INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ARTERIAL LUMEN SIZE is the fundamental parameter that determines vascular resistance and therefore blood pressure, organ blood flow, as well as cardiac work. The arterial wall presents both viscoelastic and mechanical properties, and the lumen size or inside diameter (ID) is the result of the integrated effects of transmural pressure, tethering of surrounding tissues, wall elasticity, and active responses of the vascular smooth muscle (20).

A variety of approaches have been taken to quantify the effects of physiological and pharmacological perturbations on vessel diameter. In earlier studies, large vessels (up to several centimeters in diameter) were used to study the relationship between transmural pressure and diameter utilizing pulse-wave velocity-distensibility measurements (3, 14), volume measurements as a function of transmural pressure (8, 11, 24, 28), or intraluminal saline-filled balloons with ultrasonography (30). More recently, the reactivity of small resistance arteries has been studied utilizing similar techniques (19) as well as tension-displacement measurements (22), radiological dimension measurements (10), dimension measurements utilizing photoelectric diode arrays (29) or ultrasonic echotracking devices (1, 9, 12, 33), fluorescent techniques (32), and most commonly, video-dimension analysis of transilluminated vessels (26, 34). This latter method allows diameter to be measured directly in vessels of varying sizes (25, 27) and has been utilized for studies both in vivo and in vitro.

Although vascular resistance is directly related to changes in ID, outside diameter (OD) measurements are often used because they are most easily ascertained at any intravascular pressure, and ID can be difficult to accurately measure in thick-walled or tightly constricted vessels. Biomechanical studies have shown that the blood vessel wall is minimally compressible (4, 5). Thus it has been widely assumed that the vascular wall volume is constant (16, 21, 23, 31) or that by controlling or neglecting the longitudinal extension of the vessel with pressure (8), the vessel wall cross-sectional area (WCSA) could also be considered constant (7, 10, 36). These assumptions permit calculation of the ID from OD at any pressure, assuming that at least one accurate measurement of WCSA can be made. However, the general applicability of the assumption that WCSA remains constant and that ID can thereby be determined from OD using this approach has never been rigorously assessed under a range of experimental conditions.

In the present study we have investigated whether WCSA remains constant, such that ID (and therefore vascular resistance) can be calculated directly from the OD measurement during passive and active changes in diameter. In cannulated mouse arteries, in which longitudinal extension was limited, we analyzed the relationship between measured ID and the ID calculated from OD, assuming a constant WCSA. The experiments were performed in both wild-type and elastin-mutated mice (which results in a significant difference in the vessel wall structure) (17) of two different age groups (1-3 days and 5-9 mo). Three different arteries (pulmonary, ascending aorta, and carotid artery) were used, and diameter changes were measured during both increasing and decreasing transmural pressure changes (from 0 to 175 mmHg) and during active contraction and relaxation (at a constant pressure) induced by the -adrenergic agonist phenylephrine and the endothelium-dependent vasodilator acetylcholine. These experiments were performed before and after vascular smooth muscle and endothelial cell responses were abolished by KCN treatment. Additionally, to determine the applicability of this technique to small resistance-sized arteries, measured and calculated ID values were compared in small (~120 µm ID) mesenteric arteries during active vasoconstriction with the -adrenergic agonist norepinephrine. Finally, we analyzed vessel dimension measurements reported in the literature (25, 30) to assess the relationship between measured and calculated ID in prior studies.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. Nine 5- to 9-mo-old and two 1- to 3-day-old C57B1/6J mice, as well as two 5- to 9-mo-old C57B1/6J mice in which one allele of the elastin gene exhibits a deletion in exon 1 (ELN +/ animals) have been studied. This deletion was shown to lead to structural and functional differences in the wall of elastic arteries (17). Housing and surgical procedures were in accordance with institutional guidelines.

