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BloodЦbrain barrier dysfunction in parkinsonian midbrain in vivo.

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Blood–Brain Barrier Dysfunction in
Parkinsonian Midbrain In Vivo
Rudie Kortekaas, PhD,1,2 Klaus L. Leenders, PhD,1,3 Joost C. H. van Oostrom, MD,1,3
Willem Vaalburg, PhD,5 Joost Bart, PhD,4 Antoon T. M. Willemsen, PhD,5 and N. Harry Hendrikse, PhD5
Parkinson’s disease (PD) is associated with a loss of neurons from the midbrain. The cause of PD is unknown, but it is
established that certain neurotoxins can cause similar syndromes. The brain is normally protected from these noxious
blood-borne chemicals by the blood–brain barrier which includes specialized proteins on the inside of blood vessels in
the brain. These act as molecular efflux pumps and P-glycoprotein (P-gp) is an abundant representative. Vulnerability to
PD appears codetermined by the genotype for the P-gp gene. We hypothesized that PD patients have reduced P-gp
function in the blood–brain barrier. We used positron emission tomography to measure brain uptake of [11C]-verapamil,
which is normally extruded from the brain by P-gp. Here, we show significantly elevated uptake of [11C]-verapamil
(18%) in the midbrain of PD patients relative to controls. This is the first evidence supporting a dysfunctional blood–
brain barrier as a causative mechanism in PD.
Ann Neurol 2005;57:176 –179
Parkinson’s disease is associated with neuronal death in
several cell groups in the midbrain that normally produce large amounts of neurotransmitters such as dopamine (substantia nigra pars compacta, ventral tegmental area, retrorubral field), noradrenaline (locus
coeruleus), serotonin (dorsal raphe nucleus), and acetylcholine (Edinger–Westphal nucleus).1 Clinical
symptoms include rigidity, hypokinesia, and tremor.
Despite a huge research effort during the last decades,
the cause of PD remains unknown.
The observation that the neurotoxin 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes a
parkinsonian syndrome similar to Parkinson’s disease
(PD) has led to the hypothesis that environmental toxins similar to MPTP could play an important role in
the pathogenesis of PD.2 Many pesticides fit the profile
in that they induce oxidative stress, mitochondrial
damage, and apoptosis3,4 and cause damage to the
brain’s dopaminergic cell groups.5,6 Also, the chemical
structure of the widely used pesticide paraquat is strikingly similar to that of MPTP. Indeed, exposure to
pesticides is a risk factor for PD,7 and the association
between PD and rural residence and well-water drinking8 might also be mediated by pesticides. A postmortem study found detectable levels of pesticides in the
substantia nigra of PD patients that were higher than
in controls,9 suggesting that these toxins are able to
gain access to the brain.
Because not everyone who is exposed to pesticides
develops PD, there is likely to also be a genetic component that determines vulnerability. The blood–brain
barrier (BBB) gene MDR1 encodes P-glycoprotein (Pgp), a 170kDa peptide with ATPase activity that actively transports a wide range of molecules from the
brain side to the blood side. A recent study found that
PD patients exposed to pesticides were five times more
likely to carry the 3435T allele of the MDR1 gene,10
which presumably results in reduced pump function.
In 1996, we reported the synthesis and initial in vivo
evaluation of [11C]-verapamil,11 showing that it can be
used as a positron emission tomography (PET) probe
for P-gp function in vivo. We further characterized its
distribution behavior in rats,12,13 normal and MDR1
knockout mice,14 and humans,15 confirming that in
vivo uptake and distribution of [11C]-verapamil is a
sensitive measure of P-gp function.
On the basis of the above, we hypothesized that PD
patients would have reduced P-gp function in the
brain.
From the Department of 1Neurology and 2Anatomy and Embryology, 3Movement Disorders Unit, 4Department of Pathology, and
5
PET-centre, Groningen University Hospital, Groningen, The
Netherlands.
Published online Jan 26, 2005, in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.20369
Received Jul 12, 2004, and in revised form Sep 8 and Oct 23.
Accepted for publication Oct 23, 2004.
176
Subjects and Methods
Subjects
Subjects were five nondemented PD patients (mean age ⫾
standard deviation [SD], 64.8 ⫾ 7.4; one woman, four men)
who were undergoing treatment at the Movement Disorders
Unit of the Neurology Clinic of the Groningen University
Address correspondence to Dr Leenders, Groningen University Hospital, Hanzeplein 1, 9700 RB, Groningen, The Netherlands.
