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A simplified autoradiographic method with α-[14C]methyl-tryptophan to measure serotonin synthesis rate in the rat brain
Nuclear Medicine and Biology 31 (2004) 1013 – 1019 www.elsevier.com/locate/nucmedbio
A simplified autoradiographic method with a-[14C]methyl-tryptophan to measure serotonin synthesis rate in the rat brain Hidehiko Okazawa, M.D., Ph.D.a,b,*, Sadahiko Nishizawa, M.D., Ph.D.a,b,c, Tatsuro Tsuchida, M.D., Ph.D.a, Yoshiharu Yonekura, M.D., Ph.D.a, Mirko Diksic, Ph.D.b b
a Biomedical Imaging Research Center, University of Fukui, 23-3 Shimoaizuki Matsuoka-cho, Fukui 910-1193, Japan Cone Laboratory for Neurosurgical Research, Montreal Neurological Institute, 3801 University Street Montreal, Quebec, H3A 2B4 Canada c Hamamatsu Medical Imaging Center, Hamamatsu Medical Photonics Foundation, 5000 Hirakuchi, Hamakita, Shizuoka, 434-0041 Japan Received 7 June 2004; received in revised form 29 July 2004; accepted 7 August 2004
Abstract Background: To minimize blood sampling necessary for the autoradiographic (ARG) method using a-[14C]methyl-tryptophan (a-MTrp), a simplified method using a standard exposure time (h S) was proposed and the accuracy of the method was evaluated. Methods: A total of 168 rats from three sets of experiments with different serotonergic drugs to evaluate changes in cerebral serotonin (5hydroxytryptamine; 5-HT) synthesis rate were used. In the acute treatment study, rats received a drug injection 30 min before the tracer experiment. In the repeated treatment study, the same dose of drug was injected for 7 days. The two-time point method provided a parameter of brain trapping constant (K*) from the slope of linear regression of the Patlak plot, from which 5-HT synthesis rate is calculated. The measured exposure time (h M) for 60 and 150 min after the tracer injection obtained from individual blood sampling in the original method and the h S calculated from h M were used for evaluation of differences in K* values. Results: No significant difference in h M was noted among different experiments, although plasma radioactivity at the end of experiment was significantly different between the acute and the repeated treatments for one of the three drugs. No difference in K* for each treatment was observed between the original method and the simplified single blood sampling method because there was no difference in h M among three experiments and between h M and h S. Conclusion: The simplified a-MTrp ARG method, which uses h S and a single arterial blood sample at the end of each experiment, can be used for the measurement of 5-HT synthesis rate in the rat brain. D 2004 Elsevier Inc. All rights reserved. Keywords: a-[14C]methyl-tryptophan; Two-time point method; Autoradiography; Serotonin (5-HT); Standard exposure time
1. Introduction The brain serotonergic system has been presumed to be related to various types of neuropsychiatric disorders [1–4]. Evaluation of disturbance in serotonergic neurotransmission is very important for treatment of those psychiatric disorders, as well as the investigation of effects of psychopharmaceuticals on serotonin (5-hydroxytryptamine; 5-HT) synthesis in the brain. a-[14C]methyl-l-tryptophan (a-MTrp) was developed as a tracer for measuring 5-HT * Corresponding author. Biomedical Imaging Research Center, University of Fukui, Matsuoka-cho, Fukui 910-1193, Japan. Tel.: +81 776 61 8431/8491; fax: +81 776 61 8170. E-mail address: [email protected] (H. Okazawa). 0969-8051/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.nucmedbio.2004.08.002
synthesis rates in living mammals [5–7]. Use of the autoradiographic (ARG) method with this tracer permits a direct assessment of 5-HT synthesis in the brain, and the effects of drugs on the serotonergic system can be evaluated with alteration in 5-HT synthesis rates without using auxiliary drugs as reported in the previous studies [8–14]. Although several methods have been developed to estimate the rate of 5-HT synthesis, these methods generally require some kind of pharmacological treatment using auxiliary drugs, which may themselves interfere with 5-HT synthesis [15,16]. Measurement of 5-HT synthesis rate using a-MTrp can avoid this possible confounding effect as it is performed in conscious animals without auxiliary drugs. The 5-HT synthesis rate can be directly measured in vivo with this
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method instead of measuring 5-HT release obtained by the microdialysis method [15–17] or metabolic rate of glucose in the brain [18,19]. The ARG two-time point method using a-MTrp has been successfully used to measure 5-HT synthesis rate in the rat brain. In this method, rats were killed at two time points, 60 and 150 min, after the tracer injection, which were determined in the previous investigations [5,6]. Design of this method was based on the unidirectional uptake of aMTrp, which is transported into the brain and has been shown to be converted, in part, to a-methyl-5-HT [5,20]. However, the method requires frequent arterial blood sampling to obtain an arterial input function [6], which is labor intensive and time consuming. A one-time point method may simplify the two-time point method using estimated apparent distribution volume (Vapp) [21]; however, this method requires multiple arterial blood sampling as in the original two-time point method. Experiments with multiple blood sampling take time for the procedure of plasma separation and pipetting, which allows only a single set of 60- and 150-min experiments at a time. The one-time point method would also enable to complete only a couple of rats a day because multiple blood samples are required. However, the simplified method proposed here using a single blood sample should enable several sets of experiments to be carried out at one time. The objective of the present simplified method was to achieve stable measurements of 5-HT synthesis rate with minimal number of blood samples and reducing the time for the analysis of blood samples. Multiple blood samples may introduce additional experimental errors caused by pipetting plasma samples and radioactivity measurements. Furthermore, blood in rats may be diluted toward the end of experiment with the saline required for flushing catheters in the multiple blood sampling method. We evaluated the simplified method in which a standard exposure time (h S) determined from average of individual measured exposure time (h M) was used for the analysis instead of an individual input function.
2. Materials and methods 2.1. Animals Sprague–Dawley male rats (Charles River Laboratories) weighing between 200 and 230 g were used in the acute treatment study and those weighing between 170 and 210 g before starting drug treatment were used in the repeated treatment study. Rats were housed in an animal facility (room temperature 228C; 12-h day–night cycle) for at least 48 h before use in an experiment. The rats were fasted overnight with water supply ad libitum. To avoid any influence of circadian rhythm on the results, the tracer was injected between noon and 2:00 p.m. and all rats were killed between 2:00 and 4:00 p.m. All of the experimental procedures were approved by the Animal Care Committee of the Montreal Neurological Institute and the University of Fukui.
