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Neurobiology of Disease 35 (2009) 348–351 Contents lists available at ScienceDirect Neurobiology of Disease The blood–brain barrier is intact after levodopa-induced dyskinesias in parkinsonian primates—Evidence from in vivo neuroimaging studies Arnar Astradsson a,d, Bruce G. Jenkins a,b, Ji-Kyung Choi b, Penelope J. Hallett a,d, Michele A. Levesque a,d,Jack S. McDowell a,d, Anna-Liisa Brownell a,c, Roger D. Spealman a,d, Ole Isacson a,d,⁎a Harvard University and McLean Hospital, NINDS Udall Parkinson's Disease Research Center of Excellence, Belmont, MA, USAb Massachusetts General Hospital (MGH) Nuclear Magnetic Resonance Center, Athinoula A. Martinos Center for Biomedical Imaging, Boston, MA, USAc MGH Positron Emission Tomography Center, Massachusetts General Hospital, Boston, MA, USAd New England Primate Research Center, Harvard Medical School, Southborough, MA, USA It has been suggested, based on rodent studies, that levodopa (L-dopa) induced dyskinesia is associated with Received 12 February 2009 a disrupted blood–brain barrier (BBB). We have investigated BBB integrity with in vivo neuroimaging Revised 6 May 2009 techniques in six 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesioned primates exhibiting L- Accepted 28 May 2009 dopa-induced dyskinesia. Magnetic resonance imaging (MRI) performed before and after injection of Available online 6 June 2009 Gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) revealed an intact BBB in the basal gangliashowing that L-dopa-induced dyskinesia is not associated with a disrupted BBB in this model.
2009 Elsevier Inc. All rights reserved.
Levodopa (L-dopa) is currently the primary treatment of motor Induction of parkinsonian and dyskinetic symptoms symptoms in Parkinson's disease (PD). However, a major limitation ofchronic L-dopa treatment is the development of dyskinesias after Six adult male macaque monkeys (Macaca fascicularis), aged 6– years of treatment (Fahn, 2003; Olanow et al., 2004). The pathophy- 8 years and weighing 6–7 kg, were included in this study. Animals siological mechanisms of L-dopa-induced dyskinesia are poorly were housed in individual home cages at the New England Primate understood, though non-physiological release of synaptic dopamine Research Center (NEPRC). All studies were approved by the Harvard is likely to play a major role in its development (Obeso et al., 2000; Medical School Institutional Animal Care and Use Committee (IACUC).
Olanow et al., 2004; Olanow and Obeso, 2000). Recently, it has been Parkinsonism was induced by weekly intravenous administration of suggested, based on studies in rodents, that L-dopa-induced dyskine- low doses of MPTP (Sigma-Aldrich®) diluted in normal saline. Doses sia may be associated with a disrupted blood–brain barrier (BBB) were given initially at 0.30 mg/kg to all animals but in some instances (Westin et al., 2006) and that this may in turn contribute to its subsequently reduced to 0.15 mg/kg, due to symptom severity and pathophysiology, by further exacerbating dyskinesia (Westin et al., individual sensitivity. Parkinsonian motor symptoms were rated weekly during and after MPTP administration on a Parkinson's Rating The purpose of the present study was to investigate the integrity of Scale (PRS) as developed for macaques (Imbert et al., 2000) and the BBB using in vivo neuroimaging techniques in 1-methyl-4-phenyl- modified from the motor subscale of the Unified Parkinson's Disease 1,2,3,6-tetrahydropyridine (MPTP) lesioned parkinsonian primates Rating Scale (UPDRS) (Fahn, 2003), ranging from 0 to 24, with 24 exhibiting L-dopa-induced dyskinesias.
being most severe. Stable PRS scores were obtained off L-dopa at least3 months after the last MPTP dose and were considered stable ifstandard deviation did not change more than ±2 over 6 weeks ⁎ Corresponding author. Center for Neuroregeneration Research, McLean Hospital/ (Table 1). All animals displayed a stable parkinsonian syndrome, Harvard Medical School, MRC 130, 115 Mill St, Belmont, MA 02478, USA.
including tremor, rigidity, bradykinesia, hypokinesia and posture/ E-mail address: [email protected] (O. Isacson).
