Mouse Inguinal Lymph Node Volume Analysis: An Ultra-high-frequency Ultrasound Imaging Technique to Determine the Volume of Inguinal Lymph Node in Mouse Model

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

1. Imaging procedure

1. Place the mouse in an induction chamber for gas anesthesia and supply 3% isoflurane in pure oxygen until the animal is fully anesthetized. Verify the depth of anesthesia by lack of response to paw pinch.
2. Transfer the animal to a heated board – a constitutive part of the UHFUS imaging station – holding the animal in a supine position. Use a rectal probe lubricated with petroleum jelly to measure the body temperature. Adjust the board temperature to maintain the mouse's body temperature in the physiological range (36 ± 1°C).
3. Moisten the mouse's eyes with vet ointment to prevent dryness during anesthesia. Supply narcotic gas (1.5% isoflurane in pure oxygen) through a mouse's nose mask. Adjust the percentage of isoflurane to maintain the correct depth of anesthesia.
4. Coat the fore and hind paws with conductive paste and tape them to the ECG plate electrodes embedded in the board. Check that the physiological parameters (heart rate, respiration signal, and core body temperature) are correctly acquired and displayed.
5. Remove hair from both inguinal areas by applying a depilatory agent and coat them with an acoustic coupling medium.
6. Clamp the UHFUS linear probe (40 MHz center frequency) into a specialized 3D motor embedded in the UHFUS imaging station, allowing automated and stepwise movement of the probe.
7. Properly orient and adjust the position of the ultrasound probe to obtain short-axis images of the inguinal lymph node (left/right), and place the region of interest in the focal zone.
8. Scan the entire volume of the inguinal lymph node as a sequence of 2D B-mode images, as described previously. Acquire images at multiple levels of the lymph node by linear movement of the transducer with step sizes on a micrometer scale, to generate 3D data in terms of automatically respiration- and cardiac-gated cine loops.
9. Set the image recording with the following parameters: scan distance ranging between 2 and 5 mm (depending on lymph node size); step size 44 µm, with an outcome of 46-114 scan steps/lymph node slices and an acquisition time of 1-3 min per animal. Digitally store the acquired images in raw format (DICOM) for further offline analyses.