Fig. 1.1
The endovascular injury of the murine femoral artery
Either the left or right femoral artery was exposed by blunted dissection and looped proximally and distally with a 6-0 silk suture for temporary vascular control during the procedure. A small branch was isolated and ligated distally. Transverse arterioctomy was performed in the muscular branch (a). Microsurgery forceps were used to extend the arterioctomy through which a 0.015 in. straight wire was inserted for more than 5 mm into the femoral artery toward the iliac artery (b, d). The wire was left in place for 1 min to denude and dilate the artery (e). After removal of the wire, the proximal portion of the arterial branch was tied off. Blood flow of the femoral artery was restored (c, f). Anti-CD31 immunostaining revealed that the endothelium was completely denuded (g, h). Bar, 50 μm (Reproduced from Ref. [17] with permission)
3.
Either the left or right femoral artery is exposed by blunted dissection. The accompanying femoral nerve is carefully separated, but the femoral vein is not isolated from the artery.
4.
The femoral artery and vein are looped together proximally and distally with 6-0 silk suture (Natsume Co., Tokyo) for temporary vascular control during the procedure.
5.
A small branch between the rectus femoris and vastus medialis muscles is isolated and looped proximally and ligated distally with 6-0 silk sutures.
6.
Veins and connective tissues around the artery are carefully removed with microsurgery forceps (No. 11253–20, Dumont S.A., Switzerland).
7.
The exposed muscular branch artery is dilated by topical application of one drop of 1 % lidocaine hydrochloride for 1 min.
8.
Transverse arterioctomy is performed in the muscular branch with Vannas style iris spring scissors (No. 15000–00, Fine Science Tools, Inc., Foster City, CA).
9.
Microsurgery forceps (No. 11253–25, Dumont S.A., Switzerland) are used to extend the arterioctomy through which a straight spring wire (0.38 mm in diameter, No. C-SF-15-15, COOK, Bloomington, IN) is carefully inserted into the femoral artery for more than 5 mm toward the iliac artery.
10.
The wire is left in place for 1 min to denude and dilate the artery.
11.
Then, the wire is removed, and the silk suture looped at the proximal portion of the muscular branch artery is secured.
12.
Blood flow in the femoral artery is restored by releasing the sutures placed in the proximal and distal femoral portions.
13.
The skin incision is closed with a 5-0 silk suture (Natsume Co., Japan).
14.
The mice are euthanized by intraperitoneal administration of an overdose of Nembutal at the time points indicated.
15.
At death, the mice are perfused at a constant pressure (80 mmHg) via the left ventricle with 0.9 % NaCl solution followed by perfusion fixation with freshly depolymerized 4 % paraformaldehyde in PBS (pH 7.4). The femoral artery is carefully excised, postfixed in 4 % paraformaldehyde overnight at 4 °C, and embedded in paraffin.
1.2.2 Morphometric Analysis
Cross sections (5 μm) are deparaffinized, stained with hematoxylin and eosin, and mounted with MOUNT QUICK mounting media (DAIDO, Tokyo). The image is digitized by a digital camera on a PROVIS AX80 microscope (Olympus, Tokyo). Digitalized images were analyzed using software (Image J, NIH). The lumen, internal elastic lamina, and external elastic lamina were defined. The intimal (tissue between the lumen and internal elastic lamia) and medial (tissue between the internal elastic lamina and external elastic lamina) areas were measured, and neointima/media ratio was calculated. All data are expressed as mean ± SEM.
1.2.3 TUNEL Staining
The 4 % paraformaldehyde-fixed paraffin-embedded sections (5 μm) are deparaffinized and rehydrated. The tissue is permeabilized with 20 mg/ml proteinase K for 30 min. Terminal deoxynucleotidyl transferase enzyme and dUTP conjugated to a fluorescein cocktail are added to the tissue sections according to the manufacturer’s specifications (Roche Molecular Biochemicals, in situ death detection kit). Nuclei are counterstained with Hoechst 33258 (Sigma, St. Louis, MO) and mounted with VECTASHIELD mounting media (Vector Laboratories, Inc., Burlingame, CA). Specimens are examined and photographed on a PROVIS AX80 microscope (Olympus, Tokyo) equipped with an epifluorescence optical lens. Pictures are recorded on a CCD camera.
