The study involved 25 patients with idiopathic Parkinson's disease (IPD; age: 51–75 years, mean ± SD: 64 ± 7 years, 12 women, 13 men). Four patients were at Hoehn and Yahr (H&Y) stage 1, 13 at H&Y stage 2, and eight at H&Y stage 3. No patient was at H&Y stage 4 or 5. Concerning the predominant motor type, 13 patients belonged to the akinetic-rigid type, five patients to the tremor-dominant type, and seven patients to the equivalence type. The motor deficits of the patients reached 9–51 points (23 ± 9 points, mean ± SD) in the motor part of the UPDRS score during an ‘off’ phase (12 h off drugs; cabergoline, pergolide, and prolonged formulations of ropinirole and pramipexole were discontinued at 72 h prior to the SPECT). The L-dopa equivalent daily dose (LEDD) was 100–1280 mg/day (563 ± 339 mg/day, mean ± SD). The duration of Parkinson's disease amounted to 3–17 years (7.7 ± 3.7 years, mean ± SD).
All patients had to fulfill three inclusion criteria prior to enter this study: (i) have a diagnosis of IPD according to the criteria of the UK Parkinson's Disease Society Brain Bank , (ii) have a normal cranial magnetic resonance imaging, and (iii) be aged 50–75 years old. We used an upper age limit of 75 years because the frequency of vascular or neurodegenerative lesions (unrelated to IPD), which may interfere with cerebral nicotinic binding, increases exponentially with age. The size of the study group was based upon a power analysis, with the assumption of a correlation coefficient r = 0.5, an error of 1st degree = 0.05, and an error of 2nd degree = 0.20. No healthy controls were included in the study.
Patients were excluded from the study if they were taking cholinergic or anticholinergic drugs. Further exclusion criteria were pregnancy or breastfeeding, a partner who was capable of childbearing, smoking in the last five years, actual or previous cerebral disease (except IPD), psychiatric disease, or severe internal disease. Apart from cholinergic or anticholinergic drugs, other antiparkinsonian medication was to be continued (‘on’ state) during the CERAD testing, as most previous studies had not shown a significant effect of dopaminergic treatment on cognitive functions in IPD [16-18].
The study protocol was approved by the Ethics Committee of the University of Würzburg, Germany, and by the German Federal Office for Radiation Protection (Bundesamt für Strahlenschutz, Salzgitter, Germany). All participants gave written informed consent prior to enter this study.
5-I-A-85380 SPECT imaging
Preparation and radiolabeling of 5-I-A-85380 was performed in house in the radiopharmaceutical facilities of the Department of Nuclear Medicine of the University of Wuerzburg under GMP (good manufacturing practice) conditions. The radiosynthesis provided 5-I-A-85380 in form of a carrier-free radiotracer with a highest possible specific activity. The specific activity – as determined from the ultraviolet absorbance at 254 nm – exceeded 125 GBq/μmol (the detection limit of our system).
Patients fasted at least 4 h before 5-I-A-85380 administration. Possible iodine uptake by the thyroid was blocked by oral medication with sodium perchlorate (Irenat®; Bayer, Leverkusen, Germany). Patients received 185 MBq of freshly prepared 5-I-A-85380 intravenously. The approximate administrated mass of an injectable solution with 185 MBq of 5-I-A-85380 was <0.001 nmol.
Cerebral SPECT imaging was acquired with a dual-head rotating gamma camera (E. Cam Duet, Siemens Medical Solutions; Hoffman Estates, Illinois, USA) equipped with medium energy collimators. At 4 h after injection of the radiotracer, one hundred and twenty 40 s views were acquired over a 360° circular orbit into a 128 × 128 matrix with a pixel size of 3.9 mm. Imaging at 4 h after injection was chosen in accordance with previous kinetic modeling data in healthy volunteers (Mamede et al. 2004, Fujita et al. 2003) and for practical reasons concerning scanning the patients. Images were reconstructed with filtered back-projection with a Butterworth filter (order 8, cutoff 0.4) followed by attenuation correction according to the Chang method , with an attenuation coefficient of 0.11/cm to generate the transversal slices. The resulting spacing between slices was 3.9 mm.
For further data analysis, the reconstructed transverse sections were transferred to a Hermes workstation (Hermes Medical Solutions, Stockholm, Sweden). Brain regions were analyzed using the brain analysis program BRASS (version 3.5; Hermes Medical Solutions). Each image volume was recorded to match to the built-in ECD template using an affine transform (nine parameters). Manual fitting was necessary, because of the low background uptake resulting in insufficient contrast for delineating the brain contour.
