear in one stifle joint.

Control samples were taken from joints, which were determined clinically healthy based on

clinical examination and anamnesis. All control horses submitted more than one sample,

originating from different joints. One horse, horse 8 gave samples both for control and study

group.

3.1.4. Synovial sampling and storage

Synovial samples were collected in an aseptic manner co-joined with intra-articular

interventions of disease. Tapping the joint was performed with needle and synovia either

dripped free flow or was aspired to syringe. Control samples were collected after euthanasia.

All samples were transferred to the identified Eppendorf tube and frozen within 4h of

sampling. Storage temperature was held below -20°C until laboratory analysis.

3.2. Description of study population

A total of 12 horses were enrolled in the study. Enrolled horses were allocated to the study

or control group by the inclusion criteria. The study group included 11 samples which were

collected from 10 horses allocated to the pathologic group. Horse 8 donated samples on two

different occasions for the pathologic group. The control group consisted of 8 samples,

collected from 3 horses. The survey was conducted by attending veterinarians at enrolment.

The collected data consisted of age, gender, lameness score, affected joint, study group

allocation and diagnosis. Descriptive data about the enrolled animals are presented in table

3.

The age of the study subjects varied from 1 year to 15 years old. The average age was 6.7

years. The most prevalent gender in the pathologic group was gelding (n = 6). Other

27

pathologic group horses included two mares and two stallions. The volume of samples

originating from mares (4 samples) was larger than samples originating from stallions (2

samples). Two horses in the control group missed information about gender.

The most commonly affected joint in the study group was the stifle joint (8 samples).

Secondly there were three samples from the tarsal joint, and thirdly one sample from fetlock

in the pathology group. Control group had three samples from stifle, three samples from

tarsus and two from fetlock, respectively. Pathologic group had four different branches of

aseptic joint disease present: Meniscal tear of the stifle joint, osteoarthritis, osteochondrosis

and chronic suspensory ligament injury. The solitary suspensory ligament injury sample is

taken from a co-joining fetlock joint, which swells and is treated with corticosterone

injection time to time. The injury is located on a lower branch which is co-joining with the

synovial structure of fetlock. All control group joints were described as clinically unaffected.

Table 3. Descriptive data about the study population

1 Study group allocations: 1 = pathologic sample; 2 = control sample

ID Age Gender Joint Study group1 Breed Horse 1 6 Gelding Stifle 1 Warmblood trotter Horse 2 4 Gelding Stifle 1 Warmblood trotter Horse 3 11 Mare Stifle 1 Half-blood riding Horse 4 5 Gelding Stifle 1 Warmblood trotter Horse 5 9 Gelding Stifle 1 Half-blood riding Horse 6 1 Stallion Tarsal 1 Warmblood trotter Horse 7 9 Stallion Tarsal 1 Half-blood riding Horse 8 2 Mare Stifle 1 Warmblood trotter Horse 8 2 Mare Tarsal 1 Warmblood trotter Horse 8 2 Mare Stifle 2 Warmblood trotter Horse 8 2 Mare Stifle 2 Warmblood trotter Horse 8 2 Mare Fetlock 2 Warmblood trotter Horse 8 2 Mare Fetlock 2 Warmblood trotter Horse 9 15 Gelding Stifle 1 Half-blood riding

Horse 10 12 unknown Tarsal 2 Half-blood riding Horse 10 12 unknown Tarsal 2 Half-blood riding Horse 11 5 unknown Stifle 2 Warmblood trotter Horse 11 5 unknown Tarsal 2 Warmblood trotter Horse 12 10 Gelding Fetlock 1 Warmblood trotter

28

3.2.1. Description of control group

The two horses included in the control group were euthanized due to complicated eye

disease. Anamnesis or diagnostic descriptions were not submitted with these individuals,

because they fit in the initial inclusion criteria for the control group. Eye disease in equines

can persist a very long time, and some horses do not respond to treatment, after which

options are surgical enucleation or euthanasia. It is not known, whether these horses were

trial treated topically for a long period before euthanasia, but is the most likely scenario.