Surgical procedure and mounting of vessel. The animals were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg). The vessel (ascending aorta, left carotid artery, superior mesenteric artery, or left pulmonary artery) was quickly excised and placed in a physiological buffer (pH 7.4) of the following composition (mM): 135 NaCl, 5 KCl, 1.6 CaCl₂, 1.17 MgSO₄, 0.44 KH₂PO₄, 2.6 NaHCO₃, 0.34 Na₂HPO₄, 5.5 D-glucose, 0.025 EDTA, and 10 HEPES. The vessel was cleaned of adhering connective tissue and fat and then cannulated and mounted onto a pressure arteriograph (Living Systems Instrumentation, Burlington, VT), as previously described (2, 26). The experiments were performed in an organ bath filled with physiological buffer at 37°C. The bath was placed on an inverted microscope, and a computerized image analysis system was used for measurement of ID and OD in the transilluminated vessels as previously described (2). A digital image was used for analysis with this system, and the inherent error in any measurement was therefore ±1 pixel. This corresponded to an intrinsic error of ±1-2% of the measured ID and OD values.

Experimental protocol. In the ascending aortas and carotid arteries, following a 30-min equilibration period, intravascular (transmural) pressure was increased from 0 to 175 mmHg by steps of 25 mmHg (at least 1 min per step) and then symmetrically decreased (following the same steps and timing) back to 0 mmHg. ID and OD were recorded continuously. Integrity of smooth muscle function was assessed by bath application of the -adrenergic vasoconstrictor phenylephrine (PE, 10⁵ M) and endothelial cell integrity was tested by the addition of the endothelium-dependent vasodilator ACh (10⁵ M). These latter studies were done at a constant pressure of 75 mmHg in systemic arteries or 20 mmHg in pulmonary arteries. The vessel was then treated with KCN (13 mM) in physiological buffer for 45-60 min (at 0 mmHg) to abolish smooth muscle and endothelial cell function as verified by the subsequent lack of response to PE. The response to increases (0175 mmHg) and decreases (1750 mmHg) in pressure was then retested after KCN treatment. The procedure was identical when using the pulmonary arteries except that the intravascular pressure range was 2-50 mmHg, by steps of 10 mmHg (except for the first step which was 2-10 mmHg). A starting pressure of 2 mmHg was necessary in the thin pulmonary vessels to maintain the cylinder shape, since these vessels tend to collapse at 0 mmHg. For each vessel type studied, all experiments were performed in at least two separate vessels: 2 newborn left pulmonary arteries, 2 adult left pulmonary arteries, 4 adult left carotid arteries, 2 adult wild-type ascending aortas, and 2 elastin-mutated ascending aortas. In addition, to assess the accuracy of the ID calculations in resistance arteries during active (i.e., agonist-induced) vasoconstriction, three small mesenteric artery segments (from two separate animals) were studied at constant pressure (40 mmHg) before and after treatment with norepinephrine (NE, 10⁵ M) and NE + ACh (10⁵ M).

Measurements in ascending aorta. In larger arteries, the vessel wall is often too thick to allow the edge corresponding to the ID to be visualized accurately by transillumination. This is most often a problem at lower transmural pressures (0-100 mmHg) because passive thinning of the vessel wall with increases in diameter usually allows measurement of ID by transillumination at higher pressures (125-175 mmHg). The carotid artery was variable in this regard because direct measurement of ID by transillumination across the whole pressure range (0-175 mmHg) was possible in some carotid arteries but not in others. We describe measurements performed by transillumination of carotid arteries only in arteries where we could easily detect ID by image analysis at any pressure. The wall of the ascending aorta was always too thick to allow direct ID measurement by transillumination across the lower pressure range. To measure the ID of the ascending aorta over the entire range of pressures, we filled the vessel lumen with buffer containing 25 mg/ml dextran-coupled FITC (FITC-dextran, molecular mass  500 kDa). The large size of FITC-dextran prevents it from crossing the vessel wall (32), and it therefore remains in the lumen throughout the experiment. The FITC was excited (at 420-450 nm), and ID was determined from the resulting FITC-dextran epifluorescence signal (>520 nm) corresponding to the vessel lumen by image analysis. To calibrate the ID obtained by the FITC method, the edge of the epifluorescence signal was adjusted (by adjusting the detection threshold) so that the ID measured by both transillumination and epifluorescence at 175 mmHg (where both could be measured) were identical.