2.2. Autoradiographic procedure The rats were cannulated with plastic catheters in the right femoral artery and vein under light halothane (0.5– 1.0%) anesthesia. After surgical treatment, the posterior limbs of cannulated rats were fixed using a loose-fitting plaster cast and the rats were then allowed to regain consciousness. The body temperature of the rats was kept at 378C using a heat lamp. In the acute treatment study, a dose of three drugs in 0.2 ml of saline was injected subcutaneously 2 h after surgical treatment. The injected drugs were 7-trifluoromethyl-4-(4-methyl-1-piperazinyl) pyrrolo[1,2-a]-quinoxaline dimaleate (CGS12066B; 5 mg/ kg), WAY100635 (1 mg/kg) and anpirtoline (0.5 mg/kg). These drugs are known to act on the brain serotonergic receptors. CGS12066B is an agonist of 5-HT1B/1D receptors [22], and it was shown to decrease 5-HT synthesis in the serotonergic neurons especially after the chronic treatment [13]. WAY100635 is a selective high-affinity 5-HT1A antagonist, and it is expected to increase global synthesis of 5-HT in the acute treatment and reduce it in the chronic treatment [14]. Anpirtoline is predominantly a 5-HT1B agonist and described to have antiaggressive effects [23], and it is expected to reduce 5-HT synthesis after acute administration despite very little influence in the chronic treatment. The same volume of saline was injected to control rats in the same manner. Thirty minutes after the subcutaneous injection of these drugs, 30 ACi (1110 kBq) of a-MTrp in 1 ml of saline was injected at a constant rate through a catheter into the femoral vein over 2 min by an injection pump (Harvard Apparatus, Model 55-2226). In the repeated treatment group, the same dose of drug was injected subcutaneously at a volume of 0.2 ml for 7 days. The same volume of saline was injected to control rats in the same manner. Details of each treatment have been described elsewhere [13,14]. Fourteen rats were treated in each drug group of acute and repeated treatment. Two hours after surgical treatment of femoral cannulation, a-MTrp was injected as in the acute treatment study. Arterial blood samples in a volume of approximately 50 Al at each point were taken at progressively increasing time intervals, starting from immediately after the tracer injection up until rat decapitation. Blood samples were centrifuged for 5 min at 9300 rpm, and 20 Al of plasma was taken for liquid scintillation counting to measure the plasma radioactivity needed for determination of arterial input function. Blood physiological parameters of arterial samples ( p O2, p CO2, pH and hematocrit) were measured using a blood analyzer (Ciba Corning, Model 248) in each experiment. Plasma concentrations of total and free tryptophan were also measured during the experiment using the method described elsewhere [5–11]. Rats were killed using a guillotine 60 or 150 min after the tracer injection. The brains were removed, frozen in cold isopentane (208C) and sliced into 30-Am thicknesses in a cryostat (Leica CM3000 cryostat) at about 208C. Brain
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slices were mounted on glass slides and exposed to X-ray films along with a 14C polymer standard (American Radiolabel; calibrated to 30-Am thickness of the dry tissue) for 3 weeks to obtain autoradiograms. The films were developed, and radioactivity concentrations in different structures were identified with reference to a rat brain atlas using a Microcomputer Imaging Device (MCID/M4-Image Analysis System, Imaging Research, Canada) consisting of a video camera, a frame grabber and the appropriate software. Optical density was converted into tissue tracer concentration based on an appropriate standard calibration curve obtained with the 14C standards exposed with brain slices. 2.3. Calculation of brain trapping constants of a-[14C]MTrp The method uses graphical analysis based on a threecompartment model. The equation used in the analysis is expressed as follows: DV ðhÞ ¼ K4h þ Vapp ;
ð1Þ
where DV(h) and h represent volume of distribution of the R tracer [C b(t)/C p(t)] and exposure time [ C p(t)dt/C p(t)], respectively [24,25]. The variable h is defined as the time necessary to expose brain tissue with plasma with a unit concentration to achieve the same volume of distribution. C b(t) and C p(t) are radioactivity in brain tissue and arterial plasma blood at time t. Influx rate constant K* (brain trapping constant; Al/g/min) is determined from the slope of linear regression derived from Eq. (1) [5,6,24]. A total of 168 rats from three sets of experiments were analyzed in this study. Each of the three experiments corresponded to an acute or repeated treatment study using one of three drugs. Each experiment consisted of control and treatment groups. In each group, two subgroups of six to eight rats underwent 60- or 150-min experiments after the a-MTrp injection with multipoint arterial blood sampling. In the simplified method, we assumed that a h S obtained from the mean of individual h M for each time point (60 and 150 min) could be used for calculation of K* values with corresponding plasma radioactivity at the end of experiment. In the graphical analysis, h is assumed to be unique for each time t if the injection dose and injection speed are constant. Therefore, we tested whether h S at two time points and C p(t) individually determined by a single arterial blood sample at the end of each experiment could be used to calculate reliable K* values. The aim of the method proposed here was to establish stable and reproducible estimates of K* over repeated experiments by reducing experimental errors that may be introduced by multiple blood sampling. Thus, three sets of data obtained during a short period were used for this evaluation to reduce systematic errors associated with different researchers. To validate the simplified method, measured exposures time at 60 and 150 min (h M60 and h M150) in each experiment were compared among 12 rat groups of control
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and drug treatment for the acute and repeated treatment experiments with three different drugs. After the comparison of each h M, K* values (K S*) for one drug were calculated using h S (h S60 and h S150), which was obtained from mean of h M in the experiments with the other two drugs. K S* values thus obtained for three different drugs were compared with K* values (K M*) calculated using individual h M60 and h M150. Both K M* and K S* values were calculated for 10 brain structures (see Table 2) in each group. 2.4. Statistical analysis Data were expressed as meanFS.D. A single-factor analysis of variance (ANOVA) with multiple comparisons using the Tukey test was used to test if there were any differences in h M and K* values among groups. Linear regression analysis and the Bland–Altman plot analysis were used to compare K S* and K M* [26]. The degree of agreement between the simplified and the original ARG two-time points methods was determined as the mean difference (bias or systematic error), standard deviation of the difference (random error), limits of agreement (meanF2S.D.), which represents 95% confidence interval. In the Bland–Altman plot, the correlation between the difference and average of corresponding K S* and K M* should not be significant and the deviation of difference from zero in the y-axis should not be significant if the results of the two methods are equivalent. 3. Results The mean body weights of rats were 214F8 g in acute treatment and 245F18 g at the time of tracer experiments in repeated treatment studies. The weight of rats in acute treatment study was not different between control and drug administration groups. In the 7-day repeated treatment group, the body weight was significantly greater than rats in the acute treatment group at the time of the tracer experiment ( Pb.0001; two-tailed t test). The mean increase of body weight during the 7-day treatment was not
Fig. 1. Representative time–activity curve for the 150-min experiment and corresponding h as a function of time. The shape of the time–activity curves and h were similar in all rats because of the same injection dose and speed of the tracer.
