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Accurate live tumor imaging in mice is now possible by means of high-resolution positron emission tomography (micro-PET) and X-ray computed tomography (micro-CT). By providing a powerful tool to examine biological samples with complex structure in vivo, this technology generated a significant advance in the cancer research field, particularly regarding the ability to perform longitudinal studies in combination with a therapeutic intervention. Here, we describe methods to optimize visualization of murine lung tumors by micro-PET, micro-CT and combined micro-PET-CT.

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Analysis of Murine Lung Tumors by Micro PET-CT Imaging
微型PET-CT成像分析鼠肺部肿瘤

癌症生物学 > 通用技术 > 肿瘤形成
作者: Chiara Ambrogio*
Chiara AmbrogioAffiliation: Experimental Oncology, Molecular Oncology Programme, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain
Bio-protocol author page: a2813
Juan Antonio Cámara*
Juan Antonio CámaraAffiliation: Molecular Imaging Unit, Biotechnology Programme, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain
Bio-protocol author page: a2814
Patricia Nieto
Patricia NietoAffiliation: Experimental Oncology, Molecular Oncology Programme, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain
Bio-protocol author page: a2815
David Santamaría
David SantamaríaAffiliation: Experimental Oncology, Molecular Oncology Programme, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain
Bio-protocol author page: a2816
 and Francisca Mulero
Francisca MuleroAffiliation: Molecular Imaging Unit, Biotechnology Programme, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain
For correspondence: fmulero@cnio.es
Bio-protocol author page: a2817
 (*共同第一作者)
Vol 5, Iss 24, 12/20/2015, 2693 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1692

[Abstract] Accurate live tumor imaging in mice is now possible by means of high-resolution positron emission tomography (micro-PET) and X-ray computed tomography (micro-CT). By providing a powerful tool to examine biological samples with complex structure in vivo, this technology generated a significant advance in the cancer research field, particularly regarding the ability to perform longitudinal studies in combination with a therapeutic intervention. Here, we describe methods to optimize visualization of murine lung tumors by micro-PET, micro-CT and combined micro-PET-CT.
Keywords: Cancer(癌症), Imaging(成像), CT(计算机断层扫描), Lung cancer(肺癌)

[Abstract]

Part I. Imaging by positron emission tomography (micro-PET)

The imaging of tumors at inner body locations in living animals is more challenging than the imaging of subcutaneous tumors. We have optimized the procedures outlined in the following protocol in order to study lung tumors in genetically modified mice and orthotopic models. Briefly, the mice are anesthetized prior to the administration of the [18F]-Fluorodeoxigluycose (18F-FDG) dose, and kept under anesthesia during the whole period of probe uptake and imaging, ensuring at all times that the mice are warm. The standardization of mouse handling and of anesthesia usage is essential to ensure data reproducibility and comparability.

Materials and Reagents

  1. Lacryvisc Gel 10 G (3 mg/ml carbomere in benzalconium chloride) (Alcon) (http://www.alcon.com)
  2. 1-cc tuberculin syringes (B. Braun España, model: Omnifix-F )
  3. 30-G needles (B. Braun España, model: Sterican )
  4. Heating pads [e.g. Gaymar Mul-T-Pads (Gaymar industries) (http://www.gaymar.com/)]
  5. Heating pump to maintain temperature of heating pads [e.g. Gaymar TP600 (Gaymar industries) (http://www.gaymar.com/)]
  6. Genetically modified mouse models bearing endogenous lung tumors (i.e. our own K-RasG12V inducible mouse model, Guerra et al., 2003), lung orthotopic implantation of primary tumors (Ambrogio et al., 2014), or tail-vein injected lung tumor cells (Ambrogio et al., 2014) (Figure 1)


    Figure 1. Types of murine lung tumors detectable by PET-CT scan. PET-CT technology can be used to detect lung tumors of different origins: from the left, endogenous lung tumors (i.e. tumors induced by a K-RasG12V resident knock-in allele in a genetic engineered mouse model), tail vein-injected lung cancer cell lines or orthotopically implanted lung tumors (either murine or human lung adenocarcinomas). Scale bar: 200 μM.

  7. Special mouse diets as necessary (Cussó et al., 2014)
  8. Inhalational anesthesia:
    Sevoflurane [e.g. Sevoflo (Abbott Laboratories, catalog number: 05458-02) (http://www.abbottanimalhealth.com/veterinary-professionals/products/anesthesia/sevoflo.html)]
  9. Oxygen obtained from an O2 concentrator
  10. [18F]FDG (0.01 to 0.1 μg/mCi), delivered daily from a local cyclotron (e.g. 40 mCi of [18F]FDG of 95% to 99% radiochemical purity in 1 ml of physiological saline solution buffered at pH 6.0, for ~10 micro-PET scans)
  11. Physiological saline: 0.9% (w/v) NaCl (B. Braun España)
  12. Lacryvisc Gel 10 G (3 mg/ml carbomere in benzalconium chloride) (Alcon) (http://www.alcon.com)

Equipment

  1. O2 concentrator (Eickemeyer Veterinary Equipment, model: Oxymat e3 )
  2. Infrared heating lamp Philips PAR38 IR 175W E27 (Royal Philips Electronics)
  3. Sevofluorane/oxygen-based anesthesia system fitted with an induction chamber and inhalation masks for mice McKinley, Type 2 (Everest tecnologia veterinaria)
  4. Dose calibrator (also known as activimeter) [e.g. VDC-505 dose calibrator (Veenstra Instruments) (http://www.dosecalibrator.com/)]
  5. micro-PET-CT imaging system [e.g. eXplore Vista PET-CT (GE Healthcare) (Figure 2); Argus PET-CT (SEDECAL) (http://www.sedecal.com/)]


    Figure 2. Micro-PET-CT machine (Argus SEDECAL) for mice

  6. Workstation (e.g. Dell PowerEdge) for image acquisition, processing, and analysis meeting the following specifications:
    1. PE1950 Xeon 5120 1.86 GHz/4 MB 1066 FSB processor
    2. PE1950 PCIX Riser (2 slots)
    3. PE1950 Bezel Assembly
    4. 2 GB FB 667 MHz Memory (2 x 1 GB dual rank DIMMs)
  7. Alienware Dell Studio XPS Desktop 435 MT PC (for 3DOSEM image reconstruction) meeting the following specifications:
    1. Processor: Intel Core i7 Quad CPU 940 4 x 2.93 GHz
    2. Memory: 6144 MB (6 x 1,024) 1067 MHZ DDR3
    3. Graphics: ATI Radeon HD 3450 256 Mb GDDR2

Software

  1. eXplore Vista PET-CT MMWKS software (Desco et al., 2005) or AMIDE software (Loening and Gambhir, 2003) for image acquisition, processing, and analysis

Procedure

  1. Prepare, sedate and anesthetize mice
    1. Diet conditions. For the detection of thoracic tumors, mice (as described in Materials and Reagents, Pag. 1) can be fed a specifically formulated commercial high-fat diet (such as, for example, diet D12451 from Research Diets, with 45% of total calories from fat) or, alternatively, sunflower seeds, which are rich in vegetable fats, during the 24 h prior to the analysis. This will decrease glucose uptake by the myocardium, eliminating, to a great extent, the interferences arising from the high uptake of the heart under standard feeding conditions (Cussó et al., 2014).
    2. Transport mice to the imaging unit the day before the exploration, prepare the diet, and determine their weight.
    3. Place an infrared lamp above the cage to keep the mice warm prior to the injection.
    4. Hold the mouse carefully and bring its tail close to the infrared lamp (~5 cm) during 3 to 4 min. Be careful not to expose the whole animal to the lamp, in order to avoid excessive body heating.
    5. Once the tail veins have dilated, introduce the mice into the anesthesia chamber where a deep anesthesia will be induced by inhalation of 6% sevofluorane in 100% oxygen at a rate of 1.5 L/min.
      Note: Alternatively it is possible to use i.p. injection of a mixture containing ketamine (200 mg/kg) and xylazine (10 mg/kg).

