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Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:335-342

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:335.)
© 2000 American Heart Association, Inc.

Vascular Biology

Mouse Model of Femoral Artery Denudation Injury Associated With the Rapid Accumulation of Adhesion Molecules on the Luminal Surface and Recruitment of Neutrophils

Merce Roque; John T. Fallon; Juan J. Badimon; Wen X. Zhang; Mark B. Taubman; Ernane D. Reis

From the Zena and Michael A. Wiener Cardiovascular Institute, Department of Medicine (M.R., J.T.F., J.J.B., M.B.T.); the Department of Pathology (J.T.F.); the Department of Physiology and Biophysics (M.B.T.); and the Department of Surgery (W.X.Z., E.D.R.), Mount Sinai School of Medicine, New York, NY.

Correspondence to Ernane D. Reis, MD, Department of Surgery, Box 1259, The Mount Sinai Medical Center, One Gustave L. Levy Pl, New York, NY 10029-6574. E-mail ernane.reis{at}smtplink.mssm.edu


*    Abstract
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Abstract—Techniques of arterial injury commonly used in animals to mimic endovascular procedures are not suitable for small mouse arteries. This has limited examination of the response to arterial injury in genetically modified mice. We therefore sought to develop a model of transluminal injury to the mouse femoral artery that would be reproducible and result in substantial levels of intimal hyperplasia. Mice of the C57BL/6 strain underwent bilateral femoral artery denudation by passage of an angioplasty guidewire. Intimal hyperplasia was observed in 10% of injured arteries at 1 week, in 88% at 2 weeks, and in 90% at 4 weeks. The mean intimal-to-medial area ratio reached 1.1±0.1 at 4 weeks. No intimal proliferation was found in control sham-operated arteries. One hour after injury, the denuded surface was covered with platelets and leukocytes, predominantly neutrophils. This was associated with the accumulation of P-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. Expression of these adhesion molecules was not seen in the underlying medial smooth muscle cells. At 24 hours, few neutrophils remained on the denuded surface. At 1 week, macrophages and platelets were present in the vessel wall, partially covered by regenerated endothelium. Transluminal wire injury to the mouse femoral artery induces abundant intimal hyperplasia formation by 2 and 4 weeks and elicits the rapid accumulation of leukocytes and adhesion molecules on the denuded luminal surface. This model will be a valuable tool to study arterial injury in genetically modified mouse models.


Key Words: arterial injury • cell adhesion molecules • intimal hyperplasia • mice • neutrophils


*    Introduction
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Experimental arterial injury in mammals has been instrumental in advancing the understanding of the cellular mechanisms underlying the development of intimal hyperplasia and restenosis.1 2 3 4 Injury to the arterial wall triggers smooth muscle cell (SMC) proliferation, migration, and matrix secretion.5 6 The resultant intimal hyperplasia is a common histological finding in restenosis after angioplasty,7 vascular anastomotic strictures,8 and other arterial occlusive diseases. Genetically modified mice provide unprecedented opportunities to study intimal hyperplasia.9 10 For example, apo-E11 and LDL-receptor12 knockout mice provide an important resource for examining the effect of injury on animals prone to develop atherosclerosis. Similarly, injury in mice lacking important regulators of inflammation or proliferation, such as intercellular adhesion molecule-1 (ICAM-1)13 and p27,14 may help define the molecular pathology of intimal hyperplasia.

To date, there has been difficulty in establishing reproducible models of mouse arterial injury. Endoluminal manipulation of small arteries is technically difficult. This has led to the use of extraluminal approaches, such as periarterial cuff placement,15 16 perivascular electric current,17 and simple arterial ligature.18 These methods are attractive because of their relative simplicity. However, the resultant injury differs substantially from most clinical situations in which injury is produced at the luminal surface. Denudation of the mouse carotid artery with a flexible wire19 may be more relevant to clinical intra-arterial manipulations. This approach is more challenging, and the success in obtaining significant intimal lesions is variable and has been reported in some studies as low.20 21 Our group’s initial work on transluminal injury to mouse arteries involved passage of an endodontic broach into the femoral artery.22 That method appeared to be excessively traumatic and resulted in variable degrees of injury.

