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.
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Roy, M., Nugent, S., Tremblay, S., Descoteaux, M., Beaudoin, J. F., Tremblay, L., et al. A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat. J. Vis. Exp. (82), e50761, doi:10.3791/50761 (2013).
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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.
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 much higher resolution technique (0.1 mm × 0.1 mm in-plane resolution) than PET, and is needed to clearly localize anatomical brain regions required for the regional PET analysis of brain energy metabolism.
PET data are commonly expressed as standardized uptake values (SUV)2-6. SUV are the tissue activity concentration normalized by the fraction of the injected dose/unit weight, as initially proposed over 70 years ago7. These units are still widely used because they require simpler PET acquisition and image analysis methodologies. However, an important limitation is that SUV are relative not absolute units, making it difficult to compare results across different studies. This difficulty of comparison may contribute to contradictory findings in the literature on brain glucose uptake in the elderly8. Therefore, the quantitative cerebral metabolic rate (CMR; μmol/100 g/min) has particular advantages9-11. Generating CMR values requires a dynamic PET acquisition and the plasma radioactivity counts as a function of time, i.e. the plasma time-activity curve (TAC) or input function. The input function can be obtained by multiple blood samplings throughout the PET acquisition9,12 or by the image-derived input function (IDIF) method, in which a region of interest is drawn on a blood pool (heart's left ventricle or major artery)11,13-16.
The aim of our method was to quantitatively compare for the first time the uptake of the brain's two key energy substrates, glucose and ketones, using PET and MRI in rodents. 11C-AcAc and 18F-FDG were used sequentially in the same animal. The protocol was designed to measure regional uptakes in different relevant brain structures (cortex, hippocampus, striatum, and cerebellum) clearly visible in the MR images. The protocol was also specifically intended to permit quantitative analysis of tracer brain uptake, i.e. CMR of both 18F-FDG and 11C-AcAc. Although this protocol was developed to study brain energy substrates, the radiotracers we used could be replaced by others, and the same methodology can be used to study different brain functional processes.
Advantages over existing methods
PET and MRI do not require the animal to be sacrificed after the acquisition. Therefore, follow-up studies of treatments are possible. Thus, baseline data followed by an experimental condition can be measured within the same animal, thereby reducing both biological variability and the number of animals required. A key advantage of our dual tracer PET protocol is to compare both tracers uptake in the same animal under the same physiological conditions within the same imaging session, thereby reducing even more biological variability and systematic discrepancies. This dual tracer PET protocol is feasible primarily because of the short physical half-life of 11C (20.4 min) and the fast biological washout of 11C-AcAc, which leave minimal residual 11C radioactivity during the second acquisition with 18F-FDG. The MRI scan is an important feature of this protocol as it enables the tracer uptake to be studied in specific brain areas. In addition, this method enables an absolute quantitative measure of brain tracer uptake in contrast to relative units obtained by the SUV method. Finally, 11C-AcAc images have a low signal-to-noise ratio because of relatively low brain AcAc uptake under physiological conditions, which makes automatic 11C-AcAc and MR images registration challenging. Hence, because 11C-AcAc and 18F-FDG acquisitions are sequential (no motion of the animal), the 18F-FDG to MRI alignment can be applied to 11C-AcAc images.
Key papers where the protocol has been used
We have used the dual tracer PET protocol in a study involving the comparison of the fasted state and the ketogenic diet (KD) in young rats2. We showed that both fasting and the KD increase significantly both 11C-AcAc and 18F-FDG brain uptake. However, these were not quantitative results as we did not use the dynamic PET imaging and tracer kinetic modeling methodology at that time. Thereafter, we undertook a regional and quantitative study of brain metabolism in aged rats, where the effect of aging and a KD were evaluated on brain 11C-AcAc and 18F-FDG uptake11. We also showed that the percentage of distribution across brain regions was different between 11C-AcAc and 18F-FDG. Furthermore, not only the CMR of 11C-AcAc but also that of 18F-FDG was increased in the whole brain as well as in the striatum of aged rats on the KD.
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
- 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.
- Anesthetize the rat in an induction chamber. Use 2% isoflurane and 1.5 L/min oxygen throughout the protocol for anesthesia. Pinch the hind leg to ensure the animal is fully anesthetized. CAUTION: inhalation of isoflurane can cause headache, dizziness, or unconsciousness in some cases; it should always be used in the presence of an air exchange system in a suitably ventilated room.
