Brain (2000), 123, 2321–2337
The peripheral benzodiazepine binding site in the
brain in multiple sclerosis
Quantitative in vivo imaging of microglia as a measure of
disease activity
R. B. Banati,1,10 J. Newcombe,4 R. N. Gunn,1 A. Cagnin,1 F. Turkheimer,1 F. Heppner,3,11 G. Price,7
F. Wegner,9 G. Giovannoni,5 D. H. Miller,5 G. D. Perkin,3 T. Smith,4,6 A. K. Hewson,4,8 G. Bydder,2
G. W. Kreutzberg,10 T. Jones,1 M. L. Cuzner4 and R. Myers1
1MRC
Cyclotron Unit and 2Robert Steiner Magnetic
Resonance Imaging Unit, Imperial College School of
Medicine, Hammersmith Hospital, 3Department of
Neuroscience, Imperial College School of Medicine,
Charing Cross Hospital, 4Neuroinflammation Group and
5NMR Research Unit, Institute of Neurology, University
College London, 6Eisai London Research Laboratories,
University College London, London, 7Department of
Neuroscience Research, SmithKline-Beecham
Pharmaceuticals, Harlow, 8Department of Physiology,
University of Cambridge, Cambridge, UK, 9Paul Flechsig
Institute, Leipzig, 10Department of Neuromorphology,
Max-Planck-Institute of Neurobiology, Munich, Germany
and 11Department of Neuropathology, University of Zurich,
Switzerland
Correspondence to: R. B. Banati, MRC Cyclotron Unit,
Imperial College School of Medicine, Hammersmith
Hospital, Ducane Road, London W12 0NN, UK
Summary
This study identifies by microautoradiography activated
microglia/macrophages as the main cell type expressing
the peripheral benzodiazepine binding site (PBBS) at sites
of active CNS pathology. Quantitative measurements of
PBBS expression in vivo obtained by PET and [11C](R)PK11195 are shown to correspond to animal experimental
and human post-mortem data on the distribution pattern
of activated microglia in inflammatory brain disease. Film
autoradiography with [3H](R)-PK11195, a specific ligand
for the PBBS, showed minimal binding in normal control
CNS, whereas maximal binding to mononuclear cells was
found in multiple sclerosis plaques. However, there was
also significantly increased [3H](R)-PK11195 binding on
activated microglia outside the histopathologically defined
borders of multiple sclerosis plaques and in areas, such
as the cerebral central grey matter, that are not normally
reported as sites of pathology in multiple sclerosis. A
similar pattern of [3H](R)-PK11195 binding in areas
containing activated microglia was seen in the CNS
of animals with experimental allergic encephalomyelitis
(EAE). In areas without identifiable focal pathology,
immunocytochemical staining combined with highresolution emulsion autoradiography demonstrated that
the cellular source of [3H](R)-PK11195 binding is
activated microglia, which frequently retains a ramified
morphology. Furthermore, in vitro radioligand binding
studies confirmed that microglial activation leads to a
rise in the number of PBBS and not a change in binding
affinity. Quantitative [11C](R)-PK11195 PET in multiple
sclerosis patients demonstrated increased PBBS expression in areas of focal pathology identified by T1- and T2weighted MRI and, importantly, also in normal-appearing
anatomical structures, including cerebral central grey
matter. The additional binding frequently delineated
neuronal projection areas, such as the lateral geniculate
bodies in patients with a history of optic neuritis. In
summary, [11C](R)-PK11195 PET provides a cellular
marker of disease activity in vivo in the human brain.
Keywords: PK11195; microglia; peripheral benzodiazepine receptor; mitochondrial benzodiazepine receptor; PET; multiple
sclerosis; EAE; inflammation
© Oxford University Press 2000
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R. B. Banati et al.
Abbreviations: BP ⫽ binding potential; CR3 ⫽ complement receptor 3; EAE ⫽ experimental allergic encephalitis; EDSS ⫽
Expanded Disability Status Scale; GFAP ⫽ glial fibrillary acid protein; PBBS ⫽ peripheral benzodiazepine binding site;
PMA ⫽ phorbol myristate acetate; PP ⫽ primary progressive; RR ⫽ relapsing–remitting; SP ⫽ secondary progressive;
SRCC ⫽ Spearman’s rank correlation coefficient; TAC ⫽ time–activity curve(s)
Introduction
In brain lesions without direct damage to the blood–brain
barrier, the predominant cell type expressing binding sites
for the isoquinoline PK11195 is activated microglia, the
brain’s intrinsic macrophage (Banati et al., 1997). Originally discovered as an additional binding site of certain
benzodiazepines, such as diazepam, the binding site for
PK11195 is abundantly expressed in peripheral organs and
haematogenous cells and is often referred to as the ‘peripheral
benzodiazepine binding site’ (PBBS) (for review, see Hertz,
1993), although it is structurally and functionally unrelated
to the central benzodiazepine receptor associated with GABAregulated channels.
Labelled with carbon-11, PK11195 has previously been
used in a small number of patients to image focal accumulations of brain macrophages by PET (Benavides et al., 1988;
Ramsay et al., 1992; Banati et al., 1999). There is increasing
awareness that microglia are early sensors of brain pathology
(Kreutzberg, 1996), and [11C](R)-PK11195 PET offers the
prospect of measuring microglial activation in the living
human brain in diseases such as multiple sclerosis, in which
activated microglia may contribute to tissue destruction and
disease progression (Cuzner, 1997; Sriram and Rodriguez,
1997). However, a number of fundamental methodological
issues have so far limited the interpretation of [11C](R)PK11195 imaging data in brain inflammation. Whereas there
is evidence that areas with increased PK11195 binding contain
activated microglia (Dubois et al., 1988; Myers et al., 1991a;
Stephenson et al., 1995; Conway et al., 1998), the cell type
itself has not yet been shown directly (i.e. by double-labelling
in situ) at the single-cell level to express the [11C](R)PK11195 binding site in inflammatory tissue. It is also not
yet established whether an increase in microglial PK11195
binding would reflect a rise in the number of binding sites
or a change in their affinity. Furthermore, unlike the situation
with other ligands used in PET, the near absence of significant
PK11195 binding in the normal brain makes it difficult to
establish a typical or normal pattern of PK11195 binding and
predict its change in CNS disease. To this end, in vitro
data from experimental allergic encephalomyelitis (EAE), an
animal model of multiple sclerosis, and from post-mortem
CNS tissue of multiple sclerosis patients are used to interpret
in vivo data of [11C](R)-PK11195 binding in multiple sclerosis
patients. The kinetic behaviour of [11C](R)-PK11195 in
disease tissue is quantitatively assessed by a method that
accounts for, for example, regional blood flow-dependent
changes in ligand delivery, as they may be expected in
inflammatory pathology, and is relevant to the issues that
arise from the unstable stable behaviour of the ligand in
the vascular (plasma) compartment and the absence of an
anatomically definable reference tissue in multiple sclerosis.
