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Cardiovascular Research 2003 57(3):843-852; doi:10.1016/S0008-6363(02)00736-8
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Copyright © 2003, European Society of Cardiology

Intramyocardial blood volume, perfusion and transit time in response to embolization of different sized microvessels

Stefan Möhlenkampa,1, Patricia E Beighleya, Eric A Pfeiferb, Thomas R Behrenbeckc, Patrick F Sheedy, IId and Erik L Ritmana,*

aDepartment of Physiology and Biophysics, Alfred 2-409, Mayo Clinic and Foundation, 200 First Street SW, Rochester, MN 55905, USA
bDepartment of Anatomic Pathology, Mayo Clinic and Foundation, Rochester, MN 55905, USA
cDivision of Cardiovascular Diseases and Internal Medicine, Mayo Clinic and Foundation, Rochester, MN 55905, USA
dDepartment of Diagnostic Radiology, Mayo Clinic and Foundation, Rochester, MN 55905, USA

elran{at}mayo.edu

* Corresponding author. Tel.: +1-507-255-1939; fax: +1-507-255-1935.

Received 30 July 2002; accepted 18 October 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
Objective: To study the role of the coronary microcirculation in response to different-sized microemboli, we measured changes in intramyocardial microvascular blood volume (Bv), perfusion (F) and transit time (TT) and also microvascular patterns of injury. Methods: Bv, F and TT were quantitated in 24 pigs at baseline and again 2 min after repeat injections of 10- or 100-µm microspheres at rest or during intracoronary adenosine infusion. The association of Bv and TT was assessed in the microsphere pigs and in nine control pigs. Microvascular injury was studied on gross-pathologic and histologic samples. Results: At rest, initial injection of 10-µm microspheres led to increases in Bv and F, but progressively decreased with additional injections. In contrast, even small numbers of 100-µm microspheres always led to decreases in Bv and F. Injection of microspheres during adenosine-induced vasodilation always resulted in decreases in peak Bv and F irrespective of their diameters, but microvascular TTs remained unaltered. In control pigs, however, TTs were inversely related to adenosine-induced changes in Bv. Histologically, 100-µm microspheres resulted in patchy distribution of microcirculatory plugging, while 10-µm microspheres induced contiguous hemorrhagic myocardial injury. Conclusion: Microsphere-induced changes in intramyocardial Bv and F and the associated pattern of myocardial injury are related to the size of embolized microvessels and the initial perfusion state. Microvascular functional volume reserve mechanisms appear to play a key role accompanying flow- and TT-preservation.

KEYWORDS Bv, myocardial blood volume (ml g–1); F, myocardial perfusion (ml g–1 min–1); TT, transit time (s); EBCT, electron beam computed tomography


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
Emerging data from large clinical trials suggest that embolization of plaque material or microthrombi from dysfunctional epicardial arterial endothelium into the distal coronary microcirculation has long been an underappreciated, possibly common event [1–3] particularly during acute coronary syndromes [4,5] and intravascular coronary interventions [6,7]. Biochemical markers in blood have been used to identify and quantify myocardial ischemia and necrosis in these settings [1]. Inflammatory markers, in particular tumor necrosis factor-{alpha}, have been suggested to be responsible for the associated impairment of contractile function [3,8]. However, the mechanisms responsible for characteristic epicardial flow phenomena following coronary microembolization have received little attention.

Microembolization may elevate resting epicardial arterial blood flow in the embolized region of myocardial tissue despite being functionally ‘frustrate’ [9], resulting in a ‘perfusion-contraction-mismatch’ [3,10]. The increase in coronary flow was attributed to vasodilation of adjacent nonembolized vessels in response to adenosine release from the embolized myocardium [9,11]. It may be accompanied by a reduction in maximum achievable epicardial coronary blood flow despite normal or restored coronary artery lumen diameter [12], suggesting a role of the coronary microvasculature for these observations. However, the degree to which epicardial flow phenomena reflect microvascular flow dynamics and the role of the coronary microvasculature in the observed changes in epicardial flow under various conditions has not been studied.

