Nuclear Medicine Methods in Idiopathic Parkinsonism: Pre- and Postsynaptic Dopamine SPECT

Umeå University Medical Dissertations, New Series No 1550

Susanna Jakobson Mo

Department of Radiation Sciences, Diagnostic Radiology Umeå 2013

Responsible publisher under Swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN (printed version): 978-91-7459-557-4 ISBN (digital version): 978-91-7459-646-5 ISSN: 0346-6612 New series No:1550 Cover picture: S Jakobson Mo; SPECT image of normal 123I-FP-Cit uptake and cartoon of the brain with the basal ganglia Electronic version available at http://umu.diva-portal.org/ Printed by: Print & Media Umeå, Sweden 2013

To Erik, Nils and Job

Table of Contents

Table of Contents i Abstract iii Abbreviations iv Sammanfattning på svenska vi Original papers ix Introduction 1 Nuclear medicine and imaging 1 The history behind nuclear medicine today 2 Discovery of radioactivity 2 Technical advancements in imaging 3 Development of radionuclides for imaging 4 Scintigraphy and SPECT 5 Radionuclides 5 The gamma camera and the generation of an image 6 What is SPECT? 7 Reconstruction methods 8 Attenuation correction 9 Correction for scatter 10 Image evaluation 11 What is parkinsonism? 12 Rating scales of parkinsonism 14 Idiopathic parkinsonian diseases 14 Parkinson’s disease 15 Atypical parkinsonian diseases 17 Multiple system atrophy 19 Progressive supranuclear palsy 20 Dopamine and the nigrostriatal pathway 22 Dopamine 22 DA neurons and the SNc 23 Basal ganglia 24 Movement control by dopamine 25 Effects of dopamine depletion 27 D1 and D2 receptors 27 The dopamine transporter 28 Imaging of DAT and D2/D3 receptors 30 123I-FP-Cit 30 123I-IBZM 31 Aims of the study 32 Materials and methods 33 Patients and healthy volunteers in the SPECT study 33

i Study Protocol 35 SPECT procedures 36 Gamma cameras 36 Radioligands 37 Image reconstruction and evaluation 38 Reconstruction 38 Correction for radius of rotation 38 Image evaluation 40 ROI method 40 VOI method 41 Statistics 42 Ethics 43 Materials and methods in papers I-IV 43 Paper I 43 Paper II 43 Paper III 44 Paper IV 45 Results 46 Paper I 46 Paper II 46 Paper III 46 Paper IV 47 Discussion 47 General aspects 47 Main findings 48 Future prospects 54 Main conclusions 55 Acknowledgements 56 References 57 Appendix 1 Normal SPECT uptake ratios, 123I-FP-Cit 1 ROI method 1 Normal SPECT uptake ratios, 123I-IBZM 1 ROI method 1 Normal SPECT uptake ratios, 123I-FP-Cit 2 VOI method 2 Normal SPECT uptake ratios, 123I-IBZM 2 VOI method 2

ii Abstract Background: Single photon emission computed tomography (SPECT) with dopamine transporter (DAT) and dopamine D2 receptor (D2R) ligands can visualise the integrity of the nigrostriatal dopamine system. Parkinson’s disease (PD) and the atypical parkinsonian diseases (APD), progressive supranuclear palsy (PSP) and multiple system atrophy (MSA), have similar symptoms and dopamine depletion, but differ in pharmacological response and prognosis. Clinical differentiation between PD and APD is often difficult in the early stages. The aims of the thesis were to evaluate the differential diagnostic and prognostic value of SPECT in early PD, MSA and PSP, to map the pattern of progression with dopamine SPECT, and map the pattern of dopamine SPECT in non-affected elderly volunteers with a prospective approach. Also, we evaluated the methodological aspects of dopamine SPECT with respect to image evaluation tools, reconstruction parameters and gamma cameras. Methods: 172 patients, included in an on-going clinical prospective study on idiopathic parkinsonism, participated in the SPECT study. Also, 31 age-matched healthy controls (HC) were followed within this study. SPECT was done with 123I-FP-Cit (DAT SPECT) and 123I- IBZM (D2R SPECT). Regions of interest (ROI) were used as a standard method for semi-quantitative image analysis. Results: SPECT uptake ratios from different gamma cameras could be equalised through correction equations derived from images of a brain-like phantom, provided that attenuation correction was applied. The ROI method had high reproducibility. SPECT uptake in HC, measured with the ROI method and a volume based (VOI) method rendered similar trends, but gender and age differences in SPECT uptake were more marked with the VOI method, and less pronounced in DAT SPECT compared to D2R SPECT with both methods. The DAT SPECT uptake was significantly reduced in very early disease stage of PD and APD compared to HC. DAT SPECT uptake was more reduced in PD with postural and gait disturbance (PIGD) compared to tremor-dominant PD. Decline in DAT SPECT uptake during the first year was more pronounced in PD and PSP compared to HC. D2R SPECT uptake overlapped between untreated PD and APD. After initiated treatment, the D2R SPECT uptake was significantly higher in MSA patients compared to PD, PSP and HC. Decline in D2R SPECT uptake during the first year was not significantly different between patients or compared to HC. Conclusions: 123I-FP-Cit SPECT is a valuable and sensitive method to detect early stage idiopathic parkinsonism. A different level of uptake between PIGD-PD compared to TD-PD indicates a prognostic potential. It is not possible to differ between PD, MSA and PSP in early stage with 123I-FP- Cit SPECT and no differential diagnostic value was found using 123I-IBZM SPECT in the early, untreated stage of PD, MSA and PSP. A different pattern of uptake of this ligand in MSA compared to PD and PSP during the first years of L-dopa treatment may, however, indicate a diagnostic value during the follow-up period. Keywords: Nuclear medicine, SPECT, 123I-FP-Cit, 123I-IBZM, dopamine, parkinsonism, Parkinson’s disease, multiple system atrophy, progressive supranuclear palsy

iii Abbreviations

α, β, γ alpha, beta, gamma

β- , negative beta particle = electron

β+ positive beta particle= positron

APD Atypical parkinsonistic disease

CNS Central Nervous System

CT Computed tomography

DA Dopamine

DAT Dopamine transporter

D2R Dopamine receptor type D2

FBP Filtered back projection

GPe Globus pallidus external segment

GPi Globus pallidus internal segment

HC Healthy controls

H&Y Hoehn & Yahr scale of disease stage in Parkinson’s disease

123I-FP-Cit 123I-Ioflupane, DaTSCAN™

123I-IBZM 123I-Iolopride

LB Lewy Body

MRI Magnetic resonance imaging

MSA Multiple system atrophy

MSN Medium spiny neurons

iv OSEM Ordered subset expectation maximisation

PD Parkinson’s disease

PET Positron emission tomography

PIGD Postural instability and gait difficulty

PSP Progressive supranuclear palsy

ROI Region of interest

SNc Substantia nigra pars compacta

SNr Substantia nigra pars reticulata

SPECT Single photon emission computed tomography

STN Nucleus subthalamicus

TD Tremor dominant

UK PDSBB United Kingdom Parkinson’s Disease Society brain bank

UPDRS-III Unified Parkinson’s disease dating scale -Motor part

VOI Volume of interest

v Sammanfattning på svenska Bakgrund: Funktioner i kroppen kan avbildas med nuklearmedicinska metoder. Detta sker genom att man tillför kroppen ett ämne (en ligand) som på ett naturligt sätt kan ingå i en biologisk process eller fästa vid en receptor eller ett protein. Liganden är märkt med ett radioaktivt ämne (radionuklid). Genom den radioaktiva märkningen kan fördelningen av ämnets upptag i ett organ spåras. En s.k. gammakamera används för att registrera den strålning som radionukliden sänder ut. Enkelt kan man likna detta med en ”omvänd röntgenundersökning”. Metoden för avbildning kallas SPECT (single photon emission computed tomography). I digitala bilder kan aktivitetsmängden i varje bildenhet (en tvådimensionell pixel eller tredimensionell voxel) kvantifieras genom att rita in regioner i bilden. Man använder s.k. regions of interest (ROI) för att mäta upptag i pixlar, eller volumes of interest(VOI) för att mäta i voxlar. De två ligander som studerats i denna avhandling binder till ett transportprotein för dopamin respektive en dopaminreceptortyp i hjärnan. Båda dessa finns rikligt i speciella områden belägna djupt i hjärnan (nucleus caudatus och putamen, gemensam beteckning: striatum), som ingår i de basala ganglierna vilka bl.a. styr våra rörelser. I denna process spelar signalsubstansen dopamin en central roll. Liganderna är märkta med radionukliden 123I för att kunna spåras. Den ena liganden, 123I-FP-Cit, binder till dopaminets transportprotein (DAT) som suger tillbaka dopamin efter frisättning vid en nervimpuls. Detta protein finns i ändarna på de dopaminproducerande nervcellernas långa utskott i striatum. Den andra liganden, 123I-IBZM, fäster på en viss typ av dopaminreceptorer som tillhör gruppen dopaminreceptor typ 2 (D2R), vilka främst finns på mottagande nervceller i striatum. Parkinsons sjukdom (PD) och två andra ovanligare sjukdomar, multipel systematrofi (MSA) och progressiv supranukleär paralys (PSP), har likartade sjukdomstecken, parkinsonism. Parkinsonism innebär långsamma och uttröttbara rörelser, stelhet och skakningar Det utmärkande för dessa sjukdomar är att dopamin-producerande nervceller successivt går under, vilket leder till dopaminbrist som i sin tur orsakar symptomen. Den utlösande orsaken till detta är ännu okänd. Med dopamincellerna försvinner även DAT, och därmed kan man mäta graden av brist på dopaminceller med SPECT. Vid MSA och PSP försvinner dessutom nervceller i striatum som har D2R, vilket är anledningen till att vi använt 123I-IBZM SPECT i studien. I början kan PD, MSA och PSP vara svåra att skilja åt kliniskt, och det tar ofta flera år innan säker diagnos kan fastställas. Samtidigt är det viktigt att korrekt diagnos ställs så tidigt som möjligt så att rätta behandlingsinsatser kan sättas in. Exempelvis hjälper oftast inte vanliga parkinsonläkemedel vid MSA och PSP. Istället kan andra insatser behövas för att stötta patienter

vi med MSA och PSP. Dessa sjukdomar har också ofta ett snabbare sjukdomsförlopp. Andra svåra symptom utvecklas relativt tidigt och överlevnaden efter diagnos är i genomsnitt ofta kortare än vid PD. Idag finns ingen botande eller bromsande behandling för någon av dessa sjukdomar. Genom ökad kunskap om dessa sjukdomar, blir också möjligheterna större att utveckla en bromsande eller botande behandling. När sådana behandlingar i framtiden kanske utvecklas, är en tidig diagnos givetvis mycket viktig. Syfte: Studierna i den här avhandlingen syftade till att testa möjligheten att med 123I-FP-Cit och 123I-IBZM SPECT tidigt upptäcka och särskilja diagnoserna PD, MSA och PSP. I detta arbete ingick också att utvärdera mätmetoderna samt att studera det normala upptaget av liganderna hos friska personer i samma ålder för jämförelse. Metod: Mellan år 2004 och 2009 inkluderades 186 patienter med nydebuterad parkinsonism i en stor pågående långtidsstudie, som kommer att avslutas år 2017. Medelåldern vid inkludering var drygt 70 år. Enbart kliniska kriterier används i studien för att ställa diagnos. Under uppföljningstiden, som är 8 år, följs patienterna inom studien med olika undersökningar. Av de 186 patienterna har 173 patienter gjort SPECT- undersökningar. Dessa utgör studiepopulationen i denna avhandling. SPECT gjordes vid startpunkten (innan behandling påbörjats) och upprepades sedan vid uppföljningstillfällen efter 1, 3 och 5 år. Ett antal friska kontrollpersoner, i samma ålder som patienterna, har deltagit för jämförelse och dessa har undersökts vid startpunkten samt efter 3 och 5 år. När den kliniska diagnosen har fastställts under uppföljningstiden, har vi gått tillbaka till de SPECT-undersökningar som utförts för att dra slutsatser om SPECT- upptaget vid tidig PD, MSA och PSP. SPECT-bilderna har utvärderats med en ROI-metod som standard. Resultat: ROI-metoden gav stabila värden vid upprepade mätningar. En alternativ VOI-baserad metod gav liknande tendenser som ROI-metoden vid mätning av SPECT-upptaget hos friska, men gav något större utslag för ålder. VOI-metoden visade även ett något lägre upptag hos friska kvinnor än män. Olika gammakameror ger olika värden vid mätning i bilder, beroende på en rad olika faktorer. Vi testade en metod för omräkning av mätvärden för 123I- IBZM SPECT mellan två olika gammakameror. Detta skedde genom att avbilda ett kärl innehållande kända mängder radioaktivitet (ett s.k. fantom) med SPECT i två olika gammakameror. Genom att studera relationen mellan den sanna mängden radioaktivitet i fantomet och mätvärden i bilderna beräknades ekvationer. Dessa användes sedan för omräkning av mätvärdena i bilderna. Omräknade värden överensstämde betydligt bättre mellan