Material and methods
Human post-mortem issue
Post-mortem brain and spinal cord tissue from 11 patients
with clinically and histopathologically confirmed multiple
sclerosis (mean age 57 years, range 35–63 years; mean
disease duration 18 years, range 1–28 years; mean interval
from death to snap-freezing 41 h, range 21–61 h) and nine
control subjects without neurological disease (mean age
55 years, range 37–72 years; mean interval from death to
snap-freezing 38 h, range 14–82 h) were obtained from the
Multiple Sclerosis Society Tissue Bank at the Institute of
Neurology. Immunostaining, histological grading and film
autoradiography were performed on a total of 52 tissue blocks
from different areas of brain and spinal cord.
Adoptive transfer of EAE
Adult female Lewis rats (190–220 g) were purchased from
Charles River (Margate, Kent, UK) and housed in pairs in a
standard animal facility for at least 1 week before
experimentation. EAE was adoptively transferred to naı̈ve
animals by intraperitoneal injection of 4 ⫻ 107 splenocytes
cultured for 3 days with myelin basic protein (MBP) taken
from animals 11 days after sensitization with MBP and
complete Freund’s adjuvant (Hewson et al., 1995).
Histology and immunocytochemistry
Histopathological evaluation of snap-frozen multiple sclerosis
tissue samples was carried out using cresyl violet, haematoxylin–eosin and oil red O. For immunocytochemistry, 10 µm
cryostat sections of human CNS and rat brain and rat lumbar
spinal cord (between levels L3 and L5) were processed and
stained with a panel of antibodies using the avidin–biotin–
horseradish peroxidase method as described (Smith et al.,
1996). For human tissue the following antibodies were
used at appropriate dilutions: EBM11 (1 : 100) (Dako, High
Wycombe, UK) and HLA class II DQ (1 : 500) (a gift from
Professor F. Vartdal and Dr S. Funderud, Oslo, Norway) as
markers of microglia/macrophages; CD2 for lymphocytes
(1 : 100) (Dako); polyclonal anti-GFAP (glial fibrillary acidic
protein) for astrocytes (1 : 400) (Dako); and 14E for oligodendrocytes and a reactive astrocyte subpopulation (1 : 10 supernatant) (Newcombe and Cuzner, 1988). Rat CNS sections
were stained with markers for microglia [OX-42 recognizing
Peripheral benzodiazepine binding in CNS pathology
CR3 complement receptor (1 : 3000), Dako], macrophages
[ED1 (1 : 500), Dako] and astrocytes [polyclonal anti-GFAP
(1 : 400)]. Controls included omission of the primary antibody
and replacement with a control IgG1 (Dianova, Hamburg,
Germany).
Autoradiography
Film autoradiography and photoemulsion microautoradiography (Banati et al., 1997) were performed on unfixed
cryostat sections of snap-frozen CNS tissue from human
post-mortem material and animals with EAE, using customsynthesized single enantiomer [3H]R-PK11195 (Amersham,
UK), which in previous studies has been shown to possess
higher affinity for the PBBS than the commonly used racemate
of PK11195 (Shah et al., 1994). Tritium standards (Amersham
International, Little Chalfont) co-exposed on each film were
used to quantify the autoradiographically measured binding.
If not specified otherwise, mean binding values were derived
from at least 10 sampled areas. The cell type binding [3H](R)PK11195 was identified by further immunohistochemistry
carried out immediately after the development of the microautoradiographic label using markers for microglia and macrophages (for human brain, EBM11; for rat brain, OX-42) and
astrocytes (GFAP).
In vitro radioligand binding
To assess whether increased ligand binding reflects an
increase in the number of binding sites or a change in the
affinity of the binding site, additional measurements of in vitro
radioligand binding with [3H](R)-PK11195 (1 nM, specific
activity 84 Ci/mmol) in the presence of unlabelled PK11195
(concentration range 0.3 nM to 1 µM) were carried out on
standard membrane preparations (15 mg/ml) of an
immortalized cell line with properties of activated microglial
cells (Bocchini et al., 1992), applying established binding
protocols (Itzhak et al., 1993). Maximal binding (Bmax) and
affinity (pKi) were measured before and after maximal
stimulation with 1 mM PMA (phorbol myristate acetate).
Subjects of imaging study
Twelve patients with multiple sclerosis according to the
criteria of Poser and colleagues (Poser et al., 1983) (eight
women, four men; age range 28–66 years, average age
46.3 years; for diagnosis, disease duration, disability scores
and treatment at the time of investigation, see Table 1) and
eight healthy normal volunteers (three women, five men; age
range 34–70 years, average age 51.75 years) were studied.
Eight patients were considered to have relapsing–remitting
(RR) multiple sclerosis, i.e. they had stable disability between
relapses. One patient had secondary progressive (SP) multiple
sclerosis with increased disability over 6 months without
superimposed relapses. Three patients had progressive disease
without intermittent remission and were classified as having
2323
primary progressive (PP) multiple sclerosis (Lublin and
Reingold, 1996). Except for one patient (Patient 5), who
received interferon β-1b treatment, none of the patients
was under any concurrent anti-inflammatory or immunosuppressive therapy. Informed written consent was obtained
from all subjects. Ethical approval and permission was given
by the Hammersmith Hospital ethics committee and the
Administration of Radioactive Substances Advisory
Committee of the Department of Health (ARSAC), UK.
MRI
Close to the time of the PET study (i.e. within 7 days), each
subject underwent a three-dimensional T1-weighted MRI scan
[voxel size 1 ⫻ 1 ⫻ 1.3 mm; 128 contiguous slices; repetition
time (TR) 35 ms, echo time (TE) 6 ms, flip angle 35°]. These
were obtained from a 1.0 T Picker HPQ MRI scanner for
the purpose of co-registration with the PET image (Studholme
et al., 1997) and exclusion of incidental pathology. In the 12
multiple sclerosis patients, additional 2D proton density spinecho (TR 2500 ms, TE 20 ms) and T2-weighted spin echo
images (TR 2500/TE 80) were acquired. The T1-weighted
3D spin echo images were repeated after contrast
enhancement with intravenous dimeglumine gadopentate
(Gd-DPTA, 0.1 mmol/kg).
PET
Data acquisition
The PET study was performed on a CTI/Siemens
ECAT 953B PET scanner operated in 3D acquisition
mode. [11C](R)-PK11195 was injected as a bolus 30 s after
the acquisition scan started. The mean tracer dose was
360 ⫾ 30 MBq with a specific activity of 37 ⫾ 1 GBq/mmol.
