Optimization of the Longa Middle Cerebral Artery Occlusion Method for Complete Reperfusion

Welcome to our video guides to transient middle cerebral artery occlusion in mice. My name is Rok Ister, and this experiment took place at Laboratory for Regenerative Neuroscience at the University of Zagreb School of Medicine.Introduction. Transient middle cerebral artery method, or tMCAO for short, is the most commonly used animal model of ischemic stroke.

It's predominantly done in rodents, such as rats and mice. Two main operative methods, common carotid artery and external carotid artery method, differ mainly by the arteriotomy site for filament insertion. In this video, we'll be presenting our modifications, and some key points to ensure complete reperfusion using the external carotid artery method.

Starting from recommended tools, you'll need two pieces of Dumont type 5 tweezers, Dumont type 7 tweezers, and Dumont type N0 self-closing tweezers, a mini-colibri retractor, and straight micro spring scissors for arteriotomy. For hemostasis, we'll be using two pieces of vascular micro clamps together, with a respective applicator. Surgical scissors, needle holder, and surgical forceps, we'll use for opening and closing of the surgical wound.

For materials, we'll use 5/0 monofilament suture for surgical wound, and 6/0 braided silk suture for artery ligatures, surgical tape for securing of the animal, eye ointment gel for animals'eye protection, and lidocaine gel for additional local anesthesia. Lastly, for draining of excess blood, we'll use pointed pieces of a paper towel. A stereo operating microscope is crucial for a microsurgical procedure like this one.

Anesthetized rodents are prone to hypothermia, so we'll be using a temperature monitor. On the operating side for the animal, you'll need an easy-to-clean, heated surface, together with anesthesia gas supply tube, and a way to reliably monitor animal's temperature during the procedure. On the table to the left, there are some more tools and materials we'll be using.

Measuring the duration of certain parts of the procedure is very important for reproducibility reasons, as well as data analysis later on. Betadine skin disinfectant and loaded syringes for quick and easy use. Animal hair clipper for shaving of the animal's fur.

A weighing scale, important for correct dosing of anesthesia. And lastly, an animal gas anesthesia system capable of delivering nitrogen-oxygen gas mixture in ratio 2:1, together with a knockout box for anesthesia inducement. All animal handling and procedures presented in this video were approved by the Ethics Licensing Committee of the University of Zagreb School of Medicine, and done by trained and licensed personnel.Protocol.

Preparation of the animal and the surgical site. Position the animal on the heated operating table, and bring its nose into the anesthesia mask. Apply eye ointments to animal's eyes to protect them from the hydration and use of fluorine gas.

Turn the animal on its back, and extend it neck by placing a small pillow, made from surgical tape, beneath the animal's neck. Secure the animal's limbs in place using surgical tape. Take care not to extend the forelimbs too much, not to inadvertently cause shoulder joint dislocation.

Lubricate the rectal probe using petroleum white jelly, and insert it into the rectum for continuous body temperature measurement. Apply a preoperative injection of saline and buprenorphine, intraperitoneally, to keep the animal well-hydrated and in no pain during the procedure. Shape the animal's fur in the neck region using the cordless animal clipper.

Collect all shaven fur using pieces of surgical tape. To make the area completely free of fur, additionally shape the region with the razor. Place a clean surgical drape over the operating table.

Apply a drop of Betadine to the animal's shaven skin. Use a cotton swab to rub the disinfectant into the skin in a circular fashion from the inside out. Afterwards do the same with an ethanol-soaked cotton swab.

Repeat this step three times with a fresh pair of sterile cotton swabs for each repetition. Apply a dab of lidocaine gel to the disinfected region of the future incision site for local wound analgesia. Ischemia induction surgery.

Making an initial incision on the disinfected skin using a scalpel, keep the number of skin incision strokes at a minimum to make the surgical wound heal easier. Using type 7 and 5 forceps, tear away the superficial fascia, and detach the salivary glands from the underlying tissue. Place the wire retractor in its initial position while making sure the salivary glands don't get in the way of subsequent steps.

Remove the deep neck fascia using type 7 forceps, and detach sternocleidomastoid muscles from the carotid region to reposition the retractor. Reposition the retractor to reach under the sternocleidomastoid muscles to enable access to the carotid region. Resect the omohyoid muscle to allow for a clear visual and easier approach to carotid region.

