External irradiation models for intracranial 9L glioma studies
© Vinchon-Petit et al; licensee BioMed Central Ltd. 2010
Received: 2 April 2010
Accepted: 8 November 2010
Published: 8 November 2010
Radiotherapy has been shown to be an effective for the treatment human glioma and consists of 30 fractions of 2 Gy each for 6-7 weeks in the tumor volume with margins. However. in preclinical studies, many different radiation schedules are used. The main purpose of this work was to review the relevant literature and to propose an external whole-brain irradiation (WBI) protocol for a rat 9L glioma model.
Materials and methods
9L cells were implanted in the striatum of twenty 344-Fisher rats to induce a brain tumor. On day 8, animals were randomized in two groups: an untreated group and an irradiated group with three fractions of 6 Gy at day 8, 11 and 14. Survival and toxicity were assessed.
Irradiated rats had significantly a longer survival (p = 0.01). No deaths occurred due to the treatment. Toxicities of reduced weight and alopecia were increased during the radiation period but no serious morbidity or mortality was observed. Moreover, abnormalities disappeared the week following the end of the therapeutic schedule.
Delivering 18 Gy in 3 fractions of 6 Gy every 3 days, with mild anaesthesia, is safe, easy to reproduce and allows for standardisation in preclinical studies of different treatment regimens glioma rat model.
Studies using radiation therapy rat model in combination with anticancer therapeutic agents
Tumor Cell line
Number of fractions
Roullin VG (6)
Complete response : 8%
Graf MR (7)
35 days (median)
Kimler BF (8)
Kimler BF (9)
Kimler BF (10)
38.5 days (mean)
Kimler BF (11)
Lamproglou I (12)
Olson JJ (13)
29.7 days (mean)
All experiments have been conducted under good experimental practices. All animal handling was carried out according to the European Community regulations and French Ministry of Agriculture regulations.
20 females Fischer-344 rats were used for this study (Charles River, Cleon, France). Rats were ten weeks-old, and weighed 150 to 200 grams. They were housed in groups of 4 in cages according to the standards of the directives of the European Union. Animal handling was conducted by the animal facility of the Faculty of Medicine of Angers, approved according to French law.
Rat 9L-glioma cells (European Collection of Concealment Culture, n° 94110705, Salisbury, U.K.) were cultured in "DMEM" medium ("Dulbecco's Modified Eagle's Medium", Biowhittaker, Verviers, Belgium) with 10% foetal calf serum (FBS, Biowhittaker) and a mixture of antibiotics: penicillin (100 UI/ml), streptomycin (0.1 mg/ml) and amphothericin B (25 μg/ml) (ABS, Sigma, Saint Quentin Fallavier, France). Cells were maintained in a balanced wet atmosphere (37°C and CO2 5%).
Animals were anesthetized with an intraperitoneal injection of 0.75-1.5 ml/kg of a solution containing 2/3 ketamine (100 mg/ml) (Clorketam®, Vétoquinol, Lure, France) and 1/3 xylazine (20 mg/ml) (Rompun®, Bayer, Puteaux, France). Rats were placed in a small-animal stereotaxic frame (Kopf Instruments, Phymep, France). After shaving and disinfection of the skin, a sagittal incision of 2 cm was made to expose the skull, followed by a burr hole 0.5 mm anterior and 3 mm lateral from the bregma using a small drill.
Following trypsinisation (trypsin/EDTA (Sigma)) and resuspension in "EMEM" ("Eagle's Minimum Essential Medium", Biowhittaker), 10 μl of 103 9L-cells in suspension were implanted 5 mm deep in the right striatum (according to the Paxinos atlas) using a 10 μl -26G Hamilton syringe (Harvard Apparatus, Ulis, France). After waiting 5 minutes, the needle was removed and the wound was sutured with absorbable surgical thread.
Rats bearing 9L tumor were randomized to either the "untreated" group (group A) or the group irradiated by a whole-brain irradiation (WBI) to a total dose of 18 Gy (group B). The radiotherapy started on day 8 after the tumor cell implantation when the tumor size was 10-15 μl .
Rats staging (data not published)
+ but not spontaneous
Survival was calculated from the day of the tumor implantation and presented as median and mean ± SE (Standard Error). Increase of life span (ILS) was calculated as follows: (Mean Survival Max - Mean Survival Min)/Mean Survival Min × 100. A Student t-test was performed to compare mean survival in the two groups, using SPSS® software and tests were considered as significant with p values < 0.05. Any rat surviving longer than 120 days was defined as a 'long survivor'. The Kaplan-Meier method was used to plot animal survival. Animals that died during anesthesia were not included in the survival analysis.