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Table 1 Means of plasma activity and h M at the end of each experiment (meanFS.D.) Acute treatment
Repeated treatment
Control
Treatment
Control
Treatment
CGS12066B h M60 (min) C p60 (nCi/ml) h M150 (min) C p150 (nCi/ml)
83F3 133F8 201F9 99F8
83F1 139F9 203F8 100F10
87F5 104F9* 211F8 72F5*
86F4 102F10* 207F11 73F4*
WAY100635 h M60 (min) C p60 (nCi/ml) h M150 (min) C p150 (nCi/ml)
84F3 139F16 208F13 109F13
87F3 131F16 206F7 106F10
85F3 124F4 207F9 93F11
87F1 124F15 207F8 99F10
Anpirtoline h M60 (min) C p60 (nCi/ml) h M150 (min) C p150 (nCi/ml)
85F4 131F10 210F9 91F9
84F3 133F14 206F7 98F6
87F3 121F10 207F4 89F5
86F3 118F5 210F7 93F12
* Pb.0001 in comparison of plasma activity using ANOVA with post hoc Tukey test.
significantly different between drug-treated rats (41F5 g) and saline-injected controls (45F6 g). There was no significant difference in other physiological parameters as well as plasma free and total tryptophan concentration between different experimental groups and drug groups. Fig. 1 shows the representative time–activity curve for the 150-min experiment and corresponding h M as a function of time. The shape of the time–activity curves and h M were similar in all rats because drugs were administered with equal injection dose and speed. Mean of plasma activity (C p60 and C p150) and measured exposure time (h M60 and h M150) 60 and 150 min after the tracer injection are presented in Table 1. Although the C p at the end point of each experiment was different between acute and repeated treatment studies in the experiment using CGS12066B ( P b.0001, ANOVA with post hoc Tukey test), no significant difference in C p was observed in studies of other drugs.
Fig. 2. Results of multiple comparisons for h M60 and h M150 among 12 groups in three drug experiments. Significant difference was not observed between any pair of groups. Ctl=control; Tr=treatment; Acute, acute treatment; Repeat, repeated treatment.
Table 2 K* values in experiment with CGS12066B Regions
Acute treatment
Repeated treatment
Control
Treatment
Control
Treatment
K M * (ll/g/min) DR 7.8F0.5 MR 4.7F0.5 Fcx 1.5F0.3 Scx 1.5F0.3 CPm 2.4F0.3 1.8F0.2 CPl GP 1.6F0.2 Amy 1.9F0.3 DTh 1.5F0.3 VTh 1.6F0.3
6.6F0.5 3.8F0.5 1.7F0.3 1.6F0.3 2.3F0.3 2.0F0.3 2.0F0.3 2.2F0.2 1.7F0.3 1.9F0.3
7.7F0.7 4.7F0.5 1.1F0.1 1.0F0.2 2.4F0.2 1.6F0.2 1.3F0.2 1.6F0.3 1.0F0.3 1.2F0.2
7.2F0.5 4.0F0.3 0.7F0.3 0.5F0.3 1.5F0.3 1.1F0.2 1.1F0.3 1.5F0.3 0.7F0.2 0.9F0.2
K S * (ll/g/min) DR 7.5F0.5 MR 4.4F0.5 Fcx 1.4F0.3 Scx 1.3F0.3 CPm 2.2F0.4 1.7F0.3 CPl GP 1.5F0.3 Amy 1.8F0.4 DTh 1.4F0.3 VTh 1.5F0.3
6.6F0.5 3.8F0.5 1.7F0.3 1.6F0.3 2.3F0.3 1.9F0.3 1.9F0.3 2.2F0.3 1.7F0.3 1.8F0.3
7.9F0.7 4.9F0.5 1.1F0.2 1.0F0.2 2.4F0.3 1.6F0.2 1.4F0.2 1.7F0.3 1.0F0.3 1.2F0.2
7.3F0.4 4.1F0.3 0.7F0.3 0.5F0.3 1.5F0.3 1.1F0.2 1.1F0.2 1.5F0.3 0.7F0.2 0.9F0.2
DR = dorsal raphe; MR = median raphe; Fcx = frontal cortex; Scx = sensorimotor cortex; CPm = medial caudate putamen; CPl = lateral caudate putamen; GP = globus pallidus; Amy = amygdala; DTh = dorsal thalamus; VTh = ventral thalamus.
h M60 and h M150 also did not show a difference between the various conditions (Table 1). Mean of h M60 and h M150 obtained from all experiments were 85.3F3.3 and Table 3 K* values in experiment with WAY100635 Regions
Acute treatment
Repeated treatment
Control
Treatment
Control
Treatment
K M * (ll/g/min) DR 7.0F1.1 MR 4.5F0.8 Fcx 1.3F0.4 Scx 1.2F0.3 2.1F0.4 CPm CPl 1.6F0.3 GP 1.4F0.3 Amy 1.6F0.3 DTh 1.2F0.3 VTh 1.3F0.3
7.5F0.9 5.4F0.6 1.1F0.3 0.9F0.3 2.0F0.4 1.5F0.4 1.6F0.3 2.2F0.3 1.3F0.3 1.3F0.3
6.0F0.7 3.7F0.6 1.2F0.3 1.2F0.3 1.8F0.3 1.4F0.3 1.6F0.2 1.9F0.3 1.3F0.3 1.3F0.3
7.4F0.9 4.3F0.6 0.9F0.4 0.9F0.3 1.5F0.4 1.3F0.3 1.4F0.2 1.6F0.3 0.8F0.3 0.9F0.3
K S * (ll/g/min) DR 7.4F1.1 MR 4.8F0.8 Fcx 1.4F0.4 1.2F0.4 Scx CPm 2.2F0.4 CPl 1.6F0.3 GP 1.4F0.3 Amy 1.7F0.3 DTh 1.3F0.3 VTh 1.4F0.3
7.3F1.0 5.2F0.6 1.1F0.3 0.8F0.3 2.0F0.4 1.5F0.4 1.6F0.3 2.1F0.3 1.2F0.3 1.3F0.3
6.1F0.7 3.9F0.6 1.2F0.4 1.2F0.3 1.8F0.4 1.3F0.3 1.6F0.3 1.9F0.3 1.3F0.3 1.3F0.3
7.2F1.0 4.2F0.7 0.9F0.4 0.8F0.3 1.5F0.4 1.3F0.3 1.3F0.2 1.6F0.3 0.8F0.3 0.9F0.3
See Table 2 for abbreviations of brain structures.
