[Show abstract][Hide abstract] ABSTRACT: Purpose: Photodynamic therapy (PDT) induced singlet oxygen (1O2) can cause rapid necrosis of solid tumors. Using a new near-infrared photomultiplier tube system, the 1O2 production was monitored in real-time for HSC-3 tongue cancer cells and in vivo tissue, and the relationship between the 1O2 production and anti-tumor effects was investigated.
Experimental Design: Using 5-aminolevulinic acid (5-ALA), 1O2 generation was induced with a variety of laser parameters (fluence and irradiation) in vivo. During this time, tumor death was evaluated. Based on the 1O2 production, the optimal irradiation conditions and the long-term outcome were investigated on a subcutaneous tongue cancer model over 90 days.
Methods and Results: The 1O2 production showed a strong correlation with the laser output in a dose-dependent manner. At high laser output, the 1O2 levels were high and caused gradual but strong anti-tumor effects, whereas at low laser outputs, the 1O2 levels were low and the anti-tumor effects were rapid but superficial. We then performed PDT in an experimental tongue cancer model at an irradiance lower than 150 mW/cm2, which was identified in the first part of the study as the optimal irradiance value based on the 1O2 production. Within the 90 days post PDT, PDT-induced hemorrhagic necrosis was observed in 8 mice out of 10 within 5 days. In the other 2 mice, no sign of necrosis was seen until 25 days after PDT. The tumor volume of the PDT group continued to decline except for 2 mice which showed some slight growth over 70 days after PDT. In the control group, tumor size increased dramatically over 70 days.
Conclusions: The present studies established 1O2 levels as an indicator for optimal irradiation during PDT, and showed a relationship between the 1O2 production and the photodynamic effects. Although 5-ALA mediated PDT showed a strong anti-tumor effect on larger subcutaneous tongue tumors, it might be more effective for small tumors of oral cavity.
[Show abstract][Hide abstract] ABSTRACT: Neural and mesenchymal stem cells have extensive tropism for malignant glioma. The tumor tropism of induced pluripotent stem (iPS) cells was tested using the Matrigel invasion assay. Mouse iPS cells showed a significant tropism to the conditioned media prepared from six rodent and human glioma cell lines and this tropism to the glioma conditioned media was partially blocked by the neutralizing antibodies for four major tumor-associated growth factors [stem cell factor (SCF), platelet-derived growth factor BB (PDGF-BB), stromal-derived factor-1α (SDF-1α) and vascular endothelial growth factor (VEGF)], which are secreted from the malignant gliomas. The tropism of the iPS cells was enhanced by the growth factors in a concentration-dependent manner from 0.1 to 100 ng/ml. The receptors for those growth factors (c-Kit, ICAM-1, CXCR4 and VEGFR2), measured by reverse transcriptase-polymerase chain reaction, were highly up-regulated in the mouse iPS cells compared to the mouse fibroblasts. The results showed that the specific growth factors secreted from the gliomas strongly attracted the iPS cells. Therefore, gene therapies using iPS cells as vectors to deliver anti-tumor agents are novel strategies for the treatment of malignant gliomas that deeply infiltrate the brain.
[Show abstract][Hide abstract] ABSTRACT: We examined reactive oxygen species (ROS) generation on cerebral ischemia/reperfusion by intravital fluorescence imaging.
In anesthetized adult rats, a fluorescent dye (5 microL), MitoSOX (5 micromol/L) for superoxide radical (.O2-), and hydroxyphenyl fluorescein (20 micromol/L) for hydroxyl radical (.OH), was injected into cortices by a pressurized bolus. Through a closed cranial window, fluorescent images were taken with a confocal microscope on 10-minute forebrain ischemia. Because hemoglobin absorbs excitation and emission lights, ischemia may affect the change in fluorescence intensity (FI) inside the brain. To examine the effects of ischemia on the FI change, fluoromicrospheres (0.2-microm diameter) were used to mimic a dye and FI was analyzed in the same manner as when using ROS indicators. Their FI increased to 129% during ischemia (n=3/mimicking each dye), and based on the results, FI of ROS indicators was corrected.
After correcting the FI of MitoSOX and hydroxyphenyl fluorescein, they showed no change during ischemia, whereas the raw data showed the increase. In the early period of reperfusion, FI significantly (n=5/each, P<.01) increased (to 183% in MitoSOX and to 189% in hydroxyphenyl fluorescein), and these increases were significant in the areas adjacent to the arteries. To test the feasibility of our imaging, edaravone (3.0 mg/kg) was used. The treatment completely scavenged .OH, but did not do so in .O2- generation.
