Science, Irving K. Barber Faculty of (Okanagan)
Computer Science, Mathematics, Physics and Statistics, Department of (Okanagan)
DSpace
UBCO
Fuentes, Alejandra Maria
Master of Science - MSc
University of British Columbia
Purpose: Accidental exposure to high-dose ionizing radiation may lead to significant injuries to multiple organs. Herein, this thesis established a mouse model of high-dose partial-body irradiation with minimal bone marrow shielding to model the progression, severity, and clinical features of lung injury consequent of an accidental scenario and determine whether radiation-induced hematopoietic system depletion affects the pulmonary response. Methods: Female C3H/HeJ mice were irradiated in a single fraction, using a 6 MV Flattening-Filter-Free beam on a Varian TrueBeam LINAC. The following experimental groups were assessed: (1) 8 Gy partial-body irradiation (PBI), sparing the tibiae, ankles, and feet plus an 8 Gy simultaneous-infield-boost (SIB) to the lung to elicit lung damage, (2) 16 Gy Whole-thorax lung irradiation (WTLI), (3) 8 Gy PBI, and (4) untreated controls. Mice were monitored weekly and euthanized upon the presentation of respiratory distress or at the 16-week experimental endpoint. At sacrifice, blood and lung tissue were collected for Complete Blood Count and histopathology. Additionally, subsets of mice were sacrificed one-week post-treatment to assess acute hematopoietic cell depletion. Results: Treatment with 8 Gy partial-body irradiation depleted hematopoietic cell counts, specifically leukocytes and platelets, measured at one week post-irradiation, significantly more than 16 Gy WTLI. The survival time of mice treated with 16 Gy WTLI was not significantly different from that of mice given 8 Gy PBI plus 8 Gy lung SIB (p=0.17). Lung histology revealed that mice of both 16 Gy WTLI and 8 Gy PBI plus 8 Gy lung SIB groups developed a diffuse pneumonitis response; the severity score was not significantly different between these treatment groups (p=0.96). Finally, partial-body irradiation significantly reduced blood monocyte counts of mice at respiratory distress compared to levels in local whole-thorax irradiated mice but did not influence neutrophil counts, which were increased at distress for both treatment groups relative to measures in control mice. Conclusion: Radiation-induced acute hematopoietic injury did not affect the onset or severity of radiation pneumonitis in a mouse model of partial-body irradiation with minimal bone marrow shielding.
Radiation pneumonitis assessed in amouse model of partial-bodyirradiation with minimal bone marrowsparingbyAlejandra Maria FuentesB.Sc., Acadia University, 2018A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinThe College of Graduate Studies(Medical Physics)THE UNIVERSITY OF BRITISH COLUMBIA(Okanagan)September 2020c© Alejandra Maria Fuentes, 2020The following individuals certify that they have read, and recommendto the College of Graduate Studies for acceptance, a thesis/dissertation en-titled:Radiation pneumonitis assessed in a mouse model of partial-body irradiationwith minimal bone marrow sparingsubmitted by Alejandra Maria Fuentes in partial fulfilment of the re-quirements of the degree of Master of ScienceDr. Christina Haston, Irving K. Barber Faculty of ScienceSupervisorDr. Michelle Hilts, Irving K. Barber Faculty of ScienceSupervisory Committee MemberDr. Derek Hyde, Irving K. Barber Faculty of ScienceSupervisory Committee MemberDr. Neil Eves, Faculty of Health and Social DevelopmentUniversity ExamineriiAbstractPurpose: Accidental exposure to high-dose ionizing radiation may leadto significant injuries to multiple organs. Herein, this thesis established amouse model of high-dose partial-body irradiation with minimal bone mar-row shielding to model the progression, severity, and clinical features of lunginjury consequent of an accidental scenario and determine whether radiation-induced hematopoietic system depletion affects the pulmonary response.Methods: Female C3H/HeJ mice were irradiated in a single fraction, us-ing a 6 MV Flattening-Filter-Free beam on a Varian TrueBeam LINAC. Thefollowing experimental groups were assessed: (1) 8 Gy partial-body irradia-tion (PBI), sparing the tibiae, ankles, and feet plus an 8 Gy simultaneous-infield-boost (SIB) to the lung to elicit lung damage, (2) 16 Gy Whole-thoraxlung irradiation (WTLI), (3) 8 Gy PBI, and (4) untreated controls. Micewere monitored weekly and euthanized upon the presentation of respira-tory distress or at the 16-week experimental endpoint. At sacrifice, bloodand lung tissue were collected for Complete Blood Count and histopathol-ogy. Additionally, subsets of mice were sacrificed one-week post-treatmentto assess acute hematopoietic cell depletion.Results: Treatment with 8 Gy partial-body irradiation depleted hematopoi-etic cell counts, specifically leukocytes and platelets, measured at one weekpost-irradiation, significantly more than 16 Gy WTLI. The survival time ofmice treated with 16 Gy WTLI was not significantly different from that ofmice given 8 Gy PBI plus 8 Gy lung SIB (p=0.17). Lung histology revealedthat mice of both 16 Gy WTLI and 8 Gy PBI plus 8 Gy lung SIB groupsdeveloped a diffuse pneumonitis response; the severity score was not signif-icantly different between these treatment groups (p=0.96). Finally, partial-body irradiation significantly reduced blood monocyte counts of mice atrespiratory distress compared to levels in local whole-thorax irradiated micebut did not influence neutrophil counts, which were increased at distress forboth treatment groups relative to measures in control mice.Conclusion: Radiation-induced acute hematopoietic injury did not affectthe onset or severity of radiation pneumonitis in a mouse model of partial-body irradiation with minimal bone marrow shielding.iiiLay SummaryAccidental exposure to high-dose ionizing radiation may lead to sig-nificant injuries to multiple organs. Characterizing these effects requiresappropriate animal models predictive of the human response. Herein, amouse model of high-dose partial-body irradiation with minimal bone mar-row shielding was established to characterize radiation pneumonitis in thecontext of accidental exposure and to determine whether radiation-induceddamage to the hematopoietic system affects the lung radiation response com-pared with local whole-thorax irradiation models. This thesis found thatinjury to the hematopoietic system did not affect the onset or severity of ra-diation pneumonitis in the C3H/HeJ mouse strain. Finally, increased bloodneutrophil counts were significantly associated with respiratory distress.ivPrefaceThe experiments presented in this thesis were designed by Miss AlejandraFuentes (the author) as well as Dr. Christina Haston and Dr. Derek Hyde.The concept formation, experimental timeline, and treatment groupspresented in Chapter 2 were established by Dr. Haston and the author.The radiation treatment plans described in Chapter 2 were developed bythe author and Dr. Hyde at BC Cancer, Kelowna.The author was the lead investigator for Chapters 3 and 4, where she wasresponsible for mouse irradiation, dosimetry, health monitoring, euthanasia,and tissue collection. Mouse irradiation was performed at the BC CancerCentre with the collaboration and supervision of Dr. Hyde, who operatedthe Linear Accelerator, and Ian Johnson, who assisted with anesthetizingthe animals for treatment. Blood draws in the first part of the experiment(Section 4.2) were done with the collaboration of Ian Johnson.All data analysis, figure generation, and manuscript composition weredone by the author with the guidance of Dr. Haston, the thesis supervisor.All animal experiments were approved by the UBC Animal Care Com-mittee (certificate # A18-0140).vTable of ContentsAbstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iiiLay Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ivPreface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vTable of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . viList of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viiiList of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ixList of Acronyms . . . . . . . . . . . . . . . . . . . . . . . . . . . xivAcknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . .xviiDedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xviiiChapter 1: Introduction . . . . . . . . . . . . . . . . . . . . . . . 11.1 Acute Radiation Syndrome . . . . . . . . . . . . . . . . . . . 21.1.1 Hematopoietic System . . . . . . . . . . . . . . . . . . 31.1.2 Gastrointestinal System . . . . . . . . . . . . . . . . . 41.1.3 Neurovascular System . . . . . . . . . . . . . . . . . . 41.2 Medical Management and Delayed Effects of Acute RadiationExposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41.3 Radiation-Induced Lung Injury . . . . . . . . . . . . . . . . . 51.3.1 The alveolar structure . . . . . . . . . . . . . . . . . . 61.3.2 Mechanisms of radiation-induced lung damage . . . . 61.4 The role of immune cells in radiation-induced lung injury . . 10Chapter 2: Experimental Design . . . . . . . . . . . . . . . . . 142.1 The mouse as a model of radiation-induced lung injury . . . . 14viTABLE OF CONTENTS2.2 The minimum sparing model . . . . . . . . . . . . . . . . . . 152.3 Model design . . . . . . . . . . . . . . . . . . . . . . . . . . . 172.4 Experiment Timeline . . . . . . . . . . . . . . . . . . . . . . . 172.4.1 Part I: Hematopoietic system depletion assessed atone week post-irradiation . . . . . . . . . . . . . . . . 172.4.2 Part II: Partial-body irradiation survival study . . . . 192.4.3 Part III: Experiment completion . . . . . . . . . . . . 212.5 Radiation treatment plan . . . . . . . . . . . . . . . . . . . . 212.5.1 CT simulation . . . . . . . . . . . . . . . . . . . . . . 222.5.2 Treatment planning . . . . . . . . . . . . . . . . . . . 23Chapter 3: Materials and Methods . . . . . . . . . . . . . . . . 293.1 Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293.2 Irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293.3 Dosimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303.4 Health Monitoring . . . . . . . . . . . . . . . . . . . . . . . . 323.5 Peripheral Blood Counts . . . . . . . . . . . . . . . . . . . . . 323.6 Lung Histology . . . . . . . . . . . . . . . . . . . . . . . . . . 323.7 Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . 33Chapter 4: Results . . . . . . . . . . . . . . . . . . . . . . . . . . 344.1 Relative Body Weight and Survival at one-week post treatment 344.2 Hematological measures at one-week post-treatment . . . . . 364.3 Survival and relative body weights for 8 Gy partial-body ir-radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404.4 Experimental model: Survival time and Relative Body weights 404.5 Hematological measures at respiratory distress . . . . . . . . 434.6 Lung Histopathology . . . . . . . . . . . . . . . . . . . . . . . 47Chapter 5: Conclusion . . . . . . . . . . . . . . . . . . . . . . . . 50Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61viiList of TablesTable 2.1 Experiment part I: Hematopoietic depletion at oneweek post-irradiation . . . . . . . . . . . . . . . . . . . 20Table 2.2 Experimental phase III . . . . . . . . . . . . . . . . . . 21Table 3.1 Dose to tibiae measurements by TLDs. . . . . . . . . . 31viiiList of FiguresFigure 1.1 Structure of the pulmonary alveolus. Adapted fromLierova and others[38]. . . . . . . . . . . . . . . . . . 7Figure 1.2 Radiation interactions with biological tissue and en-ergy deposition process. Adapted from Johns andCunninghams[29]. . . . . . . . . . . . . . . . . . . . . 8Figure 1.3 Sequence of events leading to radiation induced lunginjury. Adapted from Giuranno and others[18]. . . . . 9Figure 2.1 Experimental design. To determine the effect of radiation-induced hematopoietic cell depletion on radiation lungdisease, mice received a partial-body exposure withminimal bone marrow shielding followed by a boostfield to the whole thorax to accomplish a total lungdose of 16 Gy. This dose is above the threshold forlung injury in mice. . . . . . . . . . . . . . . . . . . . 18Figure 2.2 CT simulator room at BC Cancer Agency. . . . . . . 23Figure 2.3 EclipseTMTPS interface for external beam planning.Panels (a), (c), and (d) display the mouse’s recon-structed CT images in the axial, coronal and sagittalplanes, respectively; the prescription point was fixedat the thorax’s midcoronal level, coincident with themachine’s isocentre at 100 cm SAD. Panel (b) showsthe 3D field configuration. Treatment technique con-sists of two static equally weighted photon fields di-rected along the prescription point’s axis from op-posite sides (anterior, gantry angle zero; posterior,gantry angle 180) of the mouse’s volume. . . . . . . . 24ixLIST OF FIGURESFigure 2.4 Beam’s eye view digitally reconstructed radiographof the anterior field for the partial-body irradiationplan. Shown in red are the contouring of the mouse’slungs and the portion of bone marrow to be shielded.Shown in yellow are the rectangular fields defined bythe collimator jaws (X1, Y1, X2, Y2) for (a)body(b)lung SIB, and the shielding customization pro-vided by the MLC (small rectangles defined on themouse’s legs). . . . . . . . . . . . . . . . . . . . . . . 25Figure 2.5 Anterior field dose buildup calculation by the treat-ment planning system. The irradiation jig provides awater equivalent depth of 1.45 cm so that the maxi-mum dose depth coincides with the mouse’s midcoro-nal level. This provides a homogenous dose distribu-tion across the thorax. . . . . . . . . . . . . . . . . . 26Figure 2.6 Treatment planning system statistics for the 8 GyPBI + 8 Gy SIB lung plan: (a) Axial view of isodoseline distribution and prescription point; (b) DVH forlung, body, and shielded tissue; (c) Coronal view ofisodose line distribution and prescription point; (d)Sagittal view; (e) Dose coverage, minimum, maxi-mum and mean dose to structures of interest. . . . . . 28Figure 3.1 TrueBeam Linear Accelerator. The Perspex jig onthe treatment coach contains the mouse correctly po-sitioned for treatment delivery. . . . . . . . . . . . . . 30Figure 3.2 In vivo dosimetry with TLDs on mouse tibiae. . . . . 31Figure 4.1 Mean body weight relative to pre-irradiation weight± SEM over one week post-treatment; n=4-10 miceper group. Red * indicates significant difference com-pared with untreated controls, blue * indicates signif-icant difference compared with 16 Gy WTLI accord-ing to Tukey’s post hoc test (significance given by p≤ 0.05). . . . . . . . . . . . . . . . . . . . . . . . . . . 35xLIST OF FIGURESFigure 4.2 Average (a) total WBC, (b) neutrophil, (c) lympho-cyte and (d) monocyte counts ± SEM; n=4-9 miceper group. The line above the groups indicates sig-nificance level according to one-way ANOVA. # Indi-cates statistically significant difference compared withcontrols, * indicates significant difference comparedwith 16 Gy WTLI according to Tukey’s post hoc test(p ≤ 0.05). . . . . . . . . . . . . . . . . . . . . . . . . 37Figure 4.3 Average (a) eosinophil and (b) platelet counts± SEM;n=4-9 mice per group. Line above the groups indi-cates significance level according to one-way ANOVA.# Indicates statistically significant difference com-pared with controls, * indicates significant differencecompared with 16 Gy WTLI according to Tukey’spost hoc test (p ≤ 0.05). . . . . . . . . . . . . . . . . 38Figure 4.4 Average (a)total RBC counts, (b) Hemoglobin levels,(c) Mean Corpuscular Volume, (d) Mean CorpuscularHemoglobin ± SEM; n=4-9 mice per group. The lineabove the groups indicates significance level accordingto one-way ANOVA. # Indicates statistically signif-icant difference compared with controls, * indicatessignificant difference compared with 16 Gy WTLI ac-cording to Tukey’s post hoc test (p ≤ 0.05). . . . . . 39Figure 4.5 Mean body weights relative to pre-irradiation weights± SEM; n=5-12 mice per group. (a) * indicates sig-nificant difference between 8 Gy PBI treated miceand controls euthanized at eoe according to Tukey’spost hoc test. (b) Pink * indicates significant dif-ference between controls and 8Gy PBI plus 8Gy lungSIB; blue * significant difference between controls and16Gy WTLI according to Tukey’s test. . . . . . . . . 41Figure 4.6 Percent survival of mice exposed to 8 Gy PBI pluslung SIB (solid line) and 16 Gy whole-thorax lungexposure (dashed line). Mice exposed to 8 Gy PBI aredisplayed as controls for partial body dose (double-dashed line). . . . . . . . . . . . . . . . . . . . . . . . 