Kazuhiro Koshino

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.

Multicenter Evaluation of a Standardized Protocol for Rest and Acetazolamide Cerebral Blood Flow Assessment Using a Quantitative SPECT Reconstruction Program and Split-Dose 123I-Iodoamphetamine

SPECT can provide valuable diagnostic and treatment response information in large-scale multicenter clinical trials. However, SPECT has been limited in providing consistent quantitative functional parametric values across the centers, largely because of a lack of standardized procedures to correct for attenuation and scatter. Recently, a novel software package has been developed to reconstruct quantitative SPECT images and assess cerebral blood flow (CBF) at rest and after acetazolamide challenge from a single SPECT session. This study was aimed at validating this technique at different institutions with a variety of SPECT devices and imaging protocols. Methods: Twelve participating institutions obtained a series of SPECT scans on physical phantoms and clinical patients. The phantom experiments included the assessment of septal penetration for each collimator used and of the accuracy of the reconstructed images. Clinical studies were divided into 3 protocols, including intrainstitutional reproducibility, a comparison with PET, and rest–rest study consistency. The results from 46 successful studies were analyzed. Results: Activity concentration estimation (Bq/mL) in the reconstructed SPECT images of a uniform cylindric phantom showed an interinstitution variation of ±5.1%, with a systematic underestimation of concentration by 12.5%. CBF values were reproducible both at rest and after acetazolamide on the basis of repeated studies in the same patient (mean ± SD difference, −0.4 ± 5.2 mL/min/100 g, n = 44). CBF values were also consistent with those determined using PET (−6.1 ± 5.1 mL/min/100 g, n = 6). Conclusion: This study demonstrates that SPECT can quantitatively provide physiologic functional images of rest and acetazolamide challenge CBF, using a quantitative reconstruction software package.

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.

Experimental Pig Model of Old Myocardial Infarction with Long Survival Leading to Chronic Left Ventricular Dysfunction and Remodeling as Evaluated by PET

A pig model of reduced left ventricular (LV) function and remodeling or chronic heart failure with long survival after myocardial infarction (MI) has not been established. The aim of this study was to evaluate the pathophysiologic status of a pig model of old MI using a series of PET studies. Methods: Twenty-seven male farm pigs were divided into 2 groups: 7 animals in the control group and 20 animals that underwent a proximal coronary artery (CA) occlusion using an ameroid constrictor after distal CA ligation. A series of PET examinations was performed to assess LV volumes, LV functions, myocardial perfusion response to adenosine, and viability as water-perfusable tissue index. Results: The distal CA ligation inhibited arrhythmia during and after the operation, and a transmural anteroseptal MI, with an infarction area of 27% ± 5% of the whole left ventricle, was generated with a survival rate of 75% at 4 mo. Wall motion evaluated by 18F-FDG PET was diffusely reduced, including the noninfarcted wall. Global LV ejection fraction as assessed by gated C15O PET was reduced (39% ± 16%) in the group undergoing occlusion, compared with the control group (66% ± 16%, P < 0.05). LV end-systolic (31.4 ± 9.2 cm3) and end-diastolic (52.7 ± 10.2 cm3) volumes were increased, compared with controls (15.2 ± 9.4 cm3, P < 0.01, and 41.7 ± 11.5 cm3, P < 0.05, respectively). Histology showed hypertrophy and development of microscopic fibrosis in noninfarcted myocardium. PET demonstrated the reduced myocardial perfusion response to adenosine and also reduced water-perfusable tissue index in remote segments. Conclusion: The pig model of old MI generated by the chronic proximal CA obstruction after distal ligation was characterized by LV dysfunction and remodeling, with a high survival rate.

Validity of using a 3-dimensional PET scanner during inhalation of 15O-labeled oxygen for quantitative assessment of regional metabolic rate of oxygen in man

