Name: pet and mri guided irradiation of a glioblastoma rat model using a micro-irradiator
Uploaded: 2017-12-28T14:00:00
Description: Watch this Scientific Journal Video about PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator at JoVE.com

The authors would like to thank Stichting Luka Hemelaere and Soroptimist International for supporting this work.

The study was approved by the ethics committee for animal experiments (ECD 09/23 and ECD 12/28). All commercial details can be found in Table of Materials.
1. F98 GB Rat Cell Model
<ol>
	<li>Culture the F98 GB cells, obtained from ATCC, in monolayers using Dulbecco&#39;s modified Eagle Medium, 10% calf serum, 1% penicillin, 1% streptomycin, 1% L-glutamine, and 0.1% amphotericin b, and place in a CO2 incubator (5% CO2 and 37 &#176;C).</li>
	<li>Inoculate...

<ol>
	<li>Stupp, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promising+survival+for+patients+with+newly+diagnosed+glioblastoma+multiforme+treated+with+concomitant+radiation+plus+temozolomide+followed+by+adjuvant+temozolomide.">Promising survival for patients with newly diagnosed glioblastoma multiforme treated with concomitant radiation plus temozolomide followed by adjuvant temozolomide.</a> J Clin Oncol. 20 (5), 1375-1382 (2002).</li><li>Dhermain, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiotherapy+of+high-grade+gliomas:+current+standards+and+new+concepts,+innovations+in+imaging+and+radiotherapy,+and+new+therapeutic+approaches.">Radiotherapy of high-grade gliomas: current standards and new concepts, innovations in imaging and radiotherapy, and new therapeutic approaches.</a> Chin J Cancer. 33 (1), 16-24 (2014).</li><li>Ahmed, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+gliomas:+current+perspectives+in+diagnosis,+treatment,+and+early+response+assessment+using+advanced+quantitative+imaging+methods.">Malignant gliomas: current perspectives in diagnosis, treatment, and early response assessment using advanced quantitative imaging methods.</a> Cancer Manag Res. 6, 149-170 (2014).</li><li>Verhaegen, F., Granton, P., Tryggestad, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+radiotherapy+research+platforms.">Small animal radiotherapy research platforms.</a> Phys Med Biol. 56 (12), R55-R83 (2011).</li><li>Kinsella, T. J., Vielhuber, K. A., Kunugi, K. A., Schupp, J., Davis, T. W., Sands, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+toxicity+and+efficacy+study+of+a+14-day+schedule+of+oral+5-iodo-2-pyrimidinone-2-deoxyribose+as+a+prodrug+for+5-iodo-2-deoxyuridine+radiosensitization+in+U251+human+glioblastoma+xenografts.">Preclinical toxicity and efficacy study of a 14-day schedule of oral 5-iodo-2-pyrimidinone-2-deoxyribose as a prodrug for 5-iodo-2-deoxyuridine radiosensitization in U251 human glioblastoma xenografts.</a> Clin Cancer Res. 6 (4), 1468-1475 (2000).</li><li>Vellimana, A. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+method+for+reproducible+and+accurate+image-guided+stereotactic+cranial+irradiation+of+brain+tumors+using+the+small+animal+radiation+research+platform.">An integrated method for reproducible and accurate image-guided stereotactic cranial irradiation of brain tumors using the small animal radiation research platform.</a> Transl Oncol. 5 (4), 230-237 (2012).</li><li>Grosu, A. -. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+IMT-SPECT+for+postoperative+radiotherapy+planning+in+patients+with+gliomas.">Implications of IMT-SPECT for postoperative radiotherapy planning in patients with gliomas.</a> Int J Radiat Oncol Biol Phys. 54 (3), 842-854 (2002).</li><li>Butterworth, K. T., Prise, K. M., Verhaegen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+image-guided+radiotherapy:+Status,+considerations+and+potential+for+translational+impact.">Small animal image-guided radiotherapy: Status, considerations and potential for translational impact.</a> Br J Radiol. 88 (1045), 4-6 (2015).</li><li>Aird, E. G. A., Conway, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CT+simulation+for+radiotherapy+treatment+planning.">CT simulation for radiotherapy treatment planning.</a> Br J Radiol. 75 (900), 937-949 (2002).</li><li>Baker, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization:+Conventional+and+CT+simulation.">Localization: Conventional and CT simulation.