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Tremor Is Associated with PET Measures of Nigrostriatal Dopamine Function in MPTP-Lesioned Monkeys
Experimental Neurology 165, 342–346 (2000) doi:10.1006/exnr.2000.7470, available online at http://www.idealibrary.com on
Tremor Is Associated with PET Measures of Nigrostriatal Dopamine Function in MPTP-Lesioned Monkeys J. L. Eberling,* ,† P. Pivirotto,* J. Bringas,* and K. S. Bankiewicz* ,‡ *Center for Functional Imaging, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Mail Stop 55-121, Berkeley, California 94720; †Department of Neurology, University of California, Davis, California; and ‡Molecular Therapeutics Section, Laboratory of Molecular Medicine and Neuroscience, NINDS, NIH, Bethesda, Maryland Received October 15, 1999; accepted April 27, 2000
Unilateral intracarotid artery (ICA) MPTP infusion, along with sequential systemic doses of MPTP, produces near complete degeneration of the nigrostriatal pathway on the side of infusion (ipsilateral) and variable levels of damage in the contralateral hemisphere accompanied by varying levels of parkinsonism (overlesioned hemiparkinsonian model). Positron emission tomography and the dopamine (DA) metabolism tracer [ 18F]6-fluoro-L-m-tyrosine (FMT) were used to evaluate the relationship between DA metabolism and clinical features of parkinsonism in 14 overlesioned hemiparkinsonian monkeys. Monkeys were rated on a parkinsonian scale that included ratings of bradykinesia, fine motor skills (FMS), and rest tremor. Because the monkeys tended to show more severe clinical signs on the side of the body contralateral to ICA MPTP infusion, we calculated asymmetry scores for each of the clinical features as well as for FMT uptake (K i) in the caudate and putamen. Tremor asymmetry was associated with FMT uptake asymmetry in the putamen. No such relationship was observed for FMS or bradykinesia. The overall severity of tremor (mild, moderate/severe) was associated with FMT uptake in the caudate and putamen. Postmortem biochemical analysis for a subset of monkeys showed that the monkeys with moderate/severe tremor had significantly lower DA levels in both caudate and putamen than those with mild tremor. In addition, K i values were significantly correlated with DA levels in both caudate and putamen. These findings support the idea that nigrostriatal degeneration contributes to rest tremor. ©
can be used to mimic many of the neuropathological, physiological, and clinical features of PD. Using this model, we reported that dopamine (DA) function, as measured by positron emission tomography (PET) and the DA metabolism tracer [ 18F]6-fluoro-L-m-tyrosine (FMT), was associated with the clinical severity of parkinsonism such that more severely parkinsonian animals showed less FMT uptake in the caudate and putamen (7). Similar findings have also been reported in human PD patients using PET and [ 18F]6-fluoro-Ldopa (FDOPA) (8, 14, 17, 22). While neuroimaging measures of DA function appear to be tightly linked to clinical severity, the relationship between specific clinical features and DA function has not been fully explored. We used PET and FMT to determine if DA function was associated with three clinical features of PD, bradykinesia, fine motor skills (FMS), or tremor, in overlesioned hemiparkinsonian monkeys. METHODS
Fourteen Macaca mulatta were studied, 7 males and 7 females, with a mean age of 5.14 ⫾ 3.00 years (SD). All monkeys received unilateral ICA infusions (4 ml/ min) of 60 ml of saline containing 2.5 mg of MPTP–HCl along with 2–14 sequential iv doses (0.3 mg/kg over a 2to 7-week period) producing severe parkinsonism on one side of the body (contralateral to the ICA infusion) and mild to moderate parkinsonism on the other side of the body (7).
2000 Academic Press
Key Words: MPTP; tremor; Parkinson’s disease; PET; primate; dopamine.