Horse number 8 gave samples for both pathologic group and non-pathologic group. Horse

no 8 originally had osteochondrotic lesion in the tarsal joint, which was sampled and locally

treated. Other joints were also investigated intensely compared to eye-diseased counterparts.

Somewhat later on the same year, the horse no 8 got also meniscal rupture of the stifle, which

was severe enough to be the cause of euthanasia for this horse. The stifle joint was sampled

on the onset of the lameness, and the patient was followed for a short period in hopes of

improvement. No improvement was observed after which the horse was euthanized and

joints deemed healthy were sampled after euthanasia.

3.3. Laboratory analysis

SAA concentrations of the synovial samples were determined by using a commercially

available analysis kit (PhaseTM Range Multispecies SAA Elisa, Tridelta Development Ltd,

Ireland). Assay is based on chromatographic change of the reagent wells on identifying the

tested substance. This kit uses a method referred to as “sandwich” ELISA-method.

Manufacturer instructions for equine serum and plasma samples were used.

All samples were thawed to room temperature before analysis. Initially dilution 1:100 was

used for all synovial samples. Two samples results were over the highest standard (2.0 mg/l

with dilution 1:100) and these samples were re-assayed using sample dilution 1:50. The

results were read with a spectrophotometer at 450 nm, using 630 nm as reference. The

detection limit of the assay was 0.1 mg/l (reported by the manufacturer for serum samples).

29

3.4. Statistical analysis

Initially synovial SAA concentrations were compared between joint pathology groups.

Synovial samples were dived into five groups according to the joint pathologies (table 4 and

figure 2). Samples from horses 1, 2, 5, 9 and 12 (solitary sample of suspensory ligament

injury of horse 12) were included in the osteoarthritis group (n = 5). Horse 2 had a

combination pathology of osteoarthritis and meniscal tear. This case was handled more as

osteoarthritis as it was the most acute problem of this horse. Samples from horses 3, 4 and 8

were included in a group of meniscal tears (n = 3). Osteochondrosis lesions were found from

horses 6, 7 and 8 forming third group. Four samples from 2 horses euthanized due to eye

disease (horses 11 and 12) were treated as a separate group. Non-parametric Kruskal-Wallis

test followed by Tukey post-hoc pairwise comparison was used to compare synovial SAA

concentrations between described joint pathology and no-pathology groups. Wilcoxon rank-

sum test was used to compare no pathology group (n = 4) with all joint pathology synovial

samples (expect 4 samples from horses 11 and 12; n = 11).

Synovial samples were also divided into 4 groups by lameness score (L-score)(table 4 and

figure 3). These groups were sound group (n = 6), L-score 1 (n = 3), L-score 2 (n = 4) and

L-score 3 (n = 2). Horses 11 and 12 samples were excluded from this analysis and the

Kruskal-Wallis test followed by Tukey post-hoc pairwise comparison was used to compare

the groups’ synovial SAA concentrations. Wilcoxon rank-sum test was used to compare no

lameness group samples (n = 6) with all samples from L-score 1, 2 and 3 groups (n = 9).

Samples from horses 11 and 12 were not included in this analysis.

Statistical software WINKS SDA 7.0 (TexaSoft, Cedar Hill, USA) was used for described

nonparametric tests. The level of significance was set at 5% (p ≤ 0.05).

30

4. RESULTS

Concentrations of SAA were detected from both the study group and the control group (table

4). Synovial SAA concentrations in all samples and lameness scores by joints are presented

in table 4. All horses in the study group were determined lame spontaneously, or had a

positive flexion test. Only two horses administered in the study group were not

spontaneously lame and both of those horses had osteochondrosis lesions.

