Janet Sharplin

A quantitative histological study of strain-dependent differences in the effects of irradiation on mouse lung during the intermediate and late phases.

Strain differences in the intermediate and late phases of the radiation response of mouse lung were investigated histologically. The proportion of lung impairment in mice at 28 and 52 weeks postirradiation and in mice dying of respiratory insufficiency was assessed by scoring lung acini as nonfunctional due to lesions which obstructed airflow, or open and presumably functional. The nine strains tested were divided into three groups on the basis of the late fibrotic response. Group 1 mice, three C57 strains, developed extensive contracted fibrosis and usually showed enough damage to explain late deaths. Group 2, SWR, A, and BALB/c strains, developed foci of contracted fibrosis. Group 3, CBA and two C3H strains, did not form fibrotic scars. Mice in Groups 2 and 3 that died with no pleural effusions appeared to have insufficient late lung damage to account for respiratory distress. Problems with pulmonary blood flow were indicated by evidence of loss of fine vasculature and right ventricular hypertrophy. In nondistressed, late-stage mice in Groups 2 and 3, loss of capillary perfusion in lung parenchyma free of obvious lesions was demonstrated by infusion of colloidal carbon. In one strain, A, an estimate of the proportion of nonperfused lung was made on distressed late-stage mice. Almost 50% of lung acini were nonfunctional as a result of nonperfusion, and an additional 9% of acini were nonfunctional due to lesions obstructing ventilation. It is suggested that nonperfusion of apparently normal lung acini is a major factor in late-phase deaths in those mouse strains which show little or no fibrosis.

Immunohistochemical Localization of Transforming Growth Factor β and Tumor Necrosis Factor α in the Lungs of Fibrosis-Prone and "Non-Fibrosing" Mice during the Latent Period and Early Phase after Irradiation

To evaluate the possibility that TGF-beta and TNF-alpha are involved in fibrosis induced in mouse lung by irradiation, the proportion of cells immunoreactive for each was compared in two strains of mice. C3HeB/FeJ mice develop only classical pneumonitis during the early phase, whereas C57L/J mice develop small, tightly packed areas of inflammation which undergo fibrosis during the latent period, and exhibit progressive fibrosis of large regions of intense inflammation during the early phase. Very few cells were immunoreactive for an antibody to the latency-associated peptide (LAP) of TGF-beta during the latent period in C3HeB/FeJ mice, and no cells were positive during the early phase. In contrast, between 0.7 and 10% of cells were positive in C57L/J mice in lesions without fibrosis and in lesions in the early stages of fibrosis. Fibroblasts positive for LAP were seen only in lesions containing fibrosis. A similar pattern of immunoreactivity was seen in C57L/J mice using an antibody which recognizes active TGF-beta, with the exception that positive fibroblasts were observed within areas of inflammation without fibrosis. Thus the association of active TGF-beta with fibroblasts might be a characteristic of the initiation of fibrosis in this model. TNF-alpha was detected in macrophages in all classes of lesions, and minor differences between the strains did not appear to be biologically meaningful.

A quantitative histological study of strain-dependent differences in the effects of irradiation on mouse lung during the early phase

Strain differences in the radiation response of mouse lung during the early phase (before 28 weeks postirradiation) were investigated histologically. The nine strains tested were divided into three groups on the basis of the nature of the edema present, the occurrence of hyaline membranes, and the presence of fibrosis. Group 1 mice, three C57 strains, developed hyaline membranes, focal fibrosis, and a protein-rich edema containing fibrin. Group 3, CBA and two C3H strains, had only a protein-poor edema with little fibrin and developed no visible fibrosis. Group 2 mice had both types of edema and small quantities of focal fibrosis. The degree of lung impairment in mice dying of respiratory insufficiency was assessed by scoring lung acini as nonfunctional or open and presumably functional. Over 70% of acini were nonfunctional as a result of airflow obstruction. This was considered sufficient to account for death. Carbon perfusion immediately before sacrifice indicated that all types of lesions were at least partially perfused with blood. Pleural effusions were found in some individuals of two strains. The proportion of nonfunctional acini was similar in mice of the same strain with and without effusions, which would not be expected if the effusions contributed appreciably to respiratory distress in the early phase.