INTRODUCTION
The overlesioned hemiparkinsonian MPTP primate model of Parkinson’s disease (PD), in which monkeys are given both unilateral intracarotid artery (ICA) infusions of MPTP and subsequent additional iv doses, 0014-4886/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
PET Methods Prior to PET, all animals underwent magnetic resonance (MR) imaging using a 1.5-T magnet. Both MR and PET imaging were performed on animals positioned in a stereotaxic frame. Animals received PET scans 7–17 weeks after the last dose of MPTP at a time when they were clinically stable. Animals were considered to be clinically stable if clinical measures did not change for a period of 4 weeks. PET scans were per-
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formed on a Siemens-CTI ECAT EXACT HR 47-slice scanner in 2D acquisition mode (24) using arterial catheterization in order to determine blood input functions and metabolite correction factors for each animal (12). Animals were anesthetized with an im injection of ketamine (15 mg/kg), intubated, and placed on methoxyflurane anesthesia. All animals were pretreated with an im injection of the peripheral decarboxylase inhibitor benserazide (2 mg/kg) 30 min prior to FMT injection. Polyethylene catheters were inserted into the femoral vein and artery for venous FMT injection and arterial blood sampling, respectively. Animals were placed in the stereotaxic frame and positioned in the PET scanner in order to obtain images in the coronal plane. The femoral catheter was connected to a scintillation detector in order to determine online radioactivity (arterial time–activity curves). Continuous arterial blood sampling was obtained using a peristaltic pump for 3 min beginning at the time of FMT injection. Additional arterial blood samples were obtained by syringe at 10, 20, 30, 60, and 90 min in order to determine plasma radioactivity throughout the study. In addition, arterial blood samples obtained at 5, 10, 30, 60, and 90 min were used to analyze peripheral metabolism and calculate an exponential function for blood FMT metabolism (12). Once positioned in the scanner, a 20-min transmission scan was performed to correct for photon attenuation using a rotating 68Ge source consisting of three rods of approximately 2 mCi/ rod. Following the transmission scan, each animal was injected with 10 –15 mCi of FMT and emission imaging began simultaneously. Emission data were collected for a total of 90 min. The PET and MR data sets were coregistered and regions of interest (ROIs) were drawn on PET data collected at 60 to 70 min with reference to the coregistered MR images. ROIs were drawn in both hemispheres for the caudate nucleus and putamen on each slice on which they appeared by an operator (J.L.E.) blinded to the status (severity of clinical features) of the monkeys. The identification of anatomical regions was aided by the use of a 3D cursor that was simultaneously displayed in three projections (coronal, axial, and sagittal). In addition, the cursor was displayed on the corresponding MR slices. After a series of 2D ROIs were drawn (for multiple regions), the surfaces of the ROIs were tiled together into a closed triangular mesh polyhedral surface model, defining a 3D region, or volume of interest (VOI) (13). Regional activity density curves were constructed for each VOI using the dynamic PET data collected from 0 to 90 min from the original PET sinogram. A single exponential function for blood FMT metabolism, obtained from a fit of the blood metabolism of FMT for each animal, was applied to a decay corrected curve of total blood 18F activity for each animal. As previously described (12), a three-compartment three-kinetic-rate
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constant model was constructed describing forward (k 1 ) and reverse (k 2 ) transport of FMT from blood into brain, as well as FMT metabolism (k 3 ) in compartment 3 (FMT metabolites in brain). These rate constants were used to calculate an uptake constant for each VOI, describing the rate of FMT uptake (K i) during the 0- to 90-min period after FMT injection. The K i uptake rate constant is equivalent to the net rate of tracer influx (i.e., K i ⫽ (k 1 ⫻ k 3 )/(k 2 ⫹ k 3 )), representing its metabolism in brain as well as its bidirectional exchange between blood and brain. K i asymmetry ratios for the caudate and putamen were constructed by subtracting the K i value for the contralateral side (side opposite ICA MPTP administration) from the K i value for the ipsilateral side (side of ICA MPTP administration) and dividing by the average K i values of the ipsilateral and contralateral sides. Clinical Rating A parkinsonian rating scale (PD scale) was utilized to quantify the clinical status of the monkeys (7). The monkeys were rated at least once a week. The scale includes ratings of 10 parkinsonian features (tremor, posture, locomotion, hypokinesia, bradykinesia, balance, fine and gross motor skills, startle response, and freezing). Because animals showed the greatest amount of variability in tremor, bradykinesia, and FMS, these features were used to determine the relationship between individual clinical features and PET measures of DA activity. Asymmetry ratios were calculated for these features by subtracting the score for the contralateral side of the body from score for the ipsilateral side and dividing by the average of both sides. Biochemical Assay Eleven of the monkeys were sacrificed 2– 4 weeks following the PET procedure for postmortem biochemical and histochemical analysis. Monkeys were deeply anesthetized with sodium pentobarbital (25 mg/kg iv) and then transcardially perfused with 500 ml of saline. The brains were rapidly removed and sliced into 2-mm coronal sections at the level of the midstriatum using a calibrated brain slice apparatus. The slices were then frozen in dry ice and kept in a ⫺80°C freezer for biochemical assay. Specific regions in the midstriatum were later punched from the frozen slices. Punch diameter was 1.5 mm. Punched regions were as follows: dorsolateral and ventromedial caudate nucleus (DC and VC, respectively), dorsolateral and ventromedial putamen (DP and VP, respectively), and nucleus accumbens. Each sample was sonicated in 200 l of homogenizing solution containing 0.1 M perchloric acid, 0.005% EDTA, and dihydroxybenzylamine (1 ng per 100 l) as an internal standard and then centrifuged at 15,000 rpm
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FIG. 1. Correlation between tremor asymmetry and FMT uptake (K i) asymmetry in the putamen. Asymmetry ratios were calculated for both tremor and K i by subtracting the value for the contralateral side from the score for the ipsilateral side (side of MPTP infusion) and dividing by the average of both sides. Thus, more severe tremor on the contralateral side of the body, as indicated by lower asymmetry values, was associated with less FMT uptake in the ipsilateral putamen, as indicated by lower asymmetry values. Spearman rank correlation was used to evaluate this relationship in all 14 monkeys.