Table 4. Synovial serum amyloid A (SAA) concentrations found in samples, presented with lameness score and joint pathology

ID Joint L-score SAA mg/l Joint pathology Horse 1 Stifle 2 0.31 Osteoarthritis Horse 5 Stifle 1 0.60 Osteoarthritis Horse 9 Stifle 2 0.43 Osteoarthritis Horse 2 Stifle 1 0.10 Osteoarthritis, meniscal tear

Horse 12 Fetlock 2 0.31 Chronic suspensory injury1 Horse 3 Stifle 3 0.61 Meniscal tear Horse 4 Stifle 1 0.44 Meniscal tear Horse 8 Stifle 3 0.36 Meniscal tear Horse 6 Tarsal 0 0.20 Osteochondrosis Horse 7 Tarsal 2 0.19 Osteochondrosis Horse 8 Tarsal 0 0.28 Osteochondrosis Horse 8 Stifle 0 0.14 No pathology Horse 8 Stifle 0 0.15 No pathology Horse 8 Fetlock 0 0.10 No pathology Horse 8 Fetlock 0 0.22 No pathology

Horse 10 Tarsal 0 0.40 No pathology2 Horse 10 Tarsal 0 0.72 No pathology2

Horse 11 Stifle 0 3.24 No pathology2

Horse 11 Tarsal 0 3.13 No pathology2

1 Horse 12 was allocated into group osteoarthritis due to nature of the pathology, which is similar to osteoarthritis 2 Samples were excluded from the statistical analysis, since the horses 10 and 11 are likely to have acute phase response systemically from the stress and pain caused by ocular disease

31

Synovial SAA concertation varied within the study group from smaller than the detection

limit of 0.10 mg/l to 0.61 mg/l. All the horses can be divided into smaller populations by

pathology group; Arthritis, meniscal tear, osteochondrosis and no-pathology. In the arthritis

group (n = 5), horse 2 had a result of 0.10 mg/l which could be interpreted as a value under

the detection limit. Other horses gained higher values, scoring values from 0.31 mg/l to 0.60

mg/l. All samples with osteoarthritis diagnosis originate from the stifle, except for one

sample from the fetlock joint of horse 12. The osteoarthritis group had the largest range of

results in the samples. In the meniscal tear group (n = 3) results had a range from 0.36 to

0.61 mg/l. The lamest horses were found in the meniscal tear group. Osteochondrosis group

(n = 3) results were more homogenous and varied from 0.19 mg/l to 0.28 mg/l (table 4, figure

2).

Figure 2. Synovial concentrations of serum amyloids A (SAA) by joint pathology groups: 0 - no pathology; 1 - osteoarthritis; 2 - meniscal injury; 3 - osteochondrosis; 4 - no pathology, euthanasia due to eye disease.

Control group involved 3 horse individuals and eight samples. Two horses, both euthanized

due to eye disease, got relatively high concentrations in all samples. Synovial SAA

concentrations in two tarsal joints from horse 10 were 0.40 mg/l and 0.72 mg/l. The SAA

concentrations in two samples from the horse 11 had the highest results of the study (3.24

mg/l and 3.13 mg/l in stifle and tarsal joints respectively). Because of the highest

0 1 2 3 40.0

0.2

0.4

0.6

0.8

1.02.03.04.0

Single horses

Horse nr 10Horse nr 8

Horse nr 11

Joint pathology group

Syno

vial

SAA

(mg/

l)

32

concentrations, these 4 samples were grouped into a separate study group (no pathology,

euthanasia due to eye disease group, figure 2) and were not considered as control samples.

The rest of the control samples (no pathology group) consisted of horse 8 synovial fluid

samples (n = 4), which had relatively low concentrations of SAA. Horse 8 had 2 samples,

both from stifle and 2 samples both from fetlock joints, with an average concentration of

0.15 mg/l. The lowest value in this group was 0.10 mg/l, and this result should be interpreted

as below detection limit (table 4).

Overall Kruskal-Wallis test was significant (p = 0.014) when all joint pathology groups’

synovial SAA concentrations were compared. In post-hoc pairwise comparison only

significant difference was between no pathology and no pathology, euthanasia due to eye

disease group (p ≤ 0.05). There was a significant difference (p = 0.037) when comparing

synovial SAA concentrations between joint pathologies (all pathology groups, n = 11) and

no pathology group (n = 4, samples from two horses euthanized due to eye disease were not

included in this comparison) with Wilcoxon rank-sum test (figure 2).

