Development and Standardization of a Non-Clinical Rabbit Model

Disseminated intravascular coagulation; Development and standardization of a non-clinical rabbit modelPh.D. Thesis Line Olrik Berthelsen, DVMDepartment of Small Animal Clinical Sciences Faculty of Life Sciences Copenhagen University&Haemostasis Pharmacology Novo Nordisk A/SCopenhagen, Denmark 2010 TABLE OF CONTENTS:PREFACE.……………………………………………………………………………………………………………………..3 ABBREVIATIONS…………………………………………………………………………………………………………….4 SUMMARY (ENGLISH)………………………………………………………………………………………………………5 SAMMENDRAG (DANSK)…………………………………………………………………………………………………...71 INTRODUCTION, HYPOTHESES AND OBJECTIVES...... 9 2 BASIC MECHANISMS OF HAEMOSTASIS AND THROMBOSIS...... 112.1 PHYSIOLOGIC HAEMOSTASIS...... 11 2.2 REGULATION OF COAGULATION...... 13 2.2.1 Anticoagulation...... 13 2.2.2 Fibrinolysis...... 14 2.2.2.1 Activation of plasmin...... 14 2.2.2.2 Regulation of the fibrinolytic system...... 14 2.3 ACQUIRED PROCOAGULANT DISORDERS OF HAEMOSTASIS...... 15 2.4 DISSEMINATED INTRAVASCULAR COAGULATION...... 15 2.4.1 Aetiology...... 15 2.4.2 Pathophysiology...... 16 2.4.3 Interaction between inflammation and haemostasis...... 16 2.4.4 Diagnosis of DIC...... 17 2.4.4.1 Detection of Activation of Coagulation...... 17 2.4.4.2 Inhibitor consumption...... 17 2.4.4.3 Fibrinolytic activity...... 18 2.4.4.4 ISTH classification of overt and non-overt human DIC...... 18 3 EXPERIMENTAL THROMBOSIS AND DIC IN ANIMAL MODELS...... 193.1 METHODS IN DIAGNOSIS OF EXPERIMENTAL MICROTHROMBOSIS...... 19 3.2 STANDARDIZATION AND TRANSLATIONAL ASPECTS OF DIC IN ANIMAL MODELS...... 20 3.3 ANIMAL MODELS OF DIC AND THEIR RELEVANCE TO HUMAN DIC – A SYSTEMATIC REVIEW ...... (PAPER I)...... 20 4 DEVELOPMENT OF A RABBIT MODEL OF DIC AND IMPLEMENTATION OF NEW PARAMETERS FOR EARLY DIAGNOSIS OF MICROTHROMBOSIS IN DIC...... 214.1 VALIDATION OF A PULMONARY FUNCTION TEST IN A RABBIT MODEL OF EMBOLISATION MIMICKED BY MICROSPHERES...... 22 4.1.1 Background...... 22 4.1.1.1 Multiple Inert Gas Elimination Technique (MIGET)...... 22 4.1.2 Materials and methods...... 23 4.1.2.1 Animal procedures...... 23 4.1.2.2 Microspheres...... 23 4.1.2.3 Ventilation-perfusion relationships...... 24 4.1.2.4 Experimental protocol...... 24 4.1.2.5 Preparation of tissue specimens...... 24 4.1.3 Results...... 25 4.1.3.1 Histological examination...... 26 4.1.4 Discussion and conclusion...... 26 4.1.5 Cardiovascular and haemostatic changes in a rabbit microsphere model of pulmonary thrombosis (Paper II)...... 27 4.2 IMPLEMENTATION OF NEW PARAMETERS IN EARLY DIAGNOSIS OF DIC...... 28 4.2.1 Background...... 28 4.2.2 Materials and methods...... 28 4.2.2.1 Animals...... 28 4.2.2.2 Animal procedures...... 29 4.2.2.3 Cardiac troponin I (cTnI) measurements...... 29 4.2.2.4 Thromboelastography (TEG)...... 29 4.2.2.5 Experimental design...... 30 4.2.3 Results...... 30 4.2.4 Discussion and conclusion...... 32 4.3 ESTABLISHMENT OF A RABBIT MODEL OF THROMBOPLASTIN INDUCED DIC...... 332 4.3.1 Background...... 33 4.3.2 Development of a model of thromboplastin induced DIC in rabbits...... 33 4.3.2.1 Background...... 33 4.3.2.2 Materials and methods...... 34 4.3.2.2.1 Experimental design...... 34 4.3.2.3 Results...... 34 4.3.2.4 Conclusion...... 35 5 IMPLEMENTATION OF THE ISTH CLASSIFICATION OF NON-OVERT DIC IN A THROMBOPLASTIN INDUCED RABBIT MODEL (PAPER III)...... 35 6 CHARACTERISATION AND PURIFICATION OF THROMBOPLASTIN...... 366.1 BACKGROUND...... 36 6.2 MATERIALS AND METHODS...... 37 6.3 RESULTS...... 37 6.4 CONCLUSION...... 37 7 PURIFIED THROMBOPLASTIN CAUSES HAEMOSTATIC ABNORMALITIES BUT NOT OVERT DIC IN AN EXPERIMENTAL RABBIT MODEL (PAPER IV)...... 38 8 DISCUSSION AND CONCLUSION...... 39 9 PERSPECTIVES...... 42 10 REFERENCES...... 44 11 PUBLICATIONS...... 54 Paper I: “Animal models of DIC and their relevance to human DIC – A systematic review” (Submitted) Paper II: “Cardiovascular and haemostatic changes following microsphere injection in a rabbit model of acute pulmonary microvascular thromboembolism” (Submitted) Paper III: “Implementation of the ISTH classification of non-overt DIC in a thromboplastin induced rabbit model”. (Thrombosis Research, 2009; Vol. 124, Issue 4, Pages 490-497) Paper IV: “Purified thromboplastin causes haemostatic abnormalities but not overt DIC in an experimental rabbit model” (Thrombosis Research, 2010; in Press; DOI: 10.1016/j.thromres.2010.06.022)3 Preface“Whether you believe you can do a thing or not - you’re right” Henry FordThis thesis is the culmination of years of hard work, ups and downs and believing.Early on in life I discovered my passion for the detail. I take every chance I get to learn and understand what is possible on a specific subject. However, the thought of doing a PhD did not settle in my mind until almost the finishing of my master studies (probably because I was too focused on the details of my master studies), but to be given the opportunity to take this detailed journey into PhD and DIC land has been fantastic. It has also been hard work - hard in other ways than I imagined. I expected it to be mostly scientifically challenging, but realized that working with my own response to set backs, frustrations and worries has been the hardest - and most rewarding.“There are two kinds of problems; those you can solve - so don’t worry about them, and those you can’t solve - so don’t worry about them” Author unknownI want to thank my colleagues at Novo Nordisk. Everywhere I went I was met by open minds and an eagerness to help. I specifically wish to thank my supervisors. Thanks to Henrik Duelund Pedersen (Novo Nordisk A/S), my supervisor for the first 1½ years, for teaching me focus on the experiments and the data and not worry so much about forms and conformities and for advancing me from student to colleague. Thanks to Annemarie (KU Life) for many high-level, high-speed meetings with extremely valuable professional inputs. Mikael (Novo Nordisk A/S) - in a rather chaotic switch from one department to another you quickly identified my strengths and weaknesses and used this constructively to create solutions where I could do my best. Thanks for always being there, being ready, listening and taking action. My family and friends have had to put up with my absence in periods - especially during this spring, where the work has been most intense. Thank you for your patience.And a special thank goes to my fiancé Martin and our son Viktor, who have felt the tough times as much as I have. Martin thank you for pushing, lifting and nursing me through the hard times of this PhD project - without you I wouldn’t have come this far (No, I wouldn’t !).4 AbbreviationsABP Arterial blood pressure APC Activated protein C aPTT activated partial thromboplastin time AT Antithrombin cTnI Cardiac troponin IDIC Disseminated intravascular coagulationECG ElectrocardiogramFDP Fibrin degradation products FV Factor V FVa Activated Factor V FVII Factor VII FVIIa Activated Factor VII FVIII Factor VIII FVIIIa Activated Factor VIII FIX Factor IX FIXa Activated Factor IX FX Factor X FXa Activated Factor X FXI Factor XI FXIa Activated Factor XIHE Haematoxylin eosinISTH International Society on Thrombosis and HaemostasisLPS LipopolysaccharideMIGET Multiple inert gas elimination techniquePaCO2 Arterial CO2 tension PaO2 Arterial O2 tension PAI-1 Plasminogen activator inhibitor 1 PC Protein C PS Protein S PT Prothrombin time PTAH Phosphotungstic acid haematoxylinRVP Right ventricular pressureTAT Thrombin-antithrombin TEG Thromboelastography TF Tissue factor TFPI Tissue factor pathway inhibitor TM Thrombomodulin tPA tissue plasminogen activator uPA urokinase type plasminogen activatorV/Q Ventilation-perfusion ratio vWF von Willebrand factor5 Summary (English)Disseminated intravascular coagulation (DIC) is a syndrome occurring secondary to a wide range of predisposing diseases. DIC is a pathological process with widespread thrombosis in the microcirculation causing organ damage and having a poor prognosis. Comparison of animal models of DIC to human DIC is crucial in order to translate findings in research models to treatment modalities for DIC in humans. The aims of the current thesis were to establish a rabbit in vivo model of thromboplastin induced DIC and evaluate the implementation of a wide panel of markers for pulmonary, cardiovascular and haemostatic function in the early identification of microthrombosis. It was hypothesized that a DIC scoring system can be applied to a rabbit model of thromboplastin induced DIC to standardize and score the rabbit model of DIC according to the ISTH scoring system of DIC in humans.This thesis is composed of a general introduction to coagulation and its regulation and the syndrome DIC including an overview of animal models of DIC presented in paper I. The core work of the project is then presented including the methods applied and results obtained. These include the use of microspheres to establish a mechanical model of pulmonary embolism which is further described in paper II and a number of pilot studies developing a thromboplastin induced rabbit model of DIC with determination of a wide panel of markers for cardiovascular and haemostatic function including early markers of microthrombosis and finally characterising non-washed and purified thromboplastin. The validation and applicability of these methods are described in the set up of a non- clinical rabbit model of DIC in paper III and IV. Finally the conclusion and perspectives are presented based on four accompanying papers.Four papers are included in the thesis:Paper I “Animal models of DIC and their relevance to human DIC – A systematic review” (submitted for publication) provides an extensive literature search on established animal models of DIC. An overview of distribution of animal species, inducers, measurements and treatments are given for the many identified studies. Generally a high variability was found and it was concluded that standardization of animal models of DIC by for example application of a DIC score as has been developed for the diagnosis of human DIC by ISTH is recommendable. Furthermore it was identified that the majority of studies testing treatment of DIC only evaluated the prophylactic effect of such, though prophylaxis is in general irrelevant for cases of human DIC. The large amount of data compiled in this study is useful for the interpretation and comparison of responses in animal models of DIC and implementation of the recommendations may significantly improve the clinical relevance of animal models within this research area.In paper II “Cardiovascular and haemostatic changes in a rabbit microsphere model of pulmonary thrombosis” (submitted for publication), microspheres were used to validate the response of6 cardiovascular parameters (RVP and systemic blood pressure) to fixed size pulmonary thrombi in the rabbit. Haemostatic parameters were evaluated in vivo and in vitro to test whether the microspheres are truly inert. A dose dependent response on cardiovascular parameters was observed in vivo, cumulating in a lethal effect of the highest in vivo dose of microspheres. An in vitro setup evaluating thromboelastographic effects of microspheres in rabbit whole blood spiked with equivalent doses to the in vivo study demonstrates that the highest dose resulted in a procoagulant effect. Haemostatic parameters showed no significant procoagulant effect of any of the doses of microspheres in vivo and it was concluded that non-lethal doses of microspheres result in inert pulmonary fixed size emboli.Paper III “Implementation of the ISTH classification of non-overt DIC in a thromboplastin induced rabbit model” [1] describes the establishment of a rabbit model of thromboplastin induced non-overt DIC. Bolus injections of 1.25 and 2.5 mg thromboplastin/kg non-washed thromboplastin in rabbits resulted in non-overt DIC diagnosed by a modified score based on the ISTH score of non-overt DIC in humans, injection of higher thromboplastin doses were lethal and induction of overt DIC was not accomplished in this study. In paper IV “Purified thromboplastin causes haemostatic abnormalities but not overt DIC in an experimental rabbit model” [2], it is reasoned that the purification of thromboplastin would decrease variability and enable injection of higher non-lethal doses resulting in overt DIC. However injection of a 2.5 mg/kg bolus followed by a 1.25 mg/kg infusion of thromboplastin resulted in less severe procoagulant activation than for the previous study, though a lethal effect was also observed within this study indicating an even more narrow window between procoagulant and lethal effect than for the injection of non-washed thromboplastin. These results underscores the difficulties in comparisons between animal models using different inducers of DIC and emphasizes that induction of DIC, experimentally as well as clinically, may call for different treatment approaches. 