Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

The overall goal of this procedure is to model spontaneous intra cerebral hemorrhage in the mouse to study the enduring pathology and neurobehavioral outcomes in vivo. This is accomplished by first preparing the mouse for surgery. The ventral tail and scalp are shaved and cleaned.

Next, using the landmarks of the skull, a precisely placed bur hole is made through the skull. Thirdly, blood is withdrawn from the tail artery and is injected stereotactically into the basal ganglia. The injection needle is then carefully removed and the incision is closed.

Ultimately, results can be obtained that show localized inflammation, cell death, and a neurological deficit through immunohistochemistry, flow cytometry and functional testing among other methods. In general, investigators new to the procedure will struggle with blood clotting because the small amount of blood injected will clot in the system in the time it takes to do the injection. After inducing anesthesia with isof fluorine, shave the scalp and wipe down the skin with Betadine three times.

Next, coat the animal's eyes with sterile petroleum jelly. After the scalp has dried intraperitoneal, inject a postoperative analgesia using a sterile scalpel. Make a generous one centimeter midline sagittal incision into the scalp.

The incision should expose all of the skull landmarks. Now prepare the tail beginning one centimeter from the base of the tail shave one centimeter along the ventral surface. Wipe down the shaved region with Betadine three times, the prepared mouse can now be firmly secured in a stereotaxic frame.

The surface of the skull should be parallel with the base of the frame. There should also be excellent exposure of the brima and at least three millimeters to the right of brima. Keep the mouse anesthetized and use a thermistor controlled heating pad to maintain its temperature at 37 degrees Celsius.

Monitor the temperature using a rectal thermometer. Now proceed with the intrus orreal hemorrhage surgery. Begin by attaching a sterile 27 gauge needle to the one CC syringe attached to a stereotaxic arm.

On the frame. Position the needle so it is 2.5 millimeters, positively lateral to the BMA and at the surface of the skull. Now rotate the syringe to start a bur hole on the surface of the skull.

Apply gentle downward force to study the frame. Take care not to completely perforate the skull. Once the hole is started, remove the needle from the arm and complete the process manually to minimize the risk of pushing the needle into brain parenchyma or until the sensation of perforating the inner table of the skull is felt.

Then stop. Next, take a blood sample from the tail by placing paraffin wax paper under the tail and making a transverse incision along the shaved surface. Collect two to three large drops of arterial blood on the wax paper.

Then quickly stop the bleeding by applying pressure with sterile gauze. Draw up 17 microliters of the blood sample into a Hamilton syringe. Attach the syringe to a pump and secure it to the stereotaxic arm.

On the frame. Angle the stereotaxic arm to five degrees medially relative to the vertical axis. Position the arm, so the tip of the needle is over the bur hole and lower the needle.

3.5 millimeters into the brain. Wait two minutes, then withdraw the needle. Half a millimeter so that the tip is three millimeters deep.

Wait another five minutes to allow the brain to re-expand around the needle, thus minimizing risk of blood reflux up the needle insertion track during injection After five minutes, inject 7.5 microliters of the blood at a rate of one microliter per minute. After injection, wait another five minutes to allow for initial blood clotting and tissue shifts. This weight essentially minimizes the inevitable rise in intracranial pressure.

Now inject another 7.5 microliters of blood at the same rate of one microliter per minute. Leave the needle in place for 25 minutes to allow the blood to clot. Otherwise, blood will reflux up the needle insertion site when the needle is withdrawn.

After the blood has had time to clot, slowly withdraw the needle. If you hope to reuse the needle immediately rinse it out with hot water or any blood in, it will clot and it will be ruined. Now remove the mouse from the frame.

Close the tail and scalp incisions with veterinary surgical glue. Turn off the anesthesia and place some wet food pellets on the bottom of the cage to give the mouse easier access to food. Allow the animal to awaken in a warmed chamber with free access to food and water.

When the mouse is ready, return it to its home cage. 50 mice underwent the ICH surgery and had their brain sectioned postoperatively. Overall, the success rate was 80%and there was no mortality in non sacrificed animals.

Cylinder testing demonstrated that after right basal ganglia, ICH mice had a left hemiparesis. Note that the animal only places its right fall limb on the wall. Comparing five mice that underwent the surgery with four shams.

The difference was easily scored. The graft shows the laterality of the initial placement of the four limbs on the wall of the cylinder. This behavior was tallied over multiple trials and three days of testing on each mouse.

After watching this video, I hope you have a good idea of how to model spontaneous intracerebral hemorrhage using autologous blood injection. This technique eliminates all anticoagulation expandable tubing and dead space from the system. In order to reproducibly inject 15 microliters of blood into the striatum, the investigators can then study the pathologies associated with mechanical tissue disruption, inflammation, coagulation, and iron induced injury.