Figure 3. Synovial concentrations of serum amyloids A (SAA) by lameness score (L-score): 0 = no lameness; 1 - lameness is difficult to observe, not consistently apparent; 2 - lameness is difficult to observe at a walk or when trotting in a straight line but consistently apparent under certain circumstances; 3 - lameness is consistently observable at a trot under all circumstances. Results from two horses euthanized because of chronic eye problem are not presented.

0 1 2 30.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7Single horsesHorse nr 8

L-score

Syno

vial

SAA

(mg/

l)

33

There were no significant differences when synovial SAA concentrations were compered

between L-score groups (figure 3). Significant difference was if SAA concentrations in L-

score 0 group (n = 6) were compared with all lame horses (n = 9) samples (Wilcoxon rank-

sum test, p = 0.034).

34

5. DISCUSSION

Equine OA and conditions leading to it are a common problem. The United States

Agricultural Department (2000) estimated that 60% of horses are affected by OA of different

origins, which is very high morbidity for a single pathology. The problems leading to high

morbidity lies in the changed purpose of horses, which nowadays are more high performing

athletes than companion or production animals. Similarly, it has been found that more and

more foals are diagnosed with congenital joint diseases like OCD (Wilson, 2012; Bourebaba

et al., 2019). Highest morbidities are found within restricted gene pools of closed horse

breeds like Arabic horses and quarter horses (Steel et al. 2019). Rising morbidity is implied

to be affected by narrowing of the genepool of young high performing horses, also (Russel

et al., 2016).

OA requires sensitive marker (Jacobsen et al., 2006b) and the possibility of SAA being one

was charted in this study. The main aim of this study was to investigate whether SAA

concentration would significantly differ in OA-joint from a healthy joint and if the clinical

appearance of the joint, interpreted as lameness score, would reflect to the concentrations.

SAA has established a good status being a marker of inflammation in joints (Connolly et al.,

2012; Jacobsen et al., 2016). Strong evidence is gaining how SAA is contributing to the

chain of the pathology rather than just being an innocent bystander (de Seny et al., 2013;

Jacobsen et al., 2016). Previously SAA measurement in synovia has been proven to be a

useful tool for differentiating septic joint infections from aseptic joint inflammation in

equines (Jacobsen et al., 2006b, Robinson et al., 2017). Even though the non-induced aseptic

pathologies have been studied in the same study, used test have been too robust to measure

difference between aseptic pathologies and non-pathologic joint.

Handling of the OA patients is a conundrum. Visibility of the destruction will come apparent

in radiographs after the bony structures are already exposed and cartilage is severely

destroyed. Cartilage has a poor ability to self-repair, which is why the disease progression is

hard to hinder or reverse. With early diagnosis and intervention of the OA there would be a

35

possibility to slow down the progression and thus avoid cartilage damage, which is why the

idea of creating a new tool for prognosis and monitoring of the aseptic inflammation in the

joints is intriguing. Of course, this would require a normalized scale to be used as a reference

(Jacobsen et al.,2006b). A scale of a normal joint and a diseased one would provide an easier

decision whether to intervene in a joint or not. Additionally, it could be used as a prognostic

indicator for other synovia related diseases, like meniscal tear. The definitive diagnosis of

meniscal tear of the stifle joint is impossible without keyhole arthroscopy (Dubuc et al.,

2018). Invasive procedure cause inflammation and trauma on the synovial membrane, which

has the inevitable consequence of arthritis (Allen and Kold, 1988; Dubuc et al., 2018). By

evaluating the inflammatory status of the synovial cavity, stronger evidence and confidence

can be established to perform the surgery.

Previous findings in equine and human joints (Jacobsen et al., 2010; Sokolove et al., 2013;

Jacobsen et al., 2016; Bourebaba et al., 2019) suggested that SAA will be found in synovia

of aseptically inflamed joint. Although the kit used in this study is meant for serum, it is

justified to be used with the synovial fluid as well and the results are reliable. This study

gives good insight that measurable amounts of SAA can be present in the joints, as most of

the samples did demonstrate. One sample of the study group and one from the control group

had a result of 0.10 mg/l. These should be interpreted as lower than the detection limit since

the sensitivity of the assay used in the study was 0.10 mg/l. Still, the small sample size might

have not revealed the underlying problems of using this kit, which is why results are to be

trusted with caution.