Use of 15O labeled oxygen (15O2) and positron emission tomography (PET) allows quantitative assessment of the regional metabolic rate of oxygen (CMRO2) in vivo, which is essential to understanding the pathological status of patients with cerebral vascular and neurological disorders. The method has, however, been challenging, when a 3D PET scanner is employed, largely attributed to the presence of gaseous radioactivity in the trachea and the inhalation system, which results in a large amount of scatter and random events in the PET assessment. The present study was intended to evaluate the adequacy of using a recently available commercial 3D PET scanner in the assessment of regional cerebral radioactivity distribution during an inhalation of 15O2. Systematic experiments were carried out on a brain phantom. Experiments were also performed on a healthy volunteer following a recently developed protocol for simultaneous assessment of CMRO2 and cerebral blood flow, which involves sequential administration of 15O2 and C15O2. A particular intention was to evaluate the adequacy of the scatter-correction procedures. The phantom experiment demonstrated that errors were within 3% at the practically maximum radioactivity in the face mask, with the greatest radioactivity in the lung. The volunteer experiment demonstrated that the counting rate was at peak during the 15O gas inhalation period, within a verified range. Tomographic images represented good quality over the entire FOV, including the lower part of the cerebral structures and the carotid artery regions. The scatter-correction procedures appeared to be important, particularly in the process to compensate for the scatter originating outside the FOV. Reconstructed images dramatically changed if the correction was carried out using inappropriate procedures. This study demonstrated that accurate reconstruction could be obtained when the scatter compensation was appropriately carried out. This study also suggested the feasibility of using a state-of-the-art 3D PET scanner in the quantitative PET imaging during inhalation of 15O labeled oxygen.

A physiologic model for recirculation water correction in CMRO2 assessment with 15O2 inhalation PET

Cerebral metabolic rate of oxygen (CMRO2) can be assessed quantitatively using 15O2 and positron emission tomography. Determining the arterial input function is considered critical with regards to the separation of the metabolic product of 15O2 (RW) from a measured whole blood. A mathematical formula based on physiologic model has been proposed to predict RW. This study was intended to verify the adequacy of that model and a simplified procedure applying that model for wide range of species and physiologic conditions. The formula consists of four parameters, including of a production rate of RW (k) corresponding to the total body oxidative metabolism (BMRO2). Experiments were performed on 6 monkeys, 3 pigs, 12 rats, and 231 clinical patients, among which the monkeys were studied at varied physiologic conditions. The formula reproduced the observed RW. Greater k values were observed in smaller animals, whereas other parameters did not differ amongst species. The simulation showed CMRO2 sensitive only to k, but not to others, suggesting that validity of determination of only k from a single blood sample. Also, k was correlated with BMRO2, suggesting that k can be determined from BMRO2. The present model and simplified procedure can be used to assess CMRO2 for a wide range of conditions and species.

Breath-hold CT attenuation correction for quantitative cardiac SPECT

BackgroundAttenuation correction of a single photon emission computed tomography (SPECT) image is possible using computed tomography (CT)-based attenuation maps with hybrid SPECT/CT. CT attenuation maps acquired during breath holding can be misaligned with SPECT, generating artifacts in the reconstructed images. The purpose of this study was to investigate the effects of respiratory phase during breath-hold CT acquisition on attenuation correction of cardiac SPECT imaging.MethodsA series of 201Tl-emission and 99mTc-based transmission computed tomography (TCT) scans was carried out along with CT-attenuation scans on 11 young normal volunteers using a hybrid SPECT/CT scanner. The CT scans were performed at three respiratory phases: end-inspiration (INS), end-expiration (EXP), and the midpoint (MID) between these phases. Using alignment parameters between attenuation maps and SPECT images without attenuation or scatter corrections, quantitative SPECT images were reconstructed, including corrections for attenuation and scatter. Regional radioactivity concentrations normalized by the subjects’ weights were compared between CT- and TCT-based attenuation correction techniques.ResultsSPECT images with CT attenuation maps at the EXP phase showed significant differences in regional weight-normalized radioactivity concentrations relative to the images using the other attenuation maps (p < 0.05), as well as systematic positive bias errors, compared to TCT-based images for all myocardial segments, 5.7% ± 2.7% (1.9% to 10.0%). No significant differences in regional weight-normalized radioactivity concentrations were observed between images with CT attenuation maps at MID and INS phases or between these and the TCT-based images, but regional tendencies were found: for anterior to anterolateral segment, positive bias of 5.0% ± 2.2% (1.3% to 8.1%) and 5.6% ± 1.9% (2.6% to 8.5%) and for inferior to inferoseptal segment, negative bias of −5.3% ± 2.6% (−9.1% to −1.7%) and −4.6% ± 2.5% (−8.8% to −1.5%) for the MID and INS phases, respectively.ConclusionsUse of breath-hold CT attenuation maps at INS and MID phases for attenuation and scatter corrections demonstrated accurate quantitative images that would prove beneficial in cardiac SPECT/CT studies.