H. Okazawa et al. / Nuclear Medicine and Biology 31 (2004) 1013 – 1019 Table 4 K* values in experiment with anpirtoline Regions
Acute treatment
Repeated treatment
Control
Treatment
Control
Treatment
K M * (ll/g/min) DR 7.8F0.8 MR 5.0F0.6 Fcx 1.6F0.2 Scx 1.7F0.2 CPm 2.3F0.3 1.9F0.2 CPl GP 2.0F0.2 Amy 2.4F0.2 DTh 1.7F0.2 VTh 1.8F0.3
7.5F0.8 4.8F0.5 1.6F0.3 1.4F0.3 2.1F0.3 1.8F0.3 1.8F0.2 2.1F0.2 1.4F0.3 1.5F0.3
7.5F0.7 4.7F0.6 1.2v0.3 1.3F0.3 2.0F0.3 1.5F0.2 1.5F0.2 1.8F0.2 1.2F0.2 1.2F0.2
6.6F0.7 4.4F0.6 1.2F0.3 1.1F0.3 1.8F0.3 1.7F0.3 1.6F0.3 1.7F0.3 1.1F0.3 1.3F0.3
K M * (ll/g/min) DR 8.0F0.9 MR 5.1F0.7 Fcx 1.6F0.2 Scx 1.7F0.3 CPm 2.3F0.4 1.9F0.3 CPl GP 2.0F0.3 Amy 2.4F0.2 DTh 1.7F0.3 VTh 1.8F0.3
7.6F0.8 4.8F0.5 1.6F0.3 1.4F0.3 2.1F0.3 1.8F0.3 1.8F0.3 2.1F0.2 1.4F0.3 1.5F0.3
7.4F0.6 4.6F0.5 1.2F0.3 1.2F0.3 1.9F0.3 1.5F0.2 1.5F0.2 1.8F0.2 1.1F0.2 1.2F0.2
6.8F0.7 4.6F0.6 1.2F0.3 1.1F0.3 1.8F0.3 1.7F0.3 1.7F0.3 1.8F0.3 1.2F0.2 1.3F0.2
See Table 2 for abbreviations of brain structures.
206.8F8.6 min, respectively. A multiple comparison test did not show a significant difference in h M between any pairs of groups (Fig. 2). A variation of the ratio of h M150 to h M 60 was very small among groups (h M 150/h M 60: 2.42F0.04, coefficient of variation: 1.5%) and there was no difference in h M values between control and treatment groups for each experiment. K* values for three studies with different drugs are presented in Tables 2-4. K S* values were not different compared with the corresponding K M* for each brain structure evaluated (see Tables 2-4). The result of linear regression analysis showed a good correlation between K S* and K M*, with the regression line being almost on the line of identity (Fig. 3). The mean difference between K S* and
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K M* in the Bland–Altman plot was 1.2F3.0%, with limits of agreement of 4.8% and 7.2% (Fig. 3B), which was not significantly different from 0%. 4. Discussion The simplified method proposed in this study using h S was able to estimate the brain trapping constant of K* satisfactorily with no statistically significant error range as observed in the Bland–Altman plot analysis. Because 5-HT synthesis rates are obtained from K* and plasma concentration of free tryptophan, the results of this study demonstrated that the current method can be used for the two-time point ARG method for measurement of 5-HT synthesis rate with a-MTrp [5,6]. The simplified method does not require frequent arterial blood sampling as well as measurement of plasma radioactivity in multiple sampling points. Rat arterial sampling is a delicate process dealing with very small volume of blood. The difficulty of arterial blood sampling and measurement of radioactivity to obtain an input function may cause an increase in experimental error. In the simplified method, only a single blood sample is required at the end of each experiment; this would not yield a severe experimental error because a large volume of sample is available. The original multiple blood sampling method needs 15–18 blood samples for the 150-min experiment with 50 Al for each sample. The single blood sampling method with 100 Al volume of blood in rats would reduce errors in pipetting and counting. The method may also avoid possible dilution of blood toward the end of experiment with the saline required for flushing catheters in the multiple blood sampling method. The stable measurement of C p at the end of the experiment would provide more reliable volume of distribution of the tracer in the brain. Therefore, this simplified method using a h S may yield more stable and reliable estimates of K* values than the original method. One of the three drugs studied (CGS12066B) resulted in a significant difference in plasma counts 60 and 150 min
Fig. 3. (A) Scattered plot for relationship between K* values obtained from individual true input function (K M*) and from standard exposure time (K S*). Dashed line is the line of identity. (B) Bland–Altman plot of differences against average K*. Solid line indicates mean and dashed lines are meanF2S.D. (95% confidence interval of bias, 0.8% to 1.6%; lower limit, 5.3% to 4.0%; upper limit, 6.4% to 7.8%).
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after the tracer injection when comparing acute and repeated treatment rat groups (Table 1). The plasma count may be affected by the body weight of each rat, which may vary among different treatments with different experimental protocols [11,13,14]. On the other hand, the drugs for treatment are assumed to influence on brain trapping of the tracer (C b) without affecting much on C p. The h M did not show any significant difference among groups, presumably R because both C p(t) and C p(t) were equally affected by varying body weight of rats as equal doses of the tracer were injected. All three experiments of different drugs showed quite stable h M both 60 and 150 min after tracer injection (Table 1 and Fig. 2), suggesting a single point of arterial sampling for measurement of plasma activity at the end of each experiment is sufficient for accurate estimation of K*, as well as plasma concentration of tryptophan, which is needed for calculation of 5-HT synthesis rate [5,6]. A standard input function may also be used with a body weight correction and calibration for injection dose; however, the standard input function is not necessary for this experimental protocol because only h and C p at the end of each experiment are required for K* estimation. Furthermore, a standard input function should be corrected by body weight of each rat because C p may vary depending on total plasma volume in a rat as discussed above. The estimate of C p is not simple and may include errors in measurement of body weight. A single blood sample will also calibrate radioactivity in the brain (C b), which may be influenced by body weight and injection dose. Tracer accumulation in the brain (C b) was R assumed to be related to injection dose (i.e., C p(t) and C p(t)) as well as effects of drugs used for treatment. K* values obtained using both h M and h S (K M* and K S*) were similar in every region, indicating that the C p and C b at the end of experiment are closely correlated. The same dose, volume and injection speed of tracer was used for all experiments to stabilize the tracer time–activity curves in the blood and brain. However, a small difference in injection dose would not significantly affect the h M, C b/C p ratio and K*, because the differences in injection dose and body weight would be canceled out in the division process. Bias of the small difference in the injection dose would be smaller than the experimental error anticipated due to frequent arterial blood sampling. Even so, to use this simplified method with the h S, a consistent injection method should be predetermined to reduce experimental errors. A previously reported single-point method with estimated Vapp may also simplify measurement [21]; however, the method requires multiple arterial blood sampling as does the original two-time point method. It should be noted that the single-point method is based on the assumption that Vapp should be constant between animals treated with different drugs. The method proposed in this study only requires a single blood sample before decapitation. This method enables concurrent experiments with multiple rats, which would help to make the ARG method more time
efficient. In conclusion, use of the simplified a-MTrp ARG method with the h S and a single arterial blood sample will be useful for the measurement of 5-HT synthesis rate in the rat brain. Acknowledgment This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (14207039, 14370275), 21st Century COE Program (Medical Science) and the Japan Epilepsy Research Foundation and a grant from the Canadian Institutes of Health Research. MD is a Killam scholar at the Montreal Neurological Institute of McGill University.