42xiLIST OF FIGURESFigure 4.7 Average (a) White Blood Cell and (b) Lymphocytecounts ± SEM; n=3-8 mice per group. The line abovethe groups indicates significance level according toone-way ANOVA. # indicates significant differencewith controls according to Tukey’s post hoc test; *significant difference with 8 Gy PBI plus 8 Gy lungSIB sacrificed at 14 weeks (nondistress), + significantdifference between 8 Gy PBI plus 8 Gy lung SIB and16 Gy whole-thorax irradiation sacrificed at distressby Student’s t-test (p ≤ 0.05). . . . . . . . . . . . . . 44Figure 4.8 Average (a) Neutrophil and (b) Monocyte counts ±SEM; n=3-8 mice per group. The line above thegroups indicates significance level according to one-way ANOVA.# indicates significant difference withcontrols by Tukey’s post hoc test; * significant dif-ference with 8 Gy PBI plus 8 Gy lung SIB sacrificedat 14 weeks (nondistress), + significant difference be-tween 8 Gy PBI plus 8 Gy lung SIB and 16 Gy whole-thorax irradiation sacrificed at distress by Student’st-test (p ≤ 0.05). . . . . . . . . . . . . . . . . . . . . . 45Figure 4.9 Average (a) Eosinophils, (b) Red Blood Cells, (c)Platelets, (d) Mean Corpuscular Volume± SEM; n=3-8 mice per group. The line above the groups indicatessignificance level according to one-way ANOVA, NS= not significant. # indicates significant differencewith controls by Tukey’s post hoc test; * significantdifference with 8 Gy PBI plus 8 Gy lung SIB sacri-ficed at 14 weeks (nondistress), + significant differ-ence between 8 Gy PBI plus 8 Gy lung SIB and 16Gy whole-thorax irradiation sacrificed at distress, byStudent’s t-test (p ≤ 0.05). . . . . . . . . . . . . . . 46Figure 4.10 Mean pneumonitis scores ± SEM; n=3-9 mice pergroup. The pneumonitis score was based on semi-quantitative assessment of H&E stained lung sections.The line above the groups indicates significance levelaccording to one-way ANOVA. # indicates significantdifference with controls by Tukey’s post hoc test; *significant difference with 8 Gy PBI plus 8 Gy lungSIB sacrificed at 14 weeks (non distress) (p ≤ 0.05). . 48xiiLIST OF FIGURESFigure 4.11 Left-lung sections stained with H&E illustrating theinflammatory cell infiltrates and thickened alveolarwalls of pneumonitis; representative example. Mag-nification=10x. . . . . . . . . . . . . . . . . . . . . . . 49xiiiList of AcronymsAEC Alveolar Epithelial CellANOVA Analysis of varianceARS Acute Radiation SyndromeBALF Bronchoalveolar Lavage FluidCBC Complete Blood CountCT Computed TomographyDRR Digitally Reconstructed RadiographDVH Dose Volume HistogramEDTA Ethylenediaminetetraacetic acidEOE End of experimentFDA Food and Drug AdministrationFFF Flattening-Filter-FreeFOV Field of ViewG-CSF Granulocyte colony-stimulating factorGI GastrointestinalGy GrayxivList of AcronymsHCT HematocritHSC Hematopoietic Stem CellH&E Hematoxylin and eosinIL InterleukinLD Lethal doseLiF Lithium FluorideLINAC Linear AcceleratorMCH Mean Corpuscular HemoglobinMCHC Mean Corpuscular Hemoglobin ConcentrationMCV Mean Corpuscular VolumeMLC Multiple-leaf collimatorMU Monitor unitNHP Nonhuman primateNK Natural killerOSL Optically stimulated luminescencePBI Partial body irradiationRBC Red Blood CellROS Reactive Oxygen SpeciesRP Radiation pneumonitisSAD Source-to-axis distance (SAD)xvList of AcronymsSEM Standard error of the meanSIB Simultaneous in-field boostTGF Transforming growth factorTLD Thermoluminescent dosimeterTPS Treatment planning systemWBC White Blood CellWED Water equivalent depthWTLI Whole-thorax lung irradiationxviAcknowledgementsFirst, I would like to express my heartfelt gratitude to my supervisor, Dr.Christina Haston. Her invaluable guidance, patience, encouragement, andfeedback throughout both my coursework and the completion of my thesishave been paramount to this endeavor. Her meticulous revisions helped meachieve the highest quality of writing.I would also like to thank my supervisory committee members, Dr.Michelle Hilts and Dr. Derek Hyde, for their contributions both as instruc-tors and as scientists in my academic formation. Their thoughtful questionsand comments during our committee meetings inspired me to perform acomprehensive literature search so that I could have a better understandingof my thesis topic and be prepared for my defense. Also, their suggestionshave tremendously helped me improve the quality of my slides. Thanks toDr. Hilts for being an amazing teacher. I loved the radiobiology course!Thanks to Dr. Hyde, who without his help, the completion of this projectwould have not been possible. I will be forever grateful for his availabilityand collaboration with the mouse irradiations.Next, I would like to extend my gratitude to the amazing professors andinstructors from the Medical Physics Department/BC Cancer.I must also acknowledge the contribution of Ian Johnson, for his tremen-dous help with mouse transportation, anesthesia, and blood collections. Iappreciate his assistance in helping me improve my technique for variousmouse procedures.To the wonderful friends I made at UBC Okanagan, thank you for theamazing memories of good times, emotional support during challengingtimes, continuous encouragement, and sincere wishes.Finally, many thanks to mom and dad. Your unconditional love, contin-uous support, wisdom, and faith in my capacity has helped me become whoI am today. Thanks for encouraging me in my dream of studying Physicsin Canada, for teaching me to be ambitious and work hard to accomplishmy goals, and for being a safe and non-judgemental space to whom I can behonest about my fears, struggles, and doubts. I love you.xviiDedicationTo my wonderful parents. I am blessed to have you in my life.xviiiChapter 1IntroductionThe Twentieth Century was characterized by the rapid proliferation ofradioactive materials and their applications in medical imaging, cancer treat-ment, industry, and energy. Unfortunately, events like the Hiroshima andNagasaki Atomic bombings (1945), the Chernobyl Nuclear plant disaster(1986), and the Fukushima Daiichi accident (2011)[48] unveiled the destruc-tive power of ionizing radiation and raised global concern about the pos-sibility of a large-scale radiological threat. Around the world, many acci-dents involving exposure from abandoned sources sold as scrap metal[39], nu-clear power plants[25], unshielded sources[19], bypassing operational safetyguidelines[2], and mechanical failures of medical and industrial irradiators[2]have been documented. The health consequences for victims range frommild to lethal injuries to multiple tissues; with the hematopoietic, gastroin-testinal, pulmonary, cardiac, cutaneous, and renal systems considered mostradiosensitive[24][21][75].Currently, the ever-increasing use of radiological materials in disciplineslike energy production, construction, medicine, imaging, and research makesit imperative that effective medical countermeasures are readily available totreat workers and civilians exposed to potentially harmful doses of radiation.The rationale for directing research towards the development of radiationmitigators (administered after exposure and before the onset of symptoms toprevent or ameliorate radiation damage[66]) and treatments comes from evi-dence that standard supportive care alone can significantly enhance survivaland recovery from high-dose radiation exposure. Based on data collectedfrom the Japanese atomic bomb and the Chernobyl disaster, it has been es-timated that the mean lethal dose required to kill 50% of humans within 60days (LD50/60) increases from 3.25 Gy to 7 Gy when medical care includingantibiotics and blood transfusion is provided[74]. Also, the off-label adminis-tration of hematopoietic growth factors in many incidents has helped injuredvictims make a complete recovery from acute radiation-induced bone marrowdamage[19][21][62]. Finally, experiments on lethally whole-body irradiatedanimals have confirmed the effect of supportive care on increasing survivaltime and reducing morbidity to the gastrointestinal[46] and hematological11.1. Acute Radiation Syndromesystems[55][6]. These remarkable results have driven several researchers tocharacterize the effects of high-dose ionizing radiation on various organ sys-tems, as well as to develop and test safe and effective novel pharmaceuticalsto treat radiation-induced injuries.The lung is a very radiosensitive organ, with little regenerative capac-ity and high susceptibility to inflammatory damage[38]. This sensitivity isevident in patients receiving thoracic radiotherapy for cancer care[71], bonemarrow transplant recipients[45], and in a few radiation incidents wherevictims recovered from early bone marrow injury and later developed pul-monary complications[2]. Radiation exposure to the lung can lead to pneu-monitis and fibrosis, and these may result in impaired quality of life ordeath[18]. There is strong evidence from clinical and preclinical data thatthe biological basis of these complications involves an excessive immune-mediated inflammatory and tissue repair reaction to damage to alveolar ep-ithelial and vascular endothelial cells[18]; these responses are characterizedby recruitment of immune cells[34] and active cell-cell communication andsignal transduction via cytokine expression[4][38]. Ultimately, these changeshave been attributed to the activation of genes associated with the produc-tion of these cellular and molecular components[52]. However, the definitepathways leading to pneumonitis and fibrosis are yet to be fully understood.Furthermore, there is incomplete understanding as to how radiation in-juries to other organs may affect the lung, and of systemic effects of radiationon the lung[75]. As it is discussed below, significant radiation exposure maytrigger a variety of toxicities to different organ systems that manifest withinsimilar timelines. For that reason, a better characterization of the mecha-nisms and features of radiation-induced pulmonary effects following survivalfrom hematopoietic and gastrointestinal system injuries, as in the radiationincident scenario, is essential for the development and assessment of effec-tive medical countermeasures to treat the exposed lung as well as to evaluatethe effects of agents targeting other organ systems on the lung. Thus, inthis thesis, a mouse model of high-dose partial-body irradiation suitable toreproduce an accidental exposure, has been established to assess the devel-opment and severity of radiation lung sequelae and correlate to injury to thehematopoietic system.1.1 Acute Radiation SyndromeThe consequences of ionizing radiation have been characterized throughanimal experiments, and human data obtained from patients receiving radio-21.1. Acute Radiation Syndrometherapy for cancer and victims of radiological incidents. Immediate effects ofhigh-dose ionizing radiation include the Acute Radiation Syndrome[21][3].Later sequelae include injuries to multiple organs that may resolve in long-term functional impairments, immunosuppression, tissue fibrosis, organ fail-ure, and death[25][73][27]. Finally, carcinogenesis may manifest years fol-lowing the incident, as seen in survivors of the Chernobyl disaster[78].In humans, total body irradiation or significant partial body irradiationat doses greater than 1 Gy delivered at a relatively high dose rate (0.05Gy/h or higher) induces a series of clinical syndromes that manifest within apredictable range of doses and timing, collectively known as Acute RadiationSyndrome (ARS)[63]. The main mechanism of ARS is attributed to therapid depletion of the highly radiosensitive immature parenchymal stemcells in tissues with a high cell turnover rate. Without enough stem cells toreplicate, differentiate and replace dead cells for tissue renewal, impairmentsto tissue architecture and function occur, and illness manifests. The majorclinical components of ARS are the hematopoietic, gastrointestinal, andcerebrovascular subsyndromes[22].1.1.1 Hematopoietic SystemThe hematopoietic syndrome appears after total-body or near total-body irradiation greater than 1 Gy. Radiation causes death or sterilizationof hematopoietic stem and progenitor cells, which are responsible for theproduction and replenishment of all blood cell populations and sustainingnormal hematopoietic system function[7]. Histological analysis reveals de-creased myeloid, erythroid, and megakaryocytic cells in bone marrow, anddepletion of lymphocytes in lymph nodes, spleen, and thymus[21][10]. Asa result, the supply of red blood cells, white blood cells, and platelets isdiminished. Once the mature circulating cells die off (approximately 3-6weeks post-exposure) and there are insufficient new cells from the depletedprecursors, illness manifests. Clinical signs of the hematopoietic syndromeinclude blood cytopenia, myelosuppression, impairment of the immune func-tion, anemia, and poor wound healing from platelet depletion. These maylead to vulnerability to infections, uncontrolled hemorrhage, sepsis, anddeath[3][63]. Survival from this subsyndrome requires prompt reconstitu-tion of the hematopoietic system, accomplished through intensive supportivecare, administration of blood stem cell products, and hematopoietic growthfactors[62].31.2. Medical Management and Delayed Effects of Acute Radiation Exposure1.1.2 Gastrointestinal SystemExposures exceeding 10 Gy including the gastrointestinal system resultin the gastrointestinal subsyndrome. Irradiation causes apoptosis of theintestinal clonogenic crypt epithelial stem cells. In the healthy intestine,these cells continuously divide, migrate to the villi, differentiate, and be-come functioning cells; the villi are continuously replaced by cells that orig-inate in the crypts. Radiation-induced crypt depletion causes shrinkingof the villi until the lining of the intestine is completely denuded, and themucosal epithelial barrier is compromised[22]. Other mechanisms contribut-ing to morbidity include radiation-induced protein alterations, the releaseof pro-inflammatory cytokines, and differences in redox status. Damageprogresses to impaired nutrient absorption, ulceration of the GI tract, andmicrobial infiltration through the weakened mucosal barrier[63]. Victimsof this syndrome present abdominal pain, diarrhea, nausea, vomiting, andvulnerability to infections[21]. Death usually comes between 3 and 10 daysof exposure due to emaciation, malnutrition, dehydration, cardiovascularcollapse, electrolyte imbalance, gastrointestinal bleeding, and sepsis[22].1.1.3 Neurovascular SystemThe cerebrovascular syndrome occurs at whole-body exposures well above10 Gy and it results in lethal injury to the cerebrovascular system. Sincedeath is certain to occur within 24-48 hours of exposure at such high doses,this subsyndrome is considered untreatable[63]. Hence, it will not be furtherdiscussed in this thesis.1.2 Medical Management and Delayed Effects ofAcute Radiation ExposureThe hematopoietic and gastrointestinal systems are the most radiosen-sitive and earliest-responding tissues in the human body. Consequently, inthe event of accidental exposure involving low to moderate doses, damageto these systems will become the primary cause of morbidity and mortal-ity, followed by prolonged impaired function and susceptibility to infections.Patients exposed at the lower end of the doses causing bone marrow and gas-trointestinal effects are more likely to respond positively to therapeutic mea-sures. Therefore, researchers have devoted their work to the developmentand testing of therapeutic countermeasures for radiation injuries targetingthese specific systems.41.3. Radiation-Induced Lung InjuryExamples of promising agents for the management of hematological acuteradiation syndrome are hematopoietic colony-stimulating growth factors.Colony-stimulating factors are endogenous glycoproteins that promote pro-liferation and differentiation of bone marrow progenitors into mature bloodcells (e.g. neutrophils)[62]. Following radiation-induced myelosuppression,the administration of these drugs stimulates the proliferation, differentiation,maturation, and function of neutrophils. The efficacy of several growth fac-tors has been well-demonstrated in experiments with rodents, canines, andnonhuman primates receiving lethal doses of whole-body irradiation. Treat-ment with these agents enhanced survival and accelerated recovery fromneutropenia in various species[55][62]. Currently, the US Food and DrugAdministration has approved three pharmaceuticals to treat patients acutelyexposed to myelosuppressive doses of radiation: Neupogen (filgrastim), Neu-lasta (pegfilgrastim) and Leukine (sargramostim)[13]. Also, several othercompounds are currently being investigated and showing promising resultsin preclinical studies[6].The administration of appropriate countermeasures against