molecules
Article
[18F]fallypride-PET/CT Analysis of the Dopamine
D2/D3 Receptor in the Hemiparkinsonian Rat Brain
Following Intrastriatal Botulinum Neurotoxin
A Injection
Teresa Mann 1, *, Jens Kurth 2 ID , Alexander Hawlitschka 1 , Jan Stenzel 3 , Tobias Lindner 3
Stefan Polei 3 , Alexander Hohn 2 , Bernd J. Krause 2 and Andreas Wree 1
1
2
3
*
ID
,
Institute of Anatomy, Rostock University Medical Center, Gertrudenstrasse 9, 18057 Rostock, Germany;
alexander.hawlitschka@med.uni-rostock.de (A.H.); andreas.wree@med.uni-rostock.de (A.W.)
Department of Nuclear Medicine, Rostock University Medical Centre, Gertrudenplatz 1, 18057 Rostock,
Germany; jens.kurth@med.uni-rostock.de (J.K.); alexander.hohn@med.uni-rostock.de (A.H.);
bernd.krause@med.uni-rostock.de (B.J.K.)
Core Facility Multimodal Small Animal Imaging, Rostock University Medical Center, Schillingallee 69a,
18057 Rostock, Germany; Jan.Stenzel@med.uni-rostock.de (J.S.); tobias.lindner@med.uni-rostock.de (T.L.);
stefan.polei@gmx.de (S.P.)
Correspondence: teresa.mann@med.uni-rostock.de; Tel.: +49-381-494-8433
Received: 30 January 2018; Accepted: 4 March 2018; Published: 6 March 2018
Abstract: Intrastriatal injection of botulinum neurotoxin A (BoNT-A) results in improved
motor behavior of hemiparkinsonian (hemi-PD) rats, an animal model for Parkinson’s disease.
The caudate–putamen (CPu), as the main input nucleus of the basal ganglia loop, is fundamentally
involved in motor function and directly interacts with the dopaminergic system. To determine
receptor-mediated explanations for the BoNT-A effect, we analyzed the dopamine D2 /D3
receptor (D2 /D3 R) in the CPu of 6-hydroxydopamine (6-OHDA)-induced hemi-PD rats by
[18 F]fallypride-PET/CT scans one, three, and six months post-BoNT-A or -sham-BoNT-A injection.
Male Wistar rats were assigned to three different groups: controls, sham-injected hemi-PD rats, and
BoNT-A-injected hemi-PD rats. Disease-specific motor impairment was verified by apomorphine
and amphetamine rotation testing. Animal-specific magnetic resonance imaging was performed for
co-registration and anatomical reference. PET quantification was achieved using PMOD software
with the simplified reference tissue model 2. Hemi-PD rats exhibited a constant increase of 23% in
D2 /D3 R availability in the CPu, which was almost normalized by intrastriatal application of BoNT-A.
Importantly, the BoNT-A effect on striatal D2 /D3 R significantly correlated with behavioral results in
the apomorphine rotation test. Our results suggest a therapeutic effect of BoNT-A on the impaired
motor behavior of hemi-PD rats by reducing interhemispheric changes of striatal D2 /D3 R.
Keywords: D2 /D3 receptors; hemiparkinsonian rat model; Botulinum neurotoxin A; basal ganglia;
striatum; Parkinson’s disease; small animal imaging; PET/CT; [18 F]fallypride; MRI
1. Introduction
Positron emission tomography (PET) using the radioligand [18 F]fallypride enables in vivo
detection of disease-specific alterations of the dopaminergic system, more precisely of D2 /D3 receptor
(D2 /D3 R) availability. Small animal PET hybrid tomographs allow imaging and quantification of
D2 /D3 R binding by the use of [18 F]fallypride in rodent models of neurodegenerative disorders like
Parkinson’s disease (PD) [1–3]. [18 F]fallypride is characterized by high sensitivity and selectivity for
D2 R; for instance, administration of the D2 R antagonist haloperidol blocked specific [18 F]fallypride
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Molecules 2018, 23, 587
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binding in the mouse caudate–putamen (CPu) by 95% [4,5]. Besides specific binding to D2 R,
[18 F]fallypride displays affinity to the D2 -like D3 R, about 20% of the radioligand bind to D3 R
in vivo in small animals [6]. In PD a drastic loss of striatal dopamine (DA) caused by progressing
degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) results in
imbalanced neurotransmitter systems and underlies motor complications. Moreover, due to DA
depletion and the subsequent missing inhibition of striatal cholinergic interneurons, hypercholinism is
an additional feature of PD exacerbating motor impairment [7,8]. Interestingly, there are physiological
bidirectional regulating effects of the cholinergic and dopaminergic system [9] via complex involvement
of muscarinic and nicotinic receptors on DA release from dopaminergic terminals [10,11].
For causal analysis and development of novel therapeutics for PD in preclinical research the
experimental hemiparkinsonian (hemi-PD) is an accepted animal model [12]. Unilateral injection
of 6-hydroxdopamine (6-OHDA) into the medial forebrain bundle (MFB) of rats provokes rapid
dopaminergic depletion by auto-oxidation and consequent oxidative stress [13]. The near-complete
loss of dopaminergic neurons in the SNpc via retrograde axonal transport of the toxin 6-OHDA after
stereotaxic injection into the MFB mimics a late stage of PD [14]. Current therapeutic strategies for
PD focus primarily on compensation of DA in the striatum (caudate–putamen, CPu) by either DA
precursors [15,16] or DA receptor agonists [17]. Though, clinical efficiency is limited and chronic
administrations leads to severe side effects like motor fluctuations and dyskinesia [18,19]. Other
therapeutic options target the cholinergic system mostly by blocking muscarinic receptors or by
inhibition of cholinesterase [20,21]. Systemic administration of anticholinergic substances causes
severe side effects like confusion, dry mouth, blurred vision, and cognitive impairment [22].