</a> Br J Radiol. 79 (Spec No 1). , S36-S49 (2006).</li><li>Corroyer-Dumont, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI-guided+radiotherapy+of+the+SK-N-SH+neuroblastoma+xenograft+model+using+a+small+animal+radiation+research+platform.">MRI-guided radiotherapy of the SK-N-SH neuroblastoma xenograft model using a small animal radiation research platform.</a> Br J Radiol. 90 (1069), 20160427 (2017).</li><li>Bolcaen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI-guided+3D+conformal+arc+micro-irradiation+of+a+F98+glioblastoma+rat+model+using+the+Small+Animal+Radiation+Research+Platform+(SARRP).">MRI-guided 3D conformal arc micro-irradiation of a F98 glioblastoma rat model using the Small Animal Radiation Research Platform (SARRP).</a> J Neurooncol. 120 (2), 257-266 (2014).</li><li>Niyazi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FET-PET+for+malignant+glioma+treatment+planning.">FET-PET for malignant glioma treatment planning.</a> Radiother Oncol. 99 (1), 44-48 (2011).</li><li>Grosu, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+experience+with+I-123-alpha-methyl-tyrosine+SPECT+in+the+3-D+radiation+treatment+planning+of+brain+gliomas.">First experience with I-123-alpha-methyl-tyrosine SPECT in the 3-D radiation treatment planning of brain gliomas.</a> Int J Radiat Oncol Biol Phys. 47 (2), 517-526 (2000).</li><li>Ling, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+multidimensional+radiotherapy+(MD-CRT):biological+imaging+and+biological+conformality.">Towards multidimensional radiotherapy (MD-CRT):biological imaging and biological conformality.</a> Int J Radiat Oncol Biol Phys. 47 (3), 551-560 (2000).</li><li>Wahl, R. L., Jacene, H., Kasamon, Y., Lodge, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+RECIST+to+PERCIST:+Evolving+Considerations+for+PET+response+criteria+in+solid+tumors.">From RECIST to PERCIST: Evolving Considerations for PET response criteria in solid tumors.</a> J Nucl Med. 50 (5), 122S-150S (2009).</li><li>Daisne, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+volume+in+pharyngolaryngeal+squamous+cell+carcinoma:+comparison+at+CT,+MR+imaging,+and+FDG+PET+and+validation+with+surgical+specimen.">Tumor volume in pharyngolaryngeal squamous cell carcinoma: comparison at CT, MR imaging, and FDG PET and validation with surgical specimen.</a> Radiology. 233 (1), 93-100 (2004).</li><li>Grosu, A. -. L., Weber, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET+for+radiation+treatment+planning+of+brain+tumours.">PET for radiation treatment planning of brain tumours.</a> Radiother Oncol. 96 (3), 325-327 (2010).</li><li>Banissi, C., Ghiringhelli, F., Chen, L., Carpentier, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treg+depletion+with+a+low-dose+metronomic+temozolomide+regimen+in+a+rat+glioma+model.">Treg depletion with a low-dose metronomic temozolomide regimen in a rat glioma model.</a> Cancer Immunol Immunother. 58, 1627-1634 (2009).</li><li>Robinson, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+alternative+temozolomide+schedules+on+glioblastoma+O(6)-methylguanine-DNA+methyltransferase+activity+and+survival.">Effect of alternative temozolomide schedules on glioblastoma O(6)-methylguanine-DNA methyltransferase activity and survival.</a> Br J Cancer. 103, 498-504 (2010).</li><li>Espa&#241;a, S., Marcinkowski, R., Keereman, V., Vandenberghe, S., Van Holen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DigiPET:+sub-millimeter+spatial+resolution+small-animal+PET+imaging+using+thin+monolithic+scintillators.">DigiPET: sub-millimeter spatial resolution small-animal PET imaging using thin monolithic scintillators.</a> Phys Med Biol. 59 (13), 3405 (2014).</li></ol>

To achieve accurate irradiation of the glioblastoma tumor target in the rat brain, the micro-irradiator&#39;s on-board CT guidance was not sufficient. Brain tumors are hardly visible due to insufficient soft tissue contrast, even if contrast enhancement would be used. As such, MRI needs to be included to allow more precise irradiation. Using a sequential MR acquisition on a 7 T system and a CT acquisition on the micro-irradiator we were able to target the dose to the contrast-enhancing tumor tissue in the brain and calcu...

