Hepatobiliary and Liver Transplant Laboratory, Departments of
1 Surgery,
2 Pathology, and
4 Immunology, Leukocytes
recruited during ischemia-reperfusion to the liver are
important mediators of injury. However, the mechanisms of leukocyte
adhesion and the role of adhesion receptors in hepatic vasculature
remain elusive. L-selectin may critically contribute to injury, priming
adhesion for later action of intercellular adhesion molecule-1
(ICAM-1). Paired experiments were performed using mutant mice
(L-selectin
gene-targeted deficient mice; hepatic ischemia-reperfusion
injury; survival; no reflow
ISCHEMIA-REPERFUSION (I/R) injury occurs when blood
flow to an organ or tissue is interrupted for some period and
subsequently reestablished. The sequence of events that leads to tissue
injury in this situation is incompletely understood but involves
adhesion and infiltration of polymorphonuclear leukocytes (PMN) as an
early step (1, 9, 22, 34, 35, 41, 47). Subsequently, the PMN may elicit
tissue damage by diverse processes ranging from direct elaboration of
cytotoxic mediators (17, 44) to mechanical obstruction of blood flow
(10). In extreme cases, thrombosis of vascular channels may totally
abrogate perfusion of the affected tissue, a condition that has been
termed the "no-reflow" phenomenon (25).
Adhesion of PMN is enabled by a multistep process mediated by a variety
of cell surface molecules. The selectin family of adhesion receptors
(P-, E-, and L-selectin) supports rapid capture of leukocytes and
subsequent rolling along the vascular endothelium before firm adhesion
and migration. L-selectin, a receptor expressed constitutively on
quiescent PMN, is involved in the very initial process of rolling.
L-selectin has been shown to solely mediate cell arrest under
conditions of shear flow (14). Once the slowly rolling PMN becomes
activated, In I/R injury, PMN adhesion is accompanied and followed by a complex
sequence of hemodynamic events. Pressures and flow may vary
dramatically once reperfusion occurs in the injured liver. Alterations
in the hemodynamics of the hepatic microvasculature are also influenced
by PMN adhesion to the sinusoidal lining. L-selectin receptors play a
role in propagating adhesion and PMN plugging in the microvasculature
through interaction between previously adherent and flowing PMN (27).
Platelets and other blood substances may also adhere to stationary PMN,
producing partial or complete plugging that leads to a reduction or
cessation of blood flow.
The liver is particularly susceptible to I/R injury, which is evident
after conditions such as shock, trauma, transplantation, and surgical
hepatectomy. As in other organs, PMN contribute significantly to liver
injury as shown in experimental models of inflammation (24, 28, 48) and
some models of I/R injury (21, 45). The liver provides an ideal
environment for upregulation of adhesion receptors with an enormous
capillary surface, the sinusoidal endothelium. ICAM-1 expression has
been shown to be constitutively present in normal human livers on the
sinusoidal lining cells, including endothelial and Kupffer cells, with
strong inducibility under appropriate stimuli such as acute rejection
(43). The liver also contains the largest population of macrophages in
the body, providing a large source of mediators, including reactive
oxygen intermediates (2, 7, 19), eicosanoids (40), and acute reactant
cytokines (6, 20), able to readily induce expression of numerous
adhesion molecules.
The role of ICAM-1 in hepatic I/R injury has been examined in several
studies, most of which employed monoclonal antibodies against ICAM-1 as
a means of blocking PMN adhesion (11, 12, 26, 29). However, the
contribution of L-selectin to I/R injury in the liver and its possible
interactions with ICAM-1 in this process have not been explored in
detail. A powerful tool for analyzing these complex events is provided
by the recent development of knockout mice lacking one or both of these
adhesion molecules (1, 42).
Our objective was to assess the independent role as well as the
additive effects of deficits for L-selectin and ICAM-1 receptors in the
reperfused ischemic liver. We initially developed models of hepatic
ischemia in the mouse to evaluate neutrophil-mediated injury.
Because the degree of PMN adhesion and reperfusion injury is strongly
dependent on the duration of ischemia, various periods of
ischemia were studied. The degree of PMN infiltration and
microvascular failure was evaluated by histological examination of
tissue samples and by intravital microscopy. Liver injury was
quantified by release of transaminases, tissue necrosis, and animal survival.
