In vivo detection of hyperoxia-induced pulmonary endothelial cell death using (99m)Tc-duramycin

Abstract

Introduction: (99m)Tc-duramycin, DU, is a SPECT biomarker of tissue injury identifying cell death. The objective of this study is to investigate the potential of DU imaging to quantify capillary endothelial cell death in rat lung injury resulting from hyperoxia exposure as a model of acute lung injury.

Methods: Rats were exposed to room air (normoxic) or >98% O2 for 48 or 60 hours. DU was injected i.v. in anesthetized rats, scintigraphy images were acquired at steady-state, and lung DU uptake was quantified from the images. Post-mortem, the lungs were removed for histological studies. Sequential lung sections were immunostained for caspase activation and endothelial and epithelial cells.

Results: Lung DU uptake increased significantly (p<0.001) by 39% and 146% in 48-hr and 60-hr exposed rats, respectively, compared to normoxic rats. There was strong correlation (r(2)=0.82, p=0.005) between lung DU uptake and the number of cleaved caspase 3 (CC3) positive cells, and endothelial cells accounted for more than 50% of CC3 positive cells in the hyperoxic lungs. Histology revealed preserved lung morphology through 48 hours. By 60 hours there was evidence of edema, and modest neutrophilic infiltrate.

Conclusions: Rat lung DU uptake in vivo increased after just 48 hours of >98% O2 exposure, prior to the onset of any substantial evidence of lung injury. These results suggest that apoptotic endothelial cells are the primary contributors to the enhanced DU lung uptake, and support the utility of DU imaging for detecting early endothelial cell death in vivo.

Keywords: Acute lung injury; Apoptosis; Lung imaging.

Fig. 1

Planar images of a normoxic rat imaged 19 min following a first injection of ^99mTc-duramycin, DU, (left) and then five minutes later with a subsequent injection of ^99mTc-MAA (right). Lung (LU) ROI is identified in the MAA image, with the dashed horizontal lower boundary to avoid liver contribution. The ^99mTc-MAA generated lung ROI mask was then superimposed on the DU image yielding the lung DU ROI.

Fig. 2

Representative images of normoxic (A), 48-hr hyperoxic (B), and 60-hr hyperoxic (C) H&E stained lung slices. Insets at higher power illustrate structural features. Red arrows point to perivasuclar edema in 60-hr slice. Blue arrows identify the alveolar capillary membrane. Black arrows point to inflammatory cells within the perivascular spaces. Representative images of normoxic (D), 48-hr hyperoxic (E) and 60-hr hyperoxic (F) lungs where myeloperoxidase positive cells appear brown

Fig. 3

Planar images of ^99mTc-duramycin, DU, distribution in a normoxic (top), 48-hr hyperoxic (middle) and 60-hr hyperoxic (bottom) rat 19 min following DU injection. Lung (LU) ROI is determined from the MAA image with the dashed horizontal lower boundary to avoid liver contribution, and background (BG) ROIs from upper forelimbs

Fig. 4

A. Representative normoxic ^99mTc-duramycin, DU, time-activity curves acquired from lung and background ROIs. B. Average (mean ± SD) DU time-activity curves acquired from lung and background ROIs, and corresponding lung uptake (lung-to-background ratio) of the ten normoxic rats imaged

Fig. 5

Lung uptake (lung-to-background ratio) of ^99mTc-duramycin in normoxic (n = 10), 48-hr hyperoxic (n = 7), and 60-hr hyperoxic (n = 7) rats imaged. Values are mean ± SEM

Fig. 6

^99mTc-duramycin, DU, lung uptake of normoxic (n =10), 48-hr hyperoxic (n =7), and 60-hr hyperoxic (n = 7) rats. Values are mean ± SEM. One-way ANOVA followed by Tukey’s range test (p < 0.05) was used to evaluate differences among means of the three groups

Fig. 7

Correlation between ^99mTc-duramycin, DU, lung uptake and corresponding number of cleaved caspase 3 (CC3) positive cells per high-power field in lung slices (6 slices per lung) for each of 7 rats (two normoxic, two 48-hr hyperoxic, and three 60-hr hyperoxic). Values of CC3 positive cells are mean ± SD. Coefficient of determination = 0.82 (Pearson Product Moment Correlation test, p = 0.005)

Fig. 8

Panels A, B: Images of lung from a 60 hr hyperoxic rat stained with cleaved caspase 3 were selected blindly. Positive cells identified by brown stain are denoted by black circles. Consecutive slices were stained with a primary antibody for CD31 for endothelial cells. Analogous cells positive for cleaved caspase 3 were identified on CD31 slices, and marked as CD 31 positive (green circles) or negative (red circles). Of 85 cleaved caspase 3 positive cells, 45 (53%) were positive for CD 31. Panels C, D: Analogous to panels A&B except sequential slices were stained with a primary antibody for keratin 7 for epithelial cells. Of 59 CC3 positive cells, 10 (17%) were keratin 7 positive (green circle) and the remainder negative (red circles).