Male wild-type (C57BL/6) mice (8-12 week old) were purchased from the Jackson Laboratory. All animals were maintained in a laminar-flow, specific pathogen-free atmosphere at the Eulji University School of Medicine. The protocol for animal experiment in this article was previously approved by the Institutional Animal Care and Use Committee of the Eulji University School of Medicine (EIACUC 09-01). All subsequent animal experiment adhered to the "Regulation for Animal Expermentation" of the University.

A nonlethal model of segmental (70%) hepatic warm ischemia was used (15). The I/R protocol was initiated with the abdominal wall being clipped of hair and cleansed with betadine. Under sodium pentobarbital (40 mg/kg, intraperitonal) and methoxyflurane (inhalation) anesthesia, a midline laparotomy was performed. With the use of an operating microscope, the liver hilum was dissected free of surrounding tissue. All structures in the portal triad (hepatic artery, portal vein, and bile duct) to the left and median liver lobes were occluded with a microvascular clamp (Fine Science Tools) for 60 min, and reperfusion was initiated by removal of the clamp. This method of segmental hepatic ischemia prevents mesenteric venous congestion by permitting portal decompression through the right and caudate lobes. We have previously conducted a time course experiment to determine the optimal ischemia time period for the induction of hepatic injury. Less than 60 min of ischemia produced only minimal plasma transaminase elevations, whereas over 75 min of ischemia was poorly tolerated with gross evidence of poor reperfusion of the ischemic lobes. A reproducible level of hepatic damage was observed using 60 min of ischemia and was thus used for our study. After application of the clamp, the abdomen was covered with a sterile plastic wrap to minimize evaporative loss. Throughout the ischemic interval, evidence of ischemia was confirmed by visualizing the pale blanching of the ischemic lobes. The clamp was then removed and gross evidence of reperfusion based on immediate color change was assured before closing the abdomen with a continuous 4-0 diameter polypropylene suture. Either the absence of ischemic color changes or the lack of response to reperfusion was a criterion for immediate sacrifice and exclusion from further analysis. Temperature was monitored by rectal temperature probe and was maintained at 37℃ by means of a warming pad and heat lamp. At the end of the observation period following reperfusion, the mice were anesthetized with inhaled methoxyflurane and were sacrificed by exsanguination. Sham animals underwent anesthesia, laparotomy, and exposure of the portal triad without hepatic ischemia. Animals were sacrificed at predetermined time points (6-72 hr) after reperfusion to obtain serum and liver samples. Antibody-treated mice received 16 hr before surgery a single intraperitoneal injection of 100 µg of anti-CD44 mAb (16) (clone IM 7, purified rat anti-mouse CD44; BD Pharmingen, San Diego, CA, USA) that interacts with the HA-binding site of CD44 and may induce shedding of CD44 or control IgG (normal rat IgG; Santa Cruz, CA, USA).

Blood samples were collected from the right ventricle via median sternotomy in a sterile heparinized syringe containing 50 µL of heparin (100 USP units/mL). The blood samples were centrifuged and plasma were collected and stored at -80℃ until further use. Portions of the ischemic and non-ischemic liver lobes were fixed in buffered 10% formalin, embedded in paraffin, and used for hematoxylin and eosin (H&E) staining and immunohistochemical study. Other portions of ischemic and non-ischemic liver lobes were snap frozen in liquid nitrogen and stored at -80℃, until use for western blotting analysis.

To assess hepatic function and cellular injury following liver ischemia, serum alanine aminotransferase (ALT) levels were measured using the Opera Clinical Chemistry System (Bayer, Munich, Germany).

Plasma TNF-α, IL-6, and MCP-1 levels were determined in a 96-well Nunc-Immuno microplate (VWR Scientific, Chicago, IL, USA), using a sandwich enzyme-linked immunosorbent assay (ELISA). The capture antibody was a polyclonal anti-mouse TNF-α, IL-6, or MCP-1 specific goat IgG (R&D Systems, Minneapolis, MN, USA) and the detection antibody was a biotinylated polyclonal anti-mouse TNF-α, IL-6, or MCP-1 specific goat IgG (R&D Systems). All plasma samples were tested in duplicate. The minimal detectable protein concentration was 20 pg/mL.

