실험동물을이용한분자영상 (In Vivo Molecular Imaging)
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1 실험동물을이용한분자영상 (In Vivo Molecular Imaging) Laboratory Animal Medicine May
2 Contents 1. Tissue sampling for in vivo experiment 2. What s the molecular imaging 3. Molecular imaging modalities 4. In vivo molecular imaging: MRI/fMRI/MRS
3 What is the molecular Imaging?
4 What is the molecular imaging Molecular imaging is a new biomedical research discipline enabling the visualization, characterization, and quantification of biologic processes taking place at the cellular and molecular levels within intact living subjects including patients. The society for molecular imaging
5 Marriage of; Imaging technology In vivo molecular imaging Molecular biology Non-invasive and repetitive imaging of target macromolecules (cells) and biological processes (cellular processes) in living organism The in vivo characterization and measurement of biological process at the molecular & cellular level.
6 In Medicine In Classical Method for Assessing Disease Anatomic changes and Physiologic changes that are a late manifestation of the molecular changes that truly underlie disease Using Molecular Imaging Method Focus on to probe the molecular abnormalities that are the basis of disease than to image the end effects of these molecular abnormalities
7 Translational Research: bench-to-bedside (i) Basic Scientists, Who discover new genes & their functions Who discover new materials Imaging Scientists, Who could transform these discoveries into non-invasive imaging method Clinical Scientist, Who transfer the above into patients care
8 Translational Research: bench-to-bedside (ii) Biophysics Molecular biology Radiology Biomathematics Cell biology Medicine Molecular Imaging Lab Chemistry Veterinary medicine Pharmacology Bioinformatics Computer science
9 The GOALS of the field are; Early diagnosis Life science medicine Gene therapy Targeted cancer drug Molecular Imaging Stem cell research The ultimate outcome of Drug this technique Clinical should be for early diagnosis, development and pre-disease state therapeutic response pathology at the molecular level
10 Micro US Micro MR In Vivo Animal Imaging Modalities Micro PET-CT Micro CT Optical Imaging Micro PET
11 Commonly used 1. MR Imaging / Spectroscopy small animal imaging - at high field ( > 4.7T) human scanner field (1.5T ~ 3.0T) 2. PET Imaging 3. Optical Imaging bioluminescence imaging fluorescence imaging tomographic imaging.
12 Detection range of imaging modalities Things to be considered: 1. Spatial & temporal resolution 2. Depth 3. Sensitivity 4. Type of molecular probe 5. Perturbation of biological system
13 Magnetic Resonance in Molecular Imaging MRI (magnetic resonance image) MRS (magnetic resonance spectroscopy) fmri (functional MRI)
14 Anatomic MR Gross morphology Specific target Metabolic MR Tissue functionality MRI/MRS/fMRI Functional MR Function Activity Molecular MR Target imaging Probe development
15 Gyromagnetic Ratio Nucleus Spin Quantum Number Gyromagnetic Ratio (MHz/1T) Relative Sensitivity at Constant Field Natural Abunda nce (%) 1 H 1/ C 1/ P 1/ Na 3/ F 1/ Li 3/ K 3/ ex: 1 H, 3T MHz, 1H, 11.7T 500 MHz
16 WHAT IS fmri? Functional magnetic resonance imaging (fmri) is a relatively new procedure that uses MR imaging to measure the tiny metabolic changes that take place in an active part of the brain (Ogawa, et al, 1990 a and b, 1992, 1993; Belliveau, et al, 1990, 1991). Functional MRI is based on the increase in blood flow to the local vasculature that accompanies neural activity in the brain. Since deoxyhemoglobin is paramagnetic, it alters the T2* weighted magnetic resonance image signal
17 In vivo magnetic resonance spectroscopy
18 In vivo MRS provide a noninvasive window into brain In vivo magnetic resonance spectroscopy (MRS) directly measures chemically specific information. It is the only noninvasive technique for measuring concentration of metabolites from the living brain. Our MRS development at 11.7 Tesla, the highest field strength at which in vivo MRS has ever been attempted, has allowed, for the first time, detection of GABA turnover in vivo. In addition to the static levels of metabolites and metabolic fluxes measured by proton or 13 C MRS, our recent discovery of 13 C magnetization (saturation) transfer effect of specific enzyme reactions has made it possible to probe the action of several enzymes in vivo, pointing to new directions in 13 C MRS technology development and applications.