Dynamic data were collected over 60 min as 18 temporal
frames. Attenuation correction factors were determined using
a 15 min transmission scan acquired before the dynamic scan.
Scatter correction was achieved using a dual-energy window
method (Grootoonk et al., 1996). Data were reconstructed with
a ramp filter at Nyquist cut-off, producing an image resolution
of 5.8 mm (full-width at half maximum) at the centre of the
field of view.
Kinetic modelling and analysis
As the kinetic behaviour of [11C](R)-PK11195 in the plasma
compartment was found to be unstable, probably because of
high and variable plasma protein binding, the regional uptake
of [11C](R)-PK11195, extraction of which into the brain is
similar in areas with and without blood–brain barrier (Price
et al., 1990; Cremer et al., 1992), was assessed by using a
basis function implementation of a simplified reference tissue
model (Lammertma and Hume, 1996; Gunn et al., 1997;
Banati et al., 1999) and calculating the following rate
constants: RI, the ratio of delivery of radioligand between
2324
R. B. Banati et al.
Table 1 Patient data
Patient Age Sex Diagnosis Clinical course
1
2
3
4
5
6
7
8
9
10
11
12
38
55
28
51
51
20
53
34
53
46
47
66
F
F
F
M
F
F
F
F
M
M
F
M
RRMS
RRMS
RRMS
RRMS
RRMS
RRMS
RRMS
RRMS
SPMS
PPMS
PPMS
PPMS
EDSS
Subscores
Disease Last relapse Total
duration
(years)
Pyramidal Cerebellar
10
5
6
6
26
5
6
2
9
10
2
8
0
3–4
1
3
3–4
2
1
2
5
3
3
3
1 month
1 month
3 months
3 weeks
21 months*
5 months†
6 months
5 months†
5 months
–
–
–
2
4.5
2.5
3.5
4
2
3
3
7
5.5
3.5
6.5
0
2
2
3
0
1
2
2
5
2
1
1
Brainstem Sensory Bowel/ Visual
bladder
1
1
1
1
1
1
1
1
4
1
1
1
2
4
1
0
4
2
3
2
0
2
3
1
0
1
2
2
0
0
0
2
3–4
1
0
1
0
1–2
0
0
1
0
0
1
2
0
0
0
Cerebral
0
1
1
1
0
2
0
0
2
0
0
0
*Interferon β-1b treatment for 20 months; †in relapse at time of scan. RRMS⫽ relapsing–remitting multiple sclerosis; SPMS ⫽ secondary
progressive multiple sclerosis; PPMS ⫽ primary progressive multiple sclerosis.
the target and reference tissues; k2, the efflux rate constant
from the target tissue; and the binding potential (BP) which
is a measure of ligand binding. In the reference tissue
model, the reference input kinetic is usually derived from an
anatomical structure that is devoid of specific ligand binding,
such as the cerebellum. However, in multiple sclerosis,
the often widespread tissue pathology may not allow the
anatomical definition of unequivocally normal reference
tissue. Therefore, cluster analysis (Ashburner et al., 1996;
Gunn et al., 1998) was employed as an alternative, ‘dataled’ approach for the extraction of a normal ligand kinetic
to serve as the reference input function. In brief, voxels in
the raw dynamic data were segmented into 10 clusters
distinguished by the shape of their concentration time–
activity curves (TACs), thereby associating each dynamic
image voxel with one of the cluster curves according to the
likelihood with which the TAC of the voxel belongs to a
given cluster (Gunn et al., 1998). In normal brain, the
majority (~90%) of the voxels segregated into two clusters,
one representing the TAC mainly from the skull and scalp
and one representing the TAC of voxels mostly located in
the cerebral cortex (Fig. 1). The latter cluster was compared
with a normalized mean TAC (population input kinetic)
previously created from the normal ligand kinetics identified
by cluster analysis in 14 healthy subjects (eight males and
six females; age range 32–80 years, mean age 57.3 years).
Whether a TAC extracted by cluster analysis from the raw
dynamic data of a patient was suitable to serve as the patient’s
normal ligand kinetic was assessed by testing for dissimilarity
with the previously established normal population input
kinetic (χ2 test, P ⬍ 0.05) (Fig. 1).
Definition of volumes of interest
To allow anatomical localization of the regional [11C](R)PK11195 binding, the binding potential images were co-
registered and overlaid onto the individuals’ own MRIs
(Studholme et al., 1997). For the calculation of regional
mean BP values, the following volumes of interest (VOIs)
were defined anatomically on the individuals’ volumetric
MRI and applied to their parametric BP image: right and left
thalamus; brainstem (between the upper and lower borders
of the pons); and right and left hemisphere excluding the
thalamus. The cerebellum, seen within the restricted field of
view (10.65 cm) of the PET camera only to a varying extent,
was excluded from formal analysis.
Additional VOIs were determined for the multiple sclerosis
patients according to the volume of pathological tissue
revealed by proton-density, T2-weighted and T1-weighted
MRI. Analyze™ software (Robb and Hanson, 1991) was
used to outline MRI lesions (Fig. 6E and F) by a local
threshold technique and for the calculation of volume (mm3).
The definition of the MRI-defined lesions followed
established criteria (Filippi et al., 1998). The MRI lesion
mask for the lesions seen in the T1-weighted image volume
included all hypointense voxels, except those with signal
intensity close to that of cerebrospinal fluid. The latter were
included in a mask of ‘black holes’ (Truyen et al., 1996)
(Fig. 6B). The mean percentage difference (i.e. inter-rater
variability of the MRI lesion load calculation) between three
raters, who were blinded to the clinical data, was ⬍6% for
the T2-weighted lesion loads and ⬍5% for the T1-weighted
lesion loads. Comparison of the intensity distribution of the
voxels included in the group of black holes against the
remaining hypointensities of the T1-weighted MRI showed
that both lesion types were separated accurately, i.e. they
were significantly different with essentially no overlap
(P ⬍ 0.0001).
By analogy with the MRI lesion loads, hemispheric
[11C](R)-PK11195 lesion loads for both hemispheres were
calculated as described previously (Banati et al., 1999) by
Peripheral benzodiazepine binding in CNS pathology
2325
Fig. 1 The majority of the time–activity curves extracted by cluster analysis from the dynamic [11C](R)PK11195 PET data of a normal brain fell into two clusters, one localizing to extracerebal structures (A) and
the other to healthy brain tissue (B). The latter represents the normal reference kinetic that would have been
obtained from an anatomically defined mask similar to that seen in B. The population input kinetic (solid
line, with standard deviations) was used to decide whether an individual dynamic data set (here from
Patient 9) contained a time–activity curve suitable to serve as the individual’s reference input function
(dotted line and arrow).
including only those voxels in the CNS that had a value
above a threshold BPT (BP ⬎ BPT ⫽ 2 SD of background).