We now report a relatively simple approach to intra-arterial injury of mouse femoral arteries. This technique produced significant intimal hyperplasia at 2 and 4 weeks. This model was also used to examine early events occurring on the denuded luminal surface. The mouse femoral arterial injury was associated with the deposition, at 1 hour, of adhesion molecules (P-selectin, ICAM-1, and vascular cell adhesion molecule-1 [VCAM-1]), platelets, and leukocytes on the deendothelialized luminal surface. These adhesion molecules were not expressed in the underlying media.


*    Methods
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Animals
Mice of the C57BL/6 strain (mean age, 13±0.4 weeks; mean weight, 24±0.5 g), were purchased from Taconic Farms, Germantown, NY, and were housed at the Center for Laboratory Animal Sciences at the Mount Sinai Medical Center, New York, NY. Mice received standard rodent chow (PMI Nutrition International) and tap water ad libitum. Procedures and animal care were approved by the Institutional Animal Care and Use Committee and were in accordance with the Guide for the Care and Use of Laboratory Animals.23

Surgical Procedures
One hundred male and female mice were studied. Eighty-five underwent bilateral femoral artery injury, 12 were sham-operated, and 3 were uninjured controls. Endoluminal injury to the common femoral artery was produced by either 1 or 3 passages of a 0.25-mm-diameter angioplasty guidewire (Advanced Cardiovascular Systems). These 2 injury protocols were used to evaluate different degrees of injury to the vessel wall. General anesthesia was achieved with intraperitoneal pentobarbital sodium injection (Nembutal, Abbott Laboratories), 40 mg/kg body weight. While being viewed under a surgical microscope (Carl Zeiss), a groin incision was made. The femoral artery was temporarily clamped at the level of the inguinal ligament, and an arteriotomy was made distal to the epigastric branch. The guidewire was then inserted, the clamp removed, and the wire advanced to the level of the aortic bifurcation and pulled back. After removal of the wire, the arteriotomy site was ligated. The same injury protocol was carried out on the contralateral side. Control sham-operated arteries underwent dissection, temporary clamping, arteriotomy, and ligature, without passage of the wire. Nonoperated normal femoral arteries were also used as controls.

To determine the time course of neointima formation, injured arteries (n=164) were harvested at 1 hour, 24 hours, 1 week, 2 weeks, and 4 weeks after injury. To evaluate possible sex-related differences, 25 female mice were included in the 3-passage group.

Histology and Immunohistochemistry
For harvesting of specimens, perfusion-fixation was done with 4% paraformaldehyde in PBS at 100 mm Hg for 15 minutes and followed by en bloc excision of both hindlimbs. Specimens were fixed overnight in 4% paraformaldehyde in PBS and decalcified in 1% formic acid. Two 2-mm-thick transverse segments were cut from each artery at the level of injury in the common femoral artery and processed for paraffin embedding. Histological sections were cut from both segments, corresponding to the midportion of each paraffin bloc, and stained with modified elastic tissue–Masson’s trichrome and hematoxylin-eosin.