Largest challenge in the study was to collect suitable samples. As said before, aseptic joint

diseases are very common. Too often lame horses, other than high performance athletes, are

not radiographed and diagnosis is only presumed. Large-scale study should be conducted in

a clinical setting with larger patient inflow, owners committed to diagnostics and

veterinarians skilled to perform them and interventions required. Control samples were even

harder to obtain. Full orthopedic diagnostic procedure and exclusion of pathology in the

sampled joints are not economically and ethically feasible because the control subjects are

to be euthanized. Moreover, the euthanized horses are stressed, diseased and might have

APR systemically affecting the results from synovial samples. For these reasons study

population in the study was unfortunately very restricted in size and thus the results cannot

be interpreted as statistical evidence. Restricted sample size and narrow scale of

36

concentration of SAA found in the samples would require a large sample size to be able to

show a significant difference. In the statistical analysis, all samples were handled as

independent from each other, but since there were multiple samples from some individuals

this conjecture is not fully correct.

This brings us to question of the horses 10 and 11. These horses were euthanized due to eye

disease unresponsive to treatment. Both horses gained very high results in all synovial

samples, the results of these horses can be viewed in figure 2. In both cases, a similar level

of results were obtained from two different asymptomatic joints within the same individual.

It is highly unlikely to get high results by local production of SAA within the joint without

septic involvement (Jacobsen et al., 2006b) and even more unlikely to get similar levels in

both sampled joints with separate pathology. These results should thus be interpreted as

systemic APR involvement in the synovia, rather than local inflammation. Equine as flight

animals depends on the vision very much and it could be concluded that loss of sight causes

very high stress to the animal. Ocular injuries are also very painful. This combination can

very much be enough to initiate APR in horse. Samples from horses 10 and 11 were thus

excluded from aseptic joint pathology statistical analysis, since the condition of these horses

was systemically affecting the joint cavity. These horses are perfect example of error which

can be obtained by using euthanized animals in the control group.

Rest of the control samples originated from horse 8. Horse number 8 is a very interesting

individual in this study. Within the same individual we have samples from OC joint,

meniscal tear in stifle joint and multiple normal control samples. The OC lesion was first

pathology diagnosed and sampled in this horse. All sites commonly affected by OC were

radiographed and examined after the first lesion was found. Thus, the horse 8 was very well-

investigated compared to horses 10 and 11. All the control samples taken from horse 8 had

similar low results. Horse 8 has thus strong evidence that the joints categorized as unaffected

by pathology match that description and the samples were used as a controls for other horses.

Ideally, all study group samples should have a matching sample from a healthy joint. Best

comparison is always a sample from the same patient, because it limits possible variables

caused by individual differences. With horse number 8, we can cross-reference the study

samples and control samples without the error caused by variables. Pathologic samples

obtained from horse 8 had a higher concentration of SAA than the unaffected joints did,

which does support the study hypothesis.

37

The rest of the study population included four different pathologies: OA, stifle meniscal tear,

OC lesions and chronic suspensory ligament injury with fetlock swelling and lameness. All

of these pathologies can be included in the aseptic joint diseases and all of these cause pain

and inflammation in the synovial compartment (Dubuc et al., 2018; McIlwraith et al., 2018;

Bourebaba et al., 2019).

The group of osteoarthritis samples and meniscal tear samples had very similar results. Both

groups had higher values than the control samples on average. The nature of both pathologies

is related to trauma and destruction of cells within the synovial compartment. This is a very

predictable trend since, as mentioned before in the literature review, SAA production is

strongly triggered by exposed cell components (Rosenberg et al., 2017). The meniscal tear

could have even higher values because it is a pronouncedly acute and traumatic event to the

synovial compartment, but in this study such spiked values were missed. The reason could

lie in the anatomy of the equine stifle. It is a very stable structure and the meniscal tear can

persist in the joint for some period before signs get alarming the owner. This could slow the

process of getting diagnosis or interventions, which is why the local APR and top APP values

have passed before sampling.

