First international workshop for small animal arthroscopy : matkakertomus

Jarmo Rintasalo, Pentti Tapio

Small animal endoscopy

Kirjallisuusarvostelu

Portfolio kliinisen eläinlääketieteen erikoistumiskoulutuksen apuvälineenä

Summary: Professional development portfolio as a tool for veterinarians specializing in small animal diseases, equine diseases and production medicine

Tietojohtamista tukevan toiminnanohjausjärjestelmän vaatimuksia erityistoimialalla: tapaustutkimus eläinterveydenhuollon toimialalla

Veterinary Health Services are following in many areas the practices and medical direction of human medicine and health services. They are reaching for improved efficiency, quality and precision. Competitive position may be improved and productivity increased by specializing and focusing efforts at the practice. This thesis focuses in small animal practices and their needs for ERP (Enterprise Resource Planning) systems in Germany. As a result requirements for ERP solution supporting knowledge management in the small animal practice is presented. Veterinary Health Services is knowledge-intensive business, where written information and tacit knowledge is increasingly bound together with deepening expertise and specialization. Veterinarian is even legally obliged to develop and maintain her professional skills. The current ERP solutions concentrate in the treatment process of veterinary practice. Customer relationship management is left aside. As the competitive situation is getting tighter in veterinary services also the customer relationship management needs to get into the focus and interest to the wider network support in knowledge sharing should take steps forward. Taking into account the requirement of continuous development of professional skills ERP system at the veterinary practice should also be seen as knowledge management tool. It should provide the possibility to create, store, share and use knowledge. The study is conducted first by studying the AS-IS situation of ERP use and market in veterinary health services and then drawing the requirements of TO-BE situation by studying literature and the results of semi qualitative study conducted for German veterinary practices. A group of veterinarians were interviewed, market and network analysis was done and the understanding of market was deepened in two veterinary conferences in Germany. This theses work is requested by Finnish software company Finnish Net Solutions, which is the leading supplier of Veterinary Practice Management software in Finland. The company plans to expand to European market with Cloud based service. Target of the theses is to create understanding of the requirements of German veterinary market to develop ERP solution supporting Knowledge Management in Veterinary Practice.

Real-time Imaging and Mosaicking of Planar Surfaces

Laajojen pintojen kuvaaminen rajoitetussa työskentelytilassa riittävällä kuvatarkkuudella voi olla vaikeaa. Kuvaaminen on suoritettava osissa ja osat koottava saumattomaksi kokonaisnäkymäksi eli mosaiikkikuvaksi. Kuvauslaitetta käsin siirtelevän käyttäjän on saatava välitöntä palautetta, jotta mosaiikkiin ei jäisi aukkoja ja työ olisi nopeaa. Työn tarkoituksena oli rakentaa pieni, kannettava ja tarkka kuvauslaite paperi- ja painoteollisuuden tarpeisiin sekä kehittää palautteen antamiseen menetelmä, joka koostaaja esittää karkeaa mosaiikkikuvaa tosiajassa. Työssä rakennettiin kaksi kuvauslaitetta: ensimmäinen kuluttajille ja toinen teollisuuteen tarkoitetuista osista. Kuvamateriaali käsiteltiin tavallisella pöytätietokoneella. Videokuvien välinen liike laskettiin yksinkertaisella seurantamenetelmällä ja mosaiikkikuvaa koottiin kameroiden kuvanopeudella. Laskennallista valaistuksenkorjausta tutkittiin ja kehitetty menetelmä otettiin käyttöön. Ensimmäisessä kuvauslaitteessa on ongelmia valaistuksen ja linssivääristymien kanssa tuottaen huonolaatuisia mosaiikkikuvia. Toisessa kuvauslaitteessa nämä ongelmat on korjattu. Seurantamenetelmä toimii hyvin ottaen huomioon sen yksinkertaisuuden ja siihen ehdotetaan monia parannuksia. Työn tulokset osoittavat, että tosiaikainen mosaiikkikuvan koostaminen megapikselin kuvamateriaalista on mahdollista kuluttajille tarkoitetulla tietokonelaitteistolla.