vii kamerorna. Därmed kan värden från undersökningar som är utförda på två olika gammakameror göras jämförbara. Patienter som nyligt fått symtom hade redan signifikant lägre upptag av 123I- FP-Cit i både caudatus och putamen jämfört med friska. PD-patienter med stelhet och gångsvårigheter hade lägre 123I-FP-Cit-upptag i putamen än de som främst hade skakningar. Upptaget av 123I-Fp-Cit vid det första undersökningstillfället efter sjukdomsdebut skilde sig inte signifikant åt mellan patienter med PD, MSA och PSP. Minskningen av upptaget av 123I- FP-Cit under det första året var dock snabbare hos patienter med PD och PSP jämfört med friska. Vid det första undersökningstillfället skiljde sig inte upptaget av 123I-IBZM åt mellan obehandlad PD, MSA, PSP och upptaget var jämförbart med det hos friska personer. Efter 1 år, när behandling hade påbörjats, fann vi dock att patienter med MSA hade signifikant högre 123I-IBZM-upptag jämfört med PSP-patienter, men både patienter med MSA och PSP visade tecken på en bristande symptomlindring av behandling. Upptaget var också högre än hos PD och friska personer efter 3 år. Patienter med PSP hade tydligt lägre upptag av båda substanserna jämfört med patienter med MSA vid uppföljning efter 1 och 3 år, men skillnaden jämfört med patienter med PD var inte signifikant. I kontrollgruppen av friska personer undersöktes hur upptaget av 123I-FP-Cit och 123I-IBZM varierade med ålder och kön. Med ROI-metoden fann vi, i ett tvärsnittsperspektiv, en åldersberoende upptagsminskning av liganderna som i snitt motsvarade ca 0.6% per år hos kvinnorna, men någon signifikant minskning med ålder kunde inte påvisas hos männen. Vi fann att den relativa årliga minskningen av 123I-IBZM-upptaget hos patienterna under första året inte signifikant skiljde sig från den genomsnittliga årliga minskningen av upptaget hos jämnåriga friska personer som undersökts vid startpunkten och sedan efter 3 år. Slutsatser: Våra studier bekräftar att 123I-FP-Cit SPECT är en känslig metod för att tidigt verifiera dopaminbrist vid PD, MSA och PSP. Däremot fann vi inga belägg för att upptaget av 123I-Fp-Cit och 123I-IBZM SPECT skiljer sig mellan dessa diagnoser i ett tidigt, obehandlat sjukdomsskede. Orsaken till att patienter med MSA hade högre 123I-IBZM upptag än patienter med PD och PSP efter 1 resp. 3 år är oklar, men kan möjligen utgöra ett särskiljande tecken mot PD och PSP. Värdet av 123I-IBZM SPECT vid tidig parkinsonistisk sjukdom kommer att studeras ytterligare under den resterande tid som projektet pågår.

viii Original papers

This thesis is based on the following papers, referred to in the text by their roman numerals

I: Mo SJ, Linder J, Forsgren L, Larsson A, Johansson L, Riklund K. Pre-and postsynaptic dopamine SPECT in the early phase of idiopathic parkinsonism: a population-based study. Eur J Nucl Med Mol Imaging 2010; 37(11):2154-64.

II: Mo SJ, Larsson A, Johansson L, Stenlund H, Forsgren L, Riklund K. Cross-camera comparison of ROI-based semi- quantitative 123I-IBZM SPECT data in healthy volunteers using an anthropomorphic phantom for calibration. Acta Radiol. 2013 Mar 5. (E-pub ahead of print) PMID: 23463862

III: Mo SJ, Larsson A, Linder J, Birgander R, Edenbrandt L, Stenlund H, Forsgren L, Riklund K. 123I-FP-Cit and 123I-IBZM SPECT uptake in a prospective normal material analysed with two different semi-quantitative image evaluation tools. Submitted manuscript

IV: Mo SJ, Linder J, Holmberg H, Larsson A, Forsgren L, Riklund K. Pre- and postsynaptic dopamine SPECT in idiopathic parkinsonian diseases: a follow-up study. Submitted manuscript

Paper I and II are reprinted with kind permission from the copyright holders

Introduction

Nuclear medicine and imaging Nuclear medicine imaging techniques visualise physiological and biochemical processes in the body. This is done by the injection or ingestion of a small quantity of radioactivity-labelled molecules that will be involved in a physiological or pathological process in the body. These molecules can be traced owing to the radioactive labelling and the emitted radiation can be visualised with gamma cameras as in single photon emission computed tomography (SPECT) and positron emission tomography (PET). High sensitivity is achieved using radioactive-labelled receptor ligands, and very specific processes on the molecular level can be imaged. It is also possible to administer goal-seeking agents for internal radiation treatment.

Fig. 1: Different imaging methods contribute information on morphology and physiology on different levels.

With current imaging methods, we can truly depict the body in a range from the macroscopic view to the molecular level, schematically illustrated in Figure 1. With X-ray and CT scans we get excellent morphological information. MRI and ultrasound are likewise outstanding morphological

imaging methods that can illustrate certain functional aspects such as movement in organs and fluid or blood flow. Cognitive processes and other brain activities can be visualised with functional MRI (fMRI) indicated by changes in the level of blood oxygenation. Metabolism in a structure, e.g. in a tumour, can be measured with MR spectroscopy (MRS). With SPECT, functional events can be shown with high sensitivity, such as inflammation (by tracking activated white blood cells), osteoblast activity in bone turnover (due to cancer, fracture healing or normal growth) and regional blood flow in the brain (as a proxy for metabolism). Different PET ligands can demonstrate physiological and pathological glucose or fatty acid metabolism in cells. Receptor ligands can be used with SPECT and PET to visualise the amount and distribution of different receptors or receptor function; the latter has the greatest spatial and temporal resolution, sensitivity and specificity. Thus, with the combination of nuclear medicine and radiological imaging techniques in the same machine (i.e. SPECT-CT, PET-CT and PET- MR cameras), both morphology and function are displayed in the same image, which in many cases increases the diagnostic potential.

The history behind nuclear medicine today

Since the discovery of radioactivity in the late 1890s and through the subsequent amazing scientific, technical and medical development of the twentieth century, nuclear medicine has evolved into a wide-ranging diagnostic and therapeutic medical speciality.

Discovery of radioactivity

Henri Becquerel (1852-1908), a French physicist, was studying the fluorescence of uranium. In 1896, by chance, he discovered that salts of uranium, like X-rays, had the ability to blacken photographic films without being previously targeted by light. X-rays had been discovered the year before by the German Wilhelm C Roentgen (1845-1923), and these new mystical rays were initially called Becquerel rays. The Polish-French physicist and chemist Marie Skłodowska-Curie (1867-1934), Fig. 2, and her husband, the French physicist Pierre Curie (1859-1906) coined the term

radioactivity for this spontaneously occurring emission and discovered the radioactive substances polonium and radium in 1898.

Fig. 2: Marie Skłodowska-Curie (Image from http://en.wikipedia.org/wiki/Marie_Curie)

Together, Becquerel and the Curie couple received the Nobel Prize in Physics in 19031 for their pioneering work on radioactivity. In 1911 Madame Marie Skłodowska-Curie received her second Nobel Prize in Chemistry2 for the discovery of polonium and radium. In 1908 a renowned physicist from New Zealand, Ernest Rutherford (1871-1937), was awarded the Nobel Prize in Chemistry3 for his work on radioactive decay and the discovery of α and β radiation. In 1913, he discovered and coined the term gamma(γ)-radiation.4

Technical advancements in imaging

In 1958, the American engineer and biophysicist Hal Oscar Anger presented a scintillation camera (also called a gamma camera, which is the term that will be used henceforth) that he had constructed the year earlier.5 This device could detect radiation from γ-emitting radioactive isotopes which could be used for medical purposes. In the early 1960s David Kuhl and Roy Edwards developed axial reconstruction techniques and established the fundamental prerequisite for the later use of CT, SPECT and PET.

In the late 1970s and early 1980s Ronald Jaszczak and co-workers in the USA and Stig Larsson in Europe6 constructed SPECT cameras, principally by arranging one or two gamma cameras on a frame that could be rotated around the patient to measure the γ-radiation from several angles.

Development of radionuclides for imaging

Carlo Perrier and Emilio Segrè have been attributed the discovery of the synthetic element technetium (Tc), named after the Greek word for artificial, in 1937.7 The metastable nuclide 99mTc (half-life 6.0 hours) emits γ-radiation with about the same wavelength as X-rays. This short-lived radionuclide cannot be stored for a long time, but is extracted from the decay of molybdenium-99 (99Mo). In 1970 “kits” with 99mTc-labelled radiopharmaceuticals for diagnostic use were introduced by Eckelman et al.,8 making it feasible to perform clinically relevant gamma camera examinations on different systems in the body. A 99Mo source is today used locally at the clinical department as a 99mTc-generator (a “Mo-cow”). Due to the feasibility of extraction and its physical advantages, 99mTc is by far the most common radionuclide used in everyday clinical nuclear medicine, used with different ligands in a vast variety of purposes, for example bone scans, lung ventilation and perfusion scans, thyroid scans and SPECT of regional blood flow in the brain.

Iodine was discovered incidentally in 1811 by a French chemist called Bernard Courtois,9 who named it from the Greek word for violet due to its violet colour in the gas form. It was characterised as an element by the English chemist Sir Humphry Davy shortly after. 123Iodine (123I, half-life 13.2 h) is one of a large number of isotopes of iodine (of which 131I has some applications in nuclear medicine therapy); however, it has to be produced in a cyclotron and is expensive and difficult to transport due to the relatively short half-life. It is used coupled to ligands that bind to dopamine transporters and dopamine receptors, or coupled to noradrenaline-like molecules for functional studies of rare neuroendocrine tumours (i.e. pheochromocytoma and neuroblastoma). Since the thyroid takes up iodine, a protective blocking agent must be administered before and after a 123I examination.

Scintigraphy and SPECT Radionuclides Both γ and X-rays are ionising (i.e. they interact with electrons in an atom or molecule). While X-rays are generated by changes in, or interactions with, the electron cloud surrounding the nucleus, γ-rays (γ-photons) originate from the nucleus. The γ-radiation used in nuclear medicine has about the same wavelength as X-rays. Isotopes of an element have the same number of protons in the nucleus, but differ in the number of neutrons; thus, the mass of an atom can vary. There are stable and unstable isotopes. Unstable isotopes are radioactive and are called radionuclides. By spontaneous decay and transformation to a more stable state, energy is discharged in the form of emitted particles (α, β- or β+) and electromagnetic radiation in the form of γ-radiation. For diagnostic purposes, radionuclides emitting minimal levels of radiation other than γ-radiation are preferable in terms of the radiation dose to the patient. The γ-photons that are emitted have specific photon energies depending on the radionuclide. For diagnostic purposes the energy of the radiation is of importance for optimal imaging quality (i.e. the energy should not be too high) and minimal absorption in the body (i.e. the energy should not be too low). 99mTc is ideal with its relatively pure γ-radiation of 140 keV. In addition, the relatively short half-life of 6.0 h is advantageous, allowing the measurement of various physiological processes without excess radiation dose to the patient. When the radionuclide part (such as 123I or 99mTc) of a radiopharmacon decays in the body, the emitted γ-radiation can be detected from within the body with a gamma camera. Fundamentally, the same phenomenon occurs in a gamma camera as in the photographic films that were used in the first experiments with radioactivity: the imprint on a film or detector shows the intensity/concentration of emitted radiation from a certain region.

Fig. 3: Schematic illustration of the gamma camera and generation of an image

The gamma camera and the generation of an image

The gamma camera (Fig. 3) has a scintillation detector (in Latin scintilla means spark or flash), which is a light-isolated thin layer of crystal composed of sodium iodide with traces of thallium, the NaI(Tl) crystal. A collimator, virtually a slab of lead with a large number of holes, is used to “guide” or select γ-photons perpendicular to the detector surface. Different collimators must be used depending on the specific energy; for higher energies, thicker lead septa are needed. If the γ-photons are not perpendicular to the crystal surface they will be absorbed by the lead. When a γ-photon interacts within the crystal, energy is emitted in the form of a flash of light, i.e. a very large amount of light photons are generated. These light photons in turn pass through a light-leading shield of glass, a light-guide, and hit the light-

sensitive photocathodes of a number of photomultiplier tubes (PMT) which are arranged in a specific pattern in the camera. The PMTs are connected to a high voltage power supply. On the photocathode, light photons will be converted to electrons (Fig. 4). Within the PMTs, the light-induced signal will be proportionally amplified and converted to a measurable electrical pulse. The PMTs in turn are connected to an electronic network. In this way, electrical pulses are eventually generated which are needed to build up an image. One pulse is proportional to the energy released by the interaction in the crystal by the γ-photon that was originally emitted from the source of decay. Two position pulses are also generated which provide information on the X and Y coordinates for the detected event in a matrix. This information can in turn be displayed as a two-dimensional image through conversion to digital signals.

