Whole-thorax Irradiation Research Articles

A Further Comparison of Pathologies after Thoracic Irradiation among Different Mouse Strains: Finding the Best Preclinical Model for Evaluating Therapies Directed Against Radiation-Induced Lung Damage

The human lung is among the most sensitive and critical tissues of concern in localized and systemic radiation exposures, and it is a subject of active preclinical research for evaluating mitigating therapies within the radiation countermeasures program. Our previous study comparing C57BL/6, CBA and C57L mice after whole-thorax irradiation pointed to the problems of late pleural effusions that prevented the full development of lung injury in C57BL/6 mice and suggested that the CBA and C57L strains are more favorable for modeling lung injury in humans (Jackson et al., Radiat. Res. 173, 10-20, 2010). We extended these comparisons to include three other mouse strains (BALB/c, C57BR/J and A/J mice) irradiated with 10, 12.5 or 15 Gy. Most of these mice were unable to survive the first 6 months and presented with a mixture of lung injury and pleural effusions as determined from gross pathology, histology and micro-CT. The independent and varying development of compressive pleural effusions of ill-defined etiology represents a concern for these strains in that they may not satisfy the preclinical requirements for approval of medical countermeasures (e.g. radiation mitigators) for human use. Thus, among the various different mouse strains studied so far for these pathologies, only three (CBA, C3H and C57L) appear to be desirable in exhibiting an early wave of pulmonary dysfunction attributed exclusively to radiation pneumonitis and for further assessment of radioprotective and mitigating therapies. C57L mice are particularly relevant in that they show significant lung damage at lower radiation doses that are closer to what is predicted for humans.

Identifying the High Radiosensitivity of the Lungs of C57L Mice in a Model of Total-Body Irradiation and Bone Marrow Transplantation

Pulmonary tissue is sensitive and often treatment-limiting in patients exposed to total-body irradiation (TBI) in preparation for hematopoietic stem cell transplantation. Many rodent strains, however, exhibit a relatively high resistance to radiation lung damage that often requires extra radiation doses to be delivered locally to the thorax to generate significant levels of pulmonary injury. The present study compared the effects of TBI and bone marrow transplantation (BMT) on two mouse strains that are known to differ in lung radiosensitivity after whole-thorax irradiation, namely the relatively resistant CBA mice and the sensitive C57L mice. Evaluation by survival, microcomputerized tomography (micro-CT), lung tissue weights and histopathology showed that the C57L mice responded with severe lethal radiation pneumonitis at 4 months after 12.5 Gy while CBA mice showed only minimal sublethal damage at this dose. C57L mice receiving 10 Gy TBI also had focal fibrotic lesions in the lungs out to 8 months. The manifestation of both pneumonitis and focal fibrosis in the lungs of C57L mice at relatively low radiation doses points to the merits of using this strain in further studies aimed at exploring and ameliorating the high susceptibility of the lung as encountered in clinical TBI.

Revisiting Strain-Related Differences in Radiation Sensitivity of the Mouse Lung: Recognizing and Avoiding the Confounding Effects of Pleural Effusions

The mouse has been used extensively to model radiation injury to the lung, a major dose-limiting organ for radiotherapy. Substantial differences in the timing and sensitivity of this tissue between mouse strains have been reported, with some strains, including C57BL/6, being designated as "fibrosis-prone". Pleural effusions have also been reported to be a prominent problem in many mouse strains, but it remains unclear how this affects the lung function and survival of the standard C57BL/6 mouse. The purpose of this investigation was to re-evaluate this strain in comparison with C57L and CBA mice after whole-thorax irradiation at doses ranging from 10 to 15 Gy. Breathing rate measurements, micro-computerized tomography, lung tissue weight, pleural fluid weight and histopathology showed that the most prominent features were an early phase of pneumonitis (C57L and CBA) followed by a late incidence of massive pleural effusions (CBA and C57BL/6). A remarkable difference was seen between the C57 strains: The C57L mice were exquisitely sensitive to early pneumonitis at 3 to 4 months while C57BL/6 mice showed a delayed response, with most mice presenting with large accumulations of pleural fluid at 6 to 9 months. These results therefore caution against the routine use of C57BL/6 mice in radiation lung experiments because pleural effusions are rarely observed in patients as a consequence of radiotherapy. Future experiments designed to investigate genetic determinants of radiation lung damage should focus on the high sensitivity of the C57L strain (in comparison with CBA or C3H mice) and the possibility that they are more susceptible to pulmonary fibrosis as well as pneumonitis.