Representative sections were immunohistochemically stained for {alpha}-actin (alkaline phosphatase–conjugated monoclonal anti–{alpha}-smooth muscle actin, 1:100; Sigma), factor VIII–related antigen (rabbit anti-human von Willebrand factor, 0.57 µg/mL; Dako), macrophages (rabbit anti-mouse macrophage, 0.5 µg/mL; Accurate Chemical & Scientific Corp; and MOMA-2, rat anti-mouse macrophages/monocytes, 2 µg/mL; Serotec), platelets (adsorbed rabbit anti-mouse thrombocyte, 1:20 000; Inter-Cell Technologies), VCAM-1 (rat anti-mouse CD106/VCAM-1, 10 µg/mL; Southern Biotechnology Associates),24 ICAM-1 (monoclonal anti-mouse ICAM-1, 3 µg/mL; Seikagaku),25 fibrinogen (rabbit anti-mouse polyclonal antibody, 1:2000; Boehringer), platelet–endothelial cell adhesion molecule-1 (PECAM-1; purified rat anti-mouse CD31 PECAM-1 monoclonal antibody, 5 ng/mL; Pharmingen),26 and P-selectin (purified rabbit anti-human polyclonal antibody, 5 µg/mL; Pharmingen).27 Sections were deparaffinized, rinsed in xylene, and rehydrated. They were blocked with 3% H2O2, washed in water, treated with 2% ovalbumin in PBS, washed in PBS, and incubated with the primary antibodies at 37°C for 2 hours. After being washed in PBS, the primary antibody was detected by using a biotinylated anti-mouse or anti-rabbit IgG for 30 minutes at room temperature. Sections were washed in PBS, reacted with horseradish peroxidase–conjugated streptavidin, and developed with 3,3'-diaminobenzidine. After being washed in distilled water, all sections were counterstained with hematoxylin. Negative controls were prepared by substitution of the primary antibody with an irrelevant antibody. Representative sections from early time points were also stained with an IgG isotype antibody to assess specificity. All specimens were analyzed by an investigator blinded to the study design.

Morphometry
Arterial specimens were blindly analyzed by computerized morphometry (NIH Image 1.60 software). Endothelial coverage was assessed by examination of factor VIII–related antigen staining: low-profile cells with central nuclei that were positively stained were identified as endothelial cells. The 2 sections obtained from each artery were examined, and the section with maximal luminal narrowing was selected for measurements of luminal area, medial area, intimal area, vessel area, and lengths of the internal elastic lamina (IEL) and external elastic lamina. The percentage of luminal narrowing and the intima-to-media (I/M) ratio were calculated as previously described.28 Intraobserver and interobserver variation coefficients for serial measurements of morphometric parameters were <0.5%. Arteries with occlusive thrombus were not included in the morphometric analysis.

Statistical Analysis
All data are expressed as mean±SEM. A 2-tailed unpaired t test was used to compare intimal area, medial area, I/M ratio, percentage of luminal narrowing, and IEL length between male and female, 2- and 4-week, and 1- and 3-passage groups. Probability values <0.05 were considered significant.


*    Results
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Injury Technique
The operative time for bilateral femoral injury ranged from 20 to 30 minutes. Clamping of the femoral artery did not exceed 1 minute, thereby allowing introduction of the wire after arteriotomy without significant blood loss. Three animals (3%) died postoperatively at 1 day, 4 days, and 3 weeks. Autopsy did not reveal their cause of death.

Histological Findings
Sham-operated arteries (n=24) examined at 1 hour, 1 week, and 2 weeks had a normal appearance. The endothelium was intact, and there was no evidence of thrombus, arterial wall damage, or intimal thickening. Histology of injured arteries is summarized in Table 1Down. After injury, complete endothelial denudation was found in 100% of specimens examined at 1 hour. Regenerated endothelium covered 7±0.2% of the luminal surface at 24 hours, 28±3.7% at 1 week, 49±1.7% at 2 weeks, and 91±1.8% at 4 weeks. Occlusive thrombosis was found in 5% of injured arteries at 1 week, in 21% at 2 weeks, and in 12% at 4 weeks (P=NS). Intimal hyperplasia, defined as any proliferative lesion within the IEL circumference, covered 87±4.3% of the IEL length at 2 weeks and 89±6% at 4 weeks. Intimal proliferation was found in 10% of arteries at 1 week, in 88% at 2 weeks, and in 90% at 4 weeks. {alpha}-Actin staining confirmed that vascular SMCs were the main cellular component of intimal proliferative lesions. The time course of neointima formation is illustrated in Figures 1ADown through 1F.


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  		Table 1. Morphological Analysis of Mouse Femoral Arteries



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  		Figure 1. Proliferative response to arterial injury of mouse femoral arteries. A, Uninjured artery; B, 1 hour after injury; C, 24 hours after injury: thin layer of platelets; D, 1 week after injury; E, 2 weeks after injury; and F, 4 weeks after injury. All sections were stained with combined Masson’s trichrome–elastic and photographed at x200 magnification.