Recently, we demonstrated that local injection of the anticholinergic Botulinum neurotoxin A
(BoNT-A) significantly improved D2 R agonist-induced asymmetric rotational behavior in hemi-PD
rats [23–26]. BoNT-A acts mainly on cholinergic neurons and inhibits distribution of acetylcholine into
the synaptic cleft via cleavage of the synaptosomal-associated protein of 25-kDa (SNAP25) [27–29]. The
intracerebral injection of BoNT-A avoids severe side effects in both the central and peripheral nervous
system [23]. Notably, intrastriatal application of BoNT-A does not cause cytotoxicity [30] or impaired
cognition [24] in rats. As known from other medical implementation, BoNT-A demonstrates a transient
therapeutic effect in hemi-PD rats that lasts up to six months post-injection [23,31]. To examine the
longitudinal cellular mechanisms of the positive BoNT-A effect on receptor level, we performed
[18 F]fallypride-PET/CT scans one, three, and six months post-BoNT-A or -sham-BoNT-A injection
and quantified D2 /D3 R availability in controls, sham-injected hemi-PD rats, and BoNT-A-injected
hemi-PD rats.
2. Results
D2 /D3 R availability was analyzed longitudinally in controls (sham-6-OHDA + sham-BoNT-A,
n = 9), sham-injected hemi-PD rats (6-OHDA + sham-BoNT-A, n = 7) and BoNT-A-injected hemi-PD
rats (6-OHDA + BoNT-A, n = 10) by dynamic [18 F]fallypride-PET/CT scans.
2.1. Immunohistochemistry and Behavioral Testing
To qualitatively verify successful 6-OHDA-induced dopaminergic deafferentation we performed
tyrosine hydroxylase (TH) immunostaining (for dopaminergic neurons). TH-reaction in the left
and right CPu and SN of control rats (sham-6-OHDA + sham-BoNT-A) showed no loss of
TH-reaction (Figure 1a,b). In hemi-PD rats (6-OHDA + sham-BoNT-A) an ipsilateral loss of almost
all TH-immunoreactivity was visible in the CPu and SN, indicating dopaminergic deafferentation in
the CPu due to dopaminergic cell loss in the SN (Figure 1c,d), and BoNT-A injection in hemi-PD rats
(6-OHDA + BoNT-A) did not demonstrate an additive effect on these reaction patterns (Figure 1e,f).
−
−
−
−
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(a)
(b)
(c)
(d)
(e)
(f)
Figure 1. TH-immunoreactivity in the telencephalon (left column) and the mesencephalon (right
column) of (a,b) controls (sham-6-OHDA + sham-BoNT-A) (c,d) sham-injected hemi-PD rats (6-OHDA
+ sham-BoNT-A) and (e,f) BoNT-A-injected hemi-PD rats (6-OHDA + BoNT-A). 6-OHDA or
sham-6-OHDA was unilaterally injected into the MFB of the right hemisphere and BoNT-A was
injected ipsilateral at two sites into the CPu. Controls showed symmetric TH pattern in the CPu (a,c,e;
black (left) and white (right) arrow) and SN (b,d,f; black (left) and white (right) arrow), sham-injected
hemi-PD rats demonstrated an almost complete loss of TH-positive cells in the CPu and SN and BoNT-A
injection did not influence these findings in hemi-PD rats. The scale bar applies for a–f = 1 mm.
Asymmetric rotations of hemi-PD rats were tested using apomorphine- and
amphetamine-induced rotations one month after 6-OHDA lesion. Also, the positive effect
of intrastriatally injected BoNT-A on drug-induced rotations was analyzed two weeks
after administration.
All hemi-PD rats exhibited distinct apomorphine-induced rotations
contralateral to the 6-OHDA lesion before BoNT-A or sham-BoNT-A injection of 8.2 ± 3.6 rpm
(6-OHDA + sham-BoNT-A) and 9.2 ± 3.0 rpm (6-OHDA + BoNT-A), controls did not show rotational
behavior (sham-6-OHDA + sham-BoNT-A) (Figure 2a). Sham injection in hemi-PD rats slightly
decreased rotations to 4.6 ± 2.5 rpm (6-OHDA + sham-BoNT-A) and did not affect the behavior
of controls (sham-6-OHDA + sham-BoNT-A). Following BoNT-A injection rotational behavior was
reversed in hemi-PD rats to 2.2 ± 2.1 rpm (6-OHDA + BoNT-A). The positive motor effect of BoNT-A
in hemi-PD rats was significant compared to sham injections (p = 0.021) (Figure 2a). Before BoNT-A or
sham-BoNT-A injection amphetamine administration caused ipsilateral rotations in hemi-PD rats
of −7.3 ± 3.4 rpm (6-OHDA + sham-BoNT-A) and −6.6 ± 2.9 rpm (6-OHDA + BoNT-A) but not
Molecules 2018, 23, 587
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in controls (sham-6-OHDA + sham-BoNT-A) (Figure 2b). After BoNT-A or sham-BoNT-A injection
asymmetric rotational behavior was with −12.0 ± 6.3 rpm (6-OHDA + BoNT-A) and −10.3 ± 3.5 rpm
(6-OHDA + sham-BoNT-A) further increased and BoNT-A effect was compared to sham injection not
significantly abolished. Sham-BoNT-A injection in controls (sham-6-OHDA + sham-BoNT-A) did not
result in ipsilateral rotations (Figure 2b).