Radiation-Induced Lung Response of AcB/BcA Recombinant Congenic Mice

The genetic factors that influence the development of radiotherapy-induced lung disease are largely unknown. Herein we identified a strain difference in lung response to radiation wherein A/J mice developed alveolitis with increased levels of pulmonary mast cells and cells in bronchoalveolar lavage while the phenotype in C57BL/6J mice was fibrosis with fewer inflammatory cells. To identify genomic loci that may influence these phenotypes, we assessed recombinant congenic (RC) mice derived from the A/J and C57BL/6J strains for their propensity to develop alveolitis or fibrosis after 18 Gy whole-thorax irradiation. Mouse survival, lung histopathology and bronchoalveolar lavage cell types were recorded. Informative strains for each of mast cell influx, bronchoalveolar cell numbers, alveolitis and fibrosis were identified. In mice with the A/J strain background, the severity of alveolitis correlated with increased mast cell numbers while in C57BL/6J background strain mice fibrosis was correlated with the percentage of neutrophils in lavage. The data for RC mice support the association of specific inflammatory cells with the development of radiation-induced lung disease and provide informative strains with which to dissect the genetic basis of these complex traits.

Functional and Histological Changes in Rat Lung after Boron Neutron Capture Therapy

The motivation for this work was an unexpected occurrence of lung side effects in two human subjects undergoing cranial boron neutron capture therapy (BNCT). The objectives were to determine experimentally the biological weighting factors in rat lung for the high-LET dose components for a retrospective assessment of the dose to human lung during cranial BNCT. Lung damage after whole-thorax irradiation was assessed by serial measurement of breathing rate and evaluation of terminal lung histology. A positive response was defined as a breathing rate 20% above the control group mean and categorized as occurring either early (<110 days) or late (>110 days). The ED(50) values derived from probit analyses of the early breathing rate dose-response data for X rays and neutrons were 11.4+/-0.4 and 9.2+/-0.6 Gy, respectively, and were similar for the other end points. The ED(50) values for irradiation with neutrons plus p-boronophenylalanine were 8.7+/-1.0 and 6.7+/-0.4 for the early and late breathing rate responses, respectively, and 7.0+/-0.5 Gy for the histological response. The RBEs for thermal neutrons ranged between 2.9+/-0.7 and 3.1+/-1.2 for all end points. The weighting factors for the boron component of the dose differed significantly between the early (1.4+/-0.3) and late (2.3+/-0.3) breathing rate end points. A reassessment of doses in patients during cranial BNCT confirmed that the maximum weighted doses were well below the threshold for the onset of pneumonitis in healthy human lung.

Structural and functional alterations in the rat lung following whole thoracic irradiation with moderate doses: Injury and recovery

Purpose: To characterize structural and functional injuries following a single dose of whole-thorax irradiation that might be survivable after a nuclear attack/accident.Methods: Rats were exposed to 5 or 10 Gy of X-rays to the whole thorax with other organs shielded. Non-invasive measurements of breathing rate and arterial oxygen saturation, and invasive evaluations of bronchoalveolar lavage fluid, (for total protein, Clara cell secretory protein), vascular reactivity and histology were conducted for at least 6 time points up to 52 weeks after irradiation.Results: Irradiation with 10 Gy resulted in increased breathing rate, a reduction in oxygen saturation, an increase in bronchoalveolar lavage fluid protein and attenuation of vascular reactivity between 4–12 weeks after irradiation. These changes were not observed with the lower dose of 5 Gy. Histological examination revealed perivascular edema at 4–8 weeks after exposure to both doses, and mild fibrosis beyond 20 weeks after 10 Gy.Conclusions: Single-dose exposure of rat thorax to 10 but not 5 Gy X-irradiation resulted in a decrease in oxygen uptake and vasoreactivity and an increase in respiratory rate, which paralleled early pulmonary vascular pathology. Vascular edema resolved and was replaced by mild fibrosis beyond 20 weeks after exposure, while lung function recovered.

Radiation-induced strain differences in mouse alveolar inflammatory cell apoptosis

Whole-thorax irradiation results in the development of the diffuse inflammatory response alveolitis in C3H/HeJ (C3H) mice and a milder alveolitis with fibrosis in C57BL/6J (B6) mice. In this study, we investigate if this mouse strain difference in response to radiation is due to differences in lung inflammatory cell apoptosis. Mice of the C3H and B6 strains were given a radiation dose of 18 Gy to the thorax and the animals were sacrificed at 11 or 18 weeks following exposure or when they were moribund. Active caspase-3 staining was used to identify apoptotic cells in the alveolar space of histological lung sections from the mice. The apoptotic index of B6 mice was greater than that of C3H mice at 11 weeks postirradiation (17.8% of airspace cells vs. 7.8%, p = 0.028) and in mice sacrificed because of illness (27.3% vs. 14.4%, p = 0.036). No C3H mice survived to the later time point. The inflammatory cells undergoing apoptosis in the mouse lungs were morphologically consistent with alveolar macrophages. We conclude that a difference in inflammatory cell apoptosis may contribute to the disparate pulmonary radiation response of these mouse strains.