7 Sammendrag (Dansk)Dissemineret intravaskulær koagulation (DIC) er et syndrom, der forekommer sekundært til en bred vifte af predisponerende sygdomme. DIC er en patologisk proces med udbredt trombose i microcirkulationen, hvilket forårsager organskade og giver en dårlig prognose. Sammenligning af dyremodeller for DIC med human DIC er altafgørende for at kunne translatere fund i forsøgsmodeller til behandlingsmodaliteter for human DIC. Målsætningen for denne afhandling var at etablere en kanin in vivo model for thromboplastin induceret DIC og evaluere implementeringen af et bredt panel af markører for pulmonær, kardiovaskulær og hæmostatisk funktion i den tidlige identifikation af microtrombose. Hypotesen var, at et DIC scoringssystem kan anvendes i en kaninmodel for thromboplastin-induceret DIC til at standardisere og score kaninmodellen for DIC ifølge ISTH scoringssystemet for human DIC.Denne afhandling omfatter en generel introduktion til koagulation og regulering af koagulationen og syndromet DIC inkluderende et overblik over dyremodeller for DIC præsenteret i artikel I. Kernearbejderne i dette projekt præsenteres herefter og inkluderer de anvendte metoder og opnåede resultater. Disse inkluderer anvendelsen af microspherer til etablering af en mekanisk model for pulmonær embolisme, som beskrives nærmere i artikel II, samt et antal pilot studier med udvikling af en thromboplastin induceret kaninmodel for DIC med bestemmelse af et bredt panel af markører for kardiovaskulære og hæmostatiske funktioner inkluderende tidlige markører for microtrombose og endeligt karakterisering af ikke-vasket samt renset thromboplastin. Valideringen og anvendelsen af disse metoder er beskrevet i artikel III og IV. Endelig præsenteres konklusionen og perspektiveringen baseret på 4 vedlagte artikler.4 artikler indgår i afhandlingen:Artikel I ”Animal models of DIC and their relevance to human DIC – A systematic review” (indsendt) omfatter et bredt litteraturstudie af etablerede DIC dyremodeller. Fordeling af dyrearter, induktionsmetoder, målinger og behandlinger oplyses for de mange identificerede studier. Generelt sås en høj variation i henhold til disse parametre og det blev konkluderet at standardisering af dyremodeller for DIC ved for eksempel anvendelse af en DIC score som tilsvarende er udviklet af ISTH til diagnosticering af human DIC anbefales. Ydermere bliver der vist, at hoveddelen af studierne, der tester behandling af DIC, udelukkende evaluerede den profylaktiske effekt af disse, selvom profylakse generelt er irrelevant for tilfælde af human DIC. Den store mængde data indsamlet til dette studie er brugbar for fortolkning og sammenligning af respons i DIC dyremodeller og implementering af disse anbefalinger kan signifikant forbedre den kliniske relevans af dyremodeller indenfor dette forskningsområde.I artikel II ”Cardiovascular and haemostatic changes in a rabbit microsphere model of pulmonary thrombosis” (indsendt), anvendes microspherer til at validere det kardiovaskulære respons (RVP og 8 systemisk blodtryk) til pulmonære tromber af fast størrelse i kaninen. Hæmostatiske parametre blev evalueret in vivo og in vitro for at teste om microsphererne med sikkerhed er inerte. Et dosis-afhængigt respons af kardiovaskulære parametre observeredes in vivo, kumulerende til en letal effekt af den højeste in vivo dosis af microspherer. Et in vitro setup, evaluerende tromboelastografisk effekt af den højeste in vivo dosis af microspherer i kaninfuldblod spiked med doser equivalent til in vivo studiet, demonstrerede at den højeste dosis resulterede i en prokoagulant effekt. Hæmostatiske parametre viste ingen signifikant prokoagulant effekt for nogen af microsphere-doserne in vivo og det konkluderedes, at non-letale doser af microspherer resulterer i inerte pulmonære emboli af fast størrelse.Artikel III ”Implementation of the ISTH classification of non-overt DIC in a thromboplastin induced rabbit model” beskriver etableringen af en kaninmodel for tromboplastin-induceret non-overt DIC. Bolusinjektioner af 1.25 og 2.5 mg tromboplastin/kg ikke-vasket tromboplastin i kaniner resulterede i non-overt DIC diagnoseret af en modificeret score baseret på ISTH scoren for non-overt human DIC, injektioner af højere tromboplastin-doser var letale og induktion af overt DIC blev ikke opnået i dette studie.I artikel IV ”Purified thromboplastin causes haemostatic abnormalities but not overt DIC in an experimental rabbit model” argumenteredes det, at rensning af tromboplastin ville mindske variationen og muliggøre injektion af højere non-letale doser resulterende i overt DIC. Men injektion af 2.5 mg/kg bolus fulgt a 1.25 mg/kg infusion af tromboplastin resulterede i svagere prokoagulant aktivering end i det tidligere studie, selvom en letal effekt også observeredes i dette studie, hvilket indikerer et endnu smallere vindue mellem prokoagulant og letal effekt end for injektionen af ikke-vasket tromboplastin. Disse resultater understreger problemerne ved sammenligning af dyremodeller, der bruger forskellige inducere af DIC og underbygger, at induktion af DIC, eksperimentelt som klinisk, kan kræve forskellige behandlingsstrategier.9 1 Introduction, hypotheses and objectivesDisseminated intravascular coagulation (DIC) is a complex and dynamic haemostatic syndrome occurring secondary to a wide range of underlying diseases [3-5]. DIC is characterized by variable imbalances of the components of the coagulation and fibrinolytic systems and the clinical signs vary considerably ranging from no overt signs of disease (non-overt DIC) [6;7], accompanied by minor changes in haemostatic parameters, to clinical symptoms of organ failure, associated with microvascular thrombosis in vital organs, to fulminant DIC with bleeding symptoms (overt DIC) [6]. Diagnosis of DIC early in the non-overt stage may increase the chances of survival in the DIC patient, because early and aggressive intervention through supportive and antithrombotic therapy, besides treatment of the underlying disease, may minimize or prevent thrombo-embolic complications and progression to overt DIC [8]. Clinically relevant animal models of DIC are crucial in the research and development of novel therapeutic interventions as well as in understanding the pathogenesis of DIC. Standardization of animal models of DIC is necessary in order to compare findings between species and to increase the translational aspect in non-clinical testing of therapeutics for DIC in humans. Simple diagnostic scoring systems for diagnosis of DIC in humans, such as the algorithms for overt and non-overt DIC developed by the International Society on Thrombosis and Haemostasis (ISTH) [7;9] are valuable standardized approaches to the characterization of DIC in humans and have been proven to have a high diagnostic accuracy [10-12]. As the approach taken by ISTH in scoring of DIC has been successfully applied in dogs suffering from DIC [13], application of similar scoring systems in experimental animal models of DIC may help to increase the clinical relevance and standardization of these non-clinical models.The overall hypothesis of the ph.d.-study was that a DIC scoring system can be applied to a rabbit model of thromboplastin induced DIC. Since a similar scoring system for diagnosis of DIC in man has been validated as an accurate diagnostic tool, such an approach could lead to a standardized and clinical relevant animal model of non-overt and overt DIC. 10 Furthermore, it was hypothesized that determination of several cardiovascular and haemostatic parameters would help to diagnose DIC in this model at an early stage and that the use of pulmonary function tests would add in the early diagnosis of microthromboembolism, an important complication of DIC. The specific objectives of the proposed investigations were:  To develop and standardize a non-clinical rabbit model of DIC with determination of a wide panel of markers for cardiovascular and haemostatic function including early markers of microthrombosis  To investigate whether a rabbit model of DIC could be standardized through application and scoring of the observed changes in laboratory markers according to the ISTH scoring system of DIC in humans11 2 Basic Mechanisms of Haemostasis and Thrombosis Knowledge of basic mechanisms of haemostasis are important to understand the complexity of thrombo-hemorrhagic disease processes, to interpret laboratory testing modalities and to understand the mechanism of action of pharmacological agents being tested in non-clinical animal models.2.1 Physiologic HaemostasisCoagulation is a complex process resulting in formation of clots. It is an important part of haemostasis, wherein a damaged blood vessel wall is covered by a platelet and fibrin-containing clot to stop bleeding and begin repair of the damaged vessel wall [14;15]. Haemostasis starts almost instantly after damage to the endothelium as exposure of the blood to proteins such as tissue factor (TF) initiates changes to platelets and fibrinogen [16]. Platelets immediately form a plug at the site of injury [17], simultaneously coagulation proteins in the blood plasma respond to form fibrin strands, which strengthen the platelet plug [18;19].The revision of the cascade model of coagulation [20], which explains fibrin formation by two different pathways; the extrinsic and intrinsic pathway, led to the suggestion that TF is the initiating factor [21] and to the development of a cell-based model of haemostasis by Hoffman and Monroe [22], where haemostasis is divided into three overlapping stages – initiation, amplification and propagation. Initiation: Coagulation is initiated by activation of FVII to FVIIa via TF from TF-bearing cells exposed to the blood during vessel wall damage. FVII binds to TF and is activated to FVIIa by several of the coagulation proteases when coagulation is ongoing [23]. Initially however, small trace amounts of FVIIa circulating in the blood [24] functions as an autoactivator of FVII complexed with TF [25]. The TF/FVIIa complex activates FIX to FIXa and FX to FXa. FXa can combine with FVa and produce small amounts of thrombin [22;26] (figure 1).Figure 1 Initiation of coagulation. TF-bearing cells exposed to the blood bind factor VIIa. The TF:VIIa complex activates factor X to Xa, which together with factor Va produce small amounts of thrombinAmplification: Platelets adhere to collagen and von Willebrand Factor (vWF) at the site of vascular injury [27]. Thrombin generated during initiation amplifies the coagulation by activation of platelets via protease-12 activated receptors (PAR) [28]. Thrombin also binds to non-PAR platelet receptors and activates FV released from the alpha granules of the activated platelets [29]. Furthermore circulating FVIII bound to vWF attaches to the platelets, where thrombin cleaves FVIII from vWF and activates it to FVIIIa (figure 2). Importantly, the procoagulant negatively charged phospholipid, phosphatidyl-serine becomes increasingly available during the platelet activation process [30].Figure 2 Amplification of coagulation. Platelets are activated by the thrombin generated during initiation. Thrombin furthermore dissociates FVIII from vWF and activates FVIII, FV and FXI on the surface of the platelets Propagation: During the propagation stage, the generated FIXa move from the TF-bearing cell and bind to the negatively charged surface of the activated platelet [31] as does FXI from plasma [32]. Thrombin activates FXI to FXIa [33] that further activates FIX on the platelet surface. FIXa bind to FVIIIa in what is called the ‘tenase’ complex and further activate FX. The ‘prothrombinase’ complex is formed between FXa and FVa on the platelet surface; it cleaves prothrombin and leads to a burst of thrombin, which then cleaves fibrinogen leading to the formation of a haemostatic fibrin clot [22] (figure 3).Figure 3 Propagation of coagulation. The tenase complex (IXa:VIIIa) lead to the formation of the prothrombinase complex (Xa:Va) via FX activation which facilitates the thrombin burst13 2.2 Regulation of Coagulation2.2.1 Anticoagulation Anticoagulation and fibrinolysis keep platelet activation, coagulation and clot formation in balance and restricted to the site of vascular injury to prevent inappropriate escalation, leading to systemic coagulation and disseminated fibrin formation [34]. Three pathways: the antithrombin (AT) pathway, the tissue-factor pathway inhibitor (TFPI) pathway and the protein C (PC) pathway are the main players in anticoagulation.Antithrombin (AT): The serine protease inhibitor AT is a major inhibitor of coagulation proteases such as thrombin [35], FXa [36], FIXa, FXIa [37] and FVIIa in complex with TF [38]. The serine proteases bind irreversibly to AT, and the complexes are subsequently removed from the circulation by clearance in the liver [36]. The inhibitory effect of AT is markedly enhanced in presence of heparin [39]. Tissue-factor pathway inhibitor (TFPI): TFPI is a Kunitz-type plasma protease inhibitor that regulates FVIIa/TF activity by inhibition of FXa [40]. TFPI is produced by endothelial cells and can be bound hereto; however TFPI mainly circulates as a complex with plasma lipoproteins [41]. The inhibition of FXa and FVIIa/TF is a two step process. TFPI directly inhibits FXa by binding near its active site. Secondly, this binding induces conformational changes which allow the TFPI-FXa complex to bind to FVIIa/TF [42]. TFPI thereby rapidly down regulates the direct activation of FXa by FVIIa/TF preventing thrombogenesis [43].Protein C (PC)/protein S: The antithrombotic serine protease PC is converted to activated PC (APC) by thrombin bound to thrombomodulin (TM), an endothelial cell surface receptor [44]. This interaction blocks the clot promoting capacity of thrombin and enhances the specificity of thrombin to protein C [45]. In conjunction with protein S, the activated PC (APC) inhibits the tenase and prothrombinase complex formed in the propagation stage by inactivation of FVa and FVIIIa [46]. 14 Figure 4 Anticoagulation. Antithrombin (AT), protein C (PC) and tissue-factor pathway inhibitor (TFPI) keep the coagulation in balance by inhibiting key factors of the coagulation. Dotted lines indicate inhibition, double lines indicate activation. AT inhibits several serine proteases such as thrombin and factor VIIa, IXa, Xa and XIa. Thrombin activates PC to activated PC (APC), which inhibits factor VIII and V activation. TFPI binds and inhibits factor X, which leads to the binding and inhibition of TF:FVIIa 2.2.2 Fibrinolysis The formed clot is reorganised and removed from the injured tissue by a process referred to as fibrinolysis. Fibrinolysis is initiated already while the fibrin clot is being formed and the central factor in the fibrinolytic system is plasmin, which is responsible for the degradation of fibrin. 