Efficacy of the brain irradiation
Descriptive and statistical data from the survival study depending on groups of treatment
Median of survival (days)
Mean time of survival (days) ± SE
Mean ILS (%)
Long term survivors
Maximal time of survival (days)
Group A « untreated » (n = 8)
28.1 ± 1.3
Group B « WBI » (n = 10)
59.9 ± 8.2
Even though single-fraction irradiation was reported to be well tolerated in the literature, we decided to use a fractionated radiotherapy protocol to irradiate rats, as this is closer to clinical practice and more adapted for a preclinical study, especially with daily concomitant chemotherapy as defined by Stupp for human gliomas . In the literature, from 5 to 20 fractions have been delivered in the preclinical studies we reviewed (Table 1) [[6, 8, 9] and ]. One potential limitation of fractionated radiotherapy for small animals is the reproducibility of positioning. In these small animal models, rats have to be anesthetized, especially if one hemi-brain irradiation is required. However, most drugs used for anaesthesia have effects on blood brain pressure, which is already high when a brain tumor grows, or are known to be radioprotective for the normal brain parenchyma. Ketamine, which is commonly used for anaesthesia of rodents, induces a general increase in cerebral blood flow at anaesthetic concentrations . Some authors reported that pentobarbital protects against radiation-induced damage to normal rat brain. Even though there is no conclusive evidence for either radioprotection or significant improvement of radiotherapeutic efficacy, in 9L rat brain tumor model pentobarbital could potentially induce the selective protection of normal brain [11, 13]. In our model, anaesthesia with isoflurane is easy to use every three days, is well tolerated by rats with a complete and immediate recovery after irradiation and does not interfere with normal or brain tumor cells.
The optimal dose per fraction to treat a rat brain glioma is not well defined. Our protocol was selected based on the linear-quadratic formula with α/β of 10 for the tumor and α/β of 3 for the normal tissue. The effective biological dose for the normal tissue is 32 Gy and 27 Gy for the tumor. These doses correspond to the dose received in clinical practice for a whole brain irradiation. 9L cells are classified as a radioresistant cell line especially compared to other rodent glioma cell lines . Bencokova described a surviving fraction at 2 Gy (SF2) of 71.9% for 9L cells against 53.0 and 41.4% for C6 and F98 cell lines respectively . According to this, the dose to deliver by fraction must be higher than 2 Gy. The dose per fraction in literature ranges from 2 to 40 Gy (Table 1). For Kimler, the survival improvement was limited by the development of normal tissue toxicity at high doses . Kim observed that 35 Gy produced severe optic neuropathy . In his study, he tested a single high dose of radiation (ranging from 20 to 45 Gy) with radiosurgery in a limited volume. Previously, we investigated a radiation therapy schema in 3 fractionated doses of 6 Gy a week in vitro on 9L cell lines without and with concomitant chemotherapy . The results showed that cell death was most important as the number of fractions increased from 1 to 3 and the increase was higher for the schemas associated with chemotherapy. For all the conditions tested, the greatest cell death was obtained after the first fraction (60-75% cell death), and was slightly reduced after the second and the third fraction. On the other hand, the most important observation was the synergistic effect between chemotherapy (CT) and RT which was most evident after the third fraction, as cell death increased from 5.3% to 38.2% for the cells treated with RT alone versus CT + RT, respectively. After the third fraction, the cell percentage still alive was mainly due to the radioresistance mechanism described above. Taking these findings into consideration, with the aim of finding a treatment protocol that is efficacious but not toxic, we chose to deliver 3 fractions of 6 Gy in our model of rat glioma. With this schedule we noted a mild and transitory toxicity which was quickly reversible after treatment.
Two rats in the WBI group lived more than 120 days. They were sacrificed and their brain was removed; there was no sign of tumor. It is not possible to determine whether there was a technical problem during the tumor cells implantation or if the animals achieved a complete response after irradiation.