Imaging techniques applied for detection of secretion, transgene delivery and G proteincoupled receptors in reproductive and endocrine cells in vivo and in vitro

Post-testicular sperm maturation occurs in the epididymis. The ion concentration and proteins secreted into the epididymal lumen, together with testicular factors, are believed to be responsible for the maturation of spermatozoa. Disruption of the maturation of spermatozoa in the epididymis provides a promising strategy for generating a male contraceptive. However, little is known about the proteins involved. For drug development, it is also essential to have tools to study the function of these proteins in vitro. One approach for screening novel targets is to study the secretory products of the epididymis or the G protein-coupled receptors (GPCRs) that are involved in the maturation process of the spermatozoa. The modified Ca2+ imaging technique to monitor release from PC12 pheochromocytoma cells can also be applied to monitor secretory products involved in the maturational processes of spermatozoa. PC12 pheochromocytoma cells were chosen for evaluation of this technique as they release catecholamines from their cell body, thus behaving like endocrine secretory cells. The results of the study demonstrate that depolarisation of nerve growth factor -differentiated PC12 cells releases factors which activate nearby randomly distributed HEL erythroleukemia cells. Thus, during the release process, the ligands reach concentrations high enough to activate receptors even in cells some distance from the release site. This suggests that communication between randomly dispersed cells is possible even if the actual quantities of transmitter released are extremely small. The development of a novel method to analyse GPCR-dependent Ca2+ signalling in living slices of mouse caput epididymis is an additional tool for screening for drug targets. By this technique it was possible to analyse functional GPCRs in the epithelial cells of the ductus epididymis. The results revealed that, both P2X- and P2Y-type purinergic receptors are responsible for the rapid and transient Ca2+ signal detected in the epithelial cells of caput epididymides. Immunohistochemical and reverse transcriptase-polymerase chain reaction (RTPCR) analyses showed the expression of at least P2X1, P2X2, P2X4 and P2X7, and P2Y1 and P2Y2 receptors in the epididymis. Searching for epididymis-specific promoters for transgene delivery into the epididymis is of key importance for the development of specific models for drug development. We used EGFP as the reporter gene to identify proper promoters to deliver transgenes into the epithelial cells of the mouse epididymis in vivo. Our results revealed that the 5.0 kb murine Glutathione peroxidase 5 (GPX5) promoter can be used to target transgene expression into the epididymis while the 3.8 kb Cysteine-rich secretory protein-1 (CRISP-1) promoter can be used to target transgene expression into the testis. Although the visualisation of EGFP in living cells in culture usually poses few problems, the detection of EGFP in tissue sections can be more difficult because soluble EGFP molecules can be lost if the cell membrane is damaged by freezing, sectioning, or permeabilisation. Furthermore, the fluorescence of EGFP is dependent on its conformation. Therefore, fixation protocols that immobilise EGFP may also destroy its usefulness as a fluorescent reporter. We therefore developed a novel tissue preparation and preservation techniques for EGFP. In addition, fluorescence spectrophotometry with epididymal epithelial cells in suspension revealed the expression of functional purinergic, adrenergic, cholinergic and bradykinin receptors in these cell lines (mE-Cap27 and mE-Cap28). In conclusion, we developed new tools for studying the role of the epididymis in sperm maturation. We developed a new technique to analyse GPCR dependent Ca2+ signalling in living slices of mouse caput epididymis. In addition, we improved the method of detecting reporter gene expression. Furthermore, we characterised two epididymis-specific gene promoters, analysed the expression of GPCRs in epididymal epithelial cells and developed a novel technique for measurement of secretion from cells.

