Inducing Massive Pontine Hemorrhage in a Rat Model by Autologous Blood Injection

Begin with an anesthetized rat secured on a stereotaxic frame holding an anticoagulant-coated Hamilton syringe.

The rat’s skull is exposed, with a hole over the brain’s pontine region.

Insert an acupuncture assembly into the tail vein, withdraw blood, and transfer it to a tube.

Set the syringe to withdrawal mode and aspirate the blood.

Switch to infusion mode and insert the syringe through the hole, reaching the pontine region.

Inject a small volume of the autologous blood. Hold the syringe to prevent backflow. The injected blood clots over time.

After an interval, retract the syringe to the original position. Inject the remaining blood into the same region, holding for another interval before retracting.

The dual-injection method prevents blood from spreading to the surrounding area.

The infused blood enlarges the clot, exerting localized pressure on the surrounding vessels, causing them to rupture.

This results in significant blood loss and induces a massive pontine hemorrhage.

Begin by placing a surgical drape over the mouse. Make an incision with a scalpel along the marked midline. Use a cotton swab to remove any potential blood. Then place a piece of forceps on each side of the scalp flap to expose the skull. Dip a cotton swab in 0.9% saline and gently remove the connective tissues from the skull bone.

Use a marker pen to mark the central point of the bregma as the origin point. Perform a craniotomy using a micro drill. Proceeding carefully. When finished, remove the micro drill from the stereotaxic frame. Put a 100-microliter Hamilton syringe into the stereotaxic holder.

Turn on the injection pump switch. Click the Rapid Inhalation button, then aspirate heparin solution to 100 microliters, and drain it completely. Apply chlorhexidine and 75% alcohol surgical scrub to the whole tail from root to tip at least three times to disinfect the skin, soften the horniness, and dilate the tail vein, to increase the success rate of injection.

Attach a scalp acupuncture to a 1-milliliter syringe, then insert the scalp acupuncture into a lateral tail vein 3 centimeters from the tail tip, and take 150 microliters of blood. Remove the scalp acupuncture from the 1-milliliter syringe and transfer the blood into a tube, working quickly to prevent blood clotting. Change surgical gloves.

Select the withdraw mode. Set the volume to 100 microliters, and set the speed to 200 microliters per minute. Click the Run button, and aspirate 100 microliters of blood into the Hamilton syringe. Switch to the infuse mode and set the speed to 1 microliter per minute.

Advance the syringe until the tip reaches 9.2 millimeters below the surface of the brain, and inject the first 10 microliters of blood. Click on the Run button. Stop the injection, and leave the syringe in position for 20 minutes to prevent blood from flowing into the subarachnoid space.

Retract the syringe until the tip arrives at 9 millimeters below the surface of the brain. Then restart injection at the same speed of 1 microliter per minute until the residual blood has been injected completely. Leave the syringe in position for 10 minutes to avoid blood backflow. Remove the syringe from the brain slowly.