(a)
(b)
Figure 2. Results of the rotational behavior in (a) apomorphine- and (b) amphetamine-induced testing
for controls (sham-6-OHDA + sham-BoNT-A), sham-injected hemi-PD rats (6-OHDA + sham-BoNT-A)
and BoNT-A-injected hemi-PD rats (6-OHDA + BoNT-A) displayed after 6-OHDA or sham-6-OHDA
and BoNT-A or sham-BoNT-A injection. Rotations contralateral to the injection side (clockwise)
are displayed with negative algebraic signs, anti-clockwise rotations with positive algebraic signs.
Controls did not demonstrate designated rotational behavior, hemi-PD rats exhibited strong asymmetric
drug-induced rotations that were almost completely abolished after BoNT-A injection in apomorphine
testing and slightly abolished in amphetamine testing. Significance is displayed as ** p < 0.01.
2.2. Striatal D2 /D3 R Availability
Qualitative analysis with parametric mapping of non-displaceable binding potential (BPnd )
revealed no obvious interhemispheric differences for controls (sham-6-OHDA + sham-BoNT-A).
However, increased signals in the right CPu of hemi-PD rats were visible (6-OHDA + sham-BoNT-A)
compared to the unaffected side. This visual right–left difference was diminished after BoNT-A
injection into the right CPu of hemi-PD rats (6-OHDA + BoNT-A) (Figure 3a–c).
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(a)
(b)
(c)
Figure 3. Qualitative analysis using pixel-wised parametric mapping for the parameter BPnd showing
the left and right CPu in transversal sections for (a) controls (sham-6-OHDA + sham-BoNT-A)
(b) sham-injected hemi-PD rats (6-OHDA + sham-BoNT-A) and (c) BoNT-A-injected hemi-PD rats
(6-OHDA + BoNT-A) one month after BoNT-A or sham-BoNT-A injection. A representative animal
of each experimental group was used. Controls did not reveal visual side differences, an increased
signal of BPnd distribution in the right CPu of hemi-PD rats was clearly visible and BoNT-A injection
normalized the increased signal in hemi-PD rats. The right CPu is marked with a red arrow.
For quantification the simplified reference tissue model 2 (SRTM2) was applied and BPnd was
estimated separately for the left and right striatum. Controls (sham-6-OHDA + sham-BoNT-A) revealed
no relative interhemispheric right–left differences: mean BPnd of 4.2 ± 0.8/4.2 ± 0.8 (left/right
CPu) one month post-sham-BoNT-A injection, 3.8 ± 0.8/3.7 ± 0.9 (left/right CPu) three months
post-sham-BoNT-A injection and 4.1 ± 0.7/4.1 ± 0.7 (left/right CPu) six months post-sham-BoNT-A
injection were found (Figure 4a,c; Table 1). Hemi-PD rats that received sham-BoNT-A injection
(6-OHDA + sham-BoNT-A) exhibited strong interhemispheric right–left differences of about 23%:
kinetic analysis resulted in mean BPnd values of 4.4 ± 0.6/5.5 ± 0.9 (left/right CPu) one month
post-sham-BoNT-A injection, 4.4 ± 1.1/5.5 ± 1.3 (left/right CPu) three months post-sham-BoNT-A
injection, and 4.8 ± 1.0/5.7 ± 1.0 (left/right CPu) six months post-sham-BoNT-A injection. The
contralateral CPu was never affected and displayed very stable BPnd throughout all experimental
groups and scanning time points (Figure 4a–c; Table 1). The increase in BPnd in the right CPu of
hemi-PD rats (6-OHDA + sham-BoNT-A) was significant compared to the left CPu of the same
experimental group one month post-sham-BoNT-A injection (p = 0.047) (Figure 4a) and compared to
the right CPu of controls (sham-6-OHDA + sham-BoNT-A) at all 3 examined time points (p = 0.03,
p = 0.064, p = 0.039) (Figure 4a–c). BoNT-A injection in hemi-PD rats (6-OHDA + BoNT-A) reduced
relative interhemispheric right–left difference to about 13.4%: quantification revealed mean BPnd of
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4.7 ± 0.4/5.3 ± 0.5 (left/right CPu) one month post-BoNT-A injection, 4.4 ± 1.0/5.0 ± 1.2 (left/right
CPu) three months post-BoNT-A injection and 4.4 ± 1.0/5.1 ± 1.1 (left/right CPu) six months
post-BoNT-A injection (Figure 4a–c; Table 1). The BoNT-A effect was significant compared to
the ipsilateral CPu of controls (sham-6-OHDA + sham-BoNT-A) one month post-BoNT-A injection
(p = 0.0087) and showed a transient course throughout the timeline (Figure 4a–c). A list of all individual
values for BPnd expressing D2 /D3 R availability separately for the left and right CPu and the relative
interhemispheric right–left difference in each of the 26 analyzed rats is displayed in Table 1.
(a)
(b)
(c)
Figure 4. Box plots for BPnd values of D2 /D3 R depicting median and interquartile ranges
separately for the contralateral (dark grey) and ipsilateral (light grey) CPu for controls
(sham-6-OHDA + sham-BoNT-A), sham-injected hemi-PD rats (6-OHDA + sham-BoNT-A) and
BoNT-A-injected hemi-PD rats (6-OHDA + BoNT-A) (a) one month, (b) three months, and (c) six
months post-BoNT-A or -sham-BoNT-A injection. D2 /D3 R availability was consistently symmetric
in controls, increased in sham-injected hemi-PD rats at all analyzed time points and was reduced to
nearly normal values after BoNT-A injection in hemi-PD rats. Significance is displayed as * p < 0.05,
** p < 0.01.
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Table 1. Summary of all single BPnd values of D2 /D3 R for the left and right CPu and the interhemispheric difference relative to the left hemisphere in (%) analyzed in
controls (sham-6-OHDA + sham-BoNT-A), sham-injected hemi-PD rats (6-OHDA + sham-BoNT-A) and BoNT-A-injected hemi-PD rats (6-OHDA + BoNT-A). Data
are shown for all three PET/CT scans (PET/CT 1: one month post-BoNT-A or sham-BoNT-A, PET/CT 2: three months post-BoNT-A or -sham-BoNT-A, PET/CT 3:
six months post-BoNT-A or -sham-BoNT-A). * indicate that no data were analyzed due to incorrect tracer injection or no data acquisition.
