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Yong
Jiang, Ai-Hua Liu, Ke-Seng Zhao, Chinese
PLA Key Laboratory for Shock and Microcirculation, The First Mil
itary medical University Guangzhou 510515, China
Dr. Yong
Jiang,
male born on 1964-10-25
in Henan Province, graduated a
nd got a Ph.D. degree from the Academy of Military Medical Sciences
in 1997, now
professor of pathophysiology, majoring shock and cellular signal
transduction,
having more than 50 papers published in international or national
major journals
.
Supported by National Natural Science Foundation of Chin
a, No. 39270852
Correspondence to: Dr Yong
Jiang,
Chinese PLA Key Laboratory for
Shock and Microcirculation, The First Military Medical University,
Guangzhou 51
0515, China.
Telephone:
+86-20-85148376，Fax.
+86-20-87705671
Received:
1999-01-20
Subject
headings: microcirculation;
leukocyte; leukocyte-endo
thelium
interaction; mesentery
Jiang
Y,Liu AH, Zhao KS. Studies on the flow and distribution of
leukocytes in
mesentery microcirculation of rats.
World J Gastroentero, 1999;5(3):231-234
Abstract
AIM:
To study the effect of leukocyte-endothelium
interactio
n (LEI) on the flow and distribution of leukocytes in
microcirculation under physiological condition.
METHODS: A
microcirculation image multiple parameter computer an
alysis system (MIMPCAS) was used to study the flow and distribution
of leukocyte
s in mesentery microcirculation of rats in vivo.
RESULTS: The
difference of visible leukocyte flux (VLF) was as
high as 131 times in the arterioles and venules with similar
diameter and blood
velocity. The visible leukocytes rolled along the blood vessel wall
as a “jerky
”
movement. The frequency distribution of the visible leukocyte
velocity (VLV
) showed a “two
peak”
curve. The low peak value was on 10μm/s-15μm/s
while the high peak fell between 25μm/s-30μm/s.
With the increase of diameter of venules, VLF increased while the
VLV remained at the same level. With the increa
se of RBC velocity, VLV trends to elevate and VLF to fall down.
CONCLUSION: The
results herein might provide a basic theory for
the study on the mechanism of LEI under physiological condition and
novel metho
ds for the prevention and treatment of high LEI in many pathological
processes.
INTRODUCTION
Leukocyte-endothelium
interaction (LEI) exists in many pathophysiological proce
sses, such as inflammation, burns, tumor and shock［1,2］.
In the recent two decades, quantitative studies on the interaction
of leukocyte-endothelium
have been carried out and the change of the flow and distribution of
leukocytes is the basis for the abnormal increase of LEI［3-5］.
High level LEI would b
ring about the blockage of blood vessels and decrease of blood
perfusion［6,7］.Therefore,
it is important to study the flow and distribution of leukocytes in
microcirculation. We used a microcirculation image multiple
parameter computer analysis system (MIMPCAS)［4］to
study the characteristic of the flow and distribution of leukocytes
in mesentery microcirculation of rats, and analyzed its influencing
factors.
MATERIALS AND METHODS
Five Sprague-Dawley
rats were anesthetized with a mixture of 133g/L ure
thane and 10g/L chloralose (6mL/kg, im)［4］.
The abdomen of r
ats was open by the incision on the midline under the xiphoid. Small
intestinal loops were pulled out and the mesentery was mounted on a
hollowed transparent pedestal for observation. The specimen was
suffused with a balanced 37℃
Kreb′s
solution to maintain relatively normal condition in temperature and
environment.
An
Olympus microscope with a halogen lamp and Leitz long distance lens
(20×)
was used to observe the third order arterioles and venules. Being
transmitted through a low-light
level camera of model 1319, the signal was displayed on a Hitac
hi color monitor. A JVC recorder was used for off
line measurement. Each specimen was
recorded no more than 30 minutes so as not to affect the mesentery
microcirculation［3］.
The MIMPCAS was used to measure the diameter (D) of blood vessels,
the velocity of red blood cells (Vrbc), the visible
leukocyte velocity (VLV), the visible leukocyte flux (VLF) and the
adhesive leukocyte count (ALC) following the procedure described
previously［4］.
The
following formula was used to calculate the parameters of
microcirculation［4,6,9］:
1. mean blood velocity,
Vmean=Vrbc/1.6;
2. flow volume of blood, F=Vmean×π×D2/4;
3. shearrate, γw=8×(Vmean/D);
4. total leukocyte flux, TLF=(60×F)×K×10-6,
K is the amount of leukocytes in the blood［7］;
5. invisible leukocyte flux, ILF=TLF-VLF.
The
results were represented by mean ±
standard deviation (x-±s)
an
d the significance of difference was judged by Student′s
t test.