- Position the rat on the MRI examination table in the head-first prone position with a nose cone for isoflurane. Position the respiration and rectal probes. Monitor the respiration rate during the experiment and maintain body temperature at 37 °C with an automated air warming system.
- Acquire T2-weighted MR images using a fast spin-echo pulse sequence with acquisition parameters as detailed previously11. Place the rat on a heated mat during recovery from anesthesia.
2. Dual Tracer PET Acquisitions
- Use a small-animal PET scanner equipped with avalanche photodiode detectors, an axial field of view of ~7.5 cm and an isotropic spatial resolution of 1.2 mm17. Due to its short physical half-life, prepare 11C by proton bombardment of natural nitrogen (through the 14N(ρ,α)11C nuclear reaction) in a cyclotron on-site before each experiment.
- Fast the rat for 18 hr prior to PET scanning. Fasting allows higher brain uptake of 11C-AcAc and 18F-FDG, thereby improving signal-to-noise ratio.
- Anesthetize the rat in an induction chamber. Transfer the rat onto a heating mat with a nose cone for isoflurane. Start 11C-AcAc synthesis at this time. The synthesis takes 18 min from the end of bombardment, as previously described1.
- Position the rat on its side. Prepare a PE50 polyethylene catheter filled with heparinized 0.9% sodium chloride solution (saline). Install the catheter in the tail vein for tracer injection. Install a second catheter in the mid-ventral tail artery for blood sampling throughout the acquisitions.
- Rapidly transfer the rat to the scanner table in the head-first prone position with a nose cone for isoflurane. Position the respiration and rectal probes. Monitor the respiration rate during the experiment and maintain body temperature at 37 °C with an automated warming air system. Move the scanner table forward into the scanner to ensure the appropriate imaging of the brain and the heart simultaneously. As an anatomical landmark, position the edge of the field of view on the rat's eyes (with the help of laser lines).
- Using a concentrated 11C-AcAc solution (~1 GBq/ml), prepare a syringe of ~50 MBq of radioactivity (a range of 45-55 MBq is acceptable). Adjust the volume to 300 μl with saline. CAUTION: Radioactivity can be harmful for the health. It should always be handled behind a lead shield. The experimenter should be wearing a body and ring dosimeter. A Geiger counter should also be on during the experiment.
- Install the syringe on an injection pump. Start the bolus injection of 11C-AcAc at a rate of 1 ml/min (injection duration: ~19 sec). On a second pump, immediately after terminating the 11C-AcAc injection, start the injection of 300 μl of saline at a rate of 1 ml/min, which is important for an optimal injection of tracer into the blood circulation.
- Start a dynamic PET data acquisition 30 sec before starting the bolus injection for a total duration of 20.5 min. The 30 sec data acquisition before the tracer injection provides a measure of the ambient background to be subsequently subtracted from the PET data. Set the regular sampling mode and the energy window at 250-650 keV.
- Collect two 200 μl blood samples at ~15 and 18 min after starting the 11C-AcAc injection. Inject heparinized saline in the catheter after each sampling to avoid blood clotting. Take note of the time at the beginning and the end of blood sampling and use the mean time. Centrifuge at 6,000 RPM for 5 min and collect plasma. Measure radioactivity counts in plasma using a gamma counter calibrated with the PET scanner.
- After the 11C-AcAc scan, allow a waiting period of 20 min to ensure that most of radioactivity has decayed. The period can vary from 15-30 min. Do not move the scanner bed and leave the rat under isoflurane throughout this period.
- During this time, using a concentrated 18F-FDG solution (~5.5 GBq/μl), prepare a syringe of ~50 MBq. Proceed exactly as mentioned in steps 2.6 and 2.7. Start a dynamic acquisition of a total duration of 40.5 min, including the 30 sec before the injection. Collect two 200 μl blood samples at ~30 and 35 min after starting the 18F-FDG injection.
- At the end of the 18F-FDG acquisition, take one final 200 μl blood sample. Centrifuge and collect plasma. Keep plasma samples at -80 °C for glucose and AcAc analysis.
- Finally, place the rat on a heating mat during the waking period. Keep the animal for a longitudinal follow-up, or alternatively, euthanize the rat for brain sampling and further biochemical studies.
- Reconstruct the PET images according to the following time frame sequences: 1 × 30 sec; 12 × 5 sec; 8 × 30 sec; and n × 300 sec, where n = 3 for 11C-AcAc acquisition and n = 7 for 18F-FDG acquisition.