Animals
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
/
, ICAM-1
/
, and
L-selectin/ICAM-1
/
) and wild-type mice (C57BL/6) to
investigate leukocyte adhesion in the ischemic liver. Leukocyte
adhesion and infiltration were assessed histologically. Aspartate
aminotransferase levels were significantly reduced (2- to 3-fold) in
mutant vs. wild-type mice in most groups but most significantly after
90 min of partial hepatic ischemia. Leukocyte adhesion was
significantly reduced in all mutant mice. Areas of microcirculatory
failure, visualized by intravital microscopy, were prevalent in
wild-type but virtually absent in L-selectin-deficient mice. After
total hepatic ischemia for 75 or 90 min, survival was better in
mutant L-selectin and L-selectin/ICAM-1 mice vs. wild-type mice and
ICAM-1 mutants. In conclusion, L-selectin is critical in the
pathogenesis of hepatic ischemia-reperfusion injury. Poor
sinusoidal perfusion due to leukocyte adhesion and clot formation is a
factor of injury and appears to involve L-selectin and ICAM-1 receptors.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
2-integrin receptors
are upregulated, and L-selectin is shed from the PMN membrane (23).
Firm adhesion then occurs through interaction with intercellular
adhesion molecule-1 (ICAM-1), a major counterreceptor for
2-integrins (39). ICAM-1, a
member of the immunoglobulin superfamily, is constitutively present at
low levels on most endothelial cells (3) and is upregulated maximally
over a 8- to 24-h period at sites of inflammation. Thus L-selectin and
its counterendothelial receptor act in an integrated fashion with the
2-integrin/ICAM-1 complex, a
pathway thought to be an essential step for flattening and migration of PMN into the extracellular matrix (15).
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
Model of Partial Hepatic Ischemia
Mice were anesthetized by inhalation of metofane. A midline incision was made extending from the xiphisternum to the pubis. The liver was exposed completely with retractors placed in the flanks and a clamp was attached to the xiphisternum, which was elevated. The ligamentous attachments of the lateral left lobe were carefully divided, and the lobe was mobilized and freed. The blood supply to this lobe was interrupted by applying a microclamp (FD562; Aesculap, South San Francisco, CA) to the vascular pedicle. The ischemic lobe corresponded to 39.0 ± 1.0% (mean ± SE, n = 5) of the total hepatic mass. Using an intravenous dye (trypan blue), we confirmed that the lateral left lobe was completely ischemic, whereas the other lobes were homogeneously perfused.Model of Total Hepatic Ischemia
To evaluate animal survival, we developed a new and simple model of total hepatic ischemia in the mouse (50). Mice were anesthetized with metofane. A midline incision was made, and bowel loops were exteriorized, placed on saline-soaked gauze, and covered with plastic wrap/clear film. The quadrate and the papillary process of the liver were ligated with 6.0 silk suture and resected followed by the lateral right and the caudate lobes. The amount of tissue resected corresponded to 29.2 ± 1.2% (mean ± SE, n = 4) of the total liver mass (Fig. 1). Total ischemia of the remaining liver tissue, consisting of the median and lateral left lobes, was achieved with the use of vascular microclamps. In preliminary studies, total ischemia was confirmed by infusion of trypan blue into the portal vein. None of the dye was able to enter the liver. Congestion of the gut was completely absent in this model due to collateral vessels providing a shunt from the portal vein to the inferior vena cava. Survival was assumed to be permanent if animals were alive 30 days after surgery.