Calculated ID from vessel ring wall thickness. In two carotid arteries and two ascending aortas, IDs were directly measured by transillumination or the FITC method, respectively, as described above. In addition, at the end of the experiment, wall thickness was directly measured using image analysis of a cross section (ring) of the studied vessel, which was simply cut and measured using the image analysis system. Subtracting twice the measured wall thickness of the ring from the measured OD at 0 mmHg provided another direct measurement of ID at 0 mmHg. The ID determined by this method was compared with directly measured ID using transillumination or the FITC method and was then used to calculate ID values throughout the pressure range studied. These calculated ID values were then compared with the measured ID values obtained by transillumination or the FITC method.

Calculated ID from direct measurement of WCSA by transillumination in deeply constricted mesenteric arteries. In the small mesenteric arteries, pressure was maintained at a constant level (40 mmHg), and IDs were directly measured by transillumination, as described above, before and after addition of the vasoconstrictor NE (10⁵ M) followed by the addition of the endothelium-dependent vasodilator ACh (10⁵ M). IDs directly measured during application of NE and ACh were then compared with IDs that were calculated using the WCSA value obtained from direct ID and OD measurement at 40 mmHg (before addition of vasoactive agents).

Chemicals. All the chemicals were obtained from Sigma Chemical (St. Louis, MO), except NaCl, which was obtained from Fisher Scientific (St. Louis, MO).

Analysis of previously published data. OD and ID values of vessels were derived directly from the processed data presented in the articles cited using the corresponding transformation formulas given by the authors. The calculated and measured ID values were then analyzed and compared using the same methods as those used for the other experiments reported here.

Because of the inherently large error in the measured and calculated ID values at 0 mmHg (and 2 mmHg in the pulmonary artery), these values were excluded from the error ranges and mean errors reported in this paper (see DISCUSSION).

Analysis of relation between OD and ID. For each vessel, the ID was calculated at each pressure step (Px) based on the measured OD at the same pressure and on the WCSA at a reference pressure (Py), with the assumptions that WCSA was constant and that the vessel was a perfect cylinder. The formulas used were as follows

(1)

(2)

(3)

(4)

These formulas permit ID at any pressure (ID_Px) to be calculated from the measured OD at that pressure (OD_Px) assuming a constant WCSA, which has been calculated from a direct measurement of both ID (ID_Py) and OD (OD_Py) at some reference pressure (Py). To verify the general applicability of this approach, we determined reference WCSA (WCSA_Py) at five reference pressures (Py): 0, 25, and 175 mmHg as the pressure was raised, and 25 and 0 mmHg as the pressure was lowered in the systemic vessels; and 2, 10, and 50 mmHg as the pressure was raised, and 2 and 10 mmHg as the pressure was lowered in the pulmonary vessels. For the small mesenteric arteries, the reference WCSA was calculated at 40 mmHg.

In the analysis of the data from the literature, WCSA_Py was calculated at 0, 25, and 125 mmHg for the data published by Osol and Cipolla (25) and 0, 5, 25, 190 mmHg for the data published by Storkholm et al. (30).

The calculated ID (ID_c) and measured ID (ID_m) were compared at each pressure step, and percent error (E) was calculated as

(5)

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Adult mouse left pulmonary artery. The measured versus calculated ID values obtained from one adult pulmonary artery are shown before (Fig. 1A) and after treatment with KCN (Fig. 1B). The ID data obtained during pressure increases (solid lines) and decreases (dotted lines) from 2 to 50 mmHg are shown. As can be seen in Fig. 1, the measured ID and calculated ID using WCSA references at 2, 10, and 50 mmHg are nearly identical. Including the values from the two separate vessels studied, at all five of the reference WCSAs tested (both before and after treatment with KCN), the mean error (168 calculated ID values) was 0.63% with an error value range of 3.7% to +2.4%. Of these calculated values, 90% (151 of 168) were within an error range of 2.1% to +2.1%.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Relation between measured inner diameter (ID) and calculated ID in adult mouse pulmonary and carotid arteries. Figure is based on results from a single left pulmonary artery before (A) and after (B) treatment with KCN and on a single carotid artery before (C) and after (D) treatment with KCN. Results are representative of the results obtained in 2 separate pulmonary arteries and 4 separate carotid arteries. Solid lines, increasing pressure phase; dotted lines, decreasing pressure phase. Calc. ID (WCSAy mmHg, where y represents the pressure value), calculated ID from vessel wall cross-sectional area at y mmHg.