There is a paucity of experimental data in literature on rat radiobiology. Different energy sources are used. Some groups work with a dedicated irradiator for small animals in their laboratory. This type of irradiator uses 137Cesium or 60Cobalt source and delivers gamma-rays [[9, 19, 20] and ]. As Lamproglou, even though his work was on normal brain , we decided to treat our rats with linear accelerator used in clinical practice. Animal irradiation may be difficult to manage because of the limited availability of accelerators but the main advantage is to deliver the same energy type as in clinical practice. There are other advantages of using a nonradioactive x-ray-producing irradiator such as avoiding the increasing number of radioprotection controls as well as the potential source hazard, disposal and replacement; nonetheless the expected efficacy is the same whatever the radiation source chosen.
This work does not answer the crucial question of optimal therapeutic regimen as it was conducted before our studies into the efficacy of local chemotherapy concomitant to radiation therapy in 9L glioma . Another study confirms the reproducibility of the model as we obtained the same improvement in survival in the radiation group compared to the untreated group . Therefore, this radiation therapy protocol has the potential to induce strong tumor debulking and facilitate concomitant chemotherapy treatment.
Many models of radiation therapy for rat glioma are available, with different schedules. We describe a reproducible paradigm of fractionated radiotherapy for rat bearing a brain tumor, which reflects clinical practice, with a good compromise between feasibility and adaptation to chemotherapy radiosensitization studies.
The authors would like to thank Pierre Legras and Jerome Roux (Service Commun d'Animalerie Hospitalo-Universitaire, Angers, France) for skillful technical support with animals and the Radiotherapy Department of Paul Papin Center for technical help. Special thanks to Rachel Holden for her precious help. This work was supported by "La Fondation pour la Recherche Médicale".
- Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO, European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups; National Cancer Institute of Canada Clinical Trials Group: Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005, 352 (10): 987-96. 10.1056/NEJMoa043330.View ArticleGoogle Scholar
- Kristiansen K, Hagen S, Kollevold T, et al: Combined modality therapy of operated astrocytomas grade III and IV. Confirmation of the value of postoperative irradiation and lack of potentiation of bleomycin on survival time: a prospective multicenter trial of the Scandinavian Glioblastoma Study Group. Cancer. 1981, 47 (4): 649-52. 10.1002/1097-0142(19810215)47:4<649::AID-CNCR2820470405>3.0.CO;2-W.View ArticleGoogle Scholar
- Laperriere N, Zuraw L, Cairncross G: Cancer Care Ontario Practice Guidelines Initiative Neuro-Oncology Disease Site Group: Radiotherapy for newly diagnosed malignant glioma in adults: a systematic review. Radiother Oncol. 2002, 64 (3): 259-73. 10.1016/S0167-8140(02)00078-6.View ArticleGoogle Scholar
- Cairncross G, Berkey B, Shaw E, et al: Phase III trial of chemotherapy plus radiotherapy compared with radiotherapy alone for pure and mixed anaplastic oligodendroglioma: Intergroup Radiation Therapy Oncology Group Trial 9402. J Clin Oncol. 2006, 24 (18): 2707-14. 10.1200/JCO.2005.04.3414.View ArticleGoogle Scholar
- Kantor G, Laprie A, Huchet A, Loiseau H, Dejean C, Mazeron JJ: Radiation therapy for glial tumors: Technical aspects and clinical indications. Cancer Radiother. 2008, 12 (6-7): 687-94.View ArticleGoogle Scholar
- Roullin VG, Mege M, Lemaire L, Cueyssac JP, Venier-Julienne MC, Menei P, Gamelin E, Benoit JP: Influence of 5-fluorouracil-loaded microsphere formulation on efficient rat glioma radiosensitization. Pharm Res. 2004, 21 (9): 1558-63. 10.1023/B:PHAM.0000041448.22771.48.View ArticleGoogle Scholar