3. Plasma Glucose and AcAc Analysis
- Measure plasma glucose and AcAc with a clinical chemistry analyzer. Perform assays within 48 hr of the blood sampling to minimize AcAc decarboxylation into acetone. Measure glucose concentration with DF40 kit as previously described18 and AcAc with an open channel2. This type of analyzer is more accurate than strips (0.1 μM) and has a broad detection window.
4. Quantitative PET Analysis
- Use PMOD software or an equivalent system for small-animal PET image analysis.
- Plasma time-activity curve (TAC)
- Load 18F-FDG data. Sum image frames of the first 60 sec following injection (when tracer is mainly in blood).
- Using a manual drawing tool, draw a volume of interest (VOI) on the left ventricular cavity blood pool (a large pool centered in the bottom part of images). Draw the VOI ~1 mm inside of the edge of the blood pool (red voxels) to ensure no inclusion of tissue and avoid tissue radioactivity spill into the blood pool (Figure 1A). Copy the VOI to the entire dynamic image series and generate a curve of radioactivity as a function of time (TAC).
- In a spreadsheet, use radioactivity counts of the two plasma samples taken during the acquisition to correct plasma TAC (Figure 1B). Use the following equation:
Some smoothing of the plasma TAC can be performed.
- Verify if residual radioactivity from 11C-AcAc is present in the first 30 sec of 18F-FDG scan, prior to injection. If so, subtract the following factor from all the subsequent time frames of the TAC:
where R is the residual radioactivity, t1/2 is the half-life of 11C (20.4 min) and T is the time frame2,13. Residual radioactivity from 11C-AcAc after a waiting period of 20 min can be up to 1.5% of maximal 18F-FDG counts.
- 18F-FDG/MRI coregistration
- Apply coregistration obtained with 18F-FDG images to the11C-AcAc images. This is necessary because 11C-AcAc images have a low signal-to-noise ratio and automatic coregistration is difficult.
- Load the corresponding MRI data. Load all 28 18F-FDG time frames in the same orientation as the MR images (on three axes).
- Compute the 18F-FDG summed image of the 28 time frames. This generates an image with higher counts, so it is easier to work with for coregistration with MRI than individual image frames.
- Perform the automatic coregistration of the 18F-FDG summed image on MR images (Figure 2). Apply the transformation to all individual 18F-FDG image frames. Save the transformation which will later be applied to 11C-AcAc images.
- Volumes of interest (VOIs)
- Using segmentation software such as PMOD, select MRI data and choose preferred plane for manual drawing. Choose the manual or the semi-automatic drawing tool, depending on brain region size and image contrast. Locate brain structures according to a standard rat brain atlas19.
- Draw the VOIs. As shown in Figure 3, segment the whole brain, cortex, hippocampus, striatum and cerebellum, but other regions could be chosen. Save the VOIs, which will be applied to the coregistered 18F-FDG and 11C-AcAc images.
- Apply VOIs to 18F-FDG coregistered images. Select the VOI statistics to visualize brain TAC. If needed, correct TAC by subtracting the decay-corrected mean radioactivity in the first 30 sec from all the subsequent time frames.
- Cerebral metabolic rate calculation
- Load brain and the corresponding plasma TACs.
- Select Patlak plot kinetic model20,21. Set the lumped constant to 0.4822 and the corresponding plasma glucose concentration (Figure 4B). Set the maximal relative deviation from the Patlak plot to 5%. Fit the data to the model.
- 11C-AcAc images
- Compute the 18F-FDG summed image of the 24 first time frames to have the same number of frames as the 11C-AcAc scan. Otherwise, 11C-AcAc/MRI coregistration fails. Perform the automatic coregistration of 18F-FDG on MRI. Save the transformation process which will be used for 11C-AcAc image coregistration.
- Repeat steps 4.2 and 4.3 for 11C-AcAc images. Use 18F-FDG transformation in step 4.6.1 and apply it to all individual 11C-AcAc image frames. Repeat steps 4.4 and 4.5 and use 18F-FDG saved VOIs. Set the lumped constant to 1.
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 move due to an alignment in the axial plane.
We use the Patlak kinetic model20,21 to determine brain uptake, which has been previously used for 11C-ketone and 18F-FDG kinetic modeling9,12,24,25. This model uses equation 1, where the measured PET activity (CTissue) is divided by plasma activity (CP) and plotted at a normalized time. The variable Κ represents brain influx and V is the tracers distribution volume. After brain uptake steady state is achieved, the curve results in a straight line (Figure 4A). The slope represents brain influx (K). Typical values of brain influx (K) and distribution volume (V) in the whole brain for 18F-FDG are ~0.0165 min-1 and ~0.6425 ml blood/ml tissue, respectively (Figure 4B). As for 11C-AcAc, typical values are ~0.0325 min-1 and ~0.5315 ml blood/ml tissue.
