Heavy-ion beams have the feature to administer a large radiation dose in the vicinity of the endpoint in the beam range, its irradiation system and biophysical characteristics are different from ordinary irradiation instruments like X-rays or gamma-rays. In order to get clarify characteristic effects of heavy-ion beams on the brain, we have developed an experimental system for irradiating a restricted region of the rat brain using heavy-ion beams. The left cerebral hemispheres of the adult rat brain were irradiated at dose of 50Gy charged carbon particles (290MeV/nucleon  5mmspread-out Bragg peak). After irradiation, the characteristics of the heavy-ion beams and the animal model were studied. Histological examination and measurement showed that extensive necrosis was observed between 2.5mmand7.5mmdepth from the surface of the rat head, suggesting a relatively high dose and uniform dose was delivered among designed depths and the spread-out bragg peak used here successfully and satisfactorily retained its high-dose localization in the defined region. We believe that our experimental model for irradiating a restricted region of the rat brain using heavy-ion beams is a good model for analyzing regional radiation susceptibility of the brain

Cui, Chun

Fukui, Yoshihiro

Kubota, Yoshihisa

Nojima, Kumie

Sun, Xue-Zhi

Takahashi, Sentaro

Zhang, Rui

Tokushima University Institutional Repository

INTRODUCTIONAs part of the 10-year Strategy for Cancer Controlimplemented by the Japanese government, the clinicaltrials using HIMAC (Heave IonMedical Accelerator inChiba) for cancer treatment have been performedmorethan 7 years in Research Center for Charged ParticleTherapy of National Institute of Radiological Sciences.The HIMAC is the world’s only heavy-ion acceleratorcomplex dedicated to medical use in a hospital envi-ronment. Over a thousand patients have been treatedusing heavy-ion beams. The safety and efficacy of heavyions have been demonstrated to a great extent (1-4).For instance, brain tumors treated by heavy-ion beamsbecame smaller or disappearance. However, funda-mental research related to such clinical phenotypesand their underlying mechanisms are little known.HIMAC can accelerate ion particles (carbon) and ithas the feature to administer a large radiation dose inthe vicinity of the endpoint in the beam range (Braggpeak)(5-8). Thus, its irradiation system and biophysicalcharacteristics are different from ordinary irradiationinstruments like X-rays or gamma-rays.In order to get clarify characteristic effects of heavy-ORIGINALExperimental model for irradiating a restricted region ofthe rat brain using heavy-ion beamsXue-Zhi Sun, Sentaro Takahashi, Yoshihisa Kubota, Rui Zhang＊, Chun Cui＊,Kumie Nojima＊＊ and Yoshihiro Fukui＊Environmental and Toxicological Sciences Research Group, National Institute of Radiological Sciences,Chiba, Japan; ＊Department of Anatomy and Developmental Neurobiology, The University of TokushimaSchool of Medicine, Tokushima, Japan ; and ＊＊Research Laboratory of Space Radiation, National Instituteof Radiological Sciences, Chiba, JapanAbstract : Heavy-ion beams have the feature to administera largeradiation dose in the vicinityof the endpoint in the beam range, its irradiation system and biophysical characteristicsare different from ordinary irradiation instruments like X-rays or gamma-rays. In order to getclarify characteristic effects ofheavy-ion beams on thebrain,wehavedevelopedan experimentalsystem for irradiating a restricted region of the rat brain using heavy-ion beams. The leftcerebral hemispheres of the adult rat brain were irradiated at dose of 50 Gy charged carbonparticles (290 MeV/nucleon ; 5mm spread-out Bragg peak). After irradiation, the characteristicsof the heavy-ion beams and the animal model were studied. Histological examination andmeasurement showed that extensive necrosis was observed between 2.5mm and 7.5mm depthfrom thesurfaceof therat head, suggesting a relatively high doseand uniform dosewas deliveredamong designed depths and the spread-out bragg peak used heresuccessfully and satisfactorilyretained its high-dose localization in the defined region. We believe that our experimentalmodel for irradiating a restricted region of the rat brain using heavy-ion beams is a goodmodel for analyzing regional radiation susceptibility of the brain. J. Med. Invest. 