Group
PET/CT 1
PET/CT 2
PET/CT 3
BPnd Left
BPnd Right
(%)
BPnd Left
BPnd Right
(%)
BPnd Left
BPnd Right
(%)
sham-6-OHDA + sham-BoNT-A
sham-6-OHDA + sham-BoNT-A
sham-6-OHDA + sham-BoNT-A
sham-6-OHDA + sham-BoNT-A
sham-6-OHDA + sham-BoNT-A
sham-6-OHDA + sham-BoNT-A
sham-6-OHDA + sham-BoNT-A
sham-6-OHDA + sham-BoNT-A
sham-6-OHDA + sham-BoNT-A
5.06
2.92
4.53
4.58
4.03
*
*
*
*
4.98
2.92
4.53
4.59
3.80
*
*
*
*
−1.66
−0.01
−0.04
0.28
−5.71
*
*
*
*
2.21
3.74
2.87
4.25
4.24
3.81
4.73
4.27
4.23
2.07
3.93
2.78
4.15
3.93
3.46
4.89
4.32
4.13
−6.35
5.21
−3.37
−2.28
−7.22
−9.32
3.34
1.04
−2.48
*
4.56
3.29
4.65
4.07
4.68
2.80
4.50
4.07
*
4.44
3.23
4.53
4.02
4.52
2.97
4.66
4.30
*
−2.66
−1.84
−2.46
−1.36
−3.47
6.23
3.60
5.66
|Mean| ± SD
4.2 ± 0.8
4.2 ± 0.8
1.4 ± 2.5
3.8 ± 0.8
3.7 ± 0.9
2.4 ± 4.9
4.1 ± 0.7
4.1 ± 0.7
0.4 ± 4.0
6-OHDA + sham-BoNT-A
6-OHDA + sham-BoNT-A
6-OHDA + sham-BoNT-A
6-OHDA + sham-BoNT-A
6-OHDA + sham-BoNT-A
6-OHDA + sham-BoNT-A
6-OHDA + sham-BoNT-A
4.86
5.41
3.77
4.08
3.96
4.49
*
6.53
6.65
4.67
4.71
4.79
5.54
*
34.37
22.90
23.84
15.53
21.02
23.50
*
6.11
*
3.35
4.44
*
4.74
3.53
7.67
*
4.54
4.88
*
5.64
4.71
25.53
*
35.56
10.04
*
18.80
33.46
4.11
4.66
5.45
5.21
*
4.33
*
4.59
5.54
7.26
5.83
*
5.37
*
11.53
19.07
33.13
11.98
*
23.95
*
|Mean| ± SD
4.4 ± 0.6
5.5 ± 0.9
23.5 ± 6.1
4.4 ± 1.1
5.5 ± 1.3
24.7 ± 14.8
4.8 ± 1.0
5.7 ± 1.0
19.9 ± 11.4
6-OHDA + BoNT-A
6-OHDA + BoNT-A
6-OHDA + BoNT-A
6-OHDA + BoNT-A
6-OHDA + BoNT-A
6-OHDA + BoNT-A
6-OHDA + BoNT-A
6-OHDA + BoNT-A
6-OHDA + BoNT-A
6-OHDA + BoNT-A
4.58
4.54
5.07
5.09
4.85
4.37
4.59
3.88
5.30
5.10
5.33
4.90
5.54
5.42
5.43
5.15
5.12
4.28
6.22
5.59
16.19
7.94
9.23
6.39
11.80
17.86
11.55
10.26
17.22
9.74
*
5.47
4.41
2.43
*
*
3.88
4.33
4.94
5.32
*
6.10
5.15
2.79
*
*
4.06
5.13
5.26
6.41
*
11.59
16.68
14.93
*
*
4.77
18.59
6.49
20.38
3.30
*
2.63
4.74
5.88
4.86
3.63
5.00
4.58
5.01
3.92
*
3.13
5.25
6.38
6.03
3.87
5.93
5.23
5.78
18.62
*
19.11
10.77
8.56
24.05
6.45
18.53
14.22
15.38
|Mean| ± SD
4.7 ± 0.4
5.3 ± 0.5
11.8 ± 4.0
4.4 ± 1.0
5.0 ± 1.2
13.4 ± 6.0
4.4 ± 1.0
5.1 ± 1.1
15.1 ± 5.7
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2.3. Correlation of D2 /D3 R Side Differences and Behavior
A possible correlation of the degree of interhemispheric differences in D2 /D3 R
availability and apomorphine-induced rotations for controls (sham-6-OHDA + sham-BoNT-A),
sham-injected hemi-PD rats (6-OHDA + sham-BoNT-A) and BoNT-A-injected hemi-PD rats
(6-OHDA + BoNT-A) was examined one month post-BoNT-A or -sham-BoNT-A injection. Controls
(sham-6-OHDA + sham-BoNT-A) did not demonstrate right–left differences or rotational behavior.
With increasing right–left differences contralateral rotations of hemi-PD rats (6-OHDA + sham-BoNT-A)
increased and also the normalizing effect on interhemispheric D2 /D3 R differences after BoNT-A
injection (6-OHDA + BoNT-A) was connected with behavior. A highly significant relationship between
increasing right–left differences, expressing a higher D2 /D3 R availability in the right CPu, and the
apomorphine-induced rotational behavior was found (p = 0.0007) (Figure 5).