In summary, each time a γ-photon within a defined energy window interacts with the crystal material is considered a “count”. Many counts together build up an image with information on the magnitude and location of a functional process in the body, which is quantitatively measurable.

Fig. 4: The principle of the PM tube. Triggered by electrons released from the photocathode an augmenting number of secondary electrons will be released from the material in the dynodes, in an avalanche-like manner.

What is SPECT?

In SPECT, one to three gamma camera detectors rotate around the patient. The emitted γ-radiation is registered in typically 60-128 angles, i.e.

projections, for about 30-60 seconds each. A number of counts will be detected and through reconstruction of the collected projection data in a computer, a three-dimensional image of the activity distribution in the body part being studied can be created.

Reconstruction methods

In SPECT there are two main types of reconstruction methods used to obtain the three-dimensional image of the collected activity data. Either (filtered) back projection, or an iterative reconstruction method can be used.10,11

In back projection reconstruction, the (two-dimensional) projection data from each angle are re-projected to a matrix in the directions/angles from which the data was registered. An image will be built up by the sum of these projections. In filtered back projection (FBP), the information that was registered in each projection in the gamma camera is first filtered. This reduces image noise and blurriness and sharpens the image.11 Even if the data is pre-filtered, artefacts will usually appear, especially in the form of streaks.

In iterative reconstruction a simple assumption is first made of the distribution of the activity. This assumption is then projected, based on knowledge of how radiation interacts with different material or tissue, and this is then compared with the measured projection in each angle.10,12,13 In the comparison, the difference between the projected assumption and the measured projections renders a ratio that is used in the next step. An updated, better assumption is calculated and a new estimated image is projected. A predetermined number of cycles, i.e. iterations, are made and should result in a small difference between the estimated and true projections. The iterative reconstruction method that is described above in a simplified way illustrates the maximum likelihood expectation maximisation (MLEM)12 which requires many iterations and takes considerable time and computer resources. A variant of this iterative method that is more commonly used is called ordered-subset expectation maximisation (OSEM), which compares only parts, subsets, of the number of measured projections14

and takes less time. As in FBP, different types of filters are used for reconstruction, but are usually applied after reconstruction.

The advantage of the iterative reconstruction method is that it is possible to incorporate different factors such as collimator properties, the effect of scattered radiation and attenuation correction in the model used for the estimation of the activity distribution.13

Attenuation correction

Radiation is absorbed and spread, attenuated, when it passes through tissue. Different tissues have different attenuation coefficients. This is the basis for X-ray imaging, in which radiation passes through the body and hits a detector. The amount of radiation that hits the detector will be different depending on tissue, and the pattern will generate a grey-scale image of the part of the body. A CT image (i.e. a three-dimensional X-ray image) is built up based on different attenuation coefficients in each three-dimensional image unit, i.e. voxel. Conversely, in gamma camera imaging, the registered radiation emanates from within the body. Due to attenuation on the way out towards the detector, the true concentration and distribution of activity will be underestimated especially in central parts of the body. In SPECT imaging of the brain, particularly the skull bone attenuates some of the emitted radiation from within the brain. Therefore, a more correct visualisation and measurement of the activity distribution can be achieved by correcting for attenuation. There are different methods to calculate the amount of γ- radiation from a radiopharmaceutical that would be attenuated on the way towards the detector.

One option for calculation is to use scaled attenuation coefficients for different tissues derived from a CT image. With a hybrid gamma camera system (i.e. a gamma camera + a CT scanner), a SPECT study and a CT scan are performed in the same session. This allows the computation of attenuation values and correction of the attenuation of the γ-photons in each voxel of the investigated area. This is called a non-uniform attenuation

correction. It renders a differentiated and individual correction of the activity distribution, provided the patient’s position remains unaltered during the entire examination. In addition, the CT image also serves as a map of the anatomical location of the tracer uptake.

Another method for attenuation correction is based on an estimate of the average attenuation in the body. This is called uniform attenuation correction. The most frequently used uniform attenuation correction technique in brain SPECT, especially when FBP is used, is the Chang method,15 named after its originator. With this method, an ellipse is estimated in the image around the head surface. The uptake (counts) in each digital image unit, i.e. pixel, within this area is, in simple terms, adjusted by a coefficient derived from a two-step correction algorithm.

Correction for scatter

When γ-photons interact with tissue in the body, the collimator or other parts of the camera, or even with other equipment in the room, they can change direction, i.e. they become scattered. When this happens, energy is lost as well (i.e. the Compton effect). However, the energy may still be within the radionuclide specific pre-set energy window of the non-scattered photons and will thus be registered (Fig. 5). Another scatter-related phenomenon may occur in SPECT with 123I, in which a small amount of γ- photons have higher photon energies than the main 123I-photon energy of 159 keV. These may penetrate the septa between the holes in the collimator without being absorbed and can, after scattering, become registered.

Since scattered photons have deviated from the source of emission, they will give false information on the position of origin. Scatter leads to degraded image quality and quantitative measurements will be impaired.16 There are different methods for scatter correction,16 which improve the image quality and semi-quantitative measurements.17 In this thesis, a subtraction method, the triple energy window method is used in OSEM reconstructed images.18 Here, the estimation of scatter is based on slight

differences of photon energies in a spectrum. The main “energy window” is focused to include the 159 keV photons in every pixel, supposedly representing non-scattered photons (primary photons). This energy window will, however, also include some lower photon energies, which may represent scattered photons, but also some scattered high-energy photons. The amount of scattered photons is estimated from the counts in two additional windows on each side of the main window, where photons with lower and higher energies are detected. By withdrawing the scattered counts from the main window in each pixel, the resulting image quality will be improved.

Fig. 5: Schematic illustration of the principles of scatter and attenuation. The γ-photons perpendicular to the detector will fall within the pre-set energy window for the specific radionuclide and will carry the correct information regarding the position of decay in the organ. Some γ-photons will be absorbed in the tissue or collimator. Some γ-photons that interact with tissue or gamma camera components will keep enough energy to become registered, however, since they are scattered, they will degrade the image and cause artefacts.

Image evaluation

Most nuclear medicine images can be analysed visually. In SPECT images, different colours represent variations in intensity, and there are many diverse colour scales with varying steps between intensity for each shade of

colour. In research, however, quantification is needed. A semi-quantitative measurement can be made by dividing the uptake in a region or volume of interest (an ROI or a VOI) applied to the region to be measured, with the uptake in a reference region in the blood pool background as illustrated in Fig. 6.

Fig. 6: ROIs applied to striatal regions and to the reference region in the occipital lobe. A SPECT image of a parkinsonian patient is displayed.

What is parkinsonism?

Parkinsonism is an umbrella term for symptoms that may occur in various diseases and conditions. However, it mainly signifies the cardinal symptoms of Parkinson’s disease (PD). It encompasses the symptoms bradykinesia, tremor and rigidity.

Bradykinesia is the main feature of parkinsonism and is a generic term for hypo-and akinesia. Bradykinetic symptoms occur due to a dopamine deficiency. The term denotes slowness or poverty of movement and reduced control of voluntary movements. Characteristically, in repetitive movements the amplitude and speed is diminished after a few rapid repetitions. Also, there is a difficulty in initiating movements such as walking and shifting directions. Moreover, the motion of the vocal cords may be affected leading to changes in the voice and contributing to difficulties in swallowing.

The concept of hypokinesia implies scarcity of movement and is manifested as a lack of facial expressions and reduced spontaneous concomitant movements, such as swinging of the arms while walking. Akinesia is an inability to initiate movement because of difficulty in selecting or activating motor programs in the central nervous system and can be seen in severe cases of Parkinson’s disease. The gait and posture may also be affected in parkinsonism. Classically, walking is affected, with a slow pace, small shuffling steps and stooping posture. Balance may also be affected.

Rigidity means stiffness. This is a clinical finding. Rigidity manifests as an increased resistance to passive bending and stretching of the elbow joint of the patient’s relaxed arm. This resistance is independent of the speed of the passive movement, and is often referred to as “lead-pipe” rigidity. Muscle tone can vary and give rise to a jerky phenomenon with passive flexion and extension of a limb, which is called the “cogwheel” phenomenon. This phenomenon can be provoked or increased if one extremity is passively moved while the patient is voluntarily moving another extremity. The combination of hypokinesia and rigidity can lead to pain in the back or an extremity, which is sometimes the first symptom of PD.

Tremor can be of various types. The most typical tremor seen in PD is often a unilateral, slow rest tremor, called the pill-rolling tremor. It can disappear or be alleviated by a voluntary movement. As with other tremors, it is increased by anxiety and affect. However, other types of tremor can be present in PD. The definite underlying cause of tremor is still largely unknown; however, the involvement of several interlinked neuronal circuits has been proposed.19–21

Rating scales of parkinsonism

The severity of Parkinson’s disease can be measured and graded by rating scales of various types. The most utilised scale for assessment of the symptoms in PD is the Unified Parkinson’s Disease rating Scale (UPDRS). This scale is comprised of four sub-sections. The third section (UPDRS-III) ranks the motor symptoms in various aspects from 0-4 points per item, such as speech, facial expression, tremor at rest and in action, rigidity, gait, posture etc. The maximum sub-total of this part of the scale is 108; zero points mean no symptoms. To describe the stage of disease, the Hoehn and Yahr (H&Y) scale is used. This is a scale that originally had five stages, but also stages 1.5 and 2.5 have been introduced. The first stage of PD represents disease with symptoms restricted to one side only, and the fifth stage represents a wheel-chair bound, severely disabled stage. There are also other scales, such as the scale for activities of daily living (ADL), where 100% means that the patient manages all ADL activities autonomously. These scales have been used for assessment of both PD and the atypical parkinsonian diseases discussed in this project.

Idiopathic parkinsonian diseases

Clinical diagnosis of parkinsonian diseases basically relies on different symptom-based criteria and the observation of the course of the disease. Often several years of observation are needed before a firm diagnosis is set. However, a definite diagnosis cannot be confirmed without post-mortem histopathological examination of the brain. Follow-up studies with histopathological post-mortem or long-term clinical follow-up have shown that up to 25% of patients with parkinsonism are misdiagnosed in life despite being investigated by a neurologist.22–24 Modern imaging techniques as well as neurophysiological tests may serve as supplementary diagnostic tools.

Parkinson’s disease

PD is the most common idiopathic parkinsonian disease. The prevalence has been estimated at around 200/100,000,25 or 22,000 persons in Sweden.26 The incidence has been reported to be as high as 21.5/100,000,27 equal to almost 2000 new cases annually in Sweden. There is a slight male predominance in PD, however, in a study conducted in northern Sweden, the differences in gender-specific incidence rates were small.27

Histopathology and aetiology The main histopathological characteristic of PD is a reduction in the number of dopaminergic cells in the substantia nigra pars compacta (SNc) and the presence of Lewy body inclusions (LBs) in neurons and Lewy neurites in axons.28 In the central nervous system (CNS), these are found in certain vulnerable cell populations in the brainstem and cortex, among which not only dopaminergic neurons in the SNc are affected29 (Fig. 7). The neuropathologist H. Braak performed post mortem studies on the amount and distribution of LBs in a large number of PD cases in different stages of the disease. Braak proposed six different stages based on the distribution and extent of LBs in the CNS and correlated these to different phases of PD. He concluded that in PD, the first appearance of LBs in the CNS is in the olfactory bulb and the motor nucleus of the vagus nerve. In this first stage, the disease is considered asymptomatic.30 Typical motor symptoms appear in stages 3-4, when LBs are found in the dopaminergic nerves of substantia nigra and in the cholinergic cells of the nucleus of Meynert, which is followed by progressive nerve loss. Based on histopathological studies it has been estimated that, at the onset of motor symptoms in PD, over 50% of the dopamine producing nerve cells in the SNc have degenerated, and that the duration of the pre-symptomatic phase is at least 5 years.31,32 It is also typical in Parkinson’s disease that the degenerative process and symptoms begin on one side, and subsequently affect the other side.

Studies have also shown LBs and phosphorylated α-synuclein aggregates in the peripheral nervous system (sympathetic and parasympathetic ganglia),

enteric nervous system33–36 and in the cardiac sympathetic nervous system37 in PD. This leads to the question of whether PD actually begins in the CNS or elsewhere in the body. These findings indicate that PD may actually be a multi-organ disease.38

Onset is predominantly late in life, around 60-70 years. Age is a dominant risk factor,39 even if age per se is not thought to cause PD.32 With age, DA neurons are lost, but the rate and pattern of loss is different in PD.32,40,41 In normal ageing, a linear pattern of approximately 5% loss per decade is seen and preferentially neurons in the dorsal regions of caudal SNc are lost. In PD, cell loss has been reported at an exponential rate of 45% during the first 10 years of disease, being greatest in the beginning32. In PD, mainly the cells in the caudal ventrolateral part of the SNc, projecting to the putamen are affected. Also neurons linked to the dorsal rostral parts of the caudate nucleus are lost,32,40,42 but to a lesser extent. Most PD cases are sporadic, but rare forms of inherited PD exist.43 Combinations of intracellular and extracellular cascades of events involving environmental factors or toxins, oxidative stress, inflammatory reactions, mitochondrial dysfunction and protein degradation abnormalities and gene mutations have been discussed as possible causes.44–48

Subtypes and treatment The disease was first described by Dr James Parkinson in 181749 under the descriptive name “shaking palsy”. The denomination “Parkinson’s disease” was first coined by Jean-Martin Charcot in 1872,50 since, according to Charcot, the word palsy was misleading as patients were not weak but slow.