51:103-107,February, 2004Keywords : cerebral hemisphere, heavy-ion beam, irradiationReceived for publication December 8, 2003 ; accepted January 13,200４.Address correspondence and reprint requests to Dr. Xue-Zhi Sun,Environmental and Toxicological Sciences Research Group, Na-tional Institute of Radiological Sciences, Chiba, Japan and Fax :+81-43-251-4853.The Journal of Medical Investigation Vol. 51 2004１０３ion beams on the brain, we have developed an experi-mental system for irradiating a restricted region of therat brain using heavy-ion beams. The characteristicsof the heavy-ion beams and the animal model werestudied in the present work.MATERIALS AND METHODSAnimals and heavy-ion beamsTwenty-eight adultmale Sprague-Dawley rats, aged12 weeks andweighing 260-340g (Shizuoka LaboratoryAnimal Center, Hamamatsu, Japan) were used. Animalswere kept in a controlled atmosphere of 23±1℃ with55±5℃ humidity under a 12-h dark/light cycle. Theywere given free access to commercial laboratory pelletfood and tap water throughout the study. During ex-periment, animals were divided into two groups as fol-lows : twenty rats were irradiated with carbon beamsat 50 Gy, which is usual dose to be used in heavy ionbeam therapy ; eight were sham-irradiated animals.Rats were deeply anesthetized 10-15 minutes beforeirradiation with ketamine (40 mg/kg) and xylazine(10 mg/kg), immobilized in a specifically designedjig, and irradiated with 290 MeV/nucleon chargedcarbon beams in a dorsal-to ventral direction.The depth-dose distribution of the heavy ion beamwas modified tomake a spread-out bragg peak of 5mmwide with a rangemodulator, and the rat left hemispherewas irradiated, that was confirmed with a left collima-tion (Fig.1). The area of the left brain was subjectedto a single irradiation at dose of 50 Gy equivalent witha linear energy transfer of approximately 50 Ke V/µm.The position of the spread-out bragg peakwas adjustedto be at 2.5 -7.5 mm depth from the surface of the headwith a binary filter inserted before the target.All of the animal experiments were carried out withpermission and under regulation of the InstitutionalCommittee for Animal Safety andWelfare of the Na-tional Institute of Radiological Sciences, and in accor-dance with Regulations onAppropriate AnimalBreedingand Treatment,MinistryOffice of Japan. All efforts weremade tominimize the number of animals used and theirsuffering.Histological treatments and examinationsBrain samples were obtained from 4, 8, 16, 32 weeksafter exposure. Five rats treated with the heavy ionbeam and two controls were randomly selected forstudy in each experimental period. Animals in eitherthe irradiated or control groups were deeply anesthe-tized with ether, and perfused with Zamboni’s fixitivesolution (4% formaldehyde and 0.2% picric acid in0.1 M phosphate buffer, pH7.3) with a rotary pumpvia the left cardiac ventricle. Following perfusion, thebrain was removed and immersed in freshly madeZamboni’s fixitive solution for 1 week. After a grossanatomical examination, tissue samples were embed-ded in paraffin and sectioned coronally at 6 µmwitha microtome. Particular attention was given to thedissection and orientation of the tissue blocks in thisstudy. The tissue blocks through the full thicknessof the cerebral cortex were dissected as nearly as pos-sible perpendicular to the longitudinal axis of the cere-bral hemisphere. For each brain, every fourth sectionwas kept from the serial sections, and then stainedwith hematoxylin and eosin.Measurement of the radiation field and depth & behav-ioral observationThe field and depth of the heavy-ion beams werechecked by an observing outline of depilation on thesurface of the brain, and a measuring paraffin sectionof rat brain with different thickness.Behavioral changes were observed twice a weekafter irradiation. Animals were removed from theircages, placed on a table, and scored for balance usinga subjective scoring scale (9, 16).RESULTSNo obvious behavioral and histological changes be-fore 7 weeks of exposure. Loss of hairs was found inthe left brain by 7 weeks after irradiation. The shapeand size of depilation (Fig. 2) were almost same to theFig. 