Adult mouse left carotid artery. In Fig. 1, C and D, the measured versus calculated ID values obtained from the adult carotid artery are shown before and after KCN treatment, respectively. For these experiments, ID was studied during pressure increases (solid lines) and decreases (dotted lines) from 0 to 175 mmHg. Again, the measured ID and calculated ID values are nearly identical at each WCSA reference shown in Fig. 1 (0, 25, and 175 mmHg). Including the values from the four separate vessels, at all five of the reference WCSAs studied (both before and after KCN), the mean error (496 calculated ID values) was 0.89% with an error value range of 4.9% to +4.9%. Of these calculated values, 90% (446 of 496) were within an error range of 3.2% to +3.2%.

Newborn mouse left pulmonary artery. The difference between measured ID and calculated ID was again quite low in the newborn pulmonary artery (Fig. 2). This was true at all pressures tested (2-50 mmHg), whether pressure was increasing (solid lines) or decreasing (dotted lines), using any of the five reference WCSAs before and after treatment with KCN. Including values from the two separate vessels studied, the mean error (168 calculated ID values) was +0.1% with a error value range of 4.8% to +2.9%. Moreover, >90% (151 of 168) of these calculated values were within an error range of 1.6% to +1.6%.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Relation between measured ID and calculated ID in newborn mouse pulmonary artery. Results shown are from a single left pulmonary artery before (A) and after (B) treatment with KCN and are a representative example of the results obtained in 2 separate vessels. Solid lines, increasing pressure phase; dotted lines, decreasing pressure phase.

Adult mouse ascending aorta. As shown in Fig. 3, some differences in size and response to pressure changes were observed between vessels from elastin-deficient (ELN +/) and wild-type animals, but the difference between measured ID and calculated ID was again quite low in the ascending aorta from both animals. For all five reference WCSAs studied in the four separate vessels tested (two wild-type and two ELN +/), both before and after KCN (496 calculated ID values), the mean error was +0.32% with a range of 4.2% to +4.6%. Moreover, >90% (446 of 496) of these calculated values were within an error range of 2.7% to +2.7%.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Relation between measured ID and calculated ID in the adult mouse ascending aorta. Figure is based on results from a single wild-type ascending aorta before (A) and after (B) treatment with KCN and on a single heterozygous elastin-mutated ascending aorta before (C) and after (D) treatment with KCN. Results are representative examples of the results obtained in 2 separate wild-type and 2 separate elastin-mutated ascending aortas. Solid lines, increasing pressure phase; dotted lines, decreasing pressure phase.

Action of vasoactive agents on vessels. The effects of active contraction and relaxation of the vessel wall on differences between measured and calculated ID were studied utilizing the vasoconstricting agonist PE (10⁵ M) followed by the addition of the endothelium-dependent vasorelaxing agent ACh (PE + ACh, 10⁵ M). In Fig. 4, where the response of a single representative vessel for each vessel type is presented, the measured ID values for each treatment are shown together with the calculated ID values, utilizing the WCSA reference at each of the pressures indicated. Again, there is little difference between the measured ID and the calculated ID during these treatments using WCSA at any of the reference pressures. Pooling the data from all 12 vessels used (2 newborn pulmonary arteries, 2 adult pulmonary arteries, 4 carotid arteries, 2 wild-type ascending aortas, and 2 ELN+/ ascending aortas) before and after treatment with PE or PE + ACh, the mean error (180 calculated ID values) was 0.12% with a range of 4.4% to +4.9%. Moreover, >90% (162 of 180) of these calculated values were within an error range of 3.4% to +3.4%.

View larger version (55K): [in this window] [in a new window]   Fig. 4.   Effect of vasoactive agents on the relation between measured ID and calculated ID. Figure is based on results from a single vessel of each type. Similar results were obtained from 2 separate newborn pulmonary arteries, 2 separate adult pulmonary arteries, 4 separate adult carotid arteries, and from 2 separate wild-type (WT) and 2 separate elastin-mutated adult ascending (HET) aortas. Control transmural pressure was 75 mmHg in systemic vessels and 20 mmHg in pulmonary vessels. Phenylephrine (PE) and acetylcholine (ACh) concentrations were 105 M.