  2. Administer the radiolabeled probe
    1. Transfer anesthetized mice to the injection area and, using a tuberculin shielded syringe and a 30-G needle, inject them with [18F]FDG in the tail (Figure 3) at a dose of 18.5 MBq in a volume of 0.2 ml physiological saline (0.9% NaCl), calibrating the dosage using an activimeter.


      Figure 3. Administration of the probe. Tail vein injection of the radioactive probe into an anesthetized mouse using a shielded syringe.

    2. Once injected, maintain mice under anesthesia providing 3% sevofluorane in 100% oxygen at 0.2 L/min (to avoid muscle uptake due to mice movement) during the [18F]FDG uptake time, which is 30-45 min.
      Note: For micro-PET-CT imaging, the micro-CT image acquisition (5 min) is carried out during the [18F]FDG uptake period. Wait for 25-40 min after [18F]FDG administration and then carry out the micro-CT study; the micro-PET study will be performed immediately after the micro-CT one is finished. The anesthesia concentration required during the acquisition of micro-PET-CT imaging (0.2 L/min) is lower than the concentration used for anesthesia induction (1.5 L/min).

  3. Perform image acquisition
    1. Before placing mice on the exploration table, administer eye lubricant (Lacryvisc Gel 10G) to avoid lesions in the cornea while the mice are anesthetized, since under these conditions the blinking reflex is lost. During image acquisition, mice must be anesthetized with a mask providing 3% sevofluorane in 100% oxygen at 0.2 L/min.
    2. Set the heating pads on the exploration table of the micro-PET machine at a constant temperature of 37 °C.
    3. Place mice on the exploration table in a prone position as stretched out as possible to minimize organ superposition.
    4. Perform image acquisition as described below.
      1. Perform a blank test. The blank test is an empty acquisition without any radiation. It must be done daily, before any real work is carried out, to assess the correct functioning of the glass detectors of the micro-PET equipment. If the system is working properly, as determined by the blank test, then the user can start the normal operation of the micro-PET. The software program MMWKS VISTA CT (Gleason et al., 2007; Pascau et al., 2012) is launched, the option “PET ACQ” is ticked, and a static study of 5 min duration is selected, without any isotope.
      2. Select the thoracic area making sure that all lung lobules will be acquired and start the acquisition of the first bed position: the PET scan is acquired as a series of distinct bed motions, called bed positions (usually 1-3 for a standard body scan). At each bed position, the scanner acquires during a set amount of time, then moves approximately 1 cm and begins acquiring the next bed position. All of these individual frames are then knitted together to form the final image, which should take between 10 and 30 min. If we don’t want to see distant metastasis we can avoid the whole body acquisition and reduce the total scan time.
      3. Divide the acquisition into two parts. Select or create the folder where the study will be saved and create what is known as a scout (a delimitation of the area to be studied), or use a scout previously created (if a micro-CT of the same mouse has already been performed, a scout for this given mouse will already be available). Once the thoracic area has been delimited, select the kind of micro-PET study to be carried out, define the duration of the procedure, the isotope to be used, and the energy window of the isotope of choice (see protocol selection for lung in next point).
      4. Select the predefined micro-PET acquisition protocol named “static”. This protocol allows for one bed position; it requires previous knowledge of the anatomical region of interest (in this case, thoracic region). This is the protocol of choice for standard lung tumor studies.
      5. Perform the micro-PET exploration setting the type of isotope to 18F and set the lower energy threshold to 150 KeV. While acquisition is in progress, make sure that the number of coincidences is between 200 and 500. This serves as a quality control for the injection of the radiolabeled compound. The number of coincidences is equivalent to the number of detected photons arising from radioactive disintegrations. If the number of coincidences is not within this range, it will not be possible to perform micro-PET acquisitions. In this last instance, the user should inject another dose of the probe again, so as to have sufficient coincidence events to be detected.
    5. Once micro-PET acquisition is finished, retrieve mice out of the equipment, disconnect the anesthesia, and take mice to a warm cage where they will wake up on their own.

  4. Reconstruct micro-PET image
    1. Choose an option for image reconstruction. Three options for image reconstruction are available in the eXplore Vista PET-CT equipment: 2D-FBP (filtered back projections), 2D-OSEM (ordered-subsets expectation maximization) and 3D-OSEM. The author’s preference is 3D-OSEM with the number of iterations set to 80 and employing random attenuation and scatter correction. In this manner, we achieve both proper imaging quality and compensation of spatial signal positioning with reasonable computing resources and an acceptable calculation running time.
      Note: An independent PC is needed to run 3D-OSEM. The average running time for a 3D-OSEM calculation (80 iterations, random and scatter correction) in a Dell Studio XPS Desktop 435MT PC is <2 min.

  5. Quantify the data
    1. Once the total scan volume is reconstructed, inspect the images until a positive [18F]FDG signal is identified in an anatomical position consistent with a lung tumor, and distinct from the normal organs uptake (e.g., heart, urinary bladder, kidneys, and to a lesser extent brown adipose tissue) (Figure 4).
    2. Manually draw a region of interest (ROI) around the perimeter of the tumor. Subsequently, inspect adjacent sections where the software has automatically drawn the same ROI; if adjustment is needed, it can be done manually.
      Note: The 3D-OSEM reconstruction algorithm employs cubic voxels. The software predetermines the size of the reconstructed cubic voxel; in our equipment, its value is of 0.7 x 0.7 x 0.7 mm. Therefore, tumor volumes are built in volume units of cubic voxels. It has to be verified that all the tumor activity has been included. The quantification of [18F]FDG uptake contained in the ROI is performed automatically, expressed in MBq/cc.
    3. From the quantification described under the previous step, calculate the Standardized Uptake Value (SUV).
      Note: Two types of SUV measurements can be calculated using the average SUV (SUVave) and the maximum SUV (SUVmax) formulas below:
      SUVave = [uptake (MBq)/volume of the ROI (cc)]/[mouse weight (g) x injected [18F]FDG dose (MBq) x calibration factor]
      The calibration factor is obtained from acquiring a phantom filled by radiation and is specific for each PET machine. The performance is described in the user manual of each device.
      SUVmax = [uptake in the voxel with maximal activity (MBq)/volume of the voxel(cc)]/[mouse weight (g) x injected [18F]FDG dose (MBq) x calibration factor]
      SUVave reflects the amount of radioactivity per unit of volume across the entire ROI volume. SUVmax reflects the radioactivity per unit of volume at the voxel, within the ROI, with the maximal value of uptake. In the particular case of mice, due to the great variability in radiolabeled compound distribution, it is advisable to normalize the results of the ROI of interest to a reference/background ROI in normal lung. Data, for example, can be expressed as the ratio of tumor to background (TBR). In Figure 4 a representative image from a lung tumor is depicted, with SUVmax values around 1 to 3 MBq/cc, and SUVmean values around 0.5 to 1 MBq/cc.