ROS production increased in the early period of reperfusion but not during ischemia, which was location selective, being significant in the areas adjacent to the arteries. Our method was useful for investigating intracellular in situ ROS production.
[Show abstract][Hide abstract] ABSTRACT: In our previous study, we successfully treated an established C6 brain tumor using neural stem cells transduced with the herpes simplex virus-thymidine kinase gene (HSVtk) and ganciclovir in the rat. In the present study, we investigated the use of mesenchymal stem cells (MSCs), obtained from adult rats and transduced with HSVtk (MSCtk cells), instead of neural stem cells because MSCs are much easier to obtain from the adult subjects. Those cells were used for in vitro co-culture study and in vivo co-implantation study with C6 rat glioma cells to examine bystander tumoricidal effect, which revealed a sufficient bystander effect and only 1/32 MSCtk cells were needed for complete tumor eradication. In vitro bystander effect was also observed in a real-time fashion using a culture microscope and it was shown that only tumor cells that had contact with MSCtk cells died. In vivo treatment study of an established C6 brain tumor with an intratumoral injection of MSCtk cells followed by systemic ganciclovir administration demonstrated a significant reduction of the tumor size and a significant survival prolongation. The treatment strategy using MSCtk and ganciclovir (MSCtk therapy) is more feasible and practical for clinical application than the method using neural stem cells.
International Journal of Oncology 12/2009; 35(6):1265-70. DOI:10.3892/ijo-00000443 · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The neuroprotective effects and mechanism of action of GIF-0173, a Delta12-prostaglandin J analogue, were investigated in the early phase of cerebral ischemia. GIF-0173 was administered intravenously immediately following middle cerebral artery occlusion (MCAO) in photochemically induced thrombosis model of rat. Neurological scores and infarct sizes were examined at 24 h after MCAO. Cerebral blood flow (CBF) was monitored by laser-Doppler flowmetry for 1 h after MCAO. In cultured cortical neurons obtained from 1-day-old rats, the effects of GIF-0173 on the excitotoxicity induced by glutamate were examined. Morphological changes, neuronal death, and changes in intracellular calcium concentration ([Ca(2+)](i)) were also examined. GIF-0173 improved neurological scores and reduced the infarct size in a dose-dependent manner following MCAO. But GIF-0173 did not improve CBF after MCAO. GIF-0173 also prevented glutamate-induced neuronal death and acute cellular swelling in primary cultures in a dose-dependent manner, indicating that it inhibited neuronal necrosis. GIF-0173 dose-dependently suppressed the glutamate-induced increase in [Ca(2+)](i), but could not inhibit NMDA-induced calcium influx. The effects of GIF-0173 against glutamate-induced [Ca(2+)](i) increase were reversed by addition of non-specific prostaglandin D (PGD(2)) receptor antagonist and were comparable to the effects of PGD(2) DP1 receptor agonist, which prevented [Ca(2+)](i) increase and neuronal death. We conclude that GIF-0173 reduces cerebral infarction and protects cultured neurons against glutamate-induced excitotoxicity by inhibiting [Ca(2+)](i) increase through DP1 receptor activation.
[Show abstract][Hide abstract] ABSTRACT: In the PDT practice for tumor patients, the dose and irradiation time for the treatment are chosen by experience and not by real need. To establish advanced PDD-PDT model system for patients, we developed a method for monitoring the cell-death based on a spectrophotometric real-time change in fluorescence in HeLa-tumors during PhotofrinÂ®-PDT and ALA-PDT. Here, we describe the results of application of the new PDD-PDT system to human tumors. The fluorescence spectra obtained from human tumors were analyzed by the differential spectral analysis. The mass-spectral changes of tumor tissues during PDD-PDT were also examined by MALDI-TOF-MS/MS. The first author's seborrheic keratosis was monitored with this system during the PDD-PDT with a topically applied ALA-ointment. The changes in fluorescence spectrum were successfully detected, and the tumor regressed completely within 5 months. The differential spectral analysis of PDD-PDT-fluorescence monitoring spectra of tumors and isolated mitochondria showed a marked decrease of three peaks in the red region indicative of the PDD (600 - 720 nm), and a transient rise followed by a decline of peaks in the green region indicative of the PDT (450 - 580 nm). The MALDI-TOF-MS analysis of PDD-PDT HeLa-tumors showed a consumption of Photofrin-deuteroporphyrin and ALA-PpIX, and decreases in protein mass in the range of 4,000 - 16,000 Da, m/z 4929, 8564, 10089, 15000, and an increase in m/z 7002 in a PhotofrinÂ® PDD-PDT monitoring tumor.