Figure 5.
Linear correlation of right–left differences of D2 /D3 R availability in (%) and
apomorphine-induced rotations one month after BoNT-A or sham-BoNT-A injection for controls
(sham-6-OHDA + sham-BoNT-A), sham-injected hemi-PD rats (6-OHDA + sham-BoNT-A)
and BoNT-A-injected hemi-PD rats (6-OHDA + BoNT-A). Controls did neither demonstrate
interhemispheric differences nor rotational behavior. With increasing right–left differences also
asymmetric rotations increased in hemi-PD rats and both was normalized after BoNT-A injection.
Significance is displayed as *** p < 0.001.
3. Discussion
In this study we examined cellular mechanisms of the positive motor effect of intrastriatally
injected BoNT-A by [18 F]fallypride PET/CT scans in hemi-PD rats, as BoNT-A was previously
demonstrated to abolish apomorphine-induced rotational behavior in 6-OHDA-lesioned [23–26].
The control group respected the entire surgical procedure as the minimal lesion caused by the
insertion of the syringe could lead to changes in receptor binding sites [32,33]. We did not include an
experimental group studying BoNT-A in sham-lesioned rats in our design as we assumed that BoNT-A
would not alter per se the expression of D2 /D3 receptors. Indeed, we have previously performed
extensive in vitro analysis of D2 /D3 receptors as well as apomorphine-induced rotational behavior in
BoNT-A-injected rats earlier and did not find major effects [31].
Hemi-PD rats demonstrated a constant contralateral rotational behavior after apomorphine
injection and rather inconsistent amphetamine-induced rotations four weeks after 6-OHDA lesion
(Figure 2a,b). A period of four weeks before behavioral testing was left to ensure maximum
dopaminergic deafferentation, as dopaminergic cell death [34] as well as consequent plasticity
effects [31] last up to four weeks after injection of 6-OHDA. Notably, increasing right–left differences of
D2 /D3 R availability significantly correlated with increasing asymmetry in apomorphine-induced
rotations (Figure 5). Rotational tests using the D2 agonist apomorphine or the DA releaser
amphetamine are commonly used to detect the degree of dopaminergic deafferentation in hemi-PD rats.
As apomorphine acts on increased striatal D2 /D3 R in the DA-depleted CPu of hemi-PD rats [35–37],
Molecules 2018, 23, 587
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it leads to a larger inhibition of the right CPu and as a consequence to an elevated motor urge to
the contralateral side. Resulting rotations to the left of more than four rotations per minute confirm
dopaminergic degeneration of more than 90% [25,38–40]. Amphetamine induces DA release form
nerve terminals and strongly affects the non-lesioned hemisphere of hemi-PD rats, which begin to turn
to the ipsilateral side of the 6-OHDA lesion [41]. In line with our findings, another study demonstrated
that apomorphine but not amphetamine is a reliable indicator for maximal dopaminergic cell death in
hemi-PD rats [42].
Dynamic [18 F]fallypride PET/CT scans over 90 min revealed an increase of 23% in D2 /D3 R
availability being consistent up to six months post-6-OHDA lesion and a normalization of this
pathological imbalance after BoNT-A injection into the CPu of hemi-PD rats (Figures 3 and 4; Table 1).
Unlike [11 C]raclopride, [18 F]fallypride is not easily displaced by endogenous DA, as demonstrated
in monkey [43], human [44] and rat brain [45]. To cover the transient effect of BoNT-A demonstrated
previously [23], we performed longitudinal measurements using [18 F]fallypride in the same rodent.
This seemed feasible as repeated measurements with [18 F]fallypride PET/CT exhibited only small
variations in mice [5] and also in our study, controls (sham-6-OHDA + sham-BoNT-A) did not show
variations in BPnd comparing the three PET/CT scanning time points (Figures 3a and 4a–c ; Table 1).
Our finding of a constant increase of about 23% in D2 /D3 R availability in hemi-PD rats is in line
with a number of similar studies both in vitro and in vivo. Unilateral injection of 6-OHDA into the MFB
or SNpc of rats resulted in a consistent increase of D2 R density of 20% to 40% subject to the injected
dosage and survival time analyzed using in vitro autoradiography [35,46–50]. Also, in vivo PET/CT
analyses with [11 C]raclopride and [18 F]fallypride are in accordance with our results. [11 C]raclopride
PET demonstrated an ipsilateral increase of 17% to 27.7% [2] and approximately 35% [51] in D2 R
availability in hemi-PD rats after MFB injection and an increase of 23% [52] and 16.6% [53] after
6-OHDA lesion of the SNpc. [18 F]fallypride PET/CT scans revealed an 12% increase in D2 /D3 R
availability in hemi-PD rats after injection of 6-OHDA into the CPu [2].
Intrastriatally injections with BoNT-A significantly reduced the pathologically increased D2 /D3 R
availability in hemi-PD rats (Figures 3c and 4) and significantly abolished apomorphine-induced
rotations (Figure 2a). Apomorphine-induced rotations were also moderately decreased after
sham-BoNT-A injection in hemi-PD rats. This effect is likely to be caused by minimal mechanical
damage caused by insertion of the cannula into the CPu and injection of sham solution. One might
argue that the positive BoNT-A effect is caused by simple striatal cell death after BoNT-A injection,
as D2 /D3 R are localized on medium spiny neurons (MSN), presynapses of cholinergic interneurons
and boutons of dopaminergic afferents in the CPu [54–56]. Previously we demonstrated that BoNT-A
injection did neither cause striatal neuronal loss or reduced volume [26] nor death of cholinergic
interneurons in the CPu [30]. The positive BoNT-A effect diminished with increasing post-injection
time. The BoNT-A effect on D2 /D3 R in hemi-PD rats has been analyzed before in a quantitative
in vitro autoradiography study demonstrating a normalizing effect, which significantly correlates with
apomorphine-induced rotations [31].