High-grade glioma is the most common and most aggressive malignant brain tumor in adults with a median survival of 1 year despite current treatment modalities. The standard of care includes maximal surgical resection followed by combined external beam radiation therapy (RT) and temozolomide (TMZ), followed by maintenance TMZ1,2,3. Since the introduction of TMZ now more than 15 years ago, no significant improvements have been made in the treatment of these tumors. Therefore, the implementation of new therapeutic strategies is urgent but should be first investigated in small animal cancer therapy models (mostly mice and rats). Tumor-bearing rodent models can be used to investigate the efficacy of new and complex radiation protocols, possibly combined with other (new) treatment agents, to assess radiation response or to investigate radio-protective agents. A major advantage of preclinical radiation research is the ability to work under controlled experimental conditions using large cohorts resulting in accelerated data yield due to the shorter lifespans of rodents. The preclinical findings should then be translated into a clinical trial in a much faster and more efficient way than in current practice4.
Small animal radiation experiments in the last decades have typically been achieved using fixed radiation sources5,6,7, e.g., 137Cs and 60Co, isotopes, or linear accelerators intended for human clinical use, applying a single radiation field with MV X-rays6,8,9,10,11. However, these devices do not reach sub-millimeter precision, which is required for small animals12. Furthermore, MV X-rays have characteristics unsuitable for irradiating small targets, such as a dose build-up at the air-tissue interface in the entrance region of the beam with an extent in the order of the animal size itself4,6,8,9,10,11. The latter makes it quite challenging to deliver a uniform dose to a tumor while sparing surrounding normal brain tissue4,8,9,10,11. Hence, it is unclear to which extent current animal studies still are relevant for modern RT practice12. In this respect, recently developed three-dimensional (3D) conformal small animal micro-irradiators are promising to bridge the technological gap between advanced 3D image-guided RT techniques, such as intensity modulated radiation therapy (IMRT) or conformal arcs used in humans and current small animal irradiation4,13. These platforms make use of a kilovoltage (kV) X-ray source to obtain sharp penumbras and to avoid dose build-up. These platforms include a computer-controlled stage for animal positioning, a kV X-ray source for imaging and radiation treatment, a rotational gantry assembly to allow radiation delivery from various angles, and a collimating system to shape the radiation beam4. In 2011, a micro-irradiator was installed at the preclinical imaging lab of Ghent University (Figure 1). This system is similar to modern human radiotherapy practice and enables a wide variety of preclinical experiments, such as the synergy of radiation with other therapies, complex radiation schemes, and image-guided sub-target boost studies.
Treatment planning on these micro-irradiators is based on CT, which is equivalent to human planning systems14,15. For CT imaging, an on-board X-ray detector is used in combination with the same kV X-ray tube that is used during treatment. CT imaging is used as it allows for accurate animal positioning and provides information necessary for individual radiation dose calculations via segmentation. However, due to the low soft-tissue contrast in CT imaging, tumors in the brain of small animals, such as high-grade glioma, cannot be easily delineated. The incorporation of multi-modality imaging is therefore necessary for an accurate target volume delineation. Compared to CT, MRI provides vastly superior soft-tissue contrast. This makes it much easier to visualize lesion boundaries that will result in a much better delineation of the target volume, helping to better irradiate the lesion and avoid surrounding tissue, as illustrated in Figure 24,16. An additional advantage is that MRI uses non-ionizing radiation, unlike CT that is using ionizing radiation. The major disadvantages of MRI are the relatively long acquisition times and high operational costs. It is important to note that MRI scans cannot be used for dose calculations, as they do not provide the required electron density information, although progress is being made in this field, too with the recent development of MR-LINACS. As such, a combined CT/MRI dataset is the method of choice for planning the irradiation of malignant glioma, containing both the information required for targeting (MRI-based volumes) and for dose calculations (CT-based electron density).
To decrease the gap between small animal irradiation and clinical routine, MRI clearly needs to be integrated into the work flow of the micro-irradiator, requiring a correct registration between MRI and CT, which is far from trivial. In this paper, our protocol for MRI-guided 3D conformal irradiation of F98 glioblastoma in rats is discussed, which has been published recently17.
Although incorporating CT and MRI in the workflow of the micro-irradiator is a clear step forward in small animal irradiation research, these anatomical imaging techniques do not always allow a full definition of the target volume. Pathological changes in the brain on CT and MRI are characterized by increased water content (edema) and leakage of the blood-brain barrier or contrast enhancement. However, both contrast-enhancement and hyper-intense areas on T2-weighted MRI are not always an accurate measure of tumor extent. Tumor cells have been detected far beyond the margins of contrast-enhancement12. Also, none of these techniques can identify the most aggressive parts within the tumor, which may be responsible for therapeutic resistance and tumor recurrence. Therefore, additional information from molecular imaging techniques like PET may have an added value for RT target volume definition because these techniques enable to visualize biologic pathways in vivo12,18,19.
In 2000, Ling et al. introduced the concept of biological target volume (BTV) by integrating anatomical and functional imaging into the radiotherapy workflow, leading to what they called multidimensional conformal radiotherapy20. This creates the possibility to improve dose targeting by delivering a non-uniform dose to a target region using for example PET images. The most widely used PET tracer for tumor staging and to monitor treatment response is fluor-18 (18F) labeled fluorodeoxyglucose (FDG), which visualizes the glucose metabolism21. In head and neck cancer, previous studies have shown that the use of 18F-FDG PET led to a better estimate of the actual tumor volume, as defined by the pathologic specimens, compared with CT and MRI22. In primary brain tumors, where FDG is not useful due to the very strong background signal from the normal brain, amino acids, such as 11C-methionine and more recently 18F-fluoroetthyltyrosine (FET), have been investigated for GTV delineation with often marked differences between amino-acid PET and MRI-based GTVs23. However, no prospective trial investigating the meaning of this finding has been performed yet. In this study, we selected the amino-acid tracer 18F-FET and the hypoxia tracer 18F-fluoroazomycin-arabinoside (18F-FAZA). 18F-FET and 18F-FAZA were selected because an increased amino-acid uptake is strongly correlated with the proliferation rate in GB tumors, whereas uptake of a hypoxia PET-tracer is correlated with resistance to (chemo)radiotherapy18,23. Sub-volume boosting using the micro-irradiator was optimized by giving an additional radiation dose to a PET-defined part of the F98 GB tumor in rats.