We recently demonstrated that intramyocardial microvascular function can reliably be quantified minimally-invasively in vivo by measuring intramyocardial blood volume (Bv) and perfusion (F) using electron-beam CT (EBCT) [13,14]. In this study we used this technique to investigate the impact of different amounts of 10- and 100-µm polymer microspheres on porcine coronary microcirculatory function in order to enhance our understanding of the pathophysiologic consequences of embolization of 10- and 100-µm intramyocardial arteries and arterioles in vivo. The in vivo CT-based functional data were supplemented with gross pathologic and histologic patterns of myocardial injury.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusion
 References
 
2.1 Instrumentation
The study was approved by the Mayo Foundation's Institutional Animal Care and Use Committee in accordance with the National Institutes of Health Guidelines. All animals were male domestic crossbred pigs (n=33, weight 55±4 kg). Anesthesia was initiated with ketamine 12.5 mg/kg, i.m. and xylazine 2 mg/kg, i.m. and maintained by a titrated infusion of 100 mg ketamine i.v. in 500 ml normal saline. All animals were intubated, ventilated and placed supine in a cast for the duration of the EBCT study. A bipolar pacing catheter and a pig-tail catheter were inserted via the jugular veins into the coronary sinus and the superior vena cava, respectively, to permit injection of contrast agent and atrial pacing if needed. A left coronary guide catheter was placed in the left main coronary artery through the external carotid artery for coronary angiography and monitoring proximal coronary artery pressure. The tip of a 2.2-F dual lumen infusion catheter was placed in the proximal left anterior descending artery (LAD) to selectively infuse drugs and microspheres.

2.2 Electron beam CT studies
EBCT studies were performed as previously described [14]. In brief, short axis transaxial images were obtained at a mid-left-ventricular level at 80% of the RR-interval (field of view 26–30 cm, pixel size 0.52–0.69 mm2, voxel size 3.64–4.83 mm3, 7 mm slice thickness, acquisition time 50 ms). Initially, contrast agent was injected selectively into the LAD (5 ml over 1.3 s) to highlight the cross-sectional LAD perfusion territory. The initial scan sequence was followed by a series of flow studies, each performed with a bolus injection of the contrast agent iopamidol (0.33 ml kg–1 over 2 s) into the superior vena cava. Each sequence included 40 scans each obtained every 1–2 heart beats and was followed by 20-min washout periods with intracoronary infusion of saline at 1 ml min–1. Hemodynamic data were recorded just prior to each EBCT study. The first scanning sequence was a study under resting conditions (selective intracoronary infusion of normal saline at 1 ml min–1) followed by scanning sequences according to experimental protocol 1–5 (see below). On completion of the scans all surviving pigs were given a fatal intravenous dose of SleepawayTM (Fort Lodge Labs., Fort Lodge, IA, USA).

2.3 Image analysis
EBCT scans were reconstructed by using the vendor-supplied algorithm. The resulting tomographic images were transferred to a Unix-based workstation (Sun Microsystems, Palo Alto, CA, USA). Images were evaluated using an image analysis software package (ANALYZE, Biomedical Imaging Resource, Mayo Foundation, Rochester, MN, USA). On tomographic images showing peak attenuation of the left ventricular chamber, one region of interest in the anterior cardiac wall and a second region of interest in the left ventricular chamber were outlined. For the region of interest that outlines the LAD perfusion territory, we used the image sequence in which contrast agent was selectively injected into the LAD. The same region of interest as in the initial scan sequence was used for all consecutive scanning sequences in which contrast agent was injected into the superior vena cava. Average pixel intensities were calculated for the regions of interest in the left ventricular chamber and in the LAD-perfused anterior cardiac wall on every image of the sequence. These data were transferred to a PC-based software program (KALEIDAGRAPH, Synergy Software, Reading, PA, USA) and plotted to generate time-intensity curves, which are needed to obtain indices of intramyocardial perfusion (F, in milliliters per gram per minute), blood volume (Bv, in milliliters per gram of myocardial tissue) and transit time (TT, in seconds) as previously described [12,15].

