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Department of Biomedical Engineering, University of California, Irvine, California 92697
Submitted 10 September 2003 ; accepted in final form 5 January 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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injury; zero-stress state; balloon injury; endothelin
We hypothesize that the approach proposed in this study will allow removal of the endothelium without injury to the media. We examined this hypothesis structurally by studying histological sections under light and scanning electron microscopy; functionally by inducing acute contraction with [K+], endothelin, and norepinephrine; and biomechanically by studying the zero-stress state and the stress-strain relationship. The present model will be useful for future studies on endothelial regeneration, atherogenesis, and remodeling.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The animals were anesthetized with pentobarbital sodium (60 mg/kg ip). Surgical anesthesia was maintained with pentobarbital sodium (20 mg·kg–1·h–1 ip). Arterial pressure was measured through a cannulated carotid artery. A jugular vein was cannulated for the administration of heparin (100 U/kg) for anticoagulation. A heating pad was used to maintain the body temperature of the animal. An incision was made on the skin, and the fat was resected away from the femoral artery. After exposure, the common femoral arteries in both legs were isolated along their entire lengths. The proximal femoral arteries were closed with arterial clamps. An arteriotomy was made on the distal femoral arteries where the arteriotomy area was in the near proximity of the bifurcation of superfacial femoral artery to obtain a sufficient specimen for the mechanical experiment. Figure 1 shows a schematic of a specifically designed catheter (outer diameter: 0.4 mm) with a stainless steel tube (diameter: 0.4 mm, length: 1 mm) fixed on a nylon suture that passes through the catheter (top). The catheter was inserted into the right femoral artery through the arteriotomic mouth (Fig. 1, bottom). Tyrode solution containing 0.14 M KCl was dripped on the external surface of the artery to induce contraction onto the catheter. The diameter of the catheter was 
90% of the vessel lumen diameter, which was chosen so that during the contraction the endothelial surface comes in contact with the catheter. The stainless steel tube was moved back and forth, three times, along the length of the artery. The arterial lumen was washed with saline containing heparin (50 U/ml) after removal of the tube. In the same animal, a 2-F Fogart balloon was inserted into the left common femoral artery. The balloon was pressurized to 1.05 atm and then moved back and forth, three times, along the lumen of the artery. The balloon pressure was subsequently removed, and the arterial lumen was washed with saline containing heparin (50 U/ml). The arteriotomy of both femoral arteries was sutured using 10-0 nylon monofilament sutures. The arterial clamp on each proximal artery was removed to restore blood flow. The skin on both incisions was closed, and the animal was allowed to recover. Water and food were replenished as needed. The sham-operated animals were treated identically including the creation of arteriotomy but no insertion of catheter.
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Functional assessment. After 2 days, the incisions were reopened, and the femoral arteries were exposed. Tyrode solution containing 0.14 M KCl (endothelial independent) was dripped onto the outer surface of the arterial wall to elicit contraction (4). The arterial diameter was recorded during contraction. This procedure was repeated three times to obtain stable measurements. The KCl was washed away with saline for five times at the end of each contractile stimulation. This procedure was repeated with Tyrode solution containing KCl (0.14 M) and endothelin (20 pM). The vessel was again washed several times with saline. The vessel was then cannulated, and 1 ml of norepinephrine (2 µM) was administered intraluminally. The resulting diameter changes were recorded via video camera and subsquently measured with image-analysis software (SigmaScan Pro.5).
Pressure-diameter relationship. At the conclusion of the contractile experiments, the artery was cannulated from the angiotomic mouth and ligated proximally. Pressure-diameter measurements were made at a series of pressures (e.g., from 30 to 150 mmHg in increments of 30 mmHg). The outer diameter was videotaped at each corresponding pressure. The outer diameter measurements were made with image analysis software (SigmaScan Pro.5).
Determination of zero-stress state. Evans blue was injected into the jugular vein to stain the endothelium-denudated region for 1 h (14). The animal was then killed with an overdose of anesthesia. The femoral arteries in both legs were excised, and the arterial axial lengths both in vivo and in vitro were recorded to compute the longitudinal stretch ratio. Four rings were cut from the arterial segment to obtain the dimension of the artery at the no-load and zero-stress states, including inner and outer circumferences and wall area.