<table>
	<tbody>
		<tr>
			<td>GB RAT model</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>F98 Glioblastoma cell line</td>
			<td>ATCC</td>
			<td>CRL-2397</td>
			<td></td>
		</tr>
		<tr>
			<td>Fischer F344/Ico crl Rats</td>
			<td>Charles River</td>
			<td>N/A</td>
			<td>http://www.criver.com/products-services/basic-research/find-a-model/fischer-344-rat</td>
		</tr>
		<tr>
			<td>Micropump system</td>
			<td>World Precision Instruments</td>
			<td>UMP3</td>
			<td>Micro 4: https://www.wpiinc.com/products/top-products/make-selection-ump3-ultramicropump/#tabs-1</td>
		</tr>
		<tr>
			<td>Stereotactic frame</td>
			<td>Kopf</td>
			<td>902</td>
			<td>Model 902 Dual Small Animal Stereotaxic frame</td>
		</tr>
		<tr>
			<td>diamant drill</td>
			<td>Velleman</td>
			<td>VTHD02</td>
			<td>https://www.velleman.eu/products/view/?id=370450</td>
		</tr>
		<tr>
			<td>Bone wax</td>
			<td>Aesculap</td>
			<td>1029754</td>
			<td>https://www.aesculapusa.com/products/wound-closure/hemostatic-bone-wax</td>
		</tr>
		<tr>
			<td>Insulin syringe Microfine</td>
			<td>Beckton-Dickinson</td>
			<td>320924</td>
			<td>1 mL, 29G</td>
		</tr>
		<tr>
			<td>InfraPhil IR lamp</td>
			<td>Philips</td>
			<td>HP3616/01</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethilon</td>
			<td>Ethicon</td>
			<td>662G/662H</td>
			<td>FS-2, 4-0, 3/8, 19 mm</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>14040-091</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilline-streptomycine</td>
			<td>Invitrogen</td>
			<td>15140-148</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030-032</td>
			<td></td>
		</tr>
		<tr>
			<td>Fungizone</td>
			<td>Invitrogen</td>
			<td>15290-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25300-062</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>14040-224</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcons</td>
			<td>Thermo Scientific</td>
			<td>178883</td>
			<td>175 cm2 nunclon surface, disposables for cell culture with filter caps</td>
		</tr>
		<tr>
			<td>Cell freezing medium</td>
			<td>Sigma-aldrich</td>
			<td>C6164</td>
			<td>Cell Freezing Medium-DMSO, sterile-filtered, suitable for cell culture, endotoxin tested</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Animal irradiation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-irradiator</td>
			<td>X-strahl</td>
			<td>SARRP</td>
			<td></td>
		</tr>
		<tr>
			<td>software for irradiation</td>
			<td>X-strahl</td>
			<td>MuriPlan</td>
			<td>pre-clinical treatment planning system (PCTPS), version 2.0.5.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Small animal PET</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microPET system possibility 1</td>
			<td>Molecubes</td>
			<td>B-Cube</td>
			<td>http://www.molecubes.com/b-cube/</td>
		</tr>
		<tr>
			<td>microPET system possibility 2</td>
			<td>TriFoil Imaging, Northridge CA</td>
			<td>FLEX Triumph II</td>
			<td>http://www.trifoilimaging.com</td>
		</tr>
		<tr>
			<td>PET tracers</td>
