Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

The overall goal of the following experiment is to follow the migration and interaction of leukocytes in progressing liver inflammatory disease. To better understand the sequence of events underlying how these cells are recruited into the liver tissue and their potential involvement in the immunopathology. This is achieved by first performing a tracheotomy to gain control over the respiration of the mouse, a crucial step to ensure long-term survival of the animal.

As a second step, the mouse is laparotomies to surgically expose the liver, giving access to the organ with the multi photon microscope. Next, the liver is fixed and positioned by embedding it in arose to minimize movement artifacts. Imaging can be performed over several hours leading to high resolution, time-lapse sequences that follow the changes in recruitment, positioning and interaction of individual leukocytes.

In the process of developing liver inflammation, This method can help to answer key questions in the field of acute and chronic liver inflammation, such as leukocyte recruitment, cell cell interactions, or mechanisms of immunopathology. The main advantage of this technique compared to conventional microscopy is that multifocal microscopy allows long-term imaging with virtually no sample perturbation with low foal bleaching or phototoxicity, as well as optical slices up to 100 micrometer deep into the tissue, and this is due to the unique physics of the laser of the far red laser light. Well, this method can provide insight into liver disease.

It can easily be adopted to other systems. Since the method of stabilization and fixating, the tissue can be applied to virtually all questions of intravital microscopy, so it can easily image organs such as spleen, kidney, or even subcutaneous tumors Using fluent related cells. It is also possible to visualize all leukocyte subsets and send in their interactions and liver inflammation.

Generally, individuals new to this method will struggle because of the com extreme complexity of small animal surgery. However, due to our own experience, this is only a limiting factor for a couple of weeks and just needs some hands on routine Due to the fragility of liver tissue, the preparation steps are hard to learn, and visual demonstration of the method is essential. After setting up the anesthetics, fill the mouse aro stage with 3%aros, pouring it in at a 40 degree angle.

Then make certain that the surgical work area is fully disinfected, as are all the required instruments and so forth. Then anesthetize the mouse with an intraperitoneal injection of an anesthetic cocktail. Five minutes later, check the animal's reflexes and then disinfect the skin and apply ophthalmic ointment to the eyes.

Now, secure the mouse to the preparation table. Using tape, position it to expose the ventral side gently overstretching the neck. A rubber band hooked to the incisors can accomplish this.

Now, perform the initial incision below the chin about 0.5 to one centimeter long. Then carefully dissect the connective tissue between the salivary glands and find the muscular tube surrounding the trachea. Gently tear the muscle to expose the trachea.

Pull a thread under the trachea, then open the trachea with micro scissors using a T-shaped incision into the incision. Insert the ventilation tube about half a centimeter. Advance the tube using small anatomical forceps.

Then secure the tube with sutures. Then for added security, suture the tube to the skin, seal up the incision in the skin using cyanoacrylate and secure the tube to the animal's head. Using adhesive tape, maintain the anesthesia using a continuous flow of 2%iso fluorine.

After preparing the mouse for surgery, make a small skin incision below the sternum and extend the cut laterally below the ribs on either side. Cauterize all the exposed blood vessels in this process. To minimize bleeding, perform a small initial cut at the linear elbow to gain access to the abdominal cavity.

Extend the cut horizontally, sealing all blood vessels and giving access to the liver. Now, transfer the mouse to the agro stage and stack slides on either side of the animal to help keep it in place. These stacks should match the height of the mouse so that the stage for the liver, which will be placed later on, does not apply excessive pressure on the abdomen.

Now, using sutures in the sternum, retract the ribs. Four zero suture will suffice After retracting the ribs, cut the ligaments connecting the liver and diaphragm cut all the way to the aorta, but work with extra caution not to rupture or cut the vessel. Also cut the ligament that connects the liver and GI tract.

Turn the mouse 45 degrees to improve access to the larger liver lobe. Take a standard cover slip and tape it along its edges so that it is not sharp. Place it over the abdominal cavity.

This will serve as a stage for the liver to position the liver onto the stage. Carefully slip a double ball stylus probe, or a padded spatula below the liver and hold the top of the organ. Using a moist tissue mechanic pressure will instantly lesion the liver tissue so it is essential to handle the organ with care.

Then lift the lobe onto the slide and gently tug it and into position. The lobe will usually end up bent or folded gently try to unfold the edges by lifting the tissue with a thin spatula or a moist tissue. Next, add lateral supports in the form of cover slips or slides.

Stack the cover slips to be the same height as the liver lobe. The staging and fixation of the liver is the most crucial step in the protocol. Any mechanical stress, torsion or pressure will lead to instant tissue damage or at least impaired blood flow in the sinusoidal vessels.

Now, put a large cover slip with taped edges over the liver lobe supported by the lateral supports. Position this cover slip as level as possible. It should not squeeze the liver.

White tissue indicates impaired blood flow unless the imaging will go quickly. It is now necessary to insert two independent intraperitoneal catheters to provide long-term anesthesia and to apply G five. These are assembled from 27 gauge needles and flexible silicone tubing.

Then start their flow using syringe pumps. Insert the catheters into the lower abdomen near the hind limbs. After attaching vital monitors to the mouse, prepare 100 milliliters of 3%aros in one XPBS.

When the aros has cooled to 41 degrees Celsius, embed the liver in it using a five milliliter syringe with an 18 gauge needle. Once the aeros has cooled, use a heideman spatula to remove the excess and expose a field of view large enough to scan the liver. Then proceed with the scanning.

After the field of view is prepared, move the mouse under the microscope to select appropriate scanning areas for the time lapse imaging. The liver tissue is scanned by eye using an external white light source identifying representative fields with sufficient blood flow and minimal sample perturbation CXCR six GFP heterozygous mice were subjected to intravital T-P-L-S-M imaging as an experiment. Some were given an intraperitoneal injection of tetrachloride to induce acute liver damage.

Modal cells expressing GFP were tracked by video and their tracks were superimposed to get a snapshot of cellular mobility. The tracks were then normalized. Normal livers had cells with random migration patterns.

Livers injected with CCI four showed an impairment in cellular migration. Measuring total displacement from their origin, it was clear that there was cell trapping 36 hours after CCI four treatment at the site of hepatocellular damage. Impaired migration by the liver damage could also be shown by plotting the migration speed of individual cells over time, showing a partial arrest after 18 hours and full migratory arrest after 36 hours.

The reduction in the speed of the migrating cells could also be shown to be statistically significant when the cells were compared as populations. Thus, this approach is suitable for tracking changes in cell migration and positioning in developing liver disease. After watching this video, you should have a good understanding of how to prepare the liver of a mouse for intra vital multi photo microscopy.

The method we provide here allows us to stabilize the animal for long-term survival as well as minimize movement artifacts due to the gentle fixation method we provide. Once masked, this technique can be done in about one hour if it is performed properly During this procedure, it's important to control the vital parameters of the animals stabilizing temperature, heart rate, oxygen, and CO2 levels. This can be achieved by adapting the depth of the necrosis and respiration Following this technique.

Other procedures like flow cytometry analysis or laser capture micro dissection can be performed in order to answer additional questions like the phenotype of the infiltrating immune cells or mediators secreted by tissue or infiltrating cells After its development. This techniques allowed researchers to gain insight into the dynamics of leukocyte traffic and cellular interactions in the liver, allowing to dissect the sequence of events leading to inflammatory liver diseases. Please follow the laser security guidelines doing imaging with a high energy pulse infrared laser used for multi photo microscopy.

Ensure that no reflection of laser radiation can cause hazard to your eyes and skin while performing this experiment.