1.2.4 Transmission Electron Microscopy
The femoral artery is excised at 2 h after the injury and fixed in 2.5 % glutaraldehyde, 4 % paraformaldehyde, and 0.1 mol/L sodium cacodylate. Sections are postfixed in 1 % osmium tetroxide, dehydrated, stained en bloc with 3 % uranyl acetate and Sato lead stain, and embedded in epoxy resin (Epon 812). Thin sections are examined with a Hitachi H-7000 electron microscope.
1.2.5 Scanning Electron Microscopy
The injured arteries are opened longitudinally, washed, and fixed in 2 % paraformaldehyde and 2.5 % glutaraldehyde in PBS. The tissue is postfixed in 2 % OsO4 in PBS for 2 h, followed by 1 h in 1 % thiocarbohydrazide and finally again in 2 % aqueous OsO4 for 2 h, all at room temperature. The tissue is next dehydrated in ethanol, critical point dried with CO2, and sputter coated with a 20–30 nm layer of platinum palladium (E-1010, Hitachi). The sample is then examined with a scanning electron microscope (S-3500 N, Hitachi).
1.2.6 Immunohistochemistry
Paraffin-embedded sections (5 μm thick) are deparaffinized and blocked with 5 % goat serum and 0.01 % Triton-X in PBS for 1 h at room temperature. Distributions of endothelial cells, T lymphocytes, polymorphonuclear cells, and macrophages are revealed by anti-mouse CD31 rat monoclonal antibody (clone MEC13.3, Pharmingen, San Diego, CA), anti-CD3 rabbit polyclonal antibody (Sigma, St. Luis, MO), anti-mouse CD11b rat monoclonal antibody (clone M1/70, Serotec, Oxford, England), and anti-mouse F4/80 rat monoclonal antibody (clone A3-1, Serotec, Oxford, England), respectively, followed by the avidin-biotin complex technique and Vector Red substrate (Vector Laboratories, Burlingame, CA). Smooth muscle cells can be identified by immunostaining with an alkaline phosphatase-conjugated monoclonal antibody to α-smooth muscle actin (clone 1A4, Sigma). Sections were counterstained with hematoxylin.
1.3 Typical Histological Changes of the Injured Artery
1.3.1 A Wire Can Be Inserted into the Mouse Femoral Artery
A straight spring wire (0.38 mm in diameter) is readily inserted into the femoral artery via an arterioctomy in a small muscular branch between the rectus femoris and vastus intermedius muscles, following the protocol described in Methods (Fig. 1.1, the tutorial videos 1.1 and 1.2). The success rate of the wire insertion is more than 95 % for all mice examined at age of 6 weeks or older. The wire is left in place for one minute to denude and dilate the artery (wire/artery diameter ratio = 2.0 ± 0.1). The muscular branch is ligated, and blood flow of the femoral artery is restored. I observed neither extravasation of the wire nor rupture of the femoral artery. All mice can survive the surgery.
1.3.2 Mouse Femoral Artery Is Mechanically Distended by Intraluminal Insertion of a Large Wire
The wire insertion caused overexpansion of the artery. The media was markedly thinned and the lumen remained enlarged even after the wire was removed (Fig. 1.1g, h, Table 1.1). Anti-CD31 immunostaining revealed that the endothelium was completely denuded (Fig. 1.1g, h). We occasionally (about 6 %) observed acute thrombosis in the injured arteries. The occluded arteries were not used for the analysis.
