In this video protocol, we are going to demonstrate utilizing a cranial window to visualize the middle cerebral artery during endothelin one induced middle cerebral artery occlusion. For this cranial window preparation, a temporal c craniectomy is performed in order to view proximal portions of the middle cerebral artery. Creation of a cranial window Is a method that allows direct visualization of structures on the cortical surface of the brain.
This technique can be performed in many locations overlying the rat cerebro, but is most easily carried out by creating a C craniectomy over the readily accessible frontal or parietal bones. Most frequently we've used this technique in combination with the endothelial one induced middle cerebral artery occlusion or ET one induced MCAO model of ischemic stroke. A cranial window procedure is performed to quantify the changes in vessel diameter that occur after injection of ET one into the brain parenchyma adjacent to the proximal middle cerebral artery or MCA.
Specifically, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull. The cerebral arteries can then be visualized either with the dura intact or with the dura inized and retracted. Most commonly, we leave the dura intact during visualization.
Since ET one induced MCAO involves delivery of a vasoconstricting peptide into the brain parenchyma, this bypasses the need to incise the dura directly over the visualized vessels for drug delivery. In this video, we will show how to create a cranial window to visualize cerebral arteries in a stepwise fashion, as well as how to avoid many of the common potential pitfalls pertaining To this method. After Placement of the guide cannula, as referenced, a cranial window can be created to directly visualize proximal portions of the middle cerebral artery during a stroke procedure, first, make an incision in the scalp as shown using scissors and retract the skin to reveal the temporalis muscle.
Bisect the temporalis muscle using electrocautery, and then retract To visualize the squamous portion of the temporal bone. Obtaining adequate exposure of the temporal bone is crucial to this procedure. Be careful to avoid the orbit while cauterizing.
To improve visualization, use suture to retract the bisected Temporalis muscle. Draw a three to four millimeter square on the squamous portion of the temporal bone coddle to the orbit and superior to the base of the zygomatic process As it reflects off of the temporal bone. Use a drill bit to cut out this portion of bone.
Be careful to avoid applying too much pressure as it is possible to drill into the brain. Be sure to perform frequent rinses with sterile saline to improve visualization of the surgical field and prevent overheating of the Skull. Starting at a loose corner, carefully remove the piece of temporal bone using fine rat tooth forceps, making sure not to tear any vessels associated With the dura.
Here you can visualize a branch of the middle cerebral artery at baseline prior to injection of endothelial.One. Within minutes of endothelial one injection, the middle Cerebral artery will begin to constrict. Eventually, the branches become difficult to visualize and the brain tissue will become pale.
After about 20 minutes, the effects of endothelial one will diminish and the vessels will start to dilate gradually. Returning to baseline diameter after about 45 minutes To measure vessel diameter At different time points before and after endothelial one injection, still frames from the video are captured at one minute intervals. Using VLC media player using VLC Media player, go to tools, preferences, and then change the setting to all under show settings.
Expand the video menu in the left side bar and expand output modules. Select scene filter type scene for the file name prefix. Then add a directory path.
Next, set the record ratio for still frame capture based on your video's frame rate as explained in the written protocol. Finally, click save open. The desired video and VLC player will automatically save still frames once every one minute to measure vessel diameter.
Using these images first, open the Image J software, then open the still frames that you will measure. Go to analyze, set measurements, and unselect all boxes. Select the straight line tool, zoom in as required.
Place a line perpendicular to the vessel path To measure vessel diameter. Go to analyze, Then click measure to obtain vessel diameter. Repeat this for all desired images.
Vessel diameter at each time point should be normalized to the baseline vessel diameter so that comparisons can be made using multiple rats. To do this, use the formula shown here. In summary, this cranial window preparation technique is very versatile as it can be altered to meet the needs of many experiments with minor modifications.
For example, we have successfully monitored cerebral blood flow in specific MCA branches using laser doppler flow imagery to focus directly on a cerebral artery visualized through a cranial window. In addition, a similar preparation with the dura in size can be used with topical administration of vasoactive compounds to create an in vivo vascular reactivity bath. Several factors should be taken into consideration when preparing a cranial window in order to decrease the failure rate for this technique.
Many of these factors are related to obtaining good visualization of cerebral arteries. For example, care must be taken when creating the C craniectomy so that the dura or blood vessels overlying it are not disrupted with the drill bed. This is best accomplished by frequent washers with sterile saline to clear debris and cool the skull.
We hope that this video will assist you in designing experiments that utilize a cranial window and help you to avoid the common pitfalls that can occur during this procedure.