|
Serum Markers of Reperfusion Injury
Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were used as established markers of I/R injury (18). Blood samples were obtained from the inferior vena cava using a 24-gauge catheter via a midline incision. The samples were stored in serum separator tubes (Becton-Dickinson, Franklin Lakes, NJ) and immediately centrifuged at 11,000 rpm for 5 min. The serum transaminases analyzed at each I/R time point were determined from individual samples (1 ml/mouse). A 10-µl sample of serum was diluted with 0.9% saline and was analyzed using the serum multiple biochemical analyzer (Ektachem DTSCII; Johnson & Johnson, Rochester, NY). In preliminary experiments with wild-type mice subjected to varying periods of partial ischemia (30, 60, 90, and 120 min), peak AST and ALT levels occurred after ~6 h of reperfusion, and both transaminase levels plateaued after 24 h of reperfusion (data not shown). Blood samples were collected in separate experiments so as not to interfere with other studies.Histological Assessment of Liver Tissue Injury After Reperfusion
Ischemic and nonischemic liver tissue from each animal were fixed in buffered Formalin and processed to paraffin. Sections were stained with hematoxylin and eosin and examined by a pathologist (D. N. Howell) unaware of the duration of warm ischemia and genotype of the mouse. The degree of vascular congestion/thrombosis and hepatocyte death was used as a marker of tissue injury. Vascular thrombosis/congestion was defined as engorgement of portal venules, sinusoids, or terminal hepatic venules with erythrocytes, platelets, and/or fibrin material. Hepatocyte death was assessed by loss of nuclear detail and well-defined cellular borders. A semiquantitative scale from zero to four according to the percent of tissue involved was developed for each respective hepatic zone (zone 1: periportal; zone 2: intermediate; zone 3: around terminal hepatic venules). The scale was defined as grade 0 (<1%), grade 1 (1-10%), grade 2 (11-25%), grade 3 (26-50%), and grade 4 (>50%). For the assessment of PMN adhesion and infiltration to the hepatic vasculature, a semiquantitative scale for each hepatic zone from zero to four was developed (Table 1). The vast majority (>95%) of inflammatory cells identified in all cases were PMN.
|
Intravital Microscopy
Animals were anesthetized with metofane, and hepatic ischemia was achieved using a microvascular clamp placed on the vascular pedicle of the lateral left lobe. The ischemic liver lobe was placed on a table and fixed to the surface with glue (Krazy glue). The animal was kept anesthetized and placed on an adjustable Plexiglas stage. The liver surface was kept continuously moist with warm saline. Observations were made using an inverted intravital microscope (K2SBio/Optiphot) with a ×20 objective lens (Olympus), and, for epi-illumination, a 100-W mercury lamp (Nikon) was used. Images were recorded using a silicone intensified camera (Dage-MTI, Michigan City, IN) and were recorded on videotape using a video recorder (JVC) for evaluation of dynamic images. Observations were made for 30 min after removal of the microvascular clamp.The effect of ischemia on microcirculatory integrity was assessed by examination of 10 random epi-illuminated microscopic fields in each animal. Sinusoidal areas with dark, irregular outline were considered to be early indicators of microcirculatory breakdown, as described previously (31). Data are expressed as number of failing areas/microscopic field.
Statistical Analysis
Data are expressed as means ± SE. Paired Student's t-test was performed, and ANOVA was performed using the Statistical Analysis System to compare serum transaminase levels and the Mann-Whitney test to compare histological data. The Fischer's exact test was performed to compare survival. A P value of <0.05 was considered statistically significant. ![]() |
RESULTS |
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Effects of Hepatic Ischemia on Animal Survival
Assessment of survival after prolonged periods of hepatic ischemia requires a technique of total hepatic ischemia. However, complete occlusion of the liver is rapidly lethal due to massive mesenteric congestion unless a shunt is created between the portal and the systemic circulations. Because these shunts are difficult to perform in small animals and have not been reported in the mouse, we used our new technique of total hepatic ischemia in the mouse (50).All wild-type animals survived >30 days after being subjected to 60 min of total hepatic ischemia while all died within 24 h of
surgery after 90 min of total ischemia
(n = 7 in each group, Table
2). An intermediate time period of 75 min
of ischemia was associated with partial survival (2 out of 7).