2.2.2.1 Activation of plasmin The zymogen plasminogen is converted into the active enzyme plasmin by tissue-plasminogen activator (tPA) and urokinase type plasminogen acticator (uPA) [47]. tPA is the physiological activator of fibrinolysis and is found in most tissues, it is synthesized by endothelial cells and released slowly upon tissue damage [48]. In the presence of fibrin the affinity of tPA to plasminogen is increased and this localizes plasmin to the clot [49]. The other plasminogen activator uPA is synthesized in the liver or by macrophages and circulates freely in plasma [50]. The functions of uPA overlaps those of tPA [47], however uPA is important in activation of plasminogen in tissues and during wound healing whereas tPA is of primary importance for the lysis of fibrin clots in the circulation [51]. When fibrin is degraded by plasmin, a number of soluble fibrin degradation products (FDPs) are produced [48]. Thus, FDP measurements are used as an indicator that coagulation and fibrinolysis are activated. FDP’s compete for thrombin, and thus slow down the conversion of fibrinogen to fibrin and inhibit clot formation [48]. D-Dimers are a unique form of FDP, and products of cross-linked fibrin- derived degradation [52], thus D-Dimer more specifically signify clot lysis.2.2.2.2 Regulation of the fibrinolytic systemThe fibrinolytic system is primarily regulated by 2-antiplasmin and plasminogen activator inhibitor 1(PAI-1) [47]. The 2-antiplasmin is secreted by the liver and binds and neutralizes plasmin rapidly when circulating in plasma, it is also cross-linked to fibrin in the clot [53], where however the inhibition of plasmin is less effective [48]. PAI-1 is released from the endothelium upon vascular injury and15 inhibits tPA and uPA [47]. The close proximity of plasminogen activator, plasminogen and fibrin in the clot prevents inhibition of fibrinolysis by plasminogen activator inhibitor 1 (PAI-1) [48]. Furthermore, thrombin-activatable fibrinolytic inhibitor (TAFI) is activated by thrombomodulin-bound thrombin [54] and can prevent plasmin to bind to fibrin strands by cleaving plasminogen binding sites and thereby slow down the fibrinolysis rate [53;55].In summary, normal haemostasis is dependent on localization of both pro- and anti-coagulant activities, with perfect maintenance of the delicate balance between coagulation and fibrinolysis.2.3 Acquired Procoagulant Disorders of Haemostasis Acquired procoagulant disorders of haemostasis covers any insult that tips the balance between coagulation and fibrinolysis towards thrombosis [56;57], i.e. surgery, trauma [58], cancer [59], obstetrical complications [60;61], infections [62] etc. Thrombosis is the pathological and uncontrolled development of blood clots occurring as a result of impairment of the systems normally restricting clot formation to the local site of vascular injury [17]. In classical terms, thrombosis is caused by one of the corners of Virchow’s triad: hypercoagulability, vascular wall injury or circulatory stasis [63;64]. Clinical effects of thrombosis can range from no symptoms to lethality, dependent on the occurrence of complications, which are caused either by the effects of local obstruction of the vessel, embolization of thrombotic material or consumption of haemostatic elements [65]. Thrombosis is classified as either venous, arterial, cardiac or systemic [66]. Venous thrombi in humans usually occur in the lower limbs and can cause late complications such as pulmonary emboli [67]. Arterial thrombi usually occur in association with pre-existing vascular disease and induce tissue ischemia by obstructing flow or by embolizing into the distal microcirculation [68]. However both venous and arterial thrombosis may share a number of risk factors [69]. Intracardiac thrombi usually form on damaged valves, are usually asymptomatic, but may produce serious complications if they embolize [70]. Systemic thrombosis of the microcirculation is a complication of DIC in which microthrombi cause ischemic necrosis and/or consumption of platelets and clotting factors resulting in bleeding tendencies [66]. 2.4 Disseminated Intravascular CoagulationDIC, also known as consumptive coagulopathy, is a complex syndrome characterised by considerable activation of the coagulation and fibrinolytic system with increasing loss of localisation or compensated control [6]. However, the degree to which these systems are activated depends upon the triggering event, host response and contemporary conditions [71]. Consequently the clinical signs may range from no obvious disease to overt signs of bleeding and organ failure [72]. DIC is difficult to diagnose and treat, and is associated with a poor prognosis as it plays a significant role in organ failure and related mortality [72-74]. 2.4.1 AetiologyDIC occurs as a response to a variety of disorders and is neither characterised as a disease nor a symptom [75]. It is one of the most severe complications seen in patients suffering from sepsis [76],16 cancer [77-79], acute leukaemia [80], abruption of the placenta [81;82] and trauma [83;84] etc. Although a huge number of agents and disorders are associated with DIC [73] the basic aetiology is activation of the coagulation system by production or exposure of tissue factor [75;85-87]. 2.4.2 PathophysiologyThough the disorders predisposing to DIC are numerous, several seem to share overall mechanisms of initiation of DIC. Trauma, some malignancies and obstetric calamities may cause a sudden release of large amounts of tissue factor or placental tissues [6]. Infections cause a proinflammatory cytokine release and tissue factor expression from macrophages, monocytes and endothelial cells [88], thus activation of the coagulation system is mediated. Lipopolysaccharides has also been shown to induce tissue factor expression in experimental animal models [86;87;89;90]. More rare conditions such as snake bits and heat stroke initiate DIC via procoagulant or profibrinolytic mediators [91;92]. Regardless of the type of underlying disease or mechanism, continuous TF expression from either endogenous production or endothelial damage, massive enough to overwhelm the anticoagulant devices, seem to be the most important activator of blood coagulation in DIC [93;94]. The enhanced and widespread fibrin formation and deposition cause systemic microthrombosis, which trap platelets and coagulation factors [94]. The status of the fibrinolytic system plays a significant role in the fate of the formed fibrin and thereby the clinical picture of DIC [47]. If sufficient tPA is released from injured endothelium or other tissues rich in tPA such as placenta [95] and brain tissue [96] clots may be dissolved in the microcirculation by plasmin, leading to increase in circulating FDPs. On the other hand, if more thrombin than plasmin is present, lodging of clots in the microvasculature leads to tissue ischemia and organ failure [69]. The latter is typically seen in patients with septic DIC, where an initially profibrinolytic response is immediately followed by a suppression of fibrinolytic activity due to a sustained increase in plasma levels of PAI-1 [97] as confirmed in a chimpanzee model of endotoxemia [98]. The complexity of DIC is therefore caused by the variations in dysregulation of both coagulation and fibrinolysis, which challenges the diagnosis and treatment of this syndrome.2.4.3 Interaction between inflammation and haemostasisOnce activated, the inflammatory and coagulation pathways interact with one another [99;100]. Cytokines and pro-inflammatory mediators stimulate pro-coagulants (increase fibrinogen levels and induce TF expression) [88], inhibit anticoagulants (down regulate thrombomodulin) [101] and inhibit fibrinolysis (by PAI-1 release) [97;98]. Thrombin as well as FXa and probably tissue factor/FVIIa complex can interact with protease-activated receptors on cell surfaces to promote further activation and additional inflammation. Thus thrombin stimulates cytokine production, has chemotactic effect on monocytes and mitogenic effect on lymphocytes [102]. This vicious circle between inflammation and coagulation further amplifies the response to initiation of DIC. The complexity of these interactions may limit treatment effects in the clinic and affect the outcome of experimental trials if underestimated [103].17 2.4.4 Diagnosis of DICThe overall progressive but dynamic complex patho-physiologic changes in DIC calls for a panel of diagnostic markers as, until now, no single laboratory test has been able to establish or rule out the diagnosis of DIC [104]. The classic DIC patient often presents with bleeding as the predominant symptom and concurrent micro-thrombosis may be missed [6]. Thus, the whole clinical picture, such as established diagnoses, clinical condition and laboratory results, must be taken into account.2.4.4.1 Detection of Activation of Coagulation Activation of coagulation can be detected by either decreases in the level of coagulation factors or abnormalities in global coagulation tests such as prothrombin time (PT) and activated partial thromboplastin time (aPTT).PT and aPTT: The PT and aPTT, measures of tissue factor induced clotting or phospholipid and calcium induced clotting respectively, are often prolonged at some point during the course of DIC, mainly due to the consumption of coagulation factors, but may also be a result of inflammation [105]. The PT and aPTT can provide important evidence of the degree of coagulation factor consumption and activation, though interestingly PT is only prolonged in 50-75% of cases and aPTT in 50-60% of cases, and hence normal or even shortened values can not be used to rule out the diagnosis of DIC [6].Platelet count: Thrombocytopenia is found in up to 98% of DIC cases, the platelet count is even <50 x 10 9/l in approximately 50% of these [106]. Serial measurements of platelet count increases the sensitivity of this marker in the diagnosis of DIC, because a single measure of the platelet count within the normal range can be difficult to interpret in the dynamic context of DIC. A continuous drop in the platelet count, even within a normal range, indicates active thrombin generation, whereas a stable platelet count suggests that thrombin formation has stopped. Although a decrease in platelet count has a high sensitivity of DIC it is not very specific; a decrease in platelet count is associated with many of the diseases predisposing to DIC, and often thrombocytopenia is present without the development of DIC [107].Fibrinogen: Decreased levels of fibrinogen are often used as a measure of coagulation factor consumption [108]. However, fibrinogen acts as a positive acute-phase reactant [109;110], hence its plasma concentration increases during inflammation and cytokine release, which can cause the plasma fibrinogen levels to remain within the normal range despite considerable coagulation activation and fibrin formation. Thus, hypo-fibrinogenemia is only common in severe cases of human DIC [108]. 2.4.4.2 Inhibitor consumption The inhibitors of coagulation; AT and PC previously described are the markers most commonly evaluated for determination of inhibitor consumption [108].18AT/TAT: AT forms irreversible complexes with the generated thrombin, as well as other serin proteases, this causes the AT levels to rapidly decline during DIC, whereas thrombin-antithrombin (TAT) complex levels increase [111]. The persistent decrease in AT levels has been shown to have a very high prognostic value for prediction of death in human septic DIC [112]. TAT is an early marker of in vivo thrombin generation, but not a specific marker of DIC; it can however be useful in the early diagnosis of DIC when evaluated in combination with other markers of coagulation and fibrinolysis.Decreased PC/PS: PC is rapidly converted to APC in DIC and then consumed during its regulation of fibrinolysis. This is reflected in a decrease in endogenous PC levels and transient increase in endogenous APC levels as observed in both human DIC patients [113] and an animal model of DIC [114]. The reduction of PC levels is consistent and can be used prognostically in human DIC patients [112]. 