In addition to the motor symptoms, PD patients have disturbances in balance and postural stability, as well non-motor symptoms such as sleep disturbances, cognitive disturbances and other more vague symptoms reflecting the wide distribution of neuronal systems involved in the disease. Different subtypes of PD have been recognised, based on symptoms and the clinical course of the disease, that correlate to histopathological features, namely an early onset variant, a tremor dominant (TD) and a postural

instability and gait disorder (PIGD) variant and a late onset variant.51 The early onset and TD subtypes of PD are suggested to be more benign, and the PIGD and late onset more aggressive.51 In the latter, the risk of developing Parkinson’s disease dementia is increased.

The treatment is symptomatic, based mainly on dopamine replacement with L-Dopa alone or in combination with other drugs that enhance dopamine levels or act directly on dopamine receptors. In some cases with predominant and disabling tremor, neurosurgical treatment by deep brain stimulation provides relief. Also, transplantation of dopaminergic neurons has been tried, but this option is still a work in progress.52

Atypical parkinsonian diseases Atypical parkinsonian diseases (APD) are less frequent than PD. These disorders have parkinsonism plus other “atypical” symptoms that are not seen as frequently or as early in PD, e.g. severe autonomic failure, as in multiple system atrophy (MSA) and oculomotor disturbances as in progressive supranuclear palsy (PSP). APD have a poorer prognosis with a shorter survival from the onset of symptoms, and treatment with L-dopa is typically not effective in alleviating the symptoms. Corticobasal atrophy and Lewy body dementia are other atypical parkinsonian diseases that will not be further discussed in this thesis. Fig. 7 summarises the distribution of the histopathological features of PD, MSA and PSP.

Fig. 7: Summary of the main neuropathological features of PD (after Halliday51 and Dickson28).

Multiple system atrophy

MSA is much less common than PD; the prevalence has been estimated at approximately 4/100,000.53 The annual incidence has been reported at about 2-3/100,000.27,54 However, since the differential diagnosis is difficult especially relative to PD55 and the median survival time from onset is relatively short (8-10 years),54,56 incidence and prevalence figures are uncertain.57 Disease onset is reported to be 55-65 years.56,58,59 Different central nervous systems are involved in MSA, but the clinical diagnostic criteria for MSA60,61 are largely focused on the motor aspect of the symptom complex. Hence, preceding non-motor symptoms may delay diagnosis and skew the age of onset.62 On the other hand, in a histopathological study, the majority of patients with a post-mortem diagnosis of MSA with onset at ages >65 were clinically misdiagnosed as having PD.63

Histopathology and aetiology MSA is, like PD, classified as a synucleinopathy with accumulation of aggregated α-synuclein in the CNS combined with neuronal degeneration. The aggregates in MSA, called Papp-Lantos inclusions, are different from PD and are found in the oligodendroglia, leading to myelin depletion and neuronal cell death. The exact underlying mechanisms are not fully understood.64 Also, inclusions in neurons and astroglia are found, but are not as numerous. Inclusions and degeneration is widespread involving the white matter as well as neurons in SNc, putaminal medium spiny neurons65 (MSNs, see page 25), pons, medulla, autonomic centres and cerebellum (Fig. 7). The disease is sporadic and the cause and exact mechanisms of the degenerative processes in MSA are still unknown. Variations in the α- synuclein gene (SNCA) located on chromosome 4 have been associated with MSA.66 There may be ethnic variation, since MSA with predominantly cerebellar symptoms (MSA-C) is more common in Japan,67 while in western countries, MSA with predominantly parkinsonistic symptoms (MSA-P) is dominant. Glial dysfunction and α-synuclein overexpression, secondary inflammation and cytoskeletal disturbances are thought to be involved in the process of degeneration in MSA.67,68

Subtypes and treatment MSA is a term coined in early 1970, when three different syndromes were found to share symptoms and histopathological features.57 There are two main subtypes of MSA: MSA-P, which is the most common variant in western countries,58 and MSA-C. However, parkinsonism and cerebellar symptoms, e.g. ataxia, dysmetria, nystagmus and dysartria, are overlapping and most patients develop parkinsonism at some stage of the disease. A common denominator in MSA, affecting a majority of patients, is the non- motor manifestation of autonomic failure, such as bladder dysfunction, severe orthostatic hypotension and sexual dysfunction. The progression of parkinsonism is generally much faster in MSA compared to PD.69 Unfortunately, parkinsonism in MSA can (initially) be alleviated with L-dopa in only about 1/3 of patients,70 although few patients have any long-term benefit dopaminergic drugs, and many patients instead develop severe side- effects.57 The other debilitating symptoms that develop may be treated symptomatically. However, the medical treatment options are limited and instead paramedical resources and support are of importance during the course of progressing disease.57

Progressive supranuclear palsy

PSP is also a relatively rare disease with an estimated prevalence of about 6.5/100,000.53,71 PSP does not occur before the age of 40. The overall incidence rate is estimated at 5/100,000, with a higher incidence in men.54 Incidence increases steeply with age from 1.7/100,000 under 60 years of age to almost 15/100,000 over 80 years of age.54 Mean disease onset is around 70 years.54 Survival after disease onset is reported to be 5-9 years.72,73

Histopathology and aetiology PSP is a taupathy, as is Alzheimer’s disease, fronto-temporal dementia and some other less common neurodegenerative diseases. By binding, the tau protein stabilises and brings together the microtubules in the cell. Microtubules are small tube-like structures which support the cell like a skeleton and are also important in axonal growth. In PSP, the tau protein is

dysfunctional due to hyperphosphorylation and cannot bind to microtubules, therefore undermining the stability of these structures, which is thought to be a mechanism of cell death.73 Hyperphosphorylation also makes the tau protein aggregate into insoluble fibrils, called neurofibrillary tangles. These tangles have a different distribution and morphology in PSP and Alzheimer’s disease. In PSP, these deposits are found in neurons and glial cells in subcortical structures, such as the SNc, basal ganglia (including nucleus subthalamicus, STN), the pons, medulla and dentate nucleus of cerebellum. The oculomotor nucleus is also affected (Fig. 7). The gene that codes for the tau protein is located on chromosome 17. Some PSP cases may be genetic; however most cases are incidental. The underlying cause of PSP is thought to be due to a combination of genetic and environmental effects.

Subtypes and treatment PSP was first described by Richardson et al. in 1963-64.74,75 The main subtypes in PSP are Richardson syndrome (RS) and the variant with predominantly parkinsonian features (PSP-P).76 A third sub type is called pure akinesia and gait freezing (PAGF).77 Richardson syndrome is present if oculomotor disturbances leading to impairment of downward gaze, (typical for PSP) and abnormal saccades, loss of postural and balance control with decreased postural reflexes (leading to frequent falls) and cognitive deficits manifest within the first 2 years of the disease.76 Parkinsonism, voice disturbances and difficulties in swallowing (pseudo bulbar palsy) are other symptoms of PSP. Tremor is more frequent in PSP-P. The effect of L-dopa treatment is generally moderate to poor, like in MSA. Approximately 50% of PSP-P patients respond to L-dopa in any disease stage, but the frequency is significantly less in RS, and the prognosis is worse in RS compared to PSP-P.76

Dopamine and the nigrostriatal pathway

Dopamine (DA) is involved in key CNS functions such as locomotion, motivation, reward-driven behaviour and cognitive processes, e.g. working memory. DA plays an important role in PD but is also involved in schizophrenia, attention deficit hyperactivity disorder (ADHD), impulse control disorders and addiction.78 Since the discovery of DA as a CNS transmitter substance by Arvid Carlsson et al. about 50 years ago,79 (for which he gained the Nobel Prize in Physiology or Medicine in 2000 together with Paul Greengard and Eric R. Kandel) and the invention of a fluorescence method for the visualisation of monoaminergic neurons (such as DA neurons) by Bengt Falck and Nils-Åke Hillarp in early 1960,80–82 it is no exaggeration to say that the knowledge in the field has grown exponentially.83 An enormous extent of literature has been produced relating to the dopamine systems in the brain, yet there are numerous questions that remain unanswered. Every attempt to summarise the complex and intricate systems that regulate human locomotion and motivation-influenced behaviour will probably fail to be complete. However, in relation to parkinsonian diseases and the topic of this thesis, an attempt to extract relevant, yet simplified, main outlines is inevitable.

Fig. 8: Dopamine

Dopamine

The transmitter substance dopamine is a catecholamine (Fig. 8) with the chemical name hydroxytyramine. It is produced in DA neurons from the amino acid tyrosine by the enzyme tyrosine hydroxylase, generating DOPA (dihydroxyphenylalanine), which is converted to DA via the enzyme L-amino acid decarboxylase.84 DA is found in the cell body in the SNc and in the nerve terminal (Fig 9). DA is packaged into terminal vesicles (Fig.9 and 13) by the

vesicular monoamine transporter in the nerve terminal, from where DA is released.

Fig. 9: Schematic illustration of a DA neuron. Synthesis of DA takes place in the cytosol and is packed in vesicles in the nerve terminal. DA is released from the vesicles to the synaptic cleft and cleared from the synaptic cleft by reuptake via the dopamine transporter (DAT). (Auto)receptors may be found on dendrites.

DA neurons and the SNc The greatest numbers of DA producing neurons are located in the substantia nigra pars compacta (SNc) in the mesencephalon. Their axons, stretching from the SNc to the striatum, form the nigrostriatal pathway (Fig. 10). Dopamine release in the striatum plays a crucial role in the circuit which connects the cortex with the motor nuclei of the basal ganglia and which governs motor activity. DA neurons are also located in the nearby ventral tegmental area, which predominantly innervates frontal areas as well as the nucleus accumbens in the striatum, which is involved in cognitive processes. Since, in the context of idiopathic parkinsonism, the nigrostriatal pathway is of major importance85–89 (however it is not the only system involved29,30) the focus will be on this part of the dopamine system.

The neurons in SNc are active in the resting state, releasing a “baseline” level of DA; however, the frequency of “firing” is modulated by external input.90,91 In the SNc, the pigmented DA neurons are organised in a pattern, where axons of DA neurons located in the rostral part stretches mainly to the head of the caudate nucleus and caudally located neurons project to the putamen

as well as to the body of the caudate nucleus.40,92,93 The main dopaminergic output is to the striatum (caudate nucleus and putamen). The STN, the medial prefrontal cortex and the pedunculopontine nucleus in the brainstem contribute with excitatory input and the striatum and pallidal nuclei contribute with inhibitory inputs.87,91

Fig. 10: Basal ganglia and the nigrostriatal DA pathway. In the SNc, the efferents to the striatum are organised, such that cells in the rostral region project to the head of the caudate nucleus and those in the caudal part project to the putamen. Medially located neurons in caudal SNc project to ventral parts of the putamen and laterally located neurons project to the dorsal parts of the putamen (after Kume et al.93)

Basal ganglia

The basal ganglia (Fig.10) are centrally located and interlinked grey matter structures in the brain. The motor nuclei that are linked together are the caudate nucleus and putamen, which together form the striatum, the external and internal segments of globus pallidus (GPe and GPi), the STN, and the substantia nigra pars reticulata (SNr).

The striatum receives excitatory input, by the neurotransmitter glutamate, from nearly all cortical regions. Especially projections from motor and association areas will be important for the motor circuit (Fig.11) The main cell type in the striatum is medium spiny neurons (MSNs), which express two types of DA receptors (D1R and D2R) and form two pathways, the direct and indirect pathways, for the regulation of movement (see below). The MSNs use the inhibitory transmitter substance GABA to signal to the GPe and GPi. GABA is also the main neurotransmitter of the other motor nuclei, except for the STN, which signals using the excitatory neurotransmitter glutamate. The GPi, together with the SNr, are considered the main output nuclei from the basal ganglia and exert constant inhibition (via GABA) on the motor nuclei of the thalamus (the ventral anterior and ventral lateral nuclei). The GPe acts as a “brake” on the STN. The STN excites the GPi/SNr, which increases the inhibitory influence on the thalamus (Fig. 11). In addition, the activity within the basal ganglia is modulated by DA transmission.

