An In Vivo Model to Study Shear-Dependent Bacterial Adhesion

Take an anesthetized mouse fasted overnight to reduce bowel movements.

Apply eye ointment to prevent corneal drying. Secure it on a heating pad to regulate body temperature.

Expose the right jugular vein. Insert a catheter and secure it with stitches.

Open the peritoneal cavity, then place the mouse on a transparent plate.

Spread the intestine to expose the mesenterium, a supportive membrane, to access the blood vessels.

Maintain body temperature and apply saline to prevent dehydration.

Place the mouse under a fluorescence microscope, then immobilize the vessels.

Apply an ionophore that elevates calcium levels in endothelial cells. This triggers the release of von Willebrand factor (VWF), forming coiled strands on the cell surface.

Inject fluorescently labeled bacteria through the catheter to reach the blood vessel.

The shear stress from blood flow uncoils the VWF strands, which bind to bacterial adhesins and form bacterial aggregates.

Capture time-lapse images to quantify shear-dependent bacterial adhesion.

Fast six to eight-week-old mice the night before the experiment to reduce bowel movement. The day of the experiment begin by injecting 0.1 milligrams of buprenorphine per kilogram of body weight as an analgesic. Next, anesthetize the mouse and apply ointment to the eyes.

Place the mouse on the thermo-controlled heating pad under the dissecting scope and test that the mouse shows no pedal reflex. Make a one-centimetre incision parallel to the jugular vein. Remove the right side of the cervical muscle and then isolate the jugular vein from the surrounding tissue.

Insert a two French intravenous catheter into the right jugular vein and secure it in place using stitches. Then, make a midline abdominal incision to open the peritoneal cavity. Place the mouse on its right side on a transparent plate and secure the cannula with tape.

Gently pull out the intestines using cotton swabs and spread the mesenterium to visualize the mesenteric arteriolar and venular circulation. Place a hot pack over the mouse to prevent hypothermia and drop 500 microliters of 0.9% NaCl on the intestines every 30 minutes to prevent dehydration of the tissue. Use cotton swabs to immobilize the vessels and visualize them under an inverted microscope.

Next, topically, apply five microliters of a 10 millimolar solution of calcium ionophore dissolved in DMSO. This will activate the release of von Willebrand factor from the endothelial cells in the vessels. After 10 seconds, inject 100 microliters of the fluorescently labeled bacteria through the jugular catheter. At this time, begin taking time-lapse images. Acquire time-lapse images using the acquisition tool in the toolbar, using 40 cycles of 1000 images per second. Once image acquisition is complete, euthanize the mouse according to institutional guidelines. Save the images in an appropriate image file format, and then process the images using ImageJ analysis software as previously described.