			<td>In-house made</td>
			<td></td>
			<td>18F-FDG, 18F-FET, 18F-FAZA, 18F-Choline</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Small animal MRI</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microMRI system</td>
			<td>Bruker Biospin</td>
			<td>Pharmascan 70/16</td>
			<td>https://www.bruker.com/products/mr/preclinical-mri/pharmascan/overview.html</td>
		</tr>
		<tr>
			<td>Dotarem contrast agent</td>
			<td>Guerbet</td>
			<td></td>
			<td>MRI contrast agent, Dotarem 0,5 mmol/ml</td>
		</tr>
		<tr>
			<td>rat whole body transmitter coil</td>
			<td>Rapid Biomedical</td>
			<td>V-HLS-070</td>
			<td></td>
		</tr>
		<tr>
			<td>rat brain surface coil</td>
			<td>Rapid Biomedical</td>
			<td>P-H02LE-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Water-based heating unit</td>
			<td>Bruker Biospin</td>
			<td>MT0125</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G Needle for IV injection</td>
			<td>Beckton-Dickinson</td>
			<td>305128</td>
			<td>30 G</td>
		</tr>
		<tr>
			<td>PE 10 tubing (60 cm/injection)</td>
			<td>Instech laboratories, Inc</td>
			<td>BTPE-10</td>
			<td>BTPE-10, polyethylene tubing 0.011 x .024 in (0.28 x 60 mm), non sterile, 30 m (98 ft) spool, Instech laboratories, Inc Plymouth meeting PA USA- (800) 443-4227- http://www.instechlabs.com</td>
		</tr>
		<tr>
			<td>non-heparinised micro haematocrit capillaries</td>
			<td>GMBH</td>
			<td>7493 21</td>
			<td>these capillaries are filled with water to create markers visible on MRI and CT</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>isoflurane: Isoflo</td>
			<td>Zoetis</td>
			<td>B506</td>
			<td>Anaesthesia</td>
		</tr>
		<tr>
			<td>ketamine: Ketamidor</td>
			<td>Ecuphar</td>
			<td></td>
			<td>Anaesthesia</td>
		</tr>
		<tr>
			<td>xylazine: Sedaxyl</td>
			<td>Codifar NV</td>
			<td></td>
			<td>Anaesthesia</td>
		</tr>
		<tr>
			<td>catheter</td>
			<td>Terumo</td>
			<td>Versatus-W</td>
			<td>26G</td>
		</tr>
		<tr>
			<td>Temozolomide</td>
			<td>Sigma-aldrich</td>
			<td>T2577-100MG</td>
			<td>chemotherapy</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-aldrich</td>
			<td>276855-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe Microfine</td>
			<td>Beckton-Dickinson</td>
			<td>320924</td>
			<td>1 mL, 29G</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Image analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PMOD software</td>
			<td>PMOD technologies LLC</td>
			<td>PFUS (fusion tool)</td>
			<td>biomedical image quantification software (BIQS), version 3.405, https://www.pmod.com/web/?portfolio=22-image-processing-pfus</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Anesthesia-equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthetic movabe unit</td>
			<td>ASA LTD</td>
			<td>ASA 0039</td>
			<td>ASA LTD, 5 valley road, Keighley, BD21 4LZ</td>
		</tr>
		<tr>
			<td>Oxygen generator</td>
			<td>Veterinary technics Int.</td>
			<td>7F-3</td>
			<td>BDO-Medipass, Ijmuiden</td>
		</tr>
	</tbody>
</table>

