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