2.4.4.3 Fibrinolytic activity In addition to enhanced thrombin formation, fibrinolytic activity is often also increased in DIC. However, in some cases inhibition of fibrinolysis may dominate the picture [115]. It is possible to test for specific markers of fibrinolysis such as tPA, plasminogen, 2-antiplasmin, plasmin-antiplasmin complexes, fibrin and fibrinogen degradation products (FDP) and PAI-1. Except for FDP and D-Dimer assays these tests are not commonly used in the clinic.FDP/D-Dimer: Fibrin degradation can be evaluated by assays measuring FDP levels or D-Dimer levels [52;116]. As a product of fibrin degradation, FDPs reveal that both thrombin and plasmin was present, whereas D- Dimers, from degradation of cross-linked fibrin, show that a fibrin clot was formed and degraded [117]. FDP levels are increased in 85 to 100% of DIC patients and D-Dimer assays show abnormal values in 93% of DIC patients [6]. These highly sensitive DIC parameters have a limited specificity though; indications of increased thrombin and plasmin activity are not diagnostic of thrombo-embolism and DIC, these assays therefore mainly represent a negative predictive value in the diagnosis of DIC. Soluble fibrin monomers (SF) reflect the direct action of thrombin on fibrinogen and may present an advantage in diagnosis of DIC. SF are elevated in 90-100% of DIC cases [52], its generation is limited to the intravascular compartment and therefore not influenced by extravascular fibrin formation from trauma or inflammation. However, as most other markers of DIC it has a very low specificity [52].2.4.4.4 ISTH classification of overt and non-overt human DIC Although the syndrome DIC has been known for decades, the complexity of the syndrome has brought along a lack of consensus on the definition and diagnosis of DIC. Choice of assays for monitoring imbalances of coagulation and fibrinolysis in DIC has often been random and dependent on local availability and routines. In 2001, The International Society of Thrombosis and Haemostasis (ISTH) sub-committee of the Scientific and Standardization Committee (SSC) on DIC recommended the use19 of a scoring system for overt and non-overt DIC in humans. With the lack of a gold standard for diagnosis of DIC, these recommendations are based on consensus statements from specialist sub committees [7]. The ISTH criteria utilizes a combination of platelet count, measurement of prothrombin time, measurement of fibrinogen and fibrin degradation products in a 5-step diagnostic algorithm to calculate an overt DIC score, the scoring system for non-overt DIC furthermore incorporates a scoring of abnormal trends by repeated DIC scoring and addition of specific criteria such as thrombin- antithrombin and protein C levels. Parameters included in the ISTH scoring system are based on simple laboratory tests available in most hospital laboratories [7]. Included in the recommendations are that this scoring system should not be used if no underlying disorder known to be associated with DIC is present. Prospective evaluations from pilot studies using these scoring systems have shown a strong correlation between increasing DIC score and mortality and a high sensitivity and specificity of DIC [10-12]. 3 Experimental thrombosis and DIC in animal modelsDIC affects numerous components in the blood circulation; blood cells, coagulation factors, vessel wall, blood flow and pH, and trigger compensatory capabilities of the organism (changes in respiration, heart rate and blood pressure) [6]. The complexity of DIC makes ex vivo mimicking very difficult and inconsistency in administration of treatment and supportive care causes high variability in the course of the syndrome in the human DIC patients, hence relevant and predictable animal models of DIC are extremely valuable for investigation of pathophysiologic mechanisms and treatment of the syndrome. Though no single animal model can ever duplicate all aspects of DIC occurring in humans, animal models still provide an opportunity to gain insight in pathogenesis and progression of the syndrome within a living organism, with all the complexities this gives rise to. Multiple inducers have been used in experimental animal models of DIC reflecting the many identified predisposing conditions associated with DIC in humans [6]. Thus the characteristics of the different models may vary considerably according to dynamics and end-points. Also selection of parameters for diagnosis of DIC differs between animal models of DIC; however most studies use the occurrence of micro-thrombosis as evidence of DIC, and until now this is the closest to a golden standard of DIC diagnosis in animal DIC models.3.1 Methods in diagnosis of experimental microthrombosisThe above mentioned tests of coagulation activation, fibrinolysis activation and inhibitor consumption indicates the occurrence of thrombosis and are widely used in diagnosis of DIC in animal models. In addition hereto, a great advantage of experimental animal DIC studies as compared to studies in human DIC patients is the possibility to apply more invasive methods in order to increase the diagnostic accuracy of DIC. In this context histopathology is a valuable tool for the visualization of disseminated thrombus formation, one of the key parameters of DIC.20 In animal models of DIC histopathology is often used to determine the presence and number of microthrombi. Various staining methods can be used to get a morphological overview and identify components of the fibrin clot [1;71;118-120]. Alternative ways to identify and visualize the thrombi is by radiolabelling of components included in the thrombus formation, especially fibrin [121-123] or by immunohisto/cytochemichal staining [118;122;124]. Besides identification of thrombi in different tissues, several studies furthermore quantify the microthrombi either by manual or computerized counting methods. As an example, the number of thrombi in the kidneys can be assessed by the percentage glomerular fibrin deposition (%GFD), and this is used as a DIC parameter in some experimental animal models [125;126]. Thrombosis scores based on light microscopy counts from selected tissue sections [1;127;128] or by computerized counting [129] have also been used in animal models of DIC.3.2 Standardization and translational aspects of DIC in animal modelsVarious attempts have been made to establish relevant animal models of disseminated intravascular coagulation during the last 4-5 decades. A wide range of animal species, inducers of DIC and dosing regimens have been applied in the process of imitating the various predisposing conditions and complex dynamics of DIC. To phylogenetically mimic the human situation the chimpanzee (primates) are the preferred species [130]). For physiological or cardiovascular similarities to humans the pig [131] or the rabbit [132] may be chosen and finally species may be chosen because of size or availability of genetically modified specimens (mouse [124;133;134]). Non-clinical testing in animal models of DIC is aimed at development or safety testing of products for use in human DIC patients. It is of outmost importance that non-clinical and clinical researchers strive to increase predictability and translational aspects of animal models of DIC. Standardization of DIC among several different species may be challenging due to lack of availability or access to species specific assays for the different parameters. It is important to keep in mind that cross-reactivity of immunoassays between man and animal species is generally poor [135], and species specific assays should preferably be used for evaluation of coagulation parameters in the respective animal models if no validation of human assays exist [136].Though consensus on definition and diagnosis of DIC and non-clinical testing setup is currently lacking in the work with experimental animal models of DIC, the methods for development of similar scoring systems as the ISTH human DIC score are available, as are experiences from application of such a scoring system in dogs suffering from spontaneous DIC [13].3.3 Animal models of DIC and their relevance to human DIC – A systematic review (Paper I)In paper I, the established animal models of DIC were reviewed and an overview of species, inducers, and dosing regimens was given. Furthermore diagnostic approaches were compared in the light of the21 recent ISTH scoring system for human DIC as a means to standardize experimental animal models of DIC. Finally treatments tested in animal models of DIC were reviewed. The rat is by far the preferred species amongst animal models of DIC and lipopolysaccharides (LPS) the preferred inducer of DIC; in addition, species such as rabbit, dog and mouse, and inducers such as tissue factor, live bacteria and thrombin have been used most often. The induction of DIC in animal models usually tend to imitate the pathogenesis of DIC developing from a specific predisposing disease seen in humans or to mimic a more isolated part of the general DIC pathogenesis. Confirmation of DIC in experimental animal models was mainly based on histological evidence of thrombosis and changes in a series of haemostatic parameters; however there was no agreement on which parameters should be evaluated for a definitive diagnosis of DIC in the reviewed experimental animal models. An overview of the reporting of ISTH DIC scoring parameters revealed that only about 25% of the studies measure all of the four parameters necessary for the implementation of the scoring system. About half of the studies scored 2 or 3 of the 4 parameters and could probably easily be standardized to include the parameters in the ISTH score of human DIC, which could increase the ability to compare DIC amongst animal models and also potentially improve the translational aspects of treatments tested. A large number of studies testing treatment modalities for DIC in different animal models were identified and an overview and description of the most frequently tested compounds is provided, this includes antithrombin (AT), platelet activating factor (PAF)-antagonist, thrombomodulin (TM), thrombin inhibitors, heparin, FX-inhibitor, APC, TFPI and urokinase. For a number of the compounds various effects have been reported, but all of the compounds have at some point shown a positive effect in the treatment or prevention of DIC in experimental animal models. Unfortunately these positive results are rarely replicated in human clinical trials and so far only APC has been approved for treatment of septic DIC in humans. Explanations as to why compounds effective in non-clinical animal models fail in clinical trials appear to be based in the fact that most treatment compounds tested in animal models of DIC are administered prophylactically, which is not the case in human DIC patients, and in the fact that the complexity of DIC has lead to the use of inappropriate animal models, which do not accurately reflect the complex syndrome of DIC in humans.Despite the challenges in extrapolation of data from non-clinical models to setup of human clinical trials it is believed that animal models of DIC are valuable tools investigating pathogenesis as well as treatment possibilities, however it was concluded that it is recommended that the animal models of DIC are standardized so that comparison between species may increase the translational aspects within this field.4 Development of a rabbit model of DIC and implementation of new parameters for early diagnosis of microthrombosis in DIC Based on the introduction and the review of DIC and animal models of DIC, experiments were conducted to establish an animal model of DIC with application of a standardized diagnostic scoring22 system and investigation of new markers valuable in the early diagnosis of DIC. In the following an overview of the studies performed during the ph.d. is provided.4.1 Validation of a pulmonary function test in a rabbit model of embolisation mimicked by microspheres 4.1.1 Background The developed microthrombi in the initial stage of DIC most often get caught in the lung vasculature, which acts as a fine-meshed net for all thrombi developed with embolization from the venous side of circulation, or in the renal glomeruli for thrombi formed at the arterial side of circulation [94]. Thrombi blocking the small pulmonary vessels can cause impairment of the gas-exchange and result in retention of carbon dioxide and insufficient oxygenation of arterial blood [137]. Pulmonary function testing has been used as a diagnostic tool for both respiratory and pulmonary cardiovascular dysfunction in both human patients [138] and in animal models of DIC to detect gas exchange impairment caused by pulmonary thrombi [139]. It offers the ability to measure the diffusion of gas across the alveolar capillary membrane and the perfusion of the lung by blood. Optimal oxygenation of blood, vital for maintenance of nearly all tissue function, is extremely dependent on normal physiology of both ventilation and perfusion. 4.1.1.1 Multiple Inert Gas Elimination Technique (MIGET) The multiple inert gas elimination technique (MIGET) is a pulmonary function test. MIGET measures the distribution of inert gasses in the blood entering and leaving the pulmonary circulation and in the expiratory air [140]. A ventilation-perfusion ratio (V/Q) is calculated based on these measures [141]. Hospitals equipped to run MIGET use this technique to monitor gas exchange during several pulmonary; vascular and cardiac diseases in human patients [138]. In order for a pulmonary embolus to affect the gas exchange it has to be large enough to block a vessel that supplies a functional lung unit, a term used for distal alveoli grouped so that gas can diffuse between them [142]. With microthrombosis as a key parameter of DIC success in early detection of DIC by application of MIGET depends on the size of pulmonary thrombi. MIGET has been applied to animal models of experimental pulmonary thromboembolism and proven effective in early diagnosis of pulmonary emboli of a certain minimum size dependent on the individual species, e.g. 100-150 µm in the dog [143] and about 60 µm in the pig [142]. MIGET has also been applied to rabbits, however not for detection of pulmonary emboli [144].We hypothesized that MIGET can detect very early changes in pulmonary function caused by microthrombi of rabbits. Based on the information on MIGET and as part of an overall aim to identify markers for early diagnosis of DIC, the effect of MIGET in early detection of microthrombi was validated in an exploratory pilot study, where pulmonary embolization was mimicking by injection of different sizes of microspheres to rabbits. 23 4.1.2 Materials and methods4.1.2.1 Animal procedures Six male New Zealand White rabbits (2.8-3.3 kg) were included in the study. The rabbits were sedated with Hypnorm® i.m. (0.3 ml/kg) and anaesthetized with Stesolid® i.v. (0.2 ml/kg). Anaesthesia was maintained with Stesolid®/Hypnorm® mixture (1:6). The rabbits were intubated and connected to a ventilator, and the respiratory frequency maintained at 30 breaths/min. The left ear artery was catheterized for blood sampling and measurement of arterial blood pressure (ABP). 