Name: laser doppler: a tool for measuring pancreatic islet microvascular vasomotion in vivo
Uploaded: 2018-03-08T13:00:00
Description: Watch this Scientific Journal Video about Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo at JoVE.com

This work was supported by grants from the Peking Union Medical College Youth Fund and the Fundamental Research Funds for the Central Universities (Grant no. 3332015200).

All animal experiments were executed in compliance with all relevant guidelines, regulations, and regulatory agencies. The present protocol being demonstrated was performed under the guidance and approval of the Institute of Microcirculation Animal Ethics Committee (IMAEC) at the Peking Union Medical College (PUMC).
1. Animals
<ol>
	<li>Before the start of the experiment, keep three BALB/c mice per cage, with controlled temperature (24 &#177; 1 &#176;C) and humidity (55 &#177; 5%), under a 1...

<ol>
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S., Yun, H. M., Jung, I. M., Lee, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Laser+Doppler+for+the+Evaluation+of+Pedal+Microcirculatory+Function+in+Diabetic+Neuropathy+Patients.">Role of Laser Doppler for the Evaluation of Pedal Microcirculatory Function in Diabetic Neuropathy Patients.</a> Microcirculation. 23 (1), 44-52 (2016).</li><li>Sun, P. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcirculatory+vasomotor+changes+are+associated+with+severity+of+peripheral+neuropathy+in+patients+with+type+2+diabetes.">Microcirculatory vasomotor changes are associated with severity of peripheral neuropathy in patients with type 2 diabetes.</a> Diab Vasc Dis Res. 10 (3), 270-276 (2013).</li><li>Pan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+PEMF+on+microcirculation+and+angiogenesis+in+a+model+of+acute+hindlimb+ischemia+in+diabetic+rats.">Effects of PEMF on microcirculation and angiogenesis in a model of acute hindlimb ischemia in diabetic rats.</a> Bioelectromagnetics. 34 (3), 180-188 (2013).</li><li>Jumar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Signs+of+End-Organ+Damage+in+Retinal+Arterioles+in+Patients+with+Type+2+Diabetes+Compared+to+Hypertensive+Patients.">Early Signs of End-Organ Damage in Retinal Arterioles in Patients with Type 2 Diabetes Compared to Hypertensive Patients.</a> Microcirculation. 23 (6), 447-455 (2016).</li><li>Nguyen, H. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+blood+flow+is+increased+in+type+1+diabetes+mellitus+patients+with+advanced+stages+of+retinopathy.">Retinal blood flow is increased in type 1 diabetes mellitus patients with advanced stages of retinopathy.</a> BMC Endocr Disord. 16 (1), 25-33 (2016).</li><li>Forst, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+Microcirculation+in+Type+1+Diabetic+Patients+With+and+Without+Peripheral+Sensory+Neuropathy.">Retinal Microcirculation in Type 1 Diabetic Patients With and Without Peripheral Sensory Neuropathy.</a> J Diabetes Sci Technol. 8 (2), 356-361 (2014).</li><li>Hu, H. F., Hsiu, H., Sung, C. J., Lee, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+laser-Doppler+flowmetry+measurements+with+spectral+analysis+to+study+different+microcirculatory+effects+in+human+prediabetic+and+diabetic+subjects.">Combining laser-Doppler flowmetry measurements with spectral analysis to study different microcirculatory effects in human prediabetic and diabetic subjects.</a> Lasers Med Sci. 31 (1), 1-8 (2016).</li><li>Klonizakis, M., Manning, G., Lingam, K., Donnelly, R., Yeung, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+diabetes+on+the+cutaneous+microcirculation+of+the+feet+in+patients+with+intermittent+claudication.">Effect of diabetes on the cutaneous microcirculation of the feet in patients with intermittent claudication.</a> Clin Hemorheol Microcirc. 61 (3), 439-444 (2015).</li><li>Khazraei, H., Shafa, M., Mirkhani, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ranolazine+on+cardiac+microcirculation+in+normal+and+diabetic+rats.">Effect of ranolazine on cardiac microcirculation in normal and diabetic rats.</a> Acta Physiol Hung. 101 (3), 301-308 (2014).</li><li>Fujita, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+inner+ear+susceptibility+to+noise+injury+in+mice+with+streptozotocin-induced+diabetes.">Increased inner ear susceptibility to noise injury in mice with streptozotocin-induced diabetes.</a> Diabetes. 61 (11), 2980-2986 (2012).</li><li>Wiernsperger, N., Nivoit, P., De Aguiar, L. 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In the cases that involve microvascular dysfunction (e.g., diabetes, acute pancreatitis, peripheral microvascular diseases, etc.), some diseases lead to reduced blood flow. Other than changes in blood flow, there are important indicators, such as microvascular vasomotion, that mirror the functional status of microcirculation. The specific indicator, microvascular vasomotion, is generally defined as the oscillation of the microvascular tone in microvascular beds. In the current protocol, a microvascular ...