for 20 min (the relative centrifugal force was 26,890g in 20 min) at 4°C. The separation and quantification of DA were conducted by ion pair reverse-phase highperformance liquid chromatography (HPLC) with electrochemical detection. A C18 octadecyl silica 3-m minipore (3.2 ⫻ 100 mm) column (Varian, Walnut Creek, CA) was perfused with a MD-TM mobile phase (ESA, Chelmsford, MA) at a constant rate of 1 ml/min. A Coulochem II (ESA, Chelmsford, MA) electrochemical detector applied a ⫺60 mV potential to electrode 1and a ⫹330-mV potential to electrode 2 of a coulometric analytical cell (Model 5011). Protein content in each tissue sample was determined and DA levels were expressed as nanograms per milligram of protein. Data Analysis Spearman rank correlation was used to evaluate the relationship between clinical features and striatal FMT accumulation. Group comparisons were performed using the Mann–Whitney U test. RESULTS
Eleven of the monkeys showed mild bilateral parkinsonism and three showed moderate bilateral parkinsonism. As shown in Fig. 1, tremor asymmetry ratios were significantly (r ⫽ 0.95, P ⫽ 0.0006) correlated with K i ratios for the putamen such that more severe contralateral tremor was associated with lower ipsilateral K i values and more severe ipsilateral tremor was associated with lower contralateral K i values. Caudate K i ratios were not significantly (P ⬎ 0.05) correlated with tremor asymmetry. Asymmetry ratios for FMS and bradykinesia were not significantly (P ⬎ 0.5)
correlated with K i ratios for either the caudate or the putamen. Because we were able to obtain biochemical data on 11 of the 14 monkeys, we looked at the relationship between tremor severity (mild, moderate/severe) and both K i values and dopamine levels in the caudate and putamen (averaged over right and left hemispheres). Bilateral tremor was observed in all 14 monkeys, with 8 monkeys showing mild tremor and 6 monkeys showing moderate to severe tremor. The mild monkeys did not differ significantly (P ⫽ 0.69) from the moderate/ severe monkeys in overall clinical severity (PD score), nor did the PD score correlate with either K i values or DA levels in caudate and putamen. The lack of a relationship between the PD score and both K i values and DA levels is probably due to the similarity between animals in clinical severity (PD score). The monkeys with moderate/severe tremor had significantly (P ⬍ 0.05) lower K i values in both caudate and putamen. Monkeys with moderate/severe tremor (n ⫽ 3) also showed significantly (P ⫽ 0.01) lower DA levels than mild (n ⫽ 8) monkeys in both caudate and putamen. These results, along with K i values and DA levels for normal monkeys for reference (n ⫽ 8 and n ⫽ 4, respectively), are shown in Fig. 2. K i values were significantly correlated with average DA levels in both the caudate (r ⫽ 0.79, P ⫽ 0.01) and the putamen (r ⫽ 0.65, P ⫽ 0.04). DISCUSSION
Studies in both human PD patients and MPTP-lesioned monkeys indicate that PET is useful for monitoring the severity of nigrostriatal degeneration in relation to clinical severity. Less is known about the relationship between PET measures and specific clinical features. Here, we report that asymmetry in rest tremor was associated with asymmetry in PET measures of FMT uptake (K i) in the putamen, but not the caudate. The lack of relationship in the caudate may be because the relatively small size of this brain structure makes it particularly susceptible to partial volume effects resulting in less accurate K i estimates. Monkeys with moderate to severe tremor had lower striatal K i values and lower striatal DA levels, as determined by HPLC, than monkeys with mild tremor irrespective of overall clinical severity. Rest tremor is considered to be a characteristic feature of PD, although it is not present in all patients. The existence of rest tremor in MPTP-treated monkeys remains controversial. While rest tremor has been observed following MPTP administration in African green monkeys, Macaca fascicularis, and baboons (2, 6, 11, 20, 21), the findings have been inconsistent in other species, including M. mulatta (9, 10, 15). While it has been suggested that the selective lesioning of the substantia nigra is not sufficient to produce rest tremor,
TREMOR AND NIGROSTRIATAL DOPAMINE ACTIVITY
FIG. 2. FMT uptake (K i) was greater in monkeys with mild tremor (n ⫽ 8) than monkeys with moderate/severe tremor (n ⫽ 6) in both the caudate and the putamen, and dopamine concentrations were greater in monkeys with mild tremor (n ⫽ 8) than monkeys with moderate/severe tremor (n ⫽ 3) in both the caudate and the putamen. These comparisons were made using the Mann–Whitney U test. For reference, K i values and dopamine concentrations are also shown for two separate groups of normal animals (K i values (n ⫽ 8), DA concentration (n ⫽ 4)).