Table 1.1
Morphometric changes in the injured artery
Time point | uninjured | 1D | 3D | 1 W | 2 W | 3 W | 4 W | 8 W |
|---|---|---|---|---|---|---|---|---|
EEL (mm) | 0.59 ± 0.02 | 0.91 ± 0.08 | 1.16 ± 0.08 | 0.81 ± 0.12 | 1.37 ± 0.20 | 1.11 ± 0.07 | 0.84 ± 0.06 | 0.98 ± 0.15 |
I/M ratio | 0 | 0 | 0 | 0.31 ± 0.17 | 1.25 ± 0.27 | 1.51 ± 0.36 | 1.97 ± 0.32 | 1.69 ± 0.37 |
1.3.3 The Artery Remained Dilated with Rapid Onset of Medial Cell Apoptosis
The TUNEL procedure stains nuclei that contain nicked DNA, a characteristic exhibited by cells in the early stages of apoptotic cell death. Immunofluorescent TUNEL analysis was performed on sections from uninjured and injured mouse femoral arteries. Uninjured arteries had no detectable TUNEL-positive nuclei. However, a large number of TUNEL-positive nuclei were detected in the layers of the media at two hours after injury (Fig. 1.2). At 8 h after injury, a higher level of TUNEL-positive nuclei was detected. At 17 h, the cellularity of the media appeared to decline dramatically and a few TUNEL-positive cells were detected in the media.
Fig. 1.2
The injury induces dilatation of the artery with rapid onset of apoptosis of smooth muscle cells
The injured artery was perfusion fixed with 4 % paraformaldehyde and excised carefully at the time points indicated. Paraffin-embedded cross sections (5 μm) were stained with hematoxylin and eosin (H & E), TUNEL, and Hoechst 33258. Bar indicates 100 μm (Reproduced from Ref. [17] with permission)
1.3.4 Electron Microscopic Observation of the Injured Artery
Ultrastructure was also examined by transmission and scanning electron microscopes. In uninjured arteries, the endothelium lined at the luminal side of the internal elastic lamina (Fig. 1.3a, b). At two hours after injury, the artery remained dilated with a thin media containing very few cells. Fibrin deposition and platelet accumulation were observed on the denuded luminal side (Fig. 1.3a–c). At 6 days, leukocytes including monocytes and lymphocytes homed at the luminal side (Fig. 1.3d). At 14 days, neointimal hyperplasia of SMC-like cells was observed (data not shown). Scanning electron microscope demonstrated that luminal side was partially re-endothelialized. As far as we examined, it was not observed that smooth muscle cells were migrating from the media into the luminal side across the internal elastic lamina.
Fig. 1.3
Electron microscopic observation of femoral artery after wire-mediated endovascular injury
(a) At 2 h after surgery, the injured and uninjured arteries were perfusion fixed with 2.5 % glutaraldehyde and 2 % paraformaldehyde in PBS. The arteries were postfixed in 1 % osmium tetroxide and embedded in epoxy resin (Epon 812). Thin sections were stained with 3 % uranyl acetate and examined under a transmission electron microscope (H-7000, Hitachi, Tokyo). Bar, 2 μm. (b–d) Mice were sacrificed at 2 h and 6 days after injury. The excised arteries were opened longitudinally and fixed in 2 % paraformaldehyde and 2.5 % glutaraldehyde in PBS. The tissue was postfixed in 2 % OsO4, dehydrated in ethanol, critical point dried with CO2, and sputter coated with a 20–30 nm layer of platinum palladium (E-1010, Hitachi). (b) The luminal side of uninjured and injured arteries was examined at 2 h with a scanning electron microscope (S-3500 N, Hitachi). Bar, 2 μm. (c) Platelets adhering to the luminal surface of the injured artery at 2 h. Bar, 6 μm. (d) Monocytes or granulocytes (arrowheads) were homing on the luminal side at 6 days after injury. Lymphocytes also adhered on the injured artery (arrows). Bar, 6 μm (Reproduced from Ref. [47] with permission)
1.3.5 The Enlarged Lumen Was Narrowed by Neointimal Hyperplasia Composed of Smooth Muscle Cells
The lumen of the injured artery remained dilated as determined by the circumference of the external elastic lamina (Table 1.1). Cellular constituents of the arterial wall were evaluated by immunohistochemistry. At one week, small neointima was found on the luminal side of the injured artery (Fig. 1.4a). Most of the neointimal cells expressed CD45, a marker for hematopoietic cells, but not α-SMA (Fig. 1.4b). At three weeks, large neointima had grown on the luminal side. A few CD45-positive cells were detected, particularly in the luminal side of the neointima. There were some α-SMA-positive cells (Fig. 1.4b). At six weeks, CD45-positive cells were seldom detected in neointima or in media, whereas the neointima was predominantly composed of α-SMA-positive cells. Cellular constituents of the arterial wall after wire-mediated injury were also examined by RT-PCR using RNA obtained from total homogenate of the vessel wall (Fig. 1.4c). Marked downregulation of α-smooth muscle actin (α-SMA) expression was observed at one week. α-SMA expression gradually increased at two weeks and reached to that of uninjured artery at 4 and 6 weeks.