Available online on ScienceDirect (www.sciencedirect.com).
balance disturbances (Jenkins et al., 2004). Dopamine transporter loss 0969-9961/$ – see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.nbd.2009.05.018 A. Astradsson et al. / Neurobiology of Disease 35 (2009) 348–351 chorea (rapid, random flicking movements), athetosis (sinuous, Animal characteristics, dosing and symptoms.
writhing distal limb movements) dystonia (sustained twisting movements resulting in abnormal posturing), myoclonus (jerky) orstereotypy (repetitive purposeless behavior). Severity was rated according to the Dyskinesia Disability Severity scale as described (Bezard et al., 2003; Pearce et al., 1995), ranging from 1 to 4, based on frequency and interference with normal behavior by 0 = absent; 1 = mild, fleeting and dyskinetic movements and postures (b5 in Putaminal 11C-CFT binding 10 min); 2 = moderate, more prominent and abnormal dyskinesia Daily L-dopa dose (mg/kg) but not interfering with normal behavior (∼5–20 in 10 min); Duration of L-dopa 3 = marked, frequent dyskinesia, intruding on normal behavior treatment (weeks) (21–50 in 10 min); 4 = severe, virtually continuous dyskinesia, Maximal effective L-dopa disabling the animal. Sum of dyskinesia scores (peak scores) at the Maximal effective time maximally effective dose and time point were obtained and severity point after L-dopa (min) (disability) scores were calculated by dividing the total score by the Dyskinesia peak score number of affected regions, as previously described (Sanchez- Dyskinesia severity score Pernaute et al., 2007).
MF = Macaca fascicularis; 11C-CFT = 11C-(2β-carbomethoxy-3β-(4-fluorophenyl)tropane).
MRI studies with Gadolinium-diethylenetriamine pentaacetic acid(Gd-DTPA) contrast enhancement in the posterior putamen was measured by positron emissiontomography (PET) studies and binding of the dopamine transporter After developing reproducible dyskinesias, animals underwent tracer 11C-(2β-carbomethoxy-3β-(4-fluorophenyl) tropane) (CFT) at neuroimaging studies. L-dopa was administered until the morning of the stable stage, at least 3 months after last MPTP administration, as the study. Animals were anesthetized with a Ketamine (10 mg/kg)/ previously described (Brownell et al., 1998). Animals then received Xylazine (1.5 mg/kg) combination i.m. Atropine was administered at daily intramuscular (i.m.) injections of L-dopa methylester (Sigma- 0.04 mg/kg i.m. Anesthesia was maintained with halothane (1–1.5%) Aldrich®) in combination with the peripheral decarboxylase inhibitor while the animal was intubated but free breathing. The animal was benserazide (Sigma-Aldrich®), diluted in normal saline and injected at placed in an MRI compatible head frame (Kopf Instruments®) on a water 1 ml, for the induction of dyskinesia. L-dopa was administered heating blanket to maintain body temperature. Respiratory rate, heart according to individual animal response and tolerance at 30, 60 or rate, SpO2 and body temperature were constantly monitored through- 120 mg/kg daily for 15–36 weeks. Benserazide was co-administered at out the procedure. MRI studies were performed on a 3 T Allegra system 10–15 mg/kg per dose. Dyskinesia severity was rated weekly by two (Siemens®, Erlangen, Germany) using a transmit–receive 3 inch surface independent observers at 30, 60 and 90 min after a single i.m.
coil. The animal's head was placed in the center of the surface coil such administration of L-dopa (30 or 60 mg/kg) in combination with that the coil fit over the skull, above the eyes. After collection of baseline benserazide (10–15 mg/kg). Abnormal movements were classified as images, Gd-DTPA was administered intravenously at 0.3 mmol/kg and Fig. 1. The BBB of the basal ganglia is intact as shown by Gadolinium-DTPA (Gd-DTPA) MRI studies in dyskinetic monkeys. T1 weighted axial brain MRI images before (upper panel)and after (lower panel) peripheral injection of Gadolinium-DTPA, 0.3 mmol/kg to a dyskinetic macaque. Post Gd-DTPA there is marked signal enhancement of the hypothalamic/pituitary region, structures lacking a BBB, and the sagittal sinus, but no signal enhancement of the basal ganglia, including the putamen, caudate, globus pallidus and substantia nigrais observed. Put = putamen; Cd = caudate nucleus; GPe = globus pallidus externa, GPi = globus pallidus interna SN = substantia nigra, Pit/Hyp = pituitary/hypothalamic region.