1. The rat left hemisphere was irradiated with HIMAC(Heave Ion Medical Accelerator in Chiba), and its irradiatedfield (above arrow) was confirmed with this left collimationwhich was made of lead.X. Z. Sun et al. A model of brain irradiated by heavy-ion beams１０４Table 1. Histological changes in rat brain after 50 Gy irradiation with carbon ion beamsHistological changesTime after irradiation4 weeks 8 weeks 16 weeks 32 weeksNecrosis － ± ＋ ＋Vascular dilatation － ± ＋ ＋Tissue swelling ± ± ＋ ＋Histological examination : －, no visible abnormal ; ±, sight ; ＋, obviousNumbers of animals examined in each group, n=5Fig. 2. Loss of hairs was observed in the rat left hemisphere by7 weeks after irradiation of carbon ion beams. The shape and sizeof depilation were almost same to the left collimation (Fig.1).Fig. 3. High power photomicrograph of an example of histologi-cal changes since8weeks after irradiation of carbon ion beams.The distinctive histological changes were necrosis, vascular dila-tation and tissue swelling. n : necrosis ; Arrowheads : vasculardilatation. Asterisks : tissue swelling.Fig. 4. Photomicrographs of examples of histological changesat 16 weeks after irradiation of carbon ion beams. b : Extensivenecrosis was observed between 2.5 mm and 7.5 mm depth fromthe surface of the head (between arrows), suggesting a relativelyhigh dose and uniform dose was delivered among designed depths.a : Control.The Journal of Medical Investigation Vol. 51 February 2004 １０５left collimation (Fig. 1). The skin color in the irradiatedarea was no difference as compared with the othersin the no irradiated areas. The pathological changes,such as hemorrhage, dried scab and tissue swellingwere not appearance in the cutaneous surface of theirradiated area.At 8 weeks after exposure, three rats of fourteen ex-posed to the heavy ion beam exhibited slight behav-ioral changes, either in an abnormal walking patternor rotation when suspended by their tail.With the proc-ess of time, these behavioral changes became moremarked. The animals showed total loss of their balanceboth in an abnormal walking pattern and rotation from16 weeks onwards after irradiation.The histological examination showed obvious his-tological changes since 8weeks after irradiation (Table1). The distinctive histological changeswere necrosis,vascular dilatation and tissue swelling (Fig. 3). At16and 32 weeks after irradiation, necrotic rarefactionbecame dominant at the center of the irradiated regionand enlarged blood vessels were present in the sur-rounding area (Fig. 4).The field and depth of the heavy-ion beams werechecked by a measuring paraffin section of rat brainwith different thickness. A relatively high dose wasdelivered between 2.5 mm and 7.5 mm depth fromthe surface of the rat head, which was judged by celldeath through the serial sections. Extensive necrosiswas observed between 2.5 mm and 7.5 mm depth, butsuch necrosis could not found near the surface of therat head or more than 7.5 mm depth (Fig. 4).DISCUSSIONThe shape and size of depilation found in the leftcerebral hemisphere of the rat by 7 weeks after ex-posure were almost same to the left collimation, thatwas outline of the irradiation field on the surface ofthe brain, indicating HIMAC irradiation system andthe left collimation can be satisfactory to irradiate adefined region of the brain. Histological examinationand measurement showed the distinctive features ofthe depth-dose distribution of heavy-ion beams. Exten-sive necrosis was observed betweent 2.5 mm and 7.5mm depth from the surface of the rat head, suggest-ing a relatively high dose and uniform dose was deliv-ered among designed depths and the spread-out braggpeak used here successfully and satisfactorily retainedits high-dose localization in the defined region. Thesebiophysical characteristics of HIMAC irradiation sys-tem demonstrated it is good system for cancer therapy(10, 11).Radiation therapy is a clinic way to treat brain tu-mors. However, damages of the normal brain inducedby radiation occur several months after a treatment(13-15), so radiation damage is a limiting factor to useradiation therapy. In the present study, behavioralchanges in the irradiated rats appeared both in an ab-normal walking pattern and rotation from16weeksonwards after irradiation, indicating existence of ra-diation damage after heavy ion beam exposure. Thesefacts request us to study the mechanisms of radiationdamage to the brain tissue and the radiation suscep-tibility