Method Article
Ultrasound guided injection into the left ventricle of the mouse
https://doi.org/10.21203/rs.3.pex-2336/v1
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In this protocol we demonstrate an approach to the left ventricle of the mouse for an injection of sterile saline; the injection can be done with cells, therapeutics, or other injectates, to suit research needs. This technique offers a minimally invasive yet highly reproducible approach for efficacious results with minimal impact on the mouse.
cancer
left ventricle
ultrasound
minimally invasive
orthotopic
mouse
image guided injection (IGNI)
disease models
metastasis
blood-brain barrier
Injection into the left ventricle is of interest to researchers for several reasons. Primarily, it provides a robust model for metastasis. Although intravenous tail vein injections are commonly used for metastasis models, the left ventricle injections have the advantage of the left ventricle blood pool bypassing the lungs during initial circulation. This route offers more incidence of tumor formation elsewhere, enabling the study of multi-organ metastasis1. Unlike surgical orthotopic models this approach is minimally invasive and therefore does not require a recovery period and monitoring, analgesic use, and is an animal welfare refinement. Ultrasound imaging allows for real-time visualization of the site for placement verification and subsequent monitoring for adverse outcomes. Using the left ventricle injection model our researchers have successfully assayed the metastatic potential of various cells, as well as observing the effect of gene knockout cells on metastasis. Additionally, this route provides the ability to model metastasis for cancers where launching a primary tumor in the orthotopic setting is sub-optimal for technical or humane reasons; an example of this is a uveal melanoma. As a service provider to the MIT non-clinical research community, and not a research unit ourselves, the choice of injectate is driven by our researcher collaborators. Thus, in this protocol the technique is demonstrated with an injection of sterile saline and we report expected outcomes based on feedback from the researchers’ experiences when injecting cells for metastasis. A previously published protocol2 uses a parasternal long axis approach to the left ventricle, in contrast to our parasternal short axis approach with a smaller gauge needle and using a newer model ultrasound system. We feel that these updates to the procedure offer a clearer view, easier access route, and the potential for less injury. Based on the experience with several injectionists in numerous experiments, we believe this is a robustly reproducible protocol that can be utilized for multiple research needs and can easily be adapted for mouse anatomical variability.
This procedure was performed in the AAALACi accredited imaging suite of the Preclinical Imaging and Testing Core facility or in one of the adjacent housing areas when working with animals and inoculants which require protection of a biosafety cabinet. All described steps below were performed under protocols approved by the Committee for Animal Care of the Massachusetts Institute of Technology.
Preparing the ultrasound set up and the mouse
1. Verify the micro-manipulator probe clamp is set to be adjustable in the Y-axis, then mount the MX550S probe on the clamp. Position the probe transverse to the imaging platform.
2. Select the mouse abdominal application on the ultrasound system and open the image width and depth fully. Place the ultrasound focal point at a depth of about 9mm.
3. Verify the injection system is perpendicular to the imaging platform and adjust the syringe mount angle to ~ 30o below horizontal. Ensure the needle driver is fully retracted and the ruler is at zero. Ensure the injection system Y- and Z-axis fine adjustment knobs to be in the middle of their movement range (12.5mm for Z-axis, 10mm for Y-axis).
4. Adjust the imaging platform X- and Y-axis fine adjustment knobs so they are in the middle of their movement range (12.5mm for X-axis, 10mm for Y-axis). (see USGI set up Figure 1, and Image guided injection system Figure 2)
5. Turn on the supplemental heat to the imaging platform on the THM-150, and set temperature to 37oC.
6. Under isoflurane anesthesia depilate mice on the ventral surface across its thorax. (This step can occur up to one day ahead of the following steps)
7. Position the mouse supine on the imaging platform with all four limbs slightly outstretched (this is considered to be at the 12 o’clock position for the imaging platform). Apply tape across the front limbs and across the lower jaw to the nose cone. Apply tape across the pelvis. Maintain isoflurane at 2% to the imaging platform nose cone.
8. Rotate the imaging platform to the 7 o’clock position, in this orientation the mouse’s head is closest the sonographer and it’s left flank is nearly perpendicular to the needle mount. (Refer to supplemental video 1 for animal placement steps #7-8)
Locate and inject into the left ventricle
9. Apply a generous amount of ultrasound gel to the prepared area and allow some to spill over the flank. Lower the transducer onto the area leaving roughly 2mm of gel between the transducer face and the skin line.
10. Visualize the left ventricle parasternal short axis and use the Y-axis fine adjustment knob of the imaging platform to scan several millimeters cranial-caudally to identify the widest area of the left ventricle. Typically the papillary muscle attachments are visible at the widest area of the left ventricle.
11. Ensure there are no bubbles in the needle or syringe to prevent an air embolism which can cause death. Mount the syringe in the syringe mount so that the syringe scale is visible and the needle is bevel up. Then wipe needle with alcohol.
12. Release the knob on the injection system that locks the rotation of the system assembly and then retighten to set to the default perpendicular position. (See Image guided injection system Figure 2)
13. Slide the injection system towards the mouse so the needle is just touching the mouse within the flank ultrasound gel. Using the injection system Y-axis fine adjustment knob bring the needle to be in plane and visualized by the ultrasound transducer. Adjust the needle up or down using the Z-axis fine adjustment knob to line up for insertion into the left ventricle.