0.5 ml Xylocain® was injected intracutaneously in the neck, the right internal jugular vein was catheterised and the catheter was advanced to the right ventricle to measure right ventricular pressure (RVP) and to obtain venous blood samples. Catheters were placed in the ear vein of both ears – one for infusion of dissolved inert gasses and one for the maintenance of anaesthesia. ECG electrodes were placed, allowing the recording of 3 ECG limb leads (lead I, II and III). This technique was performed in collaboration with the staff at Uppsala University Hospital, Sweden, Department of Medical Sciences, Clinical Physiology. All animal studies were conducted according to guidelines and approvals of the animal research ethical committee of Uppsala University.4.1.2.2 Microspheres From previous pilot studies, performed at Novo Nordisk A/S, testing the effect of 45 µm microspheres on blood pressure (BP) and right ventricular pressure (RVP) in rabbits, it was known that a dose of 250 µl microspheres was not lethal and caused a significant increase in RVP. Thus these data determined the starting point of this exploratory pilot study which additionally tested larger microspheres as the effect of MIGET was expected to depend on the blocking of a functional lung unit. Copolymer microspheres in aqueous suspension (Duke Scientific Corporation, Palo Alto, CA, USA) were used for embolization. Different sizes of microspheres were used (45, 98 and 222 µm) (See Table 1) and each animal received only one size of microspheres. Care was taken to shake the aqueous suspension of microspheres to prevent aggregation at the time of injection, as aggregated microspheres might give misleading results. Table 1 Microsphere dosing in six rabbits Animal ID Animal weight Microsphere Microsphere (kg) diameter (µm) volume (µl) 1 3.0 45 250 2 3.2 45 250 3 3.1 45 250 4 3.2 98 250 5 2.8 45 1800 6 3.3 222 150024 4.1.2.3 Ventilation-perfusion relationships To assess the ventilation-perfusion ratio, MIGET was used. A solution of six inert gases with different solubility in blood was dissolved in saline and infused into an ear vein. The infusion was maintained for at least 45 minutes before first measurement. The MIGET technique was conducted as previously described [142], in brief; three millilitre samples of arterial and mixed venous blood and 20 ml mixed expired gas were collected simultaneously. The blood and gas samples were collected in tight glass syringes and the retention and excretion of each gas were measured by gas chromatography [141] to calculate a V/Q ratio. In addition 2 ml blood was collected for blood gas analysis and haematology. In a normal situation ventilation and perfusion are matched to reach an optimal gas exchange, this is characterised by a V/Q equal to 1. Conditions decreasing the ventilation (i.e. asthma or pneumonia) cause decreases of the V/Q, whereas conditions decreasing the perfusion (i.e. thrombosis in DIC) cause increases of the V/Q.4.1.2.4 Experimental protocol Continuous infusion of inert gases was started once rabbits were anaesthetized and instrumented. MIGET, ABP, RVP, ECG, blood gas levels and blood cell counts were evaluated at two baseline time points and 15 and 60 minutes after microsphere injection. The rabbits were used as their own controls for evaluation of the effect of anaesthesia on the V/Q ratios by the two baseline evaluations. The rabbits were euthanized by exanguination at the end of the study (Figure 5). As the initial doses of 250 µl 45 µm microspheres failed to cause any increase in RVP two rabbits were injected with microspheres until a rise in RVP was seen in order to compare this effect of pulmonary embolism with MIGET and blood gas measures. This resulted in 3 boluses of microsphere injection for one rabbit and it was chosen to perform only one baseline measurement followed by a MIGET measurement 15 minutes following each of the bolus injections, as only 4 MIGET measures could be performed on each rabbit.Figure 5. Study time line4.1.2.5 Preparation of tissue specimens Heart, lung and kidneys were harvested and fixed with 10% buffered formalin for 4 weeks. Sections were stained with hematoxylin and eosin (HE) for general overview and with Phosphotungsten acid hematoxylin (PTAH) for demonstration of fibrin. Sections were studied under the light microscope to25 determine microsphere location, extent of microsphere clustering, and detect possible fibrin deposition around the beads.4.1.3 Results Baseline cardiovascular and blood gas levels were within reference values for rabbits [145] (data not shown). Injection of 250 µl of 45 or 98 µm microspheres had no effect on any of the measured parameters. As a consequence the approach of administering microspheres until RVP effect was taken and this is reflected in the increase in RVP seen for animal 5 and 6 (Fig. 6A). RVP seemed to depend on the dose and not the size of microspheres, a tendency supported by earlier pilot studies (unpublished by Olrik Berthelsen et al. 2009) and studies in the pig [142]. Other cardiovascular parameters were not affected by the microsphere boluses. Changes in V/Q ratios, with a dose dependent increase in occurrence of high V/Q regions, only developed in rabbit 6 receiving the largest sized microspheres (222µm), whereas none of the other animals showed any increase in ventilation of high V/Q ratios (Fig. 6B). A B60 20 ) g Animal 1 H n m o Animal 2 i s t m 15 ( a a* l e i Animal 3P 40 Animal 1 t r V a n e Animal 4 RAnimal 2 Q v / c i 10 l A l Animal 5 Animal 3 a V o t t o h s 20 Animal 6 T yAnimal 4 g* i s fH 5 o n Animal 5 a % e Animal 6 M 0 0Time (min) Time (min)*For animal 6; 15 min after bolus 2 *For animal 6; 15 min after bolus 2C D300 60 Animal 1 Animal 1 ) ) g g 50Animal 2 H Animal 2 H m * m Animal 3 Animal 3200 m 40 m ( (Animal 4 2 Animal 4 2 OO 30 Animal 5 C Animal 5 P P l l a Animal 6 Animal 6 i 100 a 20 i r r e e t t r r 10 A A 0 0Time (min) Time (min)*For animal 6; 15 min after bolus 2 *For animal 6; 15 min after bolus 2Figure 6 Effect of microspheres on A) systolic right ventricular pressure (RVP), B) presence of high V/Q areas, C) arterial PO2 and D) arterial PCO2 according to time points (min). Bolus of microspheres is injected at time point 0. For animal 6 a bolus of microspheres was given 15 minutes before each of the last three measurements. Each symbol represents one measurement in one animal26 There was a slight decrease in PaO2 and increase in PaCO2 for animal 5 and 6 following embolisation, however not until 60 minutes after injection of 45 µm microspheres and 15 minutes after the third bolus of 222 µm microsphere injection (Fig. 6C and 6D).4.1.3.1 Histological examination Microspheres were easily located in lung vessels, and 222µm microspheres were visible macroscopically as well. Microspheres were found widely distributed in the lung tissue. No microspheres were found in kidney or heart sections. In none of the sections studied microspheres were found to aggregate two or more in width. No fibrin formation was observed in relation to microspheres.4.1.4 Discussion and conclusion In the current pilot study it was possible to apply the MIGET technique to a rabbit model mimicking pulmonary embolism. Sampling for MIGET measures, blood gas analysis and hematologic evaluation in this study resulted in a removal of 8 ml of blood at each sampling. As we were able to apply MIGET measures 4 times for each rabbit, samplings resulted in removal of 19% of the total blood volume (in a 3 kg rabbit) during the study (with a circulating blood volume of 56ml/kg [146]. This is a considerable amount of blood removal and it can not be excluded that it might have affected the cardiovascular and hematologic parameters.No fibrin depositions were observed around the microspheres caught in the pulmonary vessels. Also no microspheres were found to aggregate, which would have caused blockage of vessels twice (or more) the intended size. This supports that the changes in the measured parameters relate to pulmonary emboli of the stated microsphere diameters and not larger emboli as could have been formed by either fibrin attachment or microsphere aggregation. The acute effect on gas exchanging capabilities of the lung indicates that the microspheres cause a total blockage of the pulmonary vessels, however we can not exclude that blood may be able to pass around the microsphere when for example blood pressure increases, as indicated by others [147].The blood gas measurements seem not to be as sensitive of the V/Q changes as the MIGET measurements, and the RVP for that matter, this shows, that the organism to a large extent is able to secure an optimal oxygenation of the blood despite obvious changes in the small gas exchanging functional lung units, and that changes in blood gas levels reflect serious damage to larger gas exchanging areas. It should be emphasized, that this study is an exploratory pilot study and the results described are only observed in very few animals. However, each animal was used as its own control and several samplings from the individual animals all pointed to the same conclusions, thus we believe the results are of high value.27 In conclusion it was possible to apply the MIGET technique to rabbits for detection of pulmonary emboli. It was shown that MIGET detects pulmonary emboli very early on and is more sensitive of changes in the pulmonary gas exchanging capabilities than blood gas measurements. MIGET detected instant and dose dependent changes in the V/Q-ratio following the injection of 222 µm microspheres and showed that high doses of 45 µm microspheres did not affect the V/Q ratio. We believe MIGET has high value as a clinical tool for early detection and monitoring of DIC, however due to the amount of specially modified equipment and technical expertise needed, it will probably not be used on a regular basis in pre-clinical and clinical settings.Although it was decided to discontinue the validation of MIGET for early diagnosis of microthrombosis, the use of microspheres to mimic pulmonary embolism proved very valuable in this setting and it was decided to further develop this model in the attempt to find novel markers of early DIC.4.1.5 Cardiovascular and haemostatic changes in a rabbit microsphere model of pulmonary thrombosis (Paper II) We hypothesized that a rabbit model of fixed size pulmonary thromboembolism would enable investigations of the mechanical consequences of pulmonary microvascular thrombosis in DIC and possibly the discovery of useful markers for early diagnosis hereof.In paper II the aim was to establish a mechanical model of pulmonary thromboembolism in the rabbit by venous injection of various doses of microspheres in order to test the response of cardiovascular parameters such as RVP, systemic blood pressure and ECG. Furthermore in vitro and in vivo testing of haemostatic parameters such as TEG, PT, aPTT, platelet count, fibrinogen levels, TAT and FDP were included to investigate whether the microspheres are truly inert even at high doses. Histological evaluation of kidneys, heart, brain and lung was performed to determine microsphere location and evaluate fibrin formation in relation to microspheres caught in the vessels. Microspheres of 45 µm in diameter were injected. Three groups of eight rabbits received one dose of 0.3, 6 or 16 µl microspheres/ml blood and one group of only three rabbits received one dose of 32 µl microspheres/ml blood as a lethal effect of this dose was shown. No effect on any of the parameters was shown for the small dose (0.3 ul microspheres/ml blood) of microspheres. Administration of 6 and 16 µl microspheres/ml blood resulted in minor and significant increases in cardiovascular parameters with rapid and dose dependent effects on RVP and systemic blood pressure. No changes in haemostatic parameters were shown with administration of 6 µl microspheres/ml blood whereas 16 µl microspheres/ml blood caused minor initial haemostatic changes. TEG showed minimal haemostatic in vitro changes in rabbit whole blood spiked with the highest dose of microspheres (32 µl microspheres/ml blood). Microspheres were only located in the pulmonary vasculature except in animals receiving a lethal dose of microspheres, where few microspheres were found in the myocardium and renal cortex. No fibrin formation was observed around the microspheres.28 It was concluded that administration of microspheres to the rabbit enabled early detection of changes in RVP and systemic blood pressure caused by mimicked fixed sized mechanical pulmonary thromboembolism.The results from this study of mechanical pulmonary thromboembolism showing the response of the cardiovascular parameters; systemic blood pressure and RVP, to various doses of fixed size pulmonary emboli indicated that these parameters could be valuable in early diagnosis of pulmonary microvascular thromboembolism in experimental animal models of DIC.4.2 Implementation of new parameters in early diagnosis of DIC4.2.1 Background As widespread microthrombosis in DIC can cause myocardial damage due to microthrombi caught in coronary vessels [148;149], early detection of myocardial ischemia might be valuable in an animal model of DIC. The cardiac isoform of troponin I is a recognized biomarker for acute myocardial damage with a high sensitivity and specificity [150] and the effect of cTnI measurements in detection of myocardial ischemia was tested in a pilot study of non-invasive medically induced myocardial infarcts in the rabbit by injection of Isoproterenol and Vasopressin as previously described by Pinelli A. et al. [151]. Furthermore a separate purpose of this pilot study was to evaluate different settings of TEG and determine the most optimal design for TEG measurements in a rabbit model of DIC. TEG is a global haemostatic test that has the ability to evaluate all steps of coagulation and fibrinolysis in whole blood containing all the intravascular coagulation factors, inhibitors and cells, thus only missing the contribution of the vessel wall. Parameters describing the whole blood clotting stages are attained by measuring the forces acting on a pin in the rotating TEG cup [152]. TEG has been shown valuable in the diagnosis of DIC in dogs [13;153], however the use of TEG in human patients mostly involves monitoring of haemostatic function during cardiovascular surgery [154] or management of coagulopathies following injuries [155] and though able to show haemostatic abnormalities in septic patients, the diagnostic specificity and sensitivity for DIC has not been analyzed [108].4.2.2 Materials and methods 4.2.2.1 Animals New Zealand White rabbits of 2-3 kg were obtained from Charles River, Sulzfeld, Germany. The animals were kept in a barriered facility and acclimated for a period of 5 weeks which included preventive treatment with coccidiostatic drugs during the second and fourth week (Esbetre® Vet, Novartis, Denmark). The rabbits were housed in multiple pens of 8-10 animals each with free access to drinking water and standard rodent pelleted diet (Altromin 2113, Altromin GmbH, Lage, Germany) fed ad libitum. Rabbits were used non-fasted. The study was approved by the Danish Animal Experiments Inspectorate, the Ministry of Justice.29 4.2.2.2 Animal procedures Three rabbits were pre-anaesthetized with Diazepam 5 mg/ml (Stesolid®, Alpharma, Oslo, Norway) 0.4 mg/kg and anaesthetized with pentobarbital sodium 5% in sterile water (Nomeco, Copenhagen, Denmark) intravenously through an ear vein, pentobarbital sodium was supplemented as needed. The other ear vein and an ear artery were catheterized for injection of test compound and blood sampling respectively. 