While the MRI lesion loads are reported as absolute
volumes (cm3) to allow comparison with published values,
the hemispheric [11C](R)-PK11195 lesion loads are reported
as relative volumes of pathology [i.e. as a percentage of
the sampled hemispheric volume, excluding thalamus and
brainstem, which were measured separately (see above)],
thus accounting for the different field of view of the PET
camera compared with the MRI, and hence the different
absolute sampling volume.
Statistics
Spearman’s rank correlation coefficient (SRCC) was used
to calculate correlations between disability scores, disease
duration and total [11C](R)-PK11195 lesion load (mm3) or
the [11C](R)-PK11195 lesion load (mm3) within the VOIs
defined as being abnormal by MRI (referred to as ‘[11C](R)PK11195 overlap’). Student’s t-test was used to determine
the significance of the differences in mean BP values between
normal control subjects and patients.
Z-values were calculated to determine the significance of
increased [11C](R)-PK11195 binding in brains of individual
patients compared with the group of normal control brains.
As [11C](R)-PK11195 binding shows a unidirectional change,
i.e. it only increases, a Z-value of 1.6 in a one-tailed Z-test
represents a level of significance of P ⬍ 0.05 (Snedecor and
Cochran, 1980).
Results
Human post-mortem studies: [3H](R)-PK11195
binding in normal control and multiple sclerosis
tissue
The correlation of histopathological lesion grading with
measured [3H](R)-PK11195 binding values and the expression
of immunocytochemical markers for macrophages and
activated microglia is summarized in Fig. 2. Briefly, [3H](R)PK11195 binding was slightly higher in normal control
cortical and central grey matter than in normal white matter.
Maximal [3H](R)-PK11195 binding, of more than four times
above normal control white matter, was reached within those
areas of multiple sclerosis plaques that contained oil red Opositive macrophages. Even within or around oil red Onegative but EBM11-positive areas, binding was more than
three times above the normal level. Regions of increased
[3H](R)-PK11195 binding were also seen in histologically
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R. B. Banati et al.
Peripheral benzodiazepine binding in CNS pathology
2327
normal-appearing multiple sclerosis white matter and central
grey matter where the increased [3H](R)-PK11195 binding
co-localized with EBM11-positive parenchymal microglia
(Fig. 3K and L). Apart from some binding to infiltrating cells
in perivascular cuffs, no obvious binding to any other cell
type or structure was found.
[mean background value 0.19 ⫾ 0.02 (SD)]. In normal brains,
an average 5.2 ⫾ 3.1% (SD) of all voxels (sampled from the
total volume of both hemispheres without thalamus) had
values above the threshold BPT. Some constitutive binding
was seen in normal brainstem [mean BP 0.38 ⫾ 0.05 (SD)]
and in the normal thalami [mean BP 0.40 ⫾ 0.05 (SD)].
Animal studies: [3H](R)-PK11195 binding in
EAE
Multiple sclerosis brain
In longitudinally cut spinal cords (Fig. 4) from rats with the
typical disease course of EAE, autoradiographs showed
normal [3H](R)-PK11195 binding along the spinal grey and
white matter columns up to day 4. At the peak of clinical
disease, [3H](R)-PK11195 binding increased in white and
grey matter and reached maximal values in small foci.
[3H](R)-PK11195 binding, similar to that seen in the spinal
cord, was also found in the midbrain (Fig. 4C). After clinical
recovery, binding returned to normal in white matter but
remained increased in grey matter (Fig. 4D). OX-42
immunohistochemistry demonstrated that the elevated
regional binding co-localized with perivascular infiltrates or
the cytoplasmic processes of non-phagocytic and still ramified
activated microglia along normal-appearing white matter
tracts. At this level of single-cell resolution, there was no
indication of [3H](R)-PK11195 binding to any other glial
cell type.
In vitro radioligand binding
The in vitro experiments in which [3H](R)-PK11195 was
displaced by unlabelled PK11195 (concentration range 0.3 nM
to 1 µM) before and after maximal stimulation with PMA
(1 mM) of a murine brain macrophage/microglia cell line
demonstrated a significant (P ⬍ 0.001) increase in Bmax
[unstimulated, 5.54 pM/µM (SEM 1.27, n ⫽ 3); stimulated,
9.21 pM/µM (SEM 0.53, n ⫽ 3) and no change in affinity
[unstimulated, pKi ⫽ 8.6 ⫾ 0.12 (SD); stimulated, pKi ⫽
8.5 ⫾ 0.12 (SD)] (Fig. 5).
Imaging studies
Normal brain
Binding of [11C](R)-PK11195 in the hemispheres (excluding
the thalamus) of normal brains was minimal, with poor
contrast among the grey matter, white matter and ventricles
Highly focal binding and regionally circumscribed increases
in [11C](R)-PK11195 binding were observed in the brains of
multiple sclerosis patients (data are summarized in Tables 2
and 3). In multiple sclerosis brain, the mean BP of brainstem
was 0.49 ⫾ 0.07 (SD) while the mean BP of the thalami
was 0.56 ⫾ 0.25 (SD), which is significantly higher than in
the normal control brains (P ⬍ 0.001 for brainstem, P ⬍ 0.05
for thalami). Unlike in normal brain, [11C](R)-PK11195
binding to multiple sclerosis thalami was frequently
asymmetrical. In most multiple sclerosis patients (except
Patients 4 and 8), at least one thalamus and/or the brainstem
had significantly increased signal compared with controls.
This also applied to Patients 11 and 12, who had PP multiple
sclerosis and normal MRI of the brain. Patient 11 had
an additional, clinically silent area of increased [11C](R)PK11195 binding in the right frontal lobe.
The average hemispheric [11C](R)-PK11195 lesion load in
the group of multiple sclerosis brains was approximately
twice that of normal brain [10 ⫾ 5.8% (SD)], ranging from
4 to 30%. Due to the highly regional binding pattern and the
large sampling volume, i.e. a full hemisphere, the [11C](R)PK11195 lesion load per hemisphere is a greatly diluted
measure and was significantly increased in only four patients
(Patients 5, 7, 8 and 10).
