To investigate a dynamic change of pathology in the body, we are utilizing the light for bioimaging. A qualitative diagnosis of pathology is of large importance in determining the operative therapy or the commencement, selection, method of chemotherapy in many surgical procedures. Nevertheless the research for monitoring the condition of organs and the aspect of pathology has only just begun. Novel fluorescent/luminescent molecular probes which visualize cellular or tissue environments and molecular functions, will be able to monitor the condition of the living body in response to stresses, non-invasively and continuously.
a) Imaging of molecular functions and cellular conditions
The probes for redox-sensitive, caspase-3, ER stress, probe for Akt, stress-induced protein have been developed and applied for bioimaging experiments. These probes enable us to understand the condition of organs exposed to different stresses in the same individual comprehensively. We have been analyzing in vivo dynamic state of oxidative stress and cell death by using those probes.
1) We are trying to make a use of redox-sensitive GFP protein to evaluate oxidative damage caused by ischemia/reperfusion or ablation damage found in hepatic surgery (hepatectomy or liver transplantation). Brief ischemic state followed by reperfusion (reoxygenation) is unavoidable consequence of hepatectomy or liver transplantation in clinical practices, and it is known to cause sever oxidative damage in the liver.
Redox state of organs after transplantation/ablation can be traced in real-time by monitoring stress situations non-invasively and continuously at in vivo level. We established the ischemia/reperfusion model using mouse liver introduced the gene with adenovirus vector, and the redox state at different stages (before/under ischemia and after reperfusion) as well as the extent of injury were visualized and determined.
Whereas the liver becomes necrotic without any oxidative stress under excess ischemia, oxidative stress immediately after reperfusion was increased corresponding to a period of liver ischemia and it was involved in postoperative injury. We concluded that it could be a novel marker for predicting the liver injury caused by the changes of redox state in ischemia and reperfusion.
2) An attempt to design new luminescence probe (for caspase-3) has been made to visualize a cellular damage based on the chemical reaction of luciferin/firefly luciferase. The luminescence of relatively long-wavelength (longer than 600 nm) emitted by this probe has the advantage of being able to monitor organs or biological processes in vivo. The function of caspase-3 is well studied and it is known to play an important role in induction of apoptosis.
In order to detect the activity of caspase-3, a novel probe, “circular luciferase” had been designed, it consists of N-terminal domain of intein (DnaE) connected with luciferase N-terminal, and C-terminal domain of intein (DnaE) connected with luciferase C-terminal, respectively, in between the substrate sequence (DEVD) for caspase-3 was inserted (Ozawa, University of Tokyo).
The activity of luciferase in the probe is recovered once the substrate sequence is cleaved by caspase-3. We found that the activation of caspase-3 and the induction of apoptosis after stimulating Fas pathway in AML12 hepatocyte cell line with the probe. In addition, we tried the bioimaging for activated caspase-3 in a mouse model with 70% liver ischemia/perfusion state. The results showed that the signal intensity was continuously increased in reperfusion state. The signal became large corresponding to a period of ischemia and the peak signal was delayed too.
b）Near-infrared probe and probe-conjugated antibody
We discovered that the combination of luminescent luciferase and fluorescent indocyanine green emitted long-wavelength light (near-infrared region) in the presence of substrates without UV excitation.
It enables an imaging diagnosis for the deep part of the body that has been thought to be difficult. (PNAS 2009, Luminescence 2012)
c）in vivo detection of intra/extracellular specific antigens
In order to make diagnoses of disorders safely and reliably, it is essential to evaluate the key molecules characterizing a biological property of lesion (tumor) from outside of the body. We are currently developing; 1) a method for conjugating a specific signal to antigenic molecules (new optical probes activated by antigen-antibody binding), 2) a method for delivering optical probes to intracellular space or body tissues. Since it is still ongoing project, we are afraid the all details cannot be described here yet.
d）Very early detection of pancreatic cancer
As pancreatic cancer progresses without symptoms in most cases, the detection and the treatment in an early stage are very important. We recently embarked on the development of hardware and software for detecting the lesions in the deep part of the body.