Development of Fibrosis after Lung Irradiation in Relation to Inflammation and Lung Function in a Mouse Strain Prone to Fibrosis

The development of lung fibrosis after single-dose thoracic irradiation was studied histologically in C57L/J male mice. Lung function was monitored using uptake of carbon monoxide. During the latent period (prior to 15 weeks postirradiation) mice were chosen at random, while during the early phase (15-22 weeks) mice were sacrificed when they developed a functional deficit of at least 50%. Excess mice with a deficit of 50% in the early phase were followed into the late phase and sacrificed at 31 or 40 weeks. Two scoring methods were used to quantify lung damage. Fibrotic lesions and foci of inflammation were counted for the latent period, and the proportion of nonfunctional acini was determined for the early and late phases. After a dose 1 Gy less than the LD50/180, small regions of mild inflammatory infiltration appeared at 6 weeks, and small, focal fibrotic lesions containing numerous macrophages appeared at 8 weeks postirradiation. The number of fibrotic lesions increased steadily during the latent period in a manner that is consistent with conversion of inflammatory lesions to foci of fibrosis. Mice sacrificed upon developing a 50% functional deficit during the early phase had approximately equal proportions of lung affected by fibrosis and inflammation. Those mice which developed a similar respiratory deficit in the early phase and were followed into the late phase usually showed little change in lung function. However, when sacrificed at 31 weeks they had twice as much fibrosis and very little inflammation, suggesting that the inflammatory lesions had become fibrosed. The average number of macrophages per unit area of fibrosis declined during the latent period and changed little during the early and late phases. Lymphocytes and mast cells were also quantified in fibrosed regions.

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).

The density of mouse lung in vivo following X irradiation

The lungs of mice were irradiated with single X radiation doses of 5 to 14 Gy. Six weeks after irradiation, computed tomographic (CT) scans of the mice were performed at two-week intervals. Beyond 14 weeks after irradiation, the animals were scanned at 1-week intervals. The mice irradiated to 5 and 7 Gy exhibited no change in lung density, in comparison with the unirradiated lungs of control mice up to times of 48 weeks. The mice irradiated to doses of greater than 10 Gy exhibited marked increases in lung density at 15 weeks after irradiation. Increases in density followed a similar time course for these doses, but the magnitude of the density increase was dependent on the radiation dose. An interpretation of these findings in terms of radiation pneumonitis is presented, and the possibility of using CT to monitor lung density in radiotherapy patients is discussed.

Irradiation of mouse lungs causes a dose-dependent increase in lung weight

The lungs of Balb/c mice were irradiated with doses of 200 to 1300 rad and excised and weighed three days to 20 weeks later. In all cases the wet weights were increased relative to unirradiated controls. The weight increases were dose-dependent up to 1000 rad. The largest weight increase was 28%. Comparison of the wet and dry weights of irradiated and control lungs indicated that the material responsible for the weight increase was intermediate in water content between normal lung tissue and plasma, whereas the water content of pulmonary edema fluid produced by adrenalin injection was similar to plasma. Protection of the mediastinum during irradiation did not affect the weight increase appreciably.

Assessment of Radiation-Induced Lung Injury in Mice Using Carbon Monoxide Uptake: Correlation with Histologically Visible Damage

Carbon monoxide uptake is a sensitive measure of lung injury, but its application to mice using the rebreathing technique has produced a nonlinear dependence of carbon monoxide uptake on mouse weight, in contrast to the linear relationship obtained in larger rodents using the single-breath technique. Improvements were made to the equipment and the procedures used in the rebreathing technique which resulted in linear relationships between uptake and weight in three mouse strains, CBA/J, C57BL/6J, and C57L/J. Sequential measurements were made on mice during the early and intermediate phases after irradiation of the thorax which demonstrated the development of injury in individual mice with considerable sensitivity. Estimates of the proportion of lung which was considered to be nonfunctional based on its histological appearance were obtained using alveolar ducts as sampling markers in 64 C57L/J mice between 10 and 31 weeks after irradiation. The deficit in carbon monoxide uptake was determined on the day of sacrifice for each mouse, and the results showed good correspondence to the histological estimate of the extent of damage. The correspondence between breathing rate elevation and the histological assay was not as good.