In the multiple sclerosis brains, the additionally sampled
areas of MRI-defined pathology (data are summarized in
Table 3) showed varying amounts of overlap with the areas
of increased [11C](R)-PK11195 binding: the highest overlap
was seen with gadolinium-enhancing lesions (present in
Patients 6, 8 and 9), in whom 30% of the gadoliniumenhancing volume had significantly increased [11C](R)PK11195 binding (Fig. 6G). For the group of patients with
RR multiple sclerosis who were not in relapse, the following
values for percentage overlap between MRI-defined
pathology and increased [11C](R)-PK11195 binding were
calculated: hyperintense areas in T2-weighted MRI,
10 ⫾ 3.5% (SD); hypointense areas in T1-weighted MRI
Fig. 2 [3H](R)-PK11195 binding in normal controls and multiple sclerosis. Tissue types: CeG ⫽ tissue containing central grey matter;
CoG ⫽ cortical grey matter; W ⫽ white matter; G ⫽ grey matter; Q ⫽ multiple sclerosis plaque. Areas: L ⫽ lobe; O ⫽ occipital;
P ⫽ parietal; SC ⫽ spinal cord; T ⫽ temporal; V ⫽ ventricular. Histology is scored on a scale of 0–4; the first number is the score for oil red
O-positive macrophages showing the degree of ongoing demyelination, the second is the score for perivenular inflammatory cuffing. The
histology sections were evaluated by two independent observers. [3H](R)-PK11195 binding in Bq/mg tissue was calculated using [3H]
standards co-exposed on the same autoradiographic films as the tissue sections. Binding values were derived from a minimum of 10 sampling
areas (multiple sclerosis plaque, area with oil red O-positive macrophages, n ⫽ 11; multiple sclerosis plaques, area without oil red O-positive
macrophages, n ⫽ 23; multiple sclerosis white matter containing activated microglia, n ⫽ 23; normal grey and white matter and background,
n ⫽ 10 each). *All spinal cord sections were cut transversely except sample 3, which was cut longitudinally.
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R. B. Banati et al.
Fig. 3 [3H](R)-PK11195 binding in multiple sclerosis: combined high-resolution autoradiography and immunohistochemistry. (A)
Autoradiograph taken from a section of a normal control tissue (sample 12 in Fig. 2) [binding in fmol/mg tissue, 18.8 ⫾ 2.8 (SD) in grey
matter, 11.66 ⫾ 2.3 (SD) in white matter]. The square frame delineates the area from which the photomicrographs shown in D, G and J are
taken. (B) (Upper section) [3H](R)-PK11195 autoradiograph showing increased binding [46.28 ⫾ 4.16 (SD) fmol/mg tissue] within the area
of a multiple sclerosis plaque (arrows) in the central grey matter (sample 6 in Table 1). The accentuated edges (upper arrow) of the multiple
sclerosis plaque co-localize with the presence of oil red O-positive macrophages, where binding is maximal [56.51 ⫾ 0.82 (SD) fmol/mg
tissue]. (Lower section) Parietal ventricular plaque and white matter (sample 1 in Fig. 2). The square frame is positioned over an area with
activated microglia but no oil red O-positive macrophages or perivascular infiltrates; photomicrographs of this area are shown in E, H, K and
L. (C) [3H](R)-PK11195 autoradiograph of a multiple sclerosis tissue from cervical spinal cord (sample 3 in Fig. 2). F and I are
photomicrographs of the framed area. (D–I) Staining of resting (D, G) and activated (E, F, H, I) parenchymal microglia with EBM11. F and I
show activated EBM11-positive microglia following a myelinated tract. (J–L) High-resolution photoemulsion [3H](R)-PK11195
autoradiography combined with EBM11 immunohistochemistry. In areas of activated microglia specific co-localization of silver grains with
the EBM11-positive cytoplasm of parenchymal microglia is demonstrated (arrows in K and L). Sections were lightly counterstained with
haematoxylin. OG ⫽ occipital cortical grey matter; CeG ⫽ central grey matter; PV ⫽ parietal ventricular; SC ⫽ spinal cord. Scale bar ⫽
10.7 mm for A and B, 7.2 mm for C, 1 mm for D–F, 300 µm for G–J and 180 µm for K–L.
Peripheral benzodiazepine binding in CNS pathology
2329
Table 2 Quantification of [11C](R)PK11195 binding
Patient
Binding potential
Hemispheres: Lesion load (%)
Thalamus
1
2
3
4
5
6
7
8
9
10
11
12
Right
Left
0.4 (n.s.)
1.19 (15.8‡)
0.90 (10‡)
0.34 (n.s.)
0.42 (n.s.)
0.4 (n.s.)
0.56 (3.2‡)
0.41 (n.s.)
0.62 (4.4‡)
0.61 (4.6‡)
0.49 (1.8*)
0.57 (3.4‡)
0.36 (n.s.)
1.22 (16.4‡)
0.95 (11‡)
0.33 (n.s.)
0.38 (n.s.)
0.38 (n.s.)
0.48 (1.6*)
0.36 (n.s.)
0.64 (4.8‡)
0.55 (3.6‡)
0.33 (n.s.)
0.61 (4.2‡)
Brainstem
Right
Left
0.48 (2*)
0.60 (4.4‡)
0.50 (2.4†)
0.4 (n.s.)
0.50 (2.4†)
0.4 (n.s.)
0.64 (5.2‡)
0.4 (n.s.)
0.54 (3.2‡)
0.51 (2.6†)
0.50 (2.4†)
0.58 (4‡)
9.2
5.6
5.9
10
12.6 (2.4†)
4.6
16.6 (3.7‡)
13 (2.5†)
8.3
30 (8‡)
7
6.6
9.5
6.3
6.2
9.4
12 (2.2†)
6.5
14 (2.8†)
16 (3.5‡)
6.5
21.2(5.2‡)
4
5.3
Numbers in parentheses are Z-values for regional [11C](R)-PK11195 binding or lesion loads significantly
higher than in normal controls: *P ⬍ 0.05; †P ⬍ 0.01; ‡P ⬍ 0.001. n.s. ⫽ not significant.
Table 3 MRI lesion load and overlap with [11C](R)PK11195
binding
Patient MRI lesion load (cm3)
1
2
3
4
5
6
7
8
9
10
11*
12*
MRI–PK11195 overlap (%)
T2
T1
Black holes T2
T1
Black holes
–
4
2.6
8
3
7.4
5.6
9
2.9
13.1
3.4
–
–
0.1
1.1
0.3
0.4
3.6
1.4
0.9
0.6
34
7.5
–
–
1.3
1
5.2
11
9
17.4
6.2
21
5
10.2
–
–
9.3
12.4
3
11
9.2
8
12
15
40
17
–
–
8.2
6.5
2.2
20.5
2
13.7
9.1
52
4.2
–
–
7.3
6.1
13.8
9.4
27.8
8.8
31
11.5
14.5
–
–
–
*Patients 11 and 12 had a normal MRI.
(excluding black holes), 12 ⫾ 3% (SD); black holes, 6 ⫾ 4%
(SD). For patients in acute relapse (Patients 6 and 8), the
overlap with T2-weighted hyperintense areas increased to
~30% and to ~20%, respectively, in the black holes of the
T1-weighted image, which in the remaining patients contained
the lowest amount of significant binding. The patient with
secondary progressive multiple sclerosis (Patient 9) showed
high [11C](R)-PK11195 binding co-localizing with the
hypointensities of the T1-weighted MRI (excluding black
holes), reaching 40% overlap.