Cerebral metabolic rate of glucose (CMRglc) and AcAc (CMRAcAc) are calculated according to equations 2 and 3. Brain influx and plasma concentrations are required. For CMRglc, the lumped constant (LC) is used to convert 18F-FDG uptake to glucose uptake. We use a LC value of 0.48, as previously reported in rats22. No constant is used for CMRAcAc calculation as the radiotracer has the exact same chemical formula as the endogenous molecule.
One can expect whole brain CMRglc and CMRAcAc of approximately 24 μmol/100 g/min and 3 μmol/100 g/min, respectively, in healthy adult rats fasted for 18 hr prior the PET acquisitions11. In the rat, CMRAcAc is therefore about 12% of that of CMRglc.
Figure 1. Plasma time-activity curve (TAC) assessment. A) Volume of interest drawn on the blood pool in the left ventricle of the heart using a manual drawing tool. This is a 18F-FDG image showing the average radioactivity for the first 60 sec after the injection. B) 18F-FDG plasma TAC obtained by image-derived input function (IDIF; black), the two plasma samples collected 30 min (red) and 37.5 min (orange) after the injection and 18F-FDG plasma TAC after correction with radioactivity counts of the two plasma samples (blue). Click here to view larger image.
Figure 2. PET/MRI automatic coregistration process. Axial PET and MR images before (left) and after (right) automatic coregistration with PMOD software. 18F-FDG (top) and 11C-AcAc (bottom) images are shown. 11C-AcAc/MRI coregistration is performed using the 18F-FDG/MRI transformation parameters. Click here to view larger image.
Figure 3. Volumes of interest (VOIs) drawing. VOIs drawn on axial MR images (top). VOIs applied to 18F-FDG coregistered images (bottom). WB: whole brain; Cx: cortex; Hp: hippocampus; St: striatum; Cb: cerebellum. Click here to view larger image.
Figure 4. Kinetic modeling of brain 18F-FDG uptake using PMOD software. A) Patlak plot of 18F-FDG uptake in the whole brain. Y-axis correspond to the measured PET activity (CTissue) divided by plasma activity (CP) and x-axis correspond to the normalized time. The curve results in a straight line once steady state is reached. The slope represents brain influx (K). B) Cerebral metabolic rate of glucose (MRGlu.) and plot parameters of the corresponding 18F-FDG acquisition, where Plasma Gluc. is the plasma glucose concentration, Max. Err. is the maximal relative deviation from the Patlak regression line, Slope is the brain influx (K) and Intercept is the distribution volume (V). Click here to view larger image.
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 successful PET acquisition. The catheter must be correctly inserted in the vein. Heparinized saline solution should flow into the vein smoothly without blood clotting. For a quantitative measure of brain uptake, an accurate plasma TAC is essential. When drawing the VOI on blood in the left ventricle, it is important to include the fewest possible tissue pixels. Blood samples are also crucial to correct the plasma TAC derived from the PET images. Indeed, as seen in Figure 1B, radioactivity in plasma samples may be different than in the IDIF. This is due to the spill-in effect in the last time frames (contamination of blood by radiation in adjacent tissues)26, which affects the IDIF. The brain MR image is essential for regional brain analysis. After the coregistration process, the alignment of PET and MR images of every brain slice should be verified to ensure that the subsequent PET regional brain analysis is accurate. Finally, a good fit of data to the Patlak plot is required for accurate calculation of CMR. Data should be as linear as possible; if not, the plasma and brain TAC should be rechecked.
