The overall goal of the following experiment is to enable repeated measurements of the hydraulic conductivity or water permeability of the walls of Ular microvessels in the rat mesentary under controlled experimental conditions. This is achieved by cannulating a ular micro vessels to enable the oncotic and hydrostatic pressures in the lumen to be set and to allow the chemical composition of the profuse eight and Suses eight to be precisely controlled. Next, the vessel is occluded and red cells suspended in the PERFUSE eight, which act as flow markers can be observed moving in the lumen as indicators of trans vascular fluid exchange.
Then the position and velocity of the marker red cells are recorded either towards the site of occlusion or away from the occlusion site. Finally, occlusion is released and repeated for additional measurements or the vessel is rec. Cannulated and measurements are repeated under changed conditions.
Ultimately, paired measurements of hydraulic conductivity under control and test conditions can be made to enable the most direct investigation of the factors regulating micro vessels.Permeability. An advantage of the modified landis technique to investigate the regulation of capillary permeability is the perfusion step. Micro perfusion allows the permeability of the micro vessels wall to be measured under conditions where the PERFUSE eight composition, the surface area for exchange and the microvessels pressure are all directly controlled.
This method can address questions regarding cellular and molecular mechanisms of permeability regulation in micro vessels that are representative of intact organs, and it can provide comparison with results from cultured endothelial monolayers, which often are used to investigate the details of molecular mechanisms. Demonstrating our protocol is Joyce Clark, our laboratory manager. Joyce has been responsible for many of the improvements in this protocol during her time here as our lab manager.
To begin, use an electronic puller to form several clean boro silicate glass capillary tubes into tapered halves, each with an approximately one centimeter tip length using an air driven grinding wheel and a 0.5 micron abrasive paper bathed in water and a micro pipette holder set at a 30 degree angle from the horizontal bevel, the micro pipettes. After washing and drying the micro pipettes according to the text protocol, select micro pipettes with an opening about 50 microns long and a bevel length to width ratio between 3.1 and 3.5. They should also have a sharply tapered point, which helps penetrate the tough collagen fibers in the mesentery.
To make restrainers briefly hold a non beveled pulled glass capillary near a micro flame to form a blunt end. Repeat to make several restrainers and store in a dust-free box. Then make micro occludes by holding a non beveled pulled capillary tube at an angle under a micro flame and use a tubing adapter or broken micro pipette to gently bend the tube about four millimeters from the tip at an angle close to 120 degrees.
With respect to the shaft, it is important that the tip is neither sealed in the flame nor broken. After anesthetizing a rat and arranging the mesentary tissue and the gut according to the text protocol, position the animal tray on the microscope stage so that the mesentary is visible through the eye pieces. Position a gravity fed drip line of mammalian ringer solution to continuously super fuse.
The mesentary then use gauze pads to hold the gut in place, help retain moisture and wick excess ringers off the surface, adjust the flow and aspirate the effluent to maintain a constant layer of suse eight, identify target flar microvessels, which are unbranched segments downstream of convergent flow and one or two branches distal to true capillaries. Find an unbranched vessel having brisk blood flow and free of white cells sticking on the vessel wall.Possess. Position the test micro vessels in the center of the microscope field and move the restrainer into position near the chosen cannulation site.
To fill the micro pipette, draw perfuse eight and wash erythrocytes into a syringe prepared according to the text protocol, make sure that there are no bubbles in the tubing. Advance the tubing into the large end of the micro pipette until it abuts the tapered region. Apply a gentle quick push on the syringe plunger to fill the micro pipette tip.
A tiny stream or drop will appear at the tip when it is full. While withdrawing the tubing, gently push on the plunger to fill the micro pipette shaft. Next, remove small bubbles in the shaft by gently flicking it.
Then place the micro pipette into a pipette holder with a side port which allows a continuous fluid connection to a water manometer. Ensure that the holder, which is attached to a hydraulic drive and micro manipulator, is set at a slight horizontal angle so that the edge of the micro pipette taper does not hit the gut or tray. Using a syringe or water-filled rubber bulb, adjust the hydrostatic pressure applying to the fluid in the micro pipette by changing the height of the fluid in the water column of the manometer to about 40 centimeters of water above the mesentery.
Before positioning the filled micro pipette under the microscope, gently press the restrainer onto the tissue near the micro vessels, applying sufficient force to grip the tissue, draw the restrainer back slightly, stretching the tissue in line with the micro vessels so that the stresses in the mesenteric collagen fibers are aligned with the vessel. Align the micro pipette with the vessel and lower it into view through the eye pieces. Place the tip just upstream of the chosen cannulation site and lower it onto the tissue so that it partially obstructs flow within the vessel, but does not occlude it.
Using the hydraulic drive, cannulate the vessel by slowly driving the pipette tip into the vessel lumen, taking care not to push the tip through the other side of the vessel. As the micro pipette enters the lumen, the perfuse eight will rapidly wash the blood out of the vessel lumen and enter the circulation. Lower the perfusion pressure by adjusting the manometer until the red cells gently oscillate in the vessel lumen.
This determines the balance pressure, which measures the hydrostatic micro vessels pressure or PC at the distal end of the micro vessels segment. Maintain vessel perfusion above this balance pressure to carry out micro occlusion. Place the blocker above the perfused vessel near the lower edge of the microscope field using video and audio.
Verbally record the location of the block site as seen in the I piece red. The block site is 57 With the tip of the blocker placed just above the micro vessel. Use the fine Z control of the micro manipulator to gently lower the blocker until the flow in the vessel is occluded.
Use audio to note the manometer pressure. The pressure is 50. Then apply occlusion for three to 10 seconds and release.
To restore free perfusion, move the block site up the vessel toward the pipette tip in five to 10 micron steps, carrying out no more than three to five occlusions at each location to prevent vessel wall damage at the block site. Analyze the data according to the text protocol. This time course measures the changes in hydraulic conductivity or LP.In a rat ular micro vessels successfully cannulated with four perfuse eights.
The magnitude of LP at constant pressure was used to measure changes in micro vessels. Wall permeability when perfused with 1%BSA as a control versus 10 molar of the inflammatory agent Brady Kinin or bk. As shown here, BK caused a transient increase in LP that returned to control levels within 10 to 15 minutes.
After reestablishing baseline levels with 1%BSA 10 nanomolar. BK was reperfused along with one micromolar pH scene one phosphate or S one P.The results indicate the S one P significantly attenuated the response to bk. For some experimental designs, it is important to estimate both the solut reflection coefficient for a macromolecule and the lp.
This is accomplished best by measuring JV at multiple pressures on each individual micro vessels. This experiment estimated both LP and the effective oncotic pressure of 5%albumin in the PERFUSE eight when there was no albumin in the tissue surrounding the micro vessels. JV over S was measured at three different pressures and plotted.Here.
The LP is the slope of the relation between JV over S and pressure, and the intercept on the pressure axis is the effective oncotic pressure difference across the vessel wall. When combined with other techniques such as immunofluorescent labeling and confocal microscopy, or even transmission electron microscopy, micro profusion provides powerful analysis of microvascular endothelial and function using vessels of known permeability. After watching this video, you should have a good understanding of how to make repeated measurements of the hydraulic conductivity or water permeability of ular microvessels in the rat mesentry under controlled experimental conditions.
You can use this approach in currently available rat models, but I'm particularly interested in the possibility that it can be used in new genetically modified rats that will become available with the CRISPR Cas nine technology, and I hope it is extended in this way.
