Taeyun Ku

Minimally invasive molecular delivery into the brain using optical modulation of vascular permeability

Systemic delivery of bioactive molecules in the CNS is hampered by the blood–brain barrier, which has bottlenecked noninvasive physiological study of the brain and the development of CNS drugs. Here we report that irradiation with an ultrashort pulsed laser to the blood vessel wall induces transient leakage of blood plasma without compromising vascular integrity. By combining this method with a systemic injection, we delivered target molecules in various tissues, including the brain cortex. This tool allows minimally invasive local delivery of chemical probes, nanoparticles, and viral vectors into the brain cortex. Furthermore, we demonstrated astrocyte-mediated vasodilation in vivo without opening the skull, using this method to load a calcium indicator in conjunction with label-free photoactivation of astrocytes.

Blockade of VEGF-A suppresses tumor growth via inhibition of autocrine signaling through FAK and AKT

Blockade of VEGF signaling using RNA interferences, a neutralizing antibody, an antagonizing soluble VEGF receptor, and a receptor tyrosine kinase inhibitor induced anti-tumor effects in human astrocytoma U251-MG and fibrosarcoma HT-1080 in vitro in a dose-dependent manner. Furthermore, blockade of VEGF-A using the doxycycline-inducible VEGF-A RNA interference system showed a significant anti-tumor effect in a murine HT-1080-xenograft model. Anti-tumor effect through the blockade of VEGF signaling was mediated by FAK and AKT pathway in vitro and in vivo. These results collectively indicate that VEGF-A and its receptors can act as key inducer of tumor growth as well as angiogenesis.

Label-free optical activation of astrocyte in vivo

As the most abundant cell type in the central nervous system, astrocyte has been one of main research topics in neuroscience. Although various tools have been developed, at present, there is no tool that allows noninvasive activation of astrocyte in vivo without genetic or pharmacological perturbation. Here we report a noninvasive label-free optical method for physiological astrocyte activation in vivo using a femtosecond pulsed laser. We showed the laser stimulation robustly induced astrocytic calcium activation in vivo and further verified physiological relevance of the calcium increase by demonstrating astrocyte mediated vasodilation in the brain. This novel optical method will facilitate noninvasive physiological study on astrocyte function.

Principal component analysis of dynamic fluorescence images for diagnosis of diabetic vasculopathy

Abstract. Indocyanine green (ICG) fluorescence imaging has been clinically used for noninvasive visualizations of vascular structures. We have previously developed a diagnostic system based on dynamic ICG fluorescence imaging for sensitive detection of vascular disorders. However, because high-dimensional raw data were used, the analysis of the ICG dynamics proved difficult. We used principal component analysis (PCA) in this study to extract important elements without significant loss of information. We examined ICG spatiotemporal profiles and identified critical features related to vascular disorders. PCA time courses of the first three components showed a distinct pattern in diabetic patients. Among the major components, the second principal component (PC2) represented arterial-like features. The explained variance of PC2 in diabetic patients was significantly lower than in normal controls. To visualize the spatial pattern of PCs, pixels were mapped with red, green, and blue channels. The PC2 score showed an inverse pattern between normal controls and diabetic patients. We propose that PC2 can be used as a representative bioimaging marker for the screening of vascular diseases. It may also be useful in simple extractions of arterial-like features.

Enhancement of the photocytotoxic efficiency of sub-12 nm therapeutic polymeric micelles with increased co-localisation in mitochondria

We engineered phototherapeutic sub-12 nm-sized polymeric micelles to treat malignant brain tumours (MBTs). The engineered nanoparticles in MBT cells enhanced the photocytotoxic efficiency more than 2.5-fold compared with parental and PEGylated photosensitisers (PSs). Increased subcellular co-localisation of PSs in mitochondria was observed.

Optical induction of muscle contraction at the tissue scale through intrinsic cellular amplifiers

The smooth muscle cell is the principal component responsible for involuntary control of visceral organs, including vascular tonicity, secretion, and sphincter regulation. It is known that the neurotransmitters released from nerve endings increase the intracellular Ca(2+) level in smooth muscle cells followed by muscle contraction. We herein report that femtosecond laser pulses focused on the diffraction-limited volume can induce intracellular Ca(2+) increases in the irradiated smooth muscle cell without neurotransmitters, and locally increased intracellular Ca(2+) levels are amplified by calcium-induced calcium-releasing mechanisms through the ryanodine receptor, a Ca(2+) channel of the endoplasmic reticulum. The laser-induced Ca(2+) increases propagate to adjacent cells through gap junctions. Thus, ultrashort-pulsed lasers can induce smooth muscle contraction by controlling Ca(2+), even with optical stimulation of the diffraction-limited volume. This optical method, which leads to reversible and reproducible muscle contraction, can be used in research into muscle dynamics, neuromuscular disease treatment, and nanorobot control.

