Intravital Microscopy to Analyze Blood Cell-Endothelial Interactions in an Inflammation Mouse Model

Protocolo

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Animal Handling, Preparation and Induction of Inflammation

1. Use 4 - 6 week-old male mice with a weight range of 16 - 20 g. Note: If the mice are older or heavier they present excessive fat surrounding the vessel, limiting the microscopic observation.
2. Sterilize all tools and microscope table prior to surgery.
3. Inject 20 mg/kg LPS (lipopolysaccharide) intraperitoneally 4 hr prior to microscopy into the living mouse to induce a bacterial inflammation.
4. Prewarm a 0.9% saline solution in a water bath at 37 °C to humidify the plastic chamber and the mesentery tissue.
5. Anesthetize the mouse with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (5 mg/kg) right before the microscopy procedure. Alternative anesthesia can be used, e.g., 2% isoflurane inhalation. Confirm proper anesthetization by the loss of response to reflex stimulation (toe or tail pinch with firm pressure).
6. Depilate the abdomen using a shaver and remove loose hair with gauze saturated with ethanol 70%.
7. Apply vet ointment on the eyes of the mouse to prevent dryness, while under anesthesia.

2. Surgery

1. Sterilize the abdomen using 70% ethanol. This method is not a sterile method and could be lethal at the end of the experiment.
2. Perform a median laparotomy: Open the abdominal skin using small, curved forceps and small scissors. Identify the epigastric vessels and open the peritoneum in the region of the linea alba, to protect the vessels.
3. Apply a few drops of prewarmed saline into the abdominal cavity to keep the tissue moist.
4. Label leukocytes and platelets fluorescently by injecting 50 µl rhodamine 6G (1 mg/ml) retro-orbitally.
5. Exteriorize a loop of ileum and place it in a petri dish with a diameter of 10 cm; make sure to keep the tissue moist by applying the 37 °C prewarmed saline solution (0.9%) every other minute.

3. Intravital Microscopy

1. Place the Mouse underneath the microscope and bring the mesentery vein with a diameter of 200 - 300 µm in the center of view. Choose a vessel with no visible fat surroundings.
2. Do not touch the mesentery vessels thereby avoiding stimulation of the endothelium. Handle the ileum loop cautiously.
3. Visualize blood cell-endothelial interactions with an inverted or upright microscope and a camera using microscope software. Record blood cell-endothelial interactions for 1 min in 4 different veins per mouse.
4. Euthanize the mouse by cervical dislocation after the completion of imaging experiments.

4. Analysis

1. Carry out the analysis offline and blind for all parameters. Carry out analysis using any suitable software program.
2. Confirm stable and interindividual comparable blood flow conditions in high time-resolution cine-clips (maximal frame rate) focused on intraluminal blood cell flow.
3. Quantify the number of rolling leukocytes. Therefore draw a vertical line through the vein and count all leukocytes crossing this line in 1 min.
4. Determine rolling velocity by measuring the time one single leukocyte needs to pass a distance of 50 µm while stably rolling on the endothelium. To do this, draw two vertical lines at a distance of 50 µm through the vein.
   1. Measure the time a representative leukocyte needs to get from one line to the other. Calculate the speed of the leukocytes by dividing 50 µm through the time needed (µm/s).
5. Measure leukocyte adhesion in a field of 0.04 mm². To do this, draw a square with a side length of 200 µm into the vein.
   1. Count the firm adherent leukocytes, defined as no visible movement for 30 sec, within this square.
6. Count the number of platelets bound to one leukocyte to quantify platelet-leukocyte interactions.

Materiais

Name	Company	Catalog Number	Comments
Rhodamine 6G	Sigma-Aldrich	989-38-8
Lipopolysaccharides from Escherichia coli 055:B5	Sigma-Aldrich	L2637