RESULTS
Leukocytes flow and distribution in
microcirculation of rat mesentery u
nder physiological condition
Twenty arterioles with a diameter
between 11μm-45μm
were selected for observation. Only one leukocyte rolled along the
wall an
d there was no leukocyte sticking on it in the third order
arterioles. In the 20 capillaries with an average diameter of 6.3μm±1.7μm,
the visible leukocyte flux wa
s 0.2
cells/min±0.4cells/min
and there were no plugging leukocytes. In th
e 20 venules with a diameter of 10μm-50μm,
the visible leukocyte flux was 13.3cells/min±7.2cells/min
and there were about 0.3±0.3
leukocytes sticking on the wall within a length of 94μm
of blood vessels (Table 1). Under the physiological con
dition, the flow and distribution of leukocytes in different blood
vessels varie
s to a large extent. The visible leukocyte flux (VLF) differed
significantly between the venules and arteries with comparable
diameter and blood velocity and the value of VLF in venules was as
high as 131 times that of arterioles. Due to the interaction of
leukocyte and endothelium which mainly occurred in venules, further
studies were curried out on the flow and distribution of leukocytes
in mesentery venules.
Table 1 The flow and distribution of leukocytes in the mesentery
m
icrovasculature of rats under physiological condition
|
|
Arteriole
|
Capillary
|
Venule
|
|
Number
|
20
|
20
|
20
|
|
D(μm)
|
22.6±9.1
|
6.3±1.7
|
25.1±10.6
|
|
Vmean(mm/s)
|
1.07±0.3
|
0.45±0.12
|
0.86±0.27
|
|
Flow(pL/s)
|
440.1±439.2
|
14.1±6.7
|
430.7±412.4
|
|
γ·w(s-1)
|
371.1±212.8
|
570.4±189.7
|
272.1±189.2
|
|
TLF(cells/min)
|
91.1±90.9
|
2.9±1.4
|
89.2±85.4
|
|
VLF(cells/min)
|
0.1±0.2b
|
0.2±0.4
|
13.1±7.2b
|
|
ILF(cells/min)
|
91.0±90.9
|
2.7±1.3
|
76.1±77.8
|
|
ALC(cells/94μm)
|
0
|
0
|
0.3±0.3
|
bP＜0.01,
t=8.07.
The characteristic of the flow and
distribution of visible leukocytes
The space characteristic of the flow
and distribution of visible leukocytes Ten
sampling lines on vessel with equal distance were set perpend
icular to the longitudinal vessel and the velocity of leukocyte
passing through each line was measured. The variation of the
velocity of leukocyte reflected the characteristic of its temporal
distribution. Each leukocyte passing through the sampling lines with
a large variation on velocity suggested that leukocyte rolling along
the wall of blood vessels took a “jerky”
movement (Figure 1). However, the average velocity for a leukocyte
passing through a vessel with a definite length was similar, about
20μm/s.
The time characteristic of the flow and
distribution of visible leukocyt
es For the measurement, one line was
set on a third order venule (D:
37μm)
of mesentery of rat. The velocity and flux of all the visible
leukocytes passing the measuring line were determined in 10s as
one unit, and measurements were continuously performed 6 times in 1
minute. It w
as found that visible leukocyte velocity and visible leukocyte flux
changed temp
orarily (Figure 2).
Frequency distribution of VLV The
velocities of 400 visible leu
kocytes in 30 third branch venules were measured, of which the
frequency distrib
ution is shown in Figure 3. The frequency distribution of VLV
presented with the
characteristic of double peaks. Low peak value was about 10μm/s-15μm/swhile
the high peak was around 25μm/s-30μm/s.Leukocytes
with velocity below 5μm/s
or above 50μm/s
were rarely found.
The influence of vessel diameter and
blood velocity on the flow and di
stribution of leukocytes
Visible leukocyte flux and visible
leukocyte velocity were measured in 20 capillaries of mesentery of
rats. Eight venules with similar blood velocity (Vrbc: 1.2mm/s±0.1mm/s)
and vessel diameter (D: 10μm-50
μm)
were selected for the observation of influence of vessel diameter on
the fl
ow and distribution of leukocytes. It was found that with the
increase of blood
vessel diameter, visible leukocyte flux increased while visible
leukocyte veloci
ty remained relatively stable. Twelve venules with similar diameter
(31.5
μm±1.1μm)
were selected for the observation of the influence of bloo
d velocity on the flow and distribution.The blood velocity in these
vessels ranged from 0.27mm/s
to
1.38mm
/s. Following the increase of Vrbc, visible leukocyte
velocity inc
reased while visible leukocyte flux decreased as shown in Figure 4.
Figure1(PDF)
Leukocytes
rolling along the blood vessel w
all showed a “jerky”
movement.(A. Multiple sampling scheme for the velocity determination used by MIMPCAS; B. Three leukocytes passed through 10
sampling li
nes with a large variation of velocity.)