      Figure 4. Example of PET quantification. Axial view of a lung tumor PET signal and corresponding ROI of analysis (White arrow). Yellow arrow indicates background ROI placed over the healthy lung.

Part II. Imaging by Computed Tomography (micro-CT)

Micro-CT studies are simpler than micro-PET since micro-CT requires little or no preparation. However, mice must be immobilized under anesthetics to avoid artifacts arising from movement. Likewise, in order to prevent anesthesia-induced hypothermia mice must be maintained at constant body temperature by means of a heating pad. Exogenous contrast agents are often used to improve the signal ratio between the tumor and the surrounding healthy tissue, but for the detection of lung tumors contrast is not required.

Materials and Reagents

  1. Heating pads [e.g. Gaymar Mul-T-Pads (Gaymar industries) (http://www.gaymar.com/)]
  2. Heating pump to maintain temperature of heating pads [e.g. Gaymar TP600 (Gaymar industries) (http://www.gaymar.com/)]
  3. Genetically modified mouse models bearing endogenous lung tumors (Guerra et al., 2003), lung orthotopic implantation of primary tumors (Ambrogio et al., 2014), or tail-vein injected lung tumor cells (Ambrogio et al., 2014) (Figure 1)
  4. Inhalational anesthesia:
    Sevoflurane [e.g. Sevoflo (Abbott Laboratories, catalog number: 05458-02) (http://www.abbottanimalhealth.com/veterinary-professionals/products/anesthesia/sevoflo.html)]
  5. Oxygen obtained from an O2 concentrator
  6. Lacryvisc Gel 10 G (3 mg/ml carbomere in benzalconium chloride) (Alcon) (http://www.alcon.com)

Equipment

  1. O2 concentrator (Eickemeyer Veterinary Equipment, model: Oxymat e3)
  2. Infrared heating lamp Philips PAR38 IR 175W E27 (Royal Philips Electronics)
  3. Sevofluorane/oxygen-based anesthesia system fitted with an induction chamber and inhalation masks for mice McKinley, Type 2 (Everest tecnologia veterinaria)
  4. Multiparameter Monitor for respiratory gating (Figure 5) [e.g. Vision Vet (RGB) (http://www.medicalexpo.com/prod/rgb-medical-devices/product-69843-493296.html)]
  5. Infrared heating lamp
  6. Sevofluorane/oxygen-based anesthesia system fitted with an induction chamber and inhalation masks for mice
  7. Workstation (e.g. Dell PowerEdge) for image acquisition, processing, and analysis meeting the following specifications:
    1. PE1950 Xeon 5120 1.86 GHz/4 MB 1066 FSB processor
    2. PE1950 PCIX Riser (2 slots)
    3. PE1950 Bezel Assembly
    4. 2 GB FB 667 MHz Memory (2 x 1 GB dual rank DIMMs)

Software

  1. eXplore Vista PET-CT MMWKS software (Desco et al., 2005) or AMIDE software (Loening and Gambhir, 2003) for image acquisition, processing, and analysis

Procedure

  1. Prepare, sedate and anesthetize mice
    1. Anesthetize mice: Introduce mice into the anesthesia chamber where a deep anesthesia will be induced by inhalation of 6% sevofluorane in 100% oxygen at a rate of 1.5 L/min. and maintain warm; apply some eye lubricant (Lacryvisc Gel 10G) to the cornea for protection as specified in the protocol above before placing mouse inside the scanner chamber. For micro-CT acquisition mice do not require prior preparation (special diet and sedation).
    2. Before starting the micro-CT, select the thoracic area and properly set the different parameters. The parameters for CT acquisition are usually set within the following ranges of values:
      1. Intensity of the power supply: from 140 to 1,000 mA.
      2. Number of shots: From 1 to 32.
        Note: The number of shots refers to the number of times that X-rays are emitted from the source. Although a higher number of shots results in a higher signal-to-noise ratio, it also results in larger acquisition times and greater radiation damage inflicted on mice (see step A4 below).
      3. Resolution: standard (200 μm), high (100 μm), or maximum (50 μm).
      4. Number of projections: From 360◦ to 720◦.
        Note: The number of projections refers to the number of rotations of the X-ray beam around the mice, expressed in sexagesimal degrees. Double full rotations (720◦) versus single full rotation (360◦) results in an image of higher signal-to-noise ratio, but at a cost of larger acquisition times and, consequently, greater exposure of the mice to a hazardous energy source and anesthesia.
      5. Number of bed positions: 1 to 3. One bed position for thoracic imaging is enough.
        Note: When more than one bed position is required, they superimpose on top of each other, resulting in partial image overlap.
    3. Fix mice to the exploration table with adhesive tape even if they are anesthetized, to minimize involuntary movements. Once mice are ready on the exploration table, lower down the protective shielded screen of the Vista-PET-CT and lock in the safety key to perform data acquisition.

  2. Standard micro-CT data acquisition
    1. The standard acquisition parameters are: Power supply to an intensity of 150 mA and a voltage of 45 kV for a standard resolution of (200 μM), 360◦, and 16 shots for 1 bed position, or 8 shots if performing 2 bed positions. The above acquisition parameters correspond to a radiation dose in the mouse of 0.6 Gy.
    2. In order to select the acquisition area observe the scout image generated by the micro-CT machine. This scout gives a projection where the user can see how the subject is placed.
    3. Select, with the aid of a laser, the beginning and the end of the ROI that the user would like to acquire (e.g., the thoracic region). We can also select a previous scout from a micro-PET study already finished during the same session as long as it corresponds to the same mouse and we have not altered its disposition on the exploration table.

  3. Respiratory gating for thoracic CT
    1. Obtain respiratory signals from an external monitoring device simultaneously with the micro-CT scan (Figure 5; respiratory movement artifacts are important in CT acquisition, especially in the case of thoracic studies). After acquisition, group frames according to the external signal during image processing. This generates two independent scans, corresponding to inhalation and exhalation (grouping into intermediate stages is also possible).


      Figure 5. Animal handling and monitoring. Mouse fitted with electrodes being introduced into a PET-CT machine (left panel); monitor of vital constants (VisionVet, RGB http://www.medicalexpo.com/prod/rgb-medical-devices/product-69843-493296.html) registering ECG, temperature, and respiratory frequency (right panel).

    2. Stop the anesthetic delivery once micro-CT acquisition is over, retrieve mice out of the equipment, and place into a warm cage where they will wake up on their own.