A preceding study performed [11 C]raclopride PET to analyze the effect of BoNT-A on pathological
increased D2 /D3 R and found a positive effect on pathological increased D2 R availability in
hemi-PD rats [3]. Here, we extended this experimental setup by introducing a control group
(sham-6-OHDA + sham-BoNT-A) to investigate both ipsi- and contralateral effects of tissue damage
by cannula injection and increased group size to substantiate possible significant effects. Moreover,
we used the more specific radioligand [18 F]fallypride instead of [11 C]raclopride and conducted
animal-specific MRI scans for co-registration with CT-corrected PET data to improve data analysis by
making use of the high morphologic resolution of MRI.
Quantification was subsequently performed using SRTM2 [57] for kinetic modeling, having the
advantage of no need for arterial blood sampling and being established especially for neuroimaging
studies [58]. Application of kinetic models allows quantitative determination of transfer rates and
provides in depth understanding of physiological parameters. The cerebellum was used as the
Molecules 2018, 23, 587
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reference region being devoid of D2 /D3 R [59] and being validated as a suitable reference region for
D2 /D3 R before [60–62]. As spatial resolution of PET scans is a limiting factor especially in small
animals [63], we used animal-specific MRI scans for spatial normalization, thus high precision of
Voxels of interest (VOI) delineation and almost full recovery could be guaranteed in our study. Partial
volume effects (PVE) occur mostly in structures being smaller than about 2 times of the width at half
maximum of the scanner [64,65]. As our rats exhibited striatal volumes of about 35 to 50 mm2 [26,66]
and the utilized scanner is characterized by a spatial resolution of 1.5 mm, the CPu as the target region
exceeded the critical size for the occurrence of PVE. Moreover, the CPu seems to be only minimally
effected by extracranial gland and skull activity resulting from its deep localization within the brain [6].
Due to predominantly relative comparisons between hemispheres within treatment groups in our
study setup, a potentially emerging PVE would be mathematically shortened. Altogether, in this study
we decided to omit PVE correction for quantification of [18 F]fallypride uptake in the CPu of hemi-PD
rats. Also in comparable studies, no correction of the PVE seemed necessary for quantification of
radioligands demonstrating very specific and region-limited binding kinetics like [18 F]fallypride [2] or
[11 C]raclopride [67]. Nevertheless, a methodical consideration of this aspect might be of high interest
for further investigations. Another critical point of small animal imaging is the need for anesthesia,
commonly realized by respiration of isoflurane. For analyzing the availability of D2 /D3 R effects of
isoflurane seem not pivotal, as the BP of D2 R measured with [11 C]raclopride was only marginally
changed in mice [5] and [18 F]fallypride uptake did not differ in awake rats that received late isoflurane
anesthesia compared to rats under continuous anesthesia [61].
4. Materials and Methods
4.1. Animals
Twenty-six male Wistar rats (Charles River WIGA, Sulzfeld, Germany; RRID: RGD_737929)
either assigned to controls (sham-6-OHDA + sham-BoNT-A, n = 9), sham-injected hemi-PD rats
(6-OHDA + sham-BoNT-A, n = 7) or BoNT-A-injected hemi-PD rats (6-OHDA + BoNT-A, n = 10) were
included in the experiments. Housing was conducted under standard conditions (22 ± 2 ◦ C, 12 h
day-and-night cycle) in a fully air-conditioned room with access to water and food ad libitum. The
research protocol and all experimental procedures fulfilled legal obligations of the animal welfare act
and were approved by the state Animal Research Committee of Mecklenburg–Western Pomerania
(LALLF M-V/7221.3-1-005/16, approval: 03/08/2016). The timeline of the experimental setup is
presented in Figure 6.
Figure 6. Timeline of the study design. Hemi-PD was unilaterally induced by 6-OHDA injection into
the right MFB. Controls received sham-6-OHDA injection. The degree of dopaminergic cell loss was
verified with apomorphine- and amphetamine-induced behavioral testing. Five to six weeks after
the 6-OHDA or sham-6-OHDA injection, rats obtained BoNT-A or sham-BoNT-A injection into the
ipsilateral CPu. The positive effect on the motor behavior of BoNT-A was then controlled in rotation
tests. Subsequently, each rat was scanned by [18 F]fallypride-PET/CT analysis one, three and six months
post-BoNT-A or -sham-BoNT-A injection. A final MRI scan was performed as anatomical reference for
PET/CT imaging.
μ
μ
Molecules 2018, 23, 587
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4.2. 6-OHDA and BoNT-A Injection
Animals received 6-OHDA or sham-6-OHDA injection at a weight of 285–305 g in a stereotaxic
operation and BoNT-A or sham-BoNT-A injection five to six weeks later. Anesthesia was induced
with a ketamine-xylazine mixture (ketamine: 50 mg/kg, xylazine: 4 mg/kg). Dopaminergic cell
death was provoked by unilateral injection of 24 µg of 6-OHDA (Sigma-Aldrich, St. Louis, MO, USA)
dissolved in 4 µL 0.1 M citrate buffer into the right MFB. Sham-6-OHDA animals received only citrate
buffer. Exact coordinates of 6-OHDA or sham-6-OHDA injection were anterior-posterior = −2.3 mm,
lateral = −1.5 mm and ventral = −9.0 mm [68]. BoNT-A (Lot#13029A1; List, Campbell, CA,
USA; purchased via Quadratech, Epsom, UK) supplemented with phosphate-buffered saline
(PBS) and 0.1% bovine serum albumin (BSA) was injected at two sites into the right CPu (total
dose: 1 ng). Sham-BoNT-A animals received only PBS + BSA. Exact coordinates of BoNT-A or
sham-BoNT-A injection were anterior-posterior = +1.3 mm/−0.4 mm, lateral = −2.6 mm/−3.6 mm
and ventral = −5.5 mm/−5.5 mm [68]. For application of either 6-OHDA, BoNT-A or sham solution a
5 µL Hamilton syringe was used and the respective volume was continuously delivered over a time
span of 4 min. Afterwards, the needle was left in place for another 5 min to avoid reflux.
