Noboru Teramoto

Impaired myocardium regeneration with skeletal cell sheets--a preclinical trial for tissue-engineered regeneration therapy.

Background. We hypothesized that autologous skeletal cell (SC) sheets regenerate the infract myocardium in porcine heart as a preclinical trial. Methods and Results. The impaired heart was created by implantation of ameroid constrictor on left anterior descending for 4 weeks. SCs isolated from leg muscle were cultured and detached from the temperature-responsive domain-coated dishes as single monolayer cell sheet at 20°C. The following therapies were conducted: SC sheets (SC group, n=5); sham (C group n=5). Echocardiography demonstrated that cardiac performance was significantly improved in the SC group 3 and 6 months after operation (fractional area shortening, 3 months; SC vs. C=49.5±2.8 vs. 24.6±2.0%, P<0.05) and left ventricle dilatation was well attenuated in the SC group. Color kinesis index showed that distressed regional diastolic and systolic function in infarcted anterior wall was significantly recovered (SC vs. C=57.4±8.6 vs. 30.2±4.7%, P<0.05, diastolic: 58.5±4.5 vs. 35.4±6.6%, P<0.05, systolic). Factor VIII immunostains demonstrated that vascular density was significantly higher in the SC group than the C group. And % fibrosis and cell diameter were significantly lower in the SC group. And hematoxylin-eosin staining depicted that skeletal origin cells and well-developed-layered smooth muscle cells were detected in the implanted area. Positron emission tomography showed better myocardial perfusion and more viable myocardial tissue in the distressed myocardium receiving SC sheets compared with the myocardium receiving no sheets. Conclusions. SC sheet implantation improved cardiac function by attenuating the cardiac remodeling in the porcine ischemic myocardium, suggesting a promising strategy for myocardial regeneration therapy in the impaired myocardium.

Long-term observation of auto-cell transplantation in non-human primate reveals safety and efficiency of bone marrow stromal cell-derived Schwann cells in peripheral nerve regeneration

Based on their differentiation ability, bone marrow stromal cells (MSCs) are a good source for cell therapy. Using a cynomolgus monkey peripheral nervous system injury model, we examined the safety and efficacy of Schwann cells induced from MSCs as a source for auto-cell transplantation therapy in nerve injury. Serial treatment of monkey MSCs with reducing agents and cytokines induced their differentiation into cells with Schwann cell properties at a very high ratio. Expression of Schwann cell markers was confirmed by both immunocytochemistry and reverse transcription-polymerase chain reaction. Induced Schwann cells were used for auto-cell transplantation into the median nerve and followed-up for 1year. No abnormalities were observed in general conditions. Ki67-immunostaining revealed no sign of massive proliferation inside the grafted tube. Furthermore, (18)F-fluorodeoxygluocose-positron emission tomography scanning demonstrated no abnormal accumulation of radioactivity except in regions with expected physiologic accumulation. Restoration of the transplanted nerve was corroborated by behavior analysis, electrophysiology and histological evaluation. Our results suggest that auto-cell transplantation therapy using MSC-derived Schwann cells is safe and effective for accelerating the regeneration of transected axons and for functional recovery of injured nerves. The practical advantages of MSCs are expected to make this system applicable for spinal cord injury and other neurotrauma or myelin disorders where the acceleration of regeneration is expected to enhance functional recovery.

Endogenous dopamine release induced by repetitive transcranial magnetic stimulation over the primary motor cortex: an [11C]raclopride positron emission tomography study in anesthetized macaque monkeys

