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 casein, 3 g/kg DL-methionine, 497.77 g/kg sucrose, 150 g/kg cornstarch, 50 g/kg corn oil, 50 g/kg cellulose, 2 g/kg choline bitartrate, 35 g/kg mineral mix, and 10 g/kg vitamin mix.</li>
	<li>Record arterial blood pressure and LDF readings using data acquisition software or any comparable recording method.</li>
	<li>Attach the arterial pressure transducer to one channel of the recording system and the LDF probe to the other channel on the recording system.</li>
	<li>Prior to the measurement, calibrate the laser Doppler probe to set a motility standard and ensure that the laser Doppler flowmeter is providing a steady output.</li>
	<li>Prepare additional equipment needed for the preparatory surgery and for the experiment: a dissecting microscope, a rodent ventilator, an end tidal CO2 monitor, a stereotaxic instrument to fix the rat&#39;s head in position, and a micromanipulator to locate the LDF probe over the pial microcirculation and maintain it in a steady position.</li>
</ol>
2. Surgical preparation
<ol>
	<li>Weigh the rat and anesthetize the animal in an induction chamber with 3.5% isoflurane and 30% O2 supplement.</li>
	<li>Remove the animal from the induction chamber and substitute an anesthetic mask delivering 1.5&#8211;3% isoflurane with a 30% O2 supplement.</li>
	<li>Place the rat on a circulating water blanket maintained at 37 &#176;C and check reflexes with a toe pinch to ensure that there is a withdrawal reflex. Apply sterile ophthalmic ointment to both eyes to prevent corneal desiccation.</li>
	<li>Shave the top of the cranium, ventral neck area, and femoral triangles. Remove any loose hair from those areas and clean with rubbing alcohol.</li>
	<li>Place the rat in a supine position on a heating pad with a circulating warm water pump to maintain the animal&#39;s body temperature at 37 &#176;C and temporarily secure it to the pad using medical tape.</li>
	<li>Install a tracheal cannula (PE240 polyethylene tubing) through a ventral incision in the neck as described elsewhere26.</li>
	<li>Attach the tracheal cannula to an end tidal CO2 monitor and the ventilator delivering 2.5&#8211;3.0% isoflurane (depending on the size of the animal) and a 30% O2 inhalation supplement. Make sure the respiratory rate, inspiratory time, and minute ventilatory volume are set and monitored to ensure an expired end tidal CO2 of approximately 35 mmHg throughout the experiment. 
	NOTE: This is generally achieved with a respiratory rate of approximately 48&#8211;60 breaths/min, a tidal volume of 1.70&#8211;2.30 mL, and an inspiration time of 0.50&#8211;0.60 s for a 250&#8211;300 g rat.</li>
	<li>Fill two PE50 polyethylene cannulas with 1 U/mL heparin in isotonic NaCl solution to prevent clotting and to maintain patency of the catheters. After filling, bevel the open end of each cannula with surgical scissors to facilitate insertion into the artery.</li>
	<li>Cannulate the right and left femoral arteries as described elsewhere27 to allow continuous monitoring of arterial pressure in one catheter and blood withdrawal from the other catheter.
	<ol>
		<li>After carefully separating the arteries from the surrounding tissue under a dissecting microscope, ligate the distal end of the artery and place two additional sutures around the middle and proximal ends of the artery without tightening the knots.</li>
		<li>Use the proximal suture as a lifting ligature to prevent bleeding from the artery after the incision for cannula insertion (step 2.11).</li>
	</ol>
	</li>
	<li>Insert a V-shaped wire fashioned from a paper clip under the artery in order to occlude the vessel until the cannula is secured.</li>
	<li>Under a dissecting microscope make a small incision in the femoral artery near the distal ligation using Vannas scissors. Insert the beveled end of the cannula into the incision and advance it into the femoral artery. Tighten the knot on the middle ligature to secure the cannula in place so it is not dislodged by arterial pressure when the lifting ligature or paper clip is removed.</li>
	<li>After the middle ligature is tightened, release the tension on the lifting ligature and/or remove the paper clip, and tighten the proximal ligature.</li>
	<li>Close the incision with fine sutures (3&#8211;0 silk) or a surgical staple. Alternatively, place a moist gauze over the incision site, depending on the size of the incision.</li>
</ol>
3. Skull thinning for LDF measurements
<ol>
	<li>Immediately after the cannulas are in place, place the animal in a sternal position and secure the head in a stereotaxic device, being careful not to dislodge the catheters or tracheal tube.</li>
	<li>Use surgical scissors to make an elliptical incision in the skin covering the cranium. Use a cotton swab to remove any connective tissue, ensuring that the cranium is clean and dry. Place a small elongated and rolled piece of tissue paper around the incision on the scalp to stop any bleeding.</li>
	<li>Under the dissecting microscope, use a Dremel tool or a dental drill with a 2.15 mm drill bit to thin a small area of bone (approximately 0.5&#8211;1 cm depending on the size of the rat) in the parietal area over the left or right somatosensory cortex. 
	CAUTION: 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 overheating.</li>
	<li>Once the skull has been thinned and the area has a pink appearance and/or blood vessels are visualized, cover the area 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 (Figure 1). 
	NOTE: It is essential to take LDF measurements in an area where there are no external vibrations that would interfere with the laser Doppler readings and that the probe is securely fixed over the same target area throughout the experiment.</li>
</ol>
4. Assessing cerebral vascular autoregulation
<ol>
	<li>Once the LDF probe is fixed in position, allow a 30&#8211;45 min equilibration period before beginning the experiment. After the equilibration period, measure the mean arterial pressure (MAP) and laser cerebral blood flow (LCBF) every 30 s for 2 min and average the values to obtain the baseline values for the prehemorrhage blood pressure and LCBF.</li>
	<li>To evaluate the cerebral vascular autoregulation in response to arterial pressure reduction, measure the LCBF and MAP following successive withdrawals of 1.5 mL of blood from the femoral artery11. To keep the catheter patent, ensure that a volume of heparin solution (100 U/mL in isotonic saline) approximately equal to the catheter volume is infused after each blood draw. 
	NOTE: When infusing the heparin solution to maintain catheter patency, it is important to match the volume of the heparin solution to the volume of the catheter as closely as possible to prevent the animal from receiving too much heparin, which could cause unwanted bleeding.</li>
	<li>After each blood volume withdrawal, allow the rat to equilibrate for 2 min, after which the MAP and LCBF are recorded every 30 s for 2 min. Repeat the blood volume withdrawals until the animal reaches a MAP of approximately 20 mmHg.</li>
	<li>Determine the effective autoregulatory range by identifying the range of blood pressures from the prehemorrhage MAP to the LLA (steps 4.5 and 5.3, below).</li>
	<li>Determine the LLA by identifying the lowest pressure at which LCBF still returns to within 20% of the prehemorrhage control value following blood volume withdrawal, as previously described11,28 or by identifying the intersection point of the regression lines determined during the plateau phase of autoregulation and below the LLA, where LCBF decreases with each successive blood withdrawal (step 5.3, below). 
	NOTE: The criteria for defining the LLA and autoregulatory plateau may differ between laboratories (e.g., Takada et al.28 vs. Jones et al.29) as well as procedures for reducing arterial blood pressure (e.g., withdrawal of a specific volume of blood vs. controlled hemorrhage to reach specific arterial pressure levels)11.</li>
	<li>At the end of the experiment, euthanize the animal by creating a bilateral pneumothorax while under a surgical plane of anesthesia, as approved by the IACUC.</li>
	<li>LDF values obtained in the tissue after the animal is euthanized will provide the zero baseline flow value for the experimental setup.</li>
</ol>
5. Statistical analysis
<ol>
	<li>Perform linear regression analysis to evaluate the correlation between the LDF values and their corresponding arterial pressure. Use the baseline LDF readings obtained after the animal is euthanized to ensure that there was no nonspecific LDF signal affecting the measured flow rates.</li>
	<li>Calculate the LLA using the intersection between the regression lines above and below the autoregulatory plateau. To calculate the LLA using this method, combine the two regression equations and solve the resulting equation for arterial pressure.</li>
	<li>When comparing different experimental groups, use linear regression analysis to calculate the slopes of the LDF vs. arterial pressure relationship above and below the LLA for each animal and summarize them as mean &#177; SEM for the animals in that experimental group.</li>
</ol>