As a parameter of the functional status of microcirculation, microvascular vasomotion takes responsibility for the delivery and exchange of oxygen, nutrients, and hormones and is crucial to the removal of metabolic products, such as carbon dioxide and cell waste1. Microvascular vasomotion also regulates blood flow distribution and tissue perfusion, thereby affecting local microcirculatory blood pressure and responses to inflammation, which can induce edema in many diseases. Therefore, microvascular vasomotion is extremely important to maintain the physiological function of organs2,3,4, tissues, and component cells. The impairment of microvascular vasomotion might be one of the key steps in the development of microcirculation-related diseases5.
Laser Doppler was initially developed for observation and quantification in the field of microcirculation research6. This technique, together with other technical approaches (e.g., laser speckle7, transcutaneous oxygen, etc.), has been regarded as the golden standard for assessing blood flow in microcirculation. The rationale that the blood perfusion of local microcirculation (i.e., capillaries, arterioles, venules, etc.) can be determined by apparatus equipped with laser Doppler, is based on the Doppler shift principle. The wavelength and frequency of stimulated emission light change when light particles encounter moving blood cells in microvessels, or they remain unchanged. Therefore, in microcirculation, the number and the velocity of blood cells are the key factors relating to the magnitude and frequency distribution of the Doppler-shifted light, while the direction of microvascular blood flow is irrelevant. Using different methods, a variety of tissues have been used for microcirculatory studies, including the mesenteries and dorsal skinfold chambers of mice, rats, hamsters, and even humans8. However, in the current protocol, we focus on the functional status of pancreatic islet microvascular vasomotion, which is evaluated using laser Doppler and a homemade assessment parameter system.
Pancreatic islet microcirculation is mainly composed of pancreatic islet microvessels and exhibits distinctive features. A pancreatic islet capillary network shows a five-times-higher density than the capillary network of its exocrine counterpart9. Providing a conduit for the delivery of input glucose and disseminating insulin, islet endothelial cells deliver oxygen to metabolically active cells in islet &#946; cells. Furthermore, emerging evidence also demonstrates that islet microvessels are involved not only in regulating insulin gene expression and &#946;-cell survival, but also in affecting &#946;-cell function; promoting &#946;-cell proliferation; and producing a number of vasoactive, angiogenic substances and growth factors10. Therefore, in this respect, we infer that the functional status of pancreatic islet microvascular vasomotion may affect islet &#946;-cell function and get involved in the pathogenesis of diseases such as acute/chronic pancreatitis, diabetes, and other pancreas-related diseases.
Analyzing the pathological changes of the functional status of pancreatic islet microvascular vasomotion might be a feasible strategy to determine the contributions of the pancreatic islet microcirculation to the diseases mentioned above. A detailed step-by-step procedure describing the approach to determine pancreatic islet microvascular vasomotion in vivo provide here. Typical measurements are then shown in the Representative Results. Finally, the benefits and limitations of the method are highlighted in the Discussion, along with further applications.

