The authors express their sincere thanks to Kaleigh Kozak, Megan Stumpf, and Jack Bullis for their outstanding assistance in completing this study and preparing the manuscript. Grant Support: NIH #R01-HL128242, #R21-OD018309, and #R21-OD024781.

The Medical College of Wisconsin Institutional Animal Care and Use Committee (IACUC) approved all protocols described in this paper and all procedures are in compliance with the National Institutes of Health (NIH) Office of Laboratory Animal Welfare (OLAW) regulations.
1. Experimental animals and preparation for recording
<ol>
	<li>Use 8&#8211;12-week-old male Sprague-Dawley rats weighing 250&#8211;300 g. For these experiments, feed rats a standard diet consisting of 0.4% NaCl, 200 g/kg case...

Evaluation of Tissue Blood Flow Responses with Laser Doppler Flowmetry (LDF). As noted above, the LDF signal is proportional to the number and velocity of moving particles, in this case RBC, in the microcirculation. LDF readings in different organs are well correlated with whole organ blood flow assessed by established methods such as electromagnetic flow meters and radioactive microspheres30 and are generally consistent with studies evaluating the regulation of active tone in can...

Autoregulatory mechanisms in the cerebral circulation play a crucial role in maintaining homeostasis and normal function in the brain. Autoregulation of the cerebral blood flow is affected by multiple factors including heart rate, blood velocity, perfusion pressure, the diameter of the cerebral resistance arteries, and the microcirculatory resistance, all of which play a role in maintaining the total cerebral blood flow constant in the brain over the physiological range of systemic blood pressures. When arterial pressure increases, these mechanisms constrict arterioles and resistance arteries to prevent dangerous increases in intracranial pressure. When arterial blood pressure decreases, local control mechanisms dilate the arterioles to maintain tissue perfusion and O2 delivery. Various pathological conditions such as hypercapnia, traumatic or global hypoxic brain injury, and diabetic microangiopathy1,2,3,4,5,6 may disrupt the brain&#39;s ability to autoregulate its blood flow. For example, chronic hypertension shifts the effective autoregulatory range toward higher pressures7,8,9, and a high salt (HS) diet not only interferes with normal endothelium-dependent dilation in the cerebral microcirculation10, but also impairs the ability of autoregulatory mechanisms in the cerebral circulation to dilate and maintain tissue perfusion when arterial pressure is reduced11. Cerebral autoregulation is also impaired in Dahl salt-sensitive rats when they are fed a HS diet12.
During reductions in arterial pressure, dilation of the cerebral resistance arteries and arterioles initially returns cerebral blood flow to control values despite the reduced perfusion pressure. As arterial pressure is reduced further, cerebral blood flow remains constant at the lower pressure (plateau phase of the autoregulatory response) until the vasculature can no longer dilate to maintain blood flow at the lower pressure. The lowest pressure at which an organ can maintain normal blood flow is termed the lower limit of autoregulation (LLA). At pressures below the LLA, cerebral blood flow decreases significantly from resting values and decreases in a linear fashion with each reduction in arterial perfusion pressure13,14. An upward shift in the LLA, as observed in hypertension7,8,9, may increase the risk and severity of ischemic injury during conditions where the arterial perfusion pressure is reduced (e.g., myocardial infarction, ischemic stroke, or circulatory shock).
LDF has proven to be an extremely valuable approach to evaluate the blood flow in the microcirculation under a variety of circumstances, including autoregulation of the blood flow in the cerebral circulation11,14,15. In addition to evaluating autoregulatory responses, LDF can be used to monitor the cortical blood flow when investigating metabolic, myogenic, endothelial, humoral, or neural mechanisms that regulate the cerebral blood flow and the impact of various experimental interventions and pathological conditions on cerebral blood flow10,16,17,18,19,20,21.
LDF measures the shift in reflected laser light in response to the number and velocity of moving particles--in this case, red blood cells (RBC). For studies of cerebral vascular autoregulation, arterial blood pressure is changed either by the infusion of an alpha-adrenergic agonist to increase arterial pressure (because the cerebral circulation itself is insensitive to alpha-adrenergic vasoconstrictor agonists)12,15 or via controlled blood volume withdrawal to reduce arterial pressure11,14. In the present study, LDF is utilized to demonstrate the effects of graded reductions in blood pressure on cerebral autoregulation in a healthy rat. Although open and closed skull methods have been described in the literature22,23,24,25, the present paper demonstrates a closed skull preparation, allowing cerebral blood flow to be assessed without penetrating the skull or installing a chamber or cerebral window.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-0 braided black silk suture</td>
			<td>Midwest Vet</td>
			<td>193.73000.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Arterial Pressure Transducer</td>
			<td>Merit Medical</td>
			<td>041516504A</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Data Acquisition Systems (WINDAQ &#38; BIOPAC system)</td>
			<td>DATAQ Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Pressure Display Unit</td>
			<td>Stoelting</td>
			<td>50115</td>
			<td></td>
		</tr>
		<tr>
			<td>Circulating warm water pump</td>
			<td>Gaymar Industries</td>
			<td>T-pump</td>
			<td></td>
		</tr>
		<tr>
			<td>End-tidal CO2 monitor</td>
			<td>Stoelting</td>
			<td>Capstar-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sodium</td>
			<td>Midwest Vet</td>
			<td>191.46720.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipe</td>
			<td>Fisher Scientific</td>
			<td>06-666A</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Doppler Flow Meter</td>
			<td>Perimed</td>
			<td>PeriFlux 5000 LDPM</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Doppler Refill Motility Standard</td>
			<td>Perimed</td>
			<td>PF1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene Tubing (PE240) (for trachea cannula)</td>
			<td>VWR</td>
			<td>63018-828</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene Tubing (PE50) (for femoral catheters)</td>
			<td>VWR</td>
			<td>63019-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent Ventilator</td>
			<td>Cwe/Stoelting</td>
			<td>SAR-830/P</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Midwest Vet</td>
			<td>193.74504.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague-Dawley Outbred Rats</td>
			<td>Variable</td>
			<td>N/A</td>
			<td>Rats were ordered from various companies</td>
		</tr>
		<tr>
			<td>Standard Rat Chow</td>
			<td>Dyets, Inc.</td>
			<td>113755</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic Instrument</td>
			<td>Cwe/Stoelting</td>
			<td>Clasic Lab Standard</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of cerebral blood flow autoregulation in the rat using laser doppler flowmetry