4.3. Immunohistochemistry
Serial brain sections showing CPu and SN were immunohistochemically reacted for TH to verify
successful 6-OHDA lesioning and to exclude an additive effect of BoNT-A. Brains were fixed with
3.7% paraformaldehyde overnight and stained with monoclonal mouse anti-TH antibody (clone TH2,
Sigma-Aldrich) following biotinylated horse anti-mouse lgG (Vector Laboratories, Burlingame, CA,
USA, 1:67). For details of the procedure see [69].
4.4. Behavioral Testing
The degree of dopaminergic cell loss and the positive motor effect of BoNT-A was evaluated
using apomorphine- and amphetamine-induced rotational behavior. Testing was performed in a
rotometer [41] four weeks after 6-OHDA or sham-6-OHDA injection and again two weeks after
BoNT-A or sham-BoNT-A injection (Figure 6). Drugs were solved in 0.9% NaCl and injected i.p.
(apomorphine: 0.25 mg/kg, amphetamine: 2.5 mg/kg). Following apomorphine injection and a
waiting time of 5 min to ensure cerebral uptake, rotations were monitored for 40 min. Rotational
behavior induced by amphetamine application was analyzed after a waiting time of 15 min throughout
a period of 60 min.
4.5. Radioligand Preparation and PET/CT Imaging
Synthesis of [18 F]fallypride ([18 F](S)-N-((1-allylpyrrolidin-2-yl)methyl-5-(3-fluoropropyl)-2,3dimethoxybenzamide) and semi-preparative HPLC for purification was conducted according to
the protocol of [70], followed by an extensive quality control. D2 /D3 R availability was analyzed by
dynamic [18 F]fallypride PET/CT imaging over 90 min, each animal was measured one, three, and
six months post-BoNT-A or -sham-BoNT-A injection. Anesthesia was initially administered with 5%
isoflurane (AbbVie, North Chicago, IL, USA) vaporized in oxygen gas and maintained during scanning
time with 1.5–3%. Body temperature was held constant at 38 ◦ C via a heating pad and respiration rate of
the animals was monitored throughout the PET/CT measurement. Each rat was placed in head-prone
position centered in the field of view of a commercially available preclinical PET/CT scanner (Inveon® ,
Siemens Healthcare, Knoxville, TN, USA); performance evaluation of the system was described
by [71,72]. [18 F]fallypride was injected as a bolus over 1 min via a microcatheter into the lateral tail
vein in a mean dose of 23.44 ± 1.75/24.06 ± 1.76/22.81 ± 2.03 MBq (sham-6-OHDA + sham-BoNT-A),
PET/CT 1-3), 24.52 ± 2.48, 23.84 ± 2.28, 23.88 ± 2.46 MBq (6-OHDA + Sham-BoNT-A, PET/CT 1–3)
and 22.99 ± 2.50, 22.55 ± 3.93, 21.61 ± 2.27 MBq (6-OHDA + BoNT-A, PET/CT 1–3). The acquisition
of dynamic PET as list mode data set was started immediately with the injection. PET studies were
Molecules 2018, 23, 587
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reconstructed as series 3D PET images of multiple frames with various time durations (6 × 10 s,
8 × 30 s, 5 × 300 s, 5 × 1800 s) with a voxel size of 0.86 mm × 0.86 mm × 0.79 mm using a 2D-ordered
subsets expectation maximization algorithm (four iterations, six subsets). Attenuation correction
was performed on the basis of whole body CT scan and PET studies were also corrected for random
coincidences, dead time, scatter and radioactive decay.
4.6. MRI Imaging
MRI was performed on anesthetized rats (1.5–3% isoflurane in oxygen) at least 10 days after
the last PET/CT examination and about eight months post-6-OHDA lesion. MRI of the rats was
conducted using a 7 Tesla small animal MRI scanner (BioSpec 70/30 AVANCE III, 7.0 T, 440 mT/m
gradient strength, Paravision software v6.01., Bruker BioSpin MRI GmbH, Ettlingen, Germany) with
a 1 H transmit resonator (inner diameter: 86 mm; vendor type-nr.: T12053V3, Bruker, Ettlingen,
Germany) and a receive-only surface coil array (2 × 2 array rat brain coil; vendor type-nr.: T11483V3,
Bruker) positioned on the head of the rats. The imaging protocol included 3D isotropic T1w FLASH
imaging sequences with transversal slice orientation, 8/45 ms TE/TR, 35 mm × 35 mm × 16 mm FOV,
200 µm × 200 µm × 200 µm resolution, 175 pixel × 175 pixel × 80 pixel matrix size, 20◦ flip angle,
12:36 min:sec acquisition time, one average and fat suppression.