the hematopoi-etic and gastrointestinal radiation syndromes is expected to spare individu-als from early radiation lethality. Consequently, survival from early effectswill permit the evolution and expression of late chronic sequelae consistingof progressive pathologies to multiple organs, that may resolve in perma-nent functional impairments, tissue scarring, organ failure, and death in themost severe cases[25][24][73]. Studies in nonhuman primates and rodents,whose radiation response is representative of that of humans, have identifiedlong-term residual bone marrow impairment[7], as well as lung, kidney, andcardiac injuries[43][27].The high morbidity associated with late radiation effects and the poten-tial for multiple evolving pathologies to manifest at the same time makesit necessary to obtain a complete description of the delayed effects of acuteradiation exposure. In this work, the main focus is the characterization oflung injury consequent to high-dose partial body radiation exposure in amurine model that induces early sublethal depletion of the hematopoieticsystem.1.3 Radiation-Induced Lung InjuryThe temporal progression of radiation-induced pulmonary damage isclassically described by three phases: 1) A clinically latent phase of nomanifest illness, consisting of early cellular and molecular changes in reac-51.3. Radiation-Induced Lung Injurytion to the radiation; 2) an acute inflammatory phase of pneumonitis; 3) alate phase of lung fibrosis[58].Radiation pneumonitis (RP) is excessive inflammation of the lung, par-ticularly affecting the alveoli. It typically manifests within 4 to 12 weeksfollowing chest irradiation, affecting between 5% and 24% of cancer patientstreated with thoracic radiotherapy and/or chemoradiotherapy[38]. Patho-logical features of RP include thickening of the alveolar walls, cellular in-filtrate, and exudates present in the alveolar airway space[23]. Symptomsof pneumonitis include fever, fatigue, cough, dyspnea, chest congestion, andchest pain. If this complication is not controlled, it may progress to irre-versible pulmonary fibrosis[64].Fibrosis is the permanent scarring of tissue as a result of excessive ac-cumulation of extracellular matrix proteins in the lung. This leads to animpaired ability of the lung to exchange oxygen. This condition occursbetween 6-24 months following irradiation of the lung, and it results in per-manent tissue damage, decreased lung functionality, and impaired qualityof life[38].1.3.1 The alveolar structureThe alveoli constitute the lung functional units, where oxygen and car-bon dioxide exchange occurs. Figure 1.1 illustrates the alveolar structure.The lung alveoli consist of an epithelial wall, the interstitium, and the sur-rounding capillaries. The alveolar epithelium is made up of two kinds ofcells: Type I and II pneumocytes, also called alveolar epithelial cells (AECs).Type I pneumocytes are the most abundant and radiosensitive epithelialconstituents, and they are responsible for gas exchange between the alveo-lar airspace and the capillaries. Type II pneumocytes are the precursors ofType I cells, and thus fulfill the function of reconstituting the alveolar epithe-lium after damage. Also, the epithelial layer contains resident macrophages,which are essential for sustaining tissue homeostasis, pathogen recognition,and initiation of local immune and inflammatory responses[38].1.3.2 Mechanisms of radiation-induced lung damageIonizing radiation is energy transferred in the form of energetic parti-cles that is capable of ejecting electrons from atoms as it passes through amedium. The mean energy imparted to a material per unit mass, referred toas the absorbed dose, is the quantity for ionizing radiation. The fundamentalunit of dose is the Gray (Gy), defined as joules per kilogram(J/Kg)[56].61.3. Radiation-Induced Lung InjuryFigure 1.1: Structure of the pulmonary alveolus. Adapted from Lierova andothers[38].Ionizing radiation particles deposit energy to the medium through Coulombinteractions with atomic orbital electrons they encounter, resulting in kineticenergy transfer. If the deposited energy is greater than the electron bind-ing energy, an ionization event occurs, and the electron is ejected from theatom[56]. These electrons may go on to cause further ionizations with themolecules they interact with on their path as they lose kinetic energy, givingrise to an ionization cascade. Ionizations cause the breaking of atomic andmolecular bonds, leading to biological damage[76]. Figure 1.2 illustrates theprocess of radiation interactions and energy deposition.The underlying mechanisms of radiation pulmonary injuries have beenstudied by several research groups. A strong hypothesis is that these patholo-gies are the result of an overwhelming inflammatory immune reaction toionizing radiation. Figure 1.3 summarizes the sequence of events leading topulmonary radiation damage.Exposure of the lung to ionizing radiation causes direct lesions in DNAmolecules and chemical reactions with water and other cellular components.The ionization of water molecules generates reactive oxygen species (ROS)that damage alveolar epithelial and capillary endothelial cells[34]. The ini-tial insult triggers immediate apoptosis of type I pneumocytes, which resultsin denudation of the epithelial layer. In response, type II pneumocytes pro-71.3. Radiation-Induced Lung InjuryFigure 1.2: Radiation interactions with biological tissue and energy deposi-tion process. Adapted from Johns and Cunninghams[29].liferate to restore the epithelium; however, their structure and function ofsecreting surfactant become impaired, leading to loss of pulmonary surfacetension. Apoptosis and damage to vascular endothelial cells lead to in-creased microvascular permeability[18] and blood vessel dilation, allowingfor infiltration of immune cells and protein exudates at the site of tissueinjury[26].The initial tissue damage rapidly activates an inflammatory reaction,leading to the release and maintenance of a cascade of pro-inflammatoryand pro-fibrotic cytokines[58], the expression of genes encoding proteins thatmobilize immune cells from the bloodstream into the injured tissue[52], andthe activation of collagen genes[58]. Preclinical studies in mice and can-cer patients have identified early elevations (hours to two weeks followinglung irradiation) of interleukins (IL) like IL-1α[58], IL-6[4], IL-17[54], and81.3. Radiation-Induced Lung Injurytransforming growth factor TGF-β[58]. These cytokines act as activatorsand chemotactic factors of inflammatory cells, and their function involvesthe recruitment of diverse immune myeloid and lymphoid cells (neutrophils,macrophages, lymphocytes, monocytes) that infiltrate into the damagedlung, causing tissue inflammation and remodeling. The recruited cells mayfurther sustain the inflammatory conditions through the synthesis of morecytokines, aggregating more cells, and contributing to excessive inflamma-tion. If this response is persistent, it leads to radiation pneumonitis[18].Finally, if this condition is severe or is left untreated, the inflammatory dam-age may progress into an abnormal tissue repair response. This response ischaracterized by fibroblast proliferation, excessive production and deposi-tion of extracellular matrix components in the lung interstitium, leading topermanent alterations to tissue architecture and, eventually, to pulmonaryfibrosis[38].Figure 1.3: Sequence of events leading to radiation induced lung injury.Adapted from Giuranno and others[18].91.4. The role of immune cells in radiation-induced lung injury1.4 The role of immune cells in radiation-inducedlung injuryAs described above, immune cells play a significant role in the devel-opment of radiation-induced pulmonary effects. The majority of immunecells originate from pluripotent hematopoietic stem cells (HSCs) in the bonemarrow. HSCs may also differentiate into Red Blood Cells (RBC), whichtransport oxygen throughout the body, and platelets, which help with bloodclotting in damaged tissues[49]. Since the goal of this thesis is the develop-ment of a model of high-dose partial-body irradiation to determine the effectof acute hematopoietic depletion on radiation lung injury, it is important todiscuss the role of the bone-marrow-derived immune cells on the patholo-gies of pneumonitis and fibrosis. Several studies in radiotherapy patientsand experimental animals have associated different types of inflammatorycells to radiation-induced lung disease, supporting the hypothesis that aninflammatory response orchestrated by the immune system contributes tothe development of pneumonitis and fibrosis. The most significant findingsin the literature are described below.White Blood CellsWhite Blood Cells (WBC) or leukocytes are the main effector cells ofthe immune system that protect the body from infectious agents and sub-stances detected as foreign and harmful[49]. The involvement of leukocytesin radiation lung pneumonitis has been observed in preclinical studies. In-creased WBC counts in peripheral blood and Bronchoalveolar Lavage Fluid(BALF) have been found in Gottingen minipigs following single high-doselocal whole-thorax treatment[35]. Chad Tang and others have found sig-nificant associations between high post-radiotherapy WBC counts and inci-dence of grade ≥ 2 and 3 radiation pneumonitis in a retrospective analysisof patients receiving treatment for non-small cell lung cancer[70]. Similarly,Zhou and others found a marked increase in WBCs at 12 weeks followingradiotherapy in lung cancer patients presenting grade 3 pneumonitis[79].NeutrophilsNeutrophils are the most abundant type of myeloid white blood cellsof the innate immune system and form the first line of defense againstpathogens. Neutrophils quickly recognize, engulf, and destroy a variety ofmicroorganisms by phagocytosis[49]. These cells are also involved in in-101.4. The role of immune cells in radiation-induced lung injuryflammatory responses through the production of inflammatory mediators,namely cytokines and chemokines, driving the migration of cells towardsinjured or infected tissue[28]. Furthermore, neutrophils play a crucial rolein the pathogenesis of various respiratory conditions, including Chronic Ob-structive Pulmonary Disease (COPD), asthma, and cystic fibrosis. Thesepathologies are characterized by neutrophil infiltration into the airways,chronic inflammation, and tissue damage[28].Neutrophils have been associated with radiation-induced lung disease.Higher neutrophil counts in the lung tissue and BALF of whole-thorax ir-radiated animals succumbing to radiation alveolitis have been observed forvarious species: Nonhuman primates[16], Wistar rats[68], and mice[14][34].Raises in BALF and peripheral blood neutrophils have also been linked tosymptomatic pneumonitis in radiotherapy patients[79][71]. Further support-ing the involvement of these cells in the lung response to high-dose thoracicexposure, protein expression analysis of C3H/HeJ mice sacrificed at respira-tory distress reveals increased levels of colony-stimulating factor (G-CSF),Intercellular Adhesion Molecule 1 (ICAM-1), and E-selectin, molecules thatstimulate the production, maturation, and recruitment of neutrophils[34].MonocytesMonocytes are another type of leukocytes of myeloid origin. Along withneutrophils, they constitute the main effector cells of the innate immune sys-tem. Upon tissue injury or infection, monocytes circulating in the blood mi-grate to the affected sites and differentiate into macrophages. Macrophagesare relatively long-lived cells that reside in various tissues and present highphagocytic activity, attacking pathogens and clearing dead and injured ep-ithelial cells[49].Macrophages initiate and sustain inflammatory lung responses and con-tribute to tissue repair[38]. These cells are major sources of pro-inflammatorymediators like IL-6[4], IL-1α[58][32], and IL-8[52][38], and pro-fibrotic fac-tors including TGF-β1 and IL-4[32][26]; these cytokines have been associ-ated with radiation pneumonitis and fibrosis, respectively. Finally, alveolarmacrophages have been identified as significant contributors to the severityof radiation lung disease in mice. Following high-dose whole-thorax irradia-tion, C3H/HeJ mice succumb to diffuse lethal alveolitis, whereas C57BL/6mice develop milder alveolitis with pulmonary fibrosis. The severity ofalveolitis was negatively correlated with apoptosis of inflammatory alveo-lar macrophages, with C3H/HeJ mice presenting a lower apoptotic indexthan C57BL/6 mice at 11 weeks and distress[50].111.4. The role of immune cells in radiation-induced lung injuryLymphocytesLymphocytes are white blood cells that arise from lymphoid progeni-tors. They are the effectors of the adaptive immune response and possessthe most powerful ability to recognize and target pathogens. In contrast toinnate immune cells, lymphocytes express highly specialized antigen recep-tors on their surface, by which they recognize and bind to a wide varietyof antigen-presenting microorganisms[49]. The three major classes of lym-phocytes are T cells, B cells, and natural killer (NK) cells. T lymphocytesact in antigen recognition and destruction of pathogens and infected cells,and B lymphocytes support this function by the production of cytokinesand antibodies. Finally, NK cells can induce direct cell-mediated killing ofvirus-infected and tumor cells[32].Lymphocyte infiltration is a classical feature of pneumonitis. Inflamma-tory cytokines elevated in patients developing radiation pulmonary effects,including IL-1α, are potent stimulators and chemoattractant for lymphocytes[58].Increased lymphocyte numbers in the BALF of breast cancer patients re-ceiving thoracic radiotherapy have been reported, with higher counts inpatients with RP[71][57], and also in experimental murine models[34]. T-lymphocytes, particularly, have been identified as key contributors to radia-tion pneumonitis. Removal of the thymus, a primary lymphoid organ for thematuration of T-cells, before total-body irradiation followed by bone marrowtransplant decreased the incidence of pneumonitis in C3H/Kam mice[45].Recent studies have linked various subpopulations of T-cells to the radi-ation lung response, with different types adopting pro- or anti-inflammatory,pro- or anti-fibrotic, and modulating functions. For instance, Paun and oth-ers have associated increased Th17 cell numbers radiation pulmonary fibrosisin mice, whereas Th1 cells have been attributed an anti-fibrotic effect. Th2cells have also been linked to fibrosis, and excessive Th1 cell numbers havebeen associated with pneumonitis[26][53]. Finally, Treg cells counteract andmodulate inflammatory responses[32].Eosinophils and BasophilsEosinophils and basophils are the least abundant leukocytes in the blood.Along with neutrophils and monocytes, they are part of the innate immunesystem. Eosinophils target antibody-coated bacteria and protozoa and areprimarily responsible for defense against parasitic infections[44]. They at-tack pathogens mainly through the secretion of cytotoxic enzymes storedin their cytoplasmic granules[49]. Eosinophils increase in the presence of121.4. The role of immune cells in radiation-induced lung injurycirculating allergens; thus, they have also been identified as contributors toallergic inflammatory reactions[44].Basophils participate in inflammatory responses through the secretion ofhistamine- and heparin- containing granules at the site of injury/infectionto enhance local inflammation. Histamine dilates the blood vessels to fa-cilitate the flow of leukocytes and proteins, and heparin prevents bloodclotting[44]. Neither eosinophils nor basophils have been associated withradiation-induced pulmonary injuries.13Chapter 2Experimental Design2.1 The mouse as a model of radiation-inducedlung injuryThe characterization of radiation effects and mechanisms of damage re-quires well-defined animal models that accurately represent the response inhumans. The rationale for animal use comes from the fact that conductingsuch experiments in humans would be unethical, as they would be exposedto lethal or debilitating stimuli. This is best described by the US FDA An-imal Efficacy Rule, issued to accelerate the development of agents againstchemical, biological, radiological, and nuclear threats[61]. Most importantly,the pathogenesis of radiation injuries involves a complex process where var-ious cell types, danger signaling molecules, and tissues interact in responseto insult; these pathways of damage could not be evaluated solely throughcell-based assays[59].The choice of animal species must reflect the human pathophysiologi-cal mechanisms