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A simplified autoradiographic method with a-[14C]methyl-tryptophan to measure serotonin synthesis rate in the rat brain Hidehiko Okazawa, M.D., Ph.D.a,b,*, Sadahiko Nishizawa, M.D., Ph.D.a,b,c, Tatsuro Tsuchida, M.D., Ph.D.a, Yoshiharu Yonekura, M.D., Ph.D.a, Mirko Diksic, Ph.D.b b
a Biomedical Imaging Research Center, University of Fukui, 23-3 Shimoaizuki Matsuoka-cho, Fukui 910-1193, Japan Cone Laboratory for Neurosurgical Research, Montreal Neurological Institute, 3801 University Street Montreal, Quebec, H3A 2B4 Canada c Hamamatsu Medical Imaging Center, Hamamatsu Medical Photonics Foundation, 5000 Hirakuchi, Hamakita, Shizuoka, 434-0041 Japan Received 7 June 2004; received in revised form 29 July 2004; accepted 7 August 2004
Abstract Background: To minimize blood sampling necessary for the autoradiographic (ARG) method using a-[14C]methyl-tryptophan (a-MTrp), a simplified method using a standard exposure time (h S) was proposed and the accuracy of the method was evaluated. Methods: A total of 168 rats from three sets of experiments with different serotonergic drugs to evaluate changes in cerebral serotonin (5hydroxytryptamine; 5-HT) synthesis rate were used. In the acute treatment study, rats received a drug injection 30 min before the tracer experiment. In the repeated treatment study, the same dose of drug was injected for 7 days. The two-time point method provided a parameter of brain trapping constant (K*) from the slope of linear regression of the Patlak plot, from which 5-HT synthesis rate is calculated. The measured exposure time (h M) for 60 and 150 min after the tracer injection obtained from individual blood sampling in the original method and the h S calculated from h M were used for evaluation of differences in K* values. Results: No significant difference in h M was noted among different experiments, although plasma radioactivity at the end of experiment was significantly different between the acute and the repeated treatments for one of the three drugs. No difference in K* for each treatment was observed between the original method and the simplified single blood sampling method because there was no difference in h M among three experiments and between h M and h S. Conclusion: The simplified a-MTrp ARG method, which uses h S and a single arterial blood sample at the end of each experiment, can be used for the measurement of 5-HT synthesis rate in the rat brain. D 2004 Elsevier Inc. All rights reserved. Keywords: a-[14C]methyl-tryptophan; Two-time point method; Autoradiography; Serotonin (5-HT); Standard exposure time
1. Introduction The brain serotonergic system has been presumed to be related to various types of neuropsychiatric disorders [1–4]. Evaluation of disturbance in serotonergic neurotransmission is very important for treatment of those psychiatric disorders, as well as the investigation of effects of psychopharmaceuticals on serotonin (5-hydroxytryptamine; 5-HT) synthesis in the brain. a-[14C]methyl-l-tryptophan (a-MTrp) was developed as a tracer for measuring 5-HT * Corresponding author. Biomedical Imaging Research Center, University of Fukui, Matsuoka-cho, Fukui 910-1193, Japan. Tel.: +81 776 61 8431/8491; fax: +81 776 61 8170. E-mail address: [email protected] (H. Okazawa). 0969-8051/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.nucmedbio.2004.08.002
synthesis rates in living mammals [5–7]. Use of the autoradiographic (ARG) method with this tracer permits a direct assessment of 5-HT synthesis in the brain, and the effects of drugs on the serotonergic system can be evaluated with alteration in 5-HT synthesis rates without using auxiliary drugs as reported in the previous studies [8–14]. Although several methods have been developed to estimate the rate of 5-HT synthesis, these methods generally require some kind of pharmacological treatment using auxiliary drugs, which may themselves interfere with 5-HT synthesis [15,16]. Measurement of 5-HT synthesis rate using a-MTrp can avoid this possible confounding effect as it is performed in conscious animals without auxiliary drugs. The 5-HT synthesis rate can be directly measured in vivo with this
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method instead of measuring 5-HT release obtained by the microdialysis method [15–17] or metabolic rate of glucose in the brain [18,19]. The ARG two-time point method using a-MTrp has been successfully used to measure 5-HT synthesis rate in the rat brain. In this method, rats were killed at two time points, 60 and 150 min, after the tracer injection, which were determined in the previous investigations [5,6]. Design of this method was based on the unidirectional uptake of aMTrp, which is transported into the brain and has been shown to be converted, in part, to a-methyl-5-HT [5,20]. However, the method requires frequent arterial blood sampling to obtain an arterial input function [6], which is labor intensive and time consuming. A one-time point method may simplify the two-time point method using estimated apparent distribution volume (Vapp) [21]; however, this method requires multiple arterial blood sampling as in the original two-time point method. Experiments with multiple blood sampling take time for the procedure of plasma separation and pipetting, which allows only a single set of 60- and 150-min experiments at a time. The one-time point method would also enable to complete only a couple of rats a day because multiple blood samples are required. However, the simplified method proposed here using a single blood sample should enable several sets of experiments to be carried out at one time. The objective of the present simplified method was to achieve stable measurements of 5-HT synthesis rate with minimal number of blood samples and reducing the time for the analysis of blood samples. Multiple blood samples may introduce additional experimental errors caused by pipetting plasma samples and radioactivity measurements. Furthermore, blood in rats may be diluted toward the end of experiment with the saline required for flushing catheters in the multiple blood sampling method. We evaluated the simplified method in which a standard exposure time (h S) determined from average of individual measured exposure time (h M) was used for the analysis instead of an individual input function.
2. Materials and methods 2.1. Animals Sprague–Dawley male rats (Charles River Laboratories) weighing between 200 and 230 g were used in the acute treatment study and those weighing between 170 and 210 g before starting drug treatment were used in the repeated treatment study. Rats were housed in an animal facility (room temperature 228C; 12-h day–night cycle) for at least 48 h before use in an experiment. The rats were fasted overnight with water supply ad libitum. To avoid any influence of circadian rhythm on the results, the tracer was injected between noon and 2:00 p.m. and all rats were killed between 2:00 and 4:00 p.m. All of the experimental procedures were approved by the Animal Care Committee of the Montreal Neurological Institute and the University of Fukui.