Performance and Methodological Aspects in Positron Emission Tomography

Performance standards for Positron emission tomography (PET) were developed to be able to compare systems from different generations and manufacturers. This resulted in the NEMA methodology in North America and the IEC in Europe. In practices, the NEMA NU 2- 2001 is the method of choice today. These standardized methods allow assessment of the physical performance of new commercial dedicated PET/CT tomographs. The point spread in image formation is one of the factors that blur the image. The phenomenon is often called the partial volume effect. Several methods for correcting for partial volume are under research but no real agreement exists on how to solve it. The influence of the effect varies in different clinical settings and it is likely that new methods are needed to solve this problem. Most of the clinical PET work is done in the field of oncology. The whole body PET combined with a CT is the standard investigation today in oncology. Despite the progress in PET imaging technique visualization, especially quantification of small lesions is a challenge. In addition to partial volume, the movement of the object is a significant source of error. The main causes of movement are respiratory and cardiac motions. Most of the new commercial scanners are in addition to cardiac gating, also capable of respiratory gating and this technique has been used in patients with cancer of the thoracic region and patients being studied for the planning of radiation therapy. For routine cardiac applications such as assessment of viability and perfusion only cardiac gating has been used. However, the new targets such as plaque or molecular imaging of new therapies require better control of the cardiac motion also caused by respiratory motion. To overcome these problems in cardiac work, a dual gating approach has been proposed. In this study we investigated the physical performance of a new whole body PET/CT scanner with NEMA standard, compared methods for partial volume correction in PET studies of the brain and developed and tested a new robust method for dual cardiac-respiratory gated PET with phantom, animal and human data. Results from performance measurements showed the feasibility of the new scanner design in 2D and 3D whole body studies. Partial volume was corrected, but there is no best method among those tested as the correction also depends on the radiotracer and its distribution. New methods need to be developed for proper correction. The dual gating algorithm generated is shown to handle dual-gated data, preserving quantification and clearly eliminating the majority of contraction and respiration movement

Imaging of Dopamine and Serotonin Transporters. Pre-clinical Studies with Radiotracers for Positron Emission Tomography

The action of the neurotransmitters dopamine (DA) and serotonin (5-HT) at synapses is terminated by their rapid reuptake into presynaptic nerve endings via plasma membrane dopamine (DAT) and serotonin (SERT) transporters. Alterations in the function of these transporters have been suggested as a feature of several neurological and neuropsychiatric diseases, such as Parkinson’s disease (PD), depression, and anxiety. A suitable clinical method for studying these transporters non-invasively in vivo is positron emission tomography (PET) utilizing radiopharmaceuticals (tracers) labelled with short-lived positron-emitting radionuclides. The aim of this study was to evaluate in rats two novel radiotracers, [¹⁸F]beta -CFT-FP and ¹⁸FFMe-McN, for imaging DAT and SERT, respectively, using in vitro, ex vivo and in vivo methods. Substituting an N-methyl in [¹⁸F]beta-CFT, a well known DAT tracer, with a ¹⁸Ffluoropropyl group significantly changed the properties of the tracer. [¹⁸F]beta- CFT showed slow kinetics and metabolism, and a high specific uptake in the striatum, whereas [¹⁸F]beta-CFT-FP showed fast kinetics and metabolism, and a moderate specific uptake in the striatum. [¹⁸F]betaCFT-FP was selective for DAT; but [¹⁸F]beta-CFT also bound to the noradrenaline transporter. [¹⁸F]beta-CFT-FP may be a suitable PET tracer for imaging the striatal DAT sites, but a tracer with a higher affinity is needed for imaging extrastriatal DAT sites. In rats, ¹⁸FFMe-McN showed high target-to-non-target ratios, specificity and selectivity for SERT, but slow kinetics. However, ¹⁸FFMe-McN reveals potential for imaging SERT, at least in pre-clinical studies. In addition, the sensitivities of [¹⁸F]beta CFT and [¹⁸ F]FDOPA (a precursor of DA) for detecting mild nigrostriatal hypofunction were compared in an animal model of PD. The uptake of [¹⁸F]FDOPA was significantly affected by compensatory effects in dopaminergic cells, whereas [¹⁸F]beta-CFT was more sensitive and therefore more suitable for PET studies of mild dopaminergic symptoms. In conclusion, both novel tracers, [¹⁸F]-CFT-FP and ¹⁸FFMe-McN, have potential, but are not optimal PET tracers for DAT and SERT imaging in rats, respectively. [¹⁸F]beta-CFT is superior to [18F]FDOPA for imaging mild nigral lesions in rat brains.

