Name: determining glucose metabolism kinetics using 18f-fdg micro-pet/ct
Uploaded: 2017-05-02T14:30:00
Description: Watch this Scientific Journal Video about Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT at JoVE.com

This work was supported by a National Imaging Facility Subsidised Access Grant to BJC, a National Health and Medical Research Council of Australia program grant (482800) to KAR and PJB. The authors would like to thank Andrew Arthur, Hasar Hazme and Marie-Claude Gregoire for support in developing this method.

All procedures described in this study were approved by the Sydney Local Health District and University of Sydney Animal Ethics Committees and followed the NIH Guide for the care and use of laboratory animals, Eighth edition (2011).
1. Animal Preparation
NOTE: In this protocol male db/db mice (BKS.Cg-Dock7m +/+ Leprdb/J) were maintained in group housing with ad libitum access to chow and water until 6 weeks of ...

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The protocol described here represents a robust, non-invasive methodology to determine the kinetics of glucose uptake from the blood stream into tissue and subsequent metabolism in mice.
The db/db mouse is a is a well-established animal model of type 2 diabetes14 that has been used extensively to examine insulin resistance and relevant interventions. However, previous studies have only quantified endpoint uptake in the heart15 and cardiac and ske...

The purpose of this study was to develop a positron emission tomography/computed tomography (PET/CT) based methodology to quantify the in vivo, real-time uptake of glucose from the blood stream into specific tissues in mice. This was achieved using 18F-labelled fluorodeoxyglucose (FDG) to measure glucose uptake and kinetic modelling to estimate the rates of 18F-FDG uptake from the plasma to the intracellular space, the rate of transport from intracellular space to plasma and the rate of 18F-FDG phosphorylation.
In rodents, 18F-FDG has been used in the pre-clinical assessment of numerous cancer treatments1, studies of tumor progression2 and tumor metabolism3 as well as imaging of brown fat depots4, neuroinflamation5 and brain metabolism6.
Traditional methods used to examine the tissue specific uptake of glucose in mice (and rats) generally involve treatment with 2-deoxyglucose radiolabeled with either 3H or 14C followed by euthanasia, tissue collection and measurement of radioactivity in each tissue7. The use of PET/CT allows for noninvasive determination of glucose uptake and metabolism in multiple organs and regions simultaneously in live animals. Additionally, as euthanasia is not a requirement, this technique is suitable for use in longitudinal studies.
Type 2 diabetes mellitus (T2DM) is characterized by disrupted glucose metabolism and hyperglycemia secondary to reduced tissue responsiveness to insulin (insulin resistance) and the inability of pancreatic -cells to produce adequate amounts of insulin8. Kinetic analysis of glucose uptake and metabolism can provide important insights into the mechanism of action and efficacy of therapeutic interventions as well as allow for advanced monitoring of disease progression.

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			<td height="21" style="height:21px;width:216px;">PET/CT Scanner</td>
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			<td height="21" style="height:21px;width:216px;">18F-FDG</td>
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			<td></td>
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			<td height="21" style="height:21px;width:216px;">Isoflurane</td>
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			<td></td>
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			<td height="21" style="height:21px;width:216px;">30 guage needle</td>
			<td style="width:152px;">BD</td>
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			<td height="21" style="height:21px;width:216px;">PMOD modelling software</td>
			<td style="width:152px;">PMOD Technologies</td>
			<td style="width:119px;"></td>
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			<td height="42" style="height:42px;width:216px;">BKS.Cg-Dock7m&#160;+/+&#160;Leprdb/J&#160; mice</td>
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			<td style="width:119px;">000642</td>
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			<td height="21" style="height:21px;width:216px;">Human insulin</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
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determining glucose metabolism kinetics using 18f-fdg micro-pet/ct

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). Impaired uptake and metabolism of glucose in multiple organ systems due to insulin resistance is a hallmark of type 2 diabetes. The ability of this technique to extract an image-derived input function from the vena cava using an iterative deconvolution method eliminates the requirement of the collection of arterial blood samples. Fitting of tissue and vena cava time activity curves to a two-tissue, three compartment model permits the estimation of kinetic micro-parameters related to the 18F-FDG uptake from the plasma to the intracellular space, the rate of transport from intracellular space to plasma and the rate of 18F-FDG phosphorylation. This methodology allows for multiple measures of glucose uptake and metabolism kinetics in the context of longitudinal studies and also provides insights into the efficacy of therapeutic interventions.