but rather extranigral lesions are also required, this issue remains unresolved (18, 19, 23). Inconsistent findings regarding the ability of MPTP to produce tremor may stem from a variety of factors, including differences in the route and dose of MPTP administered, as well as the chronicity and distribution of the lesion. PET FDOPA studies evaluating DA function in relation to clinical features, including tremor, have been performed in humans. While there are reports of no correlation between caudate or putamen FDOPA uptake values and tremor severity (1, 17), there are also several reports showing a relationship between striatal FDOPA uptake and tremor scores, although there are differences with respect to the relative contributions of the caudate and putamen (4, 16). Inconsistent PET findings with respect to tremor may reflect underlying pathological differences, clinical differences (i.e., hemiparkinsonism versus bilateral parkinsonism), and methodological differences. Interestingly, a patient
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with a hemorrhage involving the right superior cerebellar peduncle, red nucleus, and substantia nigra developed a left rest tremor that was reduced by levodopa and was associated with decreased right striatal FDOPA uptake (K i) (5). Thus, there is some evidence from PET studies implicating the nigrostriatal pathway in the production of tremor, although damage to structures outside of the nigrostriatal pathway may be necessary as well. There have been reports of associations between a variety of clinical features and DA function in PD patients. An early report of neurochemical changes in the striatum in relation to clinical features found a strong relationship between akinesia and striatal dopamine content, a weaker relationship for tremor, and little relationship for rigidity (3). Antonini et al. (1), using PET and FDOPA, found that both bradykinesia and rigidity scores, as well as clinical severity, correlated with FDOPA uptake in the caudate and putamen in PD patients. In addition, the degree of clinical asymmetry was correlated with K i asymmetry in the putamen. Eidelberg et al. (8) also found a correlation between bradykinesia and striatal FDOPA uptake. Nagasawa et al. (16), using PET and FDOPA in hemiparkinsonian patients, found correlations between DA activity in the caudate (but not putamen) contralateral to the affected limbs and individual clinical measures, including tremor severity. Inconsistencies in findings may stem from differences in methodology as well as clinical differences in the patient populations studied. For example, Antonini et al. (1) found a relationship between K i values averaged over right and left hemispheres and clinical features for both sides of the body (as well as overall clinical severity) but did not look at correlations between asymmetry for specific clinical features and K i asymmetry. In addition, the relationship they reported between individual clinical features and K i may relate to overall clinical severity. We chose to look at clinical and K i asymmetry scores for two reasons. First, most of the monkeys that we studied showed clinical asymmetries that would be expected to relate to asymmetry in DA function, and, second, between subject variability in K i scores is reduced by calculating asymmetry ratios making differences easier to detect. Thus, we evaluated K i asymmetry in relationship to asymmetry in individual clinical features. The tremor asymmetry scores did not necessarily reflect tremor severity and were not related to overall clinical severity, although we also found a relationship between tremor severity and K i values in the caudate and putamen. We did not evaluate the severity of bradykinesia and FMS in relation to K i values because, unlike tremor, the overall severity of these symptoms was similar between monkeys. In light of earlier findings, it is surprising that we did not observe a relationship between asymmetry scores for bradykinesia and FMS and K i asymmetry. Perhaps this was because, although animals showed
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asymmetries in bradykinesia and FMS, they were more similar in overall severity for these measures than for tremor. Previously, we reported (7) a relationship between clinical severity (PD score) and FMT uptake. We did not observe such a relationship in the current report, presumably because while the animals showed differences in individual clinical features, they were very similar in overall clinical severity (PD score). Here, we report a relationship between tremor asymmetry and striatal K i asymmetry, as well as between tremor severity and both striatal K i and striatal DA content. These findings provide support for the contribution of the nigrostriatal DA pathway in the production of tremor but do not rule out the contribution of other structures as well. It is possible that the monkeys with more severe tremor may have concomitant changes in extranigral structures that were not evaluated here. Future PET studies may shed more light on the contribution of nigrostriatal dopaminergic dysfunction to specific clinical features of PD. ACKNOWLEDGMENT
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This work was supported by US DOE Contract DE-AC0376SF00098. 16.