Survival was significantly improved in L-selectin-deficient mice
(L-selectin /
) and the combined L-selectin and
ICAM-1-deficient mice (L-selectin/ICAM-1
/
); after 75 min
of ischemia, all of these groups survived permanently (Table
2). Improved survival was also observed after 90 min of warm
ischemia in the same deficient mice, although it did not reach
significance. Survival in the ICAM-1
/
mice was
comparable to wild-type animals at all ischemia times studied.
|
L-Selectin and ICAM-1 in Hepatic Reperfusion Injury
Serum AST and ALT levels were measured as markers of hepatic injury. The baseline serum AST values in untreated mice were as follows: L-selectin
|
Wild-type mice. In each ischemia group, transaminase levels increased up to 6 h after reperfusion and then declined (Table 3). In animals subjected to 30 or 60 min of ischemia, transaminase levels were comparable at 6 h of reperfusion but were significantly higher after 24 h of reperfusion in the 60-min group (P < 0.005, unpaired Student's t-test). The greatest increase in transaminase levels was observed after 90 min of ischemia followed by 6 h of reperfusion. In this ischemia group, transaminase levels remained well above baseline after 24 h of reperfusion.
L-selectin /
. At most
ischemia times, transaminase levels were lower in L-selectin
/
mice compared with wild-type mice (Table 3). The most
striking reduction was seen in L-selectin
/
mice
subjected to 90 min of ischemia. In this group, AST levels were
two- to threefold lower in the L-selectin
/
mice after 15 min and 6 h of reperfusion compared with wild-type mice, whereas no
difference was observed at 24 h. Of note, AST levels were also significantly lower in L-selectin
/
mice after 15 min of
reperfusion compared with ICAM-1 or even L-selectin/ICAM-1-deficient mice.
ICAM-1 /
. Serum
transaminase levels were significantly lower than paired wild-type
controls after 6 h of reperfusion in most ischemia groups but
not at earlier or later stages of reperfusion (Table 3). The most
significant differences were observed in livers subjected to 90 min of
ischemia and reperfused for 6 h. In fact, ICAM-1
/
mice had the lowest levels of all animals in this cohort.
L-selectin/ICAM-1 /
.
Serum transaminase levels were significantly lower at all I/R time
points compared with wild-type mice (Table 3). Although levels were
usually comparable to those found in the L-selectin
/
mice, they were significantly lower (2- to 3-fold) in L-selectin/ICAM-1
/
livers subjected to 90 min of ischemia at 1 and
24 h of reperfusion (P < 0.05).
L-Selectin and ICAM-1 in Hepatic Tissue Injury
The extent of PMN adhesion, infiltration, and tissue damage in ischemic livers was assessed using a semiquantitative grading scale (Table 1). The nonischemic lobes in all animals that underwent the surgery were histologically unremarkable and showed no detectable signs of neutrophil adhesion, infiltration, or congestion and necrosis. In addition, sham-operated animals showed no abnormal histology. Comparisons were made between wild-type and L-selectin/ICAM-1
|
In livers subjected to 90 min of ischemia followed by 1 h of reperfusion, there was a significant difference in
infiltration of PMN between the L-selectin/ICAM-1 /
and
wild-type mice. This difference was most striking in
zones 2 and
3. In the wild-type mice, numerous PMN
were seen adherent to the endothelium of terminal hepatic venules and
within adjacent sinusoids, whereas PMN adhesion was minimal in the
L-selectin/ICAM-1
/
group (Table 4 and Fig. 2). A similar difference in PMN adhesion
was seen in the L-selectin
/
(Fig. 2) and ICAM-1
/
(data not shown) animals after the same period of
ischemia and reperfusion. After 6 h of reperfusion in the same
ischemia group (90 min ischemia), there was variable, often extensive infiltration of PMN in all three hepatic zones, and no
differences were detected between the mutant and wild-type groups
(Table 4).
|
We also looked at the degree of tissue congestion after prolonged
ischemia as a factor of tissue injury and vascular plugging. Although there were no significant differences
(P < 0.08), there was a trend toward
reduced sinusoidal congestion in the L-selectin/ICAM-1 /
mice subjected to 90 min of ischemia compared with the
wild-type mice (Table 4 and Fig. 3).
|
Extensive histological tissue/hepatocyte necrosis, evidenced by nuclear
degeneration and loss of distinct cellular borders, was present in
animals subjected to 90 min of ischemia followed by 6 h of
reperfusion. In this ischemia group, hepatocyte necrosis in
zone 1 (periportal) was significantly
less in the L-selectin/ICAM-1 /
mice than in the
wild-type group (Table 4), whereas, in zones 2 (intermediate) and 3 (around terminal hepatic venules), no significant differences were
found between the L-selectin/ICAM-1
/
and wild-type mice.