Clear and well circumscribed signals (BP ⬎ 0.9 ⬍ 2) were
seen in anatomical structures that had a normal appearance
on each of the three MRI sequences used in this study.
[11C](R)-PK11195 PET signals frequently followed neuronal
fibre tracts, for example tracking through pathways in the
brainstem (Fig. 6A).
In patients who suffered from or had a recently reported
visual dysfunction, signals were observed in anatomical
locations along the pathway of the neuronal network
controlling visual processing or eye movement. Thus, in
Patient 9, who had internuclear ophthalmoplegia, a clinical
condition caused by a lesion to the medial longitudinal
fasciculus (MLF), a circumscribed PET signal could be seen
co-localizing with the MLF at the level of the mesencephalon,
i.e. the predicted site of pathology (Fig. 7A and B). Similarly,
patients with a history of optic neuritis (Fig. 7C and D)
showed signals in the lateral geniculate bodies, to which the
optic nerve projects. Patients who had never reported any
visual dysfunction showed no [11C](R)-PK11195 binding
associated with any part of the brain’s visual pathway.
In Patient 6 (Fig. 7E–H), who, at the time of the scans,
presented with transient speech dyspraxia (reduced fluency
of spontaneous speech with only mild language disturbance)
(Mohr et al., 1978), [11C](R)-PK11195 PET revealed
increased binding in the left frontal operculum close to the
area of Broca, which contains the superior longitudinal
fascicle.
A significant positive correlation (SRCC ⫽ 0.76; P ⫽
0.012) existed between the [11C](R)-PK11195 overlap with
the T1-weighted MRI hypointensities (excluding the black
holes) and the total Expanded Disability Status Scale (EDSS)
score. This correlation was maintained at a lower level of
significance (SRCC ⫽ 0.66; P ⫽ 0.05) after exclusion of
Patient 9 (SP multiple sclerosis), who had a particularly
high [11C](R)-PK11195 overlap with the T1-weighted MRI
lesion load.
No other statistically significant correlations were found,
for example between the global hemispheric [11C](R)PK11195 lesion load and disability (total EDSS and individual
subscores), disease duration or the interval since the last
relapse or between the amount of [11C](R)-PK11195 overlap
with T2-weighted MRI lesion volume and either disability or
disease duration. There was, however, a trend for an inverse
2330
R. B. Banati et al.
correlation between the interval since the last relapse and the
[11C](R)-PK11195 overlap with the T2-weighted MRI lesion
volume, indicating that the amount of binding within the T2weighted MRI lesions gradually declines during the relapsefree time (SRCC ⫽ 0.63; P ⫽ 0.09). No such trend was seen
with respect to the amount of [3H](R)-PK11195 binding
within the T1-weighted lesions.
Discussion
Pattern of PK11195 binding in vitro in multiple
sclerosis and EAE (histology)
High-resolution microautoradiography in multiple sclerosis
and EAE tissue combined with immunohistochemical cell
identification in the same tissue section demonstrated that
the PBBS is expressed on invading blood-borne cells at sites
Peripheral benzodiazepine binding in CNS pathology
of focal inflammation and on activated microglia remote
from any obvious inflammatory pathology.
The present study extends earlier observations (Benavides
et al., 1988) of focal binding in multiple sclerosis and
corroborates indirect (Dubois et al., 1988; Myers et al.,
1991a; Stephenson et al., 1995; Conway et al., 1998) and
direct (Banati et al., 1997) evidence that activated microglia
are the primary source of PK11195 binding in vivo. In
keeping with reported data (Myers et al., 1991a, b; Stephenson
et al., 1995; Conway et al., 1998) is the absence of significant
in vivo binding of PK11195 to astrocytes, although binding
of PK11195 to astrocytes in cell culture is well described
(Hertz, 1993; Itzhak et al., 1996). Recently, it has also been
reported that, in a neurotoxic lesion model, immunoreactivity
primarily in and around the nucleus of reactive hippocampal
astrocytes can be detected by a polyclonal antibody against the
peripheral benzodiazepine receptor (Kuhlmann and Guilarte,
Fig. 5 In vitro displacement of [3H](R)-PK11195 (1 nM) with
various concentrations of unlabelled PK11195 (concentration range
0.3 nM to 1 µM) before and after maximal stimulation with PMA
(1 mM) of a murine brain macrophage/microglia cell line,
demonstrating a significant increase in maximal binding, i.e.
number of binding sites, but no significant change in affinity. Error
bars denote standard error of the mean of three independent
experiments. Open circles ⫽ unstimulated cells; closed circles ⫽
stimulated cells.
2331
2000). In our study, however, microautoradiography and
immunocytochemistry performed on the same section showed
that [3H](R)-PK11195 binding was restricted to GFAPnegative and CR3-positive cells. This included cells with a
larger soma and short, broad processes, typical of the fully
matured brain macrophages/microglia that are usually found
at later stages of brain damage, which, according to their
morphology, may resemble reactive astrocytes (Kuhlmann
and Guilarte, 2000). The failure to find complete overlap of
the reported immunocytochemical stain for the peripheral
benzodiazepine receptor and the cellular distribution of the
microautoradiographic PK11195 label may reflect sub-unit
dependent variations in either the sensitivity or the specificity
of both labels for the PBBS at its variously reported
mitochondrial, cytosolic and nuclear sites (Anholt et al., 1986;
Hertz, 1993; Hardwick et al., 1999). A further potentially
important difference from previous reports is the use of the
R-enantiomer of PK11195, which has a higher affinity for
the PK11195-binding site, rather than the commonly used
racemate (Shah et al., 1994). Our data on (R)-PK11195
binding in animal and human tissue sections show that in vivo
astrocytes are not a main contributor to regionally increased
binding, an interpretation that is supported by the lack of
significantly increased [11C](R)-PK11195 binding in
astrocyte-rich tissue, such as in patients with hippocampal
sclerosis (but infrequent seizures) (Banati et al., 1999).
An important finding of our study was the extent and the
clear demarcation of PBBS expression along anatomical
tracts by microglia that were still ramified, were not colocalized with reactive astrocytes and showed few signs of
activation. Our data suggest that during autoimmune disease
there is widespread, under-appreciated activation of microglia
in the white matter that is unrelated to autoreactive T-cells
which precedes obvious demyelination (Cuzner, 1997). It is
possible that regional microglia that have not yet developed
into full-blown macrophages but are already activated and
releasing locally active cytotoxic factors (Banati et al.,
1993; Li et al., 1996) are crucial for the process of early
demyelination leading to a ‘dying back gliopathy’ (Ludwin
and Johnson, 1981; Rodriguez and Scheithauer, 1994).
