ORIGINAL ARTICLE
Arteriovenous shunting blood ﬂow is intravitally observedin the stomach after thermal injury in rats
Masashi Yoshida, Go Wakabayashi, Hideki Ishikawa, Shigeyuki Kawachi, Minoru Tanabe, Yoshihide Otani,
Motohide Shimazu, Tetsuro Kubota and Masaki Kitajima
Department of Surgery, School of Medicine, Keio University, Tokyo, Japan(Received for publication on June 12, 2002)

Abstract. Microcirculatory disturbance of the gastric wall is a crucial factor in the development ofgastric mucosal lesions induced by Helicobacter pylori, anti-inﬂammatory drugs and stress. Opening of
the arteriovenous shunting channel after thermal injury is one of the possible mechanisms to reduce
mucosal blood ﬂow. However, no in vivo observation of arteriovenous shunting blood ﬂow in thestomach has been reported. To assess gastric microcirculatory disturbance, especially arteriovenous
shunting blood ﬂow after thermal injury by in vivo microsciopy, male Wistar rats were anesthetizedand thermal injury was inﬂicted on the back skin. Gastric microvascular images were observed by invivo microscopy. Rolling of leukocytes labelled with carboxyﬂuorescein diacetate, succinimidyl ester
were counted and blood ﬂow dynamics were observed by ﬂow of a micro dye, monastral blue B(MBB). The endothelial damage was assessed by deposits of MBB 5 min after the administration.
Arteriovenous shunting blood ﬂow is difﬁcult to detect by normal methods, but it could be observed byﬂow of MBB after thermal injury. Statistical analysis showed a signiﬁcant difference in the ratio ofarteriovenous shunting blood ﬂow detection between the control (no injury) (0%; n F 15) and thermal
injury (5 hrs after thermal injury) (28.6%; n F 14) groups. In the thermal injury group, the percentageof rolling leukocytes and the area of monastral blue B deposits increased, and the venular walls tended
to be irregular. The total length of erosion increased time-dependently after thermal injury, and thelength at 5 hrs was approximately 2 times larger than that at 2 hrs. Arteriovenous shunting blood ﬂow is
intravitally observed after thermal injury. A-V shunting blood ﬂow can be a cause of mucosal hypoperfusion. It is suggested that the microcirculatory disturbance seen 5 hrs after thermal injury is contributed to the ﬁnal step of erosion formation. (Keio J Med 51 (4): 193–200, December 2002)
Key words: burn, endothelial damage, rolling of leukocytes, arteriovenous shunt, stress

Introduction
Microcirculatory disturbance of the gastric wall
is a crucial factor in the development of gastricmucosal lesions induced by Helicobacter pylori,1–3 antiinﬂammatory drugs4,5 and stress.6,7 After thermal injury to the dorsal skin in rats, macroscopic hemorrhagic
erosion developed in 14.3% at 15 min, 42.9% at 2 hrs,
100% at 5 hrs, and 85.7% at 12 hrs.8 Macroscopic
hemorrhagic erosion is formed within 5 hours afterthermal injury. Under the stereoscopic microscope,superﬁcial gastric erosion could be observed in all

rats studied at 15 min. Gastric mucosal blood ﬂow
decreased at 15 minutes, partially improved at 2 hrs,
and decreased again at 5 hrs after thermal injury.9 Since
the blood ﬂow was depressed especially at 15 minutes
and 5 hours, we have investigated mechanisms of thedecrease in gastric mucosal blood ﬂow at 15 minutes
and 5 hours. Silicon rubber casts of the vasculature
revealed contraction of arterioles 15 min after thermal
injury to the dorsal skin in the rat model.10 We also
observed the gastric microvessels by in vivo microscopy
which was ﬁrst described by Guth and Rosenberg.11,12
In vivo microscopic observation of gastric micro-

Reprint requests to: Dr. Masashi Yoshida, Department of Surgery, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo,
160-8582, Japan
193

