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 10×50µm 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
1. De Vriese AS, Tilton RG, Stephan CC, Lameire NH: Vascular endothelial growth
factor is essential for hyperglycemia-induced structural and functional alterations of the
peritoneal membrane. J Am Soc Nephrol 12:1734-1741, 2001
2. Wiedeman MP: Dimensions of blood vessels from distributing artery to collecting
vein. Circ Res 12:375-378, 1963
3. Kurose I, Anderson DC, Miyasaka M, Tamatani T, Paulson JC, Todd RF, Rusche JR,
Granger DN: Molecular determinants of reperfusion-induced leukocyte adhesion and
vascular protein leakage. Circ Res 74:336-343, 1994
4. Bekker AY, Ritter AB, Duran WN: Analysis of microvascular permeability to
macromolecules by video-image digital processing. Microvasc Res 38:200-216, 1989
5. Harris NR, Langlois KW: Age-dependent responses of the mesenteric vasculature to
ischemia-reperfusion. Am J Physiol 274:H1509-1515, 1998
6. Yuan F, Leunig M, Berk DA, Jain RK: Microvascular permeability of albumin,
vascular surface area, and vascular volume measured in human adenocarcinoma LS174T
using dorsal chamber in SCID mice. Microvasc Res 45:269-289, 1993
7. Kurose I, Argenbright LW, Anderson DC, Tolley J, Miyasaka M, Harris N, Granger
DN: Reperfusion-induced leukocyte adhesion and vascular protein leakage in normal and
hypercholesterolemic rats. Am J Physiol 273:H854-860, 1997
8. Harris NR: Arteriovenous pairing: a determinant of capillary exchange. News Physiol
Sci 18:83-87, 2003
9. Rumbaut RE, Harris NR, Sial AJ, Huxley VH, Granger DN: Leakage responses to LNAME differ with the fluorescent dye used to label albumin. Am J Physiol 276:H333339, 1999