Animal Lovers, Bridge-Builders, and Supervising Watchdogs: Framing and Civil Society Construction by Japanese Pro-Animal Organizations in the Kansai and Tokyo Areas

Unlike their counterparts in Europe and America, the citizen organizations acting for the well-being of animals in Japan have not received scholarly attention. In this research, I explore the activities of twelve Japanese pro-animal organizations in Tokyo and Kansai area from the perspective of social movement and civil society studies. The concept of a ‘pro-animal organization’ is used to refer generally to the collectives promoting animal well-being. By using the collective action frame analysis and the three core framing tasks – diagnostic, prognostic, and motivational – as the primarily analytical tools, I explore the grievances, tactics, motivational means, constructions of agency and identity as well as framing of civil society articulated in the newsletters and the interviews of the twelve organizations I interviewed in Japan in 2010. As the frame construction is always done in relation to the social and political context, I study how the organizations construct their roles as civil society actors in relation to other actors, such as the state, and the idea of citizen activism. The deficiencies in the animal welfare law and lack of knowledge among the public are identified as the main grievances. The primary tactic to overcome these problems was to educate and inform the citizens and authorities, because most organizations lack the channels to influence politically. The audiences were mostly portrayed as either ignorant bystanders or potential adherents. In order to motivate people to join their cause and to enforce the motivation within the organization, the organizations emphasized their uniqueness, proved their efficiency, claimed credit and celebrated even small improvements. The organizations tended to create three different roles for citizen pro-organizations in civil society: reactive, apolitical and emphatic animal lovers concentrating on saving individual animals, proactive, educative bridge-builders seeking to establish equal collaborative relations with authorities, and corrective, supervising watchdogs demanding change in delinquencies offending animal rights. Based on the results of this research, I suggest that by studying how and why the different relations between civil society and the governing actors of the state are constructed, a more versatile approach to citizens’ activism in its context can be achieved.

In vivo imaging of vascular adhesion protein 1 - preclinical studies with positron emission tomography

The golden standard in nuclear medicine imaging of inflammation is the use of radiolabeled leukocytes. Although their diagnostic accuracy is good, the preparation of the leukocytes is both laborious and potentially hazardous for laboratory personnel. Molecules involved in leukocyte migration could serve as targets for the development of inflammation imaging agents. An excellent target would be a molecule that is absent or expressed at low level in normal tissues, but is induced or up-regulated at the site of inflammation. Vascular adhesion protein-1 (VAP-1) is a very promising target for in vivo imaging, since it is translocated to the endothelial cell surface when inflammation occurs. VAP-1 functions as an endothelial adhesion molecule that participates in leukocyte recruitment to inflamed tissues. Besides being an adhesion molecule, VAP-1 also has enzymatic activity. In this thesis, the targeting of VAP-1 was studied by using Gallium-68 (68Ga) labeled peptides and an Iodine-124 (124I) labeled antibody. The peptides were designed based on molecular modelling and phage display library searches. The new imaging agents were preclinically tested in vitro, as well as in vivo in animal models. The most promising imaging agent appeared to be a peptide belonging to the VAP-1 leukocyte ligand, Siglec-9 peptide. The 68Ga-labeled Siglec-9 peptide was able to detect VAP-1 positive vasculature in rodent models of sterile skin inflammation and melanoma by positron emission tomography. In addition to peptides, the 124I-labeled antibody showed VAP-1 specific binding both in vitro and in vivo. However, the estimated human radiation dose was rather high, and thus further preclinical studies in disease models are needed to clarify the value of this imaging agent. Detection of VAP-1 on endothelium was demonstrated in these studies and this imaging approach could be used in the diagnosis of inflammatory conditions as well as melanoma. These studies provide a proof-of-concept for PET imaging of VAP-1 and further studies are warranted.