BACKGROUND Repetitive transcranial magnetic stimulation (rTMS) has been used as a treatment for neuropsychiatric disorders such as depression and Parkinsons disease (PD). Despite the growing interest in therapeutic application of rTMS, precise mechanisms of its action remain unknown. With respect to PD, activation of the mesostriatal dopaminergic pathway is likely to be a candidate mechanism underlying the therapeutic effects; however, modulating effects of rTMS over the primary motor cortex (M1) on the dopaminergic system have not been studied. METHODS We used [11C]raclopride positron emission tomography to measure changes of extracellular dopamine concentration after 5Hz rTMS over the M1 in eight anesthetized monkeys. RESULTS rTMS over the right M1 induced a reduction of [11C]raclopride binding potential (BP) in the bilateral ventral striatum, including the nucleus accumbens, and a significant increase of BP in the right putamen; no significant BP reduction was found in the dorsal striatum. These data indicate that rTMS over the motor cortex induces a release of endogenous dopamine in the ventral striatum. CONCLUSIONS Our results suggest that therapeutic mechanisms of rTMS may be explained in part by an activation of the mesolimbic dopaminergic pathway, which plays critical roles in rewards, reinforcement, and incentive motivation.

Rapid Quantitative Measurement of CMRO2 and CBF by Dual Administration of 15O-Labeled Oxygen and Water During a Single PET Scan—a Validation Study and Error Analysis in Anesthetized Monkeys

Cerebral blood flow (CBF) and rate of oxygen metabolism (CMRO2) may be quantified using positron emission tomography (PET) with 15O-tracers, but the conventional three-step technique requires a relatively long study period, attributed to the need for separate acquisition for each of 15O2, H215O, and C15O tracers, which makes the multiple measurements at different physiologic conditions difficult. In this study, we present a novel, faster technique that provides a pixel-by-pixel calculation of CBF and CMRO2 from a single PET acquisition with a sequential administration of 15O2 and H215O. Experiments were performed on six anesthetized monkeys to validate this technique. The global CBF, oxygen extraction fraction (OEF), and CMRO2 obtained by the present technique at rest were not significantly different from those obtained with three-step method. The global OEF (gOEF) also agreed with that determined by simultaneous arterio-sinus blood sampling (gOEFA–V) for a physiologically wide range when changing the arterial PaCO2 (gOEF = 1.03gOEFA–V +0.01, P<0.001). The regional values, as well as the image quality were identical between the present technique and three-step method for CBF, OEF, and CMRO2. In addition, a simulation study showed that error sensitivity of the present technique to delay or dispersion of the input function, and the error in the partition coefficient was equivalent to that observed for three-step method. Error sensitivity to cerebral blood volume (CBV) was also identical to that in the three-step and reasonably small, suggesting that a single CBV assessment is sufficient for repeated measures of CBF/CMRO2. These results show that this fast technique has an ability for accurate assessment of CBF/CMRO2 and also allows multiple assessment at different physiologic conditions.

Long-term effect of motor cortical repetitive transcranial magnetic stimulation induces

Repetitive transcranial magnetic stimulation (rTMS) recently has been assessed as a noninvasive treatment modality for movement and psychiatric disorders, whereas the mechanism underlying the therapeutic effects is not fully understood. Studies in rodents showed lasting functional changes in some selected regions, such as limbic‐associated structures, but unfocused brain stimulation did not clarify the regional effects. To address the topographical and temporal profiles of the effects on glucose metabolism in primate brain, we performed rTMS and repeated 18F‐fluorodeoxyglucose positron emission tomography (FDG‐PET) before, during, and up to 16 days after rTMS in anesthetized cynomologous monkeys. We delivered a total of 2,000 pulses of 5Hz‐rTMS over the right precentral gyrus using a small‐sized eight‐figured coil that induced a localized electrical field. Voxel‐based analysis in a standard space of the macaque brain showed statistically robust changes in FDG uptake: a decrease in the motor/premotor cortices and an increase in the limbic‐associated areas involving the anterior/posterior cingulate, and orbitofrontal cortices. Interestingly, these uptake changes continued for at least 8 days and the magnitude of the lasting effects in the limbic‐related areas was negatively correlated across subjects with those in the motor/premotor cortices. The results demonstrate that motor rTMS has a long‐term lasting effect on motor‐related regions and distant limbic‐related areas via functional connections.