of disease, latency, incidence, and clinical features. Multi-ple models have been used to describe the response of various organ sys-tems. The species most frequently used to model the hematological andgastrointestinal radiation effects are the mouse, the rat, and the nonhumanprimate[43].The pulmonary response to ionizing radiation is best characterized bythe mouse. The primary advantages of this model are its genetic (over 95%shared genome), physiological and anatomical similarities to humans. Sec-ond, its small size, short lifespan, relatively low cost, and easy handlingprovide advantages associated with feasibility[61]. Furthermore, potentialconfounding variables intrinsic to human studies (e.g. sex, age, medical his-tory) are eliminated so that data is collected from a large number of geneti-cally identical animals raised in the same environmental conditions, makingresults highly reproducible. Finally, the differential response to thoracicirradiation among different strains of mice can be exploited to determinethe genetics of radiation-induced lung sequelae. To illustrate, Haston and142.2. The minimum sparing modelcolleagues have characterized the inbred strains C3H/HeJ and C57BL/6 asmodels of pneumonitis and fibrosis, respectively, and used them to investi-gate the specific genes influencing lung disease susceptibility[52][37].In this thesis, the pneumonitis-prone C3H/HeJ mouse strain was se-lected to focus on the characterization of radiation pneumonitis consequentto significant partial-body irradiation inducing early bone marrow deple-tion. Following localized whole-thorax radiation exposure (18 Gy; X-rayunit, 0.54 Gy/minute), C3H/HeJ mice develop lethal pneumonitis and suc-cumb, on average, 12 weeks post-treatment. This response is accompaniedby an increase in the percentage of lymphocytes and neutrophils in BALFand lung tissue[34].2.2 The minimum sparing modelThe most popular framework for the characterization of acute radiationsyndrome is the Total-Body Irradiation (TBI) model, consisting of deliv-ering a single high-dose uniform exposure to the experimental animals[55].This regime allows to reproduce an incident involving uniform whole-bodyirradiation, as it was the case for many individuals exposed to the JapaneseAtomic Bomb[61], and it has been extensively used to characterize acutedamage to the gastrointestinal and hematopoietic systems. A major disad-vantage of this experimental design is that high early lethality associatedwith high-dose total body irradiation prevents the progression and study oflate multi-organ injuries[40]. Also, a more realistic accidental exposure sce-nario will likely involve a poorly defined, nonuniform dose distribution, withsome parts of the body being closer to the source and with the potential forphysical shielding[47]. The type and severity of consequences following anaccidental exposure will be highly dependent on the absorbed dose, distri-bution, and rate at which it is delivered. Shielding will result in sparing oftissue from irradiation, thus influencing morbidity and survival[61].In addition to the TBI model, research groups have studied radiationeffects on various organs by employing organ-specific localized exposureregimes. For instance, the single-fraction Whole Thorax Lung Irradiation(WTLI) model is used to investigate the biological basis of lung injuries[52][53].This model is appropriate for describing the scenario relevant to clinical ra-diotherapy for thoracic cancers, where the radiation field includes only thetreatment region sparing the rest of the body. This prevents from inves-tigating the impact of multiple-organ radiation injuries on the radiationpulmonary response.152.2. The minimum sparing modelIn an accidental exposure, lung sequelae will develop in a context ofsurvival from acute hematological and gastrointestinal syndromes and oc-cur concomitant to prolonged residual bone marrow damage[40]. Recentpublications of murine studies have identified long-term consequences onthe hematopoietic system following survival from acute injury. Delayedbone marrow impairment is characterized by a significant loss of hematopoi-etic stem cell (HSC) numbers and compromised functionality, that is, self-renewal and multilineage differentiation capacity. This manifests as signif-icantly reduced production of blood cells of all hematopoietic lineages inperipheral blood, decreased bone marrow cellularity, long-term immunosup-pression, and anemia[7][8]. As it was described in Section1.4, there is evi-dence of the involvement of immune cells in radiation lung injury. Becausethese cells are originated in the hematopoietic system, it is of interest to de-termine whether radiation damage to this system influences the subsequentresponse of the lung.A novel strategy to link early and delayed multiple-organ effects of acuteradiation exposure consists of models employing animals given significantpartial body irradiation, with some degree of bone marrow shielding. Therationale for this technique comes from observations that sparing of as lit-tle as 2.5-5% of active bone marrow from radiation enhances survival fromearly hematological and gastrointestinal toxicity in irradiated animals, thuspermitting the progression of later complications[11][40]. Thomas McVit-tie and colleagues have conducted studies using nonhuman primates (NHP)exposed to high-dose partial-body irradiation (6 MV LINAC photon beam,0.80 Gy/min) with tibiae, ankles, and feet out of the radiation field, sparingapproximately 5% of the animal’s bone marrow[40][69]. Their research grouphas used such NHP experiments to model late effects on the gastrointestinalsystem, kidney[9], lung, and heart[51] resulting from accidental radiationexposure. These publications have defined the time and dose-response rela-tionships, and histopathological features of such injuries in the nonhumanprimate.Nevertheless, a comprehensive literature record of delayed pulmonarysequelae requires conducting studies on more than one animal species repre-sentative of human pathophysiology, so that results can be further comparedand validated. For this purpose, the mouse is the gold standard of smallanimal models[61]. Furthermore, there is yet to be a study specifically de-signed to determine the influence of radiation damage to early respondingtissues on the subsequent development of lung injury. Hence, this work aimsat establishing a mouse model of partial-body irradiation with minimal bonemarrow shielding to describe the natural history of lung sequelae in terms162.3. Model designof pathology, latency, severity, histological features, and associated circulat-ing blood counts. At the same time, experiments on mouse cohorts givenlocalized whole-thorax irradiation are conducted to allow for direct compar-ison with the partial-body exposure model, to determine whether early bonemarrow depletion influences subsequent lung injury. Finally, the results maybe further compared to the nonhuman primate model.2.3 Model designTo model a dose distribution representative of an accidental scenario,a regime of high-dose partial-body irradiation (PBI) was chosen. The PBIconfiguration applied in this thesis is based on the technique implemented byMcVittie and colleagues for nonhuman primates[51]. Hence, mice receiveda single near whole-body exposure with shielding on the tibiae, ankles andfeet, sparing between 5-7% of the bone marrow[5][69][60].This configuration aims to produce a sublethal bone marrow response,allowing time for the development of lung injuries. However, the body doserange that elicits survivable hematopoietic syndrome in the mouse is well be-low the threshold for pulmonary damage. According to Haston and others,whole-thorax exposure on the range of 16-18 Gy results in lethal lung pneu-monitis in 100% of irradiated C3H/HeJ mice[34][53]. On the other hand, asignificant body exposure above 14 Gy results in absolute mortality within20 days due to acute gastrointestinal syndrome[5], therefore preventing theexpression of late radiation effects.To solve this problem, the PBI field was followed immediately by a simul-taneous in-field boost (SIB) to the whole thorax. This distribution, shownin Figure 2.1, delivers a sublethal body dose to the bone marrow and a to-tal whole-thorax dose above the threshold for pulmonary injury. A criticalconsideration is that the SIB must be delivered immediately after the bodyfield to avoid dose fractionation effects[31].2.4 Experiment Timeline2.4.1 Part I: Hematopoietic system depletion assessed atone week post-irradiationThe purposes of the first part of the experiment were 1) to determinethe appropriate partial-body irradiation dose for C3H/HeJ mice that causessublethal hematopoietic system injury, 2) to determine whether partial-body172.4. Experiment TimelineFigure 2.1: Experimental design. To determine the effect of radiation-induced hematopoietic cell depletion on radiation lung disease, mice receiveda partial-body exposure with minimal bone marrow shielding followed by aboost field to the whole thorax to accomplish a total lung dose of 16 Gy.This dose is above the threshold for lung injury in mice.irradiation with minimal bone marrow shielding depletes the hematopoi-etic system more than 16 Gy-whole thorax irradiation at one week post-treatment, and 3) to find out whether the body doses tested are survivableover the one-week timepoint.Damage to the hematopoietic system was measured by a Complete BloodCount (CBC) panel, which quantifies white and red blood cells circulatingin the individual’s blood. This test is a standard tool for assessing acuteradiation bone marrow damage severity in both animal models[7][55] [17]and victims of radiation incidents[25]. Furthermore, CBC is a test readilyavailable in the clinic for patient evaluation.To investigate the partial-body dose range that results in sublethal bonemarrow injury, a literature search on mouse models of ARS was conductedon PubMed. A few of the most relevant studies are briefly discussed. Calviand others used a total body irradiation dose of 6 Gy (Cs-137 source, 1.6Gy/min) on C57BL/6J mice to characterize acute and late hematopoietic182.4. Experiment Timelinedamage throughout 26 weeks[7]. Bunin and others found that a total-bodydose of 6.8 Gy (X-ray unit, 1.09 Gy/min) induced 70% lethality within 30days on C57BL/6J mice in an experiment to test the efficacy of a noveltreatment for thrombocytopenia[6]. Ghosh and others used a TBI dose of 6Gy (Co-60 unit, 0.6 Gy/min) in C3H/HeN mice to induce rapid depletion ofblood cell counts and test the effectiveness of a novel small-molecule radio-protector against ARS; such TBI dose allowed control mice to survive for the30-day duration of the study[17]. Finally, Micewicz and others determinedan LD70/30 of 7.77 Gy for C3H mice receiving whole body irradiation[46].Based on these findings, two partial-body doses, 6 and 8 Gy were assayed.Mice were assigned one of the following treatments: (1) nonirradiatedcontrols, (2) 6 Gy partial body irradiation (PBI), sparing the tibiae, anklesand feet, (3) 6 Gy PBI plus a 10 Gy simultaneous-infield-boost (SIB) tothe lung, (4) 8 Gy PBI, (5) 8 Gy PBI plus 8 Gy SIB to lung, and (6) 16Gy whole-thorax lung irradiation (WTLI). These are summarized in Ta-ble 2.1. The PBI groups were established to determine whether the bodydoses induce significant hematopoietic depletion compared with nonirradi-ated controls as measured one-week post-treatment. The PBI plus lungSIB groups were assessed to test whether the boost dose to the lung affectshematological parameters significantly different from partial-body irradia-tion alone. The WTLI group was established to test whether PBI decreasesblood cells more than thorax only treatment. If one-week circulating bloodcell counts are significantly lower for mice treated with PBI than for thosereceiving WTLI, then the PBI model can be further applied to test theeffect of radiation-induced hematopoietic depletion on the development oflung disease. Finally, nonirradiated controls provide a baseline for normalblood parameters.The one-week post-irradiation time point was chosen to test hematopoi-etic system depletion based on the lifespan of leukocytes and the reportedtime for onset of severe leukopenia in various murine models of ARS. Insuch studies, severe leukopenia, neutropenia, and lymphocytopenia man-ifest within 6-10 days after high-dose total body irradiation, with nadirs(lowest values) for leukocyte counts occurring at such time window[55][17].Additionally, significant decline in platelet counts can be measured by day7 post-treatment[17][6].2.4.2 Part II: Partial-body irradiation survival studyAs further discussed in Chapter 4, a PBI dose of 8 Gy was chosen tocomplete this thesis. The second part of the experiment consisted of testing192.4. Experiment TimelineTable 2.1: Experiment part I: Hematopoietic depletion at one week post-irradiationTreatmentGroupPBI dose(Gy)Lung SIB dose(Gy)Purpose(1) 8 0Does the body dosedeplete hematopoietic cells?(2) 8 8Does the body + lung dose depletehematopoietic cells differentlyfrom the body dose alone?(3) 6 0Does the body dosedeplete hematopoietic cells?(4) 6 10Does the body + lung dose depletehematopoietic cells differentlyfrom the body dose alone?(5) 0 16Does the lung dose depletehematopoietic cells differentlyfrom the body dose?(6) 0 0 Untreated controlswhether the selected body dose caused only sublethal damage to the earlier-responding hematopoietic system, as determined by 30-day survival. Thismay allow the progression of later radiation lung injury on mice receivingPBI plus lung SIB. The 30-day endpoint was chosen based on literaturefindings for the time window of mortality from Acute Radiation Syndromein mice. The first period for lethality from high-dose acute radiation expo-sure corresponds to gastrointestinal damage. According to a mouse modelof high-dose partial-body exposure with 5% bone marrow sparing to char-acterize gastrointestinal syndrome, the onset of symptoms (weight loss, de-hydration, diarrhea) occurs about 4-7 days post-treatment, with mice suc-cumbing at a median time of 6-7 days[5]. Survivors of GI injury developacute hematopoietic syndrome. According to a murine model of lethal bonemarrow injury following TBI, the time window for mortality is 30 days, withmedian survival time for decedents 14.1 days[55]. This time window has alsobeen reported by other authors (Calvi and others[7], Ghosh and others[17]).Thus, a subset of mice was exposed to 8 Gy PBI and monitored forhealth status and survival for 30 days. After confirming that mice survivedpast this time point, the final part of the experiment was performed.202.5. Radiation treatment plan2.4.3 Part III: Experiment completionThe purpose was to determine the effect of hematopoietic depletion in-duced by high-dose partial-body irradiation on radiation-induced lung dis-ease in the mouse. Table 2.2 shows the treatment groups assessed in thisphase. The 8Gy PBI plus 8Gy lung SIB group was established to testwhether acute hematological cell depletion affects the development and pro-gression of radiation lung injury in mice. Second, the WTLI group servedas positive control for radiation lung response. In this manner, the lungpathology, latency, severity, and associated blood cell profile may be com-pared between PBI plus lung SIB and WTLI treatments. Next, the 8 Gy PBIwas established as positive control for the body radiation field and determinethe effect of partial-body irradiation alone on mouse health and blood cellprofile at the 16-week end of the experiment. Finally, nonirradiated controlshoused on the same conditions as the experimental mice provided normalvalues for weight, hematological measures, and lung histology.Table 2.2: Experimental phase IIITreatmentGroupPBI dose(Gy)Lung SIBdose (Gy)Exptl.endpointPurpose(1) 8 016 weeks(end of expt.)Positive control for PBI(2) 8 8RespiratorydistressTest effect of bonemarrow depletion onlung injury(3) 0 16RespiratorydistressPositive control forlung injury; comparefindings to PBI plusLung SIB model(4) 0 016 weeks(end of expt)Nonirradiated controls2.5 Radiation treatment planThe mouse radiation treatment development process was the same asfor typical clinical radiotherapy treatment and it consisted of simulation,planning, and dose delivery. The steps for treatment planning are describedin this section. These were conducted at the BC Cancer Centre (Kelowna,BC).212.5. Radiation treatment plan2.5.1 CT simulationIn clinical radiotherapy, a Computed Tomography (CT) simulator is anapparatus that reproduces the treatment patient setup to collect the con-touring, anatomical, and density information of the