Structural examination. At the conclusion of the mechanical experiments, the arterial segments were fixed in buffered formalin and processed for histological examination under light microscopy. Additional arterial segments were examined by scanning electron microscopy. Those segments were cut axially, pinned flat on piece of cork, and fixed in 6.25% buffered glutaraldehyde overnight. The specimens were subsequently fixed in 1% OsO4 for 1 h. After being washed in double distilled water twice, the tissues were dehydrated in increasing concentrations of ethyl alcohol and subsequently in increasing concentration of hexamethyldisilazane. Dried tissue was mounted on an aluminum stage covered with quick-drying colloidal silver paste. The stage was coated with gold-palladium in a sputter coater, and the tissue was viewed with scanning electron microscopy.
Biomechanical analysis. The incompressibility condition was used to compute the inner radius (ri) as follows
 | (1) |
z = l/lo, where l and lo are the vessel lengths in the loaded and no-load states, respectively.
The in vivo circumferential strain was computed according to the Green strain (
), which is defined as follows
 | (2) |
i,o is the inner or outer circumferential stretch ratio (
); ci,o is the inner or outer circumference of the vessel in the loaded or no-load state, and 
is the inner or outer circumference in the zero-stress state. Similarly, the midwall strain was computed in reference to the midwall circumferences in the loaded or no-load state in reference to the zero-stress state. In the no-load state, the strain is referred to as the residual strain.
At equilibrium, the average circumferential Kirchhoff stress for a cylindrical vessel wall (
) is given by
 | (3) |
Equation 2 can also be used to determine the inner and outer strains of the vessel wall from the respective circumferences. According to the Bernoulli-Kirchhoff hypothesis that plane sections remain plane during the deformation (inflation), the strain distribution from the inner to the outer wall must be linear (7). Hence, from the known values of strains at the inner and outer walls, we may obtain the strains in the interior of the vessel wall by linear interpolation.
The determination of transmural stress is much more difficult as there is no direct way to measure stress. A constitutive equation (stress-strain relation) is necessary to compute stress from the measured values of strain. In inflation experiments, there is deformation in the circumferential and longitudinal direction. Hence, the constitutive relation should relate the circumferential and longitudinal stresses to strains. If we assume that the longitudinal deformation is negligible and the vessel wall is homogeneous, we can approximate the transmural distribution of circumferential stress from the measured circumferential stress-strain relation.
Data analysis. Data are presented as arithmetic means ± SD unless otherwise stated. Significant differences between various parameters were determined with the use of parametric ANOVA, followed by Student's t-test. A probability of P < 0.05 was considered to be of a statistically significant difference.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The removal of endothelium was also verified with the use of norepinephrine (endothelium dependent) 2 days after the injury. Similarly, the function of the media was assessed by the contraction of the blood vessel with KCl (endothelium independent) 2 days after the injury. The degree of contraction was quantified by the ratio of diameter of the vessel relative to the homeostatic diameter. Figure 4 shows that the vessel treated with the new method or by balloon injury did not contract with norepinephrine. Figure 5A shows that the femoral artery whose endothelium was denuded by the new method can still contract with K+ (endothelium independent), whereas the balloon injury vessel cannot. The degree of contraction in the new model, however, was smaller than that of control vessels (50%). The addition of endothelin to KCl induces additional contraction of the vessels treated with the new method, as shown in Fig. 5B. The measurements shown in Fig. 5B were made 10 min after application of the vasoactive substance. We found no statistically significant difference in the degree of contraction between KCl applied to a control vessel and a combination of KCl and endothelin applied to a vessel treated with the new method. This is in contrast to the balloon injury model, which did not contract at all.