Proceedings of SPIE - The International Society for Optical Engineering 06/2009; 7380. DOI:10.1117/12.823247 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION: Under the video enhanced contrast-differential interference contrast (VEC-DIC) microscope, the intact cultured neurons show large nuclei containing an amorphous nucleoplasm, and an application of glutamate induces granular structure inside the nucleus within 20 minutes (1). This nuclear change corresponds to DNA fragmentation, indicating that the extracellular stimuli would affect the nuclear DNA in the very early stage of excitotoxicity (2). However, whether alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) could produce the comparable change remains unclear. In the present study, we sought to determine the sole effects of AMPA application on the hippocampal neurons. METHODS: Dissociated hippocampal cultures were prepared from one-day old Wister rats, and used for the experiment three weeks later. Morphological changes in hippocampal neurons induced by AMPA were observed under the VEC-DIC microscope. To detect the type of cell death, that is, necrosis or apoptosis, hippocampal neurons were stained following AMPA application with propidium iodide (PI) and fluorescein-4-isothiocyanate (FITC) conjugated annexin V (annexin V-FITC), and examined under the fluorescence microscope. DNA fragmentation was assessed using comet assay, a single cell gel electrophoresis assay. To assess the severity of DNA fragmentation, we classified DNA damage from grade 1 (no damage) to 5 (severe damage) by visual scoring. RESULTS: Under the VEC-DIC microscope, continuous exposure to higher concentration of AMPA (100 μM, 1 mM) dose-dependently showed the nuclear granulation and the cellular swelling within 5 minutes. Although 10 μM-AMPA also induced the cellular swelling, it did not produce the marked nuclear granulation, suggesting that the pathway inducing cellular swelling may be distinct from that inducing nuclear granulation. Non-competitive AMPA receptor antagonist, GYKI-52466 (50 μM), blocked these morphological changes after continuous exposure to 100 μM-AMPA for 1 h, while N-methyl-D-aspartate (NMDA) receptor blocker, MK-801 (20-200 μM), did not do so, indicating that AMPA mediated toxicity independently of NMDA receptor activation. The staining with PI and annexin V-FITC revealed that AMPA (100 μM, 1 mM) induced necrosis but not apoptosis in dose dependent manner. The treatment with AMPA (100 μM, 1 mM) for 1 h significantly increased the percentage of grade 4, severer DNA fragmentation in comet assay. COMMENTS: In hippocampal neurons, AMPA receptor stimulation alone, independently of NMDA receptor, induces the cellular swelling and the nuclear granulation in the very early stage of necrosis. The granular change in the nucleus observed under the VEC-DIC microscope corresponds to DNA fragmentation. Our results suggest that: 1) even in the process of necrosis, the extracellular stimuli to the AMPA receptor rapidly produce the nuclear signal to induce DNA fragmentation; and 2) the pathway inducing DNA fragmentation may be less sensitive to AMPA receptor activation as compared with that inducing cellular swelling.
Neuroscience Research 12/2007; 58. DOI:10.1016/j.neures.2007.06.1451 · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Our previous study demonstrated successful treatment of an established rat brain tumor through the bystander effect by intra-tumoral injection of neural stem cells transduced with herpes simplex virus-thymidine kinase gene (NSCtk) followed by systemic ganciclovir (GCV) administration (NSCtk therapy). Since glioma has a strong tendency to infiltrate into surrounding brain tissue and that is one of the main causes of local treatment failure, we, in the present study, injected NSCtk cells at distant sites of rat brain tumors and evaluated migratory potential of NSCtk toward the tumor and anti-tumor effects of the NSCtk therapy of this experimental setting. NSCtk cells were intracranially implanted either at 2mm medial in the ipsilateral hemisphere or at the mirror point in the contralateral hemisphere to the C6 rat glioma cell implantation. Active migration of NSCtk cells toward C6 cells was observed even when NSCtk cells were implanted in the contralateral hemisphere. When GCV was systemically administered, growth of intracranial tumor was markedly inhibited and the survival was significantly prolonged through the bystander effect by NSCtk cells migrated from distant injection sites of the tumor. The results of the present study suggest that NSCtk therapy is still effective in the area far from the NSCtk injection site and, therefore, suitable for treatment of malignant gliomas that deeply infiltrate and widely disseminate in the brain.