Osteoarthritis is a disease in which the inflammation cycle is going on all the time but has

variation in the intensity of the signs (Kamm et al., 2010). OA in horses is treated with anti-

inflammatory medications and the most common intervention is the intra-articular injection

of corticosteroids to suppress the inflammation and signs. In the osteoarthritis group the

lameness and other signs of inflammation are especially important because the signs indicate

that inflammation is active. If animals would have been included in the OA group only based

on radiographic findings, the possible effect of past treatments might have played a role in

the results. The interesting deviation from the results can be seen with the horse 2 sample.

The horse with both OA and meniscal tear was only subtly lame and had concentration of

SAA under detection limit, which raises the question was this horse treated with

corticosteroids intra-articularly close before sampling. This cannot be known for sure, but

yet again the clinical picture corresponded to SAA concentration within the synovial cavity.

All horses that were involved in the OA group were spontaneously lame, which reflect also

higher scores of SAA in the other than horse 2.

38

From the lameness dot analysis (figure 3) we can see that more lame horses had higher scores

of SAA. Pain and inflammation go hand in hand (Zhang et al., 2007) and SAA would seem

to follow that trend. A combination of severe clinical picture and traumatic pathology

produced the highest SAA concentrations in the synovial compartments in this study.

Systemic inflammation and stress give of course higher SAA results (Jacobsen and

Andersen, 2010), but this is most probably due to liver-origin SAA that is transported

throughout the body. To minimize the error of having to be sure that the animal is not having

concurrent systemic APR, all horses should have been also tested with SAA samples from

the serum (Witkowska-Piłaszewicz et al., 2019). These samples were not taken, because

orthopedic patients are rarely investigated with blood analysis and blood collection for study

purposes only is not justified from the ethical point of view. The way around this issue could

have been found in the analysis of synovial samples: systemic reaction is lacking probably

the extra-hepatic form of SAA which is theoretically more important in the local pathology

of the joint (Jacobsen et al. 2016), than hepatic SAA. Unfortunately, the kit used in this study

could not differentiate between extrahepatic SAA-3 and hepatic origin SAA-1 or SAA-2. By

separating these isoforms, it might have been easier to differentiate systemic effects from

local inflammation. Evidence is lacking whether synoviocytes produce SAA-3 in systemic

APR as well, since the exact details within the joint during systemic APR is not fully known.

This would be an interesting topic for further investigations.

Notably lower levels of SAA were found from OC group than from other investigated groups

with pathologies. This is an interesting finding, because generally in patients with OC lesions

like subchondral cysts, joint mice, cartilage flaps and joint wear lines seem to have a very

variable clinical picture (Steel et al., 2019). The findings raise a question, does the presence

of OC lesion cause inflammation in the joint per se? OC lesions are formed with

multifactorial origin and the adverse effect of these lesions is variable (Steel et al.,2019;

Bourebaba et al., 2019). The group with OC in this study did not generally exhibit severe

spontaneous lameness, although one of the horses had scored as high as 2. OC results were

very homogenous within the group. There was a trend of OC joints having higher SAA

concentrations than the comparison in the controls. Again, interpretation must be done

carefully because of the small sample size. This finding could suggest that the presence of

the OC lesions is not equivalent to active joint inflammation but subclinical inflammation is

smoldering in the joint. I hypothesize that with a larger sample size, a significantly higher

SAA concentration could be found when compared to normal joint controls. In individuals

39

with OC, the lesion could be considered a risk factor in the future to create a clinical

inflammation and cause of OA in the joint.