Movement control by dopamine

In the healthy state, the influence of DA on signalling in the basal ganglia is essential for moderating motor activity into smooth and intentional motion. Binding of DA facilitates and modulates movement via the two pathways mentioned above by binding to the D1 and D2 receptors (Fig. 11).94 In the direct pathway, DA binding to D1 receptors on MSNs will cause an excitation of their activity. The resulting increase in inhibitory output to the GPi and SNr will diminish their inhibitory effect on the thalamus and thus movement will be promoted. When DA acts on D2 receptors on MSNs in the indirect pathway, these are instead inhibited, leading to less inhibition of the GPe. This will in turn lead to an increase in the inhibitory influence on the STN, whose excitatory effect on the output nuclei, GPi and SNr, will be reduced. The inhibitory activity on the thalamus will then in turn diminish and result in the facilitation of motor activity.

Fig. 11: The motor circuit of the basal ganglia upon binding of DA. In the striatum, MSN activity is modulated differently by DA binding to D1 and D2 receptors to facilitate desired movements. Note that this scheme is simplified; the bilateral interconnections between the STN and GPi, as well as other outputs from the SNc to STN and pallidum are not shown. Thalamic input to the striatum is not indicated, nor is the connection between the SNr and superior colliculus and pedunculopontine nucleus in the brainstem or the output of the basal ganglia to the spinal cord.

The concept of how DA regulate movement through the direct and indirect pathways is still appreciated as a valid model for the interrelation of the basal ganglia and the regulation of intentional movement; therefore this model is used for explaining the symptoms caused by DA deficiency in PD (Fig. 12).88 However, in reality, several other different receptor-types, transmitter substances and modulators are involved in the fine tuning of movement-related actions regulated by the basal ganglia,94 for example adenosine95–97, serotonin98 and acetylcholine.99,100 The basal ganglia motor circuit is indeed very complex, and not yet fully explored.

Effects of dopamine depletion

With the degeneration of DA neurons seen in PD, the balance in the complex system of motion control is severely disturbed, which leads to a derangement in both the firing rate and pattern of firing in DA neurons and the balance between the different actions of neurotransmitters and receptor-mediated signalling.100–102 When DA is not bound to DA receptors, the resulting net effect is the inhibition of desired movements, as illustrated in Fig. 12.

Fig. 12: The motor circuit of the basal ganglia upon DA depletion. The lack of DA results in over-activation of the indirect pathway due to the loss of DA suppression of MSNs in this pathway. In combination with a loss of activation of MSNs in the direct pathway, the net effect is inhibition of voluntary movement.

D1 and D2 receptors

When DA is released into to the synaptic space, it binds to specific G protein- coupled DA receptors located on the postsynaptic neuron and the neuronal message is transferred to the next neuron (Fig.13). As mentioned above, there are two main types of DA receptors. They belong to two major groups, the D1-like (D1 and D5) and the D2-like (D2, D3, D4) groups. In the

striatum, D1R couples to a G-protein that increases levels of the intracellular second messenger cAMP, while D2R activation decreases cAMP formation. Through subsequent phosphorylation or dephosphorylation of a protein called DARPP-32 (dopamine and cAMP-regulated phosphoprotein),103 the effect will be either activation or inhibition of the MSN. D2R are found in the highest concentration in the striatum.104–107 Both D1R and D2R are mainly located postsynaptically on the “receiving” side of the synapse on the dendrites of MSNs. In addition, D2R are also found presynaptically on DA neurons, acting as auto receptors.108,109

The dopamine transporter

When DA is released, it interacts with the receptor and is subsequently cleared from the synaptic cleft by different mechanisms. This regulates the extent and duration of the DA effect on postsynaptic receptors. DA, like other monoamines, can be degraded by monoamine oxidases (MAO) located either extracellularly or in the presynaptic neuron. DA and NE can also be catabolised by cathecol-O-methyl transferase, (COMT), which in the CNS is mainly membrane-bound. The final end product through enzymatic catabolism of DA is homovanillic acid (HVA). However, the most important and rapid way of clearance from the synaptic cleft in the striatum is through re-uptake by dopamine transporters (DAT). These are membrane bound proteins, located presynaptically in the nerve terminals (Fig. 9 and 13) of the DA neurons,110 and are most abundant in the striatum.111,112 DAT is the main site of action of cocaine and amphetamine. DAT also mediates the effects of the neurotoxin MPTP, known to induce acute parkinsonism due to the degeneration of DA neurons.113 MPTP is used to produce animal models of parkinsonism. The DAT has attracted much attention in research on PD as reviewed by Storch et al.114 When DA neurons in the SNc degenerate in parkinsonian diseases, it leads to a severe loss of DAT and a lack of dopamine in the striatum. Using DAT as a marker of existing/functional DA neurons is the rationale for imaging studies of DAT in parkinsonian diseases (Fig. 14). Moreover, during normal ageing there is a loss of DA neurons32,40 and a decrease in DAT expression,115,116 however the pattern of loss is

different and the extent is less in healthy state compared to PD.32,40 In a model of PD in monkeys, the onset of motor symptoms occurred when around 40 % of the DA cells had lost their function which was related to around an 80 % loss of both DA content and DATs in the striatum.117

Fig. 13: A dopaminergic synapse in striatum, illustrating the binding sites for 123I-FP-Cit (dopamine transporter) and 123I-IBZM (D2 and D3 receptors). DA is released from vesicles into the synaptic cleft, interacts with dopamine receptors and is then cleared from the synapse by DAT. (Note: only the D2 receptor is illustrated. D1 and D3 receptors are also found in striatal synapses).

Alpha-synuclein

The synaptic protein α-synuclein is found in many brain regions in the normal state, but its exact function is not yet clearly explained. Aggregates of this protein are major components of the Lewy body inclusions (LB) which are typical histological markers of PD.118 It has been hypothesised that the soluble (normal) form of the protein α-synuclein regulates/interacts with DAT function.119–121 Increased amounts of α-synuclein have been proposed to be linked to the degeneration of DA neurons through its effect on DAT, possibly due to the resulting excess influx of free intracellular DA, leading to

subsequent cytotoxic degradation products.119–121 Especially high levels of CNS α-synuclein have been found in MSA patients.122

Imaging of DAT and D2/D3 receptors

The nigrostriatal dopamine system can be visualised with nuclear medicine procedures by using different substances suitable for SPECT or PET. Only a very small amount of the pharmacological component is needed for imaging, typically in the ranges of micro- or nanograms, hence giving no pharmacological effect.

123I-FP-Cit

The commercially available radiopharmacon 123I-FP-Cit is widely used for presynaptic SPECT in both clinical routine and research imaging in parkinsonian diseases.123

123I-FP-Cit is a cocaine analogue with high affinity for the DAT (Fig. 13). It is not entirely specific and has also some, but lower, affinity for the noradrenaline transporter (NET) and serotonin reuptake transporter (SERT). However, the ligand is in effect considered specific, since the amount of NET and SERT is low in the striatum.124 The uptake of this ligand in the striatum, where the DAT is most abundant, reflects the amount of remaining, functionally intact dopamine-producing neurons in the substantia nigra (Fig. 14 middle and right) and has potential for detecting DAT loss even in the preclinical state of parkinsonian disease.125,126 With 123I- FP-Cit, it is thus possible to distinguish between essential tremor, which is not due to dopamine deficiency,127 and idiopathic parkinsonian diseases. Also, the loss of dopamine neurons in the SNc and the diagnosis of Lewy body dementia was confirmed by post mortem histopathological examinations in patients with dementia, who had a reduced 123I-FP-Cit SPECT uptake in the striatum in vivo.128 Therefore, 123I-FP-Cit is used in the diagnosis of Lewy body dementia which, in contrast to Alzheimer’s disease, has decreased uptake.129,130

In SPECT with ligands for imaging of DAT (123I-FP-Cit and 123I-β-Cit), the uptake correlates to hypokinetic symptoms and overall disease severity, but not with tremor in PD.131,132 It has been estimated that motor symptoms occur when the uptake of 123I-FP-Cit in the putamen is reduced by approximately 46-64% compared to age matched controls.133

Studies with 123I-FP-Cit have revealed a significant age-related decline in healthy volunteers in cross-sectional studies reflecting the decline in dopaminergic function in normal ageing, reported to be between 4,1 and 9.6% per decade.134,135 Additionally, SPECT with ligands for the dopamine transporter has been used in clinical trials to assess the potential neuroprotective effect of drugs.136 There has, however, not been shown any clear-cut differences in 123I-FP-Cit uptake between PD, MSA and PSP.137

123I-IBZM

Postsynaptic dopamine D2R and D3R, expressed by MSNs in the striatum, can be visualised with 123I-IBZM SPECT (Fig. 13 and 14 left). IBZM is a benzamide dopamine antagonist with specific affinity to D2 and D3 receptors,138,139 not unlike raclopride that is used for PET.140–142 Visualisation of D2 receptors with 11C-raclopride or 123I-IBZM in other brain areas is challenging because of the relatively low concentration.105,143 Both of these radiotracers have mainly been used in psychiatric and cognitive research. Pharmacological blocking of striatal D2 receptors can cause parkinsonian symptoms. In APD, the striatal degeneration with consequent loss of postsynaptic receptor sites makes the D2 receptors of interest for differentiation between parkinsonian diseases. Thus, 123I-IBZM SPECT has been used for the differentiation between PD and APD.144–147

The images acquired with pre-and postsynaptic radioligands look similar, since both DAT and D2 receptors are concentrated to the striatum (Fig. 14). Outside the striatum, the lower activity is considered to be non-specific background brain activity.

Fig. 14: Left: 123I-IBZM image with uptake in the striatum. Middle: Normal 123I-FP-Cit image. Right: Reduced 123I-FP-Cit uptake especially in the putamen in a parkinsonian patient.

Aims of the study

The aims of this thesis were to:

. evaluate the differential diagnostic value of pre-and postsynaptic dopamine SPECT in early idiopathic parkinsonian diseases such as PD, MSA and PSP. . evaluate the prognostic value of dopamine SPECT in early symptomatic idiopathic parkinsonism. . map the pattern of progression in early symptomatic idiopathic parkinsonism with dopamine SPECT. . map the pattern of dopamine SPECT in non-affected elderly volunteers with a prospective approach. . evaluate methodological aspects of dopamine SPECT with focus on differences between image evaluation tools, reconstruction parameters and gamma cameras.

Materials and methods

To be accurate in the diagnostic work up and to assess the prognosis in early idiopathic parkinsonism, a combination of clinical diagnostic criteria and different supporting laboratory tests and imaging methods are necessary.

A large prospective long-term study of early idiopathic parkinsonism, the NYPUM (Newly Diagnosed Parkinsonism in Umeå) study, was initiated in Umeå in 200427. The aim was to investigate several relevant aspects of idiopathic parkinsonian diseases such as the diagnostic and prognostic potential of different auxiliary diagnostic tools, including neurophysiological tests, MRI and SPECT. Cognitive functions and a wide range of other important aspects of idiopathic parkinsonian disease are also studied. The study involves many different medical and paramedical professions. One hundred and eighty-six patients with a recent onset of idiopathic parkinsonism were included from 2004 to 2009. The follow-up time in the study was initially 5 years, but has been extended to 8 years. The project will be terminated in 2017. In NYPUM, the diagnoses are set by clinical examination and are strictly based on defined clinical criteria, i.e. the United Kingdom Parkinson’s Disease Society Brain Bank (UK PDSBB) criteria for Parkinson’s disease,118 and the consensus criteria for MSA60 and for PSP.148

Patients and healthy volunteers in the SPECT study

One hundred and seventy-two of the patients included in the NYPUM (101 men and 71 women) participated in the SPECT sub-study, which is the basis for this thesis. Age at inclusion was 71.0±9.3 (mean ± SD) years. Mean age at onset of symptoms was 69.1±9.2 years. The median duration of symptoms at baseline SPECT was 1.6 years. In Fig. 15, a schematic view of the projects is outlined.

Fig. 15: Schematic view of the clinical main project (NYPUM) and the SPECT study *: Identified among close to 500 subjects referred 2004-2009 because of possible parkinsonism in the population of the local catchment area of Umeå University Hospital (N=142000), Sweden

In both studies, 35 healthy volunteers (17 men and 18 women) served as healthy controls (HC), recruited by announcements in the local paper. Age and gender was matched to the first included 50 patients. The HCs were not related to the patients. The mean age at baseline was 68.5±6.7 years. Four of these HCs participated at baseline only (123I-FP-Cit SPECT), while the remaining 31 HC were followed clinically as well as with examinations according to the study protocol to ascertain that none of the HCs developed a parkinsonian disease during the course of the study. In addition, 17 healthy men, 58.8±4.8 years, included in an on-going prospective project on normal ageing, participated as controls in the SPECT study at baseline (123I-IBZM SPECT only, except one who also underwent 123I-FP-Cit SPECT). Different parts of the study group are accounted for in the papers in this thesis. Data from SPECT time points at baseline and up to 5 years of follow-up are used.