Fluorescence-based imaging of cellular defect in lysinuric protein intolerance (LPI)

Fluoresenssiperusteiset kuvantamismenetelmät lysinurisen proteiini-intoleranssin (LPI) soluhäiriön tutkimuksessa Lysinurinen proteiini-intoleranssi on suomalaiseen tautiperintöön kuuluva autosomaalisesti peit¬tyvästi periytyvä sairaus, jonka aiheuttaa kationisten aminohappojen kuljetushäiriö munuaisten ja ohutsuolen epiteelisolujen basolateraalikalvolla. Aminohappojen kuljetushäiriö johtaa moniin oirei¬siin, kuten kasvuhäiriöön, osteoporoosiin, immuunijärjestelmän häiriöihin, oksenteluun ja runsaspro¬teiinisen ravinnon nauttimisen jälkeiseen hyperammonemiaan. LPI-geeni SLC7A7 (solute carrier family 7 member 7) koodaa y+LAT1 proteiinia, joka on basolateraali¬nen kationisten ja neutraalien aminohappojen kuljettimen kevyt ketju, joka muodostaa heterodimee¬rin raskaan alayksikön 4F2hc:n kanssa. Tällä hetkellä SLC7A7-geenistä tunnetaan yli 50 LPI:n aiheut¬tavaa mutaatiota. Tässä tutkimuksessa erityyppisiä y+LAT1:n LPI-mutaatiota sekä yhdeksän C-terminaalista polypep¬tidiä lyhentävää deleetiota kuvannettiin nisäkässoluissa y+LAT1:n GFP (green fluorescent protein) -fuusioproteiineina. Tulokset vahvistivat muissa soluissa tehdyt havainnot siitä, että 4F2hc on edel¬lytyksenä y+LAT1:n solukalvokuljetukselle, G54V-pistemutantti sijaitsee solukalvolla samoin kuin vil¬lityyppinen proteiini, mutta lukukehystä muuttavia ja proteiinia lyhentäviä mutantteja ei kuljeteta solukalvoon. Lisäksi havaittiin, että poikkeuksena tästä säännöstä ovat y+LAT1-deleetioproteiinit, joista puuttui korkeintaan 50 C-terminaalista aminohappoa. Nämä lyhentyneet kuljettimet sijaitsevat solukalvolla kuten villityyppiset ja LPI-pistemutanttiproteiinit. Dimerisaation osuutta kuljetushäiriön synnyssä tutkittiin käyttämällä fluorescence resonance energy transfer (FRET) menetelmää. Heterodimeerin alayksiköistä kloonattiin ECFP (cyan) ja EYFP (yellow) fuusioproteiinit, joita ilmennettiin nisäkässoluissa, ja FRET mitattiin virtaussytometri-FRET -menetel¬mällä (FACS-FRET). Tutkimuksissa kaikkien mutanttien havaittiin dimerisoituvan yhtä tehokkaasti. Kul¬jetushäiriön syynä ei siten ole alayksiköiden dimerisaation estyminen mutaation seurauksena. Tutkimuksessa havaittiin, että kaikki mutantti-y+LAT1-transfektiot tuottavat vähemmän transfektoi¬tuneita soluja kuin villityyppisen y+LAT1:n transfektiot. Solupopulaatioissa, joihin oli tranfektoitu lu¬kukehystä muuttava tai stop-kodonin tuottava mutaatio havaittiin suurempi kuolleisuus kuin saman näytteen transfektoitumattomissa soluissa, kun taas villityyppistä tai G54V-pistemutanttia tuottavas¬sa solupopulaatiossa oli pienempi kuolleisuus kuin saman näytteen fuusioproteiinia ilmentämättö¬missä soluissa. Tulos osoittaa mutanttiproteiinien erilaiset vaikutukset niitä ilmentäviin soluihin, joko suoraan y+LAT1:n tai 4F2hc:n kautta aiheutuneina. LPIFin SLC7A7 lähetti-RNA:n määrä ei merkittävästi poikennut villityyppisen määrästä fibroblasteissa ja lymfoblasteissa. SLC7A7:n promoottorianalyysissä oli osoitettavissa säätelyalueita geenin 5’ ei-koo¬daavalla alueella sekä ensimmäisten kahden intronin alueella. LPI-taudin tautimekanismin kannalta keskeisin tekijä on kuitenkin aminohappokuljetuksen häiriö, jonka vaikutuksesta näistä aminohapoista riippuvaiset prosessit elimistössä eivät toimi normaalisti. Havaittu virheellinen y+LAT1/4F2hc kuljetuskompleksin sijainti edellyttää lisätutkimuksia sen mahdol¬lisen kliinisen merkityksen selvittämiseksi.