The presence of PBBS-expressing (i.e. activated) microglia
Fig. 4 Cellular localization of [3H](R)-PK11195 binding on activated microglia in EAE. The schematic drawing shows how spinal cords were
cut longitudinally to allow viewing of the [3H](R)-PK11195 binding pattern along the spinal columns. (A–E) [3H](R)-PK11195
autoradiographs. (A) Normal spinal cord and (B) preclinical spinal cord, i.e. 4 days after induction of EAE, showing a normal binding pattern
[grey matter 21.3 ⫾ 1.01 (SD) fmol/mg tissue, white matter 7.85 ⫾ 1.07 (SD) fmol/mg tissue]. At the peak of clinical disease (day 7) (C),
binding increased in white [17.48 ⫾ 0.93 (SD) fmol/mg tissue] and grey [46.76 ⫾ 8.48 (SD) fmol/mg] matter, with areas of maximum focal
binding [64.48 ⫾ 3.02 (SD) fmol/ mg tissue]. Similar increases are seen in the midbrain of the diseased animal (C, left). epend. ⫽ ependymal
layer showing constitutive [3H](R)-PK11195 binding. After recovery from clinical symptoms (day 10) (D), binding is decreased to normal
levels in white matter but is still slightly elevated in grey matter [33.79 ⫾ 3.09 (SD) fmol/mg tissue]. (F–M) High-resolution photoemulsion
[3H](R)-PK11195 autoradiography combined with OX-42 immunohistochemistry in EAE (7 days). The square frame in E delineates the
transition zone between white and grey matter, which is shown at higher magnification in F and G. A perivascular infiltrate (left arrows) and
an activated microglial cell (right arrow) stained with OX-42 are shown in F. When the focus is on the photoemulsion layer (G), silver grains
indicating [3H](R)-PK11195 binding can be seen within the perivascular infiltrate and on the activated microglial cell in the parenchyma. The
lower-magnification micrographs in H and K reveal that the focal binding of [3H](R)-PK11195 is predominantly to the perivascular cuffs,
while the increased [3H](R)-PK11195 binding (arrowheads in L, arrows in M) in the white matter outside the perivascular lesion is found
exclusively in OX-42-positive microglia (arrowheads in I, arrows in J). Sections were lightly counterstained with haematoxylin.
2332
R. B. Banati et al.
in normal-appearing white matter may indicate preferential
migration along white matter tracts that are ready for
demyelination. Alternatively, in situ proliferation rather than
migration may be the primary cause of the regionally
increased number of microglia in areas of subtle or imminent
brain damage. For example, in the facial nerve axotomy
models without blood–brain barrier damage or neuronal cell
death, rapid activation and proliferation of microglia occurs
along the entire pathway of the lesioned nerve and
simultaneously in the affected facial nucleus (Kreutzberg,
1996). Similarly, a peripheral lesion of the sciatic nerve
induces remote expression of microglial PBBS in the
brainstem in the nucleus gracilis via ipsilaterally ascending
nerve fibres (Banati et al., 1997). Likewise, in animals with
EAE, the inflammatory damage in the spinal cord may have
remotely induced the increased PBBS expression in the
midbrain through the affected ascending spinothalamic tracts
(Fig. 4C).
Peripheral benzodiazepine binding in CNS pathology
In vivo imaging of the glial immunopathology
in multiple sclerosis
Increased [11C](R)-PK11195-PET signals in the brainstem
and the thalamic nuclei secondary to pathology elsewhere
have been observed in patients with cortical damage due
to cerebrovascular stroke (Pappata et al., 2000) and in
experimental models of cortical damage associated with a
secondary microglial reaction in the ipsilateral thalamus
(Myers et al., 1991b; Sorensen et al., 1996). It is likely that
the phenomenon of a ‘projected neuroinflammatory’ response
in the wake of primary inflammatory lesions elsewhere along
a neural pathway also occurs in multiple sclerosis patients.
For example, in Patient 11, the increased thalamic binding
is likely to be the consequence of a cervicospinal cord lesion
seen in the MRI at level C3/C4, whereas in multiple sclerosis
patients with a recent history of optic neuritis the increased
[11C](R)-PK11195 signal in the lateral geniculate bodies is
the result of damage to the optic nerve. A recent longitudinal
MRI study of five patients with isolated neurological
syndromes suggestive of multiple sclerosis found a
distribution pattern of MRI-defined lesions that was spatially
dissociated from the primary inflammatory or demyelinating
foci but followed neural pathways in a fashion that is
characteristic of Wallerian degeneration (Simon et al., 2000).
It is unclear whether the local microglial activation in the
lateral geniculate bodies reflects synaptic terminal
degeneration or whether the transient axonal irritation of
the inflamed optic nerve is a sufficient stimulus.
As expected, areas where the blood–brain barrier is
disrupted, i.e. gadolinium-enhancing lesions in T1-weighted
MRI, which are likely to contain infiltrating mononuclear
cells (Nesbit et al., 1991), had the highest overlap with
increased [11C](R)-PK11195 binding, whereas old structural
lesions, represented by black holes (Truyen et al., 1996), had
little binding. However, during relapse the binding in black
holes more than doubled, indicating that during relapse there
is renewed disease activity in existing areas of severe tissue
destruction. No relapse-associated change was observed in
the other hypointense areas of the T1-weighted MRI excluding
black holes. In contrast, the hyperintense areas of the T2weighted MRI showed an increase of more than two-fold in
overlap with the [11C](R)-PK11195 signal during relapse, and
2333
thus they probably represent a more active disease volume,
i.e. PBBS-expressing tissue.
Despite the finding that the [11C](R)-PK11195 binding
within the T1-weighted MRI lesions (hypointensities
excluding black holes) appeared to be unrelated to acute
relapse, it correlated with increasing disability (total EDSS).
This fraction of [11C](R)-PK11195 binding within T1weighted MRI lesions may, therefore, reflect the secondary
neurodegenerative (Wallerian) pathology that underlies
progressive disability.
In concordance with the post-mortem data, our in vivo
observation of increased [11C](R)-PK11195 signals well
beyond focal lesions supports the notion that additional
mechanisms apart from the focal macrophage accumulations
found in the areas of blood–brain barrier leakage (Nesbit
et al., 1991; Gay et al., 1997) must contribute to the
progression of the disease. There is mounting evidence
that hitherto undetected disease processes in brain tissue that
appears normal in MRI may explain the imperfect correlation
between clinical neurological disability and the extent of
disease defined by different MRI spin-echo protocols (Husted
et al., 1994; Riahi et al., 1998; Gonen et al., 2000; Tortorella
et al., 2000).
