The overall goal of this procedure is to disrupt the blood-brain barrier. This is accomplished by first placing the animal on IV anesthesia with an endotracheal airway. Next, the bifurcation of the common carotid artery is exposed.
Then a catheter is inserted into the external carotid artery. Finally, mannitol is administered. Ultimately, results can be obtained that show blood-brain barrier disruption through extravasation of Evan's Blue Dye.
The main advantage of this technique over existing methods of intra arterial therapy is it allows drugs to enter the brain, which do not ordinarily cross the blood-brain barrier under physiological conditions. This has implications in the field of neuroscience because it allows us to test new therapies which have poor ability to cross the blood-brain barrier. Implications of this technique extend to neuro-oncology cause many chemotherapeutic agents do not cross the blood-brain barrier.
Well, Though this method provide us insight over new oncology, it can also provide us information on other neurological dis disorders treated by drugs with poor blood brain barrier permeability. We hope that the visual demonstration of this method will allow researchers and familiar with animal surgery to use this technique in their labs. To begin, prepare a solution of 25%mannitol and verify that there are no crystals.
If crystals are present, dissolve them by placing the vial in an 80 degree Celsius water bath and agitate. Periodically record the mass of the animal and prepare two 12 cc syringes of propofol to be delivered at a rate of 0.1 milliliters per minute. After anesthetizing the animal in an induction chamber, place it on a disposable towel and shave its neck and inner thighs.
Place the animal back in the induction chamber for three minutes. To prepare for intubation to intubate the rat, begin by preparing an endotracheal tube with an IV catheter. The tube must be large enough to allow adequate airflow, but small enough to fit into the trachea without causing damage.
To make a stylus, pull the needle out of the plastic catheter and carefully break the needle. Approximately one centimeter from its sharp tip, put the needle back into the catheter. Next, hang the rat by its teeth on an angled surface, shine a light directly against the animal's neck lined up between its ears.
Next, insert a small spatula into the oropharynx. Approximately three centimeters. Lift upward while gently pulling the tongue out of the mouth.
Visualize the opening of the trachea as a small hole that opens and closes. With each breath, gently insert the catheter into the hole, making sure not to force it into the cartilage of the sensitive trachea. Set the ventilator to one liter per minute and 60 BPM and then isof fluorine to 2%Then connect the catheter to the ventilator and tape the tube to the operating surface.
Verify that the animal is anesthetized by performing a toe pinch. Secure the route by taping all four limbs in the tail to the operating surface. Scrub the shaved neck and groin sites with chlorhexidine soap three times.
Then scrub with chlorhexidine. Rinse three times. Attach a syringe of propofol to an IV extension set and mount the syringe on the infusion pump.
Next, using a scalpel cut through the skin of the shaved thigh without cutting the muscle. Then use forceps to open the incision and to locate the femoral vein and pull away the tissue over the artery to expose the vessel. Insert a 26 G mono eject veterinary IV catheter into the vein.
Once the needle punctures the wall of the vein, it'll become easier to push. Retract the needle and push the catheter into the vessel three to five minutes. After beginning the propofol infusion, switch the anesthesia gas to a mixture of 50%O2 and 50%N two O and shut off the isof fluorine.
To expose the bifurcation of the common carotid artery, begin by using a scalpel blade to make a midline incision at the neck. Cut through the superficial facia along the midline to reach the sternal hyoid muscle over the trachea. Move the tissue away laterally to visualize a group of three muscles, the sternal hyoid, the di gastric, and the sternal mastoid that formed the rat carotid triangle with forceps or scissors.
Separate the fascia between the muscles of the carotid triangle to expose the carotid artery. Use bipolar cautery to divide minor vessels along with gauze and cotton swabs To control bleeding, use retractors to hold the muscles apart with forceps. Move the tissue off of the carotid and find the bifurcation.
Expose the occipital artery just distal to the bifurcation. Cauterize it between forceps and use an electrocautery to separate it with forceps. Fully expose the carotid bifurcation and as much of the external carotid artery or ECA as possible.
Expose the superior thyroid artery branching off the ECA distal to the occipital artery. Cauterize it between forceps and separate it to catheterize the ECA. Place two lengths of four oh silk under it.
Use a hemostat and grab one length of suture and retract the ECA back coly with the other suture. Tie it with the surgeon's knot as far cranial as possible, distal to the bifurcation and proximal to the permanent ligature. Loosely tie another surgeon's knot to relax the artery.
Irrigate it with 1%lidocaine, fill the catheter with heparinized saline. Next, place a temporary vessel clip at an angle as close as possible to the bifurcation. Then slide the loose knot toward the clip using micro scissors.
Make a small cut in the ECA just proximal to the ligature. If the clip and ligature are secure, there should be no bleeding using forceps. To control the ECA walls, feed the beveled end of the catheter into the vessel with the loose surgeon's knot.
Secure the catheter. Tie the artery around the catheter, then remove the clip and use a cotton swab to clean the area. Advance the catheter about one millimeter distal to the bifurcation.
Use forceps to hold the artery proximal to the knot to ensure the vessel is immobilized. To administer mannitol. Fill an eight inch IV extension set and a six milliliter syringe with mannitol without creating any air bubbles.
Store it at 37 degrees Celsius to visualize the blood-brain barrier disruption, or BBBD administer two milligrams per kilogram of 2%Evans Blue through a new IV site on the leg contralateral to the propofol infusion. Attach the extension set to a five micron filter and fill it with minol to remove air bubbles. Then attach it to the stop cock without creating bubbles.
Administer mannitol at 0.09 milliliters per second For 25 seconds, watch for the mannitol to reach equilibrium with the flow in the common carotid at a point just proximal to the internal carotid artery, it should appear to pulse with the rat's heart rate. Ensure that the mannitol does not flow toward the heart, which indicates the flow has overcome the rat's blood pressure and can cause serious complications. Disruption of the blood-brain barrier on one side of the brain allows Evan's blue to enter and stain the disrupted hemisphere blue, while the non-disruptive hemisphere remains unchanged.
Shown here is an example of a brain without blood-brain barrier disruption or BBBD, and here is an example of a brains stained blue after osmotic BBBD, notice that the right hemisphere is stained and that some blue is entered the left hemisphere near the medial longitudinal fissure as a result of mixing of blood flow at the circle of Willis. This technique paves the way for researchers to explore therapies which do not cross the blood-brain barrier in a rat model. After watching this video, you should be able to disrupt the blurred brain barrier by catheterizing the carotid artery and infusing a manitol solution directly into the brain.