Morphometric Analysis
Morphometric data are summarized in Tables 1Up, 2Down, and 3Down. Using pooled data from all groups, we found that the mean intimal area was 5937±681 µm2 at 2 weeks and 9655±1258 µm2 at 4 weeks (P<0.01). This measurement translated into an I/M ratio of 0.5±0.05 at 2 weeks and of 1.1±0.1 at 4 weeks (P<0.01) and a luminal narrowing of 14±2% at 2 weeks and of 23±3% at 4 weeks (P<0.01). None of the sham-operated arteries had intimal proliferative lesions; therefore, their I/M ratio was zero. There were no significant differences in vessel area, intimal area, medial area, I/M ratio, or percentage of luminal narrowing between mice injured with 1 or 3 passages of the wire or between male and female groups. A reduction in medial area was found between 2 and 4 weeks (P<0.05). Disruption of the IEL was more frequent in the 3-passage group compared with the 1-passage group; IEL length was 709±39 and 850±27 µm, respectively (P<0.01). This deeper injury did not translate into significantly different intimal areas (7297±983 versus 8937±1683 µm2, P=NS). Disruption of the external elastic lamina was not observed in any group. Despite a trend toward greater vessel size at 2 and 4 weeks after injury, vessel area and luminal area were not significantly different between normal uninjured arteries and injured arteries at the various time points.


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  		Table 2. Morphometric Analysis of Injured Male Mouse Femoral Arteries at 2 and 4 Weeks


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  		Table 3. Sex Differences in Injured Arteries at 2 and 4 Weeks (3 Passages)

Early Inflammatory Response
By 1 hour, the denuded luminal surface was covered with a monolayer of leukocytes (Figures 1BUp and 2ADown), identified by nuclear morphology as mostly neutrophils. The leukocytes were in contact with a thin layer of material adherent to the denuded surface. This layer stained positively with a polyclonal antibody directed against mouse platelets (Figure 3ADown, arrowheads). A PECAM-1 antibody did not stain the luminal surface (Figure 3CDown). Antibodies against {alpha}-actin and von Willebrand factor also did not stain the injured surface (not shown). Staining for P-selectin (Figure 3EDown), ICAM-1 (Figure 4ADown), and VCAM-1 (Figure 4CDown) was positive on the luminal surface. None of these adhesion molecules were expressed in the media. Fibrinogen staining was absent (Figure 4EDown). At 24 hours, the presence of neutrophils was markedly reduced, and the luminal surface stained positively with an antibody against platelets (Figure 2BDown). Identical results were obtained with an antibody against fibrinogen (not shown).



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  		Figure 2. Accumulation of leukocytes and platelets after mouse arterial injury. A, 1 hour after injury, showing accumulation of neutrophils (PMN); B, 24 hours after injury, showing platelets (PL; rabbit anti-mouse thrombocyte, 1:20 000) coating the luminal surface; C, 1 week after injury, macrophages (MAC; rabbit anti-mouse macrophage, 0.5 µg/mL); and D, platelets (PL). Sections A and B were photographed at x400 magnification, B and C, at x630.



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  		Figure 3. Early expression of platelet antigen, PECAM-1, and P-selectin after mouse arterial injury. Sections of mouse femoral arteries 1 hour after injury (A, C, E) and uninjured controls (B, D, F) stained with antibodies to platelet antigen (A, B), PECAM-1 (rat anti-mouse CD31 monoclonal antibody, 5 ng/mL; C, D), and P-selectin (rabbit anti-human polyclonal antibody, 5 µg/mL; E, F). Arrows indicate neutrophils (PMN), and arrowheads, the stained luminal surface. All sections were photographed at x400 magnification.