patient for planning.This information is contained in a set of CT images consisting of transverseslices of the patient’s anatomy of interest with good bone and soft tissue con-trast for dose calculations[56]. Briefly, computed tomography is an imagingmodality that consists of the formation of a photon attenuation profile ofthe patient’s anatomy revealing differences in the attenuation coefficientsof the underlying tissues as a beam of X-ray photons passes through thepatient. The CT gantry contains a rotating high-output X-ray tube and anarray of detectors to collect hundreds of attenuation profiles as the X-raytube revolves around the patient and mathematically reconstructed to forma cross-sectional image of the structure of interest with rich contrast[1].A modern CT simulator (shown in Figure 2.2) consists of a large doughnut-shaped isocentric gantry, alignment laser systems to assist correct patientpositioning and to draw set up marks for treatment, patient table, and com-puter software for image reconstruction and visualization[56]. The high-quality CT images ensure accurate target localization. Finally, the simu-lation software can generate digitally reconstructed radiographs (DRRs) ofthe treatment ports to model the beams and display the anatomy, treatmentfield size, and placement on the beam’s-eye-view[65].In this work, treatment simulation was performed on a GE LightSpeedRT 16 computer tomography (CT) simulator using a euthanized mouse asa representative subject to design a standard treatment plan. The goalswere to determine the mouse treatment position and alignment, localizethe target volume, determine the treatment field geometry, and obtain theimaging data required for planning. To accomplish these, the mouse wasplaced in a 3-mm thick Perspex box and positioned according to lines drawnon the bottom on the box. The legs were pulled apart and taped to minimizethe dose to the tibiae, fibulae and feet. The jig containing the mouse wascarefully positioned on the simulator coach with the help of the externalalignment lasers located on the walls and ceiling of the room so that theEast/West and Superior/Inferior lasers intersect at the level of the thorax.This exact configuration was later reproduced on the treatment unit forall treated mice. After positioning, a collection of transverse images of themouse’s entire body was acquired (120 kVp, 35 cm FOV, 1.25 mm slicespacing) and transferred to the treatment planning system.222.5. Radiation treatment planFigure 2.2: CT simulator room at BC Cancer Agency.2.5.2 Treatment planningTreatment plans were designed using the Varian EclipseTM computer-ized treatment planning system (TPS). The treatment plan contains thedose distribution information over a 3D matrix of points over the mouse’sanatomy. First, the mouse’s external body shape, lungs and hind limbs (i.e.tibiae, fibulae, and feet) were contoured on every slice collected. Next, theradiation fields were configured using an anterior-posterior parallel opposedpair isocentric technique with the prescription point at the mouse’s tho-rax (midline level), as shown in Figure 2.3. The major advantages of thistechnique are the simplicity and reproducibility of the configuration, andthe possibility to achieve a uniform dose distribution for the mouse’s smallthickness at low photon beam energies (4-6 MV)[33].Three treatment plans were developed: whole-thorax irradiation, partial-body irradiation with minimal bone marrow shielding (i.e. tibiae, fibulae andfeet), and partial-body irradiation plus simultaneous-infield-boost (SIB) tothe thorax. The LINAC collimator jaws provided the principal shieldingand defined the treatment field sizes: 2.4cm x 2cm for the thoracic fields,and 3.2cm x 9cm for the body fields (defined at the isocentre level). Themulti-leaf collimator (MLC) leaves were used to shield the desired portion ofthe mouse’s bone marrow, thus providing further field customization. Thesetup is displayed in Figure 2.4.232.5. Radiation treatment plan(a) (b)(c) (d)Figure 2.3: EclipseTM TPS interface for external beam planning. Panels(a), (c), and (d) display the mouse’s reconstructed CT images in the axial,coronal and sagittal planes, respectively; the prescription point was fixedat the thorax’s midcoronal level, coincident with the machine’s isocentreat 100 cm SAD. Panel (b) shows the 3D field configuration. Treatmenttechnique consists of two static equally weighted photon fields directed alongthe prescription point’s axis from opposite sides (anterior, gantry angle zero;posterior, gantry angle 180) of the mouse’s volume.242.5. Radiation treatment plan(a)(b)Figure 2.4: Beam’s eye view digitally reconstructed radiograph of the ante-rior field for the partial-body irradiation plan. Shown in red are the con-touring of the mouse’s lungs and the portion of bone marrow to be shielded.Shown in yellow are the rectangular fields defined by the collimator jaws(X1, Y1, X2, Y2) for (a)body (b)lung SIB, and the shielding customizationprovided by the MLC (small rectangles defined on the mouse’s legs).For treatment planning, a 6 MV flattening-filter-free (FFF) photon beamwas selected. The 6 MV photon energy was chosen to provide a less penetrat-ing beam (depth of maximum dose in water is 1.5 cm[56][77]), appropriate252.5. Radiation treatment planfor the small mouse’s thickness. A flattening-filter-free (FFF) beam waschosen to produce a softer (less penetrating) beam and to decrease treat-ment time. In conventional clinical linear accelerators, the flattening filteris inserted in the path of the generated megavoltage bremsstrahlung beams,which have a bell-shaped profile with the highest intensity at the center,to make beam intensity uniform across the field[33]. However, small fields,such as those used in this thesis to treat the mice, are sufficiently uniformin intensity for a given mouse’s cross-section, hence the flattening filter isnot necessary. Also, removing the flattening filter increases the incidentphoton fluence[33], as lower energy photons that would have otherwise beenattenuated now enter the beam, thus more photons are available to treat.This results in an increase in dose rate from 600 Monitor Units per minute(MU/min) (flattened beam) to 1400 MU/min [77], translating into shortertreatment times. Finally, a FFF beam reduces the out-of-field head scatter,hence reducing the dose to the out-of-field tissues.Figure 2.5: Anterior field dose buildup calculation by the treatment planningsystem. The irradiation jig provides a water equivalent depth of 1.45 cm sothat the maximum dose depth coincides with the mouse’s midcoronal level.This provides a homogenous dose distribution across the thorax.During treatment simulation, the sample mouse was placed in a 3-mmthick Perspex box to provide dose buildup for the anterior fields so that thedepth of maximum dose is brought up to the mouse’s midcoronal level. Thebuildup for the posterior fields was provided by the patient couch. Buildupwas verified in the TPS by calculating the water-equivalent distance (WED)262.5. Radiation treatment planfrom the jig (anterior field) and couch (posterior) surface to the prescriptionpoint, as shown in Figure 2.5. The values for WED determined by theTPS were of approximately 1.45 cm and 1.58 cm, respectively, hence beingcloser to the 1.5-cm depth of maximum dose in water for a 6 MV photonbeam[77][56].Finally, the treatment planning system calculated the spatial dose distri-bution, dose-volume histogram, and key statistics tools for plan evaluation,to determine whether the plan delivers a uniform dose distribution insidethe target volume and an as low as possible dose to the portions to be sparedfrom radiation. These include minimum, maximum, and mean dose to thevolume of interest[56]. The dose-volume histogram (DVH) condenses theinformation contained in the 3D dose distribution plan into a 2D frequencydistribution of dose values within a defined volume, displaying the percentvolume of the total volume of a structure (y-axis) against received dose(x-axis)[56].To illustrate, a few parameters are described for the 8 Gy PBI plus 8Gy lung SIB plan. Figure 2.6 displays the TPS interface and the statisticalparameters generated. For this model, the mean total dose to the lung was16.4 Gy (min dose 15.6 Gy, max dose 16.7 Gy). The mean dose to theshielded hind limbs was 0.4 Gy (min dose 0.095 Gy, max dose 2.30 Gy),with less than 5% of the volume of these bones receiving a dose of 1 Gyor greater as seen in the DVH, thus confirming bone marrow sparing fromexposure. Finally, the body fields provided a dose coverage of 100% of themouse’s body (excluding shielded parts), with 7.5 Gy received by 85% ofthe target volume, as indicated by the DVH.272.5. Radiation treatment plan(a) (b)(c) (d)(e)Figure 2.6: Treatment planning system statistics for the 8 Gy PBI + 8 GySIB lung plan: (a) Axial view of isodose line distribution and prescriptionpoint; (b) DVH for lung, body, and shielded tissue; (c) Coronal view ofisodose line distribution and prescription point; (d) Sagittal view; (e) Dosecoverage, minimum, maximum and mean dose to structures of interest.28Chapter 3Materials and Methods3.1 MiceFemale mice of the C3H/HeJ inbred strain were purchased from TheJackson Laboratory (Bar Harbor, Maine, USA) and housed in the animalfacility of the UBC Okanagan Arts and Sciences Centre. Animal use wasapproved by the University of British Columbia Animal Care Committee.All mice were handled according to the guidelines and regulations of theCanadian Council of Animal Care.3.2 IrradiationAt 8 weeks of age, mice were irradiated with 6 MV flattening-filter-freephotons produced by a Varian TrueBeam Linear Accelerator at BC Cancer,Kelowna. To preserve correct positioning during treatment delivery, micewere anesthetized before irradiation with an intraperitoneal injection of al-faxalone (56 mg/kg of body weight) and xylazine (3.5 mg/kg). Anesthetizedanimals were placed in the Perspex box with the shoulders properly alignedto ensure whole thorax exposure and mice receiving a body dose had theirlegs pulled apart and fixed with tape to help keep this portion of the bonemarrow outside the radiation field. The jig was positioned on the treatmentcouch and aligned as previously done in the CT simulator. This set up isdisplayed in Figure 3.1. The LINAC was operated from the control room.Before irradiation, a kilovoltage image (56 kVp, 4.38 mAs, dose ∼ 0.01cGy)of the mouse was taken to confirm that placement matches the plan posi-tioning, with lungs centered in the fields. The dose was delivered at a rateof 1400 MU/min, with output rates at the mouse’s thorax midline level ofapproximately 0.99 cGy/MU for the partial-body fields, and 0.93 cGy/MUfor the lung fields. Treatment was completed in 1820 MU for the 8 Gy PBI+ 8 Gy lung SIB plan, 910 MU for the 8 Gy PBI plan, and 1880 MU for 16Gy whole-thorax irradiation. Dose delivery times were 1.3, 0.65, and 1.34mins, respectively. Mice were observed via in-room cameras to confirm that293.3. Dosimetrythey remained immobilized throughout treatment.Figure 3.1: TrueBeam Linear Accelerator. The Perspex jig on the treatmentcoach contains the mouse correctly positioned for treatment delivery.3.3 DosimetryFor partial body irradiations, in vivo dosimetry measurements were per-formed using LiF thermoluminescent dosimeters (TLD) as done by Chua andothers[8], to verify that the tissue outside of the radiation field was shieldedand validate the treatment plan system dose calculations. TLD chips weretaped on the tibiae of a subset (n=4) of mice receiving PBI with minimalbone marrow shielding and treatment was delivered. This configuration isdisplayed in Figure 3.2.Measurement consistency of the dosimeters was verified by irradiatingseveral TLD chips under a polystyrene slab in an isocentric set-up at the fol-lowing reference conditions: field size 10 cm x 10 cm at a depth d=dmax=1.5cm. 200 MU units were delivered at a dose rate of 1 cGy/MU so that thecontrol dose to the TLDs was 200 cGy. The chips were read using a Har-shaw 5500 TLD Reader readout system. The results are shown in Table 3.1.303.3. DosimetryThe in vivo measured dose to the hind limbs ranged from 14-63 cGy, henceconfirming bone marrow shielding.Figure 3.2: In vivo dosimetry with TLDs on mouse tibiae.Table 3.1: Dose to tibiae measurements by TLDs.Mouse # Treatment Dose to tibiae1 6 Gy PBI + 10 Gy lung SIBLeft: 21.1 cGyRight: 14.2 cGy2 6 Gy PBILeft: 27.6 cGyRight: 23.4 cGy3 6 Gy PBILeft: 40.6 cGyRight: 27.4 cGy4 8 Gy PBILeft: 63.0 cGyRight: 22.2 cGy313.4. Health Monitoring3.4 Health MonitoringAfter treatment was completed, mice were allowed to recover from anes-thesia, returned to the animal facility, and monitored for health status ac-cording to a protocol approved by the UBC Animal Care Committee. Micewere examined daily for the first two weeks post-treatment and then threetimes a week. Weight, appearance, behavior, activity, respiration, and hy-dration status were scored according to the “UBC Facilities Grading systemand response for rodents” protocol. Mice showing signs of distress such aspiloerection, hunched posture, grimace, reduced activity, accelerated breath-ing, and/or over 20% weight loss were humanely euthanized with an in-traperitoneal injection of Sodium Pentobarbital (0.1 mL/kg). All survivingand nonirradiated control mice were sacrificed at an experimental time pointof 16 weeks post-treatment. In addition, a subset of animals was sacrificedat a time point of one-week post-irradiation for the preliminary study as-sessing acute damage to the hematopoietic system as described in section2.4.1.3.5 Peripheral Blood CountsFor each mouse, a minimum of 200 µL of whole blood was collectedupon euthanasia via intracardiac puncture and transferred into a lavender-top tube coated with ethylenediaminetetraacetic acid (EDTA) to preventcoagulation. Specimens were submitted to Antech Diagnostics Lab (Rich-mond, BC) for a Complete Blood Count plus White Blood Cell differential.3.6 Lung HistologyAt necropsy, lungs were collected, and the single left lobe of each mousewas inflated with 10% neutral buffered formalin. Samples were immersedin a container filled with fixative and submitted for standard histologicalprocessing.Pneumonitis severity for each mouse was determined by semi-quantitativeanalysis of hematoxylin and eosin (H&E)-stained lung sections with an op-tical microscope, using a scoring system from 0 to 6 designed by Haston andothers[36][37]. A score of 0 corresponded to no alveolitis, 1 indicated one ortwo foci of alveolar inflammatory cells, 2 several foci of alveolar inflamma-tory cells with thicker alveolar walls, 3 indicated alveolar inflammatory cellswith thicker alveolar walls found in more than 50% of the entire left-lung323.7. Statistical Analysissection, 4 these traits found in more than 75% of the lung section, 5 in morethan 90%, and 6 corresponded to extreme pneumonitis (characterized by ex-cessive thickening of the alveolar walls with cellular infiltrate and exudatespresent in the alveolar space of the entire lung section).3.7 Statistical AnalysisRelative body weights, blood cell counts, and lung pneumonitis scoresfor each treatment group were displayed as average ± standard error of themean. Statistical differences in these traits among treatment groups were de-termined using an ordinary one-way ANOVA, followed by a Tukey’s post hoctest to highlight significant differences between two specific groups. Survivaldata were plotted as Kaplan-Meier curves. Differences in survival curves be-tween treatment groups were calculated with a Log-rank test. These analy-ses were performed using GraphPad Prism software (www.graphpad.com).Blood cell count differences between the partial-body irradiation and whole-thorax irradiation groups were further assessed with two-tailed Student’st-tests performed in Microsoft Excel software. For all tests, a value of p ≤0.05 was considered statistically significant.33Chapter 4Results4.1 Relative Body Weight and Survival atone-week post treatmentTo develop a survivable mouse model of partial-body irradiation withminimal bone marrow shielding, two partial-body doses, 6 and 8 Gy, wereevaluated for effect on mouse health and hematological parameters at one-week post-treatment.To determine whether 6 or 8 Gy partial-body irradiation caused a sig-nificant decline in body weight (main indicator of health status) comparedto 16 Gy whole-thorax exposure at one week following treatment, mice re-ceived one of the following regimes: (1) 6 Gy partial body irradiation (PBI)sparing the tibiae, ankles and feet, (2) 6 Gy PBI plus a 10 Gy simultaneous-infield-boost (SIB) to the lung, (3) 8 