The overall goal of this procedure is to perform 360 degree in vivo imaging of a mouse to determine the optimal angle for fluorescence analysis. This is accomplished by first preparing the orthotopic brain tumor, engineered brain tumor, and arthritis animal models. Next SAPs C protein is produced and SAP D-O-P-S-C-V-M nano vesicles are prepared.
Then the nano vesicles are injected into the tail vein of the animals. Finally, fluorescence and x-ray images are taken and analyzed. Ultimately, the M-A-R-O-I method is used to show the optimum angle for fluorescence imaging analysis.
The main advantage of this technique over existing methods like 2D Planar fluorescence imaging, is that the M-A-O-M-A-R-O-I method allows investigators to determine the optimal angle for fluorescence imaging analysis. The implications of this technical expand toward therapy and the diagnosis of cancer and arthritis because CEP CS Vesical can target tumor and the inflammation tissue. All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati and the Cincinnati Children's Hospital Research Foundation to carry out these studies and orthotopic brain tumor mouse.
A genetically engineered brain tumor mouse and A-K-B-X-N arthritis mouse model are used According to the text protocol, use an arthritic index macroscopic scoring system to evaluate mice for arthritis as follows, zero equals no detectable arthritis, one equals swelling and or redness of paw, or one digit two equals two joints involved. Three equals three joints involved and four equals severe arthritis of the entire paw and digit produce recombinant human SAPs C protein in E coli cells before precipitating with ethanol and high performance liquid chromatography purification after lyophilization determine the concentration of dry SAP C by weight. To mix SAP C protein, combine 0.18 milligrams of DOPS and 0.03 milligrams of CVM in a glass tube and use nitrogen gas to evaporate the lipid solvents.
Then add 0.32 milligrams of SAPs sea protein powder to the mixture. Suspend the dry mixture in one milliliter of PBS buffer and bath sonicate for 15 minutes. Pass the suspension through a Cidex G 25 column to remove free CVM dye.
SAP C-D-O-P-S-C-V-M nanoparticles will have excitation and emission maxima of 653 nanometers and 677 nanometers respectively. To test the multi-angle rotational optical imaging or M-A-R-O-I method using the Mars system, after anesthetizing a mouse from one of the described mouse models with 2%ISO fluorine position the mouse in a supine position in the Mars system with its spine directed towards the camera from the preview screen of the brucker MI rotator tab, calibrate the Mars 380 degree support film. To position the mouse to administer SAPs C-D-O-P-S-C-V-M into the tail of the mouse, inject 200 microliters intravenously 24 hours later, and then again seven to nine days post injection, take fluorescence and x-ray images at 10 degree increments over a course of 380 degrees, creating a slight overlap to ensure that there are no gaps in the rotational dataset.
Using the Bruker rotation software, superimposed fluorescent images over x-ray images for anatomical localization. To analyze the images, draw a rectangular region of interest, or ROI encompassing the width of the field of view, or FOV of the disease site for brain tumor mice. Use the same ROI on each tumor model and the respective control mice for all time points using anatomical landmarks to mark the position of the ROI after automatic background subtraction.
Determine the mean fluorescence intensity for every image by using the Brucker MI software to convert the fluorescence images to photons per second per millimeter squared plot the fluorescence values as a function of the imaging angles and apply as error bars. The standard deviation of the average fluorescence values obtained from control mice. The fluorescence image of a representative orthotopic tumor bearing mouse is shown in this figure.
We demonstrate here that SAPs seed, DOPS nano vesicles labeled with a far red dye, specifically accumulate in orthotopic and spontaneous mouse brain tumors, as well as in arthritic joints of KBXN mice. The primary purpose for the Mars system is to determine the optimal angle of fluorescence so that the most accurate measurements can be taken as seen here. The optimal image angle for this animal is 10 degrees.
The position at which the fluorescence photon intensity is the greatest baseline measurements were taken before injection of SAP C-D-O-P-S-C-V-M, and 24 hours after injection tumor free control mice received a similar treatment. These figures show comparable data from the genetically engineered brain tumor mouse model. The fluorescence images and photon measurements were taken at baseline 24 hours and nine days after SAP C-D-O-P-S-C-V-M injection.
The graphs show that the optimal imaging angle in the tumor bearing animal tumor mute 49 is 20 degrees, 24 hours post injection, but changes to 10 degrees nine days post injection. This suggests that the fluorescent signal alteration correlated with morphological changes likely reflecting tumor growth. As shown in this table, the M-A-R-O-I method clearly demonstrates that the fluorescent signal decreases for projections at increasing rotation away from the optimal imaging angle.
In brain tumors, a 7%average decrease in fluorescent signal was obtained. If the animal's physical orientation was more than plus or minus 10 degrees offset from the optimal imaging angle. An average 21%decrease in fluorescent signal was measured at plus or minus 20 degrees.
Thus, relatively small offsets from the optimal angle can result in significant percent signal decreases. Utilizing the M-A-R-O-I technique for image positioning will allow investigators to produce more consistent and reliable data. The M-A-R-O-I method was finally used to assess the targeting of arthritic joints by SAPs C-D-O-P-S-C-V-M 24 hours after injection.
This animal scored three with three arthritic joints. Fluorescence images of toe and ankle of the arthritic mouse are shown here based on graphs of corresponding photon measurements At 10 degree rotations, the optimal imaging angles found for the toe and ankle are 140 degrees and 120 degrees respectively. After watching this video, you should have a good understanding of how to prepare and acquire M-A-R-O-I data that allows investigators to determine the optimal angle for fluorescence imaging analysis.