- Graf MR, Prins RM, Hawkins WT, Merchant RE: Irradiated tumor cell vaccine for treatment of an established glioma. I. Successful treatment with combined radiotherapy and cellular vaccination. Cancer Immunol Immunother. 2002, 51 (4): 179-89. 10.1007/s00262-002-0269-3.View ArticleGoogle Scholar
- Kimler BF, Martin DF, Evans RG, Morantz RA, Vats TS: Effect of spirogermanium and radiation therapy on the 9L rat brain tumor model. NCI Monogr. 1988, 115-8. 6Google Scholar
- Kimler BF, Martin DF, Evans RG, Morantz RA, Vats TS: Combination of radiation therapy and intracranial bleomycin in the 9L rat brain tumor model. Int J Radiat Oncol Biol Phys. 1990, 18 (5): 1115-21.View ArticleGoogle Scholar
- Kimler BF, Liu C, Evans RG, Morantz RA: Combination of aziridinylbenzoquinone and cis-platinum with radiation therapy in the 9L rat brain tumor model. Int J Radiat Oncol Biol Phys. 1993, 26 (3): 445-50.View ArticleGoogle Scholar
- Kimler BF, Liu C, Evans RG, Morantz RA: Effect of pentobarbital on normal brain protection and on the response of 9L rat brain tumor to radiation therapy. J Neurosurg. 1993, 79 (4): 577-83. 10.3171/jns.1993.79.4.0577.View ArticleGoogle Scholar
- Lamproglou I, Chen QM, Boisserie G, Mazeron JJ, Poisson M, Baillet F, Le Poncin M, Delattre JY: Radiation-induced cognitive dysfunction: an experimental model in the old rat. Int J Radiat Oncol Biol Phys. 1995, 31 (1): 65-70.View ArticleGoogle Scholar
- Olson JJ, Friedman R, Orr K, et al: Enhancement of the efficacy of x-irradiation by pentobarbital in a rodent brain-tumor model. J Neurosurg. 1990, 72 (5): 745-8. 10.3171/jns.1990.72.5.0745.View ArticleGoogle Scholar
- Vonarbourg A, Sapin A, Lemaire L, et al: Characterization and detection of experimental rat gliomas using magnetic resonance imaging. Magma. 2004, 17 (3-6): 133-9. 10.1007/s10334-004-0049-5.View ArticleGoogle Scholar
- Laitio RM, Kaisti KK, Låangsjö JW, Aalto S, Salmi E, Maksimow A, Aantaa R, Oikonen V, Sipilä H, Parkkola R, Scheinin H: Effects of xenon anesthesia on cerebral blood flow in humans: a positron emission tomography study. Anesthesiology. 2007, 106 (6): 1128-33. 10.1097/01.anes.0000267596.57497.92.View ArticleGoogle Scholar
- Bencokova Z, Pauron L, Devic C, et al: Molecular and cellular response of the most extensively used rodent glioma models to radiation and/or cisplatin. J Neurooncol. 2008, 86: 13-21. 10.1007/s11060-007-9433-0.View ArticleGoogle Scholar
- Kim JH, Khil MS, Kolozsvary A, et al: Fractionated radiosurgery for 9L gliosarcoma in the rat brain. Int J Radiat Oncol Biol Phys. 1999, 45 (4): 1035-40.View ArticleGoogle Scholar
- Allard E, Passirani C, Jarnet D, Petit S, Vessières A, Jaouen G, Benoit J-P: Local delivery of ferrociphenol lipid nanocapsules followed by external radiotherapy as a synergistic treatment against intracranial 9L glioma xenograft. Pharm Res. 2010, 27 (1): 56-64. 10.1007/s11095-009-0006-0.View ArticleGoogle Scholar
- Kinsella TJ, Kinsella MT, Hong S, et al: Toxicology and pharmacokinetic study of orally administered 5-iodo-2-pyrimidinone-2'deoxyribose (IPdR) × 28 days in Fischer-344 rats: impact on the initial clinical phase I trial design of IPdR-mediated radiosensitization. Cancer Chemother Pharmacol. 2008, 61 (2): 323-34. 10.1007/s00280-007-0518-4.View ArticleGoogle Scholar
- Brust D, Feden J, Farnsworth J, et al: Radiosensitization of rat glioma with bromodeoxycytidine and adenovirus expressing herpes simplex virus-thymidine kinase delivered by slow, rate-controlled positive pressure infusion. Cancer Gene Ther. 2000, 7 (5): 778-88. 10.1038/sj.cgt.7700168.View ArticleGoogle Scholar
- Yacoub A, Hamed H, Emdad L, et al: MDA-7/IL-24 plus radiation enhance survival in animals with intracranial primary human GBM tumors. Cancer Biol Ther. 2008, 7 (6): 917-33. 10.4161/cbt.7.6.5928.View ArticleGoogle Scholar
- Vinchon-Petit S, Jarnet D, Paillard A, Benoit JP, Garcion E, Menei P: In vivo evaluation of intracellular drug-nanocarriers infused into intracranial tumours by convection-enhanced delivery: distribution and radiosensitisation efficacy. J Neurooncol. 2010, 97 (2): 195-205. 10.1007/s11060-009-0012-4. Epub 2009 Sep 22View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.