The ECD template consisted of a three-dimensional region of interest (ROI) map of 46 predefined brain regions. The mean count per voxel was determined for each region in both hemispheres. To compensate for intersubject variability in global tracer uptake, the ratio of mean count per voxel to mean count per voxel of the global 5-I-A-85380 brain uptake (=average of all 46 measured brain regions) was calculated for each region and each subject, resulting in intensity-normalized data.
The initial version of CERAD, the CERAD-NP test, was developed by the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) . The CERAD-NP test was developed in the English language, but a German language version (the CERAD Plus test)  was subsequently developed and validated at the Memory Clinic of the University hospital of Basel (Switzerland). All patients in our study underwent the CERAD Plus test. We did not evaluate the Mini-Mental State Examination (MMSE), the Trail Making Test A and the Trail Making Test B due to the following reasons: In contrast to all other CERAD subtests, the MMSE does not examine one or few specific cognitive domains – as it was the goal of this study – but represents a general screening test for cognitive functions. The Trail Making Test A and B – valuable tests in the examination in patients with Alzheimer's disease – can be influenced by motor slowness and tremor in IPD patients and do therefore not allow an exact evaluation of cognitive functions in IPD patients. The remaining applied subtests concern single cognitive functions and are explained in detail below:
Verbal Fluency: The subject must name as many animals as possible within 1 min and is awarded one point for each animal. This subtest examines verbal velocity and capacity, semantic memory, speech, executive function, and cognitive flexibility.
Modified Boston Naming Test: 15 drawings of daily objects are shown, which the subject must name within 10 s. The maximum score is 15 points (one point for each correctly named drawing). This subtest examines visual perception and reveals word-finding difficulties.
Word List (Learning, Recall, Intrusions, Saving, and Recognition): In the first step (Word List Learning), ten words are read out to the subject, who must memorize them and directly reproduce the words within 90 s. This procedure is repeated three times with the same words but with the word order changed (up to 3 × 10 points). The percentage of words remembered in the three runs altogether is defined as Word List Saving. After an interval, in which another subtest is performed, the same words are asked again (Word List Recall; up to ten points). The term Word List Intrusions denotes wrongly remembered words which are produced by the subject although they have not been read out by the examiner. In a further step, 20 words, the ten words from the first step (old words) and ten new words, are read out to the subject. The subject has to identify the ten old words (up to ten points) and the ten new words (also up to ten points, Word List Recognition). The subtest Word List examines learning capacity and memory for language information.
Figure Drawing: The subject must draw four figures of increasing difficulty (up to eleven points depending on the accuracy of the drawn figure). This subtest examines visuoconstructive ability. After an interval, in which two other subtests are performed, all four figures must be drawn from memory (Figure Recall, up to eleven points). The percentage of figures remembered is defined as Figure Saving. These Figure Recall/Figure Saving subtests investigate non-verbal memory.
Phonemic Fluency ‘s’-words: Subjects must say as many words as possible beginning with the consonant ‘s’ within 1 min. For each word, the subject gets one point. This subtest examines strategy-oriented verbal fluency.
The results of the individual subtests can be expressed in two ways: as an absolute value (=raw value) or as a relative value. The absolute value gives the number of points that were obtained in a subtest. The performance (=absolute value) of each patient in each subtest was standardized according to a normal population of the same age, sex, and educational standard, resulting in a relative value. The relative value is expressed as standard deviations of the mean value of the normal population. For example, a relative value of +1.0 (standard deviations) means that the individual subject is better than 68% of the healthy volunteers of the same age, sex, and educational standard. A relative value of −2.0 (standard deviations) means that the individual subject is worse than 95% of the control group, etc. The CERAD testing was performed by a neurologist who was blinded to the results of the 5-I-A-85380 SPECT.
Descriptive data are given as mean and standard deviation (SD). Correlations were calculated using Pearsons's correlation coefficient for normally distributed data. In the case of not normally distributed data, we used the Spearman's correlation coefficient. As correlation coefficients were calculated from the same data, we performed Bonferroni correction for multiple comparisons: In a first step, we correlated the 15 subtests of the CERAD testing with 5-I-A-85380 accumulation in all studied 46 brain regions resulting in 15 × 46 = 690 correlation coefficients. In a second step, we identified the brain regions and the CERAD subtests with the highest correlation coefficients with the intention of reducing the number of correlation coefficients. Then, the P value was compared with the Bonferroni-corrected significance level as quotient 0.05/number of calculated correlations. A correlation coefficient was considered to be significant, if its P value was smaller than the Bonferroni-corrected significance level.