In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney.

The overall goal of the following experiment is to map the flow of blood in the embryonic kidney. First, an embryo is extracted from the uterus and prepared from an ultrasound guided intracardiac injection. The embryo is then injected with FSE labeled tomato lectin using ultrasound guided microinjection.

Once all the embryos are injected, the dam is sacrificed and the embryos are processed as hole mounts or sectioned for immunohistochemistry and evaluation of blood flow. The results show a dynamic pattern of blood flow throughout the developing kidney, suggesting a role for blood flow in the maturation of the various kidney compartments. So the main advantage of this technique over other techniques, such as resin cast, is the fact that you can actually visualize blood flow throughout embryonic development.

So demonstrating the technique will be Christopher Rhymer, A technician from my laboratory Begin with setting up the stage mount and probe. Next, set up the instruments and begin warming up a stock of pH 7.4 PBS to 37 degrees Celsius in a water bath. Now load the microinjection needle using a flexible 25 gauge needle.

Load it through the base with mineral oil, then attach needle to the arm mount, subsequently empty out the mineral oil and refill the needle with 2.5 microliters of TL solution. Make sure no air bubbles get trapped and then move the needle away from the stage. Now using isof fluorine, anesthetized a pregnant dam.

When she's unconscious, put her in a supine position over a temperature monitor and provide her continued isof fluorine via a nose cone. After confirming the dam's fully anesthetized state from a lack of reflex, taped down her limbs at 45 degree angles, then apply depleting cream to the lower abdomen and wipe it off. Using 70%ethanol, the skin must be completely cleaned off before the first incision.

When everything is ready, perform a laparotomy using fine forceps and scissors. Use a straight incision from above the vagina towards the ribs. Be careful to avoid damaging major vessels or visceral organs.

Next, identify the linear elbow in the subcutaneous membrane and cut along it parallel to the initial incision. Thus exposing the uterine S.Manipulate the embryos using six inch cotton tipped applicators. Begin by gently pushing the first embryo out of the incision opening.

Then after extraction of the first embryo, syl gently and slowly pull the remainder of the uterine horn through the opening and onto the mother's exterior. Avoid pulling intestine or other organs through the incision. At this stage, quantify the embryos and then gently place the left uterine horn back into the mother.

Start with the Z of the right uterine horn that is farthest from the vagina.Stop. When only two embryos remain exposed, then possession these two embryos along the incision line without cutting off the uterine circulation. Now between the limbs and bodies of these two embryos position clay blocks, the clay should be parallel with the laparotomy incision.

Then wet to fenestrated dish with warm PBS using forceps. Grip the dish. Then from the top of the dish, push a pair of closed forceps about five centimeters through the fenestration along the other side.

Open the forceps without tearing the mesh. Now by barely touching the SULs, try to slide them through the slit in the dish and set the dish onto the dam with the forceps. Manipulate the mesh to surround the embryos below them and at their sides.

Then retract the forceps. Next place a blue rubber containing wall in the dish. Avoid pinching or injuring the embryos once in place.

Make sure the clay blocks firmly balance the dish, the embryos, and the rubber containing wall. Lastly, submerge the embryos in warm PBS while checking for leaks in the fenestrated meshing. First, lower the stage using the Z adjustment.

Then position the needle until its tip is about half a centimeter from the ultrasound probe's. Tip next, raise the stage back to where it was with the ultrasound probe above the embryos, slightly submerged and just three to four millimeters from the tissue. Then adjust the position of the stage so that the probe detects the embryo's heartbeat.

On the ultrasound screen, locate the left ventricle preferably or the left atrium, and adjust the stages height and position so that the injection target is precisely at the center of the ultrasound screen. Mark this position next, using only one knob, move the embryo directly to the right until it is off screen. Then swing the microinjection needle back into position.

It should be half a centimeter from the ultrasound probe and add a right angle to it. Now manipulate the needle's position so that the tip is clearly aligned to the center marker. Then adjust the injection angle to put the tip in focus and in the correct plane.

When that is done, retract the needle using only the injection control knob. Now move the embryo directly left, bringing it back into position under the probe with the injection target centered at the right on the ultrasound screen. Now with the control needle, perform the injection slowly, puncture the embryo with the needle, bringing it gradually into the left ventricle.

Then inject the full load of TL solution. It looks like a shadow as it is released when the ejection is complete. Retract the needle.

Now, repeat the injection process on the second embryo following the second injection. Continue with the remaining embryos until ultimately after the final injection. The last two embryos are placed back into the dam's abdomen with the injections complete.

Switch the gas to the anesthesia chamber and put the mother into the chamber for 15 minutes. Then euthanize the dam by a cervical dislocation and harvest the embryos for analysis. The described method was used to gauge and analyze blood flow within the developing kidney.

Co labeling for endothelial marker pcam and TL distinguished tissues as perfused vasculature un perfused vasculature. An aberly perfused structure at E 11.5, perfused vessels encapsulating the developing kidney made a web-like pattern without actually penetrating into the organ. By E 13.5, the major vessels were perfused with the few of the smaller accessory vessels showing TL staining by E 15.5.

Major vessels were perfused. However, a large number of glomeruli could also be observed holding the TL stain, suggesting that significant filtering was occurring. A number of smaller caliber vessels also appeared perfused, although a significant portion of the outer nephrogenic zone appeared to be devoid of blood flow.

Lastly, however, by E 17.5, a majority of the kidney was perfused except for vasculature of the nephrogenic zone, which was predominantly devoid of perfusion. Thus indicating an absence of angiogenesis in the peripheral regions. Although this technique has a steep learning curve once mastered, it can be performed in about an hour per litter.

Good luck with your experiments.