Development of a GSO detector assembly for a continuous blood sampling system

A new input function monitoring system has been developed using the GSO detector assembly for both PET and SPELT quantitative studies. Due to the rapid rise time of the pulse (about 10nsec), the coincidence time window can be reduced < 1nsec, reducing contribution of randoms associated with the high background activity surrounding the detector. Large light output improved the energy resolution of approximately 11% for 511keV photons, and 20% at 140 keV, as compared with the BGO detector, enabling the use of this system also in SPELT studies. The paired assembly of crystals provided the absolute sensitivity of approximately 7% for PET and 75% for PET tracers. Multiple arrangement of the paired detectors provided possibility of correcting for the transit time of radioactivity through the catheter tube. This study demonstrated that the present system can be of use in both clinical and small animal studies using SPECT and PET tracers.

A new reconstruction strategy for image improvement in pinhole SPECT

Pinhole single-photon emission computed tomography (SPECT) is able to provide information on the biodistribution of several radioligands in small laboratory animals, but has limitations associated with non-uniform spatial resolution or axial blurring. We have hypothesised that this blurring is due to incompleteness of the projection data acquired by a single circular pinhole orbit, and have evaluated a new strategy for accurate image reconstruction with better spatial resolution uniformity. A pinhole SPECT system using two circular orbits and a dedicated three-dimensional ordered subsets expectation maximisation (3D-OSEM) reconstruction method were developed. In this system, not the camera but the object rotates, and the two orbits are at 90° and 45° relative to the object’s axis. This system satisfies Tuy’s condition, and is thus able to provide complete data for 3D pinhole SPECT reconstruction within the whole field of view (FOV). To evaluate this system, a series of experiments was carried out using a multiple-disk phantom filled with 99mTc solution. The feasibility of the proposed method for small animal imaging was tested with a mouse bone study using 99mTc-hydroxymethylene diphosphonate. Feldkamp’s filtered back-projection (FBP) method and the 3D-OSEM method were applied to these data sets, and the visual and statistical properties were examined. Axial blurring, which was still visible at the edge of the FOV even after applying the conventional 3D-OSEM instead of FBP for single-orbit data, was not visible after application of 3D-OSEM using two-orbit data. 3D-OSEM using two-orbit data dramatically reduced the resolution non-uniformity and statistical noise, and also demonstrated considerably better image quality in the mouse scan. This system may be of use in quantitative assessment of bio-physiological functions in small animals.

Absolute quantitation of regional myocardial blood flow of rats using dynamic pinhole SPECT

PET and SPECT have been widely used to investigate the physiological function of animals in vivo. However, little efforts have been done to estimate absolute physiological parameters, i.e. blood flow of small animals. The present study was aimed at the absolute quantitation of myocardial blood flow of rats by means of the dynamic SPECT fitted with a pinhole collimator with a careful determination of the arterial input function (IF). The center-of-rotation was carefully aligned to the center of the field-of-view of a fixed gamma camera with the accuracy < 0.05 mm. A rat was placed on a rotating device that fixes the rat in a stand position. The arterial blood samples were frequently collected and their radioactivity concentration was measured using a well counter cross-calibrated to the SPECT images. Dynamic SPECT (the step-and-shoot mode) was initiated at 5 min after the injection of 201TlCl into the tail vein. Acquisition period was 10 sec at each rotation angle, and 120 view projection data were obtained. The 360-degree complete data sets were obtained at approximately 20 min interval for 5 time frames. Images were reconstructed by filtered-back projection technique with Feldkamp algorithm. The cross-calibration factor was determined using a cylindrical phantom of 5 cm diameter filled with the 201TlCl solution. Regions-of-interest were placed on the left ventricular wall to generate the tissue time activity curve (TTAC). TTAC and IF were fitted to the previously validated single-tissue compartment model to estimate the regional myocardial blood flow (rMBF) and volume of distribution (Vd) of thallium. The present system provided clear images of myocardial uptake of 201TlCl, and the time-dependent change of the tissue radioactivity concentration in regional basis, which was statistically sufficient for applying the compartment model analysis. The kinetic analysis yielded the rMBF of approximately 0.77 ml/min/g, which appeared to be an acceptable value with a consideration of contribution of partial volume effect and other error sources. Vd of approximately 91.9 ml/ml was also consistent with the know value of the potassium potential across the cell membrane. These results strongly suggested the potential of the dynamic pinhole SPECT as a tool for absolute quantitation of physiological parameters in small animals.