  4. Micro-CT image reconstruction
    1. Reconstruct images using a modified version of the cone-beam (CB) algorithm of Feldkamp, Davis, and Kress (FDK, Vaquero et al., 2008) included in the software of the eXplore Vista PET-CT.
      Note: FDK is a widely used filtered-back projection algorithm for three-dimensional image reconstruction from cone-beam projections measured with a circular orbit of the X-ray source. Reconstruction time when the algorithm is run on a Dell PowerEdge workstation is <5 sec.

  5. Quantification using a ROI
    1. Identify lung tumors by visual inspection of the images.
    2. Draw manually a region of interest (ROI) around the perimeter of the tumor. Subsequently, inspect adjacent sections where the software draws the same ROI; if adjustment is needed, it can be done manually (Figure 6). The software predetermines the size of the voxels. Our reconstruction software employs cubic voxels of 50 x 50 x 50 μm to define the ROI. The volume of the selected ROI, expressed in cubic centimeters, is automatically calculated by the system.


      Figure 6. Example of ROI selection in a CT, PET and fusion images. The example shows (from left to right) axial, sagittal and coronal sections of the chest cavity. The ROI, visible in all three sections, is shown in solid yellow. H: Heart. Tumor is indicated with a white arrow. Green arrow points bone marrow and yellow arrows mark brown adipose tissue.

Part III. Imaging by multimodality (micro-PET-CT)

The immobilization of mice during the exploration is essential for image co-registration in multimodality imaging studies. If positions during micro-PET and micro-CT studies are different, the matching (co-registration) of the images obtained by micro-PET and micro-CT is not possible.

Materials and Reagents

  1. 1-cc tuberculin syringes (B. Braun España, model: Omnifix-F )
  2. 30-G needles (B. Braun España, model: Sterican )
  3. Heating pads [e.g. Gaymar Mul-T-Pads (Gaymar industries) (http://www.gaymar.com/)]
  4. Heating pump to maintain temperature of heating pads [e.g. Gaymar TP600 (Gaymar industries) (http://www.gaymar.com/)]
  5. Genetically modified mouse models bearing endogenous lung tumors (Guerra et al., 2003), lung orthotopic implantation of primary tumors (Ambrogio et al., 2014), or tail-vein injected lung tumor cells (Ambrogio et al., 2014) (Figure 1)
  6. Special mouse diets as necessary (Cussó et al., 2014)
  7. Inhalational anesthesia:
    Sevoflurane [e.g. Sevoflo (Abbott Laboratories, catalog number: 05458-02) (http://www.abbottanimalhealth.com/veterinary-professionals/products/anesthesia/sevoflo.html)]
  8. Oxygen obtained from an O2 concentrator
  9. [18F]FDG (0.01 to 0.1 μg/mCi), delivered daily from a local cyclotron (e.g. 40 mCi of [18F]FDG of 95% to 99% radiochemical purity in 1 ml of physiological saline solution buffered at pH 6.0, for ~10 micro-PET scans)
  10. Physiological saline: 0.9% (w/v) NaCl (B. Braun España)
  11. Lacryvisc Gel 10 G (3 mg/ml carbomere in benzalconium chloride) (Alcon) (http://www.alcon.com)

Equipment

  1. O2 concentrator (Eickemeyer Veterinary Equipment, model: Oxymat e3)
  2. Infrared heating lamp Philips PAR38 IR 175W E27 (Royal Philips Electronics)
  3. Sevofluorane/oxygen-based anesthesia system fitted with an induction chamber and inhalation masks for mice McKinley, Type 2 (Everest tecnologia veterinaria)
  4. Dose calibrator (also known as activimeter) [e.g. VDC-505 dose calibrator (Veenstra Instruments) (http://www.dosecalibrator.com/)]
  5. micro-PET-CT imaging system [e.g. eXplore Vista PET-CT (GE Healthcare, Figure 2); Argus PET-CT (SEDECAL) (http://www.sedecal.com/)]
  6. eXplore Vista PET-CT MMWKS software (Desco et al., 2005) or AMIDE software (Loening and Gambhir, 2003) for image acquisition, processing, and analysis
  7. Workstation (e.g. Dell PowerEdge) for image acquisition, processing, and analysis meeting the following specifications:
    1. PE1950 Xeon 5120 1.86 GHz/4 MB 1066 FSB processor
    2. PE1950 PCIX Riser (2 slots)
    3. PE1950 Bezel Assembly
    4. 2 GB FB 667 MHz Memory (2 x 1 GB dual rank DIMMs)
  8. Alienware Dell Studio XPS Desktop 435 MT PC (for 3DOSEM image reconstruction) meeting the following specifications:
    1. Processor: Intel Core i7 Quad CPU 940 4 x 2.93 GHz
    2. Memory: 6144 MB (6 x 1,024) 1067 MHZ DDR3
    3. Graphics: ATI Radeon HD 3450 256 Mb GDDR2

Procedure

  1. Prepare mice
    1. Prepare mice prior to the multimodality exploration as described for the micro-PET exploration in the corresponding protocol, steps 1 to 6.
      Note: Multimodality exploration requires extreme attention to the care of mice as well as constant monitoring of the temperature, since the study times are longer than in CT or PET alone.

  2. Acquire micro-CT image
    1. Perform the acquisition of the micro-CT image during the [18F]FDG uptake period (lasting 45 min). See micro-PET protocol, step 8, and micro-CT protocol.

  3. Acquire PET image
    1. Perform micro-PET image acquisition following the same procedure as described in the corresponding protocol, steps 7 to 13.

  4. Reconstruct images
    1. Reconstruct both images separately and use the MMWKS software to obtain the co-register (Figures 6-7).


      Figure 7. PET-CT imaging of a lung tumor. Representative axial projection of FDG-PET scans showing tumor FDG-PET signal (indicated by an arrow). H: heart, B: Bone marrow. Tumor is indicated with a yellow arrow.

  5. Quantify images
    1. Once lung tumors have been identified, quantify the micro-CT image and the micro-PET image separately, as described in the protocols above.

  6. Rendering of volumes
    1. Generate 3-D renderings of the data to obtain information on lung tumor location and volume in a more graphical way (Figure 8).


      Figure 8. Example of a longitudinal study of lung cancer. 3-D lung renderings of PET-CT studies at the indicated time-points of two individual mice carrying genetically induced lung tumors (in red), either treated with vehicle (upper panel) or with a therapeutic compound (lower panel).

Acknowledgments

This protocol has been adapted from the previously published study “Imaging Cancer in Mice by PET, CT, and Combined PET-CT” (Mulero et al., 2011). We are grateful to the Molecular Imaging Core Unit for the expertise and key contributions to the optimization of the protocols described here. This work was supported by grants from the CDTI (Spanish center of industrial and technological development Spanish Ministry of Science), AMIT Project “Advanced Molecular Imaging technologies” (5710001425) to FM. CA is the recipient of a postdoctoral fellowship from the Spanish Association Against Cancer (AECC).