of each region of the brain. Therefore, it is anecessary to develop an animal model for irradiatinga restricted region of the brain using heavy-ion beams.The experimentalmodel in this studycanbe expectedas a novel model, it hasmany distinctive features whichare different from others. Because ordinary irradiationinstruments using X-rays or gamma-rays can not con-centrate the radiation dose to a small and restrictedarea of the brain. The whole brain or a relatively largevolume of the brain were irradiated and examined, soeffects of radiationmay affect with amutual interactionwith other brain regions. Heavy ion beam provides apossible to expose a small and restricted volume ofanimal brain to a high dose of radiation. We believethat our experimental model for irradiating a restrictedregion of the rat brain using heavy-ion beams is a goodmodel for analyzing regional radiation susceptibilityof the brain.REFERENCES1. Tsujii H:Clinical evaluation and perspective ofcharged particle therapy. Nippon Rinsho 55 (inJapanese) : 1588-1595, 19972. Miyamoto T, Aoyagi H, Tujii H, Yamaguchi Y :Heavy ion radiotherapy for lung cancer. NipponGeka Gakkai Zasshi 98 (in Japanese) : 60-67, 19973. Tsujii H, Ban S : tailor-made, destination ofradiation therapy. Jitsugyo Press, Tokyo, 2003(Article in Japanese)4. Tsujii H : Report of clinical trials using HIMAC(Heave Ion Medical Accelerator in Chiba) forcancer treatment during 1994-2001. News of Na-tional Institute of Radiological Sciences 66 (inJapanese) : 1-3, 20025. Fukumura A, Hiraoka T, Omata K, TakeshitaM,Kawachi K, Kanai T, Matsufuji N, Tomura H,Futami Y, Kaizuka Y, Hartmann GH : Carbonbeam dosimetry intercomparison atHIMAC. PhysX. Z. Sun et al. A model of brain irradiated by heavy-ion beams１０６Med Biol 43 : 3459-3463, 19986. Kanai T, Endo M, Minohara S, Miyahara N,Koyama-ito H, Tomura H, Matsufuji N, FutamiY, Fukumura A, Hiraoka T, Furusawa Y, AndoK, Suzuki M, Soga F, Kawachi K : Biophysi-cal characteristics of HIMAC clinical irradiationsystem for heavy-ion radiation therapy. Int JRadiat Oncol Biol Phys 44 : 201-210, 19997. Skarsgard LD : Radiobiology with heavy chargedparticles : a historical review. PhysMed 14 : 1-19,19988. Takahashi S, Sun XZ, Kubota Y, Takai N,Nojima K : Histological and elemental changesin the rat brain after local irradiationwith carbonion beams. J Radiat Res 43 : 143-152, 20029. Mickley GA, Ferguson JL,MulvihillMA, NemethTJ : Progressive behavioral changes during thematuration of rats with early radiation-inducedhypoplasia of fascia dentata granule cells. Neu-rotoxicol Teratol 11 : 385-93, 198910. Hirao Y, Ogawa H, Yamada S, Sato Y, Yamada T,Sato K, Itano A, KanazawaM, Noda K, KawachiK, AndoM,Kanai T, KohonT, SodouM,MinoharaS, Kitagawa A, Soga F, Takada E, Watanaba S,Endo K, Kumada M, Matsumoto S : Heavy ionsynchrotron for medical use-HIMAC project atNIRS-JAPAN. Nucl Phys A 538 : 541 C-550C,199211. Kanai T, Furusawa Y, Fukutsu K, Itsukaichi H,Eguchi-Kasai K, Ohara H : Irradiation of mixedbeam and design of spread-out Bragg peak forheavy-ion radiotherapy. Radiat Res 147 : 78-85,199712. Chiang CS, McBride WH, Withers HR : Myelin-associated changes in mouse brain followingirradiation. Radiother Oncol 27 : 229-236, 199313. Sun XZ, Takahashi S, Fukui Y, Hisano S, KubodaY, SatoH, InouyeM:Differentpatternsof abnormalneuronal migration in the cerebral cortex of miceprenatally exposed to irradiation. Brain Res DevBrain Res 114 : 99-108, 199914. Sun XZ, Takahashi S, Fukui Y, Hisano S, KubotaY, Sato H, Inouye M : Neurogenesis of heter-otopic gray matter in the brain of the micro-cephalic mouse. J Neurosci Res 66 : 1083-1093,200115. Kamiryo T, Kassell NF, Thai QA, LopesMB, LeeKS, Steiner L : Histological changes in the normalrat brain after gamma irradiation. Acta Neurochir138 : 451-459, 199616. Mickley GA, Ferguson JL, Nemeth TJ,MulvihillMA, Alderks CE : Spontaneous perseverativeturning in rats with radiation-induced hippocam-pal damage. Behav Neurosci 103 :722-730, 1989The Journal of Medical Investigation Vol. 51 February 2004 １０７

Experimental model for irradiating a restricted region of the rat brain using heavy-ion beams

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Experimental model for irradiating a restricted region of the rat brain using heavy-ion beams

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