2.4 Experimental protocols
Protocol 1: Repetitive injections of 10-µm microspheres at resting conditions (n=6 pigs, body-weight 57±2 kg). Initially, a bolus of a suspension of 1–2·106 and then up to 4·106 10-µm polymer microspheres ({emptyset}: 10.0±0.7 µm, Duke Scientific, Palo Alto, CA, USA) were slowly injected selectively into the proximal LAD through the dual lumen infusion catheter during continuous infusion of normal saline (1 ml min–1). Two minutes after completion of each injection, EBCT scans were performed as described above. Microsphere-injection was repeated at 20-min intervals until the heart arrested or until the end of our allocated scanning time, which ever came first.

Protocol 2: Repetitive injections of 10-µm microspheres during 5 min intracoronary infusion of adenosine (n=6 pigs, body weight 57±6 kg). After 5 min of continuous selective intracoronary infusion of adenosine (100 µg kg–1 min–1), microspheres were injected during continuing adenosine infusion and the image sequences obtained in the same fashion as in Protocol 1.

Protocol 3: Repetitive injections of 100-µm microspheres under resting conditions (n=6 pigs, body-weight 53±3 kg): a bolus of 1–2·104 microspheres ({emptyset}: 100.0±4.2 µm) were injected selectively into the LAD during continuous infusion of normal saline (1 ml min–1) using the same image sequence as in Protocol 1.

Protocol 4: Repetitive injections of 100-µm microspheres during 5 min intracoronary infusion of adenosine (n=6 pigs, body-weight 54±3 kg): After 5 min of continuous selective intracoronary infusion of adenosine (100 µg kg–1 min–1), microspheres were injected during continuing adenosine infusion and the image sequences were obtained in the same fashion as in Protocol 1.

Protocol 5: Repetitive infusion of increasing dosages of adenosine (n=9 pigs, body weight 55±7 kg): 25, 50, 75 or 100 µg kg–1 min–1 of adenosine were injected selectively into the LAD using the same image sequence as in the above protocols to obtain Bv and F values over the entire range of values seen in Protocols 2 and 4.

2.5 Postmortem examinations
In order to delineate the perfusion territory of the embolized vessels, hearts were excised and the embolized artery injected with a colored liquid silicon polymer (Microfil®, Flow Tech, Carver, MA, USA) [16]. After subsequent formalin-fixation, hearts were cut in approximately 6-mm thick short-axis cross sectional slices parallel to the posterior atrio–ventricular groove. A mid-ventricular slice, which best corresponded to the cross-sectional EBCT-image, was chosen for evaluation. A 1-cm wide transmural sample from an area with typical macroscopic damage was dehydrated in a series of graded alcohols, cleared with xylene and embedded in paraffin.

Sections (5 µm thick) were cut from the paraffin-embedded LAD-perfused tissue for staining with hematoxylin–eosin and Goldner's Masson trichrome stain. A cardiovascular pathologist (EAP), who was blinded as to the history of each histological slide, graded the degree of necrosis in the transmyocardial layers according to a scale ranging from 0 (normal) through 1 (mild), 2 (moderate) and 3 (severe).

A transmural sample, approximately 1 cm wide, adjacent to the one chosen for histologic evaluation was cut, weighed, hydrolyzed by an alkaline solution (E-Z TracTM, Los Angeles, CA, USA) and treated according to the UltrasphereTM extraction protocol from E-Z TracTM. The number of microspheres within the tissue sample was counted using a Neubauer ruling and normalized to 1 g of tissue.

2.6 Statistical analysis
Each group's values are reported as mean±1S.D. Paired and unpaired student's t-tests were used to evaluate the significance of differences between the reported variables. For repeated measures with increasing amounts of microspheres an analysis of variance test (ANOVA) was used. To evaluate correlations between Bv and TT the Pearson correlation coefficient was calculated for each group. Differences were considered significant when P was <0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusion
 References
 
3.1 Hemodynamic variables
We observed slight increases in heart rate and decreases in blood pressure in response to adenosine infusion, attributable to spill over of adenosine into the systemic circulation during steady state conditions (Tables 1 and 2Go). However, within each group the rate–pressure product remained unaltered during injection of 10- and 100-µm microspheres with and without intracoronary infusion of adenosine. Also, injection of microspheres had no discernible impact on the rate–pressure product among the groups.