References

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  5. Guerra, C., Mijimolle, N., Dhawahir, A., Dubus, P., Barradas, M., Serrano, M., Campuzano, V. and Barbacid, M. (2003). Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context. Cancer Cell 4(2): 111-120.
  6. Loening, A. M. and Gambhir, S. S. (2003). AMIDE: a free software tool for multimodality medical image analysis. Mol Imaging 2(3): 131-137.
  7. Mulero, F., Donate, L. E. and Serrano, M. (2011). Imaging Cancer in Mice by PET, CT, and Combined PET-CT. Curr Protoc Mouse Biol 1(1): 85-103.
  8. Pascau, J., Vaquero, J. J., Chamorro-Servent, J., Rodriguez-Ruano, A. and Desco, M. (2012). A method for small-animal PET/CT alignment calibration. Phys Med Biol 57(12): N199-207.
  9. Vaquero, J. J., Redondo, S., Lage, E., Abella, M., Sisniega, A., Tapias, G., Soto Montenegro, M. L., and Deseo, M. (2008). Assessment of a new high-performance a small-animal X-ray tomograph. In: IEEE transactions on nuclear science. pp 898-905.

第一部分。正电子发射断层扫描(micro-PET)成像

在活体动物的内体位置处的肿瘤的成像比皮下肿瘤的成像更具挑战性。我们已经优化了以下方案中概述的程序,以研究基因修饰的小鼠和原位模型中的肺肿瘤。简言之,在施用[18 F] - 氟脱氧葡萄糖(18 F-FDG)剂量之前将小鼠麻醉,并在整个探测期间保持麻醉摄取和成像,确保在任何时候小鼠是温暖的。小鼠处理和麻醉使用的标准化对于确保数据再现性和可比性是至关重要的。

材料和试剂

  1. Lacryvisc Gel 10 G(3 mg/ml carbomere in benzalconium chloride)(Alcon)( http://www.alcon.com
  2. 1-cc结核菌素注射器(B.BraunEspa?a,型号:Omnifix-F)
  3. 30-G针(B.BraunEspa?a,型号:Sterican)
  4. 加热垫[例如 Gaymar Mul-T-Pads(Gaymar industries)( http://www。 gaymar.com/)]
  5. 加热泵保持加热垫的温度[例如 Gaymar TP600(Gaymar industries)( http://www.gaymar.com/)]
  6. 具有内源性肺肿瘤(即我们自己的K-Ras G12V 诱导型小鼠模型,Guerra等人,2003)的遗传修饰的小鼠模型,肺原位肿瘤的原位移植(Ambrogio等人,2014)或尾静脉注射的肺肿瘤细胞(Ambrogio等人,2014)(图1) />

    图1.可通过PET-CT扫描检测的鼠肺肿瘤的类型 PET-CT技术可用于检测不同来源的肺肿瘤:从左侧,内源性肺肿瘤(<在遗传工程改造的小鼠模型中由K-Ras 驻留敲入等位基因诱导的肿瘤),尾静脉注射的肺癌细胞系或原位植入的肺肿瘤(鼠或人肺腺癌)。比例尺:200μM
  7. 必要时使用特殊的小鼠饮食(Cussó等人,2014)
  8. 吸入麻醉:
    七氟烷[例如 Sevoflo(Abbott Laboratories,目录号:05458-02)(http://www.abbottanimalhealth.com/veterinary-professionals/products/anesthesia/sevoflo.html )]
  9. 从O 2浓缩器获得的氧气
  10. 每天从局部回旋加速器(例如<40> mCi的[18 F])中递送的[ 18 F] FDG(0.01至0.1μg/mCi) FDG,在pH6.0下缓冲的1ml生理盐水溶液中的放射化学纯度为95%至99%,用于?10个微PET扫描)
  11. 生理盐水:0.9%(w/v)NaCl(B.BraunEspa?a)
  12. Lacryvisc Gel 10 G(3mg/ml carbomere in benzalconium chloride)(Alcon)( http://www.alcon.com < a>)

设备

  1. O 2浓缩器(Eickemeyer Veterinary Equipment,型号:Oxymat e3)
  2. 红外加热灯Philips PAR38 IR 175W E27(皇家飞利浦电子)
  3. 具有诱导室和用于小鼠McKinley,2型(Everest tecnologia veterinaria)的吸入掩模的七氟烷/基于氧的麻醉系统
  4. 剂量校准器(也称为活度计)[例如 VDC-505剂量校准器(Veenstra Instruments)( http://www.dosecalibrator.com/)]
  5. 微型PET-CT成像系统[例如eXplore Vista PET-CT(GE Healthcare)(图2); Argus PET-CT(SEDECAL)( http://www.sedecal.com/)]


    图2.小鼠的Micro-PET-CT机器(Argus SEDECAL)

  6. 用于图像采集,处理和分析的工作站( Dell PowerEdge)符合以下规格:
    1. PE1950 Xeon 5120 1.86 GHz/4 MB 1066 FSB处理器
    2. PE1950 PCIX Riser(2插槽)
    3. PE1950挡板组件
    4. 2 GB FB 667 MHz内存(2 x 1 GB双列DIMM)
  7. Alienware Dell Studio XPS Desktop 435 MT PC(用于3DOSEM图像重建)符合以下规格:
    1. 处理器:Intel Core i7 Quad CPU 940 4 x 2.93 GHz
    2. 内存:6144 MB(6 x 1,024)1067 MHZ DDR3
    3. 图形:ATI Radeon HD 3450 256 Mb GDDR2

软件

  1. 用于图像采集,处理和分析的eXplore Vista PET-CT MMWKS软件(Desco等人,2005)或AMIDE软件(Loening和Gambhir,2003)

程序

  1. 准备,镇静和麻醉小鼠
    1. 饮食条件。对于胸部肿瘤的检测,小鼠 描述在Materials and Reagents,Pag。 1) 配制的商业高脂肪饮食(例如,饮食D12451 从研究饮食,45%的总卡路里来自脂肪)或, 或者,向日葵种子,其中富含植物脂肪 ?在分析前24小时。这将减少葡萄糖摄取 心肌,在很大程度上消除干扰 这是由于在标准饲喂下高的心脏摄取 条件(Cussóet al。,2014)。
    2. 运输小鼠到成像单元前一天探索,准备饮食,并确定自己的体重。
    3. 在笼子上放置一个红外灯,以保持小鼠在注射前保持温暖。
    4. 小心握住鼠标,使其尾部靠近红外线 灯(?5cm),3?4分钟。小心不要暴露整体 动物到灯,以避免过度的身体加热
    5. 一旦尾静脉扩张,将小鼠引入麻醉 ?室,其中将通过吸入诱导6%的深麻醉 七氟丙烷在100%氧气中以1.5L/min的速率。
      注意:或者也可以使用i.p.注射含有氯胺酮(200mg/kg)和赛拉嗪(10mg/kg)的混合物。

  2. 管理放射性标记的探针
    1. 转移麻醉的小鼠到注射区,并使用 结核菌素屏蔽注射器和30-G针头,向其中注射[18 F] FDG ?在尾部(图3),剂量为18.5MBq,体积为0.2ml 生理盐水(0.9%NaCl),用a


      图3.管理探针。 尾静脉 将放射性探针注射到麻醉的小鼠中 防护注射器
    2. 一旦注射,维持小鼠 麻醉,以0.2L/min在100%氧气中提供3%的七氟醚 避免在[18 F] FDG摄取期间由于小鼠运动引起的肌肉摄取) 时间,即30-45分钟。
      注意:对于微PET-CT成像, 在[ F] FDG期间进行微CT图像采集(5分钟) 吸收期。在[ 18 F] FDG管理后等待25-40分钟,然后 ?进行微CT研究;将进行微PET研究 紧接在微CT完成之后。麻醉 在获取微PET-CT成像期间所需的浓度 (0.2L/min)低于用于麻醉的浓度 感应(1.5L/min)

