Methods
Evaluation of vascular density. The microvascular density of the peritoneum could in theory change with chronic diabetes, which would affect vascular permeability. Hence measures of vascular density are important for a correct interpretation of the results. We evaluate microvascular density according to a method previously reported(1). To avoid selection bias, we perform quantification in random segment of the visceral peritoneum of three predefined ileal loops (i.e, the most three distal loops of ileum). Images are taken with a 10x objective positioned at random in the mesenteric tissue. The microscopic stage is driven by a stepping motor control MS-4000 XYZ Automated Stage (Applied Scientific Instrumentation, Eugene, OR), operated by a computer through a USB interface. With the aid of the MS-4000 XYZ, the microscopic stage is driven through a meander consisting of 6 steps of 1 mm in the X direction and 6 steps of 1 mm in the Y direction. At each of these 36 positions, the microscopic image is recorded in 10-μm intervals above, through and below the plane of focus (z-plane). Multiple z-planes are used for 2D deconvolution analysis, which removes the out-of-focus light that is prevalent in epifluorescent microscopy, thus allowing for a clear definition of the vascular structures within the mesentery. The vessel length per area is determined for each microscopic image, and the average is calculated. Capillaries, defined as vessels with a luminal diameter <5 µm(2), are analyzed separately, and their relative contribution to total vessel length is also calculated. The vessel length per area, for each microscopic image, is calculated with the use of the software Image Pro Plus v5.1,
Measurement of vascular permeability. Permeability index of post-capillary venules in aging rats is determined by intravital fluorescent microscopy using a previously described technique(3) and according to principles outlined by Bekker and coworkers(4). The intravital microscopy setup described in 1a is used for these studies. The preparation is epi-illuminated with an intensified 100-W super high-pressure mercury lamp, and fluorescence is assessed using dichroic filter cubes [TR: excitation 560, emission 630 nm]. Neutral density filters are placed in the epifluorescence light path in the in vivo experiments. Texas Red-labeled albumin (TR-BSA; Molecular Probes) is used as a tracer for microvascular permeability to micromolecules. TR-BSA is administered intravenously at a dose of 25-mg/kg body wt. For the evaluation of macromolecular leakage, venular segments with a diameter of 25 to 40 µm and an unbranched length of approximately 150 µm without any other vessels in the immediate vicinity are selected for study. When traumatic leaks are observed, the experiment is discontinued. Otherwise, epifluorescence recordings are made every 10 min for 120 min, after a 30min stabilization period. A total of 4-5 randomly selected venules are studied in each rat. On the digitized image of the venule under study, the fluorescence intensity in three 10×50-µm intravascular regions (Iv) and the intensity in three 10x50-µm perivascular interstitium regions located 10 µm from the edge of the venule (It) are measured with computer-assisted digital image analyses. The average gray scale value, ranging from 0 for black to 256 for white, will be calculated for each area using the software Image Pro Plus v5.1, Mediacybernetics. The same three tissue fields are subsequently recorded for extravasation of macromolecules as a function of time. To minimize light exposure to the tissues, each measure of albumin leakage is performed during a brief (~5-15 sec) exposure to epi-illumination, controlled with a computer-operated shutter (Uniblitz VMM-D1, Vincent Associates, Rorchester, NY). The background intensity (I0; present before injection of TR-BSA) is subtracted from both It and Iv. As the molecule leaves the circulation, the intraluminal gray scale value falls and the perivascular gray scale value rises. Macromolecular leakage is defined as the permeability index (PI) calculated as (It -I0)/(Iv-I0). To allow for a more accurate determination of Iv we follow a previously published two-step approach(5). First, intravascular TR-BSA intensity is normalized using an impermeable arteriole, because TR-BSA leakage from the venule into the tissue (in the plane of the microscope objective) can cause an overestimation of the plasma concentration of TR-BSA (given the 2-dimensional limitation in determining the location of the fluorescence). Second, because the intravascular intensity of TR-BSA is influenced by the diameter of the vessel, a calibration between the average observed diameter and fluorescence intensity is also performed, according to a previously described technique(5). We are also aware of the fact that the value of Iv-I0 could in theory be an underestimate of the actual vascular fluorescence, if erythrocytes interfere with light absorbance and emission. However, control experiments performed in our lab indicate that Iv-I0 accurately reflects the relative concentration of plasma TR-BSA obtained from blood samples.
Intravital microscopy techniques are currently used by several laboratories to measure albumin permeability in the microcirculation. Seminal studies have carefully established the possible sources of errors and variability of this technique, including vessel geometry and density, as well as tracer distribution (6). Among factors affecting assay variability, photobleaching of the albumin tracer remains a main source of potential artifact since the method requires monitoring fluorescent intensity over a long period. We used intermittent illumination to minimize the effect of photobleaching. In preliminary studies, we found that light intensity of TR-BSA in vitro would be reduced by only 9% when it is subjected to intermittent illumination. If this extent of photobleaching also existed in vivo, then its effect on the calculated PI to TR-BSA would be an underestimation of PI by 9%. However, since interstitial tracers accumulate gradually, the error induced by photobleaching it is very likely to have been smaller then 9%. In the entire study, we discarded only three experiments where the vascular fluorescence intensity was abnormally decreasing during the measurement period. Post-mortem analyses revealed that loss of tracer signals was caused by intraintestinal bleeding resulting from non-specific surgical complications Overall, the PI values obtained in this study for albumin are quite consistent with similar measurements published by others in the species rat (7-9), thus confirming that the variability of our assay is within the commonly acceptable range.
References
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