There was no evidence of light-dye injury of smooth muscle cells or endothelial cells in microvessels, despite the use of a higher concentration of FITC-dextran than previously reported (6, 32). The large elastic arteries studied here by the FITC method still responded well to the vasoconstricting and the endothelium-dependent vasodilator agonists (Fig. 4). Comparison of ascending aorta ODs with FITC filling (n = 5) to ascending aorta ODs previously studied in the same conditions without FITC filling (n = 15) showed no significant differences (by 2-way ANOVA) in PE-induced vasoconstriction or ACh-induced dilation before and after FITC. PE-induced vasoconstriction resulted in 17 ± 4% and 21 ± 2% decreases in OD with and without FITC, respectively; ACh relaxed the PE-constricted vessels by 75 ± 16% and 85 ± 9% with and without FITC, respectively.

In the small mesenteric arteries, IDs were directly measured by transillumination at a constant transmural pressure of 40 mmHg before and after addition of the vasoconstrictor NE (10⁵ M) and ACh (NE + ACh, 10⁵ M). ID values calculated from the WCSA derived from the measured ID and OD before any treatment (at 40 mmHg) were then compared with the directly measured values (Fig. 5). Again, very little difference was found between directly measured IDs and calculated IDs. With the data from all three vessel segments used pooled together, before and after treatment with NE and NE + ACh, the mean error (6 calculated ID values) was 1.3% with a range of 3.5% to +1.0%.

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Relation between measured ID and calculated ID from norepinephrine (NE)-constricted and ACh-relaxed small mesenteric artery segments. Figure is based on results from a single segment of the adult mesenteric artery, with similar results obtained in 3 separate artery segments. Solid bars, measured ID from direct measurement by transillumination. Open bars, calculated IDs from vessel wall cross-sectional area.

Calculated ID from vessel ring wall thickness. In some vessels or some experimental conditions it may not be possible to accurately measure ID and calculate a reference WCSA. Thus we investigated whether ID determined from wall thickness of a cut ring segment at the end of an experiment (see MATERIALS AND METHODS), together with measured OD at 0 mmHg (in the cannulated vessel), could be used to accurately calculate a reference WCSA that could then be used post hoc to calculate ID from OD. In the carotid arteries studied the IDs determined from measurement of the ring wall thickness were 329 and 264 µm, respectively, consistent with the measured IDs (by transillumination) of 325 and 258 µm. Similarly, the IDs in the ascending aorta ring cross sections of 781 µm for the wild-type animal and 732 µm for the mutant animal were similar to the directly measured IDs of 751 and 754 µm, respectively, using the FITC method. In all cases, the differences are in the range of one pixel, supporting the accuracy of ID measurements by all three methods (transillumination, FITC, and cross-section analysis). As shown in Fig. 6, there was little difference in the ID values calculated from the ring cross sections and in the respective ID values obtained by direct measurement in the carotid artery by transillumination (Fig. 6, A and C), or in the ascending aorta by the FITC method (Fig. 6, B and D), both before and after KCN treatment. The measurements in the two carotid arteries (52 independent ID measurements) had a mean error of 0.4% with an error range of 3.9% to +4.6%. Of these calculated error values, 90% (47 of 52) were within a range of 3.7% to +3.7%. For the two ascending aortas (52 independent ID measurements) the mean error was +0.2% with a range of 5.0% to +5.7%, with 90% (47 of 52) of these calculated values within an error range of 4.6% to +4.6%.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Relation between measured ID and calculated ID from a measurement of wall thickness in a cross section of vessel. Figure is based on results from a single adult mouse carotid artery before (A) and after (C) KCN treatment and on a single adult mouse ascending aorta before (B) and after (D) KCN treatment. Results are representative of those obtained in 2 separate adult ascending aortas (wild-type and elastin mutated) and 2 separate adult carotid arteries. Solid lines, increasing pressure phase; dotted lines, decreasing pressure phase.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Vascular resistance and associated physiological parameters are directly related to the diameter of the vessel lumen, but ID is often difficult to measure directly in intact blood vessels. As a result, many investigators have calculated ID from OD in pressurized blood vessels based on the assumption that the vessel wall volume or WCSA is constant (7, 10, 16, 21, 23, 31, 36). However, these assumptions have never been rigorously tested in physiological conditions. Our hypothesis was that a constant wall volume would result in a nearly constant WCSA under conditions in which longitudinal extension of cannulated arteries is restricted by the experimental device. This is similar to the situation in vivo where longitudinal extension of vessels is limited by the tethering of surrounding tissue. In the studies presented here, the constant relationship between OD and ID under these conditions, on the basis of the assumption of constant WCSA, was demonstrated in a wide range of physiological situations in transilluminated, pressurized, cannulated vessels from a variety of sources.