Inclusion and exclusion In Fig. 15, an outline of the number of patients, the inclusion and exclusion criteria in the study as well as the relation between the clinical main study and the SPECT sub-study is displayed. Details of the inclusion and exclusion criteria for patients and healthy controls are given below:

Inclusion criteria for patients were: . Informed consent to participate in the main study and the SPECT study . Idiopathic parkinsonism . Diagnosis from 2004-01-01 to 2009-04-30 . Resident in the Umeå hospital district at the time of diagnosis Inclusion criteria for healthy volunteers were: . Informed consent . No neurological disease or other severe disease that prevented follow-up . Resident in the Umeå hospital district at the time of inclusion Exclusion criteria for patients were: . Secondary parkinsonism . Hypersensitivity to iodide . Dementia or MMSE <24 . Disease that prevented SPECT examination (e.g. severe back pain or cardiac insufficiency) . Unwillingness to participate in the study . Pregnancy or nursing (in practice this was not applicable since female participants were in post-fertile age) Exclusion criteria for healthy volunteers: . Exclusion criteria as for patients . If the clinical examination, according to the examination protocol, revealed symptoms of parkinsonism symptoms.

Study Protocol

The patients were examined with 123I-FP-Cit and 123I-IBZM SPECT at time points coinciding with the clinical follow-up, i.e. at baseline and after 1, 3 and 5 years. The 31 healthy volunteers were examined neurologically and with SPECT at baseline, and after 3 and 5 years.

Since 123I-IBZM is not a commercially available radiopharmaceutical, 123I- IBZM SPECT in this study was initially made with individual licences, and

thereafter as an academic clinical trial (EU nr. 2009-011748-20). In the SPECT sub-study the diagnostic and prognostic value in early idiopathic parkinsonism was evaluated by comparing the baseline and follow-up SPECT images with the clinical follow-up diagnoses.

SPECT procedures

Gamma cameras

At the start of the project, a three-headed brain-dedicated gamma camera was in use for brain SPECT studies at the nuclear medicine department. This camera, the Neurocam (General Electric, Milwaukee WI, US) was equipped with three detectors (Fig. 16 left) and low energy general purpose collimators with a resolution of 8.5 mm at a distance of 10 cm from the collimator surface. The rotation radius was fixed at 12.2 cm. The pixel size was 2.0 x 2.0 mm, and image data were collected in 128 x 128 matrices. The total rotation angle was 360 degrees and image data were collected for 45 seconds from 128 angles, rendering a total acquisition time of 32 minutes.

In early 2006, the Neurocam was replaced with a multi-purpose gamma camera with two detector heads (Fig. 16 right). The Infinia (General Electric, Milwaukee, WI, US) was also equipped with low-energy general purpose collimators with a spatial resolution of 9.0 mm at 10 cm from the collimator surface. This camera was additionally equipped with a low-dose CT (“Hawkeye”), with a slice thickness of 1.0 cm (0.5 cm after an upgrade in 2008), allowing calculations for individual non-uniform attenuation correction. The equivalent radiation dose for a brain scan by this low-dose CT has been estimated to be 0.1 mSv. The scanning time for the low-dose CT was 7 minutes (3.5 min after the upgrade in 2008). SPECT image data were collected for 30 sec/angle from 120 angles, with an ideal rotation radius of 15 cm. The SPECT scanning time was 30 min and 37 min or 33.5 min including the CT scan. The 128 x 128 image matrix and a zoom factor of 1.5 gave a pixel size of 2.95 x 2.95 mm. A 20% main energy window was centred on the 123I- photon energy of 159 keV for both gamma cameras. For the Infinia, two

additional energy windows, one at each side of the main window, were included for scatter correction.

Fig. 16 Gamma cameras used in the study. Left: Three- headed brain-dedicated camera, the Neurocam. Right: Two headed hybrid camera, the Infinia Hawkeye

Radioligands

For thyroid protection, and according to clinical routine, an oral dose of 200 mg of potassium perchlorate was given before and after pre-and postsynaptic SPECT.

The presynaptic SPECT was performed with 123I-FP-Cit, 123I-Ioflupane ([123]I- N-ω-fluoropropyl-2-β-carbomethoxy-3β-(4-iodophenyl)nortropane; DaTSCAN™, GE Health B.V. Eindhoven NL). 123I-FP-Cit was administered intravenously at a dose of 185 MBq. 123I has a physical half-life of 13.2 hours. SPECT was performed 3 hours after injection. The effective dose from 185 MBq is approximately 4.4 mSv. In a dose of 185 MBq the amount of Ioflupane is equivalent to 0.175-0.325 micrograms, according to the manufacturer.

The postsynaptic SPECT was performed with 123I-IBZM, 123I-Iolopride ((S)- 2-hydroxy-3-[123I]iodo-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl)methyl]- benzamide; GE Health B.V. Eindhoven NL). This radioligand is delivered ready to use with a radiochemical purity of >95% and a specific activity of >74 GBq/µmol in a concentration of 74 MBq/ml at the time of calibration. According to the manufacturer’s specification, a dose of 185 MBq was given

intravenously and SPECT was performed 1.5 hours after injection. The amount of Iolopride in a dose of 185 MBq is equivalent to 8.5 nanograms, according to the manufacturer.

Image reconstruction and evaluation

Reconstruction

Image data acquired with the Neurocam were reconstructed with FBP without attenuation correction. For reconstruction we used a Butterworth pre-filter, with a critical frequency of 0.45 cm-1, and a power factor of 7 for noise reduction.

Image data acquired with the Infinia were reconstructed with OSEM (2 iterations, 10 subsets). We applied scatter correction18 and non-uniform attenuation correction based on the attenuation map derived from the low- dose CT images acquired within the SPECT session. For noise reduction, the reconstructed images were post-filtered with a Butterworth filter with a critical frequency of 0.45 cm-1 and a power factor of 8.

The reconstructed images were adjusted for tilt in the coronal plane. The transverse slices were constructed in the orbito-meatal plane assuring that the striata of both hemispheres were on the same level and that the striatum slices also included the occipital region. Neurocam images had a slice thickness of 2.0 mm and Infinia images had a slice thickness of 2.95 mm. In the case of Neurocam images, the six slices with the most representative uptake in the striatum were selected, and in the case of Infinia images, the four most representative slices were selected for the application of ROIs (see page 40).

Correction for radius of rotation

In gamma cameras, the spatial resolution is, among other factors, dependent on the distance of the depicted object from the detector. For the Infinia gamma camera, the distance between the detectors and the centre of the patient, i.e. the rotation radius, in dopamine SPECT was set to 15 cm for practical reasons. However, the detectors are not fixed in the Infinia camera,

and for patients with, for example, broad shoulders, a short neck, claustrophobia, or the inability to lie in a strict prone position in the camera, the distance of the camera detectors on occasions had to be increased in relation to the head of the patient. It was shown, by Monte Carlo simulation experiments, that in images acquired with a rotation radius greater than 15 cm, the resolution deteriorated and measured ratios decreased in a linear fashion. Additionally, it was shown that the VOI method was more affected by this than the ROI method. Also, images with high contrast, such as 123I- FP-Cit images, were more affected compared to those with low contrast, such as 123I-IBZM images.149 Therefore, we used correction factors derived from these experiments to correct uptake ratios derived from images with rotation radii deviating from the standard distance of 15 cm in papers III and IV.

Fig. 17: Measurement of dopamine SPECT uptake in images from the Neurocam. Left: The seven template ROIs used for measurement. A SPECT image of the anthropomorphic phantom (see Fig. 20) is shown. Right: The ROIs copied and pasted to the striatum, caudate and putamen in the in the right and left hemisphere and the occipital region. A 123I-FP-Cit SPECT image, reconstructed with FPB without attenuation correction, is shown. The image was constructed by combining six slices to a 12 mm thick slab.

Image evaluation

ROI method

In the case of Neurocam images, the six selected slices with the most representative uptake were fused into a 12 mm thick block, on which predefined rigid template ROIs were manually applied to the uptake corresponding to the caudate, putamen and striatum area in each hemisphere, as well as an ROI in the occipital lobe, serving as the reference region. The ROIs were constructed to cover the normal uptake in a 123I-FP- Cit SPECT image. The caudate ROIs covered 70 pixels, the putamen ROIs covered 80 pixels, the striatal ROI 150 pixels and the occipital ROI 230 pixels (Fig. 17). The mean count in each ROI was measured and the uptake in the caudate, putamen and striatal regions was divided by the mean count in the reference region. This provided a ratio used as a measure of the specific uptake in each region.

In Infinia images, the ROIs were made proportionally smaller compared to the “original” ROIs used for Neurocam images, due to differences in the pixel size, while keeping the proportions and geometrical shape. A semi-automatic software package in the Xeleris workstation (General Electric, Milwaukee, WI, US) was used to select the four most representative images in the stack. The template ROIs were then applied to an index image (the one with the highest uptake in the striatal regions). The positioned ROIs were then automatically copied by the software to the remaining selected slices (Fig. 18). The mean uptake in each slice and respective ROI was averaged and divided by the average uptake in the reference area and the uptake ratio for each compartment was then calculated.

Fig 18: Measurement of dopamine SPECT uptake in images from the Infinia Hawkeye. Template ROIs were applied to one of four selected images, manually adjusted in position and then automatically copied and pasted to the remaining slices 123I-FP-Cit SPECT images from a healthy individual are shown in the figure.

The ROI method is useful, as it can be applied in a clinical setting without specific commercial software. As in all manual methods, it is operator dependent. Therefore, there may be intra-and inter-individual differences in measurements. The method is time-consuming, demands precision and takes some time to learn. It is important to make sure that the reproducibility is accurate and that the measurements are reliable throughout the study. A 10-step colour scale was applied in all semi- quantitative measurements in order to sharpen the contrast between the outline of the uptake in the striatum and the background, thus facilitating the ROI positioning. In 123I-IBZM SPECT images, only the whole-striatum ROIs were applied, since the contrast in the images was considered too low to correctly apply separate ROIs for the caudate and putamen respectively.

VOI method

In paper IV, a VOI-based semi-automatic method was used as an alternative to the ROI method describe above. A novel software package (Exini DAT, Exini Diagnostics AB, Lund, Sweden) was used. This method utilises transaxial images and applies predefined three-dimensional VOIs to the image. A reference region comprising the entire cortex or the occipital cortical area may optionally be used as the reference region (Fig 19). The VOIs can be moved and rotated to fit the uptake region without a change in size. Ratios are calculated by subtracting the average uptake in the background from the average uptake in the striatal region (and the

respective sub regions) and then divided by the background. The partial volume effect is compensated for by multiplying the results by a constant factor of 2.5.

Fig. 19: The VOI method: transversal, sagittal and coronal plane. A whole-brain reference region or an occipital reference region may optionally be used.

This method has the advantage that it covers the entire striatal region and it has the potential to automatically detect and delineate the striatal and background uptake contour. However, the automatic delineation is not always perfect and in many cases there is a need for manual adjustments. The method is fast and relatively easy to use; however, when manual modifications are required, the method is affected with operator dependent variability, as is the ROI method.

Statistics

For continuous data, the independent samples t-test and one way analysis of variation (ANOVA) were used for the comparison of means. The Bonferroni post hoc test for significance was applied for comparisons of >2 groups. In small samples and in comparisons of ordinal data, non-parametric tests were used, i.e. the Mann-Whitney U test and the Wilcoxon signed-rank test. In multiple comparisons, the Kruskal-Wallis H test was used in paper I as a non-parametric analogue to one-way ANOVA. Bivariate correlation analysis was used to assess the relationship between different parameters using Pearson’s correlation coefficient or Spearman’s rho. Intra Class Correlation (ICC) analysis based on a two-way mixed model, absolute agreement, 95% CI was used for test-retest analyses. Bland & Altman plots were used for bias analysis and the limit of agreement analysis (LOA) was used in comparison of evaluation methods. Mixed model analysis was used instead of linear

regression in data sets composed of both single and longitudinal repeated measurements in the same individuals. P-values>0.05 were considered significant and reported as two-tailed. Statistical analysis was performed using the SPSS statistical package, SPSS versions 12 and 17 (SPSS Inc. Chicago Ill US) and IBM SPSS Statistics 20 (IBM, US).

Ethics

The Regional Ethical Committee of Umeå and the radiation safety committee of Norrland’s University Hospital in Umeå as well as the Swedish Medical Products Agency approved of the studies. All patients and healthy controls in the projects gave their verbal and written informed consent to participate before inclusion according to the World Medical Association Declaration of Helsinki. The principles of Good Research Clinical Practice (GRCP) were followed.

Materials and methods in papers I-IV

Paper I: Differences were evaluated in 123I-FP-Cit and 123I-IBZM SPECT uptake between early, untreated PD and APD patients and between PD patients with the TD and PIGD subtypes respectively. Possible differences in SPECT uptake between genders were also studied. Baseline 123I-FP-Cit SPECT was performed in 128 patients (72 men and 56 women) and 123I- IBZM was performed in 120 patients (68 men and 56 women) with untreated parkinsonism. Image data were related to the clinical diagnosis set after 12 months of observation and compared to corresponding SPECT data in 48 HC (34 men and 14 women), of which 16 men contributed with only 123I-IBZM SPECT.