E-mail: k.l.leenders@neuro.azg.nl
© 2005 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
Hospital. They met the Gelb criteria for “probable PD”24
and had a mean (⫾ SD) disease history of 5.1 (⫾ 1.9) years.
Four of the five were taking antiparkinsonian medication
(two were taking a dopamine agonist, one was taking a dopamine agonist plus amantadine, and one was taking L-dopa
plus amantadine). Five healthy age-matched drug-free controls (mean age ⫾ SD, 63.6 ⫾ 13.1; two women, three men)
were recruited through an advertisement. All subjects underwent a general physical and a neurological examination and
were rated according to the Unified Parkinson Disease Rating Scale and the Mini-Mental State Examination by the
same neurologist. No clinical indication of central nervous
system disease was present in the control subjects. Mean
Unified Parkinson Disease Rating Scale–III scores (⫾SD) in
patients were 20.2 (⫾ 7.9). Subjects gave written informed
consent and the protocol was approved by the internal
medical-ethical committee of the Groningen University Hospital.
Positron Emission Tomography Procedure
One cannula was inserted into an arm vein for administration of tracers, while a second, sampling cannula was inserted
into the radial artery. After a bolus injection of [15O]-H2O,
radioactivity was measured over a period of 4 minutes. [11C]Verapamil was synthesized as described previously25 and after
the [11C]-verapamil injection (111– 430MBq), ten 30-second
frames, three 5-minute frames, and three 10-minute frames
were acquired with the PET camera. We used racemic [11C]verapamil to avoid chiral purification, thus maximizing specific activity (P-gp has no stereoselectivity for [11C]verapamil26). PET data were reconstructed to a 128 ⫻
128 ⫻ 63 matrix with a plane separation of 2.425mm and a
bin size of 2.250mm. During the [11C]-verapamil PET scan,
17 arterial blood samples were drawn at 0:15, 0:30, 0:45,
1:00, 1:15, 1:30, 1:45, 2:15, 2:45, 3:45, 4:45, 7:30, 12:30,
17:30, 25:00, 35:00, and 45:00 min. Radioactivity was
counted in total blood, plasma, and in high-performance liquid chromatography–fractionated plasma. The latter method
allows for identification of labeled metabolites, and for calculation of their contribution to the total signal.
maximum, a two-sample t test was done with proportional
scaling at an analysis threshold of 0.8, global calculation:
mean voxel value. The t contrasts that were calculated were
patients minus controls and vice versa, without masking at a
threshold of an uncorrected p value less than 0.001. The individual values for the significant cluster were calculated with
SPM99.
Results and Discussion
As shown previously,15 uptake of [11C]-verapamil was
determined by the degree of vascularization and the
presence or absence of a BBB: very high in the pituitary, high in the ventricles and skin, moderate in gray
matter, and low in white matter and bone. Visual inspection of the uptake images resulted in no obvious
differences in [11C]-verapamil uptake between patients
and controls. A representative image of [11C]-verapamil
uptake is given in Figure 1.
Although visual inspection of the uptake images
themselves showed no differences, a pixel-by-pixel t test
on the two groups showed significantly increased
[11C]-verapamil uptake in the Parkinson’s disease patients. Strikingly, this increased uptake was restricted to
one area that covered most of the midbrain and part of
the dorsal pons (Fig 2). There were no brain areas in
which patients had lower uptake than controls. The
magnitude of the effect was 18%, and there was complete separation of the groups; that is, all the patients
showed higher uptake than all the controls (Fig 3).
The location of the BBB impairment found in this
study overlaps to a considerable extent the region
known to show neuronal damage in postmortem PD
brains. Of all cell groups known to be damaged,1 a
Data Analysis
Using Matlab (MathWorks, Natick, MA) and a linearization
according to Logan and colleagues27 starting at t ⫽ 5 minutes, parametric images for distribution volume (DV images)
were constructed from the [11C]-verapamil scans. This pharmacokinetic model–independent analysis calculates the influx
(KI) and distribution volume (DV) of a reversibly binding
radiotracer. For all subjects, the [11C]-verapamil scans were
summated across frames using Clinical Applications Programming Package 5 (Siemens, Erlangen, Germany). The
[15O]-H2O image was spatially normalized by Statistical
Parametric Mapping (SPM) 99 to match the SPM [15O]H2O template, and the normalization parameters were saved.