A significant limitation of PET imaging is that it provides no information on the chemical status of the radiotracer, i.e. degradation to other metabolites in the brain. Indeed,18F-FDG is taken up by brain cells, where it is trapped as 18F-FDG-6-phosphate. This contrasts with nuclear magnetic resonance spectroscopy (NMRS) where the labeled atom can be monitored in multiple metabolic pathways27-29. However, PET requires very low amounts of radiotracer (10-12-10-9 M) compared to NMRS, and therefore better reflects physiological conditions. Another limitation comes from the plasma TAC derived from the PET images, which can be inexact because of the spill-out effect from blood to tissues in the first time frames26. Indeed, the present method does not include blood sampling in the first time frames. When drawing the VOI in the left ventricle, heart tissue may be included in the VOI without noticing because of heart beat, which may affect the plasma TAC. Indeed, the present method does not include electrocardiogram-gated PET acquisitions. The multiple blood sampling method is the gold standard for the most accurate plasma TAC, but remains demanding and invasive for small animals. Indeed, this technique would require approximately 18 blood samplings per scan for both scan, which would represent a considerable volume of blood sampled. Finally, for 11C-AcAc acquisitions, no correction for loss of 11C-CO2 from the tissue is performed. However, previous 11C-ketone PET studies showed that loss of 11C-CO2 had minimal impact on data, since the Patlak plot was well described by a straight line, and did not correct data for this loss24,25.
This protocol is also applicable for the study of mice models. Fasting can be shortened to 6 hr prior to the PET scan. However, with less than 6 hr, 11C-AcAc brain uptake will not be high enough for accurate CMR computation. Indeed, the brain consumes more ketones as the fasting period is prolonged. We have previously shown that after a 48 hr fasting, brain 11C-AcAc uptake increases by 160% and that brain 18F-FDG uptake also increases by 277%2. Electrocardiogram-gated PET acquisitions can be performed and may enable a more accurate plasma TAC. One can use another type of anesthetic such as propofol or ketamine depending of the Institutional Animal Care and Use guidelines. If an injection of anesthetic needs to be done during the dual PET tracer protocol, it should be performed between the two scans and attention should be paid not to move the animal. A longer delay is also possible between the two PET scans to reduce residual radioactivity from 11C in the 18F-FDG scan. However, the total period for anesthesia should be kept as short as possible since anesthesia decreases brain metabolism30. For quantitative PET analysis, any custom software enabling coregistration process and kinetic modeling can be used. If the automatic PET-MRI coregistration process is not possible, the registration can be performed manually. It is however a longer process and prone to human error. Finally, the two-tissue compartment kinetic model is also a good model for 18F-FDG brain uptake analysis and has been previously used in animal PET studies10,12. As for 11C-AcAc, the one and two-tissue compartment models were shown to give identical results as the Patlak model24,25.
Significance of the method
The presented method permits sequential measurement of regional brain uptake of two PET tracers and, hence, their comparison in the same animal under the same conditions. Therefore, this protocol is ideal for comparative studies of brain metabolic pathways using these PET tracers. Since the animals recover from the procedure, it can be repeated after an experimental intervention. This method also enables the absolute quantitative assessment of cerebral metabolic rates (μmol/100 g/min) in contrast to relative data obtained by the SUV method.
The investigation of brain ketone and glucose metabolism using 11C-AcAc and 18F-FDG uptake by PET is relevant in many physiological and pathological processes such as aging, neurodegenerative patterns and tumor expansion or treatment. Other PET tracers can be studied with the present protocol: 11C-palmitate and 18F-fluorothioheptadecanoic acid uptake can be used to evaluate fatty acids esterification in conditions such as aging or type 2 diabetes.11C-Pittsburgh Compound-B, which binds fibrillar amyloid-β plaques, could potentially be used in conjunction with 18F-FDG for the study of Alzheimer-like brain pathologies. Furthermore, 18F-fluorodopamine is useful to evaluate dopamine circuits in disease such as Parkinson. This dual tracer PET and MRI protocol can be applied to the study of other organs such as the liver, heart, kidney, or tumors.
The authors declare that they have no competing financial interests.
This study was financially supported by the Fonds de la recherche en santé du Québec, Canadian Institutes of Health Research, Canadian Foundation for Innovation and the Canada Research Chairs Secretariat (SCC). The Sherbrooke Molecular Imaging Center is part of the FRQS-funded Étienne-Le Bel Clinical Research Center. The authors thank Mélanie Fortier, Jennifer Tremblay-Mercier, Alexandre Courchesne-Loyer, Dr. Fabien Pifferi, Dr. M'hamed Bentourkia, Dr. Otman Sarrhini, Dr. Jacques Rousseau, Caroline Mathieu, and Mélanie Archambault for generous support and technical assistance. The authors would like to thank the image analysis and visualization platform (http://pavi.dinf.usherbrooke.ca) for their help.
|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|
|Isoflurane||Abbott Laboratories, Ltd||B506|
|0.9% NaCl solution||Hospira||4888010|
|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|
|b-Hydroxybutyrate dehydrogenase||Toyobo||HBD-301||1 U/ml|
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