19 In vivo evidence for reduced cortical glutamate-glutamine cycling in rats treated with the antidepressant/antipanic drug phenelzine Yang J and Shen J. Neuroscience 135: (2005) Metabolite a Group A Group B alanine ** 0.05 ± ± 0.38 aspartate 2.85 ± ± 0.30 creatine 3.39 ± ± 0.25 GABA c** 1.02 ± ± 0.26 glutamate d** ± ± 0.28 glutamine d** 5.17 ± ± 0.34 lactate 0.59 ± ± 0.24 myo-inositol 4.52 ± ± 0.33 N-acetylaspartate ± ± 0.40 phosphocreatine 5.11 ± ± 0.25 phosphorylethanola mine 2.31 ± ± 0.33 taurine 4.94 ± ± 0.17 Among 12 metabolites, Glu, Gln, GABA and Ala were the only metabolite which showed statistically significant changes. As a result, we became the first group in the world to detect turnover of the major inhibitory neurotransmitter GABA in the brain in vivo.
20 Home-built surface radiofrequency transceiver coils 1 H: circular, diameter 15 mm 13 C: square, 25 x 25 mm 2 ) 13 C/ 1 H: 11.1 x 2.8, 24.2 x 3.2 mm (diameter x conductor width, respectively) RF coil integrated in-house-built animal handling system capable of rat head fixation, body support, physiology maintenance, coil tuning, and RF shieding.
21 Animal preparation femoral vein and artery cannulation Two femoral veins (left and right) were also cannulated for intravenous infusion of α- chloralose (initial dose: 80 mg/kg supplemented with a constant infusion of 26.7 mg/kg/hr throughout the experiment) and [1,6-13C2]glucose, respectively.
22 Animal preparation monitoring of physiological condition Arterial blood po2, pco2, mean blood pressure, and ph were maintained at approximately mm Hg, mm Hg, 150±30 mm Hg, and , respectively. Heart rate, end-tidal CO2, and tidal pressure of ventilation were monitored continuously.
23 Blood glucsoe (mm/l) Mean blood pressure (mmhg) ph pco2 (mmhg) The change of arterial ph, pco 2, glucose and mean blood pressure in each group Control PHA ACZ Time (min) Control PHA ACZ Time (min) Control PHA ACZ Control PHA ACZ Time (min) Time (min)
24 Elevated Endogenous GABA Concentration Attenuates Glutamate-Glutamine Cycling between Neurons and Astroglia J Neural Tansm 116: (2009) Jehoon Yang and Jun Shen Fig. 1. Comparison of in vivo 1 H short-te spectra from a control rat of Group I (bottom trace) and a Group III rat 24 hours after vigabatrin injection (top trace; 500 mg/kg, i.p., 24 hours prior to data acquisition). Voxel size = mm 3. Total number of scans = 128. lb = 3, gb = 0.1. In the 1 H short-te spectrum of vigabatrin-treated rat, the elevation of GABA α-methylene proton signal at 2.28 ppm, the GABA β-methylene proton signal at 1.91 ppm due to vigabatrin treatment were clearly observed.
25 Detection of reduced GABA synthesis following inhibition of GABA transaminase using in vivo magnetic resonance signal of [13C]GABA C1. J Neurosci Methods 182(2) 15: (2009) Jehoon Yang, Christopher J and Jun Shen
26
27 Fast isotopic exchange between mitochondria and cytosol in brain revealed by relayed 13 C magnetization transfer spectroscopy J Cereb Blood Flow Metab 29(4):661-9 (2009) Jehoon Yang, Su Xu and Jun Shen Fig. The relationship between the TCA cycle and V x used in brain metabolic models to describe the kinetics of 13 C label incorporation from mitochondrial TCA cycle intermediates into predominantly cytosolic glutamate and aspartate pools (adapted from Figure 43 in Siesjo (1978)). V x represents the lumped exchange between mitochondrial -ketoglutarate/oxaloacetate and cytosolic glutamate/aspartate.
28 Increased oxygen consumption in the somatosensory cortex of α-chloralose anesthetized rats during forepaw stimulation determined using MRS at 11.7 Tesla Neuroimage 32 (3) (2006) Jehoon Yang and Jun Shen Fig. Typical in vivo time course of proton-detected, 13C-edited spectra following intravenous infusion of [1, 6-13C2]glucose (TR/TE = 2000/22 ms, NS = 128 2, AQ = 196 ms, lb = 10, gb = 0.175). The [4-13C]glutamine signal at 2.46 ppm and the [2-13C]GABA signal at 2.30 ppm are spectrally resolved in vivo from the target [4-13C]glutamate signal at 2.35 ppm
29 All procedures were approved by the National Institute of Mental Health Animal Care and Use Committee and the physiological data should be recorded by certificated person.
