Takayuki Ose

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.

Autologous mesenchymal stem cell–derived dopaminergic neurons function in parkinsonian macaques

A cell-based therapy for the replacement of dopaminergic neurons has been a long-term goal in Parkinsons disease research. Here, we show that autologous engraftment of A9 dopaminergic neuron-like cells induced from mesenchymal stem cells (MSCs) leads to long-term survival of the cells and restoration of motor function in hemiparkinsonian macaques. Differentiated MSCs expressed markers of A9 dopaminergic neurons and released dopamine after depolarization in vitro. The differentiated autologous cells were engrafted in the affected portion of the striatum. Animals that received transplants showed modest and gradual improvements in motor behaviors. Positron emission tomography (PET) using [11C]-CFT, a ligand for the dopamine transporter (DAT), revealed a dramatic increase in DAT expression, with a subsequent exponential decline over a period of 7 months. Kinetic analysis of the PET findings revealed that DAT expression remained above baseline levels for over 7 months. Immunohistochemical evaluations at 9 months consistently demonstrated the existence of cells positive for DAT and other A9 dopaminergic neuron markers in the engrafted striatum. These data suggest that transplantation of differentiated autologous MSCs may represent a safe and effective cell therapy for Parkinsons disease.

The use of magnetic resonance cell tracking to monitor endothelial progenitor cells in a rat hindlimb ischemic model.

A water-soluble magnetic resonance imaging (MRI) contrast agent, Dextran mono-N-succinimidyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate-gadolinium(3+) (Dex-DOTA-Gd(3+)), was shown to enable monitoring of the anatomical migration and the survival period of transplanted stem cells for up to 1 month. Gadolinium molecules in the cells were rapidly eliminated from the site and excreted upon cell death. Endothelial progenitor cells (EPCs) transplanted into the inguinal femoral muscle of rats migrated distally through the knee in rats after hindlimb ischemia but did not migrate in non-ischemic rats. Interestingly, the survival period of transplanted EPCs was notably prolonged in the ischemic limb, indicating that EPCs are required by the ischemic tissues and that the fate of transplanted EPCs was affected by the disease. Compared to the commonly used particle type of MRI contrast agents, the system described in this study is expected to be invaluable to help clarifying the process of stem cell transplantation therapy.

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.

Quantification of regional cerebral blood flow in rats using an arteriovenous shunt and micro-PET

INTRODUCTION Measurement of regional cerebral blood flow (rCBF) in rodents can provide knowledge of pathophysiology of the cerebral circulation, but generally requires blood sampling for analysis during positron emission tomography (PET). We therefore tested the feasibility of using an arteriovenous (AV) shunt in rats for less invasive blood analysis. METHODS Six anesthetized rats received [15O]H2O and [15O]CO PET scans with their femoral artery and vein connected by an AV shunt, the activity within which was measured with a germanium ortho-oxysilicate scintillation detector. The [15O]H2O was intravenously injected either at a faster or slower injection rate, while animals were placed either with their head or heart centered in the gantry. The time-activity curve (TAC) from the AV shunt was compared with that from the cardiac ventricle in PET image. The rCBF values were calculated by a nonlinear least-square method using the dispersion-corrected AV-shunt TAC as an input. RESULTS The AV-shunt TAC had higher signal-to-noise ratio, but also had delay and dispersion compared with the image-derived TAC. The delay time between the AV-shunt TAC and image-based TAC ranged from 11 to 21 s, while the dispersion was estimated to be ∼5 s as a time constant of the dispersion model of exponential function, and both were properly corrected. In a steady-state condition of [15O]CO PET, the blood activity concentration by AV-shunt TAC was also comparable in height with the image-based TAC corrected for partial volume. Whole-brain CBF values measured by [15O]H2O were 0.37±0.04 (mean±S.D.) ml/g/min, partition coefficient was 0.73±0.04 ml/g, and the CBF varied in a linear relationship with partial pressure of carbon dioxide during each scan. CONCLUSIONS The AV-shunt technique allows less invasive, quantitative and reproducible measurement of rCBF in [15O]H2O PET studies in rats than direct blood sampling and radioassay.

Kinetics of neurodegeneration based on a risk-related biomarker in animal model of glaucoma.

