Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

The overall goal of this procedure is to observe the morphology and functionality of leukocytes inside the liver during septic shock. The main advantage of this technique is that you can obtain direct videos of leukocytes inside the liver. Prior to surgery, use 70%ethanol to disinfect the surgery table and all instruments.

Then set up the tools for liver surgery and adjust the heating plate to 37 degrees Celsius. After anesthetizing LPS or PBS injected mice according to the text protocol, apply a small amount of eye ointment to prevent the retina from drying out. Use a razor and hair removal cream to remove the hair around the abdomen.

Then use wet gauze to clear the abdomen. While carrying out a retro-orbital injection using an insulin syringe, inject 200 microliters of Texas red dextran solution. Use a marker to mark the right subcostal area from the xyphoid process to the end of the rib.

Then apply povidone iodine to the mouse's abdomen to prevent secondary infection. Use forceps and scissors to cut the epidermis. Next, using micro scissors and forceps open the abdominal cavity and carefully expose the liver.

Use cotton swabs to roll out the left lateral lobe. Then pour 500 microliters of sterile PBS on the abdominal cavity to prevent the liver from drying out. Place the mouse in the right decubitis position in a custom designed chamber and apply a small amount of tissue glue to the silicon bed.

Then use cotton swabs to carefully attach the liver. Liver tissue is extremely friable. Never use sharp or heavy tools when exposing and holding the liver.

A few seconds later, wet the liver again with 500 microliters of sterile PBS to prevent it from drying out. Then place the metal frame on the liver and use nuts to firmly fix it. Always place supporting nuts before placing the metal frame.

This will help reduce the pressure of the metal frame. To carry out intravital imaging of the liver, run the Zen software to turn on the laser. Set the wavelength to 880 nanometers and set the green and red channels.

Then adjust the laser power according to fluorescence intensity. Place the chamber on the imaging stage and position the 20 power water immersion objective lens close to the cover glass. Fix the silicone rubber heater under the mouse body to maintain its body temperature.

Next draw up 500 microliters of distilled water on the metal frame. And then position the objective lens close to the liver. Adjust the focus and determine the depth and time of the imaging area.

For long term imaging, inject half a dose of the previous prepared Zoletil and Rompun anesthetic mixture for each hour during imaging for ethical reasons. Maintain the body temperature at 37 degrees Celsius and always keep an eye to the mouse since anesthetization times differ for every mouse. To analyze the data open Velocity and import the raw data from the library.

Then remove the noise from the image and take a snapshot. Finally fit the edited items to 100%and export the files as JPEG or TIF files for snapshot images and AVI files for movies. Intravital imaging analysis of control mice showed that a small number of neutrophils were circulating in the blood vessel.

Whereas the number of neutrophils was significantly increased in LPS treated mice. In LPS condition a lot of neutrophils were circulating inside the bloodstream but they did not migrate outside the vessel. As seen in these movies, compared to control mice, neutrophils in the LPS treated mice were more firmly adhered to the luminal surface of the vessel which made neutrophils more frequently detected in the vessel.

Apart from the motility of neutrophils, liver capsular macrophages, or LCMs, in CX3CR1-GFP mice were observed on the surface of liver tissue rather than deep within the tissue. In addition, LCMs showed little movement or activation in the liver even in LPS treated mice. Once mastered this technique can be done in 30 minutes if it is performed properly.

While attempting this procedure, it's important to remember to set the incision line from xiphoid process to the end of the right subcostal abdomen. Following this procedure, other methods like treatment injection can be performed in order to answer additional questions like how the liver recovers from damage and infection. After its development this technique paved the way for researchers in the field of medicine to explore the leukocyte's reaction during sepsis and test potential drugs.

After watching this video you should have a good understand of how to expose a liver with minimal abdomen incision. Don't forget that working with lipopolysaccharide can be extremely hazardous and precautions such as sterilization and hand washing should always be taken while performing this procedure.