In vivo Bioimaging as a Novel Strategy to Detect Doxorubicin-Induced Damage to Gonadal Blood Vessels

INTRODUCTION: Chemotherapy may induce deleterious effects in normal tissues, leading to organ damage. Direct vascular injury is the least characterized side effect. Our aim was to establish a real-time, in vivo molecular imaging platform for evaluating the potential vascular toxicity of doxorubicin in mice. METHODS: Mice gonads served as reference organs. Mouse ovarian or testicular blood volume and femoral arterial blood flow were measured in real-time during and after doxorubicin (8 mg/kg intravenously) or paclitaxel (1.2 mg/kg) administration. Ovarian blood volume was imaged by ultrasound biomicroscopy (Vevo2100) with microbubbles as a contrast agent whereas testicular blood volume and blood flow as well as femoral arterial blood flow was imaged by pulse wave Doppler ultrasound. Visualization of ovarian and femoral microvasculature was obtained by fluorescence optical imaging system, equipped with a confocal fiber microscope (Cell-viZio). RESULTS: Using microbubbles as a contrast agent revealed a 33% (P<0.01) decrease in ovarian blood volume already 3 minutes after doxorubicin injection. Doppler ultrasound depicted the same phenomenon in testicular blood volume and blood flow. The femoral arterial blood flow was impaired in the same fashion. Cell-viZio imaging depicted a pattern of vessels' injury at around the same time after doxorubicin injection: the wall of the blood vessels became irregular and the fluorescence signal displayed in the small vessels was gradually diminished. Paclitaxel had no vascular effect. CONCLUSION: We have established a platform of innovative high-resolution molecular imaging, suitable for in vivo imaging of vessels' characteristics, arterial blood flow and organs blood volume that enable prolonged real-time detection of chemotherapy-induced effects in the same individuals. The acute reduction in gonadal and femoral blood flow and the impairment of the blood vessels wall may represent an acute universal doxorubicin-related vascular toxicity, an initial event in organ injury

DXR induces testicular vascular changes.

<p>Histological sections of testes from saline or DXR (5 mg/kg) treated mice were immunohistochemically stained with Hoecst 33342 (nuclei labeling; blue) and anti CD34 primary antibody (1:200) followed by alexa555 anti-rat secondary antibody (red; 1:400). Saline (A,C) and DXR (B,D) at one week (A,B) or one month (C,D) after treatment. Bar=240µm.</p

Platelet adhesion to DXR-treated aortic endothelial cells.

<p>Confluent endothelial cells were exposed to growth medium without (A) or with Doxorubicin (B; 100 µM) for 4 hr followed by exposure to whole blood for 5 minutes at 37°C under defined shear rates (750 s^-1) and then fixed and platelets were stained by immunohistochemistry kit using monoclonal antibody against the platelet integrin CD41a.</p

The effect of anti-coagulant and anti-platelet agents on testicular arterial blood flow following DXR treatment.

<p>Blood flow was measured by the PW Doppler mode and quantified by analyzing the VTI using the appropriate VisualSonics software. (<b>a</b>) Blood flow of testicular arteries was continuously monitored by PW Doppler in mice pre-treated with LMWH (clexane; 100µg/mouse), before and following DXR (8mg/kg BW; n=8, n=number of imaged arteries) injection and analyzed 3, 6, 9, 12 and 15 minutes afterwards. Graphic illustration of testicular arterial blood flow; points represent percent of control (dashed line; mean±SEM), (*)-significantly different from DXR treatment values (P<0.05). (<b>b</b>) Blood flow of testicular arteries was continuously monitored by PW Doppler in mice pre-treated with eptifibatide (integrilin; 75µg/mouse), before and following DXR (8mg/kg BW; n=9, n=number of imaged arteries) injection and analyzed 3, 6, 9, 12 and 15 minutes afterwards. Graphic illustration of testicular arterial blood flow; points represent percent of control (dashed line; mean±SEM), (*)-significantly different from DXR treatment values (P<0.05).</p

Effect of Doxorubicin on platelet aggregation and adhesion.

<p>(A) PRP was pre-incubated for 15 minutes with increasing concentrations of DXR then aggregation was induced by ADP (5 µM) and maximal aggregation is presented as mean ± SD; statistical analysis was tested by ANOVA (p<0.001; n=9). (B) Whole blood was pre-incubated for 15 minutes with increasing concentrations of DXR then subjected to the Impact-R test and both surface coverage and average size of the aggregates are presented as mean ± SD (n=5).</p