Gy PBI, (4) 8 Gy PBI plus 8 Gy lungSIB, (5) 16 Gy whole-thorax lung irradiation (WTLI), or (6) no treatment(controls), and were weighed daily for one week.To investigate significant differences in body weight among treatmentgroups, the relative weight (normalized to pre-irradiation weight) of eachmouse per day was obtained, and the daily average relative weight of eachgroup was calculated to perform ANOVA+Tukey’s tests as done by Tomko[72],Kunwar and others[34]. Relative rather than absolute weight values wereused because groups exhibited significant differences in body weight at pre-treatment (ANOVA’s p=0.0360). Hence, by setting the baseline weightsof all mouse groups to a common value of 1, pre-treatment variations maybe eliminated, so that groups can be tested for differences solely due toirradiation regime.As shown in Figure 4.1, there was a significant decline in relative bodyweight with respect to untreated controls on mice receiving 6 Gy PBI plus10 Gy lung SIB at days 2, 4, and 5. Similarly, the relative weight of micegiven 8 Gy PBI plus 8 Gy lung SIB was significantly lower at days 2 and4 post-irradiation. Finally, mice receiving 8 Gy PBI had lower relativeweights 4 days following treatment. Whole-thorax irradiation did not have344.1. Relative Body Weight and Survival at one-week post treatmenta significant effect on body weight throughout one-week post-treatment.Compared to mice exposed to 16 Gy WTLI, mice treated with 6 Gy PBIplus 10 Gy lung SIB exhibited significantly lower relative weights at days 2,3, and 5 following treatment. Likewise, 8 Gy PBI plus 8 Gy lung SIB exposedmice had significantly lower relative weights at days 2 and 3. Nevertheless,weights of partial-body irradiated mice started to recover after reaching theirnadir, and no significant differences among groups were observed by day 7.With regards to one-week post-treatment survival, all mice survived to thistimepoint with no visible signs of distress.In summary, these results demonstrate that treatment with either 6 or8 Gy partial-body irradiation is survivable with no visible signs of distressat one week following treatment. There is an initial onset of weight loss inmice receiving 6 or 8 Gy partial-body irradiation; however, rapid recoveryoccurs and no differences in relative body weight compared with nonirradi-ated controls are observed by one week.Figure 4.1: Mean body weight relative to pre-irradiation weight ± SEM overone week post-treatment; n=4-10 mice per group. Red * indicates signifi-cant difference compared with untreated controls, blue * indicates significantdifference compared with 16 Gy WTLI according to Tukey’s post hoc test(significance given by p ≤ 0.05).354.2. Hematological measures at one-week post-treatment4.2 Hematological measures at one-weekpost-treatmentTo determine whether 6 or 8 Gy partial-body irradiation depleted hematopoi-etic cell counts more than 16 Gy whole-thorax irradiation, blood was col-lected from the mice treated in section 4.1 via intracardiac puncture one-week post-treatment and analyzed through complete blood count.As shown in Figures 4.2(a) and (c), all groups receiving partial-body irra-diation (with or without SIB to the lung) had significantly lower mean totalWhite Blood Cell (WBC) and lymphocyte counts than nonirradiated controlmice at one-week post-treatment (vs controls Tukey’s p<0.0003). Similarly,both 6 Gy and 8 Gy PBI significantly decreased circulating monocytes (Fig-ure 4.2(d)) and eosinophil counts (vs controls p<0.0292) (Figure 4.3(a)).In contrast, only mice exposed to 8 Gy PBI exhibited significantly lowerneutrophil counts (vs controls p=0.0069) (Figure 4.2(b)).Treatment with 16 Gy whole-thorax irradiation did not significantly af-fect one-week total WBC counts (vs controls p=0.0715). Likewise, 16 GyWTLI did not significantly decrease circulating neutrophil, monocyte andeosinophil numbers (vs controls p>0.5747). Nevertheless, this treatment sig-nificantly decreased lymphocyte counts compared with controls (p=0.0402).When comparing leukocyte depletion between partial-body and localizedwhole-thorax irradiation, total WBC counts of mice exposed to 8 Gy PBIand 8 Gy PBI + 8 Gy lung SIB were significantly lower than of mice given16 Gy WTLI (p=0.0010 and p=0.0391, respectively). Similarly, 8 Gy PBIdepleted lymphocyte, neutrophil and monocyte counts significantly morethan 16 Gy WTLI (p<0.0382). Average total WBC and differential countsfor mouse groups receiving the 6 Gy partial-body dose were lower than ofmouse treated with 16 Gy WLTI; however, differences were not statisticallysignificant (p>0.0657), except for monocytes (p> 0.0072). Finally, no signif-icant differences in eosinophil counts between partial-body and thorax-onlytreated mice were found at either dose(p>0.1503).Platelet counts were also assessed one week following treatment. Asshown in Figure 4.3, only mice exposed to 8 Gy partial-body irradiationhad significantly lower platelet counts than of mice given 16 Gy WTLI andcontrols (p=0.0181 and p=0.0062, respectively).Finally, Red Blood Cell (RBC) parameters were also assessed at one weekpost-irradiation. As shown in Figure 4.4, there were no significant differencesin RBC counts among any of the treatment groups. Likewise, radiationtreatment did not affect hemoglobin, mean corpuscular volume(MCV),364.2. Hematological measures at one-week post-treatment(a) (b)(c) (d)Figure 4.2: Average (a) total WBC, (b) neutrophil, (c) lymphocyte and (d)monocyte counts ± SEM; n=4-9 mice per group. The line above the groupsindicates significance level according to one-way ANOVA. # Indicates statis-tically significant difference compared with controls, * indicates significantdifference compared with 16 Gy WTLI according to Tukey’s post hoc test(p ≤ 0.05).374.2. Hematological measures at one-week post-treatment(a) (b)Figure 4.3: Average (a) eosinophil and (b) platelet counts ± SEM; n=4-9mice per group. Line above the groups indicates significance level accordingto one-way ANOVA. # Indicates statistically significant difference comparedwith controls, * indicates significant difference compared with 16 Gy WTLIaccording to Tukey’s post hoc test (p ≤ 0.05).mean corpuscular hemoglobin (MCH) (also shown in Figure 4.4), meancorpuscular hemoglobin concentration (MCHC) and hematocrit (HCT) lev-els (not shown in Figure).When comparing mice treated with PBI versus PBI plus lung simultaneous-infield-boost, no significant differences in average WBC counts between thosegroups were found at either of the partial-body doses tested (6 Gy PBI vs6 Gy PBI + 10 Gy SIB p>0.9999; 8 Gy PBI vs 8 Gy PBI + 8 Gy SIBp=0.9979). Hence, the top-up lung dose did not have a significant effect onWBC depletion in mice receiving partial-body irradiation measured at oneweek post-treatment. This finding was also true for all individual leukocytepopulations assessed.These findings demonstrate that 8 Gy partial-body irradiation depletesone-week hematopoietic cell counts, specifically leukocytes and platelets,significantly more than 16 Gy whole-thorax radiation treatment. Based onthis, a partial-body dose of 8 Gy was selected to perform the rest of theexperiments.384.2. Hematological measures at one-week post-treatment(a) (b)(c) (d)Figure 4.4: Average (a)total RBC counts, (b) Hemoglobin levels, (c) MeanCorpuscular Volume, (d) Mean Corpuscular Hemoglobin ± SEM; n=4-9mice per group. The line above the groups indicates significance level ac-cording to one-way ANOVA. # Indicates statistically significant differencecompared with controls, * indicates significant difference compared with 16Gy WTLI according to Tukey’s post hoc test (p ≤ 0.05).394.3. Survival and relative body weights for 8 Gy partial-body irradiation4.3 Survival and relative body weights for 8 Gypartial-body irradiationTo assess whether the 8 Gy partial-body dose was survivable throughoutthe 30-day time window of mortality from Acute Radiation Syndrome inmice[55][17] and the 16-week end of experiment (eoe) timepoint, a set ofmice (n=9) was exposed to 8 Gy partial-body irradiation with minimalbone marrow shielding and monitored weekly for health status over suchperiod. To establish comparisons in body weight, a set of age-matchedunirradiated controls was housed under the same conditions and euthanizedat the experimental endpoint.To determine whether 8 Gy PBI significantly affected mouse body weightthroughout the study, mice were weighed weekly and the average bodyweight (relative to pre-irradiation weight) of each group was calculated to en-able testing for significant differences between nonirradiated control mice, asdescribed in section 4.1 (significant difference between 8 Gy PBI and controlgroups in pre-irradiation weights by Tukey’s, p=0.0025). As shown in Figure4.5(a), the mean relative weight of mice receiving 8 Gy PBI was significantlylower than of control mice at weeks 2,6,7,9,12, and 13 post-treatment. Nev-ertheless, no significant differences were found between these two groups atthe 16-week end of the experiment (p=0.44). Also seen in Figure 4.5(a), noweight loss of ≥20% (evidence of distress) occurred for 8 Gy PBI treatedmice at any timepoint. Finally, all 8-Gy PBI treated mice survived to theexperimental endpoint (shown in Figure 4.6).These results demonstrate that the 8 Gy partial body dose is survivablethroughout the study and that it allows for observation of lung disease inmice receiving 8 Gy body plus the lung top-up dose.4.4 Experimental model: Survival time andRelative Body weightsTo determine the effect of partial-body irradiation-induced early hematopoi-etic depletion on the development and progression of radiation lung injury,8-week old female C3H/HeJ mice were exposed to either 8 Gy PBI withminimal bone marrow shielding plus an 8-Gy simultaneous in-field boostto the thorax (total lung dose 16 Gy) or 16 Gy whole-thorax irradiation(positive control for lung disease) and euthanized at respiratory distress.404.4. Experimental model: Survival time and Relative Body weights(a)(b)Figure 4.5: Mean body weights relative to pre-irradiation weights ± SEM;n=5-12 mice per group. (a) * indicates significant difference between 8 GyPBI treated mice and controls euthanized at eoe according to Tukey’s posthoc test. (b) Pink * indicates significant difference between controls and8Gy PBI plus 8Gy lung SIB; blue * significant difference between controlsand 16Gy WTLI according to Tukey’s test.414.4. Experimental model: Survival time and Relative Body weightsTo determine whether there were significant differences in the onset ofdistress (characterized by weight loss) between irradiated groups, mice werehealth monitored and weighed weekly following irradiation until they weremoribund or at the experimental endpoint. To perform the weight analysis,the mice exposed to 8 Gy PBI and nonirradiated controls discussed in Sec-tion 4.3 were also included. Average weekly relative weights were comparedby ANOVA +Tukey’s test (ANOVA of pre-treatment weights p=0.0023).As shown in Figure 4.5(b), there were no significant differences in relativebody weight at any week between 8 Gy PBI plus 8 Gy lung SIB and 16 GyWTLI exposed mice. Compared to nonirradiated control mice, the relativebody weight of mice receiving 8 Gy PBI plus 8 Gy lung SIB was significantlylower 1, 3, 6, and 7-16 weeks following irradiation. Similarly, the relativeweight of mice receiving 16 Gy WTLI was significantly lower 2, 3, 7-9, and11-15 weeks. Also seen in Figure 4.5(b), the timeline of onset of weight losswas similar in mice exposed to either treatment (∼ 12 weeks). Thus, partial-body irradiation did not affect the onset and timeline for the progression ofradiation-induced distress.Figure 4.6: Percent survival of mice exposed to 8 Gy PBI plus lung SIB (solidline) and 16 Gy whole-thorax lung exposure (dashed line). Mice exposedto 8 Gy PBI are displayed as controls for partial body dose (double-dashedline).To investigate whether there were significant differences in survival time424.5. Hematological measures at respiratory distressbetween mice exposed to 8 Gy PBI plus 8 Gy lung SIB and mice exposedto 16 Gy WTLI, survival curves were compared by the Log-rank test. Asshown in Figure 4.6, there were no significant differences in survival betweenmice receiving 8 Gy PBI + 8 Gy lung SIB and mice given 16 Gy WTLI(p=0.1732). Hence, 8 Gy partial-body exposure did not significantly affectsurvival time in mice receiving a total lung dose of 16 Gy.4.5 Hematological measures at respiratorydistressTo determine the effect of thoracic irradiation on hematologic cell num-bers at the time of lung pneumonitis, the mice irradiated in Section 4.4 hadtheir blood collected via intracardiac puncture upon sacrifice for a completeblood count. Specifically, to determine whether partial-body irradiation sig-nificantly affected circulating blood cell levels at respiratory distress, averagecounts for mice receiving 8 Gy PBI plus 8 Gy lung SIB or 16 Gy WTLI andsacrificed at humane endpoint were compared by a Student’s t-test. Further-more, to identify the specific blood cell types associated with symptomaticlung pneumonitis, a subset of mice (n=3) exposed to 8 Gy PBI plus 8 Gylung SIB was sacrificed without presenting any sign of distress at 14 weekspost-irradiation (observed time point at which mice started to succumb torespiratory distress). The mice given 8 Gy PBI only and nonirradiated con-trols euthanized at the experimental endpoint had also their blood collectedfor analysis to provide control for partial-body irradiation and untreatedmeasures, respectively.By the 16-week experimental endpoint, there were no significant differ-ences in any cell type analysed between 8 Gy PBI exposed mice and controls(Figures 4.7, 4.8, 4.9). Hence, the partial-body dose alone did not have asignificant effect on mouse hematopoietic cell counts at 16-weeks followingexposure.As shown in Figure 4.7, radiation treatment significantly increased totalWBC counts in mice receiving 8 Gy PBI plus 8 Gy lung SIB and sacrificedat respiratory distress (vs controls p=0.0410). Average lymphocyte countswere also higher in mice given this treatment than controls; however, theeffect was not significant according to Tukey’s test (vs controls p=0.0884).The leukocyte depletion observed at one week following 8 Gy partial-bodyirradiation did not affect WBC and lymphocyte levels upon respiratory dis-tress, as no significant differences in these measures between mice exposedto 8 Gy PBI plus 8 Gy lung SIB and 16 Gy WTLI and euthanized when434.5. Hematological measures at respiratory distressmoribund were found (p>0.7044). Finally, total WBC and lymphocyte lev-els were not associated specifically with symptomatic respiratory distress,since no significant differences between moribund and nondistressed 8 GyPBI plus 8 Gy lung SIB treated mice in those parameters were observed(p>0.4989).(a) (b)Figure 4.7: Average (a) White Blood Cell and (b) Lymphocyte counts ±SEM; n=3-8 mice per group. The line above the groups indicates significancelevel according to one-way ANOVA. # indicates significant difference withcontrols according to Tukey’s post hoc test; * significant difference with 8Gy PBI plus 8 Gy lung SIB sacrificed at 14 weeks (nondistress), + significantdifference between 8 Gy PBI plus 8 Gy lung SIB and 16 Gy whole-thoraxirradiation sacrificed at distress by Student’s t-test (p ≤ 0.05).As shown in Figure 4.8, mice given 16 Gy WTLI had significantly higherneutrophil (p=0.0082) and monocyte (p=0.042) levels upon succumbing tolung pneumonitis than control mice. Partial-body irradiation did not affectthe raise in neutrophils at distress since no significant differences in neu-trophil counts between 8 Gy PBI plus 8 Gy lung SIB and 16 Gy WTLI ex-posed mice sacrificed at humane endpoint were found. However, neutrophilcounts of nondistressed 8 Gy PBI plus 8 Gy lung SIB treated mice weresignificantly lower than 16 Gy WTLI treated mice (p=0.0238). This finding444.5. Hematological measures at respiratory distresssuggests that increased blood neutrophils are a feature associated with pul-monary distress (symptomatic pneumonitis). Finally, monocyte counts werenot increased for 8 Gy PBI plus 8 Gy lung SIB exposed mice, distressed ornot. These findings suggest that increased monocyte counts was associatedwith symptomatic pneumonitis only in mice exposed to localized thoracicirradiation.(a) (b)Figure 4.8: Average (a) Neutrophil and (b) Monocyte counts ± SEM; n=3-8mice per group. The line above the groups indicates significance level ac-cording to one-way ANOVA.