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 12-h light-dark cycle. Allow the mice free access to regular food and water.</li>
	<li>Randomly divide the mice into a non-diabetic control group and a diabetic group. Accurately weigh each individual mouse and calculate the injection volume using the body mass of each mouse.</li>
	<li>Fast the mice for 4 h before streptozotocin (STZ) injection and provide regular water as normal on experimental day 1.</li>
	<li>Prepare 0.1 M sodium citrate buffer at pH 4.3. Put 1 mL of the solution into a 1.5-mL microcentrifuge tube and wrap the microcentrifuge tube in aluminum foil to avoid light exposure.</li>
	<li>Dissolve the STZ in sodium citrate buffer (pH 4.3) to a final working concentration of 5 mg/mL before use.</li>
	<li>Give the mice of the diabetic group intraperitoneal injections of STZ at a dose of 40 mg/kg using a 1-mL syringe and a 25-G needle. Inject the mice of the non-diabetic control with the same volume of sodium citrate buffer (pH 4.3).</li>
	<li>Put the mice back into the cages and supply them with regular food and 10% sucrose water.</li>
	<li>Repeat steps 1.3-1.7 on experimental days 2 to 5 (i.e., the next 4 consecutive days).</li>
	<li>Replace the 10% sucrose water with regular water after the last STZ injection.</li>
	<li>Fast the mice for 6 h, but give them free access to water, and measure their blood glucose levels nine days later (experimental day 14). Collect a blood sample from the tail vein to confirm hyperglycemia using a blood glucose monitoring system. 
	NOTE: Mice with blood glucose levels &#62; 200 mg/dL are considered diabetic.</li>
</ol>
2. Preparation of the Instrument
<ol>
	<li>Clean the optical surfaces of the probe tip and probe connector of the laser Doppler apparatus with a soft, non-abrasive cloth to remove any dust or particles. Plug the cable into the port of the instrument (Figure 1A).</li>
	<li>Assemble the calibration stand by allowing the flux standard to be in thermal equilibrium with experimental surroundings (room temperature, usually for 30 min). Shake the flux standard gently for 10 s and let it rest for 2 min.</li>
	<li>Position the flux standard container in the middle of the calibration base. Adjust the clamp to the maximum height and secure the probe in the clamp such that it points downwards to the container. Make sure the flux standard is correctly positioned underneath the probe.</li>
	<li>Slowly move the probe down until the tip is correctly submerged in the flux standard. Select and press &#34;calibration&#34; on the laser Doppler apparatus and choose the working channel that the probe is connected to. Run the calibration program until a &#34;Calibration successful&#34; notice is displayed on the screen of laser Doppler apparatus.</li>
	<li>Secure the probe using probe holders. Manually secure the probe to avoid movement.</li>
	<li>Maintain the experimental room at constant temperature (24 &#177; 1 &#176;C) and humidity (~50-60%).</li>
	<li>Turn off any external light (such as fluorescent and spot lamps) before performing the experiment to avoid external light-induced change.</li>
</ol>
3. Preparation of the Animals
<ol>
	<li>Autoclave the surgical instruments and allow them to cool to room temperature before use.</li>
	<li>Give the mice 10 min to acclimatize to the experimental environment before detecting pancreatic islet microvascular vasomotion by laser Doppler.</li>
	<li>Fill a 1-mL syringe with 1 mL of 3 % pentobarbital sodium. Inject the pentobarbital sodium solution (75 mg/kg i.p.) to anesthetize the mice.</li>
	<li>Cover the eyes of the mouse with pre-moistened medical gauze to prevent dryness.</li>
	<li>Ensure that the mouse completely loses consciousness and no longer responds to tail or hindfoot pinches with forceps.&#160;Monitor the anesthesia throughout the anesthetic and intra-operative event every 15 min. Maintain the anesthesia by supplementing with 10 % of the initial injection volume of the pentobarbital solution when necessary.</li>
	<li>Place a heating pad with a semi-insulating layer below the animal and place the animal in supine position and transfer it to the working station of the laser Doppler apparatus. Fix the mouse to the working platform with surgical tape.</li>
	<li>Swab the abdominal skin of the mouse with betadine, and then 75% ethanol is used to swab the abdominal area clean.</li>
	<li>Inject 2% lidocaine/0.5% bupivacaine (50/50) mixture subcutaneously.&#160; Cut a ~3 cm-diameter hole in the center of a gauze sponge. Cover the abdominal region with the gauze sponge.</li>
	<li>Lift the abdominal skin with forceps and make an initial vertical incision along the midline of abdomen using a scalpel or skin scissors.</li>
	<li>Grasp the underlying muscle with forceps and incise to enter the abdominal cavity. Do not injure any organs. Fold the skin and underlying muscle over the chest to reveal the abdominal cavity. Gently expose the pancreatic body and the spleen using a pair of blunt-nosed forceps.</li>
</ol>
4. Data Acquisition for Analysis
<ol>
	<li>Run the software of the laser Doppler apparatus by clicking on &#34;File&#34; &#8594; &#34;New&#34; to create a new measurement file. To configure the connected monitors, under the &#34;General&#34; tab, set up the monitoring duration to &#34;Free run.&#34; Use the factory default for the &#34;LDF Monitor&#34; tab. Click &#34;Next.&#34;</li>
	<li>Set up the graph display in the &#34;display setup dialog box.&#34; Select the &#34;Flux, Conc, Speed&#34; channels by checking the respective boxes. Select the following parameters: &#34;Data source for the channel&#34; and &#34;Label, Units and Color.&#34; Click &#34;Next.&#34;</li>
	<li>Enter user information about the subject and measurement (i.e., name and subject number, operator, monitoring time, comments, etc.) in the &#34;file information dialog box&#34; and click &#34;Next&#34; to finish the measurement configuration. 