Monte Carlo estimation of scatter effects on quantitative myocardial blood flow and perfusable tissue fraction using 3D-PET and 15O-water

In clinical cardiac positron emission tomography using (15)O-water, significant tracer accumulation is observed not only in the heart but also in the liver and lung, which are partially outside the field-of-view. In this work, we investigated the effects of scatter on quantitative myocardium blood flow (MBF) and perfusable tissue fraction (PTF) by a precise Monte Carlo simulation (Geant4) and a numerical human model. We assigned activities to the heart, liver, and lung of the human model with varying ratios of organ activities according to an experimental time activity curve and created dynamic sinograms. The sinogram data were reconstructed by filtered backprojection. By comparing a scatter-corrected image (SC) with a true image (TRUE), we evaluated the accuracy of the scatter correction. TRUE was reconstructed using a scatter-eliminated sinogram, which can be obtained only in simulations. A scatter-uncorrected image (W/O SC) and an attenuation-uncorrected image (W/O AC) were also constructed. Finally, we calculated MBF and PTF with a single tissue-compartment model for four types of images. As a result, scatter was corrected accurately, and MBFs derived from all types of images were consistent with the MBF obtained from TRUE. Meanwhile, the PTF of only the SC was in agreement with the PTF of TRUE. From the simulation results, we concluded that quantitative MBF is less affected by scatter and absorption in 3D-PET using (15)O-water. However, scatter correction is essential for accurate PTF.

Sequential PET estimation of cerebral oxygen metabolism with spontaneous respiration of 15O-gas in mice with bilateral common carotid artery stenosis

Positron emission tomography with 15O-labeled gases (15O-PET) is important for in vivo measurement of cerebral oxygen metabolism both in clinical and basic settings. However, there are currently no reports concerning 15O-PET in mice. Here, we developed an 15O-PET method applicable to mice with spontaneous respiration of 15O-gas without a tracheotomy catheter. Sequential 15O-PET was also performed in a mouse model of chronic cerebral hypoperfusion with bilateral common carotid artery stenosis (BCAS) induced by placement of microcoils. 15O-gas with isoflurane was supplied to the nose of mouse with evacuation of excess 15O-gas surrounding the body. 15O-PET was performed on days 3, 7, 14, 21, and 28 after surgery. Cerebral blood flow (CBF), cerebral blood volume, oxygen extraction fraction (OEF), and cerebral metabolic rate of oxygen (CMRO2) were calculated in whole brains. A significant decrease in CBF and compensatory increase in OEF in the BCAS group produced CMRO2 values comparable to that of the sham group at three days post-operation. Although CBF and OEF in the BCAS group gradually recovered over the first 28 days, the CMRO2 showed a gradual decrease to 68% of sham values at 28 days post-operation. In conclusion, we successfully developed a noninvasive 15O-PET method for mice.

Development of matrix metalloproteinase-targeted probes for lung inflammation detection with positron emission tomography

As matrix metalloproteinases (MMPs), especially MMP-9 and MMP-12 are involved in the pathological processes associated with chronic obstructive pulmonary disease (COPD), we developed a novel radiofluorinated probe, 18F-IPFP, for MMPs-targeted positron emission tomography (PET). 18F-IPFP was designed by iodination of MMP inhibitor to enhance the affinity, and labelled with a compact prosthetic agent, 4-nitrophenyl 2-18F-fluoropropionate (18F-NFP). As a result, IPFP demonstrated the highest affinity toward MMP-12 (IC50 = 1.5 nM) among existing PET probes. A COPD model was employed by exposing mice to cigarette smoke and the expression levels of MMP-9 and MMP-12 were significantly increased in the lungs. Radioactivity accumulation in the lungs 90 min after administration of 18F-IPFP was 4× higher in COPD mice than normal mice, and 10× higher than in the heart, muscle, and blood. Ex vivo PET confirmed the radioactivity distribution in the tissues and autoradiography analysis demonstrated that accumulation differences in the lungs of COPD mice were 2× higher than those of normal mice. These results suggest that 18F-IPFP is a promising probe for pulmonary imaging and expected to be applied to various MMP-related diseases for early diagnosis, tracking of therapeutic effects, and new drug development in both preclinical and clinical applications.