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  		Table 1 Hemodynamics before and after repetitive injections of microspheres (injection at rest)

 

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  		Table 2 Hemodynamics before and after repetitive injections of microspheres (injection during adenosine infusion)

 
3.2 Bv and F in response to 10- and 100-µm microspheres at rest
Resting intramyocardial Bv before injection of microspheres was similar among the 10- and the 100-µm microspheres groups (0.12±0.02 vs. 0.14±0.01 ml g–1, P = NS) as were F values (0.84±0.18 vs. 0.83±0.12 ml g–1 min–1, P = NS). In response to the initial injection of 10-µm microspheres, Bv and F increased above baseline (Fig. 1a). With further injections of microspheres, Bv and F ultimately decreased to values significantly below baseline (Fig. 1a). In contrast, in response to 100-µm microspheres, Bv and F decreased to values below baseline even after small numbers of microspheres and decreased further with additional injections (Fig. 1b).


Figure 1
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  		Fig. 1 Response in F and Bv to repetitive injection of (a) 10-µm microspheres and (b) 100-µm microspheres under resting conditions. While injection of 10-µm microspheres leads to an initial increase in Bv and F, and decreases with further injections of microspheres, injection of 100-µm microspheres leads to immediate decrease in Bv and F, which cannot be compensated for by microvascular recruitment.

 
3.3 Bv and F in response to 10- and 100-µm microspheres during adenosine infusion
Before injection of adenosine resting intramyocardial Bvs were similar in the 10- and the 100-µm microsphere groups (0.14±0.02 vs. 0.13±0.03 ml g–1, P = NS) as were F values (0.91±0.17 vs. 0.82±0.17 ml g–1 min–1, P = NS). In both microsphere groups Bv and F increased significantly in response to adenosine (P<0.01, respectively versus rest, Fig. 2a and b) with no significant differences between the groups. With repetitive injections of 10-µm microspheres, Bv and F decreased even with small numbers of microspheres and with further injections down to 0.16±0.07 ml g–1 (P<0.002 versus maximal Bv, Fig. 2a) and 1.27±0.71 ml g–1 min–1, respectively after highest amounts of microspheres injected (P<0.002 versus maximal F, Fig. 2a). Similarly, in response to 100-µm microspheres Bv and F decreased even with small numbers of microspheres and progressively decreased to 0.09±0.03 and 0.69±0.21 ml g–1 min–1, respectively, at the highest amounts of microspheres injected (P<0.01, respectively versus maximal Bv and F, Fig. 2b).


Figure 2
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  		Fig. 2 Response in F and Bv to repetitive injection of (a) 10-µm microspheres and (b) 100-µm microspheres during adenosine-induced maximal vasodilation. Injection of 10 µm- and 100-µm microspheres leads to immediate and continuous decreases in Bv and F.

 
3.4 Blood volume-to-transit times (TT) relationship in normal and embolized myocardium
To obtain a comparable distribution of data points across the entire range of Bv values, TT and Bv values were averaged for Bv intervals of 0.01 ml g–1 steps, i.e. 0.090–0.099 ml g–1, 0.10–0.109 ml g–1, etc. In control pigs, peak and resting Bv values were similar to peak Bv and Bv after maximal microspheres injection in 10- and 100-µm microspheres pigs, respectively (Fig. 3). In nonembolized maximally vasodilated myocardium, a progressive decrease in Bv was associated with a significant increase in TT (Fig. 3). In contrast, when intramyocardial blood volume decreased in response to microvascular embolization, TT remained unchanged (Fig. 3). The transit of Bv below 0.20 ml g–1 through the microvasculature was significantly slower in the nonembolized versus 10- and 100-µm microspheres pigs (10.7±0.6 vs. 9.0±0.9 and 9.2±0.4 s, P<0.001, respectively) while 10- and 100-µm microspheres pigs had comparable TT.


Figure 3
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  		Fig. 3 Blood volume (Bv)–transit time (TT) relationship in normal, nonembolized and in embolized myocardium. In normal, nonembolized myocardium, transit time increases when maximal functionally available blood volume decreases. However, when functionally available volume of myocardium is progressively reduced by embolization with increasing amounts of 10- and 100-µm microspheres, transit times remain unaltered, leading to ‘pseudo-preservation’ of myocardial perfusion (see text for details).