Diffusion Tensor Imaging as a Diagnostic and Research Tool: A Study on Preterm Infants

Diffusion tensor imaging (DTI) is an advanced magnetic resonance imaging (MRI) technique. DTI is based on free thermal motion (diffusion) of water molecules. The properties of diffusion can be represented using parameters such as fractional anisotropy, mean diffusivity, axial diffusivity, and radial diffusivity, which are calculated from DTI data. These parameters can be used to study the microstructure in fibrous structure such as brain white matter. The aim of this study was to investigate the reproducibility of region-of-interest (ROI) analysis and determine associations between white matter integrity and antenatal and early postnatal growth at term age using DTI. Antenatal growth was studied using both the ROI and tract-based spatial statistics (TBSS) method and postnatal growth using only the TBSS method. The infants included to this study were born below 32 gestational weeks or birth weight less than 1,501 g and imaged with a 1.5 T MRI system at term age. Total number of 132 infants met the inclusion criteria between June 2004 and December 2006. Due to exclusion criteria, a total of 76 preterm infants (ROI) and 36 preterm infants (TBSS) were accepted to this study. The ROI analysis was quite reproducible at term age. Reproducibility varied between white matter structures and diffusion parameters. Normal antenatal growth was positively associated with white matter maturation at term age. The ROI analysis showed associations only in the corpus callosum. Whereas, TBSS revealed associations in several brain white matter areas. Infants with normal antenatal growth showed more mature white matter compared to small for gestational age infants. The gestational age at birth had no significant association with white matter maturation at term age. It was observed that good early postnatal growth associated negatively with white matter maturation at term age. Growth-restricted infants seemed to have delayed brain maturation that was not fully compensated at term, despite catchup growth.

Translational models of coronary artery disease, myocardial infarction and heart failure. Development, validation and in vivo imaging studies using positron emission tomography

Coronary artery disease is an atherosclerotic disease, which leads to narrowing of coronary arteries, deteriorated myocardial blood flow and myocardial ischaemia. In acute myocardial infarction, a prolonged period of myocardial ischaemia leads to myocardial necrosis. Necrotic myocardium is replaced with scar tissue. Myocardial infarction results in various changes in cardiac structure and function over time that results in “adverse remodelling”. This remodelling may result in a progressive worsening of cardiac function and development of chronic heart failure. In this thesis, we developed and validated three different large animal models of coronary artery disease, myocardial ischaemia and infarction for translational studies. In the first study the coronary artery disease model had both induced diabetes and hypercholesterolemia. In the second study myocardial ischaemia and infarction were caused by a surgical method and in the third study by catheterisation. For model characterisation, we used non-invasive positron emission tomography (PET) methods for measurement of myocardial perfusion, oxidative metabolism and glucose utilisation. Additionally, cardiac function was measured by echocardiography and computed tomography. To study the metabolic changes that occur during atherosclerosis, a hypercholesterolemic and diabetic model was used with [18F] fluorodeoxyglucose ([18F]FDG) PET-imaging technology. Coronary occlusion models were used to evaluate metabolic and structural changes in the heart and the cardioprotective effects of levosimendan during post-infarction cardiac remodelling. Large animal models were used in testing of novel radiopharmaceuticals for myocardial perfusion imaging. In the coronary artery disease model, we observed atherosclerotic lesions that were associated with focally increased [18F]FDG uptake. In heart failure models, chronic myocardial infarction led to the worsening of systolic function, cardiac remodelling and decreased efficiency of cardiac pumping function. Levosimendan therapy reduced post-infarction myocardial infarct size and improved cardiac function. The novel 68Ga-labeled radiopharmaceuticals tested in this study were not successful for the determination of myocardial blood flow. In conclusion, diabetes and hypercholesterolemia lead to the development of early phase atherosclerotic lesions. Coronary artery occlusion produced considerable myocardial ischaemia and later infarction following myocardial remodelling. The experimental models evaluated in these studies will enable further studies concerning disease mechanisms, new radiopharmaceuticals and interventions in coronary artery disease and heart failure.