Evidence of tissue necrosis was not detectable at any earlier time
points of ischemia.
L-Selectin and ICAM-1 in Microvascular Injury Leading to Sinusoidal Perfusion Failure
The macroscopic and microscopic examination of control vs. L-selectin/ICAM-1A method of in vivo intravital microscopy was developed to further
evaluate sinusoidal areas with extensive microvascular breakdown. Dark
irregular areas correspond to petechial bleeding and perfusion failure
as established elsewhere (31; as shown in Fig.
4). Animals were subjected to 60 and 90 min
of ischemia, and analysis was performed after 30 min of
reperfusion. In wild-type mice, ischemia of 60 min produced a
significant increase in the number of sinusoidal areas with
microcirculatory failure compared with the L-selectin /
mice and L-selectin/ICAM-1
/
mice (Fig. 5). Hepatic ischemia of 90 min
produced a significant increase in the number of nonperfusing areas
with persistent difference between wild-type mice and L-selectin
/
livers (Fig. 5).
|
|
In a group of animals subjected to 120 min of ischemia with 6 h
of reperfusion, serum transaminase levels were paradoxically higher in
the L-selectin/ICAM-1 /
mice (26,268 ± 769 IU/l)
compared with the wild-type mice (18,077 ± 684 IU/l;
P < 0.05, paired Student's t-test) and ICAM-1
/
mice (data not shown). In two out of seven wild-type mice subjected to
these extreme ischemia times (90 and 120 min), there was
evidence of extensive clot formation and vascular occlusion (Fig.
6), whereas this was not seen in the
L-selectin/ICAM
/
mice. The lower transaminase levels in
the controls may reflect the poor release of enzymes into the
circulation due to occluded vessels.
|
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DISCUSSION |
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In this study using mutant mice, we provide direct evidence indicating
a pivotal role for L-selectin and, to a lesser degree, ICAM-1 in the
mechanisms of hepatic I/R injury. This study also introduces novel
models of partial and total hepatic ischemia specifically
designed to study varying periods of hepatic ischemia and
reperfusion in the mouse. In wild-type mice, manifestations of I/R
injury included early infiltration of PMN and extensive tissue injury
as assessed by histologic examination, measurement of serum
transaminase levels, and microcirculatory collapse visualized by
intravital microscopy. In mice deficient in L-selectin
alone, a wide range of protective effects on I/R injury was observed, including decreases in tissue PMN infiltration, prevention of microcirculatory failure, lower transaminase levels at a wide range of
time points, and better animal survival after total hepatic ischemia. The latter was particularly impressive after 75 and 90 min of ischemia; 90 min of ischemia was a duration
associated with 100% death in the wild-type animals. Protective
effects of ICAM-1 deficiency, although significant, were limited to
decreases in PMN infiltration and decreases in transaminase levels at a single time point of reperfusion (6 h). Survival in ICAM-1
/
mice after total hepatic ischemia was similar
to that of wild-type animals. L-selectin/ICAM-1
/
mice,
with a small number of exceptions, showed a pattern of response to I/R
injury that was similar to L-selectin
/
mice. Taken as a
whole, these experiments support a pathogenetic sequence in which PMN
adhesion promotes hepatocellular injury in a complex manner, with one
final pathway of injury involving a decrease or cessation in blood flow.
Our data demonstrate a primary role for L-selectin in mediating injury in the reperfused warm ischemic liver. Loss of the L-selectin receptor caused reduction in PMN accumulation early in reperfusion. L-selectin receptors were also essential in mediating hepatocyte injury, as documented by significant reduction in transaminase levels in most ischemia groups for the deficient mice. These beneficial effects were ultimately reflected in improved survival for the L-selectin-deficient mice compared with wild-type animals.
The beneficial effects of blocking L-selectin have been observed in animal models of inflammatory injury such as the heart (5, 32), peritoneum (38), and lung (33). However, evidence supporting a role for L-selectin receptors in the pathogenesis of liver injury has been limited to a study on cold ischemia in the rat that employed blocking antibodies (16) and one other that used a nonspecific selectin blocker, also in the rat (37). In fact, studies in nonischemic models of inflammation in the liver have suggested little or no role for selectins in mediating neutrophil interaction with the endothelium (48). The cause of this discrepancy is unclear; it may reflect differences in PMN recruitment in response to different proinflammatory stimuli.