Apoptosis and Bcl-2 Expression in Irradiated Lungs and the Effect of Pentoxifylline

We measured number of bcl-2, apoptotic, neutrophil, and surfactant apoprotein D (SP-D) positive cells in irradiated rat lungs during different time points after the sublethal whole-thorax irradiation of rats. We also investigated the influence of pentoxifylline (PTX) therapy on these markers. Wistar rats were given 15 Gy thoracic irradiation and PTX (35 mg/kg) twice a week. Animals were examined histologically and imunohistochemically at intervals from 1-12 weeks. In non-treated rats compared with treated rats, bcl-2 expression was significantly inhibited from 4 weeks after irradiation. A higher apoptosis presence in non-treated rats from 4 weeks was found and apoptosis development in PTX-treated animals was delayed and started 8 weeks after irradiation. Similar differences were measured during neutrophil granulocytes examination. Neutrophil penetration in non-treated rats was found 5 weeks after irradiation in contrast to the RP onset of PTX-treated animals 8 weeks after irradiation. The number of SP-D positive cells in non-treated rats observed until 5 weeks after irradiation was higher than in the control group. PTX-treated animals expressed higher number of SP-D positive cells during the whole experiment than the control group. We suggest that apoptosis is linked to neutrophil granulocyte actions during the RP onset and that PTX-therapy causes diminished inflammation development.

The Alveolar Septal Thickness and Type II Pneumocytes Number in Irradiated Lungs, Time Expression and the Effect of Pentoxifylline

We studied the relationship between type II pneumocytes number and alveolar septal thickness during different time after sublethal whole-thorax irradiation of rats and we investigated the influence of pentoxifylline (TNF-alpha inhibitor). Wistar rats were exposed to 15 Gy thoracic irradiation and pentoxifylline (35 mg/kg) twice a week. Lungs were examined histologically and immunohistochemically at intervals ranging from 1-12 weeks and alveolar septal thickness, number of type II pneumocytes (identified by immunoreactivity for cytokeratin 18), and neutrophile granulocytes were counted. Significant increase of alveolar septal thickness and type II pneumocytes depletion 3 weeks after irradiation were found. Correlation of these markers was r = -0.759. Pentoxifylline significantly inhibits increased alveolar septal thickness without the influence on type II pneumocytes number. Neutrophil penetration started 5 weeks after irradiation in non-treated animals, 8 weeks after irradiation in PTX-treated rats. We suggest that pneumocytes depletion is linked to increased vascular permeability, and pentoxifylline therapy does not influence on pneumocytes kinetics after irradiation.

The role of T-cells in radiation pneumonitis after bone marrow transplantation

Radiation pneumonitis is a frequent complication of bone marrow transplantation. In limiting the effective dose that can be given, it decreases the chances of cancer cell destruction and of graft acceptance. The incidence of radiation pneumonitis is increased if graft-versus-host disease or infection is present, presumably due to an interaction between T-cell-mediated and radiation-mediated damage. Even in the absence of graft-versus-host disease and infection, we have found that syngeneic T-cells can contribute to radiation pneumonitis in a bone marrow transplant setting. The incidence of radiation pneumonitis was higher after whole-body irradiation and bone marrow transplantation than after whole-thorax irradiation. In the former situation, but not the latter, prior thymectomy decreased the incidence of radiation pneumonitis. It is hypothesized that autoreactive T-cells escape induction of self-tolerance during regression of the immune system after whole-body irradiation and, because autoregulatory cells are eliminated, they can contribute to radiation pneumonitis. If this concept is correct, it provides another possible explanation for the sensitivity of the lung to post-transplant pneumonitis and suggests new strategies to limit the incidence of this serious transplant-related complication.

Whole-body hyperthermia combined with hyperfractionated irradiation of the thorax in dog: acute physiological response.