0.5 ml Xylocain® 10 mg/ml (Astra Zeneca, Albertslund, Denmark) was injected intracutaneously in the neck. The right heart ventricle was catheterized via the right internal jugular vein to measure right ventricular pressure (RVP) and the left carotid artery was catheterized for measurement of systemic blood pressure and blood sampling. ECG electrodes were placed, allowing the recording of 3 ECG limb leads (lead I, II and III). 4.2.2.3 Cardiac troponin I (cTnI) measurements Serum levels of cTnI were tested by an ELISA assay validated for rabbit cTnI (Life Diagnostics, West Chester, PA)4.2.2.4 Thromboelastography (TEG) Citrated whole blood was obtained from either the carotid artery or an ear artery and left to stabilize for 30 minutes. To determine the most optimal TEG setup for future studies 1). effects of blood sampling site and 2). of kaolin as a trigger source on the coagulation profile were evaluated. TEG analyses were performed in duplicates by adding calcium, different doses of kaolin (China Clay, Haemoscope®, USA) and citrated whole blood to the computerized thromboelastograph (TEG 5000, software version 4.1.73, Haemoscope, USA). The reaction time (R) depicts the time from placement of blood in the TEG cup until initial fibrin formation and is primarily related to plasma clotting factors and inhibitor activity. The clotting time (K) measures the time to clot formation from the end of R until predetermined amplitude of 20 mm and is related to clotting factors, fibrinogen and platelets. The angle () shows the speed of fibrin cross-linking, dependent on platelets, fibrinogen and clotting factors and the maximum amplitude (MA) represents the strength of the formed fibrin clot [156]. In this study data were recorded continuously until a value for maximum strength of the clot (MA) was reached. TEG parameters reported are reaction time (R), velocity of clot formation (angle, ) and maximum strength of the clot (MA). The tracing of TEG are depicted as exemplified in figure 7.30 Figure 7 TEG parameters. The parameters reaction time (R) (latency from placement of blood in the cup until TEG tracings reach amplitude of 2 mm), clotting time (K) (measured from R until TEG tracing amplitude reaches 20 mm), alpha angle ( ) (slope of TEG tracing from R to K) and maximum amplitude (MA) (greatest amplitude of the TEG tracing) is graphically depicted4.2.2.5 Experimental design Blood samples for thromboelastography were taken from the carotid artery before induction of myocardial infarction, which was induced as previously described [151]. Briefly Isoproterenol (Isoproterenol Hydrochloride, Sigma-aldrich, Schnelldorf, Germany) 3 mg/kg was injected i.p. at time point 0 min and Vasopressin (Arg8-vasopressin acetate, Sigma-aldrich, Schnelldorf, Germany) 0.3 mg/kg/5 min was injected i.v. at time point 10-15 min. Samples were taken at baseline and 20, 30, 40 and 55 min. (fig. 8). All animal studies were conducted according to guidelines and approvals of the Danish Animal Experiments Inspectorate, the Ministry of Justice.Figure 8 Time line for induction of myocardial ischemia and samplingDue to dramatic effect on blood pressure, heart rate and respiration, the experiment was stopped at 13 minutes for one rabbit and only half the vasopressin dose was injected to the next rabbit for which the experiment then was completed as planned. In the last rabbit the whole vasopressin dose was injected and the experiment was extended to last 5 hours with blood samples every hour.4.2.3 Results Injection of Isoproterenol caused a significant increase in heart rate and systolic blood pressure for all 3 rabbits. The following injection of vasopressin worsened the blood pressure either by increasing the diastolic pressure or lowering both systolic and diastolic blood pressure significantly. The respiration became rapid and superficial in response to the circulatory changes. 31 For the last rabbit running the full protocol, serum levels of cTnI significantly increased from 120 min till the end of the experiment (fig. 9). For the two other rabbits no increase in serum cTnI levels were measured at any time point (data not shown).6 ) l 4 m / g n (I nT 2 c0 -5 20 55 120 180 240 300 Time (min)Figure 9 Serum levels of cTnI for one rabbit receiving Isoproterenol (3 mg/kg) at time point 0 min and Vasopressin (0.3 mg/kg/5 min) at time point 10-15 min. Evaluation of TEG with regard to effect of trigger addition and blood sampling site (figure 10) showed that 1). Blood sampling from the ear artery resulted in much greater variations for the TEG parameters than blood sampling from the carotid artery and 2). different doses of kaolin all resulted in a very short reaction time leaving very little room for hypercoagulant detection, whereas a wider window was achieved by pure recalcification of the citrated blood samples. 32 25 80 ) n i m 20 (60 ) ) R g ( e 15 d e ( m e 40 i l t g 10 n n o A i t 20 c 5 a e R 0 06 ) 80 m ) m ( n i) m A (60 M ) (K 4 e ( d u e t i 40 l m i p t m g 2 A n i m t 20 t u o m l i x C 0 a 0 MCarotid artery (pure recalc) Carotid artery (kaolin 1%) Ear artery (pure recalc)Figure 10 TEG parameters R, angle, K and MA for citrated rabbit whole from either the carotid artery (pure recalcification or triggered with kaolin 1%) or the ear artery (pure recalcification)4.2.4 Discussion and conclusion No macroscopic or histopathologic evaluation was made of the hearts from the rabbits. The correlation between ischemic injury and cTnI would be highly relevant as a marker of microthrombosis in animal models of DIC; however it can be speculated if ischemic myocardial injury due to early microthrombi formation in DIC is too subtle to be detected by this assay. Anyway, in the current pilot study the rabbit specific cTnI assay detected increasing levels of cTnI in response to the non-invasive myocardial infarction. In animal models it is possible to take blood by several more or less invasive methods. Blood sampling from the ear artery is less invasive than from the carotid artery and could be valuable for sampling in non-anaesthetized rabbits. However as a consequence of the great variations in TEG parameters of blood sampled from the ear artery it was decided to use blood samples from the carotid artery for determination of TEG parameters. Furthermore, because of the significant shortening of reaction time (R) with kaolin as a trigger it was decided to run purely recalcified TEG samples in future experiments.33 4.3 Establishment of a rabbit model of thromboplastin induced DIC4.3.1 Background Few animal models of TF induced DIC have been established and the pathophysiology of these models has not been as thoroughly evaluated as for the LPS induced animal models of DIC. It has however been shown, that intravenous injection of thromboplastin gives a more immediate and fast response with a short-lasting systemic thrombin generation compared with LPS induced DIC [157].Tissue factor has been suggested as the most important factor for the onset of DIC. It has been shown to be expressed and released in cancer including certain leukemias and solid tumour cells [5]. Furthermore trauma and obstetrical diseases can also cause a sudden release of large amounts of thromboplastin like material [6;158]. The mechanisms for induction of DIC by these underlying diseases are different from DIC associated with infectious diseases such as sepsis. Thus we find it important to establish an animal model of DIC with tissue factor as the inducer to enable further investigations of this mechanism involved in the onset of DIC.Thromboplastin, a mixed product of tissue factor and phospholipids, was chosen as the inducer in the establishment of a new acute rabbit model of DIC. The rabbit is widely used in other areas of research [159;160], and therefore well characterized with respect to hematologic and biochemical values and normal physiology [145;161]. Pilot studies were performed to determine the optimal dosing regimens in the balance between no response and lethal effect of procoagulant activation. Also the thromboplastin was purified of endotoxin and both non-washed and purified thromboplastin was characterised with regard to tissue factor activity, endotoxin level and procoagulant effect. Finally, in addition to the more classical assays of coagulation, an ELISA assay for measurement of a marker of myocardial damage (cardiac troponin I) and a global haemostatic profile test (thromboelastography) were implemented to explore the applicability in the early diagnosis of DIC.4.3.2 Development of a model of thromboplastin induced DIC in rabbits4.3.2.1 Background We hypothesized that intravenous bolus injection or infusion of thromboplastin would result in widespread activation of coagulation with a concurrent decrease in platelet count. Several pilot studies were performed to optimize the dosing regimen of thromboplastin in a rabbit model of DIC. Based primarily on platelet counts, effect of different bolus doses and infusion regimens were tested in several combinations. As platelet counts above 100x10 9/l are not scored according to the ISTH score of DIC in humans it was attempted to cause the platelet count of the rabbits to decrease below this limit.34 4.3.2.2 Materials and methods Choice of reagents and initial dosing level of thromboplastin in this pilot study was based on previous pilot studies of thromboplastin administration to rabbits performed at Novo Nordisk A/S. Vials of 4 mg thromboplastin (Thromboplastin rabbit brain, Fluka, Sigma-aldrich, Switzerland) were obtained and dissolved in 2 ml of sterile physiological saline to reach a concentration of 2 mg/ml. Platelet counts were determined in EDTA stabilized whole blood by an automatic counter, Medonic CA 620 (Boule Nordic, Kastrup, Denmark).4.3.2.2.1 Experimental design New Zealand White rabbits (Charles River, Sulzfeld, Germany) were anaesthetized with Diazepam and Pentobarbital and the left carotid artery was catheterized to measure systemic blood pressure as previously described. The many different dosing regimens tested are listed in table 2, dose and length of a single bolus injection and effect of bolus injection followed by infusion were evaluated. Measurement of platelet count was generally performed at baseline and at 5, 15, 30, 60 and 90 minutes. For a few of the rabbits receiving only a bolus injection of thromboplastin, measurements were only performed at baseline and 5 min after bolus to test whether the attempt to decrease the platelet count below 100x109/l succeeded.4.3.2.3 Results Effect on platelet count within the dosing groups varied. High bolus doses of thromboplastin injected over a short time span were immediately lethal or caused so severe dyspnea that assisted ventilation was necessary for the initial survival of the rabbit (dosing group A, C and D). The non-lethal bolus doses and bolus and infusion combinations caused an overall dose dependent decrease in platelet count; however platelet counts below 100x109/l were rarely observed (table 2 and fig 11).Table 2 Overview of thromboplastin dosing testing and effects Bolus Bolus Infusion Average Dosing dose time dose Infusion Lethal platelet count group (mg/kg) (min) (mg/kg) time (min) N effect at 5 min (x109/l) A 5 0-2 . . 1 1/1 171 B 2.5 0-2 . . 2 . 116 C 3.125 0-2 . . 2 1/2 165 D 5 0-10 . . 4 1/4 179 E . . 15 0-30 1 1/1 197 F 2.5 0-2 5 10-40 3 2/3 103 G 2.5 0-2 2.5 10-40 1 1/1 255 H 2.5 0-2 1.25 10-40 2 . 114 I 3 0-2 1.25 10-40 1 . 85 J 1.56 0-0.15 . . 5 . 21935 ) l / 300 9 B (n=2) 0 1 (C (n=1) t n D (n=3) u o 200 F (n=3) c t e H (n=2) l e t I (n=1) a l 100P J (n=5)-5 5 15 Time (min)Figure 11 Immediate effects on platelet count of non-lethal boluses. Each combined pair of data points represents measurements in one rabbitA general tendency for the platelet count to slowly recover was observed with the single bolus injections. Infusions following a bolus of thromboplastin did not result in further decrease in the platelet count, but low infusion doses appeared to prevent the platelet count recovery and thereby stabilize the platelet count at the level reached by the thromboplastin bolus (data not shown). High infusion doses following a bolus injection were found to be lethal (dosing group E and F). 