pet and mri guided irradiation of a glioblastoma rat model using a micro-irradiator

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without the ability to target a specific or well-delineated tumor volume. The delivery of radiation was achieved using fixed radiation sources or linear accelerators producing megavoltage (MV) X-rays. These devices are unable to achieve sub-millimeter precision required for small animals. Furthermore, the high doses delivered to healthy surrounding tissue hamper response assessment. To increase the translation between small animal studies and humans, our goal was to mimic the treatment of human glioblastoma in a rat model. To enable a more accurate irradiation in a preclinical setting, recently, precision image-guided small animal radiation research platforms were developed. Similar to human planning systems, treatment planning on these micro-irradiators is based on computed tomography (CT). However, low soft-tissue contrast on CT makes it very challenging to localize targets in certain tissues, such as the brain. Therefore, incorporating magnetic resonance imaging (MRI), which has excellent soft-tissue contrast compared to CT, would enable a more precise delineation of the target for irradiation. In the last decade also biological imaging techniques, such as positron emission tomography (PET) gained interest for radiation therapy treatment guidance. PET enables the visualization of e.g., glucose consumption, amino-acid transport, or hypoxia, present in the tumor. Targeting those highly proliferative or radio-resistant parts of the tumor with a higher dose could give a survival benefit. This hypothesis led to the introduction of the biological tumor volume (BTV), besides the conventional gross target volume (GTV), clinical target volume (CTV), and planned target volume (PTV).
At the preclinical imaging lab of Ghent University, a micro-irradiator, a small animal PET, and a 7 T small animal MRI are available. The goal was to incorporate MRI-guided irradiation and PET-guided sub-volume boosting in a glioblastoma rat model.

To mimic the human treatment methodology for the irradiation of glioblastoma in a preclinical model, inclusion of MRI-guided radiotherapy was necessary. Using the PCTPS and the micro-irradiator interface we were able to irradiate F98 glioblastoma in rats with multiple conformal non-coplanar arcs targeting the contrast-enhanced region on T1-weighted MRI17. Rigid-body transformations in combination with a multi-modality bed were used for image registration between MR...

Watch this Scientific Journal Video about PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator at JoVE.com

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

In the past, small animal irradiation was usually performed without the ability to target a well-delineated tumor volume. The goal was to mimic the treatment of human glioblastoma in rats. Using a small animal irradiation platform, we performed MRI-guided 3D conformal irradiation with PET-based sub-volume boosting in a preclinical setting.

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without ...