Whole-body hyperthermia has potential as an adjuvant treatment with chemotherapy and radiation therapy for diseases such as lung cancer which require both local and systemic control. The acute toxicity of whole-body hyperthermia combined with whole-thorax irradiation was studied in dogs. Twenty-eight dogs received three 2-h whole-body hyperthermia (WBH) treatments at 42.0 degrees C deep rectal temperature. Twenty-four of these dogs were also randomized to receive radiation doses of 18, 22.5, 27, 31.5, 40.5 or 45 Gy. Irradiation was given in 1.5 Gy fractions over 6 weeks. Three WBH treatments were given to 28 dogs with all dogs surviving treatment. WBH was given on days 1, 22 and 40 of the 6-week interval. Thirty-one dogs received radiation doses of 18-49.5 Gy without WBH. Deep rectal temperature was maintained at 41.9 +/- 0.3 degrees C over 2 h with an average of 20 min outside the chamber for irradiation. Two dogs required intervention with emergency medications during WBH treatment. One of the two dogs developed permanent neurological injury. Continuous physiological monitoring was necessary for successful WBH. WBH plus thoracic irradiation was well tolerated. All dogs survived all treatments. A significant but transient increase in peripheral blood leucocytes and a decrease in platelet counts occurred after each WBH treatment. The addition of thoracic irradiation up to 45 Gy in 1.5 Gy fractions did not appear to alter the acute toxicity of WBH with the exception of an increase in the protein content of lung lavage fluids. In conclusion, multiple WBH treatments of 2 h at a target temperature of 42 degrees C in addition to thoracic irradiation up to 45 Gy in 1.5 Gy fractions was administered with only mild acute toxicities occurring. Core temperature could be maintained for up to 20 min outside of the WBH chamber which allowed irradiation to be given concurrently with hyperthermia at a core temperature of 42 degrees C +/- 0.1 degree C.

The Genetic Basis of Strain-Dependent Differences in the Early Phase of Radiation Injury in Mouse Lung

Substantial differences between mouse strains have been reported in the lesions present in the lung during the early phase of radiation injury. Some strains show only classical pneumonitis, while other strains develop substantial fibrosis and hyaline membranes which contribute appreciably to respiratory insufficiency, in addition to pneumonitis. Other strains are intermediate between these extremes. These differences correlate with intrinsic differences in activities of lung plasminogen activator and angiotensin converting enzyme. The genetic basis of these differences was assessed by examining histologically the early reaction in lungs of seven murine hybrids available commercially after whole-thorax irradiation. Crosses between fibrosing and nonfibrosing parents were uniformly nonfibrosing, and crosses between fibrosing and intermediate parents were uniformly intermediate. No evidence of sex linkage was seen. Thus the phenotype in which fibrosis is found is controlled by autosomal recessive determinants. Strains prone to radiation-induced pulmonary fibrosis and hyaline membranes exhibited intrinsically lower activities of lung plasminogen activator and angiotensin converting enzyme than either the nonfibrosing strains or the nonfibrosing hybrid crosses. The median time of death of the hybrids was genetically determined primarily by the longest-lived parent regardless of the types of lesions expressed.

Radiation-Induced Pulmonary Endothelial Dysfunction and Hydroxyproline Accumulation in Four Strains of Mice

C57BL mice exposed to 14 Gy of whole-thorax irradiation develop significant histologic lung fibrosis within 52 weeks, whereas CBA and C3H mice do not exhibit substantial fibrosis during this time. The purpose of the present study was to determine whether this strain-dependent difference in radiation histopathology is associated with genetic differences in pulmonary endothelial metabolic activity or in endothelial radioresponsiveness. C57BL/6J, C57BL/10J, CBA/J, and C3H/HeJ mice were sacrificed 12 weeks after exposure to 0 or 14 Gy of 300-kV X rays to the whole thorax. Lung angiotensin converting enzyme (ACE) activity and plasminogen activator (PLA) activity were measured as indices of pulmonary endothelial function; and lung hydroxyproline (HP) content served as an index of pulmonary fibrosis. Lung ACE and PLA activities in sham-irradiated C57BL/6J and CB57BL/10J mice were only half as high as those in sham-irradiated CBA/J and C3H/HeJ mice. Exposure to 14 Gy of X rays produced a slight but nonsignificant reduction in lung ACE and PLA activity in the C57BL strains, and a significant reduction in the CBA/J and C3H/HeJ mice. Even after 14 Gy, however, lung ACE and PLA activities in CBA/J and C3H/HeJ mice were higher than those in sham-irradiated C57BL/6J and C57BL/10J mice. Lung HP content in all four strains increased significantly after irradiation, but this increase was accompanied by an increase in lung wet weight. As a result, HP concentration (per milligram wet weight) remained constant or increased slightly in both C57BL strains and actually decreased in the CBA/J and C3H/HeJ mice. These data demonstrate significant genetic differences in both intrinsic pulmonary endothelial enzyme activity and endothelial radioresponsiveness among the four strains of mice. Specifically, strains prone to radiation-induced pulmonary fibrosis (C57BL/6J, C57BL/10J) exhibit only half as much lung ACE and PLA activity as do strains resistant to fibrosis (CBA and C3H).