Paper II: The differences in 123I-IBZM image data acquired with two cameras were evaluated. A method for recalculation of the measured data to outweigh dissimilarities between cameras was evaluated in HCs. A brain- dedicated gamma camera (Neurocam) and a multipurpose gamma camera equipped with a low dose CT for attenuation correction (Infinia Hawkeye) were compared. A brain-like phantom, filled with known concentrations of

radioactivity in striatal and background compartments (Fig. 22), and eleven HCs were examined with both cameras. Images from the cameras were reconstructed with protocols that were equivalent for both of the cameras (i.e. FBP with and without attenuation correction). Infinia images were also reconstructed using OSEM and attenuation correction with and without correction for scatter. By scanning the phantom, the relation between the true activity concentration and the corresponding measured striatum: background ratios in the images could be expressed by linear equations. These were used for correction of measured image data from the HC and corrected striatum:background ratios were compared between cameras. The intra-observer reproducibility of the ROI method was assessed by repeating the ROI evaluation and correction procedure twice.

Fig. 20: The anthropomorphic brain phantom used in paper II. The phantom has separate compartments, corresponding to the striatum and the background blood pool in the brain, that can be filled with different concentrations of activity. The γ-photon attenuation properties of the phantom correspond to the human head. Photo: Carina Bengtsson

Paper III: The need for age- or gender-adjusted reference value ranges in 123I-FP-Cit and 123I-IBZM SPECT in HCs was prospectively evaluated. The ROI method was compared to the voxel-based VOI method. SPECT image data was evaluated twice with both methods at separate time points for reproducibility measurements. A total of 52 healthy controls (35 from the clinical main study and 17 from a prospective study of cognitive functions in normal ageing), contributed with 123I FP-Cit and/or 123I IBZM SPECT; 24 of these were followed for 3 years and 21 were followed for 5 years. Age at baseline 123I-FP-Cit SPECT in women (n=16) was 71±4.6 years and in men

(n=14) was 63.9±8.0 years. Age at baseline 123I-IBZM in women (n=12) was 71.7±3.3 and in men (n=29) was 61.3±7.5 years (see also Appendix).

Paper IV: The potential differences and diagnostic value of 123I-FP-Cit and 123I-IBZM SPECT between HC, PD, MSA and PSP was prospectively evaluated. The 123I-FP-Cit and 123I-IBZM SPECT examinations (on the Infinia Hawkeye) at baseline and after 1 and 3 years in 28 patients with APD were compared to SPECTs in 29 age-matched clinically definitive PD patients and 16 age- matched HC. APD patients had a mean age at baseline 123I-FP-Cit SPECT of 74.3±8.8 years. The clinical diagnoses were set after a median follow-up time of 4 years in patients and the follow-up time in HCs was 5 years. The selection of patients for this paper is illustrated in Fig. 21.

Fig. 21: Selection of patients for paper IV

Results

Paper I: The baseline 123I-FP-Cit uptake in untreated (L-dopa-naïve) patients with PD and APD was significantly reduced in both the putamen and caudate nucleus on the most affected side compared to HC. DAT uptake in the putamen was lower in PD with PIGD compared to PD with TD. Uptake of 123I-IBZM was not significantly different in patients with PD compared to APD at baseline. However, half of the APD patients had severely reduced uptake (below -1 SD of normal mean) of both 123I-FP-Cit and 123I-IBZM on the most affected side, which was more uncommon in PD. No gender differences were found in patients or healthy controls.

Paper II: Significant differences were seen in non-corrected image data from the two cameras. No statistically significant differences remained between the attenuation-corrected ratios from the two cameras after correction via a brain-like phantom derived equation, making it possible to compare semi-quantitative image data between two different gamma cameras. The ROI-method had high reproducibility in 123I-IBZM SPECT.

Paper III: Image evaluation with the ROI and VOI method rendered different numerical values but similar trends in HC. However, gender differences in SPECT uptake were more pronounced with the VOI than with the ROI method. Fewer gender differences and reduced age-dependency were seen in 123I-FP-Cit SPECT compared to 123I-IBZM SPECT. With a cross- sectional approach, the age related decline rates in uptake of the ligands were within the range of previously reported rates; however, we found a significant age dependent decline only in women. In HCs followed longitudinally for 5 years, the mean relative decline in 123I-IBZM SPECT uptake was somewhat more pronounced than the cross-sectional estimation, and was without significant gender differences, while the 123I-FP-Cit uptake varied individually and was, on the group level, relatively unchanged over 5 years.

Paper IV: 123I-FP-Cit SPECT uptake was significantly reduced in PD and APD compared to HC. We found no statistical differences in 123I-IBZM SPECT uptake between HC and the L-dopa-naïve patients at baseline. In contrast, at 1 year follow-up, when pharmacological treatment was initiated, 123I-IBZM SPECT uptake was significantly higher in MSA patients compared to PSP, as well as compared to PD and HC after 3 years. The relative decline in 123I-FP-Cit SPECT uptake during the first year was significantly greater in PD and PSP, but not in MSA compared to HC. The relative decline of 123I- IBZM uptake during the first year after diagnosis was not significantly different in patients compared to HC.

Discussion

General aspects

The aim of this thesis was to evaluate the diagnostic and prognostic potentials of pre-and postsynaptic SPECT in patients with newly diagnosed, early idiopathic parkinsonian disease. A radioligand binding to dopamine transporters, 123I-β-Cit, was described in the early 1990s.150 When the prospective study started in 2004, a similar presynaptic ligand, 123I-FP-Cit, with high sensitivity (i.e. the ability to detect disease) and specificity (the ability to exclude disease) for the differentiation between essential tremor and manifest degenerative parkinsonian disease was commercially available151 and was used in this project.

123I-IBZM was used, since this ligand has been reported to have a diagnostic value in predicting responsiveness to L-dopa treatment and in differentiating manifest APD from PD.144,152 However, studies have also reported overlapping findings between PD and APD.137 The prospective study design offered an option to systematically collect clinical data during the first years of disease and an option to study the uptake of these ligands in the very early stage of symptomatic disease and at subsequent follow-up.

We chose to use a manual ROI-based evaluation method for semi- quantitative analysis, since this approach is feasible to apply in the clinical

context and can be accomplished with standard procedures in the workstations used in our department. ROI methods have been reported to be reliable and reproducible153 as well as having a good correlation with visual interpretation and with the clinical stage of the PD patients,154 thus having a good validity.

Main findings

Diagnostic aspects 123I-FP-Cit SPECT: The findings in papers I and IV of significantly reduced 123I-FP-Cit uptake in the putamen of the patients were expected. However, also the caudate uptake was significantly reduced already at an early stage of disease in a majority of the patients, which was not entirely expected. The focus in the literature has mainly been on the putaminal loss of uptake in PD, which indeed is the part of striatum most affected. Dopaminergic degeneration in idiopathic parkinsonian diseases also involves the projections to the caudate; however, in PD, the degeneration starts in the parts of the SNc that project to the posterior parts of the putamen. The putamen is the nucleus involved in motor tasks of the limbs, and the most obvious symptoms of PD are motion related,155 but the caudate nucleus is involved in motion as well, especially eye movements but may also be involved in the control of propulsion.156,157 The caudate also plays a role in cognition, due to the interconnections between cortical association areas (dorsolateral prefrontal cortex)158 and the temporal lobe.159 This will be discussed further in the context of prognostic factors. It is interesting to note that both eye movements and cognitive functions are impaired in PSP, and in a recent study, using 18F-FP-Cit (a PET ligand), DAT uptake in both caudate and putamen was more reduced in PSP patients compared to MSA and PD.160 Additionally, in a PET study using 18F- dopa (measuring presynaptic dopaminergic integrity) the uptake was shown to be equally reduced in the caudate as in the putamen in PSP patients, which was not the case in PD.161 The 18F-dopa uptake in MSA patients was shown to be intermediate compared to PD and PSP. In paper IV, we found that the 123I-FP-Cit SPECT uptake in the caudate of PSP patients was

significantly lower compared to patients with MSA after one year of follow- up. However, when PSP was compared to PD patients, who were mainly of the PIGD sub-type, there was no significant difference.

In practise, the main diagnostic challenge is to discriminate PD from the different types of APD. The MSA patients in paper IV had significantly higher uptake of 123I-FP-Cit and 123I-IBZM compared to PSP patients and higher 123I-FP-Cit uptake in the caudate compared to PD patients after one year, although the age at SPECT and duration of symptoms were not significantly different. Such findings may provide diagnostic clue in the differentiation of MSA versus PSP and PD.

123I-IBZM SPECT:

In early L-dopa-naïve state of the disease, we found that the 123I-IBZM SPECT uptake in patients with PD and APD is overlapping, and does not add diagnostic information for differentiation between these three diagnostic entities. We also found, surprisingly, that the 123I-IBZM SPECT uptake was significantly higher in MSA patients compared to PSP after 1 year, after initiated treatment with L-dopa, as well as compared to PD and HC after 3 years. This was accompanied by a reduction in presynaptic uptake (indicating progressive dopamine depletion) and clinical progression of the disease. Earlier studies have shown an up-regulation of D2R on the most affected side in early untreated PD.162–164 This up-regulation is claimed to be normalised upon dopaminergic treatment and/or progressive disease165,166 and low D2R uptake is thought to be predictive for patients with APD and for PD with a fluctuating L-dopa response.167,168 With 123I-IBZM SPECT in papers I and IV, we did not find signs of an up-regulation of the D2R in PD patients, but in paper IV, it is possible that the high 123I-IBZM uptake in MSA patients represents a persistent up-regulation of dopamine receptors which is not normalised despite exogenous dopaminergic compensation by L-dopa in the early phases of disease. This may imply other neural mechanisms underlying a poor L-dopa response in the MSA patients in this study.169 It is not entirely clear what these mechanisms might be.

Prognostic aspects The PIGD subtype was slightly more frequent compared to the tremor dominant subtype among PD patients in the NYPUM project at inclusion. We found that, despite similar duration and age of onset, 123I-FP-Cit uptake was lower in PD patients with the PIGD/akinetic rigid subtype compared to PD with tremor (TD), which is in accordance with previous findings in post mortem studies.170 The non-tremor dominant PD subtype has been suggested to have worse prognosis171 compared to tremor-dominated PD. The PIGD subtype has also been shown to be associated with a higher risk of developing dementia,172,173 and post mortem histopathological studies have shown more extensive neurodegenerative changes including LBs as well as amyloid plaques in the brains of PD patients with PIGD and old onset.51,174 Since dementia is a very common complication in later stages of PD,173 a finding of early cognitive impairment may be of prognostic importance.175–177 In the last decade, non-motor symptoms and mild cognitive impairment (MCI) in early PD have attracted attention. It has been shown that MCI may be a feature of very early PD,178,179 associated with measurable effects in the caudate nuclei and prefrontal cortex.180,181 In a recent fMRI-study on PD patients included in this project, such findings were also found to be correlated to a reduction in 123I-FP-Cit uptake in the corresponding caudate in PD patients with MCI.182

Methodology One cannot directly compare semi-quantitative values from different gamma cameras.183 There are various sources for divergences such as the number of detectors, type of collimator, variations in detector sensitivity and other system-related factors as well as the influence of different filters, reconstruction parameters and the radius of rotation. A brain-like phantom, used for calibration of cameras, provided the possibility to investigate the two cameras used in this study with respect to their performance in reproducing the true activity concentration in the phantom. This procedure has also been used in a large multi-centre study with pooling of 123I-FP-Cit images of normal uptake.184 We instead used this method in 123I-IBZM

SPECT. The correction equations derived from the phantom scans were also implemented in 123I-IBZM SPECT image data from HC that were scanned on both cameras, as described in paper II. The resulting corrected ratios corresponded well between cameras on a group level if attenuation correction was applied. We believe this is important information for future studies, which may allow pooling of data from patients and controls.

We needed to assess the reliability of the ROIs used in our study. This is especially important in long-term studies, where measurements must be made in the same way throughout. Likewise, reproducibility was important in paper II in the comparison between different cameras and reconstruction procedures. In papers II and III, the ROI method was shown to be reproducible and stable, both in analysing 123I-FP-Cit and 123I-IBZM SPECT images. It has to be kept in mind that the 123I-IBZM SPECT images have much less contrast compared to 123I-FP-Cit SPECT images, yet that did not affect the reproducibility.

Additionally, an alternative, VOI-based, method was tested in evaluation of SPECT in HCs and was compared with the ROI method. The VOI method takes into account the entire striatal uptake and not just the highest uptake in the “core” of the volume, as with the ROI method. The two methods rendered similar tendencies in a large prospective normal material, but the VOI method seemed to be more sensitive to age- and gender-related differences. This may indicate that, although the ROI method is sensitive enough for discriminating parkinsonian patients from HC, it may perhaps not detect subtle differences, since it actually does not measure the uptake in the entire volume of the striatum. In 123I-IBZM SPECT, we only used a whole-striatum ROI, which may decrease the potential of discriminating between PD, MSA and PSP, since there may be differences in uptake in the caudate and putamen between these diagnostic entities. With the VOI method, however, it is possible to measure caudate and putamen uptake separately in both 123I-FP-Cit and 123I-IBZM. This method also has the potential of being automated. However, we found that manual adjustments

were needed, which introduced some variability, and this variability was more pronounced in images with high striatal uptake. In the next step the performance of the VOI method will be tested in patient images.