Fig. 1.4
Temporal and spatial characterization of cellular constituents during neointima hyperplasia development
A large wire was inserted into the femoral artery of adult male wild-type mice. After injury, blood flow was restored and the injured arteries were harvested at the time points indicated. (a) Injured arteries were embedded in paraffin and stained with hematoxylin and eosin. Arrows indicate internal elastic lamina. Bar, 20 μm. (b) Cross sections were stained for α-smooth muscle actin (α-SMA) or CD45.using the avidin-biotin complex technique and VECTOR RED substrate. Arrows indicate internal elastic lamina. Bar, 20 μm. (c) Total RNA was prepared from the femoral artery with the use of RNazol reagent. RT-PCR was performed (Reproduced from Ref. [47] with permission)
1.3.6 The Injury Can Induce Reproducible Neointimal Hyperplasia that Is Composed of Smooth Muscle Cells
The neointimal hyperplasia continued to grow up to 3 or 4 weeks after which lesion formation did not advance further. The lesion was concentric and very homogenous in the region where the wire was inserted (Fig. 1.5a). An immunohistochemical study of α-smooth muscle actin revealed that the neointima was exclusively composed of smooth muscle cells (Fig. 1.5b). The luminal side of the intima was almost completely re-endothelialized at 4 weeks as determined by anti-mouse CD31 staining. No capillary formation was detected in the intima, excluding the possibility that neointimal formation results from recanalized thrombosis. Macrophages were detected in the adventitia, occasionally also in the intima. No polymorphonuclear cells were detected in the intima (data not shown).
Fig. 1.5
Characterization of the neointimal hyperplasia
(a) Three-dimensional characterization of the neointima. The injured femoral artery of the C57BL/6 mouse was perfusion fixed at 4 weeks after the injury. Paraffin-embedded femoral artery was sectioned every 500 μm as indicated. Sections were deparaffinized and stained with hematoxylin and eosin. The lesion formation was homogenous in the region where the wire expanded the artery. Bar, 100 μm. Arrows indicate internal elastic lamina. (b) The paraffin-embedded cross sections were stained for smooth muscle cells (SMC, α-smooth muscle actin), endothelial cells (CD31), and macrophages (MΦ, F4/80) using the avidin-biotin complex technique and VECTOR RED as substrate. Sections were counterstained with hematoxylin. The neointima was exclusively composed of smooth muscle cells. Arrows indicate internal elastic lamina. The luminal side was almost completely re-endothelialized (small arrow heads). Macrophages were detected in the adventitia, occasionally also in the neointima (large arrows), but not in the intima or in the media. Bar 50 μm. (c) The femoral artery of the inbred mice was injured by the wire and harvested at 4 weeks. The wire injury produced similar exuberant neointimal formation in all inbred mice. Bar, 100 μm. Arrows indicate internal elastic lamina (Reproduced from Ref. [17] with permission)
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