<table>
	<tbody>
		<tr>
			<td>MoorVMS-LDF2</td>
			<td>Moor Instruments</td>
			<td>GI80</td>
			<td>PeriFlux 5000 (Perimed Inc.) can be used as an alternative apparatus to harvest data</td>
		</tr>
		<tr>
			<td>MoorVMS-PC Software</td>
			<td>Moor Instruments</td>
			<td>GI80-1</td>
			<td>Software of MoorVMS-LDF2</td>
		</tr>
		<tr>
			<td>Calibration stand</td>
			<td>Moor Instruments</td>
			<td>GI-cal</td>
			<td>Calibration tool</td>
		</tr>
		<tr>
			<td>Calibration base</td>
			<td>Moor Instruments</td>
			<td>GI-cal</td>
			<td>Calibration tool</td>
		</tr>
		<tr>
			<td>Calibration flux standard</td>
			<td>Moor Instruments</td>
			<td>GI-cal</td>
			<td>Calibration tool</td>
		</tr>
		<tr>
			<td>One Touch UltraEasy glucometer</td>
			<td>Johnson and Johnson</td>
			<td>#1955685</td>
			<td>Confirm hyperglycemia</td>
		</tr>
		<tr>
			<td>One Touch UltraEasy strips</td>
			<td>Johnson and Johnson</td>
			<td>#1297006</td>
			<td>Confirm hyperglycemia</td>
		</tr>
		<tr>
			<td>Streptozotocin</td>
			<td>Sigma-Aldrich</td>
			<td>S0130</td>
			<td>Dissolve in sodium citrate buffer (pH 4.3)</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Sigma-Aldrich</td>
			<td>P3761</td>
			<td>Working concentration 3 %</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sinopharm Inc.</td>
			<td>200121</td>
			<td>Working concentration 75 %</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Amresco</td>
			<td>335</td>
			<td>Working concentration 10 %</td>
		</tr>
		<tr>
			<td>Medical gauze</td>
			<td>China Health Materials Co.</td>
			<td>S-7112</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Blunt-nose forceps</td>
			<td>Shang Hai Surgical Instruments Inc.</td>
			<td>N-551</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Surgical tapes</td>
			<td>3M Company</td>
			<td>3664CU</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Gauze sponge</td>
			<td>Fu Kang Sen Medical Device CO.</td>
			<td>BB5447</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Yu Lin Surgical Instruments Inc.</td>
			<td>175C</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Skin scissor</td>
			<td>Carent</td>
			<td>255-17</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>Ning Bo Surgical Instruments Inc.</td>
			<td>3325-77</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Syringe and 25-G needle</td>
			<td>MISAWA Inc.</td>
			<td>3731-2011</td>
			<td>Scale: 1 ml</td>
		</tr>
	</tbody>
</table>

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.

A photograph of the microvascular vasomotion measurement laser Doppler apparatus equipped with a semi-conductor laser diode is shown in Figure 1A. User interface software is presented in Figure 1B. Using the method mentioned above, the hemodynamic parameters of pancreatic islet microvascular vasomotion were detected for both non-diabetic control and diabetic mice. A variety of techniques, including laser Doppler flowmetry, reflec...

Watch this Scientific Journal Video about Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo at JoVE.com

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.

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 ...