Formalin-fixed liver samples were embedded in paraffin and cut to 5 µm thick sections. Tissues were stained with hematoxylin-eosin, and slides were assessed for inflammation and tissue injury. The percent necrotic area was estimated by random evaluation of lower power fields (× 40) of each hematoxylin-eosin-stained section. A pathologist, blinded to the experimental procedure of the mice, examined the histopathology of the hepatic tissue sections.

Tissue sections in 5 µm thickness were prepared from paraffin-embedded tissue blocks. Tissue sections were deparaffinized in xylene and rehydrated in a series of ethanol. Microwaving antigen retrieval was performed in 0.01 M citrate buffer, pH 6.0, for 10 min. Then, tissue sections were placed in 0.3% H₂O₂/methanol for 15 min to inactivate endogenous peroxidase. After washing in PBS, tissue sections were incubated in 10% normal goat serum (Chemicon International, Temecula, CA, USA) for 30 min at room temperature to block nonspecific binding of the primary antibody. Then, tissue sections were exposed to diluted primary antibody for overnight at 4℃. Proper dilutions of primary antibodies were selected from preliminary studies. We used dilutions of 1:200 for CD44 (monoclonal rat anti-mouse CD44; BD Pharmingen, Franklin Lakes, NJ, USA) and 1:25 for neutrophil (monoclonal rat anti-neutrophil; Abcam, Cambridge, MA, USA). After washing in PBS to remove excess primary antibody, tissue sections were then incubated with biotinylated goat anti-rat IgG (SC-2041, Santa Cruz, Santa Cruz, CA, USA) at 1:100 dilution for 1 hr at room temperature. Unbound secondary antibody was removed with PBS washing, and avidin-biotin peroxidase (Vector Laboratories, Burlingame, CA, USA) was placed on tissue sections for 30 min at room temperature. The positive immune-reactivity on tissue sections was detected by treatment with a mixture of 3,3'-diaminobenzidine (Sigma, St. Louis, MO, USA), 0.05 M Tris-HCl buffer, and 5% hydrogen peroxide. Then, the tissue sections were counterstained with hematoxylin, followed by dehydration in a series of ethanol. Immunostaining was examined and evaluated under light microscopy. For negative control, normal goat serum at the same dilutions in the place of primary antibody was treated on tissue sections.

After protein concentration was determined using the BCA protein assay, equal volumes of elute were fractioned by SDS-PAGE and transferred to nitrocellulose membranes for 2 hr at 250 mA in the presence of transfer buffer. The transferred membrane was blocked for 1 hr at room temperature with 5% milk in TBS-T buffer and incubated with purified rat anti-mouse CD44 mAb (KM114; BD Pharmingen), rat anti-mouse neutrophil mAb (clone 7/4; Abcam) respectively for 12 hr at 4℃. Blots were incubated at room temperature for 1 hr with a horseradish peroxidase-conjugated secondary antibody against the primary antibody. After extensive washing with TBS-T buffer, membranes were developed with the Super Signal West Pico chemiluminescent kit (Pierce Chemical Co., IL, USA) and exposed to film.

Results were expressed as the mean ± SEM. Group comparisons were performed using Student's t test or analysis of variance. Differences were considered significant at P < 0.05.

Hepatic I/R caused significant hepatocellular damage as demonstrated by plasma ALT levels. The plasma ALT levels of wild-type mice after 60 min. of ischemia followed by serial time interval of reperfusion were measured. The plasma ALT level was maximally elevated after 6 h of reperfusion following ischemic insult, which was decreased to level of sham operation after 72 hr (Fig. 1).

In order to determine whether plasma cytokine/chemokine levels correlated with tissue injury, the plasma cytokine/chemokine levels were measured using an ELISA. The plasma levels of all cytokines/chemokine (i.e., TNF-α, IL-6, and MCP-1) were significantly increased in response to I/R and reached their maximum at 6 hr of reperfusion, which then declined to baseline by 24 hr of reperfusion (Fig. 2). As Fig. 2 shows, hepatic I/R caused significant elevation of TNF-α at 6 hr of reperfusion, which was declined at 24 hr of reperfusion. This data paralleled plasma ALT data, which showed at 6 hr after ischemic insult, followed by a decrease in levels until 72 hr of reperfusion. A similar pattern was observed in plasma IL-6 and MCP-1 levels.