These were used to normalize the DV images, which then
were summed to form a [11C]-verapamil DV template. To
further reduce interindividual differences in normalization,
we normalized individual DV images again to this [11C]verapamil DV template. After spatial smoothing by convolution with a Gaussian kernel of 20mm full-width at half-
Fig 1. Uptake of [11C]-verapamil (distribution volume [DV])
in the brain. This image is representative for all subjects, irrespective of disease. n.c. ⫽ nasal cavity; L.V. ⫽ lateral ventricle; pit ⫽ pituitary; 4V ⫽ fourth ventricle.
Kortekaas et al: BBB Dysfunction in PD
177
Reduced activity or expression of molecular efflux
pump P-gp is likely to mediate the increased DV because [11C]-verapamil is a substrate for P-gp. Although
increased mesencephalic binding of [11C]-verapamil in
brain tissue past the endothelial cells theoretically could
also cause an increased DV, we consider it unlikely
that this is the case here. No evidence is available in
the literature to suggest such altered binding in PD.
With the Logan analysis, we could also exclude an increased influx (KI) of [11C]-verapamil as the cause of
the increased DV. KI (⫾SD) was of the same magnitude in both groups: 0.89 (⫾0.068) in controls and
0.95 (⫾0.068) in PD patients (t ⫽ 1.21; not significant). Unaltered KI in the PD group is also evidence
against endothelial damage and perfusion differences as
the cause of the increased DV confirming previous results.12,13 We believe based on these considerations
that the difference in DV between the groups is caused
by reduced P-gp mediated efflux in the patients.
Any effects of antiparkinsonian medication on the
uptake of [11C]-verapamil are likely to be of a global
nature. Because our data analysis includes an intrasubject numerical normalization it is insensitive to global
differences. It is unlikely that PD medication would
preferentially affect P-gp function in the midbrain.
Also, there was one unmedicated patient who showed
the same increase in DV in the midbrain.
In view of our findings, stimulation of P-gp in the
Fig 2. Increased uptake of [11C]-verapamil in Parkinson’s
disease patients. On the left are the result of a pixelwise t test.
The only cluster of pixels that reached statistical significance
was the one in the midbrain region (p ⫽ 0.02). Its location
is further clarified below by projection on a model anatomical
magnetic resonance image (T1-weighted). The two small
paired clusters in the upper panel did not reach significance
and correspond to the uncus/piriform cortex area.
large proportion and certainly the most prominent
ones (substantia nigra pars compacta, ventral tegmental
area, retrorubral field, locus coeruleus, dorsal raphe nucleus, Edinger–Westphal nucleus) fall inside the area
identified in this study (see Fig 2).
The findings of this study provide strong evidence in
favor of the environmental toxin hypothesis of PD and
suggest that in PD an impaired BBB function causes or
at least accelerates the disease progress. This is in agreement with the increased levels of pesticides in postmortem substantia nigra tissue from PD patients.9
178
Annals of Neurology
Vol 57
No 2
February 2005
Fig 3. Individual values of [11C]-verapamil uptake in the
midbrain (area highlighted in Fig 2). The controls had similar uptake as in the rest of the brain (mean, 102% of individual brain average), whereas the Parkinson’s disease patients
showed an 18% increase in [11C]-verapamil uptake (mean,
120% of individual brain average). Group means are indicated by a horizontal line.
BBB should be considered as a novel neuroprotective
strategy. Many studies have described stimulation of
P-gp activity in the gut or in cell lines in vitro by a
wide range of prescription drugs, grapefruit juice,16
and St. John’s wort.17 There are also endogenous factors that stimulate P-gp expression,18,19 such as progesterone20,21 and HSP90␤.22 Antagonists for the ␴2 receptor could also be promising because agonists reduce
transcription of the MDR1 gene which encodes P-gp.23
In summary, PD patients have reduced P-gp function in the midbrain. This suggests that P-gp dysfunction is part of PD pathogenesis.
This work was supported by a grant from the School for Behavioral
and Cognitive Neurosciences (R.K.).
We are grateful to the experimental subjects, Dr J. Pruim, and the
medical nuclear workers at the PET-centre of the Groningen University Hospital.
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Kortekaas et al: BBB Dysfunction in PD
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