Although patients with high disability scores generally had
either regionally and/or globally increased [11C](R)-PK11195
binding in our study, we found only a limited correlation
with clinical disability measured by standardized clinical
assessment scales (EDSS) (Kurtzke, 1983). This may in part
reflect the frequently discussed limitations of clinical scales
when applied to heterogeneous disease entities such as
multiple sclerosis (Riahi et al., 1998). It is also likely that,
as a marker of current disease activity, [11C](R)-PK11195
binding would relate better to parameters of clinical change
(e.g. speed of disease progression) than to cumulative
measures of long-standing and recent disability. This is
demonstrated in Patient 6, who was scanned during relapse
and suffered from transient speech apraxia (Mohr et al.,
1978). Here, the increased [11C](R)-PK11195 binding in the
vicinity of the left Broca’s area established the clinicopathological correlation which was not obvious in either the
EDSS scores or the global lesion loads, as defined by MRI
or PET imaging.
Fig. 6 MRI and [11C](R)-PK11195 PET. All images follow the radiological convention, i.e. the left side of the image corresponds to subject’s
right side. (A) Three orthogonal views of [11C](R)-PK11195 images co-registered and overlaid on the MRI of Patient 9, showing
spinothalamic tract-associated [11C](R)-PK11195 signals extending through the brainstem and pons into the thalamus. (B–D) T1-weighted (B)
and T2-weighted (C) MRI and [11C](R)-PK11195 PET (overlaid onto T1-weighted MRI) (D) of Patient 9 show lesions in all different spinecho MRI sequences that partially overlap with areas of significantly increased [11C](R)-PK11195 binding (red arrow).The white arrow points
to a ‘black hole’ in an area that appears strongly hypointense in the T1-weighted MRI and has little binding of [11C](R)-PK11195. Note,
however, that a similar black hole (yellow arrowhead) adjacent to the right occipital horn of the lateral ventricle shows significant [11C](R)PK11195 binding. (E–F) Demonstration of the definition of the MRI lesion load masks in Patient 9 (purple, T1-weighted MRI lesions
excluding black holes; blue, black hole only; green, gadolinium-enhancing areas; dark grey (in F), T2-weighted MRI lesions; red, areas of
overlap between significantly increased [11C](R)-PK11195 binding and MRI-defined areas of pathology); yellow, areas of increased [11C](R)PK11195 binding and no overlap with any MRI-defined pathology. (G) Average percentage volume of the MRI-defined lesions overlapping
with increased [11C](R)-PK11195 binding. The red square represents Patient 8 and the red triangle Patient 6, who were both in relapse at the
time of the scans. The yellow diamond represents Patient 9, who had secondary progressive multiple sclerosis. T1*, black holes.
2334
R. B. Banati et al.
Fig. 7 Focal [11C](R)-PK11195 binding. (A and B) A focus of elevated [11C](R)-PK11195 binding (arrow)
in the brainstem co-localizing with the medial longitudinal fasciculus (MLF) is seen in this patient (Patient
9), who was suffering from internuclear ophthalmoplegia at the time of the PET scan. MR ⫽ medial rectus
muscle. (C and D) Patient 2, who had a recent history of optic neuritis. There is increased [11C](R)PK11195 binding in the lateral geniculate bodies (LGB), which are more pronounced on the right side.
The schematic drawing indicates that optic neuritis caused an anterograde neuronal reaction of the optic
nerve (NII) in the lateral geniculate body. VC ⫽ visual cortex. Extracerebral signals from regions such as
the retro-orbital glands have been removed from the [11C](R)-PK11195 images for the sake of clarity.
(E–H) Patient 6, who had transient speech dyspraxia (reduced fluency of spontaneous speech with only
mild language disturbance) at the time of the scans. There was increased [11C](R)-PK11195 binding in the
left frontal operculum close to Broca’s area in the anterior portion of the large white matter tract formed by
the superior longitudinal fascicle (SLF). A small but clinically silent lesion, which is gadolinium-enhancing
in the T1-weighted MRI (E), is seen to have a similarly confined signal in the [11C](R)-PK11195 PET (red
arrow).
Peripheral benzodiazepine binding in CNS pathology
Limitation of the study and outlook
[11C](R)-PK11195
Binding of
was measured with a simplified reference region approach and cluster analysis that
permitted the robust extraction of a normal reference
kinetic without prior anatomical definition of a normal
reference region. However, one patient with SP multiple
sclerosis had to be excluded from this study since no
appropriate concentration time–activity curve could be
extracted to serve as an input function. This may indicate
that, in this patient with extremely severe brain disease,
the amount of healthy tissue from which a normal input
function could have been derived was too low.
The presence of small amounts of specifically bound
[11C](R)-PK11195 (Petit-Taboue, 1991) in normal white and
grey matter would lead to slight underestimation of the true
binding. Since similar reference tissue curves were used both
for patients and for controls, the small reduction in BP would
be consistent across all subjects.
This study has focused on the expression of the PBBS as
a marker of active disease, yet some evidence points to the
possibility that the PBBS may play a more active role in
inflammation and may thus itself be a target for therapeutic
intervention. The PBBS is known to regulate steroidogenesis
(Krueger and Papadopoulos, 1993) and modulate macrophage
functions (Pawlikowski, 1993), while some PBBS ligands
appear to possess anti-inflammatory properties (Torres et al.,
1999). Finally, microglia are activated not only in inflammatory diseases, such as multiple sclerosis, but also in chronic
neurodegenerative diseases, such as Alzheimer’s dementia,
for which the potentially beneficial effects of antiinflammatory drug treatment are already under investigation
(McGeer and McGeer, 1995). Thus, in future, a broader
concept of activated microglia as mediators of ‘neuroinflammation’ together with new methods for the in vivo
detection of microglial activation may also be applied to
primarily non-inflammatory CNS diseases.
2335
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Acknowledgements
We wish to thank our patients, who volunteered to participate
in this study, Dr V. Cunningham for advice on PET data
quantification, J. Chalcroft, D. Buringer, K. Bruckner,
M. Falkenberg and I. Milojevic for technical assistance, the
radiographers A. Blythe, D. Griffith, J. Holmes, H. McDevitt
and L. Schnorr, and Professor S. McNeish. R.B.B. is supported
by a grant from the Deutsche Forschungsgemeinschaft and
the Multiple Sclerosis Society of Great Britain and Northern
Ireland. Material and financial support was given by the
Max-Planck-Institute of Psychiatry, Martinsried and by Dr
Hammer and Dr Schneider of Boehringer Ingelheim,
Ingelheim, Germany. A.C. is supported by a Marie Curie
fellowship from the European Community in the Training and
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Received March 27, 2000. Revised June 7, 2000.
Accepted June 29, 2000