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The pressure-diameter relationship is shown in Fig. 6A for the control vessels as well as for denudated vessels with the balloon injury and new method. The pressure-diameter data are transformed into mean stress-midwall strain relationship as shown in Fig. 6B. The stress and strain were computed with reference to the zero-stress state. The zero-stress state is characterized by the opening angle, which is defined as the angle subtended by two radii connecting the midpoint of the inner wall. The data on the opening angles are shown in Fig. 7A. There was no statistically significant difference between the opening angle in the control vessels and those treated with the new method. There was a large and significant decrease, however, in the vessels treated with the balloon method. The changes in the residual strain shown in Fig. 7B were consistent with the observed changes in opening angle. Both inner and outer residual strains were significantly reduced after the balloon injury. The balloon method, however, altered the transmural distribution of strain and stress, as shown in Fig. 8. The transmural strain distribution is shown in Fig. 8A, where the inner and outer strains are linearly interpolated across the vessel wall as described in MATERIALS AND METHODS. The corresponding stress distribution shown in Fig. 8B was obtained from the stress-strain relation shown in Fig. 6B for the inner and outer strains and their interpolated values. The ratios of inner to outer strain were 1.4 ± 0.27 for control, 1.4 ± 0.25 (P > 0.05) for the new method, and 2.4 ± 0.55 (P < 0.01) for balloon injury. Similarly, the ratios of inner to outer stress were 2.2 ± 0.41 for control, 3.6 ± 0.69 (P = 0.06) for the new method, and 12.0 ± 2.9 (P < 0.001) for balloon injury. The foregoing results on stress and strain were calculated at physiological pressures (120 mmHg) in reference to the zero-stress state.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Structural validation. We confirmed that the damage to the endothelium by the new method was complete and reproducible by the extensive uptake of Evans blue and silver nitrate throughout the region of interest. The scanning electron microscopy photomicrographs also confirmed the denudation of the endothelium, as shown in Fig. 2. The media, on the other hand, showed normal structure under light microscopy, as seen in Fig. 3. The balloon injury method was equally successful in denudation but caused significant damage to the media and in particular to the internal elastic lamina (5, 6, 16, 19, 22, 24, 25). Finally, the denudation was confirmed by the lack of contraction in the presence of norepinephrine (Fig. 4).
Functional validation. The functional studies, quantified by the contraction of the media, confirmed that the vessel treated with the new method will contract to 50% of the control value 2 days after the insult (Fig. 5). This can be compared with the zero contraction for the balloon injury model. Similarly, it was found that vessels denudated with Triton can only contract to 50% of the control values 2 days after treatment (12). These investigators also verified that the media was not damaged structurally during Triton treatment. Hence, this poses the question of why an apparently normal media only contracts to 50% of control in the absence of endothelial cells.
It is well known that the endothelium plays an important role in the control of smooth muscle tone by production and release of various vasoactive substances, e.g., most notably nitric oxide and endothelin (3, 20). Hence, even normal media may not have the same vasoactive ability in the absence of endothelium. To test this hypothesis, we added an in vivo concentration of endothelin in addition to [K+] and found that the vessel increased the degree of contraction to those of control values. Hence, the role of endothelium (e.g., the release of endothelin) is essential for the normal contraction of the media.
Biomechanical validation. We verified that the circumferential mechanical properties of the vessel, expressed in terms of pressure-diameter and stress-strain relationships, are unaltered after removal of endothelium with the new method (Fig. 6). This is an expected finding because the endothelium is relatively thin in the normal artery and does not contribute significantly to the circumferential mechanical properties. The balloon model, however, overdistends the vessel and increases the diameter at any given pressure or the strain at any given stress.
We also measured the changes in the opening angle and consequently the residual strain. We found that the new method preserves the control values of opening angle and residual strain, whereas the balloon method significantly lowers both (Fig. 7). It has previously been shown that the residual strain significantly reduces the stress concentration at the inner portion of the vessel wall at the in vivo state and leads to a uniformity of transmural strain (1, 8). These features are preserved using the new method but are significantly altered with the balloon model. The balloon injury model imposes stress and strain concentration at the intima, as shown in Fig. 8, which is likely responsible for some of the remodeling observed in this model (10).
Significance of study. Arterial injury is known to initiate repair and remodeling processes that are implicated in the pathogenesis of important vascular disorders including atherogenesis and restenosis. The remodeling processes of structure and function are very likely to be injury specific. Hence, it is essential to develop models where the degree of injury is both well defined and reproducible. In the present study, we developed such a method for pure endothelial injury. In future studies, this model will be used to understand endothelial regeneration, vascular remodeling, and atherogenesis.
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ACKNOWLEDGMENTS |
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This research was supported in part by National Heart, Lung, and Blood Institute Grant 2 R01 HL-055554-06 and American Heart Association (AHA) Grant 0140036N. G. S. Kassab was the recipient of an AHA Established Investigator Award.
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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