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  		Figure 4. Early expression of ICAM-1, VCAM-1, and fibrinogen after mouse arterial injury. Sections of mouse femoral arteries 1 hour after injury (A, C, E) and uninjured controls (B, D, F) stained with antibodies to ICAM-1 (monoclonal anti-mouse ICAM-1, 3 µg/mL; A, B), VCAM-1 (rat anti-mouse CD106/VCAM-1, 10 µg/mL; C, D), and fibrinogen (rabbit anti-mouse polyclonal antibody, 1:2000; E, F). All sections were photographed at x400 magnification.

At 1 week, platelets (Figure 2CUp) and macrophages were present on the injured surface. Macrophages were identified by using a polyclonal antibody (Figure 2DUp) and MOMA-2 (not shown). Normal nonoperated arteries did not express platelet antigen (Figure 3BUp), PECAM-1 (Figure 3DUp), P-selectin (Figure 3FUp), VCAM-1 (Figure 4DUp), or fibrinogen (Figure 4FUp) in endothelial cells or SMCs. Only ICAM-1 was expressed in normal endothelium (Figure 4BUp). Staining of sections from sham-operated animals (n=24, not shown) was identical to the nonoperated controls.


*    Discussion
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This article reports a model of mouse femoral arterial injury that uses passage of an angioplasty guidewire. The operative time is short, the procedure is safe for the animals, and it can be done bilaterally. This technique results in substantial intimal hyperplasia, with I/M ratios comparable to those seen in larger animal models.

Most mouse models of intimal hyperplasia described to date use some form of external injury. With such methods, the mechanisms of injury differ from those associated with intravascular manipulations. For example, Kumar and Lindner18 reported on remodeling plus neointima formation after simple ligature of the mouse carotid artery. That method caused little mechanical trauma and no deendothelialization, and neointima formation appeared to be induced by blood stasis. Another external method, the application of electric current to the femoral artery,17 caused endothelial denudation and platelet-thrombus formation, but it was also associated with extensive necrosis of medial SMCs. The simple placement of a cuff around the mouse femoral or carotid artery can induce modest intimal hyperplasia with I/M ratios of {approx}0.3.15 16 In the first study of intravascular denudation, Lindner et al19 obtained an intimal area of 12 000 µm2 at 2 weeks in the mouse carotid artery; however, no I/M ratio was reported. The main limitations of that model appear to be the technical difficulty and a modest neointimal response, resulting in low I/M ratios.20 21

The femoral approach offers the advantage of bilateral injury and carries less risk of morbidity and mortality compared with the carotid artery. The time course of intimal hyperplasia in the present study is similar to that found in rabbit,29 pig,30 and baboon31 models. The I/M ratios obtained are comparable to those of other animal models.32 33 34 At 4 weeks, we obtained an I/M ratio of 1.1, higher than those previously reported in mice.15 20 Of note was a significant decrease in medial area between 2 and 4 weeks after injury. This decrease in medial area was of similar magnitude to the increase in intimal area and thus, contributed substantially to the marked increase in I/M ratio between 2 and 4 weeks. Potential mechanisms for the reduction in medial area include continued migration of SMCs to the neointima and ongoing medial SMC loss (due to necrosis and/or apoptosis), in the absence of continued medial SMC proliferation.35 36 Although a trend toward larger vessel size was observed at late time points after injury, no differences in vessel and lumen size were found between normal and injured arteries. This kind of arterial remodeling has been described in different animal models of arterial injury.37 38 39 Together with the reduction in medial area, this may account for the limited luminal narrowing elicited in this mouse model, despite the substantial increase in intimal area.

Although estrogens are known to be atheroprotective,40 their effect on neointima formation is not well defined. In the present study, no significant differences were found between female and male adult wild-type mice. In contrast, using the periarterial cuff injury technique, Moroi et al15 reported I/M ratios of 0.17 in female and 0.27 in male wild-type mice and virtually no neointima formation in pregnant mice. Another study showed I/M ratios of 0.04 in oophorectomized mice receiving exogenous estrogen and of 0.12 in untreated animals.20 Those studies are difficult to compare, because both the response to endoluminal injury may be different from periarterial cuff placement and estrogen replacement may have different effects than do physiological levels of hormones in normal female adult mice.