4.3.2.4 Conclusion Great variations were seen within the different dosing groups and a clear decision of optimal dosing regimen for future studies was difficult to make. As there seemed to be a very narrow window between significant procoagulant effect and mortality the dosing regimen for group B and H (receiving a single 2.5 mg/kg bolus and a 2.5 mg/kg bolus followed by a 1.25 mg/kg infusion, respectively) were determined as the most optimal in the establishment of a rabbit model of DIC.5 Implementation of the ISTH classification of non-overt DIC in a thromboplastin induced rabbit model (Paper III) We hypothesized that a rabbit model of DIC could be established by intravenous injection of thromboplastin and that it was possible to apply a non-overt DIC score based on the ISTH score of non-overt DIC in humans. We further hypothesized that measurement of thromboelastography (TEG), TAT levels, Troponin I and right ventricular pressure (RVP) would add to an early diagnosis of DIC in this rabbit model. In paper III the aim was to test cardiovascular and haemostatic effect of the determined optimal thromboplastin doses supplemented with surrounding thromboplastin doses. The applicability of a non-overt DIC score based on major and specific criteria used in the ISTH DIC score was also tested36 and finally the responses in TEG, TAT, cTnI and RVP as early markers of the initial microthrombotic stage of DIC were evaluated. Twenty-four rabbits included in the study were administered saline and different doses of rabbit brain thromboplastin. At baseline and fixed time points following thromboplastin or saline administration (5, 30, 60 and 90 minutes) evaluation of cardiovascular parameters (BP, RVP, ECG and cTnI) and haemostatic parameters (complete blood counts, PT, aPTT, TAT and FDP) were performed on all rabbits. Thromboelastographic tests on whole blood were applied to half of the rabbits in each test group. For all rabbits PT, platelet count, fibrinogen level, FDP and TAT levels were scored at all time points for diagnosis of non-overt DIC, the number of thrombi in key organs were scored by histopathology and fibrin formation described. The effect on cardiovascular and haemostatic parameters was shown to be dose dependent and resulted in a lethal effect for the highest dose group. For the two middle doses of thromboplastin the rabbits could be scored as having non-overt DIC according to the diagnostic non-overt DIC score, which was found valuable in the evaluation of severity and progression of DIC during the study.It was concluded that administration of 1.25 – 2.5 mg thromboplastin/kg to rabbits induces non-overt DIC and that the severity of non-overt DIC can be scored according to the ISTH scoring system of DIC in humans. Measurement of TAT levels was found important for the early diagnosis of non-overt DIC in combination with the other parameters of the non-overt DIC score (Platelet count, PT, Fibrinogen and FDP). TEG was considered valuable in the detection of early hypercoagulability in response to thromboplastin injection, although further investigations were found necessary due to the few TEG samples in the current study resulting in large variations. The non-washed thromboplastin was used as inducer of DIC in this study. As rabbits are generally very sensitive to endotoxin we speculated that purified thromboplastin might provide the opportunity to administer larger non-lethal doses of thromboplastin, in order to reach an overt stage of DIC in the rabbit and to further determine the contribution of the endotoxin to the current findings.6 Characterisation and purification of thromboplastin6.1 Background Rabbit brain thromboplastin is per definition hard to produce without any contamination somewhere in the process. Consequently a significant and variable endotoxin level of the thromboplastin exists and when injected in a rabbit, most likely affects the coagulation response. We hypothesized that any endotoxin contamination of the thromboplastin product could be removed by purification of the thromboplastin and aimed to produce a purified pool of thromboplastin to be compared with a non-purified product and to thoroughly characterise these with regard to tissue factor activity, endotoxin level and procoagulant effect.37 6.2 Materials and methods Thromboplastin was obtained from Sigma-aldrich, Switzerland and contains 4 mg thromboplastin. A purified thromboplastin pool was produced by centrifugation of the pooled thromboplastin vials dissolved in saline followed by removal of the supernatant and finally resuspension with sterile saline, all steps performed under aseptic conditions. The tissue factor activity was measured by a chromogenic assay using a known level of FVIIa, a modification of the method previously described [162] and endotoxin levels were determined by a lysate assay as described elsewhere [1]. Procoagulant effect of non-washed and purified thromboplastin was evaluated by thromboelastography. Citrated blood from female New Zealand White rabbits (Charles River, Sulzfeld, Germany), was spiked with 0.045 mg non-washed or purified thromboplastin or buffer. The dose of thromboplastin added was calculated to be equivalent to a final concentration of 2.5 mg thromboplastin/kg injected as a bolus in a rabbit with a blood volume of 56 ml/kg [146]. TEG was run as described previously, in brief, rabbit blood spiked with either buffer, non-washed or purified thromboplastin was placed in the TEG cup containing CaCl2. Parameters reported are reaction time (R), clotting time (K) and angle. All samples were tested in duplicates.6.3 Results Tissue factor activity of the non-washed and purified thromboplastin was determined to be 2.46 nM and 2.59 nM respectively and endotoxin levels were decreased from 454 EU/ml for the non-washed thromboplastin to <20 EU/ml for the purified thromboplastin. A significant decrease in R and K and increase in angle was determined for both non-washed and purified thromboplastin compared to control samples (fig. 12). Differences between R, K and angle values for non-washed and purified thromboplastin were insignificant, however a tendency towards a higher procoagulant effect of the non-washed thromboplastin was observed.15 *** 4 *** 100 *** ) ) n i *** n *** *** ns ns i ns m 80 m ( ( 3) ) ) R 10 g K ( ( e 60 e d e ( m2 e i m l i t t g40 n n 5 g o A i n i t t 1 c t 20 a o l e C R 0 0 0Figure 12 In vitro TEG results of procoagulant effect of non-washed and purified thromboplastin6.4 Conclusion The purification procedure reduced the endotoxin level dramatically and preserved tissue factor activity. Both the non-washed and the purified thromboplastin furthermore demonstrated a significant38 procoagulant effect in spiked whole rabbit blood measured by thromboelastography. No significant differences were determined between non-washed and purified thromboplastin.Based on these experiments and the results obtained it was decided to pursue the establishment of a thromboplastin induced rabbit model of overt DIC including evaluation of cardiovascular and haemostatic parameters and scored according to the ISTH score of overt DIC in humans.7 Purified thromboplastin causes haemostatic abnormalities but not overt DIC in an experimental rabbit model (Paper IV) We hypothesized that an overt DIC model could be established in rabbit by using purified thromboplastin as inducer and that it was possible to apply an overt DIC score based on the ISTH score of overt DIC in humans. We further hypothesized that measurement of TEG, TAT levels, Troponin I and RVP would increase the diagnostic sensitivity of early overt DIC.In paper IV the objectives were to test effect on cardiovascular and haemostatic parameters of purified thromboplastin bolus administration followed by purified thromboplastin infusion. As for the established rabbit model of non-overt DIC diagnosed by a non-overt DIC score based on ISTH parameters of human DIC an overt DIC score was accordingly implemented in this study as well as measurement of TEG, TAT, cTnI and RVP responses. Eleven rabbits included in the study were administered saline or thromboplastin as a bolus of 2.5 mg thromboplastin/kg followed by a 15 minutes infusion of 1.25 mg thromboplastin/kg. Cardiovascular and haemostatic responses and thromboelastographic profiles were evaluated at baseline and fixed time points following thromboplastin or saline administration (5, 30, 60 and 90 minutes). For all rabbits PT, platelet count, fibrinogen level and FDP were scored at all time points for diagnosis of overt DIC and key organs were evaluated by histopathology and described with regard to occurrence of microthrombi and fibrin formation. Slight effects on cardiovascular and haemostatic parameters were observed for rabbits injected with thromboplastin; however this effect was less severe than for the previous study of non-overt DIC in rabbits and none of the rabbits in the current study reached a score to be diagnosed as having overt DIC according to the ISTH scoring system.It was concluded that administration of purified thromboplastin to rabbits under the current settings can not cause overt DIC. The purified thromboplastin was described as having less procoagulant effect in vivo than the non-washed thromboplastin as indicated by the in vitro comparison of the two thromboplastin products. The implementation of TEG was found valuable as TEG parameters were able to reflect the procoagulant changes early on. 39 8 Discussion and ConclusionThe overall aim and main hypothesis of this thesis that a DIC scoring system can be applied to a rabbit model of thromboplastin induced DIC was confirmed with the establishment of a rabbit model of non- overt DIC diagnosed by a scoring system based on the ISTH scoring system of DIC in humans. The specific objectives of the current thesis were: 1. To develop and standardize a non-clinical rabbit model of DIC with determination of a wide panel of markers for cardiovascular and haemostatic function including early markers of microthrombosis. The application of a pulmonary function test revealed a high sensitivity in the early diagnosis of pulmonary thromboembolism as hypothesized, however due to practical and technical obstacles, this test was not used in the rabbit model of thromboplastin induced non-overt DIC. It was demonstrated that intravenous bolus administration of thromboplastin rapidly induced non-overt DIC in the anaesthetized rabbit; however an overt DIC stage could not be reached in the current acute setting despite exploration of numerous dosing regimes. We evaluated a number of cardiovascular and haemostatic parameters to diagnose DIC at an early stage including the classic parameters from the ISTH scoring system (platelet counts, PT, fibrinogen and FDP) as well as alternative methods including Troponin I and whole blood Thromboelastography. These parameters were applied in a model of fixed size mechanical pulmonary embolization as well as in the thromboplastin induced rabbit model of non-overt DIC. 2. To standardize and score a rabbit model of DIC according to the ISTH scoring system of DIC in humans. Application of a DIC scoring system to the established thromboplastin induced rabbit model of non-overt DIC provided a standardized and clinically relevant diagnosis and monitoring of DIC as hypothesized.The reader is kindly referred to the papers in the appendix for discussions of specific results. Here the studies of the project are generally discussed and concluded upon.Microspheres were used to establish a mechanical model of pulmonary embolism. Application of MIGET, a pulmonary function test, revealed that this method rapidly detects pulmonary thrombi of a certain size able to block a functional lung unit. MIGET was found more sensitive in detecting impaired gas exchange capabilities than blood gas measurements. Though promising, implementation of this technique in a rabbit model of DIC was not further pursued due to the challenges of access to the specialized equipment and technical expertise. Furthermore groups of rabbits receiving sizes of microspheres between 45 and 222µm as well as a higher number of rabbits in each group needs to be added for final validation of the use of MIGET in early detection of pulmonary embolism in the rabbit. Though MIGET is not a handy bed-side method, it may be a valuable parameter for monitoring DIC patients in hospitals, especially in cases dominated by thromboembolic events.The microspheres were further used to validate the response of cardiovascular parameters (RVP and systemic blood pressure) to fixed size pulmonary thrombi in the rabbit. Haemostatic parameters were40 evaluated in vivo and in vitro to test whether the microspheres were truly inert. A dose dependent response on cardiovascular parameters was observed in vivo, cumulating in a lethal effect of the highest in vivo dose of microspheres. An in vitro setup evaluating thromboelastographic effect of microspheres in rabbit whole blood spiked with equivalent doses as the in vivo study showed that the highest dose resulted in a procoagulant effect. Other haemostatic parameters showed no significant procoagulant effect of any of the doses of microspheres in vivo and it was concluded that non-lethal doses of microspheres result in inert pulmonary fixed size emboli. The finding of microspheres in the myocardium and kidneys in rabbits receiving a lethal dose of microspheres intravenously were puzzling and clarification of the mechanism enabling microspheres to transit the lung are interesting in understanding the basic physiology of the response of lung vasculature to pulmonary embolism. The use of microspheres for mimicking pulmonary emboli is debatable and the in vivo cardiovascular effect in rabbits and in vitro thromboelastographic effect in rabbit whole blood of microspheres have to my knowledge not previously been investigated. The microspheres are