Mapping Hepatic Stellate Cell Morphology in Mouse Models of Liver Fibrosis

This protocol presents a comprehensive imaging pipeline to visualize and model hepatic stellate cells morphology in intact liver tissue. Current 2D imaging misses full stellate cell morphology, limiting structural and functional insights. This protocol enable robust captures of complex 3D architecture.

To begin, pinch the palms of an anesthetized mouse to ensure proper anesthesia. Spray the abdomen with 70%ethanol to wet the fur. With a pair of forceps, pick up the skin in the middle of the abdomen aligned with the hind legs and start cutting the skin to reveal the abdomen.

Then use cotton swabs wetted with PBS to move intestines and other organs to the side and lift the left lobe. Carefully cut the membrane attached between the left lobe and the caudate lobe. Then use a suture to tie off at the top base of the left lobe.

Cut the left lobe below the suture knot and set it aside for tissue sample collection and processing. Use PBS-wetted cotton swabs to move the liver and reveal the portal vein. Then carefully insert the catheter needle into the portal vein at about halfway along its length.

Remove the catheter needle. Ensure a backflow of blood through the catheter to indicate successful insertion. Connect the filled tubing from the running perfusion pump to the catheter and perfuse the liver with 20 milliliters of PBS.

Clamp the pulmonary artery above the liver with a hemostat to stop the PBS from going through the heart. Cut the inferior vena cava to release the pressure buildup. Adjust the pump speed to five milliliters per minute.

After 15 minutes, stop the pump to change the reservoir to 4%paraformaldehyde then perfuse the liver with 25 milliliters of 4%paraformaldehyde. Once the perfusion is complete, remove the liver from the surrounding organs by cutting the connecting tissues. Place the liver in a 50-milliliter conical tube filled with 25 milliliters of 4%paraformaldehyde.

Place the tube horizontally on a bench-rocking platform overnight in a cold room at four degrees Celsius at 1.5 revolutions per minute. On the next day, wash the liver three times in PBS on a bench-rocking platform for 30 minutes each at room temperature at 1.5 revolutions per minute. Transfer the liver to 20 milliliters of PBS with 0.02%sodium azide and store at four degrees Celsius.

Cut the liver samples into pieces approximately 0.5 to 1 cubic centimeter in size. Transfer the liver samples into glass vials with screw tops. Dehydrate the samples in a water-diluted methanol series from 20%to 80%on a wheel rotator at eight rotations per minute for one hour each at room temperature.

After performing sample bleaching, permeabilization, immunolabeling and clearing, incubate the sample in dibenzyl ether overnight at room temperature before imaging. Samples can be stored in dibenzyl ether for up to one year without loss of the staining signal. For confocal imaging, place the cleared sample in a 35-millimeter glass-bottom dish with a number 1.5 glass bottom.

Fill the dish with ethyl cinnamate to completely submerge the sample. Then place the dish on the stage of the confocal microscope. Acquire a tile image on the brightest plane to capture a large area of the sample using 1024 by 1024 format and a speed of 600.

Based on the tile image, select regions of interest to collect a higher resolution Z-scan. Next, acquire a Z-stack spanning the entire depth of the cleared tissue at the highest resolution supported by the microscope using a 1024 by 1024 format and a scan speed of 600. Successful perfusion and fixation of the mouse liver resulted in a uniform pale, beige yellow color.

Improper perfusion resulted in dark red or brown regions of blood clots, which reduced optical clarity and led to a high background signal in these regions. Complete optical transparency was achieved after incubation in dibenzyl ether where the tissue appeared clear compared to before clearing. High resolution Z-stacks revealed fine dendritic processes extending from the cell body of individual hepatic stellate cells.

Both control and chronic carbon tetrachloride liver samples showed strong and stable fluorescent signals under confocal microscopy. This pipeline reproducibly map stellate cell morphology in the complex server of microenvironment, capturing dendritic processes and spatial relationships with neighboring cells. Time commitment is an important considerations when performing the protocol.

The clearing process takes approximately three weeks, which requires advanced planning. Enabling precise 3D morphometric analysis, this platform links fibroblast structure to functions, advancing insights into tissue homeostasis and a Z mechanism.