In contrast to L-selectin, ICAM-1 was less crucial in mediating tissue damage in our model. Deficiency in ICAM-1 alone significantly reduced injury only after 6 h of reperfusion and had no effects on early (1 h) or late (24 h) reperfusion injury. This time course is consistent with the known pattern of ICAM-1 upregulation after inflammatory stimuli in which peak expression occurs 6-8 h after stimulation (8). The absence of ICAM-1 also had no effect on animal survival after prolonged ischemia.
In the livers of mice lacking ICAM-1 receptors, after 90 min of
ischemia and 1 h of reperfusion, there was a significant
reduction in infiltrating PMN, similar to that seen in L-selectin
/
mice. At this time point, however, ICAM-1
/
mice showed a rise in serum transaminase levels
comparable to wild-type controls, whereas L-selectin
/
mice had significantly lower transaminase levels. This disparity
suggests that the early rise in transaminase levels seen in ICAM-1
/
mice (and, by extension, wild-type mice) may involve
factors not directly related to PMN infiltration or alternatively may
be mediated by small numbers of infiltrating PMN below the threshold of
histological detection. Discrepancies between PMN infiltration and
tissue damage have been noted in a small number of studies in I/R (4,
12, 49). By the same token, the reduced transaminase levels seen in
L-selectin
/
mice may not be a simple result of decreased
PMN infiltration. Because L-selectin, unlike ICAM-1, is expressed on
PMN themselves, it is possible that the protective effect of L-selectin
deficiency involves aspects of PMN function other than or in addition
to adhesion (e.g., release of cytokines, receptor signaling events).
Indeed, recent studies have shown an increased tendency for PMN to
undergo programmed cell death in mice that were deficient for
L-selectin receptors (30).
Blocking the function of ICAM-1 adhesion receptors with monoclonal
antibody has proven to be protective in the ischemic liver, with
documented decreases in infiltration of PMN, oxygen radical formation,
and transaminase levels (11, 12, 26, 29). These studies, although
strongly suggestive of a role for ICAM-1 in I/R injury, are hampered by
difficulties inherent in the monoclonal antibody blocking approach. The
efficacy of blocking antibodies may be diminished when there is
substantial injury to the sinusoidal lining (12, 36), and attenuation
of injury may be unrelated to the blocking function of the antibody
(46). In spite of these observations, our studies with ICAM-1
/
mice, which circumvent the complexity of monoclonal
antibody blocking experiments, support and extend the findings of the
previous studies. However, from a therapeutic point of view, our data
suggest that blocking ICAM-1 alone is unlikely to provide significant
beneficial effects in the ischemic liver.
Combined deficiencies for L-selectin and ICAM-1 receptors also markedly
diminished many of the detrimental effects of hepatic I/R injury. In
many respects, including survival, histologic manifestations of tissue
damage, and PMN infiltration, these animals resembled L-selectin
/
mice. Unlike either L-selectin
/
or
ICAM-1
/
mice, the L-selectin/ICAM-1
/
mice
enjoyed a statistically significant reduction in transaminase levels
after 24 h of reperfusion, suggesting a possible additive protective
effect. However, the discrepancy in the site of early PMN margination
(zone 3) and the eventual pattern of
tissue injury/protection seen in these mice suggest that the PMN may be
mediating damage by some mechanism other than direct cytotoxicity. One
possible mechanism is an impedance of hepatic blood flow potentiated by
the early margination of the PMN, leading to subsequent exacerbation
(through poor perfusion) of ischemic damage.
In all animals, both wild-type and mutant mice, there was extensive diffuse PMN infiltration and adhesion after 6 h of reperfusion; the abrogation of differences between groups at this time point reflects alternative pathways of PMN recruitment. These data suggest a complex role for PMN-mediated injury. It seems that the prevention of early interaction between PMN, adhesion receptors, and the endothelium is essential for an impact on persistent attenuation of injury. Our results indicate that L-selectin and ICAM-1 receptor-mediated leukocyte adhesion is one critical factor in a complex pathway for ischemic injury to the liver. However, it also seems likely that the impact of the loss of these receptors is not solely mediated through leukocyte adhesion.
