Name: laser doppler: a tool for measuring pancreatic islet microvascular vasomotion in vivo
Uploaded: 2018-03-08T13:00:00.000+00:00
Duration: 10 min 39 s
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 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>

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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...

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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

JoVE Journal

Medicine

8.2K Views.  Chinese Academy of Medical Sciences & Peking Union Medical College. Alvleesklier islet microvasculaire vasomotion regelt islet bloed verspreiding en onderhoudt de fysiologische functie van β eilandjecellen. Dit protocol wordt beschreven met behulp van een laser Doppler-monitor om de functionele status van de alvleesklier islet microvasculaire vasomotion in vivo te bepalen en te beoordelen van de bijdragen van de alvleesklier islet microcirculatie alvleesklier-gerelateerde ziekten.

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

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 ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Biology

Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared from live human and mouse pancreatic tissue embedded in agarose and cut using a vibratome. This method allows for the tissue to maintain viability and function in addition to preserving underlying pathologies such as type 1 (T1D) and type 2 diabetes (T2D). The slice method enables new directions in the study of the pancreas through the maintenance of the complex structures and various intercellular interactions that comprise the endocrine and exocrine tissues of the pancreas. This protocol demonstrates how to perform staining and time-lapse microscopy of live endogenous immune cells within pancreatic slices along with assessments of islet physiology. Further, this approach can be refined to discern immune cell populations specific for islet cell antigens using major histocompatibility complex-multimer reagents.

This study presents the application of live pancreatic tissue slices to the study of islet physiology and islet-immune cell interactions.

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared ...

Immunology and Infection

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology have been developed and utilized in vivo, they are limited in terms of allowing single cell resolution and continuous longitudinal recordings. Because of the transparency of the cornea, the anterior chamber of the eye (ACE) in mice is well suited to study human and mouse pancreatic islet cell biology. Here is a description of how this approach can be used to perform continuous longitudinal recordings of grafting and revascularization of individual human islet grafts. Human islet grafts are inserted into the ACE, using NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mice as recipients. This allows for the investigation of the expansion of recipient versus donor cells and the contribution of recipient cells in promoting the encapsulation and vascularization of the graft. Further, a step-by-step approach for image analysis and quantification of the islet volume or segmented vasculature and islet capsule forming recipient cells is outlined.

The goal of this protocol is to continuously monitor the dynamics of the human pancreatic islet engraftment process and the contributing host versus donor cells. This is accomplished by transplanting human islets into the anterior chamber of the eye (ACE) of an NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mouse recipient followed by repeated 2-photon imaging.

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology ...

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 ...

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

Investigating Novel Mechanisms of Microvascular Dysfunction in Humans

Intradermal Microdialysis: An Approach to Investigating Novel Mechanisms of Microvascular Dysfunction in Humans

The cutaneous vasculature is an accessible tissue that can be used to assess microvascular function in humans. Intradermal microdialysis is a minimally invasive technique used to investigate mechanisms of vascular smooth muscle and endothelial function in the cutaneous circulation. This technique allows for the pharmacological dissection of the pathophysiology of microvascular endothelial dysfunction as indexed by decreased nitric oxide-mediated vasodilation, an indicator of cardiovascular disease development risk. In this technique, a microdialysis probe is placed in the dermal layer of the skin, and a local heating unit with a laser Doppler flowmetry probe is placed over the probe to measure the red blood cell flux. The local skin temperature is clamped or stimulated with direct heat application, and pharmacological agents are perfused through the probe to stimulate or inhibit intracellular signaling pathways in order to induce vasodilation or vasoconstriction or to interrogate mechanisms of interest (co-factors, antioxidants, etc.). The cutaneous vascular conductance is quantified, and mechanisms of endothelial dysfunction in disease states can be delineated.

Author Spotlight: A Pharmacodissection Approach to Uncover Mechanisms in Cardiovascular Disease Risk Populations 

Intradermal microdialysis is a minimally invasive technique used to investigate microvascular function in health and disease. Both dose-response and local heating protocols can be utilized for this technique to explore mechanisms of vasodilation and vasoconstriction in the cutaneous circulation.

The cutaneous vasculature is an accessible tissue that can be used to assess microvascular function in humans. Intradermal microdialysis is a minimally ...

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails significantly fewer preparation-induced changes than isolated islets, acini, ducts, or dispersed cells described in typical in vitro studies. By combining the acute pancreatic tissue slice with live-cell calcium imaging in confocal laser scanning microscopy (CLSM), calcium signals can be studied in a large number of endocrine and exocrine cells simultaneously, with a single-cell or even subcellular resolution. The sensitivity permits the detection of changes and enables the study of intercellular waves and functional connectivity as well as the study of the dependence of physiological responses of cells on their localization within the islet and paracrine relationship with other cells. Finally, from the perspective of animal welfare, recording signals from a large number of cells at a time lowers the number of animals required in experiments, contributing to the 3R-replacement, reduction, and refinement-principle.

We present the preparation of acute pancreatic tissue slices and their use in confocal laser scanning microscopy to study calcium dynamics simultaneously in a large number of live cells, over long time periods, and with high spatiotemporal resolution.

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails ...

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

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