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Tremor Is Associated with PET Measures of Nigrostriatal Dopamine Function in MPTP-Lesioned Monkeys J. L. Eberling,* ,† P. Pivirotto,* J. Bringas,* and K. S. Bankiewicz* ,‡ *Center for Functional Imaging, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Mail Stop 55-121, Berkeley, California 94720; †Department of Neurology, University of California, Davis, California; and ‡Molecular Therapeutics Section, Laboratory of Molecular Medicine and Neuroscience, NINDS, NIH, Bethesda, Maryland Received October 15, 1999; accepted April 27, 2000
Unilateral intracarotid artery (ICA) MPTP infusion, along with sequential systemic doses of MPTP, produces near complete degeneration of the nigrostriatal pathway on the side of infusion (ipsilateral) and variable levels of damage in the contralateral hemisphere accompanied by varying levels of parkinsonism (overlesioned hemiparkinsonian model). Positron emission tomography and the dopamine (DA) metabolism tracer [ 18F]6-fluoro-L-m-tyrosine (FMT) were used to evaluate the relationship between DA metabolism and clinical features of parkinsonism in 14 overlesioned hemiparkinsonian monkeys. Monkeys were rated on a parkinsonian scale that included ratings of bradykinesia, fine motor skills (FMS), and rest tremor. Because the monkeys tended to show more severe clinical signs on the side of the body contralateral to ICA MPTP infusion, we calculated asymmetry scores for each of the clinical features as well as for FMT uptake (K i) in the caudate and putamen. Tremor asymmetry was associated with FMT uptake asymmetry in the putamen. No such relationship was observed for FMS or bradykinesia. The overall severity of tremor (mild, moderate/severe) was associated with FMT uptake in the caudate and putamen. Postmortem biochemical analysis for a subset of monkeys showed that the monkeys with moderate/severe tremor had significantly lower DA levels in both caudate and putamen than those with mild tremor. In addition, K i values were significantly correlated with DA levels in both caudate and putamen. These findings support the idea that nigrostriatal degeneration contributes to rest tremor. ©
can be used to mimic many of the neuropathological, physiological, and clinical features of PD. Using this model, we reported that dopamine (DA) function, as measured by positron emission tomography (PET) and the DA metabolism tracer [ 18F]6-fluoro-L-m-tyrosine (FMT), was associated with the clinical severity of parkinsonism such that more severely parkinsonian animals showed less FMT uptake in the caudate and putamen (7). Similar findings have also been reported in human PD patients using PET and [ 18F]6-fluoro-Ldopa (FDOPA) (8, 14, 17, 22). While neuroimaging measures of DA function appear to be tightly linked to clinical severity, the relationship between specific clinical features and DA function has not been fully explored. We used PET and FMT to determine if DA function was associated with three clinical features of PD, bradykinesia, fine motor skills (FMS), or tremor, in overlesioned hemiparkinsonian monkeys. METHODS
Fourteen Macaca mulatta were studied, 7 males and 7 females, with a mean age of 5.14 ⫾ 3.00 years (SD). All monkeys received unilateral ICA infusions (4 ml/ min) of 60 ml of saline containing 2.5 mg of MPTP–HCl along with 2–14 sequential iv doses (0.3 mg/kg over a 2to 7-week period) producing severe parkinsonism on one side of the body (contralateral to the ICA infusion) and mild to moderate parkinsonism on the other side of the body (7).
2000 Academic Press
Key Words: MPTP; tremor; Parkinson’s disease; PET; primate; dopamine.