# indicates significant difference with controlsby Tukey’s post hoc test; * significant difference with 8 Gy PBI plus 8 Gylung SIB sacrificed at 14 weeks (nondistress), + significant difference be-tween 8 Gy PBI plus 8 Gy lung SIB and 16 Gy whole-thorax irradiationsacrificed at distress by Student’s t-test (p ≤ 0.05).454.5. Hematological measures at respiratory distress(a) (b)(c) (d)Figure 4.9: Average (a) Eosinophils, (b) Red Blood Cells, (c) Platelets, (d)Mean Corpuscular Volume ± SEM; n=3-8 mice per group. The line abovethe groups indicates significance level according to one-way ANOVA, NS =not significant. # indicates significant difference with controls by Tukey’spost hoc test; * significant difference with 8 Gy PBI plus 8 Gy lung SIBsacrificed at 14 weeks (nondistress), + significant difference between 8 GyPBI plus 8 Gy lung SIB and 16 Gy whole-thorax irradiation sacrificed atdistress, by Student’s t-test (p ≤ 0.05).464.6. Lung HistopathologyFinally, as shown in Figure 4.9, radiation exposure did not significantlyalter eosinophil levels compared with controls according to ANOVA + Tukey’s(p>0.05). Eosinophil numbers were significantly higher in distressed 8 GyPBI plus 8 Gy lung SIB treated mice than mice given 16 Gy WTLI (p=0.0303).Nevertheless, eosinophil levels were not associated with symptomatic lungpneumonitis, as no significant differences between distressed versus nondis-tressed partial-body treated mice were observed (p>0.1542). Likewise, nosignificant differences among groups in platelet and Red Blood Cell param-eters were found by ANOVA test. RBC, HGB and HCT levels were sig-nificantly higher in distressed than nondistressed 8 Gy PBI plus 8 Gy lungSIB treated mice. However, these differences did not appear to be due toradiation-induced distress, since distressed mice did not differ from controlsin those parameters.4.6 Lung HistopathologyTo determine whether the lung phenotype resulting from 8 Gy partial-body irradiation plus 8 Gy lung SIB significantly differed from that of 16 Gywhole-thorax irradiation, the lung tissue of mice irradiated in Section 4.4was histologically assessed for pneumonitis severity. Similarly, histologicalanalysis of lung tissue was performed on mice treated in Section 4.3 toinvestigate whether 8 Gy partial-body irradiation alone significantly affectedthe lung phenotype compared with that of nonirradiated controls.As shown in Figures 4.10 and 4.11, mice of both 16 Gy WTLI and 8Gy PBI plus 8 Gy lung SIB groups developed significant pneumonitis (score>3) upon respiratory distress compared with lungs of untreated controls(Tukey’s p<0.0001). The phenotype was characterized by the thickening ofthe alveolar walls and cellular infiltrates into the airspaces.There were no statistically significant differences in pneumonitis scorebetween 16 Gy WTLI and 8 Gy PBI plus 8 Gy lung SIB treated mice sacri-ficed at distress (p=0.9640). This finding indicates that early hematopoieticcell depletion resulting from partial-body irradiation did not have a signifi-cant effect on pneumonitis severity.Also shown in Figures 4.10 and 4.11, mice exposed to 8 Gy PBI plus8 Gy lung SIB and sacrificed at 14-weeks experimental endpoint (nondis-tressed) had significantly lower pneumonitis score than mice euthanized atdistress (p<0.0001). Moreover, nondistressed mice did not have extensivepneumonitis (score <3).Finally, the pneumonitis score of mice exposed to 8 Gy PBI only did474.6. Lung Histopathologynot differ significantly from that of control mice (p=0.9950). This findingindicates that a total dose to the lung of 8 Gy was not sufficient to induceobservable pulmonary injuries in the C3H/HeJ mice and that partial-bodyirradiation alone does not influence the pulmonary radiation response if thedose to the lung is below the threshold for pulmonary effects.Figure 4.10: Mean pneumonitis scores ± SEM; n=3-9 mice per group.The pneumonitis score was based on semi-quantitative assessment of H&Estained lung sections. The line above the groups indicates significance levelaccording to one-way ANOVA. # indicates significant difference with con-trols by Tukey’s post hoc test; * significant difference with 8 Gy PBI plus 8Gy lung SIB sacrificed at 14 weeks (non distress) (p ≤ 0.05).484.6. Lung Histopathology(a) 16Gy WTLI (distress) (b) 8Gy PBI + 8Gy lung SIB (distress)(c) 8Gy PBI + 8Gy lung SIB (14 weeks-nondistress) (d) 8Gy PBI (16 weeks)(e) Controls (16 weeks)Figure 4.11: Left-lung sections stained with H&E illustrating the inflamma-tory cell infiltrates and thickened alveolar walls of pneumonitis; representa-tive example. Magnification=10x.49Chapter 5ConclusionThis thesis found that radiation-induced early hematopoietic cell deple-tion resulting from high-dose partial-body irradiation with minimal bonemarrow shielding did not affect the onset, progression, and severity of respi-ratory distress in a mouse model. Partial-body irradiation significantly re-duced peripheral blood monocyte counts at symptomatic respiratory distresscompared with localized whole-thorax irradiated mice, whose counts wereincreased upon humane euthanasia. Nevertheless, partial-body treatmentdid not influence total neutrophil counts, which were significantly increasedin mice sacrificed upon respiratory distress.Accidental exposure to high-dose ionizing radiation may lead to signifi-cant injuries to multiple organs. This raises the need for the characteriza-tion of high-dose acute radiation exposure effects and the development ofnovel medical countermeasures to treat injured patients. Since these stud-ies cannot be ethically conducted in humans, appropriate animal models arerequired to identify key radiosensitive organ systems, characterize radiation-induced injuries, and test the effectiveness and safety of potential radiationmitigators.The lung is especially sensitive to ionizing radiation, as most evident incancer patients receiving thoracic radiotherapy. Ionizing radiation of thelung causes the pathologies of radiation pneumonitis and fibrosis[34][14].These injuries have been characterized in animal models employing local-ized whole-thorax irradiation[35][16][52], which reproduces the radiotherapyscenario. However, the dose distribution associated with a radiation inci-dent may involve exposure to various tissues, specifically the hematopoieticsystem[47] [39]. Hence, this thesis aimed to determine the effect of radiation-induced injury to the hematopoietic system on the pulmonary radiation re-sponse consequent to a single high-dose significant partial-body exposure, adistribution likely of a radiation accident.To characterize the lung sequelae following an accidental exposure sce-nario, a mouse model of partial-body irradiation with minimal bone mar-row shielding was established to describe the progression, histological andhematological features of radiation lung pneumonitis, and link it to early50Chapter 5. Conclusionradiation-induced hematopoietic system damage.Mice were exposed to a high-dose (6 or 8 Gy) body radiation field to in-duce acute sublethal hematopoietic cell depletion, followed immediately bya thoracic simultaneous in-field boost to elicit lung damage. The body dosedistribution delivered is consistent with the model implemented by MacVit-tie and others in nonhuman primates[40] to model a realistic accidentalexposure scenario; hence mice had their tibiae, ankles, and feet shieldedfrom radiation exposure. The partial-body plus simultaneous in-field boosttechnique was employed because the partial-body dose that results in sur-vivable acute hematopoietic injury is insufficient to cause a lung response,hence, a top-up thoracic field is delivered immediately after the body fieldto bring the total lung dose to 16 Gy, which is on the range that resultsin 100% lethal lung response in mice. This model has also been used byMcBride and others, who exposed C3H/Kam mice to 10 Gy total-body irra-diation plus and additional 4.5 Gy lung only irradiation (total lung dose 14.5Gy) to characterize the pulmonary response following TBI plus bone mar-row transplant. The treated mice developed radiation pneumonitis 80-180days following treatment, with 70% of animals succumbing to pulmonarydistress[45].To verify that bone marrow shielding was accomplished in the treatmentplan and delivery, in vivo dose measurements were performed on a subset ofpartial-body treated mice during irradiations using LiF Thermo Lumines-cent Dosimeter chips attached to the animals’ tibiae. Similar measurementshave been performed by other authors in animal models of acute radia-tion syndrome. Chua and others used optically stimulated luminescence(OSL) dosimeters to verify dose homogeneity in a mouse model of delayedbone marrow damage[8]. Similarly, MacVittie and others used silicon diodedosimeters placed on the hind limbs of irradiated nonhuman primates toconfirm tissue sparing in a model of partial-body irradiation. The authorsmeasured a mean dose to the animal’s tibiae of 0.53 Gy[40]. In this thesis,the mean dose to the mouse’s tibiae ranged from 0.14-0.63 cGy, consistentwith the 0.4 Gy mean dose to the hind limbs calculated by the treatmentplanning system.Consistent with other authors[5], treatment with 6 or 8 Gy partial-bodyirradiation caused mild gastrointestinal symptoms within one week followingtreatment. There was a mild (∼ 7%) but statistically significant decrease inthe body weights of mice receiving partial-body irradiation compared withuntreated controls by days 2-5 post-treatment, followed by weight restora-tion, so that no significant differences between partial-body treated miceand controls were seen at day 7. Whole-thorax irradiation did not decrease51Chapter 5. Conclusionmouse body weights throughout the first week of treatment. The time win-dow for observed weight loss in partial-body irradiated mice is in agreementwith other studies’ reported time for onset of acute GI syndrome in rodents.Booth and others reported a mean time for onset of diarrhea (correlated withweight loss) and crypt depletion in mice of 3.5-5 days following partial-bodyirradiation with 5% bone marrow shielding, after which time animals recov-ered or deteriorated and succumbed to GI distress[5]. In a rat model of ARS,gastrointestinal toxicity manifested 5-7 days after radiation exposure, andit was characterized by early weight loss, dehydration, and reduced appetiteby one-week post-treatment, followed by weight restoration[12]. Finally, be-cause 100% of animals survived to the one-week timepoint with no apparentsigns of illness, both body doses tested allowed for one-week hematopoieticcell counts to be assessed.In agreement with other authors’ murine models of Acute RadiationHematopoietic syndrome[55], treatment with 8 Gy partial-body irradia-tion with minimal bone marrow shielding significantly depleted total WhiteBlood Cell and individual leukocyte type counts more than 16 Gy whole-thorax irradiation at one week following treatment. Also, platelet num-bers were significantly depleted. However, Red Blood Cell counts andHemoglobin measures remained unaltered at one-week post-irradiation. Plettand others characterized the hematologic profile of C57BL/6 mice exposedto 7.97 Gy TBI (Cs 137 source, 0.67 Gy/min) throughout 30 days, and founda rapid decrease in peripheral blood leukocytes by one week after treatment.Total WBC and neutrophil counts reached their nadir 6-10 days followingirradiation. Lymphocyte counts reached their nadir by day 2 and continuedto be severely depleted over 20 days. Nevertheless, platelets and RBC countswere slower in presenting significant decreases. Platelets reached their low-est point by day 17, whereas RBC reached it by day 22 post-treatment[55].Similar timelines for the depletion of blood cell types were observed byother authors. Ghosh and others reported severe WBC, neutrophil, andmonocyte decreases in 6-8 week-old C3H/HeN mice at 4 days following 6Gy TBI (Co60 source, 0.6 Gy/min). Platelet levels declined by days 7-17[17]. Finally, Bunin and others found large depletion in mouse’s WBCand neutrophil counts by 4 days after 6.5 Gy TBI (X-ray unit), and inplatelets by day 10 post-treatment, whereas depletion was more delayed inRBC counts[6].As observed in other studies[67], treatment with 16 Gy local whole-thorax exposure significantly decreased lymphocyte counts at one-week post-treatment. Nevertheless, lymphocyte depletion was more severe for the 8Gy partial body treated mice. Sutherland and others [67], reported lym-52Chapter 5. Conclusionphopenia in C57BL/6 mice following both 4 Gy TBI (single fraction) orlocal thorax irradiation (20 Gy in 5 days, X-ray unit), with the severityof lymphopenia being higher in total-body treated mice. Lower lympho-cyte counts following high-dose whole-thorax exposure have also been seenin nonhuman primate models. Ghandhi and others observed lymphope-nia in thoracic treated NHP within 9 days, followed by a quick recoveryto pre-irradiation levels [16]. Finally, 16 Gy whole-thorax exposure did notsignificantly affect the rest of hematopoietic cell types measured at one week.Because 8 Gy partial-body irradiation significantly depleted the hematopoi-etic system more than 16 Gy WTLI, this body dose was chosen to establishthe model. The following part consisted of assessing whether the 8 Gypartial-body dose was survivable so that later lung injury could be observedin mice given body plus lung boost field. For this purpose, a subset of micewas treated with 8 Gy PBI and monitored for distress for 30 days. As dis-cussed previously, the 30-day survival time point was chosen based on the10-30 day range of mortality from Acute Radiation Syndrome reported formice following high-dose total body irradiation[46].Consistent with findings in nonhuman primate models[41], a minimalamount of bone marrow sparing had a significant effect in mouse survivalfrom acute radiation-induced hematopoietic injury. By 30 days post-treatment,100% of 8 Gy partial-body irradiated mice survived with no visible signs ofdistress (facial grimace, dehydration, % 20 weight loss). MacVittie andothers found in NHP exposed to near whole-body irradiation (6 MV Linacphotons) that sparing 5% of the animal’s bone marrow reduced the severity(but not duration) of myelosuppression and increased the LD50/60 from 7.54to 11.01 Gy[41]. These results are in contrast with other authors’ murinemodels who employed similar body doses but on a total-body irradiationregime. Plett and others reported 70% 30-day mortality in C57BL/6 miceexposed to 7.97 Gy TBI (Cs137, 0.63 Gy/min)[55]. Bunin and others foundsignificant mouse mortality at a lower dose of 6.5 Gy TBI (X-ray unit)[6].Finally, Micewicz and others determined that a whole-body irradiation doseof 7.72 Gy (Cs137, 0.60 cGy/min) caused 70% mortality by 30 days followingtreatment in C3H mice[46].In agreement with other authors[7], peripheral blood cell counts recoveryin 8 Gy PBI treated mice occurred by 16-weeks post-treatment. Hematopoi-etic cell counts of mice assessed at 16 weeks following treatment revealed nosignificant differences between 8 Gy PBI treated mice and nonirradiatedcontrols. This finding is supported by other authors who have describedhematopoietic recovery from acute exposure. Plett and others reportedmouse total WBC, RBC, and platelet counts recovery to normal levels by53Chapter 5. Conclusion30 days following 7.97 Gy total-body irradiation, except for lymphocytes,which were severely depleted by the 30-day end of experiment[55]. Calviand others assessed blood cell counts in C57BL/6 mice receiving 6 Gy TBI(Cs137 source, 1.6 Gy/min), and found normal values for all parametersat 12 weeks post-irradiation. Nevertheless, bone marrow long-term dam-age characterized by decreased hematopoietic stem and progenitor cells wasseen at 12 and 26 weeks, suggesting that persistent long-term damage to thehematopoietic system continues[7].In agreement with previous studies by Haston and colleagues[52][23],the onset of distress, measured by a decline in mean relative body weightin female C3H/HeJ mice exposed to 16 Gy whole-thorax irradiation was 11weeks, with mice succumbing to pneumonitis 13.7 weeks (96 days) followingtreatment.The latency, incidence, and progression of radiation pneumonitis werenot influenced by early hematopoietic system injury in mice receiving 8 Gypartial-body irradiation plus 8 Gy lung simultaneous in-field boost (totallung dose 16 Gy). The average time of onset of distress (11 weeks) and me-dian survival time for 8 Gy PBI plus 8 Gy lung SIB treated mice (101 days,14.4 weeks) were not significantly different from mice given thoracic onlyirradiation. Hence, partial-body irradiation did not have a significant effecton duration and survival from radiation pneumonitis. This result agreeswith findings in rats and nonhuman primates. Fish and others concludedthat