Use of a compact pixellated gamma camera for small animal pinhole SPECT imaging

ObjectivesPinhole SPECT which permitsin vivo high resolution 3D imaging of physiological functions in small animals facilitates objective assessment of pharmaceutical development and regenerative therapy in pre-clinical trials. For handiness and mobility, the miniature size of the SPECT system is useful. We developed a small animal SPECT system based on a compact high-resolution gamma camera fitted to a pinhole collimator and an object-rotating unit. This study was aimed at evaluating the basic performance of the detection system and the feasibility of small animal SPECT imaging.MethodsThe gamma camera consists of a 22 × 22 pixellated scintillator array of 1.8 mm × 1.8 mm × 5 mm NaI(TI) crystals with 0.2-mm gap between the crystals coupled to a 2″ flat panel position-sensitive photomultiplier tube (Hamamatsu H8500) with 64 channels. The active imaging region of the camera was 43.8 mm × 43.8 mm. Data acquisition is controlled by a personal computer (Microsoft Windows) through the camera controller. Projection data over 360° for SPECT images are obtained by synchronizing with the rotating unit. The knife-edge pinhole collimators made of tungsten are attached on the camera and have 0.5-mm and 1.0-mm apertures. The basic performance of the detection system was evaluated with99mTc and201T1 solutions. Energy resolution, system spatial resolution and linearity of count rate were measured. Rat myocardial perfusion SPECT scans were sequentially performed following intravenous injection of201T1C1. Projection data were reconstructed using a previously validated pinhole 3D-OSEM method.ResultsThe energy resolution at 140 keV was 14.8% using a point source. The system spatial resolutions were 2.8-mm FWHM and 2.5-mm FWHM for99mTc and201T1 line sources, respectively, at 30-mm source distance (magnification factor of 1.3) using a 1.0-mm pinhole. The linearity between the activity and count rate was good up to 10 kcps. In a rat study, the left ventricular walls were clearly visible in all scans.ConclusionsWe developed a compact SPECT system using compact gamma camera for small animals and evaluated basic physical performances. The present system may be of use for quantitation of biological functions such as myocardial blood flow in small animals.

Rapid quantitative CBF and CMRO2 measurements from a single PET scan with sequential administration of dual 15O-labeled tracers

Positron emission tomography (PET) with 15O tracers provides essential information in patients with cerebral vascular disorders, such as cerebral blood flow (CBF), oxygen extraction fraction (OEF), and metabolic rate of oxygen (CMRO2). However, most of techniques require an additional C15O scan for compensating cerebral blood volume (CBV). We aimed to establish a technique to calculate all functional images only from a single dynamic PET scan, without losing accuracy or statistical certainties. The technique was an extension of previous dual-tracer autoradiography (DARG) approach, but based on the basis function method (DBFM), thus estimating all functional parametric images from a single session of dynamic scan acquired during the sequential administration of H215O and 15O2. Validity was tested on six monkeys by comparing global OEF by PET with those by arteriovenous blood sampling, and tested feasibility on young healthy subjects. The mean DBFM-derived global OEF was 0.57 ± 0.06 in monkeys, in an agreement with that by the arteriovenous method (0.54 ± 0.06). Image quality was similar and no significant differences were seen from DARG; 3.57% ± 6.44% and 3.84% ± 3.42% for CBF, and −2.79% ± 11.2% and −6.68% ± 10.5% for CMRO2. A simulation study demonstrated similar error propagation between DBFM and DARG. The DBFM method enables accurate assessment of CBF and CMRO2 without additional CBV scan within significantly shortened examination period, in clinical settings.