High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

The overall goal of this procedure is to use high frequency ultrasound to visualize mouse cervical lymph nodes and use image guided ultrasound to conduct lymph node biopsies. This is accomplished by first placing the anesthetized mouse in a supine position on the heated imaging platform and conducting all required animal specific steps to prepare for imaging. The second step is to conduct high frequency ultrasound imaging of the mouse neck region to identify and visualize cervical lymph nodes.

Next, an image guided biopsy is performed on the cervical lymph nodes of interest. Ultimately, high frequency ultrasound imaging is used to analyze mouse cervical lymph nodes and identify and quantify normal and pathological changes using longitudinal monitoring in a variety of mouse models. Visual demonstration of this method is critical as the mouse setup and biopsy steps require a fair degree of detail and precision to properly Accomplish.

We first had the idea for this method when we employed high frequency ultrasound to monitor mouse tongue tumor formation. Then realized that the method could be easily adapted to simultaneously monitor changes in the associated cervical lymph nodes. Though this method can provide insight into cancer related changes of cervical lymph nodes in mice.

It can also be used to monitor cervical lymph adenopathy in additional mouse model systems such as bacterial infection or autoimmune diseases. In preparation, preheat the imaging platform to 40 degrees Celsius and plan to maintain it within a few degrees. After anesthetizing the mouse, place it in a supine position on the imaging platform.

Next, attach a nose cone to maintain steady state sedation. Then confirm the anesthesia using a toe pinch and apply ophthalmic ointment to the eyes. Then using tape and gel, attach the paw to the ECG.

The next step is to insert a lubricated rectal temperature probe to maintain the mouse's temperature within two degrees of 36.9 degrees Celsius. Lastly, use depilatory cream to remove the fur from the neck and elsewhere. Rinse the cream and hair off with water soaked gauze.

To begin liberally apply a layer of warmed ultrasound gel to the neck area to void a fur. Do not introduce air bubbles in the gel. We have found that applying a liberal application of the ultrasound gel enhances imaging by preventing the transducer from pulling on the skin.

Next angle the imaging platform to slightly elevate the mouse's head, which will improve the mouse's respiration rate. Then place the 40 megahertz transducer transversely in the mounting system and carefully lower it until the front of the scan head is in the gel. Do not use excessive pressure, which would compromise breathing using the software control.

Adjust the brightness mode settings as follows. Set the gain to 22 decibels. Set the depth to 10.00 millimeters and set the width to 14.08 millimeters.

This will serve as the starting point. Adjust the tilt of the imaging platform to make both sides of the neck appear even in the B mode image. Now to image the lymph nodes, use the Y axis to scan the neck in a cranial to coddle manner towards the thoracic region.

Use the x axis to center the image. Identify major landmarks like the buccal cavity, tongue and thyroid gland. Next, map the lymph nodes with the 3D skin of the entire neck region from the thyroid gland to the buccal cavity tongue region.

Along the Y scale. Note the numerical location of the tongue, buccal cavity, and the thyroid gland, and take the difference to know the length of the neck. Then center the transducer at the calculated midpoint of the neck.

Once centered, press 3D and enter the calculated neck length as the scan distance. Set the 3D step size to 0.076 millimeters. Then press scan to acquire the image series stack for the entire neck region.

To elevate the mouse, either use a taller platform or raise the platform with a plastic micro fuge tube rack. Start with selecting the largest visible lymph node for biopsy and maintain a high frequency ultrasound image of it With the 50 megahertz transducer, the selected nodes should be larger than 10 cubic millimeters, or the biopsy will be very difficult to perform or form. Now, attach a one milliliter syringe to a 27 gauge half inch needle and place it into the syringe holder.

Orient the needle perpendicularly to the neck, and now follow the text protocol to adjust the image for the biopsy. Next, adjust the needle holder until the needle tip comes into view and contacts the skin. Then advance the needle with a firm fast push to puncture the skin, continue to advance the needle until the tip also punctures the capsule and is visible within the magula During biopsy.

It is important to rapidly advance the needle in order to puncture the skin. Then pause briefly before continuing with a second quick advance to puncture the lymph node capsule. Once the needle is in the lymph node, gently pull the plunger back to between the 200 to 300 microliter mark.

The biopsy material typically remains within the needle and thus is not visible within the syringe. Then gently remove the needle from the mouse and expel the contents into a 1.5 milliliter micro fuge tube. Remove the needle from the syringe and put that into the tube as well.

Then load one milliliter of biopsy media into the syringe. Reattach the needle and expel the biopsy material into the tube after the procedure. Be sure to monitor the mouse until it is fully active in the ultrasound software.

Select the image for analysis and to navigate to the image processing tab, choose load into 3D. Then select 3D reconstructed image in the top left corner and click on display single pane. Use the zoom function to enlarge the image if desired.

Then toggle the display layout button to view the image in B mode, which removes the view of the power doppler overlay. This makes it easier to see the edges of the lymph node during subsequent 3D analysis. Scroll through the image series to locate the beginning of the lymph node to circumscribe the lymph node.

Navigate to the 3D settings tab, select volume. Then the start button next to parallel by scrolling around, draw contours around the area of interest within individual images. Continue doing this until enough images are marked so that the entire lymph node is marked.

Then choose finish to complete the analysis. At the bottom of the image, the 3D volume and percent vascularity are automatically displayed. Next, toggle the display layout button to view the power doubler image overlay on the surface.

Observe a net view of the volume area of interest. Ultimately export the images in TIF format or export the 3D scans as a VI movies using the described protocol. Realtime high frequency ultrasound imaging allowed for guided fine needle biopsy of cervical nodes similar to what is conducted in clinical practice.

BMO sono graft showed the ideal needle placement prior to biopsy. The sonograph guided needle tip entry into the cervical lymph node and the sonograph guided the needle position during the biopsy C, the biopsy components were processed using cytosine. Abundant lymphoid cell clusters and associated connective tissue were found verifying a successful lymph node biopsy.

Computerated analysis of the high frequency ultrasound images provided information about the lymph node architecture volume, and vascular flow using power doppler mode and 3D volume measurements. The percent vascularity was calculated. Additionally, 3D imaging allowed for virtual lymph node reconstruction revealing the overall lymph node topography Once mastered.

This technique can be done in about 15 minutes per mouse if it is performed properly. After watching this video, you should have a good understanding of how to identify mouse cervical lymph nodes using high frequency ultrasound, and have knowledge on how to use image guided ultrasound to perform a lymph node biopsy.