Application And Value Of Micro Magnetic Resonance Imaging In The Experimental Study Of Oncology

The need of small animal imaging is largely determined by the development of new treatment such as gene therapy and biotherapy in the late years. Most of the traditional research work on oncology is confined in the laboratory. What the oncologists and biologists usually do in their researches is to try to elucidate the principles with molecular biological technique. Though it truly helps but regrettably there is still great gap between what researchers achieves from their experiments and clinical use largely due to the complex internal environment in the human body. Now we urgently need a new research method to bridge the gap between the laboratory and clinical application. Lately the prototype imaging comes to our attention. Large amount of transgenic animals or animals with gene defect for research are in need because molecular genologists try to simulate human disease in animals and pharmacy companies want to test their new drugs or treating techniques in animals before clinical use. Therefore, reliable in-vivo monitoring methods are definitely helpful especially new anticancer drugs or techniques are urgently to be developed in clinical settings. That insufficient transgenic mouse available for preclinical trials and increased fees of mouse for research application promotes researchers to consider the possibility of in-vivo imaging to replace the traditional method of sacrificing the animal for histological correlation. In order to promote the development of high-resolution imaging modalities the national institute of cancer research of America sponsored several small animal imaging programs.Obviously there are much difference between the mice of only 20 grams and human beings with 70 kilograms in some aspects such as weight, dimension, resolution and imaging time considering the imaging of small animals. The interests in small animal imaging greatly increased. Lately many animal imaging systems were developed and used in experiments such as molecular imaging and preclinical trials. But the magnetic resonance imaging (MRI) systems and coils designed specific for small animal imaging widely used abroad are too expensive for us to buy considering its high price of over one million U.S dollars and economical status of our country. That many hospitals have high-field (over 1.5 tesla ) clinicalMR scanners for routine clinical service to the patients in our country urges us to consider the possibility of using the clinical MR systems for additional small animal imaging with the aid of some new instruments. My research is to study the feasibility to replace the expensive micro-MR system with clinical MR scanner combined with additional domestic sending and receiving coil of small core based on the subjects of proton MR spectroscopy of developing rat brain with high fever convulsion and MR imaging of implanted subcutaneous tumor model of S-180 sarcoma in mouse. One of the purposes of this study is to determine whether MR spectroscopy obtained from the clinical MR imaging system combined with new small coil could detect the changes of metabolism in the rat before obvious irreversible damages to thebrain and the new MR modality could detect the nature of implanted tumor.It is of great importance in the manufacturing and improvement of anticancer drugs by in-vivo tracking and monitoring of biological activity of nano-particles targeted to cancer cells or neo-vasculatures and understanding the principles of adhesion and drug or gene release of the nano-particle vectors in the targets. As the nano-particle vectors are loaded with drugs or bioactive materials such as gene or RNA segments, pharmacologists and clinicians must know early whether the nano-particles have adhered and fixed to the targeted organs or tissues, the bioactive materials loaded in the vectors have released and reacted with the targeted tissue and how long the vectors will play their role in the tissue. So obviously it's of priority to determine whether the nano-particle vectors have arrived in the targeted tissues and adhered to them.Because of its unrivaled advantages of non invasiveness to the examinees and high soft tissue resolution MR imaging is the one of the first choices for in-vivo tracking and monitoring of nano-particle vectors. My research will focus on synthesizing new nano-particle vectors loaded with MR contrast and uncover the phenomenon of adhesion and absorption of the vectors in the colon wall in the mice with MR imaging. In this research, novel chitosan-nanoparticles and solid liposome nano-particles loaded with Gd-DTPA will be synthesized and in-vitro and in-vivo imaging will be carried out. Results obtained from the research will definitely deliver a reliable monitoring modality in the future research of developing new targeted anticancer drug and gene loaded vectors.Purpose1. To