Male Wistar rats were purchased from Charles River (Margate, Kent, UK). Fourteen rats were scanned with [11C]leucine; all animals had online and discrete blood sampling; 6 were used for baseline scan, and 3 received a pre-injection of anisomycin (PSR inhibition) prior to PET scan, and an additional 5 rats were used for measurements of [11C]leucine and LNAA concentrations in blood and plasma on the bench only (i.e. not scanned). Two male and two female Fischer-344 (WT) and TgF344-AD (TG) rats with the APPswe and PS1Δe9 mutations (purchased from the laboratory of Prof T. Town, University of Southern California) were set up as breeding pairs, housed in the Biological Services Unit at the University of Manchester for breeding purposes. Genotyping was outsourced to Transnetyx® (Cordova, USA). Male WT and TG rats were imaged via [11C]leucine PET at 6, 12 and 18 months of age (for details of n number, body weight and inclusion/exclusion, see Supplementary Tables 1 and 2). All procedures were conducted in accordance with the Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Animals. For the whole duration of the experiment, all animals were housed in groups of 2-4 per cage with individual ventilation, environmental enrichment, constant access to food and water and a 12:12-h light/dark cycle (7 AM to 7 PM). Although using other PET tracers, sample sizes were based on our experience using this [13] and other models of AD [16] and PET imaging. Investigators were not blind to the genotype of the animals. Exclusion criteria were related to potential health issues (spontaneous tumour, etc.) preventing scanning of the animals or leading to the animal to be culled to prevent unnecessary suffering. Whenever possible, animals that had died or were excluded before the 18 months time point were replaced by age-matched rats of the same genotype (for more details see Supplementary Tables 1 and 2).
Scanning Protocol and Image AnalysisL-Leucine was labelled at the carboxyl acid group 1 with 11C as previously described [17] with a purity > 98% and a 37.5 ± 7.5 GBq/µmol molar activity. Rats were anaesthetised by isoflurane inhalation (induction 4–5% and 2–2.5% thereafter) in O2/NO2 (30%/70%), monitored for respiration and temperature using a pressure-sensitive pad and a rectal probe (BioVet, m2m Imaging Corp., USA) for the duration of the scans. Imaging was carried out on a Siemens Inveon® PET/CT scanner (for full details of acquisition and reconstruction protocols, see Supplementary materials). For all scans, 300 μl of tracer (injected dose 37.7 ± 7.45 MBq, molar activity 22.7 ± 18.7 GBq/µmol at injection time) and 300 μl of saline were injected in the tail vein sequentially using injection pumps (syringe pump, Cole-Palmer®, ref. WZ-74905–02) at a rate of 1.2 ml/min in a single bolus over 30 s at the start of a 60-min PET acquisition. To scale the population-based input function (PBIF) to each individual in the longitudinal study, images were segmented automatically using local means analysis (LMA) in BrainVisa 4.1.1 (http://brainvisa.info) [18,19,20,21], and the segmented region of interest (ROI) of the heart left ventricle was then selected. Skeletal and whole-brain ROIs were defined manually in the CT images using Anatomist 4.1.1 to register PET-CT images with the rat MRI template adapted from Schwarz and colleagues [22] used thereafter for quantification of atlas-based brain ROIs.
In order to assess the sensitivity of the [11C]leucine PET to measure PSR alterations, three Wistar rats were injected i.v. with 60 mg/kg of anisomycin in saline (pH 7) [23] 10 min prior PET acquisition. Anisomycin inhibits the protein synthesis by interfering with the 80S ribosome, and the effect is rapid (within 15 min) and reversible (lasts 90 min), 60 mg/kg producing an 80-96% inhibition of the PSR [23, 24]. Images are shown as standard uptake values (uptake in Bq.cm−3 × 10−6 × body weight (g)/injected dose in MBq, hence normalising uptake for injected dose and body weight).
Input FunctionsIndividual arterial input functions (AIF) were determined for each Wistar rat (n = 6 baseline and n = 3 with anisomycin) and WT and TG rats at 12 (n = 3) and 18 months (n = 6) (due to unexpected loss of animals and other experimental constraints, the n number were low for blood data in WT and TG, and both genotypes were pooled together) (see details in Supplementary Table 3). A femoral arteriovenous shunt was connected to a Swisstrace™ Twilite® system for continuous monitoring of whole blood radioactivity (full details in Supplementary materials). Whole blood samples were collected from the shunt at 2, 5, 10, 20, 30, 40 and 60 min post-injection into heparinised Eppendorf tubes and placed immediately on ice. Aliquots of whole blood and plasma were counted using a γ-counter, and additional plasma aliquots were stored at -80 °C (full details in Supplementary materials) until sent to Alta Bioscience Ltd (Redditch, UK, https://altabioscience.com) for analysis of the large neutral amino acids (LNAA) (histidine, methionine, leucine, isoleucine, valine, phenylalanine, tyrosine and tryptophan), concentration in nmol/ml [25]. The plasma/whole blood and free/protein-incorporated [11C]leucine ratios were measured by γ-counting (full details in Supplementary materials). For the longitudinal study, the PSR was calculated using population-based values of unlabelled leucine, a population-based AIF scaled for each rat using an image-derived input function derived from quantification of the activity in the left ventricle (full details in Supplementary materials and Figs. S1 and S2) and the average plasma/whole blood ratio and free/protein-incorporated [11C]leucine in plasma ratio obtained in the F344 rats (Fig. 3D).
ModellingIn-house software MICK (Modelling, Input functions and Compartmental Kinetics), written in MATLAB, was used to determine the PSR through the calculation of the three rate constants (Fig. 1), Kcplx and λ [11]. The compartmental model used to study the [11C]leucine brain uptake is shown in Fig. 1.
Fig. 1Simplified compartmental model of leucine metabolism in the brain. The rate constants K1 and k2 represent the transfer of leucine across the blood-brain barrier from blood to the brain and vice versa. k3 represents the incorporation of leucine in proteins, and krec (recycling of leucine from the protein pool) is negligible (adapted from[11]).
λ is defined as
$$\underset}[\frac}}]$$
(1)
where Cf is the concentration of free leucine in the precursor pool in the tissue and Cp is the concentrations of leucine in plasma (cold = unlabelled leucine). Unlabelled leucine is at steady state in tissue and:
$$\frac_}_}=\frac_}_} = 0$$
(2)
and because Cf for labelled leucine is equal to
$$_=\frac\times C_} + k_)}$$
(3)
λ will be [11]
$$\lambda =\frac_}_+_}$$
(4)
The unidirectional uptake rate of plasma leucine into tissue Kcplx is calculated as follows [11]:
For [11C]leucine, it is assumed that none is recycled from radioactive proteins during the experimental time of 60 min; krec = 0. Therefore, the PSR can be estimated by the following equation:
$$PSR=_\times \frac$$
(6)
where PSR is in μM·min−1, Kcplx in min−1 and leucine[C] is the concentration of unlabelled leucine in arterial plasma in nmole·ml−1.
Statistical AnalysisGraphPad Prism 9.4 was used to analyse the data. All data are expressed as mean ± SD. Effects of anisomycin on Kcplx and PSR were analysed using a two-way ANOVA (treatment and ROIs as main factors and interaction treatment × ROIs). Two-way ANOVA (age and origin of samples (arterial vs venous) as main factors and interaction age × origin) and Šídák post hoc tests were used to compare the differences in concentration of unlabelled leucine between arterial and venous plasma samples in WT rats at 12 and 18 months. PSR and SUV in the longitudinal study were analysed using mixed effect analysis (age and genotype as main factors and interaction age × genotype) and Šídák post hoc tests.
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