Optical modulation of astrocyte network using ultrashort pulsed laser

Astrocyte, the most abundant cell type in the central nervous system, has been one of major topics in neuroscience. Even though many tools have been developed for the analysis of astrocyte function, there has been no adequate tool that can modulates astrocyte network without pharmaceutical or genetic interventions. Here we found that ultrashort pulsed laser stimulation can induce label-free activation of astrocytes as well as apoptotic-like cell death in a dose-dependent manner. Upon irradiation with high intensity pulsed lasers, the irradiated cells with short exposure time showed very rapid mitochondria fragmentation, membrane blebbing and cytoskeletal retraction. We applied this technique to investigate in vivo function of astrocyte network in the CNS: in the aspect of neurovascular coupling and blood-brain barrier. We propose that this noninvasive technique can be widely applied for in vivo study of complex cellular network.

Current Optical Imaging Techniques for Brain Tumor Research: Application of in vivo Laser Scanning Microscopy Imaging with a Cranial Window System

“Seeing is believing.” This may not be true in all areas of biomedical research, but identifying cellular characteristics of tumors and specifying their anatomical locations are the most important processes for diagnosing and treating tumors. Furthermore, various imaging techniques with various modalities have been introduced to investigate disease progression, track the pharmacokinetic behavior of drugs, and in clinical applications. Reconstructing images at the molecular level has been realized with the dramatic advancement in energy sources, detectors, computational methods, and instruments. Computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and ultrasound imaging are well-known imaging techniques that have tremendously improved not only preclinical research but also clinical treatment. Contrastenhanced CT, MRI and PET have permitted non-invasive detection of abnormal tissues, particularly for tumor research and clinical applications. Imaging techniques currently used for brain tumor research vary with purpose and imaging characteristics. The techniques can be grouped by three aspects: (1) energy, (2) spatial resolution, and (3) type of information obtained (Weissleder & Pittet, 2008). The energies generally used for these techniques are X-rays (e.g., classic X-ray imaging, CT, multidetector CT), magnetic fields (e.g., MRI and diffusion MRI), positrons (e.g., PET), sound waves (e.g., ultrasound imaging, interventional ultrasound imaging), photons (e.g., bioluminescence imaging, fluorescence reflectance imaging, fluorescence-mediated tomography, and laser scanning microscopy imaging), and combinations of such modalities (e.g., PET-CT, PET-MRI, CT, or MRI with fluorescence-mediated tomography). The techniques can also be distinguished by spatial resolution and the information obtained: (1) macroscopic, (2) mesoscopic, and (3) microscopic or (1) anatomical, (2) physiological, and (3) cellular and molecular.

Coloring brain tumor with multi-potent micellar nanoscale drug delivery system

Brain tumor, especially glioblastoma multiforme (GBM), is one of the most malignant tumors, which not only demands perplexing treatment approaches but also requires potent and effective treatment modality to deal with recurrence of the tumor. Photodynamic therapy (PDT) is a treatment which has been recommended as a third-level treatment. We are trying to investigate possibility of the PDT as an efficient adjuvant therapeutic modality for the treatment of brain tumor. Inhibition of tumor progression with photosensitizer was verified, in vitro. With micellar nanoscale drug delivery system, localization of the tumor was identified, in vivo, which is able to be referred as photodynamic diagnosis. With consequent results, we are suggesting photodynamic diagnosis and therapy is able to be performed simultaneously with our nanoscale drug delivery system.

Cerebral blood flow imaging using time-series analysis of indocyanine green molecular dynamics in mice

Measurement of cerebral perfusion is important for study of various brain disorders such as stroke, epilepsy, and vascular dementia; however, efficient and convenient methods which can provide quantitative information about cerebral blood flow are not developed. Here we propose an optical imaging method using time-series analysis of dynamics of indocyanine green (ICG) fluorescence to generate cerebral blood flow maps. In scalp-removed mice, ICG was injected intravenously, and 740nm LED light was illuminated for fluorescence emission signals around 820nm acquired by cooled-CCD. Time-lapse 2-dimensional images were analyzed by custom-built software, and the maximal time point of fluorescent influx in each pixel was processed as a blood flow-related parameter. The generated map exactly reflected the shape of the brain without any interference of the skull, the dura mater, and other soft tissues. This method may be further applicable for study of other disease models in which the cerebral hemodynamics is changed either acutely or chronically.