INTRODUCTION
The overlesioned hemiparkinsonian MPTP primate model of Parkinson’s disease (PD), in which monkeys are given both unilateral intracarotid artery (ICA) infusions of MPTP and subsequent additional iv doses, 0014-4886/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
PET Methods Prior to PET, all animals underwent magnetic resonance (MR) imaging using a 1.5-T magnet. Both MR and PET imaging were performed on animals positioned in a stereotaxic frame. Animals received PET scans 7–17 weeks after the last dose of MPTP at a time when they were clinically stable. Animals were considered to be clinically stable if clinical measures did not change for a period of 4 weeks. PET scans were per-
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formed on a Siemens-CTI ECAT EXACT HR 47-slice scanner in 2D acquisition mode (24) using arterial catheterization in order to determine blood input functions and metabolite correction factors for each animal (12). Animals were anesthetized with an im injection of ketamine (15 mg/kg), intubated, and placed on methoxyflurane anesthesia. All animals were pretreated with an im injection of the peripheral decarboxylase inhibitor benserazide (2 mg/kg) 30 min prior to FMT injection. Polyethylene catheters were inserted into the femoral vein and artery for venous FMT injection and arterial blood sampling, respectively. Animals were placed in the stereotaxic frame and positioned in the PET scanner in order to obtain images in the coronal plane. The femoral catheter was connected to a scintillation detector in order to determine online radioactivity (arterial time–activity curves). Continuous arterial blood sampling was obtained using a peristaltic pump for 3 min beginning at the time of FMT injection. Additional arterial blood samples were obtained by syringe at 10, 20, 30, 60, and 90 min in order to determine plasma radioactivity throughout the study. In addition, arterial blood samples obtained at 5, 10, 30, 60, and 90 min were used to analyze peripheral metabolism and calculate an exponential function for blood FMT metabolism (12). Once positioned in the scanner, a 20-min transmission scan was performed to correct for photon attenuation using a rotating 68Ge source consisting of three rods of approximately 2 mCi/ rod. Following the transmission scan, each animal was injected with 10 –15 mCi of FMT and emission imaging began simultaneously. Emission data were collected for a total of 90 min. The PET and MR data sets were coregistered and regions of interest (ROIs) were drawn on PET data collected at 60 to 70 min with reference to the coregistered MR images. ROIs were drawn in both hemispheres for the caudate nucleus and putamen on each slice on which they appeared by an operator (J.L.E.) blinded to the status (severity of clinical features) of the monkeys. The identification of anatomical regions was aided by the use of a 3D cursor that was simultaneously displayed in three projections (coronal, axial, and sagittal). In addition, the cursor was displayed on the corresponding MR slices. After a series of 2D ROIs were drawn (for multiple regions), the surfaces of the ROIs were tiled together into a closed triangular mesh polyhedral surface model, defining a 3D region, or volume of interest (VOI) (13). Regional activity density curves were constructed for each VOI using the dynamic PET data collected from 0 to 90 min from the original PET sinogram. A single exponential function for blood FMT metabolism, obtained from a fit of the blood metabolism of FMT for each animal, was applied to a decay corrected curve of total blood 18F activity for each animal. As previously described (12), a three-compartment three-kinetic-rate
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constant model was constructed describing forward (k 1 ) and reverse (k 2 ) transport of FMT from blood into brain, as well as FMT metabolism (k 3 ) in compartment 3 (FMT metabolites in brain). These rate constants were used to calculate an uptake constant for each VOI, describing the rate of FMT uptake (K i) during the 0- to 90-min period after FMT injection. The K i uptake rate constant is equivalent to the net rate of tracer influx (i.e., K i ⫽ (k 1 ⫻ k 3 )/(k 2 ⫹ k 3 )), representing its metabolism in brain as well as its bidirectional exchange between blood and brain. K i asymmetry ratios for the caudate and putamen were constructed by subtracting the K i value for the contralateral side (side opposite ICA MPTP administration) from the K i value for the ipsilateral side (side of ICA MPTP administration) and dividing by the average K i values of the ipsilateral and contralateral sides. Clinical Rating A parkinsonian rating scale (PD scale) was utilized to quantify the clinical status of the monkeys (7). The monkeys were rated at least once a week. The scale includes ratings of 10 parkinsonian features (tremor, posture, locomotion, hypokinesia, bradykinesia, balance, fine and gross motor skills, startle response, and freezing). Because animals showed the greatest amount of variability in tremor, bradykinesia, and FMS, these features were used to determine the relationship between individual clinical features and PET measures of DA activity. Asymmetry ratios were calculated for these features by subtracting the score for the contralateral side of the body from score for the ipsilateral side and dividing by the average of both sides. Biochemical Assay Eleven of the monkeys were sacrificed 2– 4 weeks following the PET procedure for postmortem biochemical and histochemical analysis. Monkeys were deeply anesthetized with sodium pentobarbital (25 mg/kg iv) and then transcardially perfused with 500 ml of saline. The brains were rapidly removed and sliced into 2-mm coronal sections at the level of the midstriatum using a calibrated brain slice apparatus. The slices were then frozen in dry ice and kept in a ⫺80°C freezer for biochemical assay. Specific regions in the midstriatum were later punched from the frozen slices. Punch diameter was 1.5 mm. Punched regions were as follows: dorsolateral and ventromedial caudate nucleus (DC and VC, respectively), dorsolateral and ventromedial putamen (DP and VP, respectively), and nucleus accumbens. Each sample was sonicated in 200 l of homogenizing solution containing 0.1 M perchloric acid, 0.005% EDTA, and dihydroxybenzylamine (1 ng per 100 l) as an internal standard and then centrifuged at 15,000 rpm
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FIG. 1. Correlation between tremor asymmetry and FMT uptake (K i) asymmetry in the putamen. Asymmetry ratios were calculated for both tremor and K i by subtracting the value for the contralateral side from the score for the ipsilateral side (side of MPTP infusion) and dividing by the average of both sides. Thus, more severe tremor on the contralateral side of the body, as indicated by lower asymmetry values, was associated with less FMT uptake in the ipsilateral putamen, as indicated by lower asymmetry values. Spearman rank correlation was used to evaluate this relationship in all 14 monkeys.
