This protocol is ideally suited for evaluating the ability of the cerebral microcirculation to regulate its own blood flow during blood pressure changes under normal and pathological conditions. LDF provides a relatively non-invasive method for the continuous monitoring of cerebral blood flow under a variety of conditions that's not possible with other methods. Laser Doppler flowmetry can be used for non-invasive blood flow evaluation.
For example, to assess the effects of a high salt diet on impaired blood flow regulation in healthy young women. LDF allows blood flow to be easily monitored in many different vascular beds of humans and animals, and can be used in a wide variety of studies. Demonstrating the procedure with Adrienne Allen will be Megan Stumpf, my research technologist and lab manager.
Before beginning the procedure, place the rat on a circulating water blanket maintained at 37 degrees Celsius and confirm a lack of response to pedal reflex in an anesthetized experimental rat. Apply ointment to the animals eyes and shave the top of the cranium, ventral neck area, and femoral triangles. Remove any lose hair from the exposed skin.
And disinfect the skin with rubbing alcohol. Move the rat to a heating pad with a circulating warm water pump. And use medical tape to temporarily secure the rat in the supine position.
Fill two PE 50 cannulas with one unit per mL of heparin in isotonic sodium chloride solution. And bevel the open end of the catheters with surgical scissors to facilitate insertion into the arteries. After carefully separating the femoral arteries from the surrounding tissue under a dissecting microscope, ligate the distal end of the isolated femoral artery segment and place two additional sutures around the middle and proximal ends of the artery without tightening the knots.
Insert a V-shaped wire fashioned from a paper clip under the artery to occlude the vessel until the cannula has been secured. Then use vannas scissors to make a small incision in the femoral artery near the distal ligation. Insert the beveled end of the cannula into the incision and advance the cannula into the femoral artery.
Remove the paper clip. Tighten the knot in the middle ligature over the cannula to secure the cannula in place. Release the tension on the lifting ligature, then tighten the proximal ligature and close the incision.
Immediately after the cannulas are in place, place the animal in a sternal position. And secure the head in a stereotaxic device taking care not to dislodge the catheters. Make an elliptical incision in the skin covering the cranium.
And use a cotton swab to remove any connective tissue, ensuring that the cranium is clean and dry. Place a small, rolled elongated piece of tissue paper around the incision on the scalp to stop any bleeding and under the dissecting microscope, use an appropriately sized drill with a 2.15mm drill bit to thin a 0.5 to 1 cm area of bone in the parietal area over the left or right somatosensory cortex, depending on the size of the rat. Thin the bone slowly and carefully to avoid penetrating the skull.
While performing this step, saline solution should be applied liberally to prevent the area from over heating. When the area has a pink appearance and/or blood vessels can be visualized, cover the thinned bone with mineral oil. And use a micromanipulator to position the laser doppler probe over the exposed cerebral microcirculation so that the tip of the probe is just touching the top of the pool of mineral oil.
For the initial probe placement, it's best to start with a signal of approximately 150 units to ensure that the probe is positioned over the micro circulation. When the laser doppler flowmetry probe is in place, allow the animal for rest for a 30 to 45 minute equilibrition period before withdrawing 1.5mL of blood from the appropriate femoral artery cannula over a period of 30 seconds. To keep the catheter patent, infuse 100 units per mL of heparin in isotonic saline into the cannula after each blood draw.
Allow the rat to recover for two minutes and then measure LDF and blood pressure every 30 seconds for two minutes. After the blood pressure and LDF measurements, repeat the blood volume withdrawal every two minutes until the animal reaches a mean arterial pressure of approximately 20mmHg. In these representative experiments, the mean laser cerebral blood flow of 10 Sprague-Dawley rats fed standard laboratory chow was maintained within 20%of the pre-hemorrhage value following the first three blood volume withdrawals, until the mean arterial pressure reached the lower limit of autoregulation.
Subsequent blood volume withdrawals at pressures below the lower limit of autoregulation, caused a progressive reduction of laser cerebral blood flow demonstrating that the cerebral circulation was no longer able to produce a sufficient level of vasodilation to maintain the cerebral blood flow constant at the lower profusion pressures. At pressures at or above the lower limit of autoregulation, there was no significant correlation between the laser cerebral blood flow and the arterial pressure, indicating that the laser cerebral blood flow was independent of the arterial pressure in the plateau range of the autoregulatory curve. Below the lower limit of autoregulation, the laser cerebral blood flow arterial pressure relationship had a negative slope.
And the laser cerebral blood flow was significantly correlated with the arterial pressure. The most important things to remember are to thin the skull very carefully and to make sure that the LDF probe remains firmly anchored over the same area of tissue. This procedure can be used to test the reactivity of the microcirculation of various agents after retrograde infusion via the carotid artery, or to evaluate functional hyperemia and neurovascular coupling in the cerebral circulation.
This technique has enabled investigation of the effects of different drugs and anesthetics on the cerebral circulation and the changes in cerebral autoregulation in disease models such as hypertension.