4.7. Image Analysis
For qualitative and quantitative analysis PMOD v3.7 (PMOD Technologies LLC, Zurich,
Switzerland) was used. Qualitative assessment was conducted with parametric maps of the
spatial BPnd distribution by pixel-wised calculation (extracting signals from individual pixels). For
determination of BPnd , delineation of the target region (left and right striatum) as well as the reference
region (cerebellum) was conducted using an implemented MRI-based rat brain atlas [73]. Therefore,
PET data were first transformed to the standard matrix of the animal-specific MRI (3D isotropic T1w
FLASH). Two rats did not receive a MRI scan, for transformation a representative MRI of the same
experimental group was used. Two animals died during the study course and four PET/CT scanning
time points were canceled. Six PET/CT scans were excluded from analysis due to incorrect injection of
the radioligand and one due to development of a brain tumor (Table 1). Animal-specific MRI datasets
were then transformed to Schiffer matrix and the respective transformation matrix was saved. In a
final step, PET data were transformed into a Schiffer matrix using the saved transformation matrix
to guarantee maximal resolution of PET data (Figure 7a,b). All transformations were performed
using ridged matching method implemented in PMOD. VOIs for target and reference region were
then defined with the Schiffer atlas [73] (Figure 7c,d) and time–activity curves (TAC) were extracted
from dynamic PET data. For kinetic analysis the model-driven SRTM2 [54] was applied and BPnd
was estimated, being defined as the ratio of receptor density (Bmax ) multiplied by the radioligand
affinity [74]. We assumed that receptor affinity was not changed in 6-OHDA-lesion rats compared to
controls as BP of [18 F]fallypride is resistant to DA depletion [75–77].
Molecules 2018, 23, 587
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(a)
(c)
(b)
(d)
Figure 7. (a,b) Workflow of the transformation process using rigid matching. In a first step PET (input)
was transformed to the matrix of the animal-specific MRI (reference) (a). Secondly, the animal-specific
MRI (input) was transformed to the matrix of the Schiffer atlas and the transformation matrix was
saved and finally applied to PET data (b). (c,d) Example of the delineation of the left and right striatum
(purple grid) and the cerebellum (orange grid) in PET (d) with published Schiffer atlas (c) in a control
animal (sham-6-OHDA + sham-BoNT-A).
4.8. Statistical Assessment
Statistical significance was examined using IBM SPSS Statistics software version 22. To test
for the Gaussian distribution of all reported data, a Kolmogorov–Smirnov test was performed
followed by a univariate general linear model. Between-subjects post hoc ANOVA analysis of
variance was conducted with D2 /D3 R availability of the CPu (left/right) as the dependent variable
and the experimental group as covariate. Following, Bonferroni correction with the factor group
for each of the analyzed time points was performed (df = 5; (F = 4.598 one month post-BoNT-A
or -sham-BoNT-A; F = 3.051 three months post-BoNT-A or -sham-BoNT-A, F = 2.584 six months
post-BoNT-A or -sham-BoNT-A)). Statistical significance of apomorphine- and amphetamine-induced
behavior was analyzed by the unpaired student’s t-test. To analyze correlations of D2 /D3 R availability
and rotational behavior in the apomorphine-induced rotation test linear regression followed by
a two-sided Pearson correlation test was implemented. A p-value below 0.05 was considered to
indicate significance.
5. Conclusions
We here provide a longitudinal study on changes of D2 /D3 R availability in the 6-OHDA-induced
hemi-PD rat model. We found an increase in D2 /D3 R availability of 23% up to six months post-lesion,
which was significantly reduced after striatal injection of BoNT-A. Interestingly, this decrease of
pathological D2 /D3 R imbalance by intrastriatal BoNT-A injection significantly correlated with behavior
in the apomorphine rotation test. Altogether, our results emphasize the therapeutical capability of
BoNT-A in hemi-PD rats and provide insights in the underlying mechanisms.
Acknowledgments: The authors would like to thank Anne Möller, Joanna Förster, and Susann Lehmann
for their technical assistance. We gratefully acknowledge Susann Lehmann, Iloana Klamfuß, Petra Wolff,
Robin Piecha, Mathias Lietz, and Ulf Hasse for animal housing and care. We appreciate financial support
by Deutsche Forschungsgemeinschaft and Universität Rostock/Universitätsmedizin Rostock within the funding
program Open Access Publishing.
Author Contributions: Teresa Mann, Andreas Wree, Jens Kurth, and Tobias Lindner wrote the manuscript.
Andreas Wree and Bernd Joachim Krause planned the project and Jens Kurth provided the analyzing strategy.
Andreas Wree, Alexander Hawlitschka, and Teresa Mann executed stereotaxic operations. Teresa Mann analyzed
Molecules 2018, 23, 587
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data and designed figures. Teresa Mann and Alexander Hawlitschka conducted rotation tests with apomorphine
and amphetamine. Alexander Hawlitschka performed TH immunostaining. Jan Stenzel planned and performed
PET/CT scans. Tobias Lindner and Stefan Polei determined the MRI protocol and conducted measurements.
Alexander Hohn established the synthesis protocol and synthesized the radioligand [18 F]fallypride. All authors
contributed substantially to the project and the manuscript.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
Abbreviations
BSA
BPnd
BoNT-A
CPu
D2 /D3 R
DA
hemi-PD
MFB
MRI
MSN
PD
PET
PBS
PVE
SNAP25
SN(pc)
SRTM2
TAC
TH
VOI
6-OHDA
Bovine serum albumin
non-displaceable binding potential
Botulinum neurotoxin A
caudate–putamen
D2 /D3 receptor
dopamine
hemiparkinsonian
medial forebrain bundle
magnetic resonance imaging
medium spiny neuron
Parkinson’s disease
positron emission tomography
phosphate-buffered saline
partial volume effect
synaptosomal-associated protein of 25-kDa
substantia nigra (pars compacta)
simplified reference tissue model 2
time-activity curve
tyrosine hydroxylase
voxels of interest
6-hydroxdopamine
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Sample Availability: Samples of the compounds are not available from the authors.
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