for 20 min (the relative centrifugal force was 26,890g in 20 min) at 4°C. The separation and quantification of DA were conducted by ion pair reverse-phase highperformance liquid chromatography (HPLC) with electrochemical detection. A C18 octadecyl silica 3-m minipore (3.2 ⫻ 100 mm) column (Varian, Walnut Creek, CA) was perfused with a MD-TM mobile phase (ESA, Chelmsford, MA) at a constant rate of 1 ml/min. A Coulochem II (ESA, Chelmsford, MA) electrochemical detector applied a ⫺60 mV potential to electrode 1and a ⫹330-mV potential to electrode 2 of a coulometric analytical cell (Model 5011). Protein content in each tissue sample was determined and DA levels were expressed as nanograms per milligram of protein. Data Analysis Spearman rank correlation was used to evaluate the relationship between clinical features and striatal FMT accumulation. Group comparisons were performed using the Mann–Whitney U test. RESULTS
Eleven of the monkeys showed mild bilateral parkinsonism and three showed moderate bilateral parkinsonism. As shown in Fig. 1, tremor asymmetry ratios were significantly (r ⫽ 0.95, P ⫽ 0.0006) correlated with K i ratios for the putamen such that more severe contralateral tremor was associated with lower ipsilateral K i values and more severe ipsilateral tremor was associated with lower contralateral K i values. Caudate K i ratios were not significantly (P ⬎ 0.05) correlated with tremor asymmetry. Asymmetry ratios for FMS and bradykinesia were not significantly (P ⬎ 0.5)
correlated with K i ratios for either the caudate or the putamen. Because we were able to obtain biochemical data on 11 of the 14 monkeys, we looked at the relationship between tremor severity (mild, moderate/severe) and both K i values and dopamine levels in the caudate and putamen (averaged over right and left hemispheres). Bilateral tremor was observed in all 14 monkeys, with 8 monkeys showing mild tremor and 6 monkeys showing moderate to severe tremor. The mild monkeys did not differ significantly (P ⫽ 0.69) from the moderate/ severe monkeys in overall clinical severity (PD score), nor did the PD score correlate with either K i values or DA levels in caudate and putamen. The lack of a relationship between the PD score and both K i values and DA levels is probably due to the similarity between animals in clinical severity (PD score). The monkeys with moderate/severe tremor had significantly (P ⬍ 0.05) lower K i values in both caudate and putamen. Monkeys with moderate/severe tremor (n ⫽ 3) also showed significantly (P ⫽ 0.01) lower DA levels than mild (n ⫽ 8) monkeys in both caudate and putamen. These results, along with K i values and DA levels for normal monkeys for reference (n ⫽ 8 and n ⫽ 4, respectively), are shown in Fig. 2. K i values were significantly correlated with average DA levels in both the caudate (r ⫽ 0.79, P ⫽ 0.01) and the putamen (r ⫽ 0.65, P ⫽ 0.04). DISCUSSION
Studies in both human PD patients and MPTP-lesioned monkeys indicate that PET is useful for monitoring the severity of nigrostriatal degeneration in relation to clinical severity. Less is known about the relationship between PET measures and specific clinical features. Here, we report that asymmetry in rest tremor was associated with asymmetry in PET measures of FMT uptake (K i) in the putamen, but not the caudate. The lack of relationship in the caudate may be because the relatively small size of this brain structure makes it particularly susceptible to partial volume effects resulting in less accurate K i estimates. Monkeys with moderate to severe tremor had lower striatal K i values and lower striatal DA levels, as determined by HPLC, than monkeys with mild tremor irrespective of overall clinical severity. Rest tremor is considered to be a characteristic feature of PD, although it is not present in all patients. The existence of rest tremor in MPTP-treated monkeys remains controversial. While rest tremor has been observed following MPTP administration in African green monkeys, Macaca fascicularis, and baboons (2, 6, 11, 20, 21), the findings have been inconsistent in other species, including M. mulatta (9, 10, 15). While it has been suggested that the selective lesioning of the substantia nigra is not sufficient to produce rest tremor,
TREMOR AND NIGROSTRIATAL DOPAMINE ACTIVITY
FIG. 2. FMT uptake (K i) was greater in monkeys with mild tremor (n ⫽ 8) than monkeys with moderate/severe tremor (n ⫽ 6) in both the caudate and the putamen, and dopamine concentrations were greater in monkeys with mild tremor (n ⫽ 8) than monkeys with moderate/severe tremor (n ⫽ 3) in both the caudate and the putamen. These comparisons were made using the Mann–Whitney U test. For reference, K i values and dopamine concentrations are also shown for two separate groups of normal animals (K i values (n ⫽ 8), DA concentration (n ⫽ 4)).
