Massive Pontine Hemorrhage by Dual Injection of Autologous Blood

We provide a protocol to establish massive pontine hemorrhage in a rat. This model is easy to establish, repeatable, and highly successful. Distilled injection of autologous rat method could be used to make hemorrhage in any of the brain regions.

People who want to try these techniques should learn how to use a stereotaxic frame, and be familiar with pontine anatomy. 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 to drain it completely. Apply chlorhexidine and 75%alcohol surgical scrub to the whole tale 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 one milliliter syringe, then insert the scalp acupuncture into a lateral tail vein three centimeters from the tail tip and to take 150 microliters of blood.

Remove the scalp acupuncture from the one 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 one 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 nine millimeters below the surface of the brain, then restart injection at the same speed of one 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. Then use bone cement to cover the craniotomy hole. When the cement dries suture the wound with a four zero polyamide suture filament.

After sewing three or four stitches tie 2 one-one standard surgical knots. Observe the rat every 15 minutes until it fully recovers from anesthesia, then return it to the original cage with a heating pad underneath. Provide the rat free access to food and water.

A total of 25 animals were used for control 30 microliter, 60 microliter, and 100 microliter blood injections. Behavioral tests were conducted on days one, three, seven and 14 after surgery. Pontine hemorrhage caused neurological deficits like diminished corneal reflex and circling.

Injection of 100 microliters of blood also induced the myotonia. MRI Scanning was performed 24 hours after surgery. In the blood injection groups, Hemorrhage was detected as a hypointense rim with an ISO to slightly hyperintense core in the Basiler part of the ponds.

The volume of hemorrhage increased with the injection volume. The rats were sacrificed at 24 hours and 14 days after surgery. And two millimeter thick sections were made.

Hemorrhage was detected surrounding the injection site and distributing in the base of the ponds. There was a slight edema around the hemorrhage in the 100 microliter blood injection group. Some of the rats were sacrificed three days after surgery and paraffin sections were made for HE staining.

Results showed that in the blood injection groups inflammatory cells enriched in the para hemorrhage zone the hemoglobin remained contained with intact red blood cells. When attempting this protocol make sure to remember the injection position. This method can be used to induce a hemorrhage in any specific cerebral region.