Horse number 12 was an interesting addition to the otherwise classical aseptic synovial

diseases since the suspensory ligament is not usually thought of as part of the synovial

compartment. The horse 12 had several years old suspensory ligament branch injury, which

caused intermitted lameness from time to time. Generally, the lameness was associated with

fetlock joint swelling in the patient which is why the fetlock joint was tapped and treated

with corticosteroids. The suspensory ligament (Musculus interosseous medius) runs distally

in the metacarpal /-tarsal region, arising from third metatarsal bone and distal row or carpal/

tarsal bones. In the distal quadrant of metacarpus/-tarsus, the suspensory ligament bifurcates

into the medial and lateral branch. The branches insert into proximal sesamoid groove,

intersesamoid groove, and partially blend into long extensor tendon. The primary insertions

are part of the metacarpo-/metatarsophalangeal joint capsule and are considered subsynovial

structures (Minshall et al., 2010). Injury of the synovial membrane will cause strong

inflammatory reaction in the joint, and OA in long run (Rosenberg et al., 2017). Thus,

articular involvement should be suspected with a suspensory ligament injury. This is why

the horse 12 was handled as part of OA group in the statistical analysis. In this study, only

one fetlock with suspensory injury was tested. The SAA concentration was higher than the

compared control sample from the fetlock of horse 8. More samples are required to acquire

a stronger conclusion concerning the pathology and SAA.

Although statically findings are not significant and statistical analysis has other issues like

mentioned before, some trends could be carefully established from the results: Pathology in

the joint associated with physical damage on the joint structure, like meniscal tear and OA

produced highest concentrations of SAA. Although OC samples had only moderate amounts,

healthy counterparts had even lower amounts.

40

CONCLUSIONS

In this study it was found that synovial fluid of non-pathologic and pathologic joints have

measurable concentrations of SAA. This study indicates a trend that aseptically inflamed

joints, which are clinically painful and lame, will have higher SAA concentrations in the

synovial fluid. Less lame horses with OC lesions have moderate SAA concentrations, and

difference to non-pathological group is subtle. Significantly higher values of SAA

concentrations were found from pathologic samples when compared to non-pathological

samples. When different pathologic groups were compared individually to non-pathologic

samples, no significant differences were found. Similar findings were found when

comparing non-lame horses to higher lameness scores. Samples from lame joint had

significantly higher values than non-lame samples, but the individual comparison between

scores did not yield significant differences. Due to restricted sample size the results are to

be interpret as trend. Larger sample sizes are required to give more trustworthy results in

statistical analysis.

Based on the literature review and the results of this study it can be concluded that synovial

SAA could be potentially used as diagnostic marker for equine joint disease. Further studies

must be conducted to confirm trends found in this study.

41

ÜLDKOKKUVÕTE

Sünoovia seerumi amüloid A (SAA) kui võimalik diagnostiline marker

hobuse degeneratiivsete liigesehaiguste korral

Erinevate aseptiliste põhjustega liigesehaigused on hobustel väga levinud. Üheks selliseks

liigesehaiguseks on osteoartriit (OA) mida iseloomustab paiksed põletikulised protsessid

mille tulemusena toimub liigese järkjärguline hävimine ning selliste haigusprotsesside

objektiivseks jälgimiseks on vaja mõõdetavaid markereid. Ägeda järgu valk, seerumi

amüloid A-d (SAA) toodetakse süsteemselt põletikuvastuse ajal maksas ja SAA sisalduse

mõõtmine veres on kasutusel hobuste üldise põletikuvastuse mõõdikuna. Samas toodavad

seda valku koekahjustuse korral ka paiksed koed. SAA-d toodetakse kondrotsüütide poolt

liigeses ja seega on potentsiaalselt võimalik kasutada liigesevedeliku SAA-d diagnostilise

markerina ka hobuse liigesehaiguste korral.

Antud uuringu käigus koguti liigesehaigusega hobuste liigestest 11 sünoviaalproovi ja

mittepatoloogilistest liigestest 8 proovi. Proove oli hobuste põlve- (n = 10), kanna- (n = 6)

ja randmeliigesest (n = 3). Diagnoositi 4-ja erinevat patoloogiat: osteoartriit (OA),

osteokondroos (osteochondrosis; OC), meniski rebend ja üks proov oli kroonilise

tugisidemete kahjustusega randmeliigesest. Viimati nimetatud proov liideti andmete

analüüsiks OA rühmaga.

