Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

The overall goal of this procedure is to perform intravital microscopy imaging of the mouse liver to measure the blood flow speed with an individual hepatic vessels. This is accomplished by first exposing the liver and injecting a fluorescent plasma reagent intravenously to label the liver vasculature. The liver sinusoids and the erythrocytes traveling into them were then visualized by confocal microscopy in real time.

The hepatic vessels of interest are then selected for measurement of their blood flow velocity in the final step, movies of the liver blood flow are acquired at high speed and resolution. Ultimately, the blood flow speed within individual vessels under normal physiological conditions and following leash mania infection can be quantified. The main advantage of this technique over other existing methods like TOLA ultrasound and optical coherence tomography, is that it provides greater spatial and temporal resolution combined with the capacity to measure several other parameters at the same time.

In vivo, This method can provide insight into the hemodynamics of the liver during microbial infections. It can also be applied to other system, including the study of organ physiology in cancer and autoimmune disease. Visual demonstration of this method is critical, as it is difficult to properly expose an angio liver and to rapidly choose the blood vessel exhibiting the fluorescent plasma membrane reagent staining without practical experience Before beginning the procedure, disinfect the workspace with a bio cidal disinfectant spray, and all of the surgical instruments with 70%ethanol for 30 minutes.

Then after confirming the appropriate level of anesthesia by lack of response to toe pinch, shave the animal's abdomen and clean the surgical area with povidone iodine. Next, make a small incision in the skin and musculature under the thoracic cage on the left side of the abdomen, and place a piece of moist gauze just below the opening. Extract the liver and place it on the gauze, placing a second piece of moist gauze just above the tissue.

Then place a 24 by 60 millimeter 150 micrometer thick cover slip cyanoacrylate bonded to a metal frame on top of the liver. Flip over the mouse and cover the animal's eyes with another piece of wet gauze. To measure the liver blood flow speed, open the Leica application system software and select the resonance scanner mode.

Next, click on the configuration tab and select laser and helium neon 6 33 laser. Then click settings and select 12 bits resolution. Now click on the acquisition menu and in the beam pathway settings window, select the 63 x objective and set the 6 33 laser power to 100%To activate the signal gain and offset parameters from the photo multi tube three dropdown menu, select Alexa 6 33.

In the acquire tab, select the XYT acquisition mode and set the format width to 1024 by 1024, set the speed to 8, 000 hertz and select pinhole and a zoom factor of three. Then for Vidal imaging of the liver architecture, place the mouse on the stage of the inverted confocal microscope with the cover slip and liver face down. On the objective, set the temperature of the thermostatic controlled chamber to 29 degrees Celsius.

Using the USB control panel, adjust the focus of the liver based on the fluorescence of the injected plasma reagent dye. To quantify red blood cell velocities using XYT images, first, click on the live icon to visualize the blood flow in the live mode. Next, choose the area of interest and select a specific blood vessel for analysis.

Adjust the x, y and scan field rotation coordinates on the USB control panel to adjust the vessel into a horizontal position. Then stop using the live mode and change the format set width to 1024 by 2 56. In the acquisition mode setting window, set the line and frame averages to one and set the stacks to 150.

Click start to acquire the images. When all of the images have been obtained, open the Image J software and the file of interest. Then under the plugin menu, select the loci and bio formats import options.

Set the stack viewing parameters to hyper stack and select the X-Y-Z-C-T option. A window will open containing all of the acquisition series information under the plugins menu, again, select the MT a J option, click the add icon and select a red blood cell to track. Click on the same red blood cell in each of the consecutive frames, and then click on the measure icon to measure the speed of the cell.

Track individual red blood cells within a single vessel and analyze a minimum of three individual vessels of the same size. To quantify red blood cell velocities using XT line images, click live to visualize the blood flow in the live mode and select a specific blood vessel for analysis. Move the vessel of interest into a horizontal position and set the central lumen of the selected vessel along the line scan as just demonstrated.

Then stop the live mode once the vessel is in place. Now select the XT scanning mode. Set the format width to 1024 by 512, the speed to 8, 000 hertz, the line average to 32, and the time to 512.

Then click start and acquire the XT line images to generate streaks for quantifying the red blood cell velocity. Open the Leica application system. Advanced fluorescent software, select experiments.

Open the DO LIF file and select the image of interest. A graph showing an XT line image will automatically open using the draw line tool, draw a line along the streak, which indicates the corresponding distance in micrometers and the time in seconds. Then use these values to calculate the velocity as the distance divided by the time using the MT A KJ plugin for image J software.

Quantitative analysis of the velocity of these individual red blood cells in a single vessel demonstrated that the velocity of a red blood cell migrating through small liver vessels averages between 25 to 35 micrometers per second, repeated scanning along a line parallel to the vessel wall and centered in the lumen of the vessel, however, allows the use of the orthogonal projections on the X and Y AEs to calculate the velocity of the individual red blood cells with a similar accuracy to the MT a CJ method. In these images, a typical example of a suboptimal experiment is shown in which the rate of internalization of the plasma reagent into the endothelium lining the blood vessel was not considered. Note the altered quality of the streaks in the XT image rendering the red blood cell velocity within these vessels.

Uninterpretable, in this final representative experiment, the blood flow in the liver of L Donovan I infected mice was evaluated. Note the significant increase in the blood flow velocity observed as early as one hour and up to 24 hours post-infection, validating the use of the XT line image method for gaining new insights into the influence of pathological conditions on liver hemodynamics Once mastered, this technique can be complete in two or three hours if it is performed properly After its development. This technique paved the way for researchers in the fields of physiology and pathophysiology to explore organ specific hemodynamics in mice in vivo, under physiological and pathological conditions.

After watching this video, you should have a good understanding of how to do a surgery for exposing liver and how to measure the blood flow velocity in individual hepatic blood vessels following the injection of a plasma tie.