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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P., 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=Microvascular+dysfunction:+a+potential+mechanism+in+the+pathogenesis+of+obesity-associated+insulin+resistance+and+hypertension.">Microvascular dysfunction: a potential mechanism in the pathogenesis of obesity-associated insulin resistance and hypertension.</a> Microcirculation. 19 (1), 5-18 (2012).</li><li>Nilsson, G. E., Tenland, T., Oberg, P. 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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. 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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 ...

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