L = left; R = right. Bright spots near SN are contrast filled blood vessels.
A. Astradsson et al. / Neurobiology of Disease 35 (2009) 348–351 serial gradient echo imaging was continued with a flip angle alpha of 25° including the substantia nigra, in any animal. Signal enhancement and short TE (TR/TE= 235/4.5 ms) with 30 s temporal resolution, and was observed in structures lacking a BBB, namely the pituitary/ high resolution (0.65 mm isotropic) T1-weighted sequence (TR/TI/ hypothalamus region (pit/hyp), in addition to the sagittal sinus and TE= 1910/1100/3.1 ms) were collected. MRI data acquisition occurred jaw muscles, thus serving as an internal control of Gd-DTPA delivery over a total of 20 min (Fig. 2). At the conclusion of the study, the animals (Fig. 1). A region of interest (ROI) quantitative analysis (see Methods), were extubated and placed in a warmed cage until fully recovered.
confirmed an intact BBB in the basal ganglia in all six animals. One way Regions of interest (ROIs) were hand drawn of the SN, the putamen, the ANOVA across brain regions showed that there were no significant caudate, the pituitary–hypothalamic region, the sagittal sinus and jaw differences between images of the caudate nucleus (Cd), putamen muscle on the various MRI images, and the average image intensity was (Put), SN or occipital cortex (OccCx) with either the serial gradient used for the quantitative analysis using the serial gradient echo images echo sequences (F23,3 = 1.27; p N 0.3) or the high resolution (0.65 mm as a function of time after injection of Gd-DTPA and delayed isotropic) T1-weighted sequence (F23,3 = 1.40; p N0.25) whereas enhancement was analyzed using the high resolution T1 weighted there were highly significant differences between the Cd, Put, SN or images. Statistical analyses were performed with the GraphPad Prism® OccCx and either jaw muscle, pit/hyp or sagittal sinus, as expected program, version 5.01.
(Figs. 2A and B).
Animals received a weekly low dose of the neurotoxin MPTP for a This is the first in vivo demonstration of the integrity of the BBB total of 4–34 weeks, with a total cumulative MPTP dose of 7.2– in parkinsonian primates exhibiting L-dopa-induced dyskinesia.
39.5 mg. This resulted in moderate to severe parkinsonian symptoms The induction of dyskinesia by the administration of daily high in all six animals, with an average PRS score of 18 ± 2.7 (range 14–22), dose L-dopa over several months to MPTP lesioned, parkinsonian that remained stable at least 3 months after the last MPTP dose primates, did not lead to a leaking BBB. It is conceivable that in the (Table 1). All animals displayed a significant loss of dopamine case of a disrupted BBB, this could lead to high and uncontrolled transporter binding in the putamen with an average reduction of 59 levels of L-dopa entering the brain following systemic L-dopa ± 7.4 % (t-test; p b 0.001), as measured by PET and the dopamine therapy, further exacerbating non-physiological synaptic release of transporter tracer 11C-CFT. After 15–36 weeks of daily L-dopa dopamine (Olanow et al., 2004; Westin et al., 2006). Also, the BBB is treatment, all six animals developed dyskinesias as defined by the usually impermeable to carbidopa, a peripheral L-dopa decarboxylase presence of abnormal involuntary movements, mainly choreiform, inhibitor, and if disrupted and rendered permeable, this could dystonic and stereotypic movements affecting limbs, axial body, tail, compromise physiological L-dopa decarboxylation in the brain (Carvey and orolingual muscles (Table 1).