  3. 执行图像采集
    1. 在将小鼠放在探查台上之前,给予眼睛润滑剂 (Lacryvisc Gel 10G),以避免小鼠的角膜损伤 麻醉,因为在这些条件下,闪烁反射丢失。 在图像采集期间,小鼠必须用面罩麻醉 在100%氧气中以0.2L/min提供3%的七氟醚
    2. 将微PET机探测台上的加热垫设置在37°C的恒温下
    3. 将小鼠放在探查台上,以尽可能伸展的俯卧位置,以最小化器官重叠
    4. 按如下所述执行图像采集。
      1. 执行空白测试。空白测试是没有的空采集 ?任何辐射。它必须每天做,在任何真正的工作之前 出来,以评估玻璃检测器的正确功能 微PET设备。如果系统正常工作,由确定 ?空白测试,然后用户可以开始正常操作 微PET。软件程序MMWKS VISTA CT(Gleason等人,2007; Pascau等人,2012),选项"PET ACQ"被选中,并且 选择5分钟持续时间的静态研究,没有任何同位素
      2. 选择胸部,确保所有肺小叶将 获得并开始采集第一床位置:PET 扫描被获取为一系列不同的床运动,称为床 位置(对于标准身体扫描通常为1-3)。在每个床位置, 扫描仪在设定的时间内获取,然后移动 大约1cm并开始获取下一床位置。所有的 这些单独的框架然后被编织在一起以形成最终的 图像,这应该需要10和30分钟之间。如果我们不想看 远处转移我们可以避免全身采集和减少 总扫描时间。
      3. 将收购分成两部分。 选择或创建将保存并创建研究的文件夹 所谓的侦察(被研究区域的划界),或者 使用以前创建的侦察(如果同一个鼠标的微CT 已经执行,这个给定的鼠标的侦察兵已经 可用)。一旦胸区被分隔,选择种类 的微PET研究进行,定义的持续时间 程序,要使用的同位素和同位素的能量窗 (参见下一点肺的方案选择)。
      4. 选择 ?预定义的名为"静态"的微PET采集协议。这个 协议允许一个床位;它需要以前的知识 感兴趣的解剖区域(在这种情况下,胸部区域)。这个 是标准肺肿瘤研究的首选方案。
      5. 执行微PET探索设置同位素的类型为 18 F和 ?将较低能量阈值设置为150KeV。而收购是在 进度,确保巧合数在200和之间 这用作用于注射的质量控制 放射性标记的化合物。巧合数等于 由放射性分解产生的检测光子数。如果 重合次数不在此范围内,则不会 可能执行微PET采集。在最后一个例子中, 用户应该再次注射另一剂量的探针,以便有 足够的同时检测事件。
    5. 一旦微PET 采集完成后,从设备中取出鼠标,断开连接 麻醉,并取小鼠到一个温暖的笼子里,他们将醒来 他们自己的。

  4. 重建微PET图像
    1. 选择用于图像重建的选项。图像的三个选项 重建在eXplore Vista PET-CT设备中可用: 2D-FBP(滤波反投影),2D-OSEM(有序子集期望 ?最大化)和3D-OSEM。作者的偏好是3D-OSEM与 迭代次数设置为80并采用随机衰减和 散射校正。以这种方式,我们实现两种适当的成像 质量和空间信号定位的补偿合理 计算资源和可接受的计算运行时间。
      注意:运行3D-OSEM需要独立的PC。平均运行 用于3D-OSEM计算的时间(80次迭代,随机和散射 校正)在Dell Studio XPS Desktop 435MT PC上<2分钟。

  5. 量化数据
    1. 一旦重建了总扫描体积,检查图像直到a 在解剖位置中识别正[18 F] FDG信号 与肺肿瘤一致,并且与正常器官摄取不同 ?(例如,心脏,膀胱,肾脏,以及较小程度的棕色) 脂肪组织)(图4)。
    2. 手动绘制感兴趣区域 (ROI)围绕肿瘤的周边。随后,检查相邻 软件已自动绘制相同ROI的部分;如果 需要调整,可以手动完成。
      注意:3D-OSEM 重建算法采用立方体素。软件 预先确定重构的立体体素的大小;在我们的 设备,其值为0.7×0.7×0.7mm。因此,肿瘤体积 以立方体素的体积单位建立。它必须得到验证 所有的肿瘤活性已被包括。 [
    3. 从上一步描述的定量,计算标准吸收值(SUV)。
      注意:两种类型的SUV测量可以使用平均值计算 ?SUV(SUV ave )和最大SUV(SUV max )公式如下:
      SUV ave = [摄取(MBq)/体积的ROI(cc)]/[小鼠体重[ 18 F] FDG dose(MBq)x calibration factor] 校准因子从获取填充的幻影获得 辐射并且特定于每个PET机器。性能是 在每个设备的用户手册中描述。
      SUV max = ?具有最大活动性的体素(MBq)/体素的体积(cc)]/[小鼠 重量(g)x注射[ 18 F] FDG剂量(MBq)x校准系数]
      SUV ave 反映每单位体积的放射性量 整个ROI体积。 SUV max 反映每单位的放射性 在ROI内的体素处的体积与最大摄取值。 在小鼠的特定情况下,由于大的变异性 放射性标记的化合物分布,建议标准化 在正常的参考/背景ROI的感兴趣的ROI的结果 肺。例如,数据可以表示为肿瘤与的比例 背景(TBR)。在图4中,来自肺肿瘤的代表性图像 ,其中SUV max 值为约1至3MBq/cc,SUV mean 值 ?约0.5至1 MBq/cc。


      图4. PET定量的实施例。肺肿瘤PET信号的轴向视图和相应的分析ROI (白色箭头)。黄色箭头表示背景ROI放置在 健康肺。

第二部分。计算机断层扫描(微CT)成像

微-CT研究比微PET更简单,因为微CT需要很少或没有制备。然而,小鼠必须在麻醉下固定以避免由运动引起的伪像。同样,为了防止麻醉诱导的低体温,小鼠必须通过加热垫保持在恒定的体温。外源性造影剂通常用于改善肿瘤和周围健康组织之间的信号比,但是为了检测肺肿瘤,不需要对比度。

材料和试剂

  1. 加热垫[例如 Gaymar Mul-T-Pads(Gaymar industries)( http://www。 gaymar.com/)]
  2. 加热泵保持加热垫的温度[例如 Gaymar TP600(Gaymar industries)( http://www.gaymar.com/)]
  3. 具有内源性肺肿瘤的遗传修饰的小鼠模型(Guerra等人,2003),原发性肿瘤的肺原位植入(Ambrogio等人,2014)或尾静脉注射的肺肿瘤细胞(Ambrogio等人,2014)(图1)
  4. 吸入麻醉:
    七氟烷[例如 Sevoflo(Abbott Laboratories,目录号:05458-02)(http://www.abbottanimalhealth.com/veterinary-professionals/products/anesthesia/sevoflo.html )]
  5. 从O 2浓缩器获得的氧
  6. Lacryvisc Gel 10 G(3mg/ml carbomere in benzalconium chloride)(Alcon)( http://www.alcon.com < a>)