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained using laser Doppler flowmetry (LDF). This paper demonstrates a closed skull preparation that allows cerebral blood flow to be assessed without penetrating the skull or installing a chamber or cerebral window. To evaluate autoregulatory mechanisms, a model of controlled blood pressure reduction via graded hemorrhage can be utilized while simultaneously employing LDF. This enables the real time tracking of the relative changes in the blood flow in response to reductions in arterial blood pressure produced by the withdrawal of circulating blood volume. This paradigm is a valuable approach to study cerebral blood flow autoregulation during reductions in arterial blood pressure and, with minor modifications in the protocol, is also valuable as an experimental model of hemorrhagic shock. In addition to evaluating autoregulatory responses, LDF can be used to monitor the cortical blood flow when investigating metabolic, myogenic, endothelial, humoral, or neural mechanisms that regulate cerebral blood flow and the impact of various experimental interventions and pathological conditions on cerebral blood flow.

Figure 2 summarizes the results of experiments conducted in 10 male Sprague-Dawley rats fed standard laboratory chow. In those experiments, mean LCBF was maintained within 20% of the prehemorrhage value following the first three blood volume withdrawals, until the mean arterial pressure reached the LLA. Subsequent blood volume withdrawals at pressures below the LLA caused a progressive reduction of LCBF, showing that the cerebral circulation was no longer able to produce a sufficient level o...

Watch this Scientific Journal Video about Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry at JoVE.com

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry

This article demonstrates the use of laser Doppler flowmetry to evaluate the ability of the cerebral circulation to autoregulate its blood flow during reductions in arterial blood pressure.

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained ...

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.

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry

Abstract