Our studies did not examine the possible contribution of adhesion molecules other than L-selectin and ICAM-1 in hepatic I/R injury. Expression of E- and P-selectin is reportedly lacking in the sinusoidal endothelium of human livers, even after severe inflammatory injuries such as chronic rejection and sepsis (43). Other endothelial surfaces in the liver, however, including those of hepatic arteries and portal and central veins, express these selectins in both a basal and inducible fashion (43). We are currently investigating a possible role for P-selectin in hepatic I/R injury using gene-targeted deficient mice.
Another important finding in our studies was the identification of a critical ischemia time resulting in obstruction of the hepatic sinusoids by blood elements, a phenomenon mediated by L-selectin and ICAM-1 receptors. The involvement of adhesion molecules in mediating microcirculatory disturbances due to leukostasis has remained controversial. Some authors have proposed that the development of microcirculatory occlusion is solely dependent on factors such as endothelial swelling, protrusion of blebs, and hemoconcentration (31), whereas others have suggested that PMN plugging is an important event in the occlusion of microvasculature after reperfusion (13, 25). Koo et al. (25) have described this phenomenon of no-reflow to occur as early as after 30 min of hepatic ischemia.
In our studies, gross examination of livers in wild-type mice subjected
to prolonged ischemia (90 and 120 min) showed the presence of
widespread areas of patchy mottling, a finding not observed in
L-selectin /
or L-selectin/ICAM-1
/
mice
(data not shown). Using intravital microscopy, this phenomenon of
microcirculatory failure was consistently observed after 60 min of
hepatic ischemia as areas of petechial bleeding.
Microcirculatory failure was almost absent in the L-selectin
/
and L-selectin/ICAM-1
/
livers. Under
extreme conditions, a few (2 out of 7) histological sections of control
livers subjected to 90 and 120 min ischemia, when compared with
paired experiments in L-selectin/ICAM-1
/
mice, showed extensive clot formation and vascular plugging. The lower levels of
transaminases observed in wild-type mice compared with mutant mice
after 120 min ischemia and 6 h of reperfusion may represent poor perfusion of liver tissue with the inability to release enzymes in
the circulation.
The breakdown of vascular integrity with subsequent occlusion (the
no-reflow phenomenon) occurring in livers subjected to 60 min or more
of ischemia may be critical for animal survival. Survival was
improved significantly in both L-selectin /
and L-selectin/ICAM-1
/
mice after 75 min of
ischemia. After 90 min of ischemia, survival was
achieved in the L-selectin
/
and L-selectin/ICAM-1
/
mice, whereas none of the wild-type mice survived.
These findings may have important implications, not only regarding
mechanisms of injury but also from a therapeutic perspective. Blocking
either L-selectin alone or both L-selectin and ICAM-1 may provide
significant benefits in liver function after prolonged periods of ischemia.
In summary, this study demonstrates that L-selectin mediates microcirculatory disturbances and hepatocyte injury in the ischemic mouse liver. ICAM-1, although involved independently to a lesser degree, is important in mediating injury and microcirculatory disturbances in conjunction with L-selectin. The demonstration that deficits in these adhesion receptors attenuate injury and prevent microcirculatory compromise in the liver provides opportunity for interventions to minimize hepatic injury and extend the permissible duration of ischemia. Currently, a variety of agents are available, such as antibodies, chimeric molecules, peptides or glycan moieties, and other pharmacophores, to act against selectin-mediated leukocyte adhesion. These findings open new avenues for therapeutic interventions and provide a rationale to study the exact timing of interventions on specific adhesion molecules.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. J. J. Lemasters and Robert T. Currin for help with the intravital studies and Dr. T. F. Tedder for invaluable discussion and provision of deficient mice.
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FOOTNOTES |
---|
A preliminary report of this work was presented at the American Society of Transplant Surgeons, Chicago, IL, May 1997.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: P.-A. Clavien, Dept. of Surgery, Duke Univ. Medical Center, PO Box 3247, Durham NC 27710.
Received 4 May 1998; accepted in final form 11 August 1998.
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