We have previously used the db/db mouse model to investigate the impact of increasing plasma apoA-I levels on the kinetics of glucose uptake and metabolism13. In this study we used db/db mice treated with insulin to demonstrate the utility of PET/CT imaging to monitor the uptake of 18F-FDG from the plasma into the gastrocnemius muscle in real time.
Six week old db/db mice were anesthetized and ...

Watch this Scientific Journal Video about Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT at JoVE.com

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This study describes a protocol that uses 18F-FDG and positron emission tomography/computed tomography (PET/CT) imaging, together with kinetic modelling, to quantify the in vivo, real-time uptake of 18F-FDG into tissues.

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). ...

The overall goal of this imaging protocol is to quantify the in vivo realtime uptake of glucose from the bloodstream into specific tissues in mice. This method can help answer key questions in the diabetes and metabolism field, such as the impact of any diabetic treatments on glucose uptake and metabolism. The main advantage of this technique is that experiments can be performed longitudinally to assess for example the impact of age or diet on glucose metabolism.Learning this method provides information about glucose metabolism in sclero muscle. It is also useful in other tissues such as liver and brain and is transferrable to rats. Visual demonstration of this method is critical, as the imaging acquisition and our analysis steps must be completed correctly to ensure accurate results.First, wipe down the induction chamber, the imaging bed, and the heat pad with 80%ethanol. Place the mouse subject in the induction chamber and anesthetize the mouse with 5%isoflurane and oxygen. Secure an electric heating pad and a sensor pad on the imaging bed.Place the mouse in a prone position and continue delivering one liter per minute of 1.5 to 2%isoflurane by a nose cone. Apply veterinary ointment to the mouse's eyes to prevent dryness. Then, warm the mouse's tail for one to two minutes.Once the lateral tail vein is dilated, insert a 30 gauge needle into the vein. Fix the needle in place with surgical glue and connect a catheter to the needle. Load the imaging bed into the PET/CT scanner and position the bed so the catheter is accessible from the rear of the machine.Secure a syringe containing the ten mega back roll F18-FDG dose to the catheter. After a set time following induction of anesthesia, start a one hour PET scan and inject the F18-FDG dose. Upon completion of the PET scan, perform a 10 minute CT scan.After the CT scan, move the imaging bed to its initial position and remove the mouse from the bed. Either euthanize the mouse by cervical dislocation under anesthesia, or allow the mouse to recover in single housing under observation. To begin the image processing, load the reconstructed PET and CT images.Designate the CT data as source and the PET data as target. Co-register the data and align the images as needed. Then, select ROI quantification.Use the panning and zoom tools to locate the desired region of interest in the image. Navigate to the create tab and select the paintbrush tool. Draw an ROI corresponding to the gastrocnemius muscle.Then, use the save ROI quantification function to extract the tissue time activity data as a CSV file. De-convolve the estimated system point spread function for five iterations with a Van Cittert deconvolution method. Next, draw an ROI corresponding to the vena cava.Export the blood input function time activity data from the post de-convolution images as a CSV file. Import the CSV files into spreadsheet software. Save the data as new CSV files and convert the uptake values to percent injected dose per centimeter cubed.Plot the time activity curves with the spreadsheet software. Next, save the processed vena cava data to a new CSV file. Convert the vena cava data to a plasma input function.Select kinetic in the kinetic modeling tool. Import the tissue and plasma total activity count data files. Choose a two tissue, three compartment model.Ensure that the F18-FDG dephosphorylation rate, K4, is unchecked and has a value of zero. Set the blood volume fraction, VB, to 2%To add dispersion to the vena cava derived input function, use a program such as disp4dft to create multiple input functions with different dispersion times. Then load the input functions with dispersion one by one and select fit current region.Repeat to find the input function with dispersion that has the lowest ChiSquared value. Using the optimized input function, set the blood volume fraction to a floating value. Fit the region again to determine the regional rate constants.Then calculate the regional influx constant. Six week old DBDB mice treated with either insulin or phosphate-buffered saline were imaged with this method. The insulin treated mice showed increased F18-FDG activity in the gastrocnemius muscle compared to the control mice.No increased activity was observed in the vena cava. A two tissue, three compartment model was used for kinetic modeling. No significant difference from control was observed in the rate of F18-FDG transport from arteriol plasma to the intracellular space or vice versa.However, the F18-FDG phosphorylation rate was significantly higher in the insulin-treated mice. The influx constant was correspondingly higher for the insulin-treated mice. After watching the video, you should have a good understanding of how to correctly image the mice, draw regions of interest, and calculate kinetic parameters.Don't forget that working with radiation can be hazardous and precautions such as proper training, use of shielding and monitoring of exposure should always be taken when performing this procedure.

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