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Yoshida M, et al: Arteriovenous shunting blood ﬂow

circulation showed also the contraction of arterioles
15 min after thermal injury.13 The contraction is responsible for the decrease in gastric mucosal blood ﬂow
15 min after thermal injury. Five hours after thermal
injury, in vivo microscopy showed irregular constrictionof the venules caused by suppressed production of nitricoxide.14 Another interesting ﬁnding of the microcirculation 5 hrs after thermal injury is the opening ofarteriovenous (A-V) shunting channels, as demonstrated by silicone rubber casts10,15 and in vivo microscopy.9 A-V shunting channels in the submucosa or baseof the gastric mucosa are thought to divert blood ﬂow
from the mucosa and thereby contribute to mucosal lesion formation. No study, however, has demonstrated
direct observation of A-V shunting blood ﬂow, which is
difﬁcult to identify by ordinary in vivo observation.
Monastral blue B (MBB) is a stable pigment of negligible solubility in water which deposits in the vessels
where permeability is increased.16 MBB has previously
been used to assess gastric mucosal microcirculatory
disturbance17 using permanent specimens. We applied
MBB to in vivo observation of gastric microcirculation, with the idea that observation of both deposits andﬂow of MBB would allow assessment of endothelial
damage and A-V shunting blood ﬂow. One factor
inducing endothelial damage and vascular hyperpermeability which may be assessed by MBB is leukocyteendothelial interaction including the rolling of leukocytes.18 We have previously observed the behavior of
leukocytes 5 hrs after thermal injury using acridine orange.9 However, ﬂuorescent illumination of leukocytes
after administration of acridine orange was rapidly lostand rolling of leukocytes could be counted for only 30
sec. In the present study, we attempted to observe therolling of leukocytes labelled with 5-(and 6)-carboxyﬂuorescein diacetate, succinimidyl ester (CFDASE).
CFDASE is a nonﬂuorescent precursor that diffuses
into cells and forms a stable ﬂuorochrome, carboxyﬂuorescein succinimidyl ester (CFSE), after being catalyzed by an esterase which occurs predominantly inleukocytes and platelets.19 CFSE ﬂuorescence provides
for a extended duration of in vivo observation.
The present study attempted to assess gastric microcirculatory disturbance 5 hrs after thermal injury using
MBB and CFDASE. We were particularly interested ininvestigating whether it is possible to observe A-V
shunting blood ﬂow directly using in vivo microscopy.
Materials and Methods

30% full skin-thickness dorsal scald burn was caused byplacing them in scalding water for 15 seconds as described previously.20 The animals showed no reaction
to pain. The animal experimentation guidelines of
the Keio University School of Medicine were followed.
The experiment was performed on two groups: control
group (n ¼ 15) (without thermal injury) and thermal
injury group (n ¼ 14) (5 hrs after thermal injury).
Gastric microvascular images
Microvascular images were observed with an intravital microscope as described previously.21,22 Animals
in both groups were anesthetized with sodium pentbarbital (20 mg/kg im), and laparotomy was performed
just before the observation. A portion (about 2 mm indiameter) of the serosa and muscle layer of the gastricwall was resected gently, the stomach was mounted on aplastic stage, and a light rod was inserted through an
incision in the forestomach. Microcirculatory images
were observed with 10 objective, captured with acharge-coupled video camera system (CC-980; Flovel,
Tokyo, Japan) and recorded on S-VHS videotapes.
Rolling leukocytes
Before thermal injury was inﬂicted, 0.1 ml of 15.6
mM/L 5-(and 6)-carboxyﬂuorescein diacetate, succinimidyl ester (CFDASE) (Molecular Probes, Eugene,
OR, USA) was administered intravenously to label
leukocytes. Illuminated cells were monitored by ﬂuorescence microscopy using a silicon intensiﬁer target
image tube camera (Hamamatsu Photonics, Shizuoka,
Japan) according to a method described previously.23
The leukocytes which stick to the same place for morethan 30 sec were deﬁned as sticking. Leukocytes rolling
along the wall of venules in the basal region of the gastric mucosa were observed on videotaped images. Leukocytes passing through the conﬂuence of a prevenuleand a venule were counted for 3 min in the ﬁeld
observed with a 10 objective and the percentage ofrolling leukocytes was calculated as the number of therolling leukocytes/the number of leukocytes passing
through the conﬂuence of prevenules and venules 100(%), as described previously.13 Since the number ofcirculating leukocytes can change, we decided to calculate the percentage. Leukocytes were counted at threedifferent conﬂuences in each rat and the average values
were compared.