The gold standard for the diagnosis of PD, MSA and PSP is based on neuropathological post mortem findings. However, such studies take long time and large-scale studies are very difficult to perform for practical and ethical reasons. In this project, a diagnosis based on purely clinical criteria (non-biased from previous laboratory or SPECT results) was instead used as the gold standard, against which the diagnostic performance of SPECTs, done early in the course of disease, was tested. As stated before, it may be challenging to establish a correct diagnosis in very early parkinsonism, especially in differing between PD and APD. A number of patients with idiopathic parkinsonism may be misdiagnosed during life; most commonly an MSA diagnosis is missed. With the use of established clinical criteria and follow-up, the diagnoses diagnostic accuracy can be improved to around 90%.22,24,185 Especially, in evaluating the true diagnostic potential of 123I- IBZM to differentiate between early PD and APD, accurate clinical diagnoses are of great importance.186 In the NYPUM project the clinical diagnoses were continuously re-evaluated. At each follow-up time point, the diagnosis was set before and irrespective of the result of the SPECT. Accordingly, the evaluation of the SPECT images was made without knowledge of the clinical diagnosis of the subject.

The diagnoses of some patients in the NYPUM project changed over time, which was anticipated. Of the patients participating in the SPECT study, 14 patients were diagnosed with MSA at baseline, but only 8 retained this diagnosis during follow-up and 6 patients were converted to other diagnoses (predominantly PD and occasionally PSP). During the course of the study, 5 patients, originally diagnosed as PD, were re-diagnosed as MSA, resulting in a total of 13 patients with a diagnosis of MSA after a median follow-up time of 4 years. In the case of PSP, 8 of 9 patients that were diagnosed with PSP at baseline retained this diagnosis during follow-up. Meanwhile, 8 patients

changed diagnosis into PSP during follow-up, resulting in a total of 16 patients with a diagnosis of PSP after a median follow-up time of 4 years. Moreover, many of the patients fulfilling the criteria for an APD-diagnosis also fulfilled the criteria for PD and occasionally also another APD diagnosis. This clearly implies that the later in the follow-up course of the disease the assessment of the diagnostic value of the SPECT methods is made, the better.

Parkinsonian symptoms may occur in many other conditions and diseases other than PD, PSP and MSA. It should be underlined, that the performance of 123I-FP-Cit SPECT in an unselected population of patients with parkinsonism, also including other atypical parkinsonian diseases or secondary parkinsonism such as vascular parkinsonism and others, was not studied within this project.

Occasionally, patients that according to clinical criteria, are diagnosed with PD do not have a pathological 123I-FP-Cit, neither at onset nor at follow-up, and do not exhibit significant clinical deterioration with or without therapy,187–189 thus implying another underlying cause than a dopamine- related degeneration. These are known as SWEDDs (scans without evidence of dopaminergic deficit). Since the inclusion in the NYPUM project was made based on clinical criteria only, infrequent SWEDDs were encountered in the project. These were readily discovered with 123I-FP-Cit SPECT, owing to the prospective approach and the reference values from the HC. With SPECTs and the prospective clinical evaluation of the patients, the assessment of the exact number of “true SWEDDs” will be possible when the project is finalised.

Limitations The small numbers of APD patients in the project is clearly a limitation. The incidence and prevalence of MSA and PSP is lower than that of PD and the generally worse prognosis leads to a shorter follow-up time for these patients. Additionally, in a prospective study there is a risk of a biased drop- off, i.e. only the least affected patients complete the study, which may distort

the results. In paper IV, the drop-off was analysed to reveal such tendencies, but clinical parameters as measured with the UPDRS-III, ADL and H&Y scales did not point at any such bias.

The change of equipment for SPECT was a non-desired obstacle in this prospective long-term study dependent on quantification, and normal value ranges had to be collected also for the new system. Since the image measurements from the two systems were not directly comparable, pooling of data from these cameras is not possible, thus reducing the group sizes. Images from the first camera were reconstructed with FBP without attenuation correction, which was the routine at that time. Image data from the second camera was instead reconstructed with OSEM, which improved image quality190 and correction for attenuation and scatter was applied. In paper II we found that attenuation corrected image data from the Neurocam corresponded best with image data reconstructed with OSEM and attenuation correction but without scatter correction in 123I-IBZM images. This implies that the old and new examinations still have to be reconstructed again with the parameters most favourable for comparison. Additionally, a phantom analysis has to be made for 123I-FP-Cit SPECT images before data can be pooled together.

Future prospects

The NYPUM project and the SPECT sub-study are not yet finalised, and will still generate data for some years to come.

The value of SPECT with 123I-FP-Cit and 123I-IBZM in PD and APD with respect to various aspects remains to be further studied.

The ROI method was shown to be stable, but perhaps somewhat blind to subtle differences in SPECT uptake. Image evaluation with automatic voxel based methods will be tested and possibly used in future studies in the project.

Main conclusions

The results in this thesis confirm that 123I-FP-Cit SPECT clearly is a valuable and sensitive method for detecting a dopamine deficit as the underlying cause in the early stage of suspected idiopathic parkinsonian disease. Already in the early stage of symptomatic PD, MSA and PSP, the uptake in both the putamen and caudate is significantly decreased, but differentiation between early PD, MSA and PSP is not possible with 123I-FP-Cit SPECT. We could not confirm any additional benefit of 123I-IBZM SPECT in the differentiation between the early, untreated stage of PD, MSA and PSP. On the other hand, a different pattern of 123I-IBZM SPECT uptake in MSA compared to PD and PSP during the first years of L-dopa treatment may indicate that this ligand can add diagnostic information during the follow-up period. Since the main study is not yet terminated, the final outcome remains to be assessed. 123I-FP-Cit SPECT seems to have prognostic potential, with different levels of uptake between the clinical subtypes of PD. There is a correlation between motor function and the 123I-FP-Cit SPECT uptake in untreated PD. During the first year of follow-up, the relative decline in 123I-FP-Cit SPECT uptake was greater in PD and PSP, but not in MSA, compared to age-matched HC, but the relative decline in 123I-IBZM SPECT uptake was not significantly different between patients or age-matched HC. To improve similarity between measured uptake ratios from different gamma cameras, a brain-like phantom should be scanned to determine the linear equations to be used for the calculation of corrected ratios. The ROI method used in this thesis was robust and reproducible. An alternative VOI method rendered more pronounced age and gender dependent differences in SPECT uptake than the ROI method in HCs. Thus, different evaluation tools may affect the pattern of gender and age differences in SPECT uptake. We advocate the use of age-adjusted reference values for 123I-FP-Cit and 123I-IBZM SPECT in women if using the VOI method. If using the ROI method, age adjusted reference values are not necessary for 123I-FP-Cit SPECT but may be used for 123I-IBZM SPECT.

Acknowledgements

First of all, I would like to express my profound gratitude to: My supervisor, Professor Katrine Åhlström Riklund -thank you for sharing your proficiency and enthusiasm, for all encouragement, commitment and guidance throughout these years! My co-supervisor, Dr Anne Larsson Strömvall -without your expertise in nuclear medicine physics and our close collaboration, this work would not have been possible, thank you! My co-supervisor and the primus motor of the NYPUM project, Professor Lars Forsgren -thank you for your most valuable guidance in the field of movement disorders and the rewarding collaboration! I also want to recognise the contribution and support from a number of people who, in one way or another, have been involved during the course of this work. Especially, I want to thank the participating patients and the persons volunteering as healthy controls in this project and the nurses at the department of nuclear medicine, who made all the SPECT examinations. I would also like to thank my collaborators and co-authors: Jan Linder, who examined the patients and healthy volunteers together with Lars Forsgren, Hans Stenlund and Henrik Holmberg for invaluable statistical guidance, Lennart Johansson for the invaluable contributions involving nuclear medicine physics and ROIs, Richard Birgander for evaluation of the MRI examinations and Lars Edenbrandt for the collaboration with Exini DAT. Very special thanks to Jörgen Andersson, who administered the database; Mona Edström, who administered the NYPUM project; Jan Axelsson for helping me with filing of all the image data; the engineers and technicians at the department of nuclear medicine for help with stubborn computers; Anders Hellenäs and Åsa Krantz at GE for providing the brain-phantom; Prof. Ingrid Strömberg and Dr Nina Nevalainen at the dept. of Histology and Cell Biology, for sharing your great expertise on the dopamine system and Parkinson’s disease, and for the inspiration that you and all the colleagues in your research group have given me; the researchers at the UFBI for inspiration and instructive and enjoyable lab-meetings; Börje Morian, Marie Strand and many of my colleagues at the dept. of Radiology and Nuclear medicine for encouragement and support during all this time. Last, but by all means not least, I want to thank my close friends, in particular Anna and LG, for cheerful company along the way and invaluable pep-talk; my mother Ing-Marie and my father Åke, for always being there for me with your love, encouragement, and attention; my dear Nils and Job for all the joy you bring to life and my husband Erik, for your patience and support, care and love.

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Appendix

Normal uptake ratios in 123I-FP-Cit SPECT and 123I-IBZM SPECT (From the SPECTs in paper III; mean of 1st and 2nd evaluations)

SPECT in healthy controls (baseline, 3 and 5 years) 123I-FP-Cit 123I-IBZM

Total Number of SPECTs 75 85

Age at SPECT (total) Mean 70.9 69.0

Median 72.5 71.5

Min 48.7 48.5

Max 83.5 83.6

Normal SPECT uptake ratios, 123I-FP-Cit ROI method Age at SPECT Number of Uptake ratio † SPECTs Mean 95% CI Caudate ≥48.7≤83.5 years 75 4.46 4.33 - 4.58 All Putamen ≥48.7≤83.5 years 75 4.03 3.92 - 4.14 Striate ≥48.7≤83.5 years 75 4.23 4.11 - 4.34 †Mean of first and second evaluation; left and right hemisphere uptake averaged; occipital reference region. Infinia Hwk, OSEM, CT based attenuation correction; scatter correction; corrected for radius of rotation (15 cm).

Normal SPECT uptake ratios, 123I-IBZM ROI method Age at SPECT Number of Uptake ratio †

SPECTs Mean 95% CI <72 years 45 1.81 1.76 - 1.85 All Striate >72 years 40 1.74 1.70 - 1.79* ≥48.5 ≤ 83.6 years 85 1.78 1.75 - 1.81 †Mean of first and second evaluation; left and right hemisphere uptake averaged; occipital reference region. Infinia Hwk, OSEM, CT based attenuation correction; scatter correction; corrected for radius of rotation (15 cm). * Student’s t-test: age<72 years compared to >72 years: p=0.049

Normal SPECT uptake ratios, 123I-FP-Cit VOI method Age at SPECT Number of Uptake ratio † SPECTs Mean 95% CI <72 years 13 6.54 6.09 - 6.99 Caudate >72 years 24 6.10 5.72 - 6.47* <72 years 13 5.27 4.71 - 5.82 Women Putamen >72 years 24 4.81 4.50 - 5.12* <72 years 13 5.84 5.34 - 6.34 Striate >72 years 24 5.39 5.06 - 5.73* Caudate ≥48.7 ≤ 83.5 years 38 6.6 6.27 - 6.94 Men Putamen ≥48.7 ≤ 83.5 years 38 5.35 5.09 - 5.6 Striate ≥48.7 ≤ 83.5 years 38 5.91 5.63 - 6.2 †Mean of first and second evaluation; left and right hemisphere uptake averaged; occipital reference region. Infinia Hwk. OSEM. CT based attenuation correction; scatter correction; corrected for radius of rotation (15 cm). *Student’s t-test: If age >72 years, then uptake rations in men >women: caudate p=0.033, putamen p=0.015, striate p=0.020

Normal SPECT uptake ratios, 123I-IBZM VOI method Age at SPECT Number of Uptake ratio † SPECTs Mean 95% CI <72 years 10 1.38 1.20 - 1.55 Caudate >72 years 23 1.18 1.08 - 1.28* <72 years 10 1.13 1.01 - 1.25 Women Putamen >72 years 23 0.89 0.80 - 0.97** <72 years 10 1.24 1.10 - 1.38 Striate >72 years 23 1.02 0.93 - 1.11** Caudate ≥48.5 ≤ 83.6 years 52 1.49 1.41 - 1.57 Men Putamen ≥48.5 ≤ 83.6 years 52 1.23 1.16 - 1.3 Striate ≥48.5 ≤ 83.6 years 52 1.35 1.28 - 1.42 †Mean of first and second evaluation; left and right hemisphere uptake averaged; occipital reference region. Infinia Hwk, OSEM, CT based attenuation correction; scatter correction; corrected for radius of rotation (15 cm). Student’s t-test: *caudate p=0.028, **putamen p=0.002, **striate p=0.006