2.2. Autoradiographic procedure The rats were cannulated with plastic catheters in the right femoral artery and vein under light halothane (0.5– 1.0%) anesthesia. After surgical treatment, the posterior limbs of cannulated rats were fixed using a loose-fitting plaster cast and the rats were then allowed to regain consciousness. The body temperature of the rats was kept at 378C using a heat lamp. In the acute treatment study, a dose of three drugs in 0.2 ml of saline was injected subcutaneously 2 h after surgical treatment. The injected drugs were 7-trifluoromethyl-4-(4-methyl-1-piperazinyl) pyrrolo[1,2-a]-quinoxaline dimaleate (CGS12066B; 5 mg/ kg), WAY100635 (1 mg/kg) and anpirtoline (0.5 mg/kg). These drugs are known to act on the brain serotonergic receptors. CGS12066B is an agonist of 5-HT1B/1D receptors [22], and it was shown to decrease 5-HT synthesis in the serotonergic neurons especially after the chronic treatment [13]. WAY100635 is a selective high-affinity 5-HT1A antagonist, and it is expected to increase global synthesis of 5-HT in the acute treatment and reduce it in the chronic treatment [14]. Anpirtoline is predominantly a 5-HT1B agonist and described to have antiaggressive effects [23], and it is expected to reduce 5-HT synthesis after acute administration despite very little influence in the chronic treatment. The same volume of saline was injected to control rats in the same manner. Thirty minutes after the subcutaneous injection of these drugs, 30 ACi (1110 kBq) of a-MTrp in 1 ml of saline was injected at a constant rate through a catheter into the femoral vein over 2 min by an injection pump (Harvard Apparatus, Model 55-2226). In the repeated treatment group, the same dose of drug was injected subcutaneously at a volume of 0.2 ml for 7 days. The same volume of saline was injected to control rats in the same manner. Details of each treatment have been described elsewhere [13,14]. Fourteen rats were treated in each drug group of acute and repeated treatment. Two hours after surgical treatment of femoral cannulation, a-MTrp was injected as in the acute treatment study. Arterial blood samples in a volume of approximately 50 Al at each point were taken at progressively increasing time intervals, starting from immediately after the tracer injection up until rat decapitation. Blood samples were centrifuged for 5 min at 9300 rpm, and 20 Al of plasma was taken for liquid scintillation counting to measure the plasma radioactivity needed for determination of arterial input function. Blood physiological parameters of arterial samples ( p O2, p CO2, pH and hematocrit) were measured using a blood analyzer (Ciba Corning, Model 248) in each experiment. Plasma concentrations of total and free tryptophan were also measured during the experiment using the method described elsewhere [5–11]. Rats were killed using a guillotine 60 or 150 min after the tracer injection. The brains were removed, frozen in cold isopentane (208C) and sliced into 30-Am thicknesses in a cryostat (Leica CM3000 cryostat) at about 208C. Brain
H. Okazawa et al. / Nuclear Medicine and Biology 31 (2004) 1013 – 1019
slices were mounted on glass slides and exposed to X-ray films along with a 14C polymer standard (American Radiolabel; calibrated to 30-Am thickness of the dry tissue) for 3 weeks to obtain autoradiograms. The films were developed, and radioactivity concentrations in different structures were identified with reference to a rat brain atlas using a Microcomputer Imaging Device (MCID/M4-Image Analysis System, Imaging Research, Canada) consisting of a video camera, a frame grabber and the appropriate software. Optical density was converted into tissue tracer concentration based on an appropriate standard calibration curve obtained with the 14C standards exposed with brain slices. 2.3. Calculation of brain trapping constants of a-[14C]MTrp The method uses graphical analysis based on a threecompartment model. The equation used in the analysis is expressed as follows: DV ðhÞ ¼ K4h þ Vapp ;
ð1Þ
where DV(h) and h represent volume of distribution of the R tracer [C b(t)/C p(t)] and exposure time [ C p(t)dt/C p(t)], respectively [24,25]. The variable h is defined as the time necessary to expose brain tissue with plasma with a unit concentration to achieve the same volume of distribution. C b(t) and C p(t) are radioactivity in brain tissue and arterial plasma blood at time t. Influx rate constant K* (brain trapping constant; Al/g/min) is determined from the slope of linear regression derived from Eq. (1) [5,6,24]. A total of 168 rats from three sets of experiments were analyzed in this study. Each of the three experiments corresponded to an acute or repeated treatment study using one of three drugs. Each experiment consisted of control and treatment groups. In each group, two subgroups of six to eight rats underwent 60- or 150-min experiments after the a-MTrp injection with multipoint arterial blood sampling. In the simplified method, we assumed that a h S obtained from the mean of individual h M for each time point (60 and 150 min) could be used for calculation of K* values with corresponding plasma radioactivity at the end of experiment. In the graphical analysis, h is assumed to be unique for each time t if the injection dose and injection speed are constant. Therefore, we tested whether h S at two time points and C p(t) individually determined by a single arterial blood sample at the end of each experiment could be used to calculate reliable K* values. The aim of the method proposed here was to establish stable and reproducible estimates of K* over repeated experiments by reducing experimental errors that may be introduced by multiple blood sampling. Thus, three sets of data obtained during a short period were used for this evaluation to reduce systematic errors associated with different researchers. To validate the simplified method, measured exposures time at 60 and 150 min (h M60 and h M150) in each experiment were compared among 12 rat groups of control
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and drug treatment for the acute and repeated treatment experiments with three different drugs. After the comparison of each h M, K* values (K S*) for one drug were calculated using h S (h S60 and h S150), which was obtained from mean of h M in the experiments with the other two drugs. K S* values thus obtained for three different drugs were compared with K* values (K M*) calculated using individual h M60 and h M150. Both K M* and K S* values were calculated for 10 brain structures (see Table 2) in each group. 2.4. Statistical analysis Data were expressed as meanFS.D. A single-factor analysis of variance (ANOVA) with multiple comparisons using the Tukey test was used to test if there were any differences in h M and K* values among groups. Linear regression analysis and the Bland–Altman plot analysis were used to compare K S* and K M* [26]. The degree of agreement between the simplified and the original ARG two-time points methods was determined as the mean difference (bias or systematic error), standard deviation of the difference (random error), limits of agreement (meanF2S.D.), which represents 95% confidence interval. In the Bland–Altman plot, the correlation between the difference and average of corresponding K S* and K M* should not be significant and the deviation of difference from zero in the y-axis should not be significant if the results of the two methods are equivalent. 3. Results The mean body weights of rats were 214F8 g in acute treatment and 245F18 g at the time of tracer experiments in repeated treatment studies. The weight of rats in acute treatment study was not different between control and drug administration groups. In the 7-day repeated treatment group, the body weight was significantly greater than rats in the acute treatment group at the time of the tracer experiment ( Pb.0001; two-tailed t test). The mean increase of body weight during the 7-day treatment was not
Fig. 1. Representative time–activity curve for the 150-min experiment and corresponding h as a function of time. The shape of the time–activity curves and h were similar in all rats because of the same injection dose and speed of the tracer.