et al., 2005). Finally, in gene therapy, a dysfunctional BBB could Animals then underwent an MRI brain scan with Gd-DTPA. Visual possibly result in a different distribution of secreted gene products inspection of high resolution T1 weighted images, revealed no (Isacson and Kordower, 2008) or in the case of cell transplantation, increase in signal intensity post Gd-DTPA in the basal ganglia, exposure to immune factors and rejection (Isacson and Kordower, Fig. 2. Quantitative results following injection of Gadolinium-DTPA in dyskinetic monkeys show an intact BBB of the basal ganglia. (A) Averaged plot across all animals showing theeffects of 0.3 mmol/kg Gd-DTPA as a function of time using serial gradient echo imaging with a flip angle alpha of 25° and short TE (TR/TE = 235/4.5 ms) with 30 s temporalresolution. Injections were made during serial imaging for comparison effects and are shown in the sagittal sinus vein (Sag sinus), the pituitary/hypothalamic region (Pit/Hyp) andthe substantia nigra (SN). There is no increase in the SN aside from a small contribution attributable to the intrinsic blood volume. (B) Bar plot showing the averages across all animalsfor signal enhancement at an average of 16 min after GD-DTPA injection using a high resolution (0.65 mm isotropic) T1-weighted sequence (TR/TI/TE = 1910/1100/3.1 ms). Theregions shown are the same as in (A) but also include putamen (Put), caudate (Cd), jaw muscle (muscle), and occipital cortex (OccCx) as a control gray matter region. One wayANOVA across brain regions showed that there were no significant differences between images of the Cd, Put, SN and OccCx with either the gradient echo data in (A) (F23,3 = 1.27;p N0.3) or in (B) (F23,3 = 1.40; p N 0.25). As expected, there were highly significant differences between the latter four regions and either muscle, pit/hyp or sagittal sinus.
A. Astradsson et al. / Neurobiology of Disease 35 (2009) 348–351 2008). The findings of an intact BBB in the present study may increased cerebral blood flow and dissociation of cerebral blood flow therefore have implications for existing and new therapies for PD.
and metabolism in the striatum (Hirano et al., 2008).
BBB integrity has also been studied in clinical and experimental In conclusion, in primates rendered parkinsonian with MPTP, models of Parkinson's disease. For example, a PET study of 11C- repeated L-dopa treatment or dyskinesia did not disrupt the BBB in the verapamil uptake in the brain demonstrated a decreased function of basal ganglia, as detected with MRI neuroimaging using Gd-DTPA.
the P-glycoprotein (P-gp) transporter in the BBB of PD patients These findings contrast with studies of the BBB in rodent models of L- (Kortekaas et al., 2005). Findings from a rodent study have suggested that L-dopa-induced dyskinesia may be associated with a compro-mised BBB (Westin et al., 2006). Postmortem analysis of 6-OHDA lesioned rats rendered dyskinetic after a 2 week course of L-dopa,revealed a BBB with long-term structural changes in the basal ganglia, This work was supported by the US National Institutes of Health particularly in its output regions; the entopeduncular nucleus and the NINDS Udall Parkinson's Disease Research Center of Excellence (P50 substantia nigra pars reticulata, as demonstrated by increased NS39793), The Michael Stern Foundation, the Consolidated Anti- immunostaining for albumin and a reduction in endothelial barrier Aging Foundation, the Orchard Foundation, and an NIH base grant to antigen (EBA) expression (Westin et al., 2006). However, no external NEPRC (RR00168). The authors declare no financial conflict of interest.
tracer such as horseradish-peroxide (HRP) was administered (Westin We thank Angela Carville and Shannon Luboyeski for veterinary et al., 2006). HRP is a glycoprotein with a small molecular weight that produces a fluorimetric or luminescent derivative of the labeledmolecule, and can be administered intravenously, subsequentlyallowing it to be histologically detected and quantified and has been widely used as a histological marker of BBB integrity (Harris et al., Beaumont, A., et al., 2000. The permissive nature of blood brain barrier (BBB) opening 2002). EBA is rodent specific and may not be applicable to the clinical in edema formation following traumatic brain injury. Acta. Neurochir. Suppl. 76, setting (Sternberger and Sternberger, 1987). Finally, Westin et al.
found a high rate of cell proliferation in the basal ganglia and newly Bezard, E., et al., 2003. Attenuation of levodopa-induced dyskinesia by normalizing dopamine D3 receptor function. Nat. Med. 9, 762–767.
born microvessels (Westin et al., 2006). These observations were Brownell, A.L., et al., 1998. Combined PET/MRS brain studies show dynamic and long- specifically associated with the development of dyskinesia and not L- term physiological changes in a primate model of Parkinson disease. Nat. Med. 4, dopa treatment alone (Westin et al., 2006).
