Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model

This model of subarachnoid hemorrhage helps us better understand the pathophysiological mechanisms and the consequences. This will hopefully result in generating therapies, reducing the burden of disease in afflicted patients. The main advantage of this technique is its highly reproducible, as a fixed amount of blood is injected.

Furthermore, it resembles a human subarachnoid hemorrhage in key parameters. To make the ICP probe, burn one end of a 20 millimeter piece of polythene tubing, making a circular plate and keeping the lumen open. Then slip a one millimeter silicone tubing ring over the polythene tubing before connecting a 10 millimeter piece of silicone tubing to the end of the polythene tubing.

Next, anesthetize, intubate, and catheterize a Sprague Dawley rat before placing it in a stereotaxic frame. Following administration of local anesthesia, make an eight millimeter skin incision caudally from the needle puncture in the midline. Under a dissecting microscope, dissect all the muscles bluntly in layers to identify the atlantooccipital membrane.

Then use the arm retractor to restrain the neck musculature, placing the retractor caudally if needed. After checking that the sterile ICP probe is connected to the ICP transducer, flush the ICP probe with saline, making sure that there are no air bubbles in the probe. Using a 23 gauge needle, incise the atlantooccipital membrane, making a hole to gently coax the ICP probe through the membrane.

Pull the probe lightly and ensure that it shows a pulsating curve ranging between zero to five millimeters of mercury. If not, remove the probe, recheck the connection to the transducer, and confirm the flow through the lumen. In the area where the atlantooccipital membrane wraps around the probe, apply two drops of tissue glue.

Then move the one millimeter silicone tubing forward toward the membrane, before applying additional glue to minimize the risk of ICP probe displacement. Once the ICP probe is glued, remove the retractor. Then, using a non-resorbable monofilament 4-0 suture, place one horizontal mattress suture at the caudal end of the incision and one simple interrupted suture at the cephalic end.

To place the laser-doppler probe, make a 15 millimeter incision caudally in the midline, starting just anterior to the eyes. After removing the connective tissue and the muscles with forceps, use the end of a sterile cotton swab as a rougine to identify the bregma and the coronal sutures. After placing the armed retractor, place a 25 gauge spinal needle in the stereotaxic frame exactly on the bregma and note the position.

Next, remove the needle from the bregma. Move the frame anteriorly by 6.5 millimeters. Then replace the needle in the midline to mark the side of drilling.

Drill until the dura mater is identified below the bone. Using straight forceps, gently remove the bone fragments. Then fill the cavity with bone wax.

Next, three to four millimeters lateral to the right of the bregma and just anterior to the coronal suture for the laser-doppler, drill another hole. Taking care not to penetrate the dura mater. Look for the vessels where the laser-doppler can measure the blood flow.

Place the laser doppler and check the values. A minimum value of 100 flux units is required. If the values remain acceptable after removing the microscope, add one drop of glue to fix the probe.

Recheck to confirm whether the value is above 80 flux units. To induce subarachnoid hemorrhage, insert the needle gently through the skull in the midline between the hemispheres until resistance is felt due to the base of the skull. Retract the needle by one millimeter to ensure correct placement just anteriorly to the optic chiasm.

To ensure the most homogenous result when injecting the blood, turn the needle clockwise by 90 degrees such that the needle tip points to the right. Then remove the stiletto. After a 15 minute equilibration, adjust the level of anesthesia to obtain a mean arterial blood pressure in the range 80 to 100 millimeters of mercury.

Then perform a blood gas analysis to confirm that the pH, partial pressure of carbon dioxide and partial pressure of oxygen are within physiological parameters. Next, using a one milliliter syringe with a blunt 23 gauge needle, withdraw 500 microliters of blood from the tail catheter. To avoid injection of air, fill the dead space of the spinal needle chamber with blood.

After adjusting the volume of the blood in the syringe to 300 microliters, connect the syringe to the spinal needle. Then, grasping firmly around the syringe, inject the blood manually to surpass mean arterial pressure. Observe a steep rise in the intracerebral pressure and a concurrent steep fall in the cerebral blood flow.

30 minutes after the induction of subarachnoid hemorrhage, remove the needle and the laser-doppler probe. And fill the cavities with bone wax. Using non-resorbable monofilament 4-0 suture, close the incision with two horizontal mattress sutures.

To use the ICP probe for injections into the cisterna magna, remove the silicone tubing and insert a pin port adapter to the polythene tubing. To perform an intrathecal administration while the animal is awake, place a pin port injector on a precision syringe. Then administer the treatment through the pin port adapter.

If intrathecal administrations do not have to be performed, cut the simple interrupted suture. Then, using scissors, shorten the ICP probe as much as possible and glue the end to prevent cerebrospinal fluid leakage. Close the incision with a non-absorbable monofilament 4-0 suture.

In the evaluation of gross sensory motor function using the rotating pole test, rats with experimentally induced subarachnoid hemorrhage performed significantly worse 24 and 48 hours after the surgery compared to sham animals. Sensitivity to endothelin-1, an agonist for the endothelin-1 receptor family significantly increased two days after subarachnoid hemorrhage in the basilar and the middle cerebral arteries of the experimental rats compared to those of the sham controls. Both arteries of the experimental rats also exhibited a similar increase in sensitivity to 5-carboxamidotryptamine, an agonist for the 5-hydroxytryptamine receptor family.

Following the procedure, neurobehavioral tests as well as tissue analysis including immunohistochemistry, PCR, western blotting analyzer can be performed to analyze consequences and mechanisms.