	NOTE: A measurement window is automatically created by the software (Figure 1B).</li>
	<li>Manually advance the electrode to the pancreas. Make sure the distance between the probe and pancreas tissues is within 1 mm. An inappropriate distance gives an artificially increased or decreased blood flow reading.</li>
	<li>Click the &#34;Start&#34; toolbar icon to start recording the microvascular blood perfusion units (PU) data. Collect the PU data continuously for 1 min every run. Click &#34;Stop&#34; to stop the measurement. Select &#34;File&#34; &#8594; &#34;Save as&#34; to name and save the finished measurement file.</li>
	<li>Manually reposition the probe after each run to avoid additive effects and the localized exhaustion of contractile and relaxation capacity. Repeat steps 4.1-4.4 to harvest multi-point (i.e., three randomly chosen points from pancreatic tissue) microvascular PU data for each mouse. Measure the PU data of a non-reflective plate as a baseline control.</li>
	<li>Close the abdominal muscle layer and the skin layer with a suture. Place the animals in clean cages after the experiments.</li>
	<li>Keep the animal warm by placing the recovery cage half-on the heating pad. 
	NOTE: Pay attention to warmth, hygiene, fluid and food intake, and infection. Administrate mice with 2 mg/kg Carprofen for 48 h as postoperative pain management.&#160;&#160;Perform euthanasia by injecting 150 mg/kg pentobarbital sodium i.p. when mice are observed to be in a state of severe pain or distress that cannot be alleviated.</li>
</ol>
5. Calculating the Parameters of Microvascular Vasomotion
<ol>
	<li>Use the &#34;Export&#34; command of the laser Doppler software to export the time and PU raw data as a *.xlsx file and open the file in a spreadsheet.</li>
	<li>Calculate the average baseline perfusion unit (PUb) (see step 4.6).</li>
	<li>Calculate the average blood perfusion (PUa) for 1 min of a measurement as follows: Average blood perfusion (PUa) = PU - PUb (Equation 1).</li>
	<li>Calculate the frequency (cycles/min) for each 1 min of measurement. 
	NOTE: The frequency of microvascular vasomotion is defined as the number of peaks that occurred in a microvascular vasomotion wave per minute.</li>
	<li>Calculate the amplitude (&#916;PU) for each 1 min of measurement.
	<ol>
		<li>Calculate the amplitude of microvascular vasomotion as the difference between the maximum (PUmax) and minimum (PUmin): Amplitude (&#916;PU) = PUmax - PUmin (Equation 2).</li>
	</ol>
	</li>
	<li>Calculate the relative velocity (PU) for each 1 min of measurement.</li>
</ol>

<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. 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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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The authors have nothing to disclose.

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 ...

laser-doppler-tool-for-measuring-pancreatic-islet-microvascular

Research

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Medicine

8.2K Views.  Chinese Academy of Medical Sciences & Peking Union Medical College. Pancreatic islet microvascular vasomotion regulates islet blood distribution and maintains the physiological function of islet β 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.

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

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 ...

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 cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

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

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 extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

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

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

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 ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

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 important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.

Retinal image analysis is an unobtrusive procedure for visualizing the microcirculation. The impact of cardiovascular disease risk factors can result in changes of retinal vessel calibers. The procedures to acquire fundus images and the steps for calculating the vessel calibers are described.

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

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

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

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 of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

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

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

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

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

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

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) 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 options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

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

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

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Chapters in this video

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

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

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

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

In vivo ¹⁹F MRI for Cell Tracking

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

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

A High-content In Vitro Pancreatic Islet β-cell Replication Discovery Platform

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

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