We established an organ-stabilizing system to evaluate the real-time imaging of visceral organs in actin-GFP transgenic mice using in vivo TPLSM. DSS-induced colitis showed irregularity of crypt architecture, disappearance of crypts, inflammatory cell infiltration and increased rolling of white blood cells along the vasculature. In addition, the intercellular distance of mucosal cells in the crypt and vascular endothelial cells in the intestinal wall was increased in the intestinal mucosa during DSS colitis. In DSS colitis, there was remarkable loss of mucosal and vascular endothelial ZO-1 expression, as could be seen by a decrease in ZO-1 staining. In conclusion, our observations suggested the possibility that our TPLSM imaging system can be used to clarify the pathophysiological changes in various diseases using longitudinal studies of microscopic changes in the same animal over long periods of time.

No abstract

Abstract. CD133 and CD44 have been considered as markers for colorectal cancer stem cells (CSCs). The association of CD133 and CD44 expression with radiation has not been fully examined in rectal cancer. Both CD133 (PROM) and CD44 mRNA levels were measured in post-chemoradiotherapy (CRT) specimens of 52 rectal cancer patients using real-time RT-PCR and compared to clinicopathological variables and clinical outcome. Their protein levels were examined in the radiationtreated HT29 human colon cancer cell line. Post-CRT CD133 in residual cancer cells was significantly higher than matched pre-CRT CD133 in biopsy specimens (n=30). By contrast, CD44 was significantly lower in post-CRT specimens (P<0.01). CD133 was associated with distant recurrence after CRT followed by surgery (P<0.05). Patients with elevated CD133 in residual cancer cells showed poor disease-free survival (P<0.05). No significant association between post-CRT CD44 and clinical outcome was found. The in vitro study showed that CD133 protein was increased in a radiation dose-dependent manner, despite of the decreased number of clonogenic radiationsurviving cells. CD44 protein was decreased after irradiation. CD133, but not CD44, was increased in radiation-resistant surviving colon cancer cells. Post-CRT CD133 in residual cancer cells may predict metachronous distant recurrence and poor survival of rectal cancer patients after CRT.

Although thrombus formation in vivo has recently been reported, all previous laser induced thrombus models have been associated with vessel wall disruption. This study aimed to evaluate in vivo real-time platelet aggregation after selective endothelial injury, and to visualize thrombus formation without disruption and swelling of the arterial intima induced by two-photon laser irradiation. Cecal arteriole thrombi were created in beta-actin-green fluorescent protein transgenic mice by selective endothelial injury using two-photon laser-scanning microscopy (TPLSM). The in vivo real-time process of thrombus formation was assessed. Anticoagulant drug efficiency for thrombi was also analyzed in detail. TPLSM allowed visualization of microvessel components from the arterial smooth muscle to the intimal layer. Immediately after selective laser irradiation of the intimal layer, platelet adhesion and aggregation were seen only at the area of injury of the intimal layer after forming linear adhesions downstream of the injured area. When shear stress was overcome, thrombus formation began at the downstream edge of the injured area. Thrombus volume plateaued approximately 60 min after laser irradiation. The thrombolytic effects of anticoagulant drugs were precisely assessed; therefore, our model appears the most advanced model in point of real-time imaging of pathophysiological processes in vivo currently reported. In vivo real-time imaging of thrombus formation can be achieved using TPLSM in combination with an organ stabilizing system. The high magnification and resolution of TPLSM allows investigation of the mechanisms of thrombus formation along with assessment of antithrombotic drug efficacy with little interexperimental variation.

In vivo real-time visualization of the process of angiogenesis in secondary tumors in the same living animals presents a major challenge in metastasis research. We developed a technique for intravital imaging of colorectal liver metastasis development in live mice using two-photon laser scanning microscopy (TPLSM). We also developed time-series TPLSM in which intravital TPLSM procedures were performed several times over periods of days to months. Red fluorescent protein-expressing colorectal cancer cells were inoculated into the spleens of green fluorescent protein-expressing mice. First- and second-round intravital TPLSM allowed visualization of viable cancer cells (red) in hepatic sinusoids or the space of Disse. Third-round intravital TPLSM demonstrated liver metastatic colonies consisting of viable cancer cells and surrounding stroma with tumor vessels (green). In vivo time-course imaging of tumor angiogenesis in the same living mice using time-series TPLSM could be an ideal tool for antiangiogenic drug evaluation, reducing the effects of interindividual variation.