Figure2(PDF)
The time-dependent
changes of VLV and VLF
in the third order venules of rat mesentery.
Figure3(PDF)
The frequency histogram of VLV.
Figure4(PDF)
Effect of vessel diameter and red blood cell velocity
on the flow and distribution of visible leukocytes in the venules of rats.
DISCUSSION
Recently, wide interest has been shown in the research of
leukocyte-endothelium
interaction.Studies on the rheological behavior of leukocytes in the
microcirculation have promoted the understanding on the mechanism of
cellular adhesion. These studies mainly involved two aspects, i.e.,
one is the interaction between leukocytes and red blood cells and
the other is that between leukocytes and endothelial cells［6,7］.
It was found in this study that leukocytes
scarcely rolled on the wall or firmly
sticked in arterioles. Under normal condition, sticking leukocytes
in venules were rarely observed while some leukocytes rolled along
the vessel wall in the marginal stream, suggesting that the flow and
distribution of leukocyte in venules and arterioles was
significantly different. There exist some interactions betwee
n leukocytes and endothelium in venules, for which the major
phenomenon is the leukocytes rolling on the endothelium of venule
wall. The development of leukocyte rolling and sticking on
endothelium depends on two forces: leukocyte-endotheli
um adhesive forces and hemodynamic dispersal forces, i.e., shear
stress［1,8］.
Mayrovitz had suggested that the adhesion of leukocytes on the
vessel wall might be mainly related to shear stress, for the
adhesion of leukocytes existed on the wall of post-capillary
venules［7］.
However, the results herein
showed that VLV varied to a large extent even in the arterioles and
venules with a similar shear stress, suggesting that under
physiological condition the difference of leukocyte flow and
distribution in different vessels mainly came from the
characteristic of endothelial cells and the micro-environment
around leukocyt
es［9］.
The rolling of leukocytes along the walls
presented with an uneven “jerky”
movement. The balance between adhesive forces and dispersal forces
was broken by the non-homogeneity
of endothelium and that of hemodynamic forces, which brought
about the rolling of leukocytes with the characteristic of non
stable speed
［3,8,10］.
The factors that influence homogeneity of endothelium include non
-even
surface of endothelium, local characteristic of endothelium, surface
distribution of charge, the concentration of reactive substances,
etc., while that for hemodynamic force, were temporal variation of
blood velocity and local concentration of red blood cells. The
results from the analysis on the frequency of VLV showed that the
rolling leukocytes with a velocity lower than 10μm/s
had a potential to stick on the endothelium of blood vessels, while
the rolling leukocytes with a speed higher than 30μm/s
tended to merge into the cent
ral stream of blood. The two peaks of the distribution of VLV
suggested that the
re were at least two kinds of adhesive molecules with different
property, by whi
ch two different velocities of leukocyte rolling along the walls
were mediated.
However, the adhesive molecules involved in the leukocyte-endothelium
interacti
on are waiting to be identified and it is also necessary to pay more
attention to the study on the mechanism of cellular adhesion.
In
this study, the impact of diameter of blood vessels and blood flow
of venules on the flow and distribution of leukocytes was analyzed.
The diameter of blood vessels were found to have a significant
effect on the flow and distribution of leukocytes. In the blood
vessels with a larger diameter, visible leukocyte flux (VLF)
increased significantly, but the visible leukocyte velocity (VLV)
kept stable. Atherton had suggested that the temporal contact
between leukocyte and endothelium should be taken as an inelastic
collision［3］.
The increase of adhesion force would bring about more chances of
random collision and higher degree of inelastic collision between
leukocytes and endothelium. Visible leukocyte flux mainly reflects
the random collision between leukocyte and endothelium, while
visible leukocyte velocity indicates the degree of inelastic
collision. In the larger vessels, the leukocyte flux and the area of
endothelium are also larger, so the random contact chances increase
to bring about high flux of visible leukocyte. The change of
diameter would not impact the adhesion force, so visible leukocyte
velocity was the same.
Given a certain extent of blood viscosity,
shear stress is determined by blood velocity under the condition of
definite vessel diameter［2,6,8,10］.
The shear stress is high in the blood vessels with fast flow of
blood, which will reduce the chance of collision between leukocyte
and endothelium. Therefore in the vessels with fast blood flow,
visible leukocyte flux is low while visible leukocyte velocity is
high.
In summary, the flow and distribution of
leukocytes in the mesentery microcirculation of rats was studied in
vivo, and the influencial factors on which were explored under
normal conditions. The result of this study is helpful for the
understanding of the mechanism for leukocyte endothelium interaction
in physiological state and provides theoretic basis for the study
and treatment of increased LEI in pathological processes.
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