Liigeseproovide SAA sisaldus määrati ELISA testiga mille avastamispiir oli 0.1 mg/l. OA

rühma (n = 5) ja meniski rebendi rühma (n = 3) sünoovia SAA kontsentratsioonid olid

kõrgemad, vastavalt 0,1-0,60 mg/l ja 0,36-0,61 mg/l. OC rühmas olid madalamad tulemused

(0,20-0,28 mg/l). Kahe hobuse 4 liigeseproovi eemaldati kontrollproovide rühmast

võimaliku süsteemse põletikuvastuse tõttu ja nende proovide tulemusi käsitleti eraldi

rühmana (0.4-3.24 mg/l). Ilma liigese patoloogiata rühmas (n = 4) olid madalaimad sünoovia

SAA kontsentratsioonid (0,1-0,22 mg/l). Nende rühmade SAA kontsentratsioonid ei

erinenud omavahel oluliselt, välja arvatud ilma patoloogiata liigeseproovid ja võimaliku

42

süsteemse põletikuvastusega hobuste proovid (p < 0.05). Kõigi patoloogiatega

liigeseproovide (n = 11) SAA oli kõrgem võrreldes tervete liigeste proovidega (n = 4; p =

0.037). Tugevama lonkeastmega hobuste proovides oli ka kõrgem SAA tase ning lonkavate

hobuste liigeseproovide (n = 9) SAA sisaldus oli kõrgem kui mittelonkavate hobuste

liigeseproovides (n = 6; p = 0.034).

Kokkuvõtteks võib ütelda, et sünoovia SAA-d saab potentsiaalselt kasutada hobuste

liigesehaiguste diagnostikas, kuid selle kinnitamiseks on vaja laiahaardelisemaid uuringuid.

43

ACKNOWLEDGEMENTS

Greatest thanks to my supervisor Professor Toomas Orro who has guided me from the start.

Without his involvement, I would not have any results on this study, nor would I have been

able to figure out how to make study in the first place. Secondly, I am grateful to my

colleagues Kristiina Ertola and her staff, and Paula Korpela who collected me the samples.

I would also like to thank my significant other, Isto Talvio, who always pushes me to do the

best I can do, and whom without I would have given up a hundred times before this moment.

Thanks to all of my friends who have shared this experience of becoming a veterinarian.

44

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APPENDIXES

51

Appendix 1. Data-survey used to collect anamnesis of the animals included

in the study population

Survey number (filled by the

laboratory)

Questionnaire

Horse’s name:

Date of birth:

Place of residence: Sex:

□ stallion

□ mare

□ gelding

Breed:

Filled in by the veterinarian who took the sample:

Sample collection time: ___.___._______ (date) at ___:___ (time)

Sample was taken from the ______________________ leg, ____________________ joint.

The reason for tapping the joint

was______________________________________________

_________________________________________________________________________

__

Taken sample was the sample number: _________

NB! Remember to write the sample number on the collection tube and cork!

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Appendix 2. Non-exclusive license for depositing the final thesis and

opening it for the public and the supervisor’s confirmation for allowing

the thesis for the defense Hereby I, Kia Reetta Katariina Pekkonen 07/12/1993

1. grant Eesti Maaülikool, the Estonian University of Life Sciences, a free-of-charge non-exclusive licence to store the final thesis titled Synovial serum amyloid A (SAA) as a potential marker of equine degenerative joint disease, supervised by Professor Toomas Orro for 1.1. preservation; 1.2. depositing a digital copy of the thesis in the archive of DSpace and 1.3. opening it for the public on the Web until the validity of the term of protection of copyright.

2. I am aware that the author retains the same rights as listed in point 1;

3. I confirm that by being issued the CC license no rights deriving from the Personal

Data Protection Act and the Intellectual Property Rights Act have been infringed. Author of the final thesis ______________________________ signature In Tartu, 17.05.2020 _________________________________________________________________ The core supervisor’s approval for the final thesis to be allowed for defense This is to confirm that the final thesis is allowed for defense. Toomas Orro ……………………………….. Supervisor’s name and signature Date …… ……………………