optimize the technique and to probe the feasibility of proton magnetic resonance spectroscopy (¹HMRS) in the small animal with clinical imaging scanner combined with small animal coil.2. To probe the feasibility of imaging implanted subcutaneous tumor model with clinical MR imaging system and small animal coil ,and whether micro-MR imaging could reflex the anatomical nature of the tumor and pave the way for further in-vivo imaging of small animal and tumor model.3.Purpose of this subject is to synthesize a novel vector of chitosan-nanoparticles loadedwith Gadolinium and in-vivo image the adhesion and absorption of the particles in the colon wall with MR and achieve molecular MR imaging of nano-particle vectors.4. To synthesize a novel solid liposome nano-particle vector loaded with MR contrast Gd-DTPA and fluorescence FITC and in-vivo image the adhesion and absorption of the solid liposome nano-particle vector with MR and achieve molecular imaging of liposome vectors.5. To expand the application of the clinical MR scanner to the area of small animal imaging and molecular imaging with the aid of small coil and partly take the role of the expensive micro MR scanner. Through the research of MR imaging of nanoparticle vectors, we hope that targeted diagnosis with MR and targeted delivery of drugs and genes to the cancer cell through the colon mucosa will be possible in the future.Materials and Methods1.All the MR scans were performed with a high-field 1.5T clinical MR scanner and a small animal coil.Twenty juvenile male SD rats weighted 25 grams were included. High fever(45° Celcius )water bath convulsion model was produced in 10 rats and other control rats were bathed in warm water (37° Celcius). Multi-voxel Point-resolved spectroscopy (PRESS) of ¹HMR spectroscopy and routine T2-weighted imaging were scanned in 10 high fever convulsion rats and another 10 control group. The original data were transmitted to the local workstation for further analysis to obtain spectroscopy of compounds such as N-acetyl aspartate (NAA), Choline (Cho) , Creatine (Cr) and Lactate (Lac). Values of Cho/NAA, Cr/NAA, Cho/Cr and Lac/ Cr were obtained according to the height of the spectrums. Values were compared between high fever convulsion group and control group and statistical analysis was done with pared t test. T2-weighted images were analyzed if there was any abnormal signal change in the whole brain.2. Implanted subcutaneous tumor model of S-180 sarcoma in the right groin were produced in 20 male Kunming mouse and after 7 days MR scans were carried out with high-field 1.5T MR scanner and a small animal coil of 10 cm in core as a sending and receiving coil. Axial chemical shift fat-suppressed fluid attenuation inversion recovery T1-weighted imaging (FLAIR-T₁WI) and non fat suppressed T2-weighted imaging (T2WI) were scanned. On the axial FLAIR-T₁WI images the largest dimension of the tumor was calculated. Intra-tumor necrosis/haemorrhage and invasion of the abdominal muscles were determined depending on the signal change of the tumor and margin between the mass and the abdominal muscles found on the images. After MR scans animals were sacrificed to determine if there were necrosis and/or hemorrhage in the tumor after splitting of the mass and observe the invasiveness and adhesion of the abdominal muscles and skin by the tumor.3.Gd-nanoCPS(Gadolinium chitosan nanoparticles) was synthesized with the emulsion-droplet coalescence method and there was no Gadolinium loaded in the control particles with the same procedure. Dimension of the particles and content rate of the Gd-nanoCPS were measured. MR scans with T₁WI, T₂WI, GRE-T₂^* WI and FLAIR-T₁WI sequences were performed in-vitro to confirm if Gd-DTPA was loaded in the particles. Signal intensity of the Gd-nanoCPS and non Gadolinium particles was measured and compared.Sixteen male Kunming mouse were included in the in-vivo MR imaging with 8 mice as Gd-labeled group and others as control. Suspension of Gd-nanoCPS with concentration of 7.5mg% of Gadolinium loaded was infused into the colon after fully purge of the colon and retained in the colon for 40 minutes. Before and after the infusion full purge of the colon with warm saline water was carried out. MR scans of axial chemical shift fat suppression FLAIR-T₁WI and T₂WI before, during and after infusion of the suspension were performed .The same procedures were carried out in the control 8 mice. After MR scans animals were sacrificed and samples were obtained for electron microscopy examination in reference to the enhanced areas on the MR images. Signal intensity (SI) of ROIs(region of interest) placed in the wall of rectum or colon , muscles of the pelvis near the rectum and background were calculated. So SI of the rectum or colon (SIr_pre and SIr_post for pre- and post-infusion respectively), SI of the muscle (SIm_pre and SIm_post)and SI of noise of the background ((SIn_pre and SIn_post) were obtained. SI of the rectum or colon was obtained from averaging SI in six ROIs. SIr/SIm and SIn/SIm represented the relative SI of the wall of the rectum or colon and noise of the background. The values of SIr_pre/ SIm_pre, SIn_pre/ SIm_pre,SIr_post/ SIm_post and SIn_post/ SIm_post in the Gadolinium group and control group were achieved. Relative SI values as described previously between pre- and post infusion were compared with pared t analysis. Also SI of Gadolinium group and counterpart of non- Gadolinium group were analyzed. Software used in the statistical analysis was SPSS11.5.4. A novel vector of loaded stearic acid solid liposome nano-particles (SLN) loaded with Gd-DTPA and ODA - FITC (fluorescein isothiocyanate labeled Otcadecylamine) were synthesized with the equal amount of Gd-DTPA and ODA -FITC loaded in the particles with solvent diffusion method in aqueous system. ODA-FITC was synthesized by the reaction between amino group of ODA and isothiocyanate group of FITC. The control particles only contained ODA -FITC. Dimension of the particles and content rate of the Gd- ODA -FITC were measured. MR scans with PDWI(proton density-weighted imaging ),T₂WI, GRE -T₂^* WI-(gradient recalled echo T₂ weighted imaging)and FLAIR-T₁WI(fluid attenuated inversion recovery T₁ weighted imaging ) sequences were performed in-vitro to confirm if the Gd-DTPA was contained in the Gd-ODA-FITC loaded particles. Signal intensity of the suspension of SLNs containing Gd-ODA -FITC and that of non Gadolinium particles was measured andcompared.7 mice were included in the Gd-ODA-FITC group and 5 in the control ODA-FITC group. Suspension of Gd-ODA-FITC with concentration of 10mg/ml SLN was infused into the colon after full purge of the colon and retained in the colon for 40 minutes. Before and after the infusion full purge of the colon with warm saline water was carried out. MR scans of axial chemical shift fat suppression FLAIR-T₁WI and T₂WI before, during and after infusion of the suspension were performed .The same procedures were carried out in the control ODA-FITC group in 5 mice. After MR scans were finished animals were sacrificed and samples were obtained and were frozen for fluorescent microscopy examination according to the enhanced area correponding to the MR images. Signal intensity (SI) of ROIs(region of interest) placed in the wall of rectum or colon , muscles of the pelvis near the rectum or colon and background. So SI of the rectum or colon (SIr_pre and SIr_pos for pre- and post-infusion respectively), SI of the muscle(SIm_pre and SIm_post)and SI of noise of the background ((SIn_pre and SIn_post) were calculated. SI of the rectum or colon was obtained from averaging those in six ROIs. SIr/SIm and SIn/SIm represented the relative SI of the wall of rectum or colon and noise of the background respectively. Values of SIr_pre/ SIm_pre, SIn_pre/ SIm_pre, SIr_post/ SIm_post and SIn_post/ SIm_post in the Gadolinium group and control group were achieved. Relative SI values as described previously between pre- and post infusion were compared with pared t analysis. Also relative SI of Gd-ODA-FITC group and counterpart of non-Gadolinium group were analyzed. Software used in the statistical analysis was SPSS11.5.Results1. ¹HMRs of the brain was successfully carried out in the high fever convulsion juvenile SD rats and control group. ¹HMRs clearly revealed the four spectrums commonly analyzed in the brain such as NAA, Cho, Cr and Lac obviating any disturb of cranial bone and subcutaneous fat. The typical spectrum of Lac was found in all convulsion rats and only very low level of suspect Lac was found in 4 of 10 normal juvenile rats. The values of Cho/NAA, Cr/NAA, Cho/Cr and Lac/ Cr were 1.37±0.41, 1.08±0.15, 0.83±0.23 and 0.48±0.20 (means±SD) in the convulsion group. Those of the counterparts in the control group were 1.24±.14(P=0.005), 1.44±.19(P<0.001), 1.18±.21(P=0.001) and 0.14±0.16(P<0.001).There was no abnormal signal intensity change on the routine T2-weighted images in the convulsion and control normal groups.2. MR images on the fat suppression FLAIR T₁WI and T₂WI clearly showed the tumor itself and surrounding anatomical structures. S-180 sarcoma showed medium signal intensity on FLAIR T₁ -weighted images and low signal intensity on T₂-weighted images. There were areas of high signal intensity and low on FLAIR T₁ -weighted images respectively compatible with necrosis and hemorrhage in 13 of 20 mice. The margin between the tumor and the abdominal muscles found on FLAIR T₁ -weighted images were not clear in 16 out of 20 mice. The dimension calculated on MR images were 0.82±.191cm (means ±SD) and 0.85±.231cm obtained from the real mass during autopsy with P >0.05. During autopsy there was obvious necrosis or hemorrhage in 15 out of 20 tumors and adhesion or invasion of abdominal muscles in 13 out of 20 mouse. There was no signicant