设备

  1. O 2浓缩器(Eickemeyer Veterinary Equipment,型号:Oxymat e3)
  2. 红外加热灯Philips PAR38 IR 175W E27(皇家飞利浦电子)
  3. 具有诱导室和用于小鼠McKinley,2型(Everest tecnologia veterinaria)的吸入掩模的七氟烷/基于氧的麻醉系统
  4. 用于呼吸门控的多参数监测器(图5)[例如 Vision Vet(RGB)(http://www.medicalexpo.com/prod/rgb-medical-devices/product-69843-493296.html)]
  5. 红外线加热灯
  6. 配有诱导室和小鼠吸入面罩的七氟烷/氧基麻醉系统
  7. 用于图像采集,处理和分析的工作站( Dell PowerEdge)符合以下规格:
    1. PE1950 Xeon 5120 1.86 GHz/4 MB 1066 FSB处理器
    2. PE1950 PCIX Riser(2插槽)
    3. PE1950挡板组件
    4. 2 GB FB 667 MHz内存(2 x 1 GB双列DIMM)

软件

  1. 用于图像采集,处理和分析的eXplore Vista PET-CT MMWKS软件(Desco等人,2005)或AMIDE软件(Loening和Gambhir,2003)

程序

  1. 准备,镇静和麻醉小鼠
    1. 麻醉小鼠:将小鼠引入麻醉室,其中a 深度麻醉将通过吸入6%七氟烷在100% ?氧气以1.5L/min的速率。并保持温暖;应用一些眼睛 润滑剂(Lacryvisc Gel 10G)到角膜,按规定保护 在上面的协议,然后将鼠标放在扫描仪室内。 对于micro-CT采集小鼠不需要事先准备(特殊 饮食和镇静)。
    2. 在启动micro-CT之前,选择 胸区并正确设置不同的参数。参数 用于CT采集通常设置在以下范围内 值:
      1. 电源强度:140至1,000 mA。
      2. 拍摄张数:从1到32。
        注意:镜头数指的是X射线的次数 ?从源发射。虽然更高的拍摄数量导致了 ?更高的信噪比,它也导致更大的采集 时间和更大的辐射损伤小鼠(见步骤A4 )。
      3. 分辨率:标准(200μm),高(100μm)或最大(50μm)
      4. 投影数:从360?到720?。
        注意:投影数量是指的旋转数 围绕小鼠的X射线束,以六十进制度表示。双 ?全旋转(720°)与单全旋转(360°)导致 图像具有较高的信噪比,但成本较高 获得时间,并因此,小鼠的更大的暴露 危险能源和麻醉。
      5. 床位数:1 ?一个床位置为胸部成像就足够了。
        注意:更多 比一个床位置需要,它们叠加在每个的顶部 其他,导致部分图片重叠。
    3. 修复鼠标 探测表用胶带即使他们麻醉, 最小化不自主运动。一旦小鼠准备好探索 表,降下Vista-PET-CT的保护屏蔽屏 ?锁定安全钥匙以执行数据采集。

  2. 标准微CT数据采集
    1. 标准采集参数有:电源强度为 ?150mA,标准分辨率为(200μM)的电压为45kV, 360°和16张照片为1床位置,或8张照片,如果执行2床 位置。上述获取参数对应于辐射 剂量在0.6Gy的小鼠中
    2. 为了选择采集 区域观察由微CT机生成的侦察图像。这个 侦察员给出一个投影,用户可以看到主题是如何 放置。
    3. 选择,借助激光,开始和 结束用户想要获取(例如,胸部)的ROI 地区)。我们还可以从micro-PET研究中选择以前的侦察 已经在同一会话中完成,只要它对应 同样的鼠标和我们没有改变其处置 探索表

  3. 胸部CT的呼吸道选通
    1. 从外部监控设备获取呼吸信号 同时与微CT扫描(图5;呼吸运动 人为因素在CT采集中是重要的,特别是在情况下 胸部研究)。采集后,根据 外部信号。这产生两个独立的 扫描,对应于吸入和呼气(分组 中间阶段也是可能的)。


      图5.动物处理和 ?监测。将装有电极的鼠标引入PET-CT ?机器(左图);生命常数监控(VisionVet,RGB
      http://www .medicalexpo.com/prod/rgb-medical-devices/product-69843-493296.html ) ?记录ECG,温度和呼吸频率(右图)
    2. 停止麻醉输送一旦micro-CT采集结束, 从设备中取出小鼠,并放入温暖的笼子里 他们会自己醒来。

  4. 微CT图像重建
    1. 使用修改版本的锥形束(CB)重建图像 Feldkamp,Davis和Kress的算法(FDK,Vaquero等人,2008) 包括在eXplore Vista PET-CT的软件中。
      注意:FDK是一个 ?广泛使用的滤波投影算法为三维 图像重建从锥形束投影测量与圆形 ?X射线源的轨道。算法的重建时间 在Dell PowerEdge工作站上运行时间<5秒。

  5. 使用ROI进行量化
    1. 通过视觉检查图像识别肺肿瘤。
    2. 画 手动地围绕肿瘤的周界的感兴趣区域(ROI)。 随后,检查软件绘制的相邻部分 相同ROI;如果需要调整,可以手动完成(图6)。 软件预先确定体素的大小。我们的重建 软件采用50 x 50 x 50μm的立方体素来定义ROI。的 所选ROI的体积(以立方厘米表示)为 由系统自动计算

      图6. ROI的示例 在CT,PET和融合图像中选择。示例显示(从左到右) 向右)胸腔的轴向,矢状和冠状切面。的 在所有三个部分中可见的ROI以纯黄色显示。 H:心。 肿瘤用白色箭头指示。绿色箭头指向骨髓 和黄色箭头标记棕色脂肪组织。

第三部分。多模式成像(微PET-CT)

小鼠在探索过程中的固定对于多模态成像研究中的图像共注册是必要的。如果在微PET和微-CT研究中的位置不同,通过微PET和显微CT获得的图像的匹配(共同对准)是不可能的。

材料和试剂

  1. 1-cc结核菌素注射器(B.BraunEspa?a,型号:Omnifix-F)
  2. 30-G针(B.BraunEspa?a,型号:Sterican)
  3. 加热垫[例如 Gaymar Mul-T-Pads(Gaymar industries)( http://www。 gaymar.com/)]
  4. 加热泵保持加热垫的温度[例如 Gaymar TP600(Gaymar industries)( http://www.gaymar.com/)]
  5. 具有内源性肺肿瘤的遗传修饰的小鼠模型(Guerra等人,2003),原发性肿瘤的肺原位植入(Ambrogio等人,2014)或尾静脉注射的肺肿瘤细胞(Ambrogio等人,2014)(图1)
  6. 必要时使用特殊的小鼠饮食(Cussó等人,2014)
  7. 吸入麻醉:
    七氟烷[例如 Sevoflo(Abbott Laboratories,目录号:05458-02)(http://www.abbottanimalhealth.com/veterinary-professionals/products/anesthesia/sevoflo.html )]
  8. 从O 2浓缩器获得的氧气
  9. 每天从局部回旋加速器(例如<40> mCi的[18 F])中递送的[ 18 F] FDG(0.01至0.1μg/mCi) FDG,在pH6.0下缓冲的1ml生理盐水溶液中的放射化学纯度为95%至99%,用于?10个微PET扫描)
  10. 生理盐水:0.9%(w/v)NaCl(B.BraunEspa?a)
  11. Lacryvisc Gel 10 G(3mg/ml carbomere in benzalconium chloride)(Alcon)( http://www.alcon.com < a>)