Cancer Letters 07/2007; 251(2):220-7. DOI:10.1016/j.canlet.2006.11.024 · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reactive free radical species are thought to be involved in postoperative neurologic dysfunction after antegrade selective cerebral perfusion in brains with old infarction. We assessed the brain protective effect of prophylactically administered edaravone, a free radical scavenger, for antegrade selective cerebral perfusion in brains with or without old infarction in a canine model.
A canine model of old cerebral infarction was created by injecting cylindric silicone embolus into the middle cerebral artery. Animals showing obvious neurologic deficits and surviving 4 weeks or longer were included in the model. Deep hypothermia with antegrade selective cerebral perfusion was performed in both intact (non-edaravone, group A; edaravone-treated, group B) and infarcted animals (non-edaravone, group C; edaravone-treated, group D). Serum concentrations of malondialdehyde, hexanoyl-lysine, glutamate, and venous-arterial lactate difference were measured, and central conduction time and amplitude of somatosensory evoked potentials were assessed during the operation.
Compared with the intact groups, serum concentrations of malondialdehyde and hexanoyl-lysine in group C significantly increased at the end of antegrade selective cerebral perfusion, whereas that of glutamate did so in the rewarming phase. Increases in all these biochemical parameters were suppressed in group D. In group C, the venous-arterial lactate difference was significantly greater in the rewarming phase at 28 degrees C compared with intact groups. A significant prolongation of postoperative central conduction time and decrease in neuronal activity were detected in group C, both of which recovered in group D.
Prophylactic administration of edaravone exerted a significant protective effect against postoperative neurologic dysfunction after antegrade selective cerebral perfusion in a canine model with old cerebral infarction.
The Journal of thoracic and cardiovascular surgery 04/2007; 133(3):710-6. DOI:10.1016/j.jtcvs.2006.10.032 · 4.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Due to very complex structure of nasal area that is covered by facial bones, a tracking of surgical instruments on the preoperative CT image is very important for obtaining an improved image guidance as well as preventing surgical accidents in the paranasal sinus surgery. In this contribution, we present our recently developed an efficient and compact navigation system for paranasal sinus surgery and its first clinical trial. In our system, we use an optical-based 3D range imaging device intra-operatively, in order to achieve registration and a tracking of instruments. Before the intervention, the range image of patient's face is acquired by a 3D range scanner and registered to corresponding surface extracted from the preoperative CT images. The surgical instrument fitted with spherical markers that also can be measured by range scanning device, is tracked during the procedure. The main advantages of our system are (a) markerless on the patient's body, (b) an easy semiautomatic registration, (c) frameless during surgery, thus, it is feasible to update a registration and to restart the tracking when a patient moves. In this paper, we describe a summary of used techniques in our approach including the benefits and limitations of the system, experimental results using a precise model based on a human paranasal structure and a first clinical trial in the surgical room.
Proceedings of SPIE - The International Society for Optical Engineering 03/2007; DOI:10.1117/12.709070 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Singlet oxygen ((1)O(2)) generated in photodynamic therapy (PDT) plays a very important role in killing tumor cells. Using a new near-IR photomultiplier tube system, we monitored the real-time production of (1)O(2) during PDT and thus investigated the relationship between the (1)O(2) production and photodynamic effects.
We did PDT in 9L gliosarcoma cells in vitro and in an experimental tumor model in vivo using 5-aminolevulinic acid and nanosecond-pulsed dye laser. During this time, we monitored (1)O(2) using this system. Moreover, based on the (1)O(2) monitoring, we set the different conditions of laser exposure and investigated whether they could affect the tumor cell death.
We could observe the temporal changes of (1)O(2) production during PDT in detail. At a low fluence rate the (1)O(2) signal gradually decreased with a low peak, whereas at a high fluence rate it decreased immediately with a high peak. Consequently, the cumulative (1)O(2) at a low fluence rate was higher, which thus induced a strong photodynamic effect. The proportion of apoptosis to necrosis might therefore be dependent on the peak and duration of the (1)O(2) signal. A low fluence rate tended to induce apoptotic change, whereas a high fluence rate tended to induce necrotic change.
The results of this study suggested that the monitoring of (1)O(2) enables us to predict the photodynamic effect, allowing us to select the optimal laser conditions for each patient.
