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

Summary

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.

Abstract

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.

Introduction

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 organs^2,3,4, 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 β cells. Furthermore, emerging evidence also demonstrates that islet microvessels are involved not only in regulating insulin gene expression and β-cell survival, but also in affecting β-cell function; promoting β-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 β-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.

Protocol

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

1. Before the start of the experiment, keep three BALB/c mice per cage, with controlled temperature (24 ± 1 °C) and humidity (55 ± 5%), under a 12-h light-dark cycle. Allow the mice free access to regular food and water.
2. 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.
3. Fast the mice for 4 h before streptozotocin (STZ) injection and provide regular water as normal on experimental day 1.
4. 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.
5. Dissolve the STZ in sodium citrate buffer (pH 4.3) to a final working concentration of 5 mg/mL before use.
6. 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).
7. Put the mice back into the cages and supply them with regular food and 10% sucrose water.
8. Repeat steps 1.3-1.7 on experimental days 2 to 5 (i.e., the next 4 consecutive days).
9. Replace the 10% sucrose water with regular water after the last STZ injection.
10. 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 > 200 mg/dL are considered diabetic.

2. Preparation of the Instrument

1. 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).
2. 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.
3. 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.
4. Slowly move the probe down until the tip is correctly submerged in the flux standard. Select and press "calibration" on the laser Doppler apparatus and choose the working channel that the probe is connected to. Run the calibration program until a "Calibration successful" notice is displayed on the screen of laser Doppler apparatus.
5. Secure the probe using probe holders. Manually secure the probe to avoid movement.
6. Maintain the experimental room at constant temperature (24 ± 1 °C) and humidity (~50-60%).
7. Turn off any external light (such as fluorescent and spot lamps) before performing the experiment to avoid external light-induced change.

3. Preparation of the Animals

1. Autoclave the surgical instruments and allow them to cool to room temperature before use.
2. Give the mice 10 min to acclimatize to the experimental environment before detecting pancreatic islet microvascular vasomotion by laser Doppler.
3. 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.
4. Cover the eyes of the mouse with pre-moistened medical gauze to prevent dryness.
5. Ensure that the mouse completely loses consciousness and no longer responds to tail or hindfoot pinches with forceps. 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.
6. 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.
7. Swab the abdominal skin of the mouse with betadine, and then 75% ethanol is used to swab the abdominal area clean.
8. Inject 2% lidocaine/0.5% bupivacaine (50/50) mixture subcutaneously.  Cut a ~3 cm-diameter hole in the center of a gauze sponge. Cover the abdominal region with the gauze sponge.
9. Lift the abdominal skin with forceps and make an initial vertical incision along the midline of abdomen using a scalpel or skin scissors.
10. 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.

4. Data Acquisition for Analysis

1. Run the software of the laser Doppler apparatus by clicking on "File" → "New" to create a new measurement file. To configure the connected monitors, under the "General" tab, set up the monitoring duration to "Free run." Use the factory default for the "LDF Monitor" tab. Click "Next."
2. Set up the graph display in the "display setup dialog box." Select the "Flux, Conc, Speed" channels by checking the respective boxes. Select the following parameters: "Data source for the channel" and "Label, Units and Color." Click "Next."
3. Enter user information about the subject and measurement (i.e., name and subject number, operator, monitoring time, comments, etc.) in the "file information dialog box" and click "Next" to finish the measurement configuration.
   NOTE: A measurement window is automatically created by the software (Figure 1B).
4. 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.
5. Click the "Start" toolbar icon to start recording the microvascular blood perfusion units (PU) data. Collect the PU data continuously for 1 min every run. Click "Stop" to stop the measurement. Select "File" → "Save as" to name and save the finished measurement file.
6. 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.
7. Close the abdominal muscle layer and the skin layer with a suture. Place the animals in clean cages after the experiments.
8. 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.  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.

5. Calculating the Parameters of Microvascular Vasomotion

1. Use the "Export" 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.
2. Calculate the average baseline perfusion unit (PU_b) (see step 4.6).
3. Calculate the average blood perfusion (PU_a) for 1 min of a measurement as follows: Average blood perfusion (PU_a) = PU - PU_b (Equation 1).
4. 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.
5. Calculate the amplitude (ΔPU) for each 1 min of measurement.
   1. Calculate the amplitude of microvascular vasomotion as the difference between the maximum (PU_max) and minimum (PU_min): Amplitude (ΔPU) = PU_max - PU_min (Equation 2).
6. Calculate the relative velocity (PU) for each 1 min of measurement.

Results

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...

Discussion

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 ...

Materials

List of materials used in this article

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