In all the vessels investigated, including arteries from elastin-deficient mice, under conditions where pressure was increasing or decreasing, during active contraction and relaxation, or following treatment with KCN, the difference between calculated ID and measured ID was always quite low, with mean errors generally <1%. This indicates that WCSA is relatively constant, in agreement with a prior study of WCSA during changes in flow and during NE-induced contraction in rat mesenteric arteries (15). Our data demonstrate that this can be applied to both large conductance (Figs. 1-4) and small resistance arteries (Fig. 5). With the use of this technique, ID and vascular resistance can be closely approximated in tightly constricted or thick-walled arteries in which the lumen size (ID) cannot be determined from visual inspection.

We did note some differences in the accuracy of calculated ID that depended on the pressure level at which the reference WCSA was measured. In particular, larger differences between calculated and measured ID were consistently found for IDs at 0 mmHg. Similarly, when WCSA at 0 mmHg was used as the reference to calculate ID, the differences between calculated and measured ID were larger than those obtained when a reference WCSA at a higher pressure was used. This slight discontinuity in the relation between calculated ID and measured ID between the unpressurized (i.e., 0 mmHg) and pressurized vessels is not surprising. Previous observations have demonstrated that the elastic structures of the vascular wall, including the intima, decrease in size, infold, and bulge in the collapsed vessel as intravascular pressure decreases to 0 mmHg (13, 35). Nevertheless, the differences between measured and calculated ID remained quite small, even when the reference WSCA at 0 mmHg was derived from the wall thickness at the end of the experiment (Fig. 6).

In addition to our experiments, we analyzed data from the literature where our hypothesis could also be tested (Table 1). Analyses of the diameter measurements made by Osol and Cipolla (25) in small rat uteroplacental arteries and the analysis of the data from porcine aorta made by Storkholm et al. (30) are presented in Table 1. The data shown in Table 1 include both the measured ID values (derived from the reported data) and the calculated ID values (derived from measured OD and WCSA at the pressures indicated) and the error range and mean error for the calculated values. When initially evaluating these data, it was again clear that the calculated ID values at 0 mmHg again had consistently higher errors than the calculated ID values at other pressures, presumably due to the same instability in the circular shape of the vessel at 0 mmHg as discussed above. As done elsewhere in the paper, the error values at 0-2 mmHg were thus excluded from the error ranges and means shown. Excluding these measurements (at 0 mmHg), only a few outlier error measurements >5% are evident in the raw error measurements, and mean errors varied between 4.1% and +3.4%, with the exception of one value of 7.2% in which the reference WCSA at 0 mmHg was used. The results from the literature thus lend further support to the validity of this method for calculating ID from OD measurements.

Until now, measurement of ID in cannulated arteries by transillumination has been the preferred method, but this technique is applicable to only thin-walled vessels and may even be difficult in these vessels during active contraction (18, 26). In large thick-walled arteries, direct ID measurements are often not possible because of the inability to obtain an adequately contrasted image with transillumination. Our results suggest that the FITC method can be used to measure ID in such vessels, but the fluorescence may lead to light-dye damage (6, 32), and this method still requires calibration with at least one accurate ID measurement. Alternatively, our data indicate that ID can be accurately estimated from OD values retrospectively, utilizing the wall thickness determined on a cut section of the vessel at the end of the experiment. The simple approach presented here for determining ID and related physiological parameters (e.g., vascular resistance) from the measured OD should prove particularly useful for both in vivo and in vitro studies of vascular reactivity and mechanics.