To determine if the CD44-dependent injury was associated with change in protein levels, Western blot analysis was performed on liver lysates from animals that were subjected to liver I/R (Fig. 3A). Following 60 min of warm ischemia, CD44 protein expression was up-regulated as early as 6 hr after reperfusion and then increased in a time dependent manner up to 24 hr. To quantify the amount of protein, a densitometer was used. The values of CD44 in I/R mice were higher by comparison with the values of CD44 in sham mice. The differences were statistically significant (Fig. 3B).

Mice were subjected to 60 min of ischemia followed by reperfusion at the time point of 6 hr and 24 hr. The ischemic liver sections were prepared and stained with H&E and then immunohistochemical staining of CD44 and neutrophils using specific anti-CD44 monoclonal antibody and anti-neutrophil antibody. The histopathologic injury of the liver tissue was evaluated based on sinusoidal congestion, cytoplasmic vacuolization, hepatocellular necrosis, and neutrophil infiltration. The liver sections from the sham-operated mice displayed no necrosis, similar to that of the non-operated control mice (Fig. 4A). Additionally, there was no apparent evidence of hepatic injury due to ischemia alone (i.e., at zero hour of reperfusion; image not shown). However, reperfusion of the ischemic liver induced an extensive hepatocellular necrosis, sinusoidal congestion, and neutrophil infiltration after 6 and 24 hr of reperfusion in wild-type mice (Fig. 4D, G). There was sparing of the periportal areas with progressively increased injury approaching the central vein.

CD44 expression is indicated by dark brown color stain. There was progressively increased CD44 expression approaching the central vein (Fig. 4B, E, H). A similar pattern was observed in neutrophil infiltration. The injury was associated with a marked number of neutrophils infiltrated into the midzonal region of ischemic liver after 6 and 24 hr of reperfusion, which was confirmed with in situ immunohistochemical staining of the neutrophils. Neutrophils are also indicated by dark brown color stain (Fig. 4C, F, I).

To determine if endogenous CD44 contributed to organ damage after hepatic I/R, neutralizing antibody to CD44 was administered to mice that were subjected to warm I/R. Mice received a single intraperitoneal injection of anti-CD44 mAb (100 µg per mouse) or control IgG antibody (100 µg per mouse) at 16 hr before surgery as previously described (15). Sixty minutes of warm hepatic ischemia were followed by 6 and 24 hr of reperfusion. Serum ALT levels were analyzed as a measure of hepatocellular injury. As Fig. 5A, B display, serum ALT levels in the mice with pretreatment of control IgG were significantly increased after 6 and 24 hr of reperfusion. This data paralleled in initial I/R mice experiment. However, the serum ALT levels in the mice with pretreatment of anti-CD44 mAb were declined after 6 and 24 hr of reperfusion. The differences compared with the control IgG treated mice were statistically significant (Fig. 5A, B). Treatment with anti-CD44 mAb resulted in significant protection from hepatic I/R injury at each time point.

Liver histology confirmed the serum ALT estimation of hepatocellular damage. Liver sections from mice that were pretreated by control antibody and anti-CD44 mAb before hepatic I/R injury were stained with hematoxylin-eosin. Severe sinusoidal congestion and hepatocellular necrosis was present in liver tissue from mice that were treated with control IgG, whereas minimal damage was noted in samples from anti-CD44 mAb-treated mice (Fig. 5C, D). Moreover, the disruption of CD44 and subsequent inhibition of neutrophil recruitment was confirmed in ischemic liver after 6 and 24 hr of reperfusion by specific immunohistochemical staining. After 6 and 24 hr reperfusion in hepatic I/R mice, the CD44 expression and neutrophil influx were observed in the liver sections from control antibody treated mice. The other hand, immunostaining for CD44 revealed decreased in hepatic sinusoidal area of anti-CD44 mAb-treated mice compared with given control antibody after I/R (Fig. 6A-D). The infiltration of neutrophils into anti-CD44 mAb-treated mice was impaired relative to given control antibody. This pattern of neutrophil influx was similar to that of CD44 immunostaining pattern (Fig. 6E-H).