Clinical Cancer Research 01/2007; 12(23):7132-9. DOI:10.1158/1078-0432.CCR-06-0786 · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This paper presents a segmentation method of brain tissues from MR images, invented for our image-guided neurosurgery system under development. Our goal is to segment brain tissues for creating biomechanical model. The proposed segmentation method is based on 3-D region growing and outperforms conventional approaches by stepwise usage of intensity similarities between voxels in conjunction with edge information. Since the intensity and the edge information are complementary to each other in the region-based segmentation, we use them twice by performing a coarse-to-fine extraction. First, the edge information in an appropriate neighborhood of the voxel being considered is examined to constrain the region growing. The expanded region of the first extraction result is then used as the domain for the next processing. The intensity and the edge information of the current voxel only are utilized in the final extraction. Before segmentation, the intensity parameters of the brain tissues as well as partial volume effect are estimated by using expectation-maximization (EM) algorithm in order to provide an accurate data interpretation into the extraction. We tested the proposed method on T1-weighted MR images of brain and evaluated the segmentation effectiveness comparing the results with ground truths. Also, the generated meshes from the segmented brain volume by using mesh generating software are shown in this paper.
Proceedings of SPIE - The International Society for Optical Engineering 03/2006; 6141. DOI:10.1117/12.652969 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To study cellular morphology and functions in vivo in realtime, we developed a fiber-coupled confocal microscope (FCM), and observed fluorescently-labeled cells inside the body of anesthetized rat. We developed an imaging fiber bundle (IFB), which consisted of an objective lens and a multi-fiber assembly (unit fiber: NA > 0.4, 3 micron in diameter). By combining the IFB with a real-time confocal scanner, we detected intracellular signals of the molecular messenger, and the death signals in the form of fluorescence changes even from cells located deep (> 2 mm) inside the solid organs. The FCM we developed is very promising for detailed studies in both the cell-based researches and clinical researches.
Proceedings of SPIE - The International Society for Optical Engineering 01/2006; DOI:10.1117/12.645939 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In order to study the dynamic change in the cell, we modified the evanescence microscope with an ultra high NA objective lens so as to modulate the penetration depth of the evanescent wave. We employed a galvanomirror to aim and switch the laser beam rapidly at the back focal plane near the periphery of 1.45 or 1.65 NA objectives. Under this microscope equipped with a 1.45 NA objective, images of the fluorescent bead were clearly distinguishable by the modulation of the penetration depth of the evanescent wave. Thus, translocation dynamics of protein kinase Calpha (PKCalpha) upon cell activation were compared every 0.5 s between two modes using HeLa cells expressing PKCalpha fused with the green fluorescent protein (GFP). Stimulation of the cell with phorbol ester induced a transient increase in GFP fluorescence images illuminated by the thin evanescent field, but not in the image illuminated by the thick evanescent field. Later, a persistent increase in fluorescence appeared at cell borders in the both images. Using a 1.65 NA objective, trafficking of secretory vesicles was studied in MIN6 cells expressing insulin-GFP. Occasionally, the change in fluorescence of a vesicle observed under one illumination mode appeared very different from the other, allowing unique assignments of the fluorescence change to a certain combination of vesicle movement and a chemical response of fluorescent molecules. The ultra high NA lens provides a large window for evanescent illumination with a wide range of penetration depth, thus is useful for analyzing 3D events in the cell.
Proceedings of SPIE - The International Society for Optical Engineering 01/2006; DOI:10.1117/12.647094 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neurosurgical navigation systems using preoperative images have a problem in their accuracy caused by brain deformation during surgery. To address this problem the use of laser range scanner in order to obtain intraoperative cortical surface, is under study in our currently developing neurosurgical navigation system. This paper presents preliminary results of registration of intraoperatively acquired range and color images to preoperative MR images, within the context of image-guided surgery. We register images by performing two procedures: mapping of color image on the range image; and registration between color-mapped range images and preoperative medical images. The color image is mapped on the range image using camera calibration. Point-based rigid registration of preoperative images to the intraoperative images is performed through detection and matching of common fiducials in the images. Experimental results using intraoperatively acquired range images of cortical surface demonstrated the ability to perform registrations for MR images of the brain. In the future, we will focus on incorporating the above registration results into a biomechanical model of the brain to predict brain deformation during surgical procedures.
Proceedings of SPIE - The International Society for Optical Engineering 01/2006; 6141. DOI:10.1117/12.652923 · 0.20 Impact Factor