14. Place several right-hand fingers on the mouse’s right side in a supportive position to counteract any force during needle insertion. Do not push on the mouse.
15. Insert the needle into the left ventricle with a clockwise turn of the needle driver knob. Verify the needle passes fully through the myocardium and into the ventricle lumen, approximately 5 to 7mm in depth. If the needle tip and bevel move out of plane of the transducer and are no longer visible, adjust the transducer position with the knob on the micro-manipulator ultrasound probe clamp (Y-axis probe adjustor) to bring the transducer back into plane with the needle tip and bevel.
16. Inject saline volume slowly over many cardiac cycles (~7-10 seconds) followed by several more cardiac cycles (up to 5 seconds) before retracting the needle driver with a counterclockwise turn, then slide the injection system away. Very small bubbles may be visualized during the injection as bright flecks in the lumen. This is normal and is not of concern for the health of the mouse or an indication of injection success. (Refer to supplemental video 2 for dual synched video of steps #10-16)
17. Monitor for adverse outcomes by observing if the ventricle appears as it did prior to injection. If adverse outcomes are observed prompt humane euthanasia should be considered.
a. The ventricle contraction should be uniform all the way around the myocardium, occur at regular intervals, and
at the rate observed prior.
i. If ventricle contraction is slowed or irregular, keep the left ventricle under ultrasound observation.
ii. Remove the nose from the nose cone to allow mouse to breathe room air. This should start to revive the
mouse which then increases the ventricle contractions and can correct irregularities.
iii. The mouse can then be recovered normally.
b. Assess if any free fluid is around the ventricle which would be indicative of bleeding.
c. The lumen of the ventricle should free from any bubbles visualized during the injection.
d. Occasionally a drop of blood is observed externally when the needle is retracted. This is normal and not
considered adverse and the mouse can be recovered normally.
18. Retract the transducer, un-tape and recover the mouse in its home cage.
19. Monitor the mouse by cage-side observation the next day for signs of distress.
Step Problem Possible Reason Solution
10 Left ventricle appears more Mouse anatomical variability Rotate the imaging platform towards 6
midline than the usual shallow o’clock or 8 o’clock locations to find
ventral left thoracic location the best view. And/Or: Slightly tilt the
imaging platform down on the mouse’s
left side
13 Significant lung area is present Mouse anatomical variability, Slightly tilt the imaging platform down
along needle path (bright white or deep anesthesia causing on the mouse’s left side. And/Or:
line between left ventricle and changes in lung and heart Gently massage the mouse’s rib on the
skin line, see lung in injection volumes left side with a bit of downward
path Figure 3) pressure to move lung off of the heart
15 Heart appears to move Dull needle or mouse Replace needle, slightly tilt the imaging
proximally towards midline anatomical variability platform down on the mouse’s left side,
when attempting to puncture and increase the angle of the incoming
with the needle needle
15 Needle path is difficult to Learning curve for those Turn on the Needle Guide feature to
conceptualize or not as new to the technique overlay a trace estimate of the needle
expected path on the ultrasound image to aid in
positioning for insertion.
8+ (optional) Difficult for Ergonomic issue Set up portable monitor behind the
sonographer to view ultrasound integrated rail system to view images
images and use both hands for while researcher’s head and arms are
USGI forward facing. Vevo 3100 has a
display port connection for portable
monitor.
All (optional)Biocontainment Biohazard or Immuno- Integrated rail system and all
needed for hazardous material compromised risk accompanying items can be placed
or immuno-compromised within most biosafety cabinets and the
mouse work Vevo 3100 placed adjacent to
workspace
This procedure is accomplished most efficiently and with less likelihood for errors with at least 2 people. One person’s role is sonographer/injectionist and the second person’s role is anesthesiology induction and recovery as well as injectate and syringe prep. With this arrangement and the mice fur removal occurring a day before, a throughput of 2 minutes per mouse is possible.
Researchers have reported high reproducibility across subjects on the same and subsequent days as verified with whole body luminescence value. We have performed this procedure on 6 projects for a totaling near 500 injections with roughly 10 adverse outcomes related to the procedure and 96-99% of mice having tumors develop at desired sites.
When injecting cells, concentration ranges are about 5K – 1M in 100uL of PBS
1. P. Basse, P. Hokland, I. Heron, M. Hokland, Fate of Tumor Cells Injected Into Left Ventricle of Heart in BALB/c Mice: Role of Natural Killer Cells, JNCI: Journal of the National Cancer Institute, Volume 80, Issue 9, 6 July 1988, Pages 657–665, https://doi.org/10.1093/jnci/80.9.657
2. Zhou H, Zhao D. Ultrasound imaging-guided intracardiac injection to develop a mouse model of breast cancer brain metastases followed by longitudinal MRI. J Vis Exp. 2014 Mar 6;(85):51146. Doi: 10.3791/51146. PMID: 24637963; PMCID: PMC4143100.
Thanks to Keene Abbott of the Vander Heiden lab at the Koch Institute for expected results and feedback. We are grateful to Dr. Aurora Burds Connor for their invaluable technical and collegial support.
This work was supported in part by the Koch Institute Support (core) Grant P30-CA014051 from the National Cancer Institute.
Supplementary video 1 Placement of mouse on the mouse imaging platform in steps #7-8
Supplementary video 2 Dual synched video procedure step #10-16
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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