assumed to be inert, and as histopathology confirms that the pulmonary emboli are caused purely by single microspheres we argue that this model is relevant for validation of acute cardiovascular responses to fixed size pulmonary emboli. However, though we did not observe any signs of coagulation activation in relation to the microspheres caught in the pulmonary vasculature, the decrease in platelet count, fibrinogen level and white blood cells indicate that coagulation was activated at some point, hence if this approach was to be used for imitating chronic pulmonary embolism further investigations of a possible in vivo haemostatic and inflammatory effect of high microsphere doses are necessary. One approach could be to apply scientigraphy studies to the current setting to monitor the distribution of 111In labelled platelets as previously described [163]. Furthermore, a plausible explanation to mechanisms of cardiovascular arrest in the animals injected with the highest dose of microspheres would be fatal pulmonary vascular obstruction and measurement of cardiac output might add further insight to this effect. Validation of cTnI measurements and optimization of thromboelastography as new parameters in early diagnosis of DIC was demonstrated in a rabbit model of non-invasive myocardial infarction showing an increase in cTnI within two hours of myocardial infarction. Levels of cTnI have previously been correlated by histology to the degree of myocardial ischemia induced by this non-invasive approach and cTnI levels were shown to increase within 45 minutes of vasopressin injection [151]. cTnI is regarded as the most cardiac-specific biochemical marker of myocardial injury [164]. During both myocardial infarction and thromboplastin induced DIC studies performed, no thrombi were observed in coronary or myocardial vessels and no significant increase in cTnI levels were determined in rabbits with thromboplastin induced DIC during a 90 minute time line. However in a more chronic experimental setup with long term induction of DIC, coronary thrombi might develop and result in a significant myocardial ischemia and increase in cTnI or the analysis might show insensitive for detection of microinfarcts.41 Determination of a relevant dosing regimen for thromboplastin induction of DIC was investigated in a wide-ranging pilot study. A narrow window between lethality and a significant procoagulant effect of thromboplastin injection in rabbits was found. Comparison of thromboplastin induction of DIC in animal models is difficult due to the non-standardized reagents with large variations in endotoxin levels and probably tissue factor activity and phospholipid content of the thromboplastin. Characterisation of non-washed and purified thromboplastin revealed a high endotoxin level of the non-washed thromboplastin. As many studies of tissue factor induced DIC in animal models use a source of lung or brain thromboplastin without further characterisation it is most likely that these animal models of DIC are based on a contaminated source of thromboplastin. Although these model are still relevant as models of aspects of DIC in humans, where predisposing disorders may often involve tissue factor and endotoxin exposure, thorough characterization of the inducer of DIC is important for the standardization, reproducibility and comparison of animal models of DIC.In the establishment of a rabbit model of thromboplastin induced non-overt DIC, bolus injections of 1.25 and 2.5 mg thromboplastin/kg non-washed thromboplastin in rabbits resulted in non-overt DIC diagnosed by a modified score based on the ISTH score of non-overt DIC in humans, injection of higher thromboplastin doses were lethal and induction of overt DIC was not accomplished in this study. It was hypothesized that the purification of thromboplastin would decrease variability and enable injection of higher non-lethal doses resulting in overt DIC. However, injection of a 2.5 mg/kg bolus followed by a 1.25 mg/kg infusion of thromboplastin resulted in less severe procoagulant activation than for the previous study, though a lethal effect was also observed within this study indicating an even more narrow window between procoagulant and lethal effect than for the injection of non-washed thromboplastin. These results underscore the difficulties in comparisons between animal models using different inducers of DIC and emphasize that the inducer of DIC, experimentally as well as clinically, may call for different treatment approaches. The narrow window between lethality and significant procoagulant effect observed in the pilot study testing different thromboplastin dosing regimens was also evident in these in vivo studies with injection of non-washed thromboplastin and even more so with injection of purified thromboplastin. These gathered results indicate that induction of overt DIC with the current inducer and acute setup is not possible without further pathology involved. As a DIC scoring system has not previously been applied to an animal model of DIC the present data can not be compared to previous findings. In conclusion, a rabbit model of thromboplastin induced non-overt DIC with early detection of microthromboembolism has been established and standardized according to the ISTH scoring of DIC in humans. This model can be useful in efficacy and safety testing of drugs as well as for investigation of pathophysiological events of DIC. No animal model of acute overt DIC was established and animal studies of long term induction of DIC are needed to further explore the long term aspects of thromboplastin induction of DIC.42 9 Perspectives Experiences and ideas evolved during the studies in this ph.d. are here placed in perspective and speculations on future investigations of interest based on the data obtained are given.Two overall perspectives arise from the studies performed during this project, which regards 1) the optimization and expansion of the rabbit DIC model and future implications of such and 2) the future implementation of the established rabbit model of non-overt DIC in safety or efficacy studies.Ideally animal models of DIC should reflect the different stages of human DIC; the non-overt and overt stage as defined by the ISTH scoring system. Because of the narrow window between no effect and lethal effect with thromboplastin injections a rabbit model of overt DIC appears difficult to establish, at least in the acute setting. The next step in the attempt to establish a rabbit model of overt DIC could be to include extended studies in the evaluation of long term effect of low dose thromboplastin injections in order to reach an overt stage of experimental DIC. An overt DIC stage might otherwise be established in the rabbit by two consecutive endotoxin injections, known as the Generalized Shwartzmann reaction (GSR) [165], which has been generally accepted as the counterpart of DIC in humans [90] but, to our knowledge, never evaluated according to a standardized score of DIC in humans. Due to the complexity of DIC in humans, comparison between several similarly standardized animal models of DIC would allow the thorough exploration of differences in pathogenesis and severity relating to varying predisposing diseases and underlying conditions observed with DIC in humans. In the work with experimental animal modelling of human DIC, the many different settings explored have generally been conceived as minor modifications of similar animal models of DIC. The experiences from the current studies, and the provided overview of animal models of DIC, indicate that significant differences exist in pathogenesis and severity of DIC models, depending on the inducer and species and hence, the different setups should be perceived as distinct animal models of DIC. Though some authors have published studies on experimental DIC covering the use of different species and inducers [166-168] the differences in pathophysiology between their settings have not been directly addressed, with the exception of one scientific group comparing pathophysiology of endotoxin and thromboplastin induced DIC in rats [71;157;169-172]. Further investigations into the differences in pathogenesis and severity of thromboplastin and endotoxin induced DIC in different animal species could be valuable in the understanding, standardization and comparison of animal models of DIC. Standardized diagnosis and monitoring of DIC as described in the current thesis may be transferred to other settings and species to help understand the impact of selected setup criteria on the pathophysiology of DIC in distinct animal models.A rabbit model of non-overt DIC was validated and standardized including the implementation of a non-overt DIC score based on the ISTH scoring of DIC in humans. This model and the methods applied could provide a valuable tool for safety and efficacy testing of haemostatic drugs in research and development of new therapeutic interventions in the complex pathology of DIC. 43 Safety evaluations are important in development of treatments for DIC as the dynamic properties of DIC often result in treatment schedules aimed at thrombosis and bleeding simultaneously [4], which potentially could have adverse effects if not correctly balanced. Hence, it would be interesting to implement this standardized rabbit model of DIC in non-clinical safety evaluations of new procoagulant drugs in order to increase the prediction of safe doses, toxicity values, identify potential target organs of toxicity and determine reversibility of adverse effects for patients at risk of developing DIC. A knowledge base on safety aspects of different pro-coagulant drugs in animal models of DIC with different species and settings standardized as the established rabbit model of non-overt DIC could potentially prove valuable. Optimization of efficacy of drugs in the non-clinical stage of development is primary of ethical but also economical importance as these drugs otherwise may fail in clinical trials or cause hazards to patients enrolled in these trials. Efficacy testing of both haemostatic drugs in development and reference compounds could be performed in the established rabbit model of non-overt DIC and in future modified and expanded animal models of DIC applying a similar standardized diagnostic DIC scoring. Regarding the translational aspects of drug effect evaluations in animal models of DIC, it is important to mention that testing of prophylactic drug effects are generally clinically irrelevant, rather effect of a compound on the fully developed DIC should be tested. Besides modification, with different species and inducers, including prolonged induction and observation time, this rabbit model of non-overt DIC could furthermore be adapted to include predisposing conditions of DIC and administration of supportive care. Such interventions might increase the prediction of exposure and activity of relevant drugs during various conditions in the clinic. A predisposing condition of DIC seen in humans is atherosclerosis, where rupture of atherosclerotic plaques cause release of tissue factor [173], this condition has been imitated in either a spontaneous [174] or diet-induced [175] rabbit model, it would be interesting to combine atherosclerosis and thromboplastin induction of DIC in a rabbit model to further test efficacy and safety of haemostatic drugs.44 10 ReferencesReference List1. Olrik BL, Thuri KA, Wiinberg B, et al. Implementation of the ISTH classification of non-overt DIC in a thromboplastin induced rabbit model. 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Beneficial effect of the active form of vitamin D3 against LPS-induced DIC but not against tissue-factor-induced DIC in rat models. Thromb Haemost 2001 Feb;85(2):287-90.170. Asakura H, Sano Y, Yamazaki M, et al. Role of fibrinolysis in tissue-factor-induced disseminated intravascular coagulation in rats - an effect of tranexamic acid. Haematologica 2004 Jun;89(6):757-8.171. Ontachi Y, Asakura H, Takahashi Y, et al. No interplay between the pathways mediating coagulation and inflammation in tissue factor-induced disseminated intravascular coagulation in rats. Crit Care Med 2006 Oct;34(10):2646-50.172. Suga Y, Asakura H, Yoshida T, et al. Relationship between endothelin and the pathophysiology of tissue factor-induced and lipopolysaccharide-induced disseminated intravascular coagulation in rats: a study examining the effect of an endothelin receptor antagonist. Blood Coagul Fibrinolysis 2004 Oct;15(7):593-8.173. Reininger AJ, Bernlochner I, Penz SM, et al. A 2-step mechanism of arterial thrombus formation induced by human atherosclerotic plaques. J Am Coll Cardiol 2010 Mar 16;55(11):1147-58.174. Shiomi M, Fan J. Unstable coronary plaques and cardiac events in myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits: questions and quandaries. Curr Opin Lipidol 2008 Dec;19(6):631-6.175. Yang AL, Jen CJ, Chen HI. Effects of high-cholesterol diet and parallel exercise training on the vascular function of rabbit aortas: a time course study. J Appl Physiol 2003 Sep;95(3):1194-200.54 11 PublicationsPaper I: “Animal models of DIC and their relevance to human DIC – A systematic review” (Submitted) Paper II: “Cardiovascular and haemostatic changes following microsphere injection in a rabbit model of acute pulmonary microvascular thromboembolism” (Submitted) Paper III: “Implementation of the ISTH classification of non-overt DIC in a thromboplastin induced rabbit model”. (Thrombosis Research, 2009; Vol. 124, Issue 4, Pages 490-497) Paper IV: “Purified thromboplastin causes haemostatic abnormalities but not overt DIC in an experimental rabbit model” (Thrombosis Research, 2010; in Press; DOI: 10.1016/j.thromres.2010.06.022)55

Development and Standardization of a Non-Clinical Rabbit Model

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