Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

The overall goal of this procedure is to study the cerebral vascular anatomy of mice in a reproducible manner. First, the cerebral vessels are stained with colored latex for later comparison to the carbon black ink staining. Next, the carbon black inks are mixed and the animals are perfused with P-B-S-P-F-A and the carbon black ink mixture through the left ventricle.

During the final step, the brain is collected and photos are taken for comparison and analysis of the stained cerebral vessels. Ultimately, image J software is used to analyze the anastomotic points between the middle and anterior cerebral arteries. The implications of this technique extend towards the therapy of cerebrovascular diseases, particularly stroke as it can be used to analyze vascular density and angiogenesis.

Now, this method can provide insight into the vascular system of the brain, can also be applied to other systems, such as for the visualization of micro vessels in the heart, liver, or kidney. After combining the latex with carbon black ink in an EP tube warm the mixture at 37 degrees Celsius in a water bath. Use a 23 to 25 gauge needle to collect the warmed latex in a two milliliter syringe.

Then dissolve 12.5 milligrams of papine hydrochloride powder into one milliliter of sterile normal saline, and after vortexing collect the resulting solution in an insulin syringe. Next, break the needle off of another sterile insulin syringe and place the separated needle on the end of a 20 centimeter long PE 10 tube. Then attach the PE 10 tube to the needle of the insulin syringe containing the Papine hydrochloride solution.

Now fill two more insulin syringes with one milliliter of saline, and then take all the syringes along with the sterilized dissecting instruments to the operating stage prior to anesthetization. Weigh the mouse, then position the continuously anesthetized mouse on its back. Fix the limbs and cut the skin over the left femoral vein.

Now locate the femoral vein and insert the tip of the needle attached to the PE 10 tube. Then slowly inject the Papine hydrochloride solution at a rate of about 100 microliters per minute and a dose of 50 milligrams per kilogram body weight. Following the injection, cut open the abdominal cavity and then pierce the diaphragm.

Then cut the ribs to expose the heart and clip the descending thoracic aorta with artery forceps. Next, make a sharp cut over the right atrium to drain the venous blood. Then inject two milliliters of saline into the left ventricle.

The presence of air in the Profil solutions or in the combined ink mixture can result in unstained vascular areas of the brain. To ensure success, please check very carefully that all your syringes are completely free of air bubbles. Now, gently administer the contents of the prefilled syringes, one after the other for manual injection of the colored latex over a period of 18 to 20 seconds, leave the animal for five minutes.

Then after decapitating the animal, remove the skull bone and meninges taking care not to injure the cortex or vessel architecture, and then carefully harvest the whole brain. Place the brain in a 60 millimeter cell culture dish containing six to eight milliliters of 4%PFA, and then place a measuring ruler under the dish using a camera attached to the operating microscope. Take photos at a 25 x magnification of the dorsal and the ventral surfaces of the brain at a 90 degree angle between the microscope objective and the Petri dish.

In order to quantify the original distance, begin the carbon black ink staining procedure by mixing two 100 microliter aliquots of herts stemple farba ink or CB one with two 900 microliter aliquots of pelican scribal schwartz ink or CB two in two separate 1.5 milliliter EP tubes. Collect the ink mixtures in two separate one milliliter insulin syringes. Then cap the syringes and place them into a 37 degree Celsius water bath, along with two other insulin syringes containing one milliliter of saline each.

After treating the mouse as just demonstrated, use slight pressure to inject both carbon black ink solutions into the left ventricle over 18 to 20 seconds per milliliter. After removing the brain and taking pictures as just demonstrated, place the brain in 4%PFA overnight followed by incubation in gradually increasing concentrations of sucrose until the floating brain totally submerges. Begin this step by inducing transient focal cerebral ischemia.

According to a standard protocol of intraluminal occlusion of the middle cerebral artery, at the end of the reperfusion period, stain the vasculature with the CB one and CB two inks as just demonstrated, and then stain the whole brain with 2%TTC solution at 37 degrees Celsius for five to 10 minutes. For identification of the infarct volume, the infarct area will remain white, whereas the non-ischemic area will turn red. Collect images as just demonstrated to analyze the gross anatomy of the cerebral vessels begin by opening an image of the stained surface of the brain.

With image J software brains perfused with colored latex may show variable degrees of vessel staining located on the ventral surface. While CB one plus CB two perfusion will stain all the vessels on both ventral and dorsal surfaces. Set the scale in millimeters, mark the anastomotic points between the anterior cerebral artery or a CA and the middle cerebral artery or MCA to create an imaginary line connecting all these anastomotic points.

Now, measure the distance between the anastomotic line and the midline at four, and then six millimeters coddle to the frontal pole of the brain. After analyzing three to four images per animal, calculate the mean value. Compare the values among the different groups of animals to identify the variation within the cerebrovascular anatomy.

In C 57 black six J animals, the anastomotic line between the a CA and the MCA can be traced closer to the midline at four millimeters than at six millimeters coddly from the frontal pole while APO lipoprotein e knockout mice present no significant difference in this respect. By contrast, the anastomotic line in SV 1 29 mice lies further and more parallel to the midline, both at four millimeters and six millimeters, coddly from the frontal pole for analysis of the vascular anatomy. Under ischemic conditions, identify the infarction border as the red and white colored tissue margin, which represents the metabolically active tissue, and the necrotic tissue respectively measure the distance of the infarction border from the midline at four millimeters and at the six millimeter coddle from the frontal pole of the brain.

The protocol described here overcomes the technical limitations of conventional latex based visualization of rodent cerebral vasculature. As this first image shows following perfusion of colored latex, only the large vessels on the ventral surface are stained, leaving the entire dorsal surface unstained. Conversely, CB one plus CB two perfusion results in sufficient filling of both the small and large vessels in an equal manner.

The staining is stable for seven days without any change in quality. Using the Image J program, the distance of the anastomotic points between the A CA and the MCA from the midline can be analyzed in all the different groups of mice, which have been perfused with inks to assess the differences in the regional vascular anatomy. Although knocking out of a POE does not affect the anatomical structure of the cerebral vessels in C 57 black six J mice, a significant difference can be noted between the C 57 black six J and SV 1 29 strains.

In experimental rodent stroke models, TTC staining is widely used for visualization of the infarction volume. CB one plus CCB two staining is also applicable under ischemic conditions for observing structural changes in the vasculature and demonstrates a high stability when combined with TTC staining. Note, the concurrent visualization of the cerebral vessels and the infarct volume in the ischemic brains induced by different durations of cerebral ischemia and reperfusion time points.

Analysis of the anastomotic points between the a c and the MCA after 45 or 90 minutes of ischemia matched with either one day or five day reperfusion periods shows as expected that the mean difference between the midline and the anastomotic points showed no significant difference among the groups. Further, the mean distance was in the same range as non-ischemic animals. However, the distance from the midline to the infarction border differs significantly between the groups reflecting the increased infarction volume with higher degrees of ischemia reperfusion injury.

This technique has the potential to pave the way for researchers in the field of experimental stroke in order to explore the vascular territories in the mouse brain. After watching this video, you should have a good understanding of how to study the cerebrovascular, an anatomy in mice by perfusing the brain with lax or carbon black inks, and of how to analyze the resulting stained brain tissue with the help of Image J software.