NASH is one of the most important chronic liver disease in the world. It can progress to fibrosis, cirrhosis, and eventually the liver cancer. Study the dynamic change of the ECM in the NASH can help to understand the fibrosis better.
Traditional to imaging cannot demonstrate 3D ECM structure changes, limiting the understanding of dynamic ECM remodeling process in the liver fibrosis. To begin, weigh the mouse before the procedure. After anesthetizing the mouse shave the ventral area using a hair clipper and disinfect the skin with 70%ethanol.
Place the mouse on its back and immobilize the legs on the surgical board with tape. Use Mayo scissors and Adson forceps to make a three centimeter incision laterally in the lower abdomen. And then a five centimeter incision vertically from the low abdomen to the xiphoid process without opening the chest cavity.
After opening the peritoneum, gently place the intestines to the animal's left and elevate the right lobe of the liver with a rayon tipped applicator to expose the portal vein. To catheterize the portal vein, induce a 24 gauge IV catheter into the distal end of the portal vein. Place the catheter tip before the portal vein branches out into the hepatic lobes.
Withdraw the needle immediately when the catheter enters the vein. Check that the catheter is correctly positioned inside the vein by perfusing the liver with PBS using a one milliliter syringe via the catheter. Use Dumont micro forceps, a Castroviejo microneedle, and 4-0 suture to place a stitch below the branching of the portal vein and a second stitch one centimeter below to secure the catheter.
Connect the catheter to a silicone tubing of one meter length, three millimeter inner diameter, and 4.1 millimeter outer diameter with a lure connector. Connect the silicone tubing to a peristaltic pump and a reservoir containing deionized water. Carefully remove the air bubbles inside the tube and avoid generating new bubbles during the perfusion.
Set the peristaltic pump at a flow output of 0.2 milliliters per minute. Peruse first with deionized water for two hours and the color of the liver will change from red to yellow during perfusion. Switch the perfusion solution to 0.5%sodium deoxycholate and continue overnight and the liver will become white at the end of the perfusion.
Switch the perfusion solution to deionized water and perfuse for two hours. Use Dumont micro forceps and micros springing scissors to collect the decellularized liver and wash it carefully in a Petri dish with PBS. Transfer a small piece of decellularized liver to a microscope slide and place the tissue in the middle.
Add 10 microliters of antifade mounting medium to the tissue with a pipette to avoid tissue drying. Cover the tissue with a cover glass and apply a small force to flatten the sample. Seal the edges of the cover glass with colorless and transparent nail polish.
Place a drop of a immersion oil on the top of the cover glass and place the slide on the microscope slide holder. Lower the 20X oil objective lens until it contacts the immersion oil. Switch to the violet channel of wavelength 405 nanometers.
Turn on the shutter and focus the sample. Navigate and position the area of interest for imaging. Turn off the shutter before moving to image acquisition using laser light.
To start the two photon laser, switch to the computer control and turn on the two photon laser first. Then turn on the two photon laser controller and shutter ensuring that the output power is higher than 2.5 watts. Reduce the laser power to prevent fluorescence quenching.
Select and adjust detectors both photo multipliers and the hybrid to accommodate the chosen fluorophores. Choose simultaneous or sequential image acquisitions. Adjust the pinhole to the highest value.
Adjust laser wavelengths, gain, power, offset the pixel dwell time, pixel size averaging, and zoom. Scroll the computer Z controller and set the Z dimensions. Define the start and endpoints and choose the numbers of images given within a volume.
Acquire images. After performing the two photon microscopy, collagen fibers without decellularization revealed a low resolution image of the collagen network. In the decellularized extracellular matrix, apparent differences were observed in the morphology and spatial distribution between chow and fast food diet livers.
Collagen fibers and mice on a chow diet exhibited an interweaving and well-organized network, whereas the mice on fast food diet showed collagen bundles with less connectivity. The three dimensional structure of the liver extracellular matrix shows that the collagen fibers in mice on the chow diet showed a well-organized network, but not in fast food diet mice. To confirm the quality of the fibrillar collagen in the decellularized extracellular matrix, the slides were stained with Hematoxylin Eosin and Picrosirius red.
Hematoxylin and Eosin show that all the cells have been removed. In the Picrosirius red staining images, the extracellular matrix from the chow fed mice revealed a well-organized network. And in contrast, mice on fast food diet showed denser collagen fibers.
Carefully remove the bubble inside of a tube before the perfusion. The bubble will block the vessel in the liver and affect the perfusion.