difference between MR findings and autopsy concerning necrosis/hemorrhage and abdominal invasion with P>0.05.3. Dimension of the Gd-nanoCPS was about 500nm and content rate was about 30mg%. Signal intensity of suspension of Gd-nanoCPS particles was sharply different from those of Nano-CPS particles and water on images from the sequences of T₁WI, T₂WI, GRE-T₂^* WI and FLAIR-T₁WI .The signal intensity change was comparable to the high concentration of Gd-DTPA solution. Signal intensity of the wall in rectum or colon calculated after infusion of the suspension of Gd-nanoCPS particles was higher than that before infusion, which indicated there was Gd-DTPA contained in the colon wall signaling local deposition of Gd-nanoCPS particles. There was slight to medium enhancement after infusion of suspension of Gd-ODA-FITC loaded particles along the wall of the colon or rectum. The enhancing pattern was segmental or annular. Values of SIr_pre/ SIm_pre and SIr_post/ SIm_post in the Gd-nanoCPS group was 0.84±.061 and 0.98±.086 (mean ±SD) respectively with P=0.003. Values of SIn_pre/ SIm_pre and SIn_post/ SIm_post were 0.044±.015 and 0.038±.008 (P >0.05).Values of SIr_pre/ SIm_pre, SIr_post/ SIm_post were 0.83±.042 and 0.84±.052(P >0.05).The enhancement rate in Gd-nanoCPS group was 17.5±12% which was significantly higher than that in the control Nano-CPS group(P <0.01). Under the electron microscopy Gd-nanoCPS particles were found inside the mucosal cells.4. Particle dimension, multi dispersion index and zeta potential of the SLN particles loaded with Gd-ODA-FITC was about 202.7±4.25 nm, 386±0.062 and -20.8±1.99mV. Content rate of Gd-DTPA and FITC-ODA was about 97.9 %. Signal intensity of suspension of Gd-ODA-FITC loaded particles was significantly different from those of Nano-CPS particles and water on images from the sequences of PDWI, T₂WI, GRE-T₂^* WI and FLAIR-T₁WI.The signal intensity change was comparable to the high concentration of Gd-DTPA solution. There was medium to severe enhancement after infusion of suspension of Gd-ODA-FITC loaded particles along the wall of the colon or rectum. The enhancing pattern was segmental or annular. Signal intensity of the wall in rectum or colon calculated after infusion of the suspension of Gd-ODA-FITC loaded particles was higher than that before infusion, which indicated there was a large amount of Gd-DTPA contained in the colon wall signaling local deposition of Gd-ODA-FITC loaded particles. Values of SIr_pre/ SIm_pre and SIr_post/ SIm_post in the Gd-ODA-FITC group was 0.86±.051, 1.47±.191 (means ±SD, P<0.001). Values of SIn_pre/ SIm_pre and SIn_post/ SIm_post were 0.043±.080, 0.050±.016 (P=0.831, P >0.05).Values of SIr_pre/ SIm_pre,SIr_post/ SIm_post were 0.84±.041, 0.87±.147 (P=0.654, P >0.05).The enhancement rate in Gd-ODA-FITC group was74.7±19.8% which was significantly higher than that in the control ODA-FITC group (P <0.001). Under the fluorescent microscopy green fluorescent material was found in the whole wall of the rectum or colon which was comparable to the MR findings. Linear green fluorescent material was found along the serosa and large amount of fluorescent material was observed in the intersticial space.Conclusion1. ¹HMRs of the brain in developing rat can be performed with clinical MR scanner combined with small animal coil and it can accurately reflect the metabolism of the normal brain in the juvenile rat and convulsion moddel. ¹HMRs could be a reliable method to study brain tumor model of the rat.2. MR scanning performed with clinical MR scanner and small coil can clearly show the nature of implanted subcutaneous tumor and it is a good method to in-vivo monitor the growth and anticancer treatment. Clinical MR scanner combined with small animal coil may partly replace the micro-MR scanner in the research of tumor model.3.Chitosan nano-particles can be loaded with MR contrast Gd-DTPA. MR imaging can in-vivo reveal the phenomenon of adhesion and absorption of mucosa targeted chitosan nano-particles. MR imaging based on small animal coil is a good method to in-vivo monitor the nano-particle vectors.4. Stearic acid solid liposome nano-particles loaded with Gd-ODA-FITC is a promising nano-particle vector which could be monitored by MR imaging. SLN is absorbed by the colon mucosa by intercellular infiltration. MR imaging can in-vivo monitor the adhesion and absorption of liposome particles by the colon and it is a promising technique to monitor the bioactivity of targeted liposome vectors.5. With the aid of small animal coil clinical MR scanner can still be used in the small animal imaging and molecular imaging which may expand the application of clinical MR scanner. MR imaging of mucosa targeted nanoparticle vector may eventually help the targeted delivery of drugs and genes across the mucosa of colon .Our researches also indicate that targeted diagnosis and staging of colon cancer with MR imaging is possible.