30 In vivo 13C magnetic resonance spectroscopy of human brain on a clinical 3 T scanner using [2-13C]glucose infusion and low-power stochastic decoupling. Magnetic Resonance in Medicine 62(3): (2009) Li S, Zhang Y, Wang S, Yang J, Ferraris Araneta M, Farris A, Johnson C, Fox S, Innis R, Shen J. Time course spectra of glutamate, glutamine, and aspartate turnover detected in the occipital lobe during intravenous infusion of [2-3C]glucose.
31 Weaknesses of MRS Limited sensitivity = large voxels (nucleus dependent) = high metabolite concentration (millimolar) Limited specificity = multiple species in many resources = spectral overlap may be difficult to overcome Larger data acquisition windows =spectra are the sums of large numbers at averages =scans are expensive (several hundred dollars/hour) Special equipment may be required =software ( 1 H MRS) =hardware (RF coils, amplifiers, decouplers) Local expertise critical = automated methods variably reliable = multiple issues in data acquisition, analysis
32 Molecular imaging probe
33 Future drug Therapeutic drug Multimodality Imaging Imaging Drug
34 HMON as an MRI contrast agent HMON (Hollow Manganese Oxide Nanoparticles) Newly developed T1 contrast agent Gd 3+ or Mn 2+ containing colloidal nanoparticles : recently reported as potent T1 MRI contrast agents A nanometer-sized colloid particle with small size, large surface area & internal void spaces Large water accessible surface areas Able to carry high payloads of MR-active magnetic centers with an ability to take up a large amount of drug molecules within the internal void space Cetuximab-conjugated HMON
35 Synthesis of HMON Scheme MnCl 2 + Na-Oleate Manganese oleate Thermal decomposition (used schlenk technique) Phospholipid ph4.6 MON (Manganese Oxide Nanoparticle) Mn3O4 WMON (Water soluble MON) MnO HMON (Hollow MON) MON (~20nm) HMON (dia : ~20nm, core : ~10nm) HMON in water
36 In vivo molecular MR image after treatment with Cetuximab-conjugated HMON T1-Pre T1-30min T1-1hrs T1-3hrs T1-6hrs T1-24hrs T2-Pre T2-6hrs T2-24hrs
37 표적항암제전달경로및분포의영상화기술개발 약물담체용분자영상프로브개발 HMON-(Doxorubicin) 표적항암제용분자영상프로브개발 HMON-(small molecule drug) - sorafenib: 간암치료경구용표적항암제 1. Lee JH and Lee IS et al., Angew Chem Int Ed. (2007) 2. Lee JH and Lee IS et al., Angew Chem Int Ed. (2009) Work-in-progress 2010 년하반기보건의료연구개발사업 - 구두평가 4. 연구개발내용
38 Molecular imaging probe (ii) J Control Release Oct 10;155(1):11-7. Fig. 6. T 2 *-weighted MR imaging of the rat brain with MCAO treatment and enhanced by Fe 3 O 4 -PEG-PAEA10. The polymeric micelles were dissolved in the acidic area of the ischemic brain and the Fe 3 O 4 nanoparticles were accumulated over time (arrows).
39 Ongoing sudy 100 Years of Research on Alzheimer s Disease Alzheimer s Disease (AD) First described by Dr. Alois Alzheimer on November 3rd, 1906 in Tübingen, Germany The most common cause of dementia among people age 65 and older Progressive, irreversible decline in memory, loss of orientation and changes in personality and behavior No disease-modifying therapeutics available to date Page 39
40 Detection of reduced GABA synthesis following inhibition of GABA transaminase using in vivo magnetic resonance signal of [ 13 C]GABA C1 J Neurosci Methods (2009) 182(2): , Yang J, Johnson C and Shen J Lin AP et al, 2007
41 Prospective results Metabolite Wild type AD Drug evaluation Alanine 0.05± Aspartate 2.85± Creatine 3.39± GABA 1.02± Glutamate 10.22± Glutamine 5.17± Lactate 0.59± myo-inositol 4.52± N-acetylaspartate 10.50± Phosphocreatine 5.11± Phosphorylethanolamine 2.31± Taurine 4.94± Detection of potential biomarkers for early diagnosis of AD Evaluation of the effect of new developed drugs for AD
42 Thank you for your attention!
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