Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

The overall goal of this procedure is to describe a mouse model that utilizes micro CT to compare volume retention between deferring techniques of fat graft preparation and delivery. This is accomplished by first processing human lipo aspirate to obtain fat ready for grafting. The second step is to place fat grafts into the scalps of CD one thymic nude mice.

Next, the fat graft survival is analyzed weekly by CT scanning mice, reconstructing images, and quantifying volume retention. The final step is to explan fat graft at the end of eight weeks and compare them by weight and histology. Ultimately, micro CT and histology are used to show differences in fat graft quality and survival.

The main advantage of this technique over existing methods like weighing fat grafts, is that it allows visualizing and objectively quantifying fat graft volume at multiple time points after initial grafting without having to sacrifice the study animal. We first had the idea for this method when we began using micro CT to analyze calva Defects. The implications of this technique is that it offers a surgical model for all studies evaluating fat graft, retention and viability.

Generally, individuals new to this method will struggle because containing the fat graft in the correct position above the scalp is Difficult. Visual demonstration of this technique is critical as the grafting and reconstruction steps are difficult to learn because there are numerous stages at which a small deviation in technique can alter the outcome. Using the Coleman procedure, obtain human adipose tissue from the abdomen, flank, and thigh regions of healthy female patients undergoing elective liposuction.

To process the lipo aspirate for grafting begin by allowing the fat to settle for 30 minutes. Lipo aspirate typically settles into three layers with oil at the top, fat in the middle, and blood at the bottom. Aspirate and discard the top oil layer followed by the bottom blood layer.

To further remove any remaining tumescent fluid or cell debris, centrifuge the fat for five minutes at 350 Gs and four degrees Celsius. Following centrifugation aspirate the bottom blood layer and the top oil layer proceed. To calculate the amount of fat needed for grafting as described in the text protocol, to perform fat grafting, obtain female homozygous CD one nude mice.

For the experimental study, choose mice between eight to 12 weeks of age to induce anesthesia. Place the mouse into a knockdown box with 2.5%isof fluorine oxygen mixture at two liters per minute for approximately 10 minutes When the respiration rate of the mouse has slowed. Confirm adequate sedation with a toe pinch.

Apply veterinary lubricating ophthalmic ointment to both eyes of the mouse and place the mouse's nose into a nose cone, delivering 2.5%ISO fluorine oxygen mixture at one to two liters per minute. Next, set up a sterile field under the mouse. Then sterilize a scalp with 2.5%povidone iodine, followed by 70%ethanol solution.

Repeat the sterilization two more times. Back load a one milliliter lure lock syringe with one milliliter of fat which has been equilibrated to room temperature. Connect a 14 gauge eight centimeter long fat grafting cannula to the end of the syringe.

Prime the system by depressing the syringe plunger until between 200 and 400 microliters of fat remains in the syringe. While depressing the syringe plunger, confirm that the cannula has fully filled with fat by observing fat exiting the distal cannula hole using fine forceps. Lift the dorsal skin in the midline overlying the coddle most aspect of the skull.

Make a 1.5 millimeter cut in the skin using fine scissors. Place a six oh nylon suture through the middle of the cut that will later be used to bring the wound edges together. After grafting is performed, do not tie the suture.

Then create a subcutaneous pocket over the skull by inserting the cannula through the skin incision and passing the cannula back and forth in a fan shaped pattern over the skull to free any connective tissue attachments to the overlying skin in once the pocket has been created, position the cannula in the midline of the mouse directly over the skull until the tip lies at theramos aspect of the pocket, which should be just behind a line drawn between the eyes. Slowly inject the fat in a retrograde fashion, advancing the plunger while pulling the cannula back. Using forceps, bring the wound edges together and lift them up to keep any fat from leaking out of the pocket.

Tie the suture that was previously placed, making sure that the first knot lies lightly against the skin. Tie three more square knots and cut the suture with a three millimeter tail. Remove the mouse from anesthesia and proceed with recovery as detailed in the text protocol.

Prior to performing the first scan, calibrate the micro CT with an imaging phantom that distinguishes between air, water, and bone intensity. Following calibration. Place four mice into the scanner in the ventral position with two mice on top and two on bottom.

Place onto a scanning bed. Previously constructed from 60 milliliter syringes to hold the mouse body and 10 milliliter syringes as nose cones. Confirm with a scout image that the entire skull of the mouse from nose to first cervical vertebra and from top of skull to base of skull will be imaged.

Continue with the scan as detailed in the text protocol. Open the reconstructed images with a micro CT imaging analysis software that allows the creation of regions of interest or ROIs by selecting voxels using thresholds for pixel intensity. Start by loading the reconstructed CT images in two dimensional, coronal, axial and sagittal views.

Using the axial slice as a guide, navigate to the sagittal slice that corresponds to the leftmost aspect of the fat graft. Select an upper and lower threshold for pixel intensity that captures all voxels corresponding to the fat graft, but that excludes the surrounding tissue and bone. Define an ROI in the sagittal view that corresponds to the fat graft.

Using previously defined pixel intensity thresholds, repeat this procedure every fifth sagittal slice navigating until the rightmost aspect of the graft is reached. Next interpolate Selected voxels from all 2D ROIs into a single combined 3D ROI record the ROI volume calculated by the software. Render the 3D isosurface to visualize the final fat graft volume.

In subsequent analysis, be sure to keep the pixel intensity maximum and minimum threshold values the same as those used for the baseline analysis after mice have been scanned for the week eight time point. Euthanize the mice as described in the text protocol. Place the mouse in the operative field using tenotomy scissors carefully open the pocket and the overlying skin and connective tissue attachments from the fat graft.

At this point, it may help to excise a patch of the overlying dorsal skin to aid an extraction of the graft. Stay as close to the graft as possible when excising to minimize the connective tissue taken from the graft after excising graft. Measure the mass on a teared scale that is accurate to at least 0.01 grams.

Fat grafts progressively decreased in volume over the course of the study resulting in 62.2%average survival by week eight. At the completion of the week eight scan, each fat graft was extracted in a single piece. A WIL coxin rank sum test was used to compare the difference between volume measurements of fat graft obtained by either micro CT or calculated from physical mass.

No significant difference was found between these two methods. Once mastered, this technique can be performed in two hours per session if performed properly. When attempting this procedure, it is important to remember to perform the surgeries and reconstructions the same way every time Following this procedure.

Other methods like immuno staining and Q-R-T-P-C-R can be performed to answer additional questions like how vascularity and gene expression is influenced by different methods of grafting. After watching this video, you should have a good understanding of how to process lipo aspirate for grafting serially CT scan grafts in vivo, evaluate graft survival and explan grafts for weight histology, and future analysis After its development. This technique paved the way for researchers in the field of plastic surgery to explore fat grafting in the mirroring model.