Animals and procedures

Vascular labeling with monastral blue B

Male Wistar rats weighing 220–270 g were fasted for
24 hours with free access to water before the experiments. They were anesthetized with diethyl ether, and aThree percent suspensions of MBB (Sigma ChemicalCo., St Louis, MO, USA) were administered intravenously (0.1 ml/100 g body weight) as described pre-

Keio J Med 2002; 51 (4): 193–200

195

viously.16 Deposits of MBB in venules were thenobserved intravitally with a CCD camera 5 min afteradministration. The percentage of the area of MBB
deposits in venules in two ﬁelds (6 objective) was calculated using NIH Image software version 1.56 (Aladdin systems Co, Watsonville, USA) on a Macintosh
computer.
Intravital observation of arteriovenous shunting of blood
ﬂow
MBB deposits were the results of the endothelial
damage. We carefully observed the ﬂow of MBB for thedetection of the A-V shunting blood ﬂow for 5 min afteradministration. It was reported that the mucosal venules connected with the mucosal capillary network onlyat, or very close to, the luminal surface and passed
through the mucosa without receiving further tributaries, i.e. all mucosal capillary blood must drain via
vessels close to the stomach lumen.24 Therefore, any
blood ﬂow draining at the base of the mucosa without
ﬂow into the mucosal layer can be recognized as A-V
shunting blood ﬂow. A-V shunting blood ﬂow can be
identiﬁed if a venule accepts blood ﬂow from an arteriole at the base of the gastric mucosa without ﬂow into
the mucoosal layer. A-V shunting of blood ﬂow wasrecognized only in cases in which both the outﬂow of
MBB from the arteriole and the inﬂow of the same
MBB grain into the venule were observed.
Assessment of the mucosal lesions
Animals were killed at 15 min (n ¼ 8), 2 hrs (n ¼ 8)
and 5 hrs (n ¼ 12), and the length of erosions wasmeasured with stereoscopic microscopy and the total
length of all erosions per rat (TLE) was calculated.
Statistical analysis
Data were presented as means þ S.D. Statistical signiﬁcance of the differences between two groups wasdetermined by the Mann-Whitney test and Fisher’s exact test.

Fig. 1 Gastric microvascular images. 1A: The walls of control arterioles and venules. 1B: Irregularity of the venular walls and abnormal
venules in the thermal injury group.

Results
Gastric microvascular images

Rolling leukocytes

The walls of arterioles and venules in the control
group were basically smooth and there was no evident
contraction during the experiment up to 30 min (Fig.
1A). In the thermal injury group, irregularity of thevenular walls and abnormal venular constrictions werealways observed, but arteriolar constriction was rarely
observed (Fig. 1B).

Fluorescence of leukocytes was sufﬁciently stable forlong observation and counting the number of leukocytes for 3 min was reproducible. The percentage ofrolling leukocytes which passed the conﬂuence of prevenules and venules was 1:9 þ 3:0% in the control
group and 20:8 þ 7:1% in the thermal injury group, withthis difference signiﬁcant (p < 0.01) (Fig. 2).

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Yoshida M, et al: Arteriovenous shunting blood ﬂow

Fig. 2 Percentage of rolling leukocytes. Leukocytes passing though
the conﬂuence of a prevenule and venule were counted for 3 min andthe percentage of rolling leukocytes was calculated. Values signiﬁcantly increased 5 hrs after thermal injury (** P < 0.01).

Vascular labeling with monastral blue B
Few venular deposits of MBB were seen in the control group but were evident in the thermal injury group(Fig. 3), with the percentage area of MBB deposits
in venules being 0.7 þ 1.3% in the control group and13.4 þ 12.7% in the thermal injury group (p < 0.01)(Fig. 4).
Intravital observation of arteriovenous shunting
Although it was difﬁcult to demonstrate the continuity of A-V shunting channels from arterioles to venules
by two dimensional observation, A-V shunting was
identiﬁed using MBB. Both the outﬂow of MBB froman arteriole and the inﬂow of the same grain of MBB
into a venule were observed in the thermal injury group(Fig. 5). While A-V shunting of blood ﬂow was notobserved in any animal in the control group (n ¼ 15),
arteriovenous shunting of blood ﬂow was detected in 4
of 14 rats in the thermal injury group, the difference
being statistically signiﬁcant (p < 0.05) (Table 1).
Gastric mucosal erosion
The total length of erosions was 1:80 G 1:42 mm at
15 min, 3:35 G 2:14 mm at 2 hrs and 6:33 G 5:04 mm
at 5 hrs after thermal injury. It increased timedependently after thermal injury (Fig. 6), and thelength at 5 hrs was approximately 2 times larger thanthat at 2 hrs.