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clinically applied, for the induction of dyskinesias in primates, in order Imbert, C., et al., 2000. Comparison of eight clinical rating scales used for the assessment to shorten the length of the induction phase (Sanchez-Pernaute et al., of MPTP-induced parkinsonism in the Macaque monkey. J. Neurosci. Methods 96, 2007). Nevertheless, this model may more realistically simulate the Isacson, O., Kordower, J.H., 2008. Future of cell and gene therapies for Parkinson's progressive pathogenesis of dyskinesia in clinical PD, than current disease. Ann. Neurol. 64 (Suppl 2), S122–S138.
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Ludemann, L., et al., 2002. Pharmacokinetic modeling of Gd-DTPA extravasation in brain (Ludemann et al., 2002) and head injury (Beaumont et al., 2000).
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We have chosen to use Gd-DTPA MRI to detect BBB integrity in our in Obeso, J.A., et al., 2000. Pathophysiology of levodopa-induced dyskinesias in Parkinson's disease: problems with the current model. Ann. Neurol. 47, L-dopa-induced dyskinesia of primates, as it is a well S22–S32. Discussion S32–S34.
established, clinically useful marker to evaluate BBB integrity. It has Olanow, C.W., et al., 2004. Levodopa in the treatment of Parkinson's disease: current the advantage over HRP and albumin, that it can be readily used in controversies. Mov. Disord. 19, 997–1005.
vivo, whereas the analysis of HRP and albumin leakage is suitable for Olanow, C.W., Obeso, J.A., 2000. Preventing levodopa-induced dyskinesias. Ann. Neurol.
47, S167–S176. Discussion S176–S178.
postmortem studies. Furthermore, Gd-DTPA is a much smaller Pearce, R.K., et al., 1995. Chronic L-DOPA administration induces dyskinesias in the 1- molecule than both albumin and HRP and therefore should be more methyl-4- phenyl-1,2,3,6-tetrahydropyridine-treated common marmoset sensitive to subtle BBB permeability changes (Harris et al., 2002; (Callithrix Jacchus). Mov. Disord. 10, 731–740.
Sanchez-Pernaute, R., et al., 2007. In vivo evidence of D3 dopamine receptor Schmiedl et al., 1991). Notably, if a molecule as large as albumin can sensitization in parkinsonian primates and rodents with L-DOPA-induced dyskine- leak across the BBB it must indicate a very high permeability surface sias. Neurobiol. Dis. 27, 220–227.
area product (Westin et al., 2006). Given that we could not see leakage Schmiedl, U.P., et al., 1991. MRI of blood–brain barrier permeability in astrocytic of a small molecule like Gd-DTPA in the present study, it must mean gliomas: application of small and large molecular weight contrast media. Magn.
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that there was minimal opening of the BBB in our model.
Soon, D., et al., 2007. Quantification of subtle blood–brain barrier disruption in non- While we found no evidence of BBB damage after chronic L-dopa enhancing lesions in multiple sclerosis: a study of disease and lesion subtypes.
administration in our study, it cannot be excluded that other Mult. Scler. 13, 884–894.
Sternberger, N.H., Sternberger, L.A., 1987. Blood–brain barrier protein recognized by microvascular effects of L-dopa treatment might have occurred in monoclonal antibody. Proc. Natl. Acad. Sci. U. S. A. 84, 8169–8173.
this model. For example, the possibility of L-dopa-induced micro- Wardlaw, J.M., et al., 2008. Changes in background blood–brain barrier integrity vascular proliferation and increased cerebral blood volume cannot be between lacunar and cortical ischemic stroke subtypes. Stroke 39, 1327–1332.
Westin, J.E., et al., 2006. Endothelial proliferation and increased blood–brain barrier excluded (Westin et al., 2006). Furthermore, it cannot be excluded, as permeability in the basal ganglia in a rat model of 3,4-dihydroxyphenyl-L-alanine- was recently demonstrated, that L-dopa treatment is associated with induced dyskinesia. J. Neurosci. 26, 9448–9461.

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