The authors have nothing to disclose.

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 ...

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9.2K Views.  Medical College of Wisconsin. 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.

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

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include supporting mastication and deglutition, as well as taste-sensing and phonetics. Accordingly, the strength and volume of the tongue can impact the ability of vertebrates to accomplish basic activities such as feeding, communicating, and breathing. Human patients with sleep apnea have enlarged tongues, characterized by reduced muscle tone and increased intramuscular fat that can be visualized and quantified by magnetic resonance imaging (MRI). The abilities to measure force generation and viscoelastic properties of the tongue constitute important tools for obtaining functional information to correlate with imaging data. Here, we present techniques for measuring tongue force production in anesthetized Zucker rats via electrical stimulation of the hypoglossal nerves and for determining the viscoelastic properties of the tongue by applying passive Lissajous force/deformation curves.

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include ...

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon dioxide and waste products. The impairment of microvascular vasomotion might be a crucial step in the development of microcirculation-related diseases. In addition, the highly vascularized pancreatic islet is adapted to support endocrine function. In this respect, it seems possible to infer that the functional status of pancreatic islet microvascular vasomotion might affect pancreatic islet function. Analyzing the pathological changes of the functional status of pancreatic islet microvascular vasomotion may be a feasible strategy to determine contributions that pancreatic islet microcirculation makes to related diseases, such as diabetes mellitus, pancreatitis, etc. Therefore, this protocol describes using a laser Doppler blood flow monitor to determine the functional status of pancreatic islet microvascular vasomotion, and to establish parameters (including average blood perfusion, amplitude, frequency, and relative velocity of pancreatic islet microvascular vasomotion) for evaluation of the microcirculatory functional status. In a streptozotocin-induced diabetic mouse model, we observed an impaired functional status of pancreatic islet microvascular vasomotion. In conclusion, this approach for assessing pancreatic islet microvascular vasomotion in vivo may reveal mechanisms relating to pancreatic islet diseases.

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Pancreatic islet microvascular vasomotion regulates islet blood distribution and maintains the physiological function of islet &#946; cells. This protocol describes using a laser Doppler monitor to determine the functional status of pancreatic islet microvascular vasomotion in vivo and to assess the contributions of pancreatic islet microcirculation to pancreatic-related diseases.

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is a common, noninvasive, repeatable method for assessing blood flow recovery. The technique calculates overall blood flow in the sampled tissue from the Doppler shift in frequency caused when a laser hits moving red blood cells. Measurements are expressed in arbitrary perfusion units, so the contralateral non-intervened upon leg is usually used to help control measurements. Measurement depth is in the range of 0.3-1 mm; for hindlimb ischemia, this means that dermal perfusion is assessed. Dermal perfusion is dependent on several factors&#8212;most importantly skin temperature and anesthetic agent, which must be carefully controlled to result in reliable readings. Furthermore, hair and skin pigmentation can alter the ability of the laser to either reach or penetrate to the dermis. This article demonstrates the technique of LDPI in the mouse hindlimb.

Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.