Fig. 3 Vascular labelling with Monastral Blue B (MBB). 5 min afteradministration of 3% suspension of MBB (0.1 ml/100 g body weight).
3A: Few venular deposits of MBB are seen in the control group. 3B:
Venular deposits of MBB are clearly evident in the thermal injury
group.

Discussion
Although gastric A-V shunting channels after thermal injury have been demonstrated using a silicone
rubber cast of the vasculature,15 their presence has
been regarded as controversial. A-V shunting bloodﬂow is difﬁcult to identify by ordinary in vivo observation. After the application of MBB, however, it wasKeio J Med 2002; 51 (4): 193–200

Fig. 4 Percent area of MBB deposits in venules. Percent area of
MBB deposits in venules in two ﬁelds (60) was calculated. The percentage of MBB deposits in the thermal injury group was signiﬁcantly
higher than that of the control group (p < 0.01).

possible to observe A-V shunting blood ﬂow directly inthe present study.
Because serosal resection could not be larger than 2
or 3 mm in diameter in order to avoid bleeding, we
observed only a limited area of the stomach. Notwithstanding this methodological limitation, statistical analysis showed a signiﬁcant difference in the detection of
A-V shunting blood ﬂow. The present results support
those of studies that used silicone rubber casts of thevasculature.10,15 A-V shunting were observed in bothmethods after thermal injury. A-V shunting blood ﬂow
in the basal region of the gastric mucosa or submucosal
layer would steal blood from the mucosa, supportedby the observation that gastric mucosal blood ﬂow
was decreased 5 hrs after thermal injury.25 Macroscopic hemorrhagic erosion was formed within 5 hours
after thermal injury,8 and the total length of erosions
measured using stereoscopic microscope increased
time-dependently after thermal injury. Both the incidence8 of macroscopic hemorrhagic and the total lengthof erosion at 5 hrs were 2 times larger than that at 2 hrs.
The decrease in the gastric mucosal blood ﬂow 5 hrs
after thermal injury plays a role in the ﬁnal step of
erosion formation. A-V shunting blood ﬂow is the totally abnormal blood ﬂow which does not reach most of
mucosal cells and cannot contribute to the mucosal defensive mechanism.
Our result showed that rolling of leukocytes in venules was also increased 5 hrs after thermal injury. Mulligan et al.26 reported that both vascular injury andremote lung injury after thermal injury are neutrophil-197

dependent and require the participation of E-selectinand L-selectin. These adhesion molecules are probably
responsible for the increase in rolling leukocytes
observed in our study. Administration of P-selectinneutralizing monoclonal antibody decreases plasma
free amino-nitrogen concentration in a model ofsplanchnic ischemia-reperfusion, suggesting the presence of leukocyte-dependent tissue injury.27 The addition of leukocytes stimulated by phorbol myristate
acetate to an endothelial cell monolayer causes a signiﬁcant increase in the intracellular peroxide level inthe endothelial cells after 15 min and severe endothelial
cell injury after 5 hrs, suggesting the presence of leukocyte-dependent endothelial damage.28 We previously
reported an increase in active oxygen species generated
by leukocytes obtained from the gastric vein and inferior vena cava 5 hrs after thermal injury.8 These previous studies indicated that leukocyte-dependent endothelial damage probably exists 5 hrs after thermal injury
and an important factor in formation of gastric mucosal
lesion. Therefore we attempted to show in vivo the existence of endothelial damage in the present study.
It has previously been reported that MBB is able tolabel a leaky vessel if its endothelial barrier is interrupted without being totally disrupted.16 Little venulardeposition of MBB was seen in the control group, but
was evident in the thermal injury group. This ﬁnding
indicates that the endothelial barrier of venules may beinterrupted after thermal injury due to endothelial cell
damage. In our previous reports, percentages of therolling leukocytes observed 15 min after thermal injury
were approximately 27–29%.7,13,29 The values were alittle higher than that observed 5 hrs (20.8%) afterthermal injury in the present study. However, the percentage area of MBB deposits in venules observed 15
min after thermal injury was approximately 6%.13,29
The values were lower than that observed 5 hrs (13.4%)
after thermal injury in the present study. Rolling ofleukocytes is considered to be a ﬁrst step of leukocyteendothelial interactions and subsequently followed byaggravation of the endothelial damage.
We also observed irregularity of venule walls at 5 hrs
after thermal injury. A similar condition was observed
when preparation for intravital microscopy was not successful with bleeding from the serosal incision. However, irregularity was always observed at 5 hrs after
injury with sufﬁcient preparation without bleeding. Our
previous report showed no evident change in venular
walls at 15 min after thermal injury.13 Therefore, thisirregularity represents a pathological reaction inducedby thermal injury. We thought that dehydration might
be a cause of this irregularity, and performed the same
experiment with water resuscitation. Because this venular irregularity was also observed in water resuscitatedrats,30 dehydration is not a major cause of the irregu-