The overall goal of this procedure is to determine the functional status of pancreatic islet microvascular vasomotion in vivo to assess its impact on related diseases. This method can help answer the key questions in the medical circulatory field such as whether pancreatic islet microvascular vasomotion is changed in pathological status. We first had idea for this method when we demonstrated that there's functional mech circulation that'd been evolved in the development of diabetes.The main advantage of this technique is that it can evaluate the functional status of microvascular vasomotion of tissues and organs in both clinical and laboratory\settings. This method can provide insight into pancreatic islet-related diseases. It can also be applied to other systems, such as kidney and retinal-related microvascular diseases.The applications of this technique also extend toward understanding the nature of trendings in the functional status of microvascular vasomotion. Generally, individuals new to this method will struggle because there are huge amounts of microvascular academic data derived from laser doppler during the experiments. Be sure demonstration of this method is critical because many factors can adversely affect accurate collection of microcirculation data.This protocol begins with giving mice a daily injection of STZ for five consecutive days. Dissolve the STZ in sodium citrate buffer to a working concentration of five milligrams per milliliter. Then, give intra-peritoneal injections via a one milliliter syringe and 25 gauge needle.The mice must fast for four hours prior to the injection, but their water access should not be removed. Inject the control mice with the same volume of pure sodium citrate buffer. After the injections, supply the mice with regular food and 10%sucrose water.After the fifth day of injecting STZ, replace the 10%sucrose water with normal water. Nine days later, test their blood glucose. First fast the mice for six hours with free access to water.Then, take blood from their tail veins and use a standard handheld glucometer to measure the blood. Consider any mice with blood glucose levels over 200 milligrams per deciliter diabetic. Before using the laser doppler apparatus, first clean the optical surfaces of the probe tip and probe connector with a soft, non-abrasive cloth, then plug the cable into the port of the instrument.Next, assemble the calibration stand. First let the stored flux standard reach room temperature. This takes about half an hour.Then, shake the flux standard gently for 10 seconds and let it rest on the calibration base for two minutes. Next, raise the clamp as high as possible and secure the probe into the clamp. Now slowly submerge the probe into the flux standard and secure the probe using the probe holders.To proceed, dim the room lights and press calibration on the laser doppler apparatus. Now choose the working channel that the probe is connected to and run the calibration program. Position an anesthetized mouse in a supine position on a working platform and secure it using surgical tape.Next, sterilize the abdominal skin with 75%ethanol from the cervical region to the tail, then cover the periphery of the region with a dry gauze sponge that has a three centimeter wide hole at the center. Next, lift the abdominal skin with forceps and make an initial vertical incision along the midline of the abdomen using a scalpel or skin scissors. Then grasp the underlying muscle with forceps and incise to enter the abdominal cavity.Do not injure any organs. To reveal the abdominal cavity, fold the skin and underlying muscle over the chest, then gently expose the pancreatic body and the spleen using a pair of blunt-nosed forceps. Now, in the control software for the laser doppler apparatus, create a new measurement file, then configure the connected monitors.Under the general tab, set the monitoring duration to free run and use the factory default for the LDF monitor tab. Next, open the display setup dialogue box and select the appropriate channels. Then, toggle the following parameters:data source for the channel and label, units, and color, then click next.Next, enter identifying information about the subject and measurement in the file information dialogue box and proceed by clicking next again. Now, the configuration is completed and a measurement window will pop up. Next, manually advance the electrode to within one millimeter of the pancreas.The accuracy of measurement is related to the distance between the probe and the pancreatic islet tissue. The blood flow reading will be inaccurate unless one millimeter gaps between the tissues and the probe is carefully adhered to. To begin the recording, click the start icon and the microvascular blood PU data will begin to get saved.Do this for a full minute and use the compress and expand display buttons and the increase or decrease height of graph traces buttons to adjust the displayed effect during recording and then stop the recording. Then, save the file with an appropriate name. Now, manually reposition the probe to avoid additive effects and localized exhaustion of contractile and relaxation capacity.In all, collect PU data from three random points on the pancreas for each mouse. At some point during the experiment, measure the PU data from a non-reflective plate to get a baseline control value. After taking the three measurements, use three-oh sutures to close the muscle layers and then four-oh sutures to close the skin layer.Now monitor the mouse in a recovery cage until it is ready to transfer to a clean home cage. In general, the microcirculatory condition of the pancreatic islet is represented by periodic contraction and relaxation phases. The overall hemodynamic phenomena creates a pattern of blood flow perfusion.Using the laser doppler apparatus, PU data was collected to show the distribution pattern of microvascular blood perfusion in non-diabetic and diabetic mice. The results were totally different. The rhythm of contractions and relaxations of pancreatic islet microvascular vasomotion was chaotic and irregular in STZ-induced diabetic mice, whereas non-diabetic controls had rhythmic oscillations.Next, the vasomotion was quantified using PU profiles. Compared to controls, the average blood perfusion of the pancreatic islet microcirculation was decreased in STZ-induced diabetic mice. These mice also showed significant decreases in the amplitude and frequency of this vasomotion, as well as in the relative velocity of blood perfusion to the pancreatic islet.Once mastered, this technique can be done in 30 minutes if it is performed properly. While attempting this procedure, it's important to remember to make sure the distance between the probe and the pancreas tissue is within one millimeter. An inappropriate distance gives an artificially increased or decreased blood flow reading.Following this procedure, other methods such as laser speckle and translocating oxygen can be performed in order to answer additional questions like ischemia visualization, blood flow assessment, functional state of microvascular vasomotion evaluation. After its development, this technique paved the way for researchers and clinicians in the field of endocrinology and metabolism to explore the microcirculatory mechanisms hidden behind pancreatic islet related diseases. Don't forget that working with laser instrument can be extremely hazardous due to radiation exposure and precautions such as environment side is at least 200 millimeter from the eye should always be taken while performing this procedure.

Research

JoVE Journal

Medicine

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

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 ...

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 ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an ...

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...