A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat

Summary

Small-animal positron emission tomography enables the assessment of the brain's two main energy substrates: glucose and ketones. In the present method, ¹¹C-acetoacetate and ¹⁸F-fluorodeoxyglucose are injected sequentially in each animal, and their uptake is measured quantitatively in specific brain regions determined from the magnetic resonance images.

Abstract

We present a method for comparing the uptake of the brain's two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the rat. The developed method is a small-animal positron emission tomography (PET) protocol, in which ¹¹C-AcAc and ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) are injected sequentially in each animal. This dual tracer PET acquisition is possible because of the short half-life of ¹¹C (20.4 min). The rats also undergo a magnetic resonance imaging (MRI) acquisition seven days before the PET protocol. Prior to image analysis, PET and MRI images are coregistered to allow the measurement of regional cerebral uptake (cortex, hippocampus, striatum, and cerebellum). A quantitative measure of ¹¹C-AcAc and ¹⁸F-FDG brain uptake (cerebral metabolic rate; μmol/100 g/min) is determined by kinetic modeling using the image-derived input function (IDIF) method. Our new dual tracer PET protocol is robust and flexible; the two tracers used can be replaced by different radiotracers to evaluate other processes in the brain. Moreover, our protocol is applicable to the study of brain fuel supply in multiple conditions such as normal aging and neurodegenerative pathologies such as Alzheimer's and Parkinson's diseases.

Introduction

Context and Rationale

Positron emission tomography (PET) enables the minimally-invasive study of functional processes in the brain. Glucose is the brain's main energy substrate, but in conditions of glucose deficiency, ketones (acetoacetate [AcAc] and β-hydroxybutyrate) are the main alternative energy substrates. Brain energy metabolism has been widely studied by PET using the most common PET tracer, ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG), a glucose analog. Our group recently developed a novel radiotracer -¹¹C-AcAc - to measure brain ketone metabolism¹. Magnetic resonance imaging (MRI) is a m....

Protocol

All experiments were completed in accordance with the Animal Care and Use Committee at the Université de Sherbrooke and with the Canadian Council on Animal Care. The experimental protocol was approved by the Institutional Animal Research Ethics Review Board (protocol #011-09).

1. Brain Anatomy with MRI

1. Let rats acclimatize in the animal facility for a minimum of 7 days prior to the protocol. Perform brain MRI scans 1-2 weeks prior to the dual tracer PET protocol to allow full recovery from the anesthesia.
2. Anesthetize the rat in an induction chamber. Use 2% isoflurane and 1.5 L/min oxygen throughout the protocol for ane....

Results

As seen in Figure 2,¹¹C-AcAc uptake is low within the brain itself. As mentioned earlier, ketones consumption by the brain is very low on a short-term fasting. ¹¹C-AcAc uptake is higher in the tongue and cheek muscles. Indeed, ketones are rapidly taken up by rat skeletal muscles²³. In contrast, ¹⁸F-FDG uptake is mostly in the brain and the cheek muscles. Figure 2 shows that during the coregistration process, MR images are fixed and PET images m.......

Discussion

Critical steps

A critical step in this dual tracer PET protocol is to be able to simultaneously scan the heart's left ventricle and the brain at the same time. This requires a PET scanner with a sufficient axial length, i.e. a minimum of 7.5 cm. A few test scans are needed to determine the exact position of the scanner table (x, y, and z values), where the brain and the heart are scanned correctly.

Tracer injection is also a crucial point for a succes.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MRI scanner	Varian		7 Tesla
Small-animal PET scanner	Gamma Medica		Lab-PET/-Triumph
Heat mat	Sunbeam	PN 143937
Heater system	SA Instruments	761 100 Rev B
Respiratory gating	SA Instruments	SAII's P-resp
Clinical chemistry analyzer	Siemens Healthcare Diagnosis	765000.931	Dimension Xpand Plus
Polyethylene tubing 50	Becton Dickinson	427411
Injection pump	KD Scientific	Model 210
Gamma-counter	GMI	Packard Cobra II
Centrifuge	Thermo Scientific	75002416	Heraeus Pico 21
PMOD software	PMOD Technologies		PMOD 3.2 version
Geiger counter	Fluke Biomedical	ASM-990	Advanced Survey Meter
Reagent
Isoflurane	Abbott Laboratories, Ltd	B506
0.9% NaCl solution	Hospira	4888010
Heparin	Sandoz	1004336
Isopropenyl acetate	Aldrich	11778	99%
Methyllithium	Aldrich	197343	1.6 M
THF	Aldrich	87371
Flex reagent cartridge glucose	Siemens Healthcare Diagnosis	DF40
Trizma base	Sigma	T6066-500G	prepare tris buffer 100 mM pH 7.0
Sodium oxamate	Sigma	O2751	20 mM
NADH	Roche	10128015001	0.15 mM
b-Hydroxybutyrate dehydrogenase	Toyobo	HBD-301	1 U/ml