BackgroundNeurodegenerative diseases including Parkinson’s and Alzheimer’s diseases progress slowly and steadily over years or decades. They show significant between-subject variation in progress and clinical symptoms, which makes it difficult to predict the course of long-term disease progression with or without treatments. Recent technical advances in biomarkers have facilitated earlier, preclinical diagnoses of neurodegeneration by measuring or imaging molecules linked to pathogenesis. However, there is no established “biomarker model” by which one can quantitatively predict the progress of neurodegeneration. Here, we show predictability of a model with risk-based kinetics of neurodegeneration, whereby neurodegeneration proceeds as probabilistic events depending on the risk.ResultsWe used five experimental glaucomatous animals, known for causality between the increased intraocular pressure (IOP) and neurodegeneration of visual pathways, and repeatedly measured IOP as well as white matter integrity by diffusion tensor imaging (DTI) as a biomarker of axonal degeneration. The IOP in the glaucomatous eye was significantly increased than in normal and was varied across time and animals; thus we tested whether this measurement is useful to predict kinetics of the integrity. Among four kinds of models of neurodegeneration, constant-rate, constant-risk, variable-risk and heterogeneity models, goodness of fit of the model and F-test for model selection showed that the time course of optic nerve integrity was best explained by the variable-risk model, wherein neurodegeneration kinetics is expressed in an exponential function across cumulative risk based on measured IOP. The heterogeneity model with stretched exponential decay function also fit well to the data, but without statistical superiority to the variable-risk model. The variable-risk model also predicted the number of viable axons in the optic nerve, as assessed by immunohistochemistry, which was also confirmed to be correlated with the pre-mortem integrity of the optic nerve. In addition, the variable-risk model identified the disintegrity in the higher-order visual pathways, known to underlie the transsynaptic degeneration in this disease.ConclusionsThese findings indicate that the variable-risk model, using a risk-related biomarker, could predict the spatiotemporal progression of neurodegeneration. This model, virtually equivalent to survival analysis, may allow us to estimate possible effect of neuroprotection in delaying progress of neurodegeneration.

Visualization of drug translocation in the nasal cavity and pharmacokinetic analysis on nasal drug absorption using positron emission tomography in the rat

We performed positron emission tomography (PET) using 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) to evaluate the pharmacokinetics of nasal drug absorption in the rat. The dosing solution of [(18)F]FDG was varied in volume (ranging from 5 to 25 μl) and viscosity (using 0% to 3% concentrations of hydroxypropylcellulose). We modeled the pharmacokinetic parameters regarding the nasal cavity and pharynx using mass balance equations, and evaluated the values that were obtained by fitting concentration-time profiles using WinNonlin® software. The regional nasal permeability was also estimated using the active surface area derived from the PET images. The translocation of [(18)F]FDG from the nasal cavity was visualized using PET. Analysis of the PET imaging data revealed that the pharmacokinetic parameters were independent of the dosing solution volume; however, the viscosity increased the absorption rate constant and decreased the mucociliary clearance rate constant. Nasal permeability was initially higher but subsequently decreased until the end of the study, indicating regional differences in permeability in the nasal cavity. We concluded that the visualization of drug translocation in the nasal cavity in the rat using PET enables quantitative analysis of nasal drug absorption, thereby facilitating the development of nasal formulations for human use.

Three-dimensional quantitation of regional cerebral blood flow in mice using a high-resolution pinhole SPECT system and 123I-iodoamphetamine

INTRODUCTION This study is intended to evaluate the feasibility of using a high-resolution pinhole SPECT system and iodine-123-N-isopropyl-4-iodoamphetamine ((123)I-IMP) for three-dimensional (3D) absolute quantitation of regional cerebral blood flow (rCBF) in mice. METHODS The pinhole SPECT system consists of a rotating stage and a pinhole collimator attached to a clinical gamma camera. The collimators focal length is 251 mm. Phantom studies were performed to evaluate sensitivity and full-width half-maximum (FWHM) spatial resolution. The aperture-to-object distance was 15 mm. Six mice were studied. Cerebral infarctions were induced by ligating and disconnecting the distal portion of the left middle cerebral artery. Ex vivo SPECT studies were performed using harvested brains and skulls. The CBF volumetric image was computed using the standardized input function. RESULTS Excellent spatial resolution of 0.9-mm FWHM and uniform sensitivity throughout the 3D volume were demonstrated in the phantom experiments. The CBF images showed a defect in the infarcted areas and a reduction of CBF values in the infarcted region as compared with the control region. CONCLUSIONS This study demonstrated the feasibility of the 3D quantitation of rCBF in mice using a high-resolution pinhole SPECT system and (123)I-IMP.