A significant finding in our experiments was the early margination of leukocytes, predominantly neutrophils. This phenomenon, in the absence of an endothelium, may be mediated by interactions between leukocyte receptors and adhesion molecules on the denuded surface.41 42 Among the molecules involved in these interactions are P-selectin,40 PECAM-1,41 ICAM-1, and VCAM-1.41 43 44 These adhesion molecules have been shown to be upregulated in arterial injury models45 46 and in atherosclerotic lesions.47 48 49 The present study provides evidence that adhesion molecules are present on the denuded luminal surface as early as 1 hour after injury, with an associated early accumulation of leukocytes. PECAM-1 is normally expressed on activated endothelial cells, platelets, and leukocytes.50 We did not find PECAM-1 on uninjured, normal endothelial cells or medial SMCs. In addition, no PECAM-1 was present on the injured wall. The presence of platelet antigen on the luminal surface without PECAM-1 expression suggests that platelet microparticles or nonactivated platelets, rather than intact activated platelets, may be components of the layer to which neutrophils adhere. Platelet microparticles have been reported to enhance leukocyte adhesiveness to endothelial cells.51

VCAM-1, P-selectin, and ICAM-1 were all found to "decorate" the surface of denuded vessels at 1 hour. In normal uninjured vessels, endothelial cells do not express VCAM-152 but may express low levels of ICAM-1.46 High levels of these adhesion molecules have been found 2 days after injury in regenerating endothelial cells.45 To our knowledge, no evidence for the presence of VCAM-1 or ICAM-1 has been previously reported on a deendothelialized vessel wall. The cellular expression of VCAM-1 and ICAM-1 in vivo and in vitro is observed 4 hours after stimulation and depends on mRNA and protein synthesis.41 The source of the adhesion molecules found 1 hour after injury is unclear. In the current model, the endothelium is completely denuded. Because VCAM-1 and P-selectin were not seen in normal endothelial cells, it is unlikely that injured endothelial cells are the source. In addition, no staining was seen in the media of normal or injured vessels, making this an unlikely source as well. We therefore hypothesize that these adhesion molecules are derived from the circulation. Recent data have demonstrated the presence of circulating ICAM-1, VCAM-1, and other adhesion molecules, which are upregulated under conditions of endotoxic shock,53 ischemia/reperfusion,54 55 variant angina,56 and atherosclerosis.49 57 Although these circulating molecules have been thought of as markers of cellular inflammation, it is possible that they might remain functional and serve as a nidus for early leukocyte adhesion to injured vascular surfaces.

Previous studies have shown the early recruitment of neutrophils after injury and the persistence of neutrophil products in the vessel wall at later time points.58 59 60 The contribution of neutrophils to intimal hyperplasia has not been well defined. On activation and recruitment to areas of injury, neutrophils degranulate and release a number of proteolytic enzymes (eg, elastase and collagenase), cytokines, free oxygen radicals and other products capable of promoting additional tissue injury.61 Moreover, neutrophils have been shown to stimulate SMC growth in vitro.62 Markers of neutrophil activation are correlated with the development of human restenosis after coronary angioplasty.63 64 65 66 In this regard, it should be noted that specific inhibition of leukocyte adhesion with a monoclonal antibody directed against the ß2-integrin Mac-1 reduced neointimal formation after balloon angioplasty and stent injury in a rabbit model.42 That study did not establish whether this result was due to an effect on neutrophils or macrophages.

In summary, we have established a relatively simple and reproducible model of femoral artery injury in mice. This model is associated with substantial intimal hyperplasia and a rapid inflammatory response that is similar to that found in larger animals. This model should be valuable in examining the effects of arterial injury in transgenic mouse models.


*    Acknowledgments
 
This work was supported in part by a grant-in-aid from the Spanish Heart Association to M.R. and by grants HL-43302, HL-52206, and HL-61818 to M.B.T. We thank Veronica Gulle and Ameera Ali for excellent technical assistance.

Received February 8, 1999; accepted August 10, 1999.


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