The overall goal of this methodology is to perform image-guided conformal irradiation in small animals. This method can help to answer key questions in the radiology field about how to delineate specific tumor volumes for targeted radiation therapy. The main advantage of this technique is that it mimics the human treatment of cancer and allows for cells irradiation of tumors in rats.Using PETs to guide to deradiation beams, allows to take into account cancer biology as a new and promising development in the field of small animal radiotherapy. The inclusion of a biological target volume, allows the most active and radiation resistant areas of the tumors to be targeted, resulting in better outcomes of the therapy. To inoculate the brain of an anesthetized 170 gram female fisher 5344 rat with glioma cells, female fisher 5344 rat with glioma cells, first, confirm sedation by lack of response to toe pinch, remove the hair from eye level to the back of the skull, and apply ointment to the animal's eyes.Immobilize the animal in a stereotactic device. Disinfect the exposed skin with povidone iodine, and expose the skull with a two centimeter midline scalp incision. Using a diamond drill, make a one millimeter hole two millimeters posterior and two and a half millimeters lateral to the bregma in the right frontal hemisphere.Next, load the needle of a 29 gauge insulin syringe with five microliters of cell suspension, use a microsyringe pump controller to inject the cells three millimeters deep into the skull under stereotactic guidance and withdraw the needle slowly. Close the incision with bone wax. Then, suture and disinfect the skin with more povidone iodine and use a red lamp to stabilize the body temperature of the animal post surgery with monitoring until full recovery.Eight days post inoculation, connect a 30 gauge needle to a 60 centimeter long tube. Positioned intravenously within the lateral tail vein, and place the anesthetized animal into an MRI bed. Place the bed in the holder with a fixed rat brain surface coil, and position the bed in a 72 millimeter rat hold body transmitor coil.Then, assess the tumor growth with a localizor scan, followed by a T2 weighted spin echo scan. If a tumor is confirmed, start a 12 minute dynamic contract enhanced MRI acquisition, injecting a gadolinium containing contrast agent into the intraveniously placed tubing 30 seconds after beginning the scan. To plot the signal intensity over time, use the image sequence analysis tool to select a region of interest within the suspected tumor region and analyze the shape of the resulting dynamic contract enhanced curve to confirm the presence of the glioblastoma.Then, acquire a contrast enhanced T1 weighted, spin echo sequence. For multi-modality imaging of the target volume, insert a 26 gauge catheter into the tail vein and inject 37 megabecquerels of the PET radioactive tracer of interest and 200 microliters of saline into the catheter. 15 minutes before the PET acquisition, inject the MRI contrast agent through the tail vein catheter and place the anesthetized rat on a custom made multi-modality bed.Position one multi-modality marker under, above, and on the right side of the skull. Using hook and loop fasteners, secure the rat to the bed. Place the bed in the animal holder of the MRI scanner, fix the rat brain surface coil, and position the entire setup in the 72 millimeter rat hold body transmitor coil.Obtain a localizer scan followed by a contrast enhanced T1 weighted spin echo sequence as demonstrated. At the end of the T1 scan, transfer the animal to the PET instrument and obtain the appropriate 30 minute static PET scan in list mode according to the parameters for the injected PET tracer. Then, transfer the bed onto a plastic holder secured onto the four axis robotic positioning table of the micro-irradiator.And obtain a high resolution treatment planning CT scan using an aluminum filter of one millimeter and a 20 by 20 centimeter amorphous silicon flat panel detector. Select manually gray value thresholds until you achieve a good segmentation of bone, soft tissue and air. Make sure that there is no air inside the skull.For treatment planning, import the planning computed tomography, or CT, into the pre-clinical treatment planning system, or PCTPS, and manually segment the CT image into three different tissue classes. A precise fusion can be achieved by over laying the increased signal intensity of the skull on the CT scan with the black signal on the MRI scan. Load MRI scan and co-register using rigid transformations and the multi-modality markers and the skull.Load the MRI into the PCTPS. Then first fill in the transformation matrix. Switch from CT to MRI and back to check the fusion and add left, right, posterior, anterior, and inferior, superior transformations and rotations until the perfect fusion is achieved.Then, select the target or irradiation in the center of the contrast enhancing tumor on the T1 weighted MRI. If additional PET information must be included, use the biomedical image quantification software to include a CT/MRI PETCO registration. First, load the CT scan, afterwards load the PET scan.Check the orientation of the PET scan when loading. Change the color scale of the PET image, and the orientation. Apply a gaussian filter to the image, such that the tracer uptake in the tumor becomes clearly visible.Adjust the CT contrast to start the image fusion process to achieve a PET MRI image fusion, and use the contouring tool in the quantification software. After co-registration, select the target in the center of the increased PET tracer uptake in the quantification software. Use both rotations and translations and check the fusion in all the slices of the image.Select the center of the region with the highest tracer uptake and extract the coordinates. And manually enter the coordinates into the PCTPS. If the automatic PET MRI and CT image fusion tools do not generate a good fusion, contouring tools and manually transformations can be used to improve the fusion results.Select the prescribed dose, number of arcs, arc position, rotation range of the arcs, and the collimator size and adjust the settings for the appropriate MRI or PET-MRI guided radiation therapy. For the actually irradiation, select a 0.5 millimeter copper filter, set the x-ray voltage to 220 kilovolts, and the x-ray current to 13 milliamps, and position the right collimator on the gantry. Then, transfer the appropriate beam delivery parameters from the PCTPS to the micro-irradiator to execute the radiation therapy.To mimic the human treatment methodology for glioblastoma irradiation in a pre-clinical model, the iso-center for irradiation is selected in the center of the contrast enhanced tumor region on the T1 weighted MRI, as just demonstrated. In this experiment, distributions and cumulative dose volume histograms of the mean, minimal and maximal doses of the target volume, and the normal brain tissue volumes, were calculated for five different animals. For co-registration of the MRI, and CT modalities, the bio-medical image quantification software enables the use of many tools for rigid matching.By applying a simple transformation, both the MRI and PET based iso-centers can be transferred to the PCTPS for dose calculation of the radiation within each iso-center. One small certain technique can be use to irradiate tumors in rats and mice in lesson to our if performed properly. By performing this procedure, it's important to remember to monitor the animals carefully while they are under anesthesia.After watching this video, you should have a good understanding of how to apply image guided, small animal irradiation of tumor targets of interest.

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