设备

  1. O 2浓缩器(Eickemeyer Veterinary Equipment,型号:Oxymat e3)
  2. 红外加热灯Philips PAR38 IR 175W E27(皇家飞利浦电子)
  3. 具有诱导室和用于小鼠McKinley,2型(Everest tecnologia veterinaria)的吸入掩模的七氟烷/基于氧的麻醉系统
  4. 剂量校准器(也称为活性计)[例如 VDC-505剂量校准器(Veenstra Instruments)( http ://www.dosecalibrator.com/))
  5. 微型PET-CT成像系统[例如eXplore Vista PET-CT(GE Healthcare,图2); Argus PET-CT(SEDECAL)( http://www.sedecal.com/)]
  6. 用于图像采集,处理和分析的eXplore Vista PET-CT MMWKS软件(Desco等人,2005)或AMIDE软件(Loening和Gambhir,2003)
  7. 用于图像采集,处理和分析的工作站( Dell PowerEdge)符合以下规格:
    1. PE1950 Xeon 5120 1.86 GHz/4 MB 1066 FSB处理器
    2. PE1950 PCIX Riser(2插槽)
    3. PE1950挡板组件
    4. 2 GB FB 667 MHz内存(2 x 1 GB双列DIMM)
  8. Alienware Dell Studio XPS Desktop 435 MT PC(用于3DOSEM图像重建)符合以下规格:
    1. 处理器:Intel Core i7 Quad CPU 940 4 x 2.93 GHz
    2. 内存:6144 MB(6 x 1,024)1067 MHZ DDR3
    3. 图形:ATI Radeon HD 3450 256 Mb GDDR2

程序

  1. 准备小鼠
    1. 在多模态探索之前准备小鼠,如所述 ?micro-PET探索在相应的协议,步骤1至6.
      注意:多模态探索需要极度注意护理 的小鼠以及不断监测的温度,因为 研究时间比单独使用CT或PET更长。

  2. 获取微CT图像
    1. 在[ 18 F] FDG摄取期间进行微CT图像的采集 ?(持续45分钟)。参见micro-PET方案,步骤8和显微CT 协议。

  3. 获取PET图像
    1. 按照与相应的方案步骤7至13中所述相同的程序进行微PET图像采集。

  4. 重建图像
    1. 单独重建两个图像,并使用MMWKS软件获得协同寄存器(图6-7)。


      图7.肺肿瘤的PET-CT成像。 代表轴向 FDG-PET扫描的投影显示肿瘤FDG-PET信号(由 一个箭头)。 H:心脏,B:骨髓。肿瘤用黄色表示 箭头。

  5. 量化图像
    1. 一旦肺肿瘤已经确定,量化的微CT图像和 微型PET图像,如上述方案中所述。

  6. 呈现卷
    1. 生成数据的3-D渲染,以更加图形化的方式获得肺肿瘤位置和体积的信息(图8)。


      图8.肺癌的纵向研究的实施例。 3-D肺 在指定的两个时间点的PET-CT研究的效果图 携带遗传诱导的肺肿瘤(红色)的个体小鼠, 用载体(上图)或用治疗化合物治疗 ?(下图)。

致谢

该协议已经改编自先前公开的研究"通过PET,CT和组合PET-CT成像在小鼠中的癌症"(Mulero等人,2011)。我们感谢分子成像核心单位的专业知识和对这里描述的协议的优化的关键贡献。这项工作得到了CDTI(西班牙科技部西班牙工业技术发展中心),AMIT项目"高级分子成像技术"(5710001425)到FM的资助。 CA是西班牙抗癌协会(AECC)的博士后研究员的接受者。

参考文献

  1. Ambrogio,C.,Carmona,FJ,Vidal,A.,Falcone,M.,Nieto,P.,Romero,OA,Puertas,S.,Vizoso,M.,Nadal,E.,Poggio,T.,Sanchez- Cespedes,M.,Esteller,M.,Mulero,F.,Voena,C.,Chiarle,R.,Barbacid,M.,Santamaria,D.和Villanueva, 通过原位小鼠同种异体移植建模肺癌的进展和临床前反应。癌症研究 74(21):5978-5988。
  2. Cusso,L.,Vaquero,J.J.,Bacharach,S。和Desco,M。(2014)。 在小鼠中减少心肌18F-FDG摄取的方法比较:钙通道阻滞剂与高脂肪饮食。 PLoS One 9(9):e107999。
  3. Desco,M.,Penedo,M.,Gispert,J.D.,Vaquero,J.J.,Reig,S.and Garcia-Barreno,P.(2005)。 ROC评估在[15O] -H 2中脑活动的统计小波分析 O PET扫描。 Neuroimage 24(3):763-770。
  4. Gleason,S.S.,Austin,D.W.,Beach,R.S.,Nutt,R.,Paulus,M.J.and Yan,S。(2007)。一种新的多功能多模式小动物成像平台。在: IEEE核科学研讨会会议记录。 pp 2447-2449。
  5. Guerra,C.,Mijimolle,N.,Dhawahir,A.,Dubus,P.,Barradas,M.,Serrano,M.,Campuzano,V.and Barbacid,M.(2003)。 内源性K-ras致癌基因的肿瘤诱导高度依赖于细胞环境。 em> Cancer Cell 4(2):111-120。
  6. Loening,A.M。和Gambhir,S.S。(2003)。 AMIDE:用于多模态医学图像分析的免费软件工具 Mol成像 2(3):131-137。
  7. Mulero,F.,Donate,L.E。和Serrano,M。(2011)。 通过PET,CT和组合PET-CT成像小鼠中的癌症 Curr Protoc Mouse Biol 1(1):85-103。
  8. Pascau,J.,Vaquero,J. J.,Chamorro-Servent,J.,Rodriguez-Ruano,A.and Desco,M。(2012)。 小动物PET/CT对准校准的方法。 Phys Med Biol。57(12):N199-207。
  9. Vaquero,J.J.,Redondo,S.,Lage,E.,Abella,M.,Sisniega,A.,Tapias,G.,Soto Montenegro,M.L。,和Deseo,M。(2008)。一个新的高性能小动物X射线断层扫描仪的评估。在:关于核科学的IEEE交易。 pp 898-905。
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How to cite this protocol: Ambrogio, C., Cámara, J. A., Nieto, P., Santamaría, D. and Mulero, F. (2015). Analysis of Murine Lung Tumors by Micro PET-CT Imaging. Bio-protocol 5(24): e1692. DOI: 10.21769/BioProtoc.1692; Full Text



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