early bone marrow and gastrointestinal injuries in rats given PBI didnot affect the latency, incidence, and severity of radiation lung pneumonitis.The authors exposed female rats to either 11-13 Gy whole-thorax irradia-tion (9-10 weeks old) or 11-13 Gy partial-body irradiation with only oneleg out of the radiation field (10-12 weeks old). In this study, the 11-13Gy PBI doses induced early survivable gastrointestinal and hematopoieticsyndromes on the treated rats. In both models, rats developed radiationpneumonitis 42-90 days following treatment, determined by increased respi-ratory rates, with a subset of them succumbing to pulmonary distress andthe rest recovering. Survivor rats exposed to local thorax irradiation lived tothe 448-day end-of-experiment time point while surviving PBI treated ratsdeveloped kidney injuries between 70-120 days post-treatment. The authorsreported no significant differences between treatments in the lethal dose forradiation pneumonitis LD50/90. The values determined were 12.7 Gy [12.6,12.8] for whole-thorax exposure, and 12.5 Gy [12.1, 12.9] for partial-bodyirradiation[12]. In contrast with Fish and others, our study delivered awhole-thorax dose that resulted in lethal lung pneumonitis in 100% of irra-diated mice in both radiation treatments. A strength of our study is that54Chapter 5. Conclusionall mouse treatment groups had the same age.Likewise, MacVittie and others defined the progression of pulmonaryinjury in nonhuman primates exposed to high-dose partial body irradia-tion (10-11 Gy 6 MV LINAC photons, 0.80 Gy/min) with 5% bone marrowshielding. In that study, NHPs were given a dose that resulted in signif-icant early mortality due to gastrointestinal and hematopoietic radiationsyndromes. Hence, lung injury in surviving animals developed in the contextof early and prolonged hematopoietic injuries. The lethal dose LD50/180for partial-body irradiation-induced lung injury was 9.94 Gy [9.35,10.29](n=87). This dose was not significantly different (p=0.86) from the LD50/180determined in a previous model of local whole-thorax irradiation by the sameauthor, which was 10.24 Gy [9.88, 10.52] (n=76). Median survival times ofpartial-body irradiated NHP succumbing to lung injury were 171 (10 Gy)and 105 days (11 Gy). Similar times were seen for whole-thorax exposedanimals, 163 (10 Gy) and 124 days (11 Gy). Finally, the time for onset ofrespiratory distress, as measured by the first day of respiratory rate > 80bpm was not significantly different between treatments[42].As previously discussed, a hallmark of radiation pneumonitis is infiltra-tion of immune myeloid and lymphoid cells into the lungs. For that reason,it was of interest to measure peripheral blood cell counts at the time ofrespiratory distress, to identify associations between these parameters andradiation pneumonitis, as well as to determine whether partial-body irradi-ation altered hematologic profile at respiratory distress.Increased white blood cell counts have been identified as markers ofinflammation in various studies[70]. Mice exposed to 8 Gy PBI + 8 Gylung SIB and sacrificed at respiratory distress had significantly higher WBCcounts than untreated controls. The rise in WBC counts agrees with findingsin a minipig model, in which animals presented higher WBC counts in pe-ripheral blood and bronchoalveolar lavage fluid (BALF) at 6-17 weeks afterhigh-dose local right hemithorax treatment[35]. Similarly, Tang and othersfound significant associations between high post-radiotherapy White BloodCell (WBC) counts and incidence of grade ≥ 2 and 3 radiation pneumoni-tis in a retrospective analysis of patients receiving treatment for non-smallcell lung cancer[70]. Also, the fact that no significant differences between 8Gy PBI + 8 Gy lung SIB and 16 Gy whole-thorax treated mice euthanizedat distress were found, shows that partial-body irradiation does not affectWBC increase at the onset of distress.Moreover, since WBC counts did not differ between distressed and nondis-tressed partial-body treated mice sacrificed at a similar time point (∼ 14weeks), it was concluded that WBC counts do not raise exclusively at the55Chapter 5. Conclusiontime of symptomatic illness. Similar observations were made in the minipigmodel[35], where peripheral WBC and lymphocyte counts peaked at 6 weekspost-treatment and remained high until the period of pneumonitis. How-ever, no gross signs of lung inflammation had been found in animals at 6and 12 weeks upon bronchoscopy evaluation. Kunwar and others also re-ported increases in BALF leukocyte and lymphocyte percentages, at both 8weeks (a time point before the onset of symptoms) and 12 weeks (humaneendpoint) following thoracic treatment in female C3H/HeJ[34].Contrary to other studies, blood lymphocyte counts of 16 Gy whole-thorax irradiated mice were not significantly higher than of nonirradiatedcontrols. Increased lymphocyte numbers in both BALF and lung tissue havebeen observed in other mouse models[34][30] and in radiotherapy patientsdeveloping pneumonitis[71][57], so this finding was unexpected. The lack ofstatistical significance when comparing lymphocyte counts of irradiated micewith controls may be attributed to the wide variations in lymphocyte levelsbetween individuals of a specific treatment group. For instance, lymphocytecounts for thoracic only treated mice ranged from 4.1-24.2x109 counts/L.A larger number of mice per group may be required to confirm this re-sult. Finally, consistent with the findings for WBCs, high-dose partial-bodyirradiation did not affect lymphocyte counts at respiratory distress, sinceno significant differences between 8 Gy PBI + 8 Gy lung SIB and 16 Gywhole-thorax treated mice euthanized at distress were found.Mice exposed to 16 Gy WTLI had significantly higher blood neutrophilcounts at distress. This finding agrees with Haston and others, who re-ported increased neutrophil counts in bronchoalveolar lavage of C3H/HeJmice succumbing to pneumonitis following 18 Gy WTLI, and in geneti-cally mixed mice who developed a pneumonitis response[23]. Supportingneutrophil involvement in pulmonary distress, higher neutrophil counts inthe lung tissue of mice developing radiation pneumonitis have also beenobserved[14]. Further validating the contribution of these immune cells as adriving mechanism of radiation lung disease, Kunwar and others found thatadministration of the organoselenium compound DSePA in C3H/HeJ miceafter local thorax irradiation reduced the influx of inflammatory cells, espe-cially of neutrophils, delayed the onset of respiratory distress, and decreasedseverity of pneumonitis[34].Partial-body irradiation did not affect the raise of neutrophils upon res-piratory distress, as no significant differences between thoracic and partial-body treatments were found on mice sacrificed because of respiratory symp-toms. Nevertheless, neutrophil counts in nondistressed 8 Gy PBI + 8 Gylung SIB treated mice euthanized at 14 weeks was significantly lower than56Chapter 5. Conclusionthose of distressed mice. Hence, the neutrophil increase was associated solelywith symptomatic respiratory distress. Similar observations have been re-ported by Ghandhi and others, who exposed NHPs to a single dose of 10Gy WTLI (6 MV LINAC photons, 6Gy/min), and found that neutrophilssharply increased by three times the baseline at day 66; this increase wasrelated to the pneumonitis response as confirmed by computed tomography(CT) and histology[16]. Neutrophils have been identified to contribute tothe development and progression of infectious and non-infectious pulmonaryinjuries, where disparate neutrophil increases resolve in tissue inflammationand damage. Neutrophil infiltration has also been involved in the lethal in-flammatory lung response consequent of ricin (plant-derived toxin) exposurein mice. To further demonstrate the involvement of neutrophils as a majorcause of distress in ricin-induced pulmonary injury, the authors used TBIto induce neutropenia in mice and found out reduced neutrophil infiltrationin the lungs of ricin-exposed mice, as well as a significantly longer survivaltime from 6.3 to 8.7 days [15].Local thorax irradiation significantly raised circulating blood monocytecounts in mice at respiratory distress. This finding is consistent with stud-ies associating macrophages with radiation-induced lung responses[20][50].Monocytes contribute to inflammation by migrating from the bloodstreaminto the injured tissues and differentiating into macrophages, which consti-tute major effector cells of inflammatory responses[49]. Grooves and othersfound that CCR2 (a receptor expressed in monocyte surfaces involved incell trafficking) deficient chimeric mice were spared from radiation-inducedpulmonary disease compared to inbred C57BL/6 control mice and that thisresponse was correlated with decreased numbers of infiltrating macrophagescompared to controls[20]. Their findings suggest that these cells may be criti-cal contributors to radiation-induced pulmonary disease. Likewise, Johnstonand others observed increased BALF monocyte counts beginning 16 weeks inC57BL/6 mice irradiated with a single dose of 15 Gy WTLI, and increasednumbers of macrophages in lung tissue of mice 24 weeks post-irradiation[30],confirming monocytic cell recruitment.Finally, partial body-irradiation significantly decreased circulating mono-cyte counts measured at the time window for the onset of respiratory dis-tress, as evident in the fact that 8 Gy PBI plus 8 Gy lung SIB treatedmice, distressed or not, were significantly lower than those of thorax-onlyirradiated mice.Consistent with Haston and colleagues[23], C3H/HeJ mice exposed to16 Gy whole-thorax irradiation developed a diffuse lethal pneumonitis uponrespiratory distress. Early hematopoietic cell depletion resulting from 8 Gy57Chapter 5. Conclusionpartial-body irradiation did not significantly affect the extent of pneumoni-tis, since the pneumonitis scores of mice exposed to 16 Gy WTLI and micetreated with 8 Gy PBI plus 8 Gy lung SIB were not significantly different.Mice exposed to 8 Gy PBI plus 8 Gy lung SIB and sacrificed before the onsetof distress did not exhibit remarkable pathology upon histological analysis.The mean pneumonitis score of those mice was 1.66 and it was significantlylower than that of mice of both treatment groups sacrificed at distress. Re-lating these histological findings to the hematological analysis, the fact thatneutrophil counts were significantly lower in nondistressed 8 Gy PBI plus 8Gy lung SIB treated mice, which had no significant disease than mice eu-thanized when distressed, which exhibited remarkable pathology, supportsthe hypothesis that neutrophils may be key contributors to the developmentof radiation-induced pneumonitis. Nevertheless, further investigation is re-quired to determine whether neutrophils are the principal contributors topulmonary distress.Finally, a few limitations of this thesis should be noted. First, thisthesis was limited solely to the characterization of radiation pneumonitisconsequent from high-dose partial-body irradiation, since experiments wereconducted only on one mouse strain, the C3H/HeJ mice, which is sparedfrom pulmonary fibrosis upon lung irradiation. Investigating the effect ofhigh-dose partial-body irradiation on the development and progression oflung fibrosis will require conducting the experiments on a mouse strain thatis prone to fibrosis upon local thorax irradiation, such as the C57BL/6 mice.Because this strain exhibits pulmonary distress at a longer period of 24-26weeks after 18 Gy whole-thorax irradiation[53], the study was focused onlyon the earlier responding C3H/HeJ strain.Second, the in vivo TLD measurements aimed to confirm that the mouse’stibiae were spared from radiation were only performed in a few of the animalsirradiated. Because the mouse positioning was easily reproduced accordingto setup marks placed in the buildup perspex box and a kV image of thepositioned mouse was obtained before irradiation to verify that positioningmatch that of the treatment plan, it was assumed that bone marrow sparingwould be accomplished for the rest of irradiated animals. A more rigorousverification of this would have involved performing these measurements forall animals irradiated.Third, there was a slight difference in the form the 16 Gy total lung dosewas delivered between the experimental models characterized in this thesis.The 8 Gy PBI plus 8 Gy lung SIB plan delivered the total lung dose in 4radiation fields, that is, 2 anterior-posterior beam pairs. However, the 16 GyWTLI plan delivered the total lung dose in 2 radiation fields only, that is,58Chapter 5. Conclusionone anterior-posterior beam pair. This methodology has also been applied inother studies[45] characterizing the effect of thymectomy in the developmentof radiation pneumonitis consequent of bone marrow transplant. However,an improvement to our present work would be having a third experimentalgroup of 16 Gy whole-thorax irradiation delivered in 2 anterior-posteriorpairs, to establish a stronger comparison between models.Finally, determining the optimal humane endpoint for euthanasia ishighly dependent on the observer’s qualitative judgment of mouse health,which may be fallible. Euthanizing right at the point when mice becamemoribund is crucial for significant pathology to be observed in histologicalanalysis. The main quantitative indicator for mouse health is weight. Aweight loss equal to or above 20% of the maximum weight is consideredthe main standard for euthanasia. Nevertheless, in practice, this criterionalone was fallible, as the final weight loss at which the humane endpoint wasreached varied among individual mice from 14-25% of maximum weight inthis thesis. For that reason, a thorough evaluation of mice’s signs of distressbased on qualitative signs such as piloerection, facial grimace, and hunching,was required to ensure that euthanized mice would have observable diseaseupon histology.In conclusion, this thesis characterized the lung pneumonitis responseconsequent to high-dose partial-body irradiation with minimal bone marrowshielding in the C3H/HeJ mouse strain. The latency, incidence, progression,and severity of radiation pneumonitis was not significantly different betweenmice treated with 16 Gy local whole-thorax irradiation and mice exposed to8 Gy partial-body irradiation plus 8 Gy simultaneous in-field boost to thelung. These findings suggest that radiation-induced pneumonitis developsindependently from injury to the hematopoietic system following signifi-cant radiation exposure. Furthermore, blood neutrophil counts were signifi-cantly associated with symptomatic pneumonitis in both treatment regimes,demonstrated by increased neutrophil counts at respiratory distress. Thisfinding agrees with other authors’ observations of neutrophil infiltration inthe lung tissue of rodents exposed to high-dose thoracic irradiation[14][34].The raise of neutrophil counts supports the hypothesis that an inflammatoryimmune response is involved in the development of radiation pneumonitis.The fact that neutrophil counts increased at distress in both 8 Gy PBI plus8 Gy lung SIB and 16 Gy WTLI models suggests that these cells may bemarkers of severe pneumonitis. Further investigation involving a larger sizeof mice will be required to validate this observation.Future directions suggested for this project could involve further inves-tigation of the role of neutrophils in radiation pneumonitis, specifically de-59Chapter 5. Conclusiontermining whether the increase of these cells is causative of the disease oran effect of other mechanisms driving this pathology. This may involve as-sessing the levels of specific cytokines and expression of genes involved inthe maturation and chemotaxis of neutrophils, as well as assessing the effectof neutrophil suppression on the development of RP in mouse models, par-ticularly through the administration of compounds that decrease neutrophilcounts. Finally, the results of such experiments could also be applicable inthe clinical oncology setting to identify monitoring markers of radiation lungside effects in thoracic radiotherapy patients.60Bibliography[1] International Atomic Energy Agency. Quality Assurance programmefor Computed Tomography: Diagnostic and Therapy applications. 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Thesis/Dissertation
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Medical Physics
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Radiation pneumonitis assessed in a mouse model of partial-body irradiation with minimal bone marrow sparing
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