Pinch the carotid fascia on the lateral side of the carotid triad, and gently pull it laterally to visually identify the artery, nerve, vein, and all the surrounding blood vessel branches. Completely detach the lower part of CCA from underlying tissue and fascia using curved type 7 forceps, making sure it is ready for clamping in future steps. With the CCA carefully prepared together with its two respective branches, raise the ECA using curved type 7 forceps, and clamp it with type N0 self-closing forceps With the ECA clamped, and held securely with type N0 tweezers, introduce two braided silk threads behind the ECA.

Tie down the cranial thread completely, as it is permanent, and clip the excess thread away using scissors. Tie the caudal thread into a loose securing knot. Using vascular micro clips, clamp the CCA and the ICA shut to prevent any bleeding after the arteriotomy.

Using micro spring scissors, make a small arteriotomy right beneath the ECA clamping site. With an angle matching that of the ECA, insert the filament into the arteriotomy site in a proximal direction to the CCA, passing through the loose securing knot at the branching side of the ECA, and sticking into the lumen of the CCA with the filament. Carefully open the ICA micro clip, and remove it.

With the filament partially inserted into ECA, and secured down, make a complete arteriotomy, thereby setting the ECA stump loose with the MCAO filament inside. At this point, release the type N0 self-closing forceps, and remove it. With one set of type 5 tweezers, firmly pinch the ECA stump, and raise it slightly.

While holding onto the partially inserted filament with another forceps, lower and gently pull the ECA stump to orient the inner end of the filament towards the ICA. Never pinch the silicon part of the filament. Slowly advance the MCAO filament through the Circle of Willis until you feel a sudden increase in resistance.

At this point, it's important to observe the remaining length of the filament. In an adult mouse, the filament should proceed effortlessly at least seven millimeters from the ICA branching point. Tighten the securing knot to ensure that the filament doesn't get displaced during the ischemia period.

Remove the CCA clip, and take note of the time, this marks the beginning of the ischemia onset. Remove the wire retractor, and bring the edges of the surgical wound closer to each other. Let the surgical site settle for a couple of seconds for the tissue to return to its anatomical position.

Close the surgical wound using tape wound closures to facilitate faster reopening of the wound after the ischemia period. Ischemia period. Validate the success of the surgery using MRI, or some other quantitative perfusion measuring method.

Filament withdrawal surgery. Remove the tape wound closers, and reopen the surgical wound with the wire retractor. With type N0 forceps, pinch and pull the edge of the ECA stump ventrally to put the tension on the ECA stump.

Clamp the CCA again with a macrovascular clip. Slowly withdraw the MCAO filament until the point where the silicon part of the filament starts to stick out of the ECA stump. Tighten down the securing knot slightly to prepare for complete filament withdrawal.

When close to the silicon end of the filament, tighten down the securing knot in such a way that it pushes out the MCAO filament and closes the ECA stump in a single maneuver. Tighten down the knot completely after the filament slips out. Open and remove the pinching forceps together with the CCA clip.

Suture down the surgical wound, starting from the periphery, thereby completely closing the wound without any underlying tissue visible. The number of sutures required depends on the size of the surgical wound. Clean and disinfect the surgical site using rubbing alcohol wipes.

Representative results. After a successful procedure, results from MRI scans should look similar to this. Intraoperatively, during the ischemia period, you should be able to observe a clear-cut region with ischemia on perfusion weighted images.

Apparent diffusion coefficient lesion should roughly cover the same area. Postoperatively, after the withdrawal of the filament, you should ideally observe the hyper-perfused lesion core on the perfusion weighted images. With the exception of the ischemic core, you should see recovery and slight overshoot in the ADC values in the lesion.

On the second day after the procedure, the lesion should be reperfused completely. ADC values should drop again in the ischemic lesion core due to brain edema, which should also be visible on the T2 image. In case of a filament-induced hemorrhage, no improvement is observed in perfusion or ADC maps postoperatively.

Also, a clear sign of hemorrhage can be observed on the T2 images, both intraoperatively and postoperatively.Conclusion. Thanks to recent anatomical studies of the mouse brain vasculature, we're able to adjust and modify our MCAO model based on the new knowledge. Posterior communicating arteries, painted green, are bilaterally patent in only 10%of most commonly used mouse strains.

For that reason, they can be relied upon to provide blood flow to the branches proximal from MCA. Thereby, CCA needs to be kept patent during the ischemia period, and the silicon part of the filament should not exceed three millimeters in length. We hope to have been informative and concise in this video.

Thank you for watching.