An optical system was developed to visualize hepatic microcirculation with fluorescent labeled erythrocytes and to measure the partial pressure of oxygen in the micro vessels with laser assisted phospho imagery. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter blood flow velocity, and oxygen tension. In hepatic microcirculation, blood flows from portal ES to central Es, creating an oxygen gradient between the two.
Oxygen concentration in the liver affects both enzyme expression and pathology making blood flow analysis and oxygen measurement. Important topics of research. Our experimental method involves intravenously administering red blood cells labeled with fluorescent.
In order to visualize microcirculation using fluorescence for oxygen partial pressure measurement, we intravenously administer palladium porphyrin, which emits phosphorescent in the bloodstream when excited by a laser to a degree dependent on concentration of surrounding oxygen. This process allows quantitative analysis of oxygen, partial pressure measuring hepatic microcirculation showed portal and central venal oxygen partial pressure of 59.8 and 38.9 millimeters of mercury respectively demonstrating effective measurement of oxygen concentration. Next, we applied this methodology to a hepatitis model.
We found elevated oxygen partial pressure in portal ES sinusoidal and central es, but a decreased pressure differential between portal and central es. This is likely due to decreased oxygen consumption in the central Ven area due to hepatocyte necrosis. These results show that our methodology of using an optical system to measure hepatic hemodynamics and oxygen consumption is capable of elucidating mechanisms of various liver diseases In hepatic micro circulation.
Oxygen gradient is an essential parameter involving expressions of enzymes in periportal and paracentral regions, kinds of hepatic diseases related to micro distributions, so it's important to quantify blood flow and oxygen consumption. To clarify the mechanisms, the advantage of our method is non-contact and continuous optical measurement can quantify blood flow, blast vessel diameters and oxygen gradient in hepatic micro circulation. Whole blood was withdrawn from a donor mouse and the erythrocytes were washed twice with PBS by centrifugation at 400 G for five minutes.
Next, dissolve two milligrams of FITC in one milliliter, 100 millimolar of DI sodium hydrogen phosphate before filtering through a 0.2 micron pore membrane. Next place a one milliliter FITC solution in a test tube along with a 0.15 milliliter three millimolar dexterous solution, a 0.25 milliliter 180 millimolar DI sodium hydrogen phosphate, and a 1.5 milliliter 100 millimolar DI sodium hydrogen phosphate and tap to mix. Keep it room temperature For two hours before washing the stained RBCs twice with PBS.
Next, place a small amount of RBC suspension on a glass slide and confirm both normal RBC appearance and sufficient fluorescence suspend 0.1 milliliter of RBCs in 0.9 milliliter of PBS And keep it four degrees Celsius until Use next, Dissolve 500 milligrams of BSA in 10 milliliters of PBS. Then add 30 milligrams of palladium porphyrin and agitate overnight centrifuge, the solution to remove any unsolved palladium porphyrin And Filter the SUP natin through a 0.2 micron po membrane eloquent, one milliliter of filtrate into test tubes and store at minus 20 degrees Celsius. Taken care to avoid repeated freezing and thawing.
To visualize The microcirculation microscopically, cut a 20 millimeter diameter hole in a plastic plate and cover the hole with a square cover glass 30 millimeters to a side. Spread the main hepatic lobe onto a plastic plate in a prone position after cannulating a tail vein. Next, use multiple three millimeter by eight millimeter sections of plastic food wrap to cover the area surrounding the lobe to prevent movement from respiration and drying.
Visualize the hepatic circulation with a transmission microscope and confirm there is no stasis of the blood flow in field of view for at least 15 minutes. To observe the blood flow gradually inject. Point two milliliters of fluorescently labeled RBCs.
To measure the oxygen partial pressure inject 0.2 milliliters of palladium porphyrin solution. FITC labeled RBCs will account for one 50th of all the RBCs in circulation in The visualized region, excite the FITC by Irradiating it with a mercury lamp passed through a 450 to 490 nanometer band pass filter and record the fluorescence with a CCD camera. Palladium Porphyrin phosphorescence is relatively weak and requires a high sensitivity detector and a darkened room.
Palladium porphyrin absorption peaks are around 410 and 532 nanometers, and the second harmonic wavelength of 532 nanometers is recommended. This wavelength is produced by a neodymium YAG pulse laser connect the laser beam to an inverted microscope and adjusts such that the beam illuminates the center of the focal plane. The spatial resolution of the laser is dependent on the spot size, which may be adjusted by passing the beam through a pinhole.
Attach a long pass filter and photomultiplier tube to the microscope as well to detect phosphorescence. The PMT should be sensitive to red wavelengths, especially around 700 nanometers. Sample the phosphorescent signal and calculate the decay time constant by applying an exponential function.
In this experiment, we sampled 500 points at 200 kilohertz. To calibrate, prepare two palladium porphyrin solutions with an oxygen partial pressure of 150 and zero millimeters of mercury each. For the zero millimeters of mercury solution, add 1%dihi sodium to the samples to create the condition where oxygen is absent during calibration.
Maintain a sample pH of 7.4 and a temperature of 37 degrees Celsius. Position the mouse on the microscope stage and adjust the microvessels for which you want to visualize hepatic microcirculation to the point of the laser spot fast mice with Free access to water overnight, inject acetaminophen at 200 milligrams per kilogram intraperitoneal 24 hours from injection, anesthetize the mouse and expose the liver with a median incision. If there is hepatitis, it will be easily visible to the naked Eye through necrosis in the paracentral region.
Our measurement of partial oxygen pressure and Hepatic micro vessels in mice showed mean oxygen pressures for portal es, sinus soils, and central vees of 59.8, 48.2, and 38.9 millimeters of mercury respectively. In other words, the oxygen gradient from portal to central ES indicates sufficient release of oxygen from RBCs as they pass through the sinusoids. We prepared acetaminophen induced hepatitis mice to measure hepatic microvessels.
As an example pathology model, we found that oxygen partial pressures in portal vees, sinusoidal and central Es were higher in the acetaminophen group compared to normal mice. The oxygen partial pressure differential between portal and central vees was 20.9 millimeters of mercury for normal mice and 16.9 millimeters of mercury for the hepatitis model mice. The oxygen partial pressure differential between portal and central vees was 20.9 millimeters of mercury for normal mice and 16.9 millimeters of mercury for the hepatitis model mice.
The oxygen gradient in hepatic microcirculation mainly arises due to the supply of oxygen from the portal ven that is consumed by hepatocytes, hepatocyte necrosis in the central venal area caused by acetaminophen results in decreased oxygen consumption, downstream microcirculation, and therefore issues related to hemodynamics. Oxygen supply and oxygen consumption are involved in many Forms of liver disease. We made acute hepatitis model and found increase of oxygen tension in micro vessels.
Our optical system can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, rather flow distribution and oxygen consumption.