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Table 1 Means of plasma activity and h M at the end of each experiment (meanFS.D.) Acute treatment
Repeated treatment
Control
Treatment
Control
Treatment
CGS12066B h M60 (min) C p60 (nCi/ml) h M150 (min) C p150 (nCi/ml)
83F3 133F8 201F9 99F8
83F1 139F9 203F8 100F10
87F5 104F9* 211F8 72F5*
86F4 102F10* 207F11 73F4*
WAY100635 h M60 (min) C p60 (nCi/ml) h M150 (min) C p150 (nCi/ml)
84F3 139F16 208F13 109F13
87F3 131F16 206F7 106F10
85F3 124F4 207F9 93F11
87F1 124F15 207F8 99F10
Anpirtoline h M60 (min) C p60 (nCi/ml) h M150 (min) C p150 (nCi/ml)
85F4 131F10 210F9 91F9
84F3 133F14 206F7 98F6
87F3 121F10 207F4 89F5
86F3 118F5 210F7 93F12
* Pb.0001 in comparison of plasma activity using ANOVA with post hoc Tukey test.
significantly different between drug-treated rats (41F5 g) and saline-injected controls (45F6 g). There was no significant difference in other physiological parameters as well as plasma free and total tryptophan concentration between different experimental groups and drug groups. Fig. 1 shows the representative time–activity curve for the 150-min experiment and corresponding h M as a function of time. The shape of the time–activity curves and h M were similar in all rats because drugs were administered with equal injection dose and speed. Mean of plasma activity (C p60 and C p150) and measured exposure time (h M60 and h M150) 60 and 150 min after the tracer injection are presented in Table 1. Although the C p at the end point of each experiment was different between acute and repeated treatment studies in the experiment using CGS12066B ( P b.0001, ANOVA with post hoc Tukey test), no significant difference in C p was observed in studies of other drugs.
Fig. 2. Results of multiple comparisons for h M60 and h M150 among 12 groups in three drug experiments. Significant difference was not observed between any pair of groups. Ctl=control; Tr=treatment; Acute, acute treatment; Repeat, repeated treatment.
Table 2 K* values in experiment with CGS12066B Regions
Acute treatment
Repeated treatment
Control
Treatment
Control
Treatment
K M * (ll/g/min) DR 7.8F0.5 MR 4.7F0.5 Fcx 1.5F0.3 Scx 1.5F0.3 CPm 2.4F0.3 1.8F0.2 CPl GP 1.6F0.2 Amy 1.9F0.3 DTh 1.5F0.3 VTh 1.6F0.3
6.6F0.5 3.8F0.5 1.7F0.3 1.6F0.3 2.3F0.3 2.0F0.3 2.0F0.3 2.2F0.2 1.7F0.3 1.9F0.3
7.7F0.7 4.7F0.5 1.1F0.1 1.0F0.2 2.4F0.2 1.6F0.2 1.3F0.2 1.6F0.3 1.0F0.3 1.2F0.2
7.2F0.5 4.0F0.3 0.7F0.3 0.5F0.3 1.5F0.3 1.1F0.2 1.1F0.3 1.5F0.3 0.7F0.2 0.9F0.2
K S * (ll/g/min) DR 7.5F0.5 MR 4.4F0.5 Fcx 1.4F0.3 Scx 1.3F0.3 CPm 2.2F0.4 1.7F0.3 CPl GP 1.5F0.3 Amy 1.8F0.4 DTh 1.4F0.3 VTh 1.5F0.3
6.6F0.5 3.8F0.5 1.7F0.3 1.6F0.3 2.3F0.3 1.9F0.3 1.9F0.3 2.2F0.3 1.7F0.3 1.8F0.3
7.9F0.7 4.9F0.5 1.1F0.2 1.0F0.2 2.4F0.3 1.6F0.2 1.4F0.2 1.7F0.3 1.0F0.3 1.2F0.2
7.3F0.4 4.1F0.3 0.7F0.3 0.5F0.3 1.5F0.3 1.1F0.2 1.1F0.2 1.5F0.3 0.7F0.2 0.9F0.2
DR = dorsal raphe; MR = median raphe; Fcx = frontal cortex; Scx = sensorimotor cortex; CPm = medial caudate putamen; CPl = lateral caudate putamen; GP = globus pallidus; Amy = amygdala; DTh = dorsal thalamus; VTh = ventral thalamus.
h M60 and h M150 also did not show a difference between the various conditions (Table 1). Mean of h M60 and h M150 obtained from all experiments were 85.3F3.3 and Table 3 K* values in experiment with WAY100635 Regions
Acute treatment
Repeated treatment
Control
Treatment
Control
Treatment
K M * (ll/g/min) DR 7.0F1.1 MR 4.5F0.8 Fcx 1.3F0.4 Scx 1.2F0.3 2.1F0.4 CPm CPl 1.6F0.3 GP 1.4F0.3 Amy 1.6F0.3 DTh 1.2F0.3 VTh 1.3F0.3
7.5F0.9 5.4F0.6 1.1F0.3 0.9F0.3 2.0F0.4 1.5F0.4 1.6F0.3 2.2F0.3 1.3F0.3 1.3F0.3
6.0F0.7 3.7F0.6 1.2F0.3 1.2F0.3 1.8F0.3 1.4F0.3 1.6F0.2 1.9F0.3 1.3F0.3 1.3F0.3
7.4F0.9 4.3F0.6 0.9F0.4 0.9F0.3 1.5F0.4 1.3F0.3 1.4F0.2 1.6F0.3 0.8F0.3 0.9F0.3
K S * (ll/g/min) DR 7.4F1.1 MR 4.8F0.8 Fcx 1.4F0.4 1.2F0.4 Scx CPm 2.2F0.4 CPl 1.6F0.3 GP 1.4F0.3 Amy 1.7F0.3 DTh 1.3F0.3 VTh 1.4F0.3
7.3F1.0 5.2F0.6 1.1F0.3 0.8F0.3 2.0F0.4 1.5F0.4 1.6F0.3 2.1F0.3 1.2F0.3 1.3F0.3
6.1F0.7 3.9F0.6 1.2F0.4 1.2F0.3 1.8F0.4 1.3F0.3 1.6F0.3 1.9F0.3 1.3F0.3 1.3F0.3
7.2F1.0 4.2F0.7 0.9F0.4 0.8F0.3 1.5F0.4 1.3F0.3 1.3F0.2 1.6F0.3 0.8F0.3 0.9F0.3
See Table 2 for abbreviations of brain structures.
