Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

The overall goal of this procedure is to permanently occlude a major rodent cerebral vessel. This is accomplished by first performing a two by two millimeter craniotomy above the vessel. Next, a suture is passed below and around the target vessel.

Then two ligatures are secured around the blood vessel, and finally, the vessel is transected between the suture knots. Ultimately, results can be obtained that show a blood flow reduction of greater than or equal to 75%as assessed by either laser speckle imaging or optical coherence tomography. The main advantage of this technique over existing methods, such as the monofilament occlusion method, is that you can get consistent regions of infarct in the rodent cortex, limited variability in infarct size and limited subject mortality.

The implications of this technique extend towards the diagnosis and treatment for stroke because new therapies can be evaluated during the acute onset and throughout recovery within the same subjects. To prepare for surgery, use a glass bead sterilizer to sterilize the following instruments. A dental drill with two bit and three bit drills, two 30 gauge hypodermic needles, serrated tweezers, two fine tip tweezers, a half curve reverse cutting, suture needle wire cutters, suture thread, and micro scissors.

After anesthetizing a Sprague dolly rat, according to a method approved by your institution's animal care and use committee, insert a rectal probe to measure body temperature and place the rat on a self-regulating thermal blanket. Next, place a 26 gauge needle into a dental drill. To access the middle cerebral artery or MCA, use hemostats to temporarily reflect the temporalis muscle away from the skull surface.

After locating the MCA, use the drill to create a small surgical window about three millimeters anterior and one millimeter lateral to the foramen val or the mandibular nerve. Close to the arch rostrum to estimate where the M c's cortical branch is located, follow it to the rostral ventral corner of the imaging window. Next, create a thin skull region, slightly rostral and ventral to the imaging window where the M1 segment of the MCA should be leaving an almost two millimeter gap between the imaging window and the surgical window.

Using a size three bit drill, thin the skull above the estimated M1 segment location. When the skull becomes somewhat transparent, switch to the more delicate size two bit drill, and thin the skull. Until it is completely transparent, the thickness of the skull should be similar to plastic wrap.

Next, using serrated tweezers, bend the tip of a 30 gauge needle and use the needle to carefully puncture the skull in an area not directly above an artery. Then use the hole as an entry point for tweezers. To carefully remove the thinned surgical window using a new 30 gauge needle, carefully remove the dura, which will allow the MCA to become more prominent as a result of reduced pressure.

Then locate the stem of the MCA or M1 segment. MCA will be distinct from the veins that often accompany it as the brighter red vessel. Confirm that you have exposed MCA just before cortical branching of the artery as shown here.

Assess the location of M1 and confirm that the window has two to three millimeters on either side of the length of the M1 segment. To occlude the middle cerebral artery, use wire cutters to trim a half curve reverse cutting suture needle to about three to five millimeters. Thread the trimmed suture needle ensuring that both ends of the suture are equal in length under a surgical scope.

And using the serrated tweezers, slip the suture needle under M1 about 0.5 to one millimeter away from the MCA and as shallow as possible to minimize damage to the cortex while not straining the MCA while continuing to guide and feed the needle under the MCA with the serrated tweezers. Use a fine tip tweezers to pull the needle from the opposite side. Once the needle has completely passed under the MCA, continue to pull the suture halfway through while pressing down on it.

To minimize strain on the artery, cut the thread close to the suture needle, resulting in two independent threads, roughly one millimeter apart with both fine tip tweezers. Tie two separate knots around the MCA, keeping them about one millimeter apart after the knots have been pulled tight. Use micro scissors to transect M1 between the two knots.

Once the MCA is successfully transected, keep the animals sedated, warm, and in a quiet environment for a period of five hours to ensure that stimulation induced protection from stroke does not occur. After confirming successful occlusion through laser speckle imaging or another imaging technique, the animal is allowed to recover from anesthesia and administered antibiotics and analgesia. Stroke damage will develop over the course of 24 to 48 hours.

Successful occlusion of a vessel can be confirmed using blood flow imaging techniques such as laser speckle imaging or LSI shown here are representative LSI. Images of a segment of a cortical branch of an MCA before and after MCA occlusion blood flow in the major cortical branches of MCA should drop to about 25%of baseline or less following occlusion depending on the level of noise in the recording system and sensitivity of the technique. When the described occlusion technique is applied to the MCA at the M1 segment blocking all cortical MCA branches and preventing sensory stimulation, the result following a permanent occlusion and five hours without stimulation is a cortical infarct of 28.4 plus or minus 2.4 cubic millimeters.

Once mastered, this technique can be performed in 20 minutes or less. Following this procedure, a number of other techniques may be employed such as functional imaging, laser speckle imaging, and neuronal recording in order to ask questions such as, what is the functional and structural status of the cortex post occlusion.