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Yoshida M, et al: Arteriovenous shunting blood ﬂow

Fig. 5 Intravital observation of arteriovenous (A-V) shunting. Arrows in Figure 5A, 5B, 5C and 5D show movement of the same grain of MBB
at 0.07 sec intervals. Figure 5A shows the grain of MBB in an arteriole. In Figure 5B, it has moved into the A-V shunting channel, in Figure 5C
into the venule; and in Figure 5D it has ﬂowed along the venule.

Table 1

Detection of A-V Shunting of Blood Flow
A-V shunting blood ﬂow

control groupthermal injury group
total

positive

negative

0
4*
4

15
10
25

total

15
14
29

* p < 0.05

larity of venular walls. Ohno et al.31 reported endothelin-dependent venular constriction using intravital microscopy. It was also reported that the level of endothelin-1 in gastric tissue increased 5 hrs after thermal
injury in our experimental model.32 In an ischemia/
reperfusion model in the rat mesentery, leukocyte ad-herence was seen in postcapillary venules, with subsequent elicitation of albumin leakage, and decrease inplasma nitrate/nitrite levels in the superior mesentericvein were observed.33 A decrease in the constitutive
type of nitric oxide synthase (cNOS) activity in the
rat gastric mucosa was also reported in a model ofischemia/reperfusion-induced mucosal injury,34 andleukocyte-dependent endothelial damage was regarded
as a cause of the suppressed nitric oxide production by
gastric cNOS. In the present study, we found anincreased adherence of leukocytes in venules andexaggerated permeability of venules as indicated by
MBB deposition 5 hrs after thermal injury, both ofwhich are also seen in rats subjected to ischemia/reperfusion.34 One possible cause of the venular irregularity
seen in the present study is imbalance between endothelin and nitric oxide. The presence of smooth muscle
structures in rat venules was demonstrated using scan-Keio J Med 2002; 51 (4): 193–200

199

References

Fig. 6 Total length of erosions. The length of erosions was measured
with stereoscopic microscopy and the total length of all erosions per
rat was calculated. It increased time-dependently after thermal injury,
and the length at 5 hrs was approximately 2 times larger than that at
2 hrs.

ning electron microscopy,35 suggesting that venules
have the ability to contract. However, the presence ofmuscle structures in vessels smaller than venules havenot been reported. We speculated that A-V shunting
blood ﬂow develops in a passive manner.
Davenport36 reported the disruption of the gastricmucosal barrier to hydrogen ions (Hþ ) as a causative
factor in the development of gastric mucosal lesions.
Resulting from this ‘‘Hþ back-diffusion theory’’, impairment of gastric mucosal defensive mechanisms is
accepted as a cause of gastric mucosal lesions induced
by stress. The coexistence of Hþ back-diffusion andmicrocirculatory disturbance in the stomach after thermal injury was reported in our experimental model,15
and microcirculation was accepted as an important factor in gastric mucosal defensive mechanisms.
In conclusion, we showed an increase in rolling leukocytes and observed deposits of MBB in venules afterthermal injury. Direct intravital observation of A-V
shunting blood ﬂow using MBB was also demonstrated.
A-V shunting blood ﬂow can be a cause of mucosal
hypoperfusion. It is suggested that the microcirculatory
disturbance seen 5 hrs after thermal injury contributes
to the ﬁnal step of erosion formation.
Acknowledgments: This work was supported in part by a Grant-inAid for Scientiﬁc Research from the Ministry of Education, Science,and Culture of Japan for Masashi Yoshida.

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