H. Okazawa et al. / Nuclear Medicine and Biology 31 (2004) 1013 – 1019 Table 4 K* values in experiment with anpirtoline Regions
Acute treatment
Repeated treatment
Control
Treatment
Control
Treatment
K M * (ll/g/min) DR 7.8F0.8 MR 5.0F0.6 Fcx 1.6F0.2 Scx 1.7F0.2 CPm 2.3F0.3 1.9F0.2 CPl GP 2.0F0.2 Amy 2.4F0.2 DTh 1.7F0.2 VTh 1.8F0.3
7.5F0.8 4.8F0.5 1.6F0.3 1.4F0.3 2.1F0.3 1.8F0.3 1.8F0.2 2.1F0.2 1.4F0.3 1.5F0.3
7.5F0.7 4.7F0.6 1.2v0.3 1.3F0.3 2.0F0.3 1.5F0.2 1.5F0.2 1.8F0.2 1.2F0.2 1.2F0.2
6.6F0.7 4.4F0.6 1.2F0.3 1.1F0.3 1.8F0.3 1.7F0.3 1.6F0.3 1.7F0.3 1.1F0.3 1.3F0.3
K M * (ll/g/min) DR 8.0F0.9 MR 5.1F0.7 Fcx 1.6F0.2 Scx 1.7F0.3 CPm 2.3F0.4 1.9F0.3 CPl GP 2.0F0.3 Amy 2.4F0.2 DTh 1.7F0.3 VTh 1.8F0.3
7.6F0.8 4.8F0.5 1.6F0.3 1.4F0.3 2.1F0.3 1.8F0.3 1.8F0.3 2.1F0.2 1.4F0.3 1.5F0.3
7.4F0.6 4.6F0.5 1.2F0.3 1.2F0.3 1.9F0.3 1.5F0.2 1.5F0.2 1.8F0.2 1.1F0.2 1.2F0.2
6.8F0.7 4.6F0.6 1.2F0.3 1.1F0.3 1.8F0.3 1.7F0.3 1.7F0.3 1.8F0.3 1.2F0.2 1.3F0.2
See Table 2 for abbreviations of brain structures.
206.8F8.6 min, respectively. A multiple comparison test did not show a significant difference in h M between any pairs of groups (Fig. 2). A variation of the ratio of h M150 to h M 60 was very small among groups (h M 150/h M 60: 2.42F0.04, coefficient of variation: 1.5%) and there was no difference in h M values between control and treatment groups for each experiment. K* values for three studies with different drugs are presented in Tables 2-4. K S* values were not different compared with the corresponding K M* for each brain structure evaluated (see Tables 2-4). The result of linear regression analysis showed a good correlation between K S* and K M*, with the regression line being almost on the line of identity (Fig. 3). The mean difference between K S* and
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K M* in the Bland–Altman plot was 1.2F3.0%, with limits of agreement of 4.8% and 7.2% (Fig. 3B), which was not significantly different from 0%. 4. Discussion The simplified method proposed in this study using h S was able to estimate the brain trapping constant of K* satisfactorily with no statistically significant error range as observed in the Bland–Altman plot analysis. Because 5-HT synthesis rates are obtained from K* and plasma concentration of free tryptophan, the results of this study demonstrated that the current method can be used for the two-time point ARG method for measurement of 5-HT synthesis rate with a-MTrp [5,6]. The simplified method does not require frequent arterial blood sampling as well as measurement of plasma radioactivity in multiple sampling points. Rat arterial sampling is a delicate process dealing with very small volume of blood. The difficulty of arterial blood sampling and measurement of radioactivity to obtain an input function may cause an increase in experimental error. In the simplified method, only a single blood sample is required at the end of each experiment; this would not yield a severe experimental error because a large volume of sample is available. The original multiple blood sampling method needs 15–18 blood samples for the 150-min experiment with 50 Al for each sample. The single blood sampling method with 100 Al volume of blood in rats would reduce errors in pipetting and counting. The method may also avoid possible dilution of blood toward the end of experiment with the saline required for flushing catheters in the multiple blood sampling method. The stable measurement of C p at the end of the experiment would provide more reliable volume of distribution of the tracer in the brain. Therefore, this simplified method using a h S may yield more stable and reliable estimates of K* values than the original method. One of the three drugs studied (CGS12066B) resulted in a significant difference in plasma counts 60 and 150 min
Fig. 3. (A) Scattered plot for relationship between K* values obtained from individual true input function (K M*) and from standard exposure time (K S*). Dashed line is the line of identity. (B) Bland–Altman plot of differences against average K*. Solid line indicates mean and dashed lines are meanF2S.D. (95% confidence interval of bias, 0.8% to 1.6%; lower limit, 5.3% to 4.0%; upper limit, 6.4% to 7.8%).
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after the tracer injection when comparing acute and repeated treatment rat groups (Table 1). The plasma count may be affected by the body weight of each rat, which may vary among different treatments with different experimental protocols [11,13,14]. On the other hand, the drugs for treatment are assumed to influence on brain trapping of the tracer (C b) without affecting much on C p. The h M did not show any significant difference among groups, presumably R because both C p(t) and C p(t) were equally affected by varying body weight of rats as equal doses of the tracer were injected. All three experiments of different drugs showed quite stable h M both 60 and 150 min after tracer injection (Table 1 and Fig. 2), suggesting a single point of arterial sampling for measurement of plasma activity at the end of each experiment is sufficient for accurate estimation of K*, as well as plasma concentration of tryptophan, which is needed for calculation of 5-HT synthesis rate [5,6]. A standard input function may also be used with a body weight correction and calibration for injection dose; however, the standard input function is not necessary for this experimental protocol because only h and C p at the end of each experiment are required for K* estimation. Furthermore, a standard input function should be corrected by body weight of each rat because C p may vary depending on total plasma volume in a rat as discussed above. The estimate of C p is not simple and may include errors in measurement of body weight. A single blood sample will also calibrate radioactivity in the brain (C b), which may be influenced by body weight and injection dose. Tracer accumulation in the brain (C b) was R assumed to be related to injection dose (i.e., C p(t) and C p(t)) as well as effects of drugs used for treatment. K* values obtained using both h M and h S (K M* and K S*) were similar in every region, indicating that the C p and C b at the end of experiment are closely correlated. The same dose, volume and injection speed of tracer was used for all experiments to stabilize the tracer time–activity curves in the blood and brain. However, a small difference in injection dose would not significantly affect the h M, C b/C p ratio and K*, because the differences in injection dose and body weight would be canceled out in the division process. Bias of the small difference in the injection dose would be smaller than the experimental error anticipated due to frequent arterial blood sampling. Even so, to use this simplified method with the h S, a consistent injection method should be predetermined to reduce experimental errors. A previously reported single-point method with estimated Vapp may also simplify measurement [21]; however, the method requires multiple arterial blood sampling as does the original two-time point method. It should be noted that the single-point method is based on the assumption that Vapp should be constant between animals treated with different drugs. The method proposed in this study only requires a single blood sample before decapitation. This method enables concurrent experiments with multiple rats, which would help to make the ARG method more time
efficient. In conclusion, use of the simplified a-MTrp ARG method with the h S and a single arterial blood sample will be useful for the measurement of 5-HT synthesis rate in the rat brain. Acknowledgment This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (14207039, 14370275), 21st Century COE Program (Medical Science) and the Japan Epilepsy Research Foundation and a grant from the Canadian Institutes of Health Research. MD is a Killam scholar at the Montreal Neurological Institute of McGill University.
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