but rather extranigral lesions are also required, this issue remains unresolved (18, 19, 23). Inconsistent findings regarding the ability of MPTP to produce tremor may stem from a variety of factors, including differences in the route and dose of MPTP administered, as well as the chronicity and distribution of the lesion. PET FDOPA studies evaluating DA function in relation to clinical features, including tremor, have been performed in humans. While there are reports of no correlation between caudate or putamen FDOPA uptake values and tremor severity (1, 17), there are also several reports showing a relationship between striatal FDOPA uptake and tremor scores, although there are differences with respect to the relative contributions of the caudate and putamen (4, 16). Inconsistent PET findings with respect to tremor may reflect underlying pathological differences, clinical differences (i.e., hemiparkinsonism versus bilateral parkinsonism), and methodological differences. Interestingly, a patient
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with a hemorrhage involving the right superior cerebellar peduncle, red nucleus, and substantia nigra developed a left rest tremor that was reduced by levodopa and was associated with decreased right striatal FDOPA uptake (K i) (5). Thus, there is some evidence from PET studies implicating the nigrostriatal pathway in the production of tremor, although damage to structures outside of the nigrostriatal pathway may be necessary as well. There have been reports of associations between a variety of clinical features and DA function in PD patients. An early report of neurochemical changes in the striatum in relation to clinical features found a strong relationship between akinesia and striatal dopamine content, a weaker relationship for tremor, and little relationship for rigidity (3). Antonini et al. (1), using PET and FDOPA, found that both bradykinesia and rigidity scores, as well as clinical severity, correlated with FDOPA uptake in the caudate and putamen in PD patients. In addition, the degree of clinical asymmetry was correlated with K i asymmetry in the putamen. Eidelberg et al. (8) also found a correlation between bradykinesia and striatal FDOPA uptake. Nagasawa et al. (16), using PET and FDOPA in hemiparkinsonian patients, found correlations between DA activity in the caudate (but not putamen) contralateral to the affected limbs and individual clinical measures, including tremor severity. Inconsistencies in findings may stem from differences in methodology as well as clinical differences in the patient populations studied. For example, Antonini et al. (1) found a relationship between K i values averaged over right and left hemispheres and clinical features for both sides of the body (as well as overall clinical severity) but did not look at correlations between asymmetry for specific clinical features and K i asymmetry. In addition, the relationship they reported between individual clinical features and K i may relate to overall clinical severity. We chose to look at clinical and K i asymmetry scores for two reasons. First, most of the monkeys that we studied showed clinical asymmetries that would be expected to relate to asymmetry in DA function, and, second, between subject variability in K i scores is reduced by calculating asymmetry ratios making differences easier to detect. Thus, we evaluated K i asymmetry in relationship to asymmetry in individual clinical features. The tremor asymmetry scores did not necessarily reflect tremor severity and were not related to overall clinical severity, although we also found a relationship between tremor severity and K i values in the caudate and putamen. We did not evaluate the severity of bradykinesia and FMS in relation to K i values because, unlike tremor, the overall severity of these symptoms was similar between monkeys. In light of earlier findings, it is surprising that we did not observe a relationship between asymmetry scores for bradykinesia and FMS and K i asymmetry. Perhaps this was because, although animals showed
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asymmetries in bradykinesia and FMS, they were more similar in overall severity for these measures than for tremor. Previously, we reported (7) a relationship between clinical severity (PD score) and FMT uptake. We did not observe such a relationship in the current report, presumably because while the animals showed differences in individual clinical features, they were very similar in overall clinical severity (PD score). Here, we report a relationship between tremor asymmetry and striatal K i asymmetry, as well as between tremor severity and both striatal K i and striatal DA content. These findings provide support for the contribution of the nigrostriatal DA pathway in the production of tremor but do not rule out the contribution of other structures as well. It is possible that the monkeys with more severe tremor may have concomitant changes in extranigral structures that were not evaluated here. Future PET studies may shed more light on the contribution of nigrostriatal dopaminergic dysfunction to specific clinical features of PD. ACKNOWLEDGMENT
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This work was supported by US DOE Contract DE-AC0376SF00098. 16.
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