 
3.5 Postmortem examinations
In myocardial samples adjacent to the ones used for histologic evaluation we found 1.0±0.8·107 10-µm microspheres and 1.3±1.0·104 100-µm microspheres per gram tissue, with slightly higher counts in the adenosine studies, respectively. The main gross pathologic finding was a consistently patchy distribution of microvascular filling with microfil® of nonembolized microvessels in 100-µm microspheres pigs, while 10 µm microsphere pigs presented with predominantly contiguous areas of hemorrhagic injury with lack of filling. These patterns were markedly different from the homogeneous filling of myocardial microvessels with the colored polymer in normal hearts (Fig. 4).


Figure 4
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  		Fig. 4 Gross pathology of a representative normal heart (left panel), a heart after repetitive injections with 10-µm microspheres (middle panel) and a heart after repetitive injections with 100-µm microspheres (right panel). Hearts were excised and injected with microfil (yellow), which filled arteries, capillaries and veins. Note the hemorrhagic injury after injection of 10-µm microspheres, while injections with 100-µm microspheres leads to a characteristic patchy pattern of injury (arrows).

 
Histologically (Fig. 5), ten out of twelve (83%) 100-µm microsphere pigs had signs of primarily nontransmural necrosis. In all 100-µm microsphere pigs we observed a characteristic patchy pattern of injury. Two out of twelve pigs showed mild hemorrhagic infiltration within the myocardial areas of injury. In 10-µm microsphere pigs all but one animal showed moderate to severe degree of hemorrhage in association with varying degrees of necrosis.


Figure 5
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  		Fig. 5 (a) Histologic section of myocardium after repetitive injections with 10-µm microspheres. Diffuse interstitial hemorrhage with a border zone towards normal tissue (left). Minimal, focal myocyte necrosis and disseminated 10-µm microspheres (right). H&E, 100x (left) and 400x (right) original magnification. (b) Histologic section of myocardium after repetitive injections with 100-µm microspheres. Single 100-µm microsphere lodged in microvessel (left). Magnification shows early contraction band necrosis (black arrow), with minimal interstitial hemorrhage (right). H&E, 100x (left) and 400x (right) original magnification.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusion
 References
 
The main findings in this study were: (1) resting Bv and F initially increased after injection of 10-µm microspheres and then decreased with further injections to values significantly below baseline. With injection of 100-µm microspheres resting Bv and F decreased even after injection of small numbers of microspheres. (2) Maximal Bv and F decreased after injection of even small amounts of microspheres independent of the size of microspheres. The reduction in Bv and F was in proportion to the number of injected microspheres. (3) A decrease in maximal Bv in nonembolized myocardium is associated with an increase in microvascular TT. A decrease in maximal Bv in response to microembolization is associated with unchanged TT irrespective of microsphere size. (4) On postmortem analysis, 100 µm microsphere pigs showed a patchy pattern of injury while 10-µm microsphere pigs showed contiguous areas of injury with predominantly hemorrhagic myocardial necrosis.

4.1 Bv and F dynamics at rest and during adenosine infusion
The increase in resting Bv and F in response to 10-µm microspheres is consistent with previous findings from our laboratory using fast CT but intraaortic injection of contrast agent [17] and others using open-chest, electromagnetic flow probe-based methods [9,11]. This increase was observed only at low amounts of microspheres but after additional injections of microspheres decreased to values below baseline. Grund et al. also reported an increase in porcine coronary blood flow with injection of 15-µm microspheres into the left porcine atrium, but in their study flow did not decrease with additional microspheres injections [12]. The total microsphere-dose injected was substantially lower than the amount of microspheres injected into the LAD-perfusion territory in our study and the effect of higher amounts of microspheres on coronary blood flow was not studied [12].

After the injection of small amounts of 10-µm microspheres at rest, an initial brief decrease in coronary blood flow is followed by a hyperemic response over several minutes as observed in our experiments [3]. In a canine model, this hyperemic response was also progressively diminished with increasing doses of microspheres [18]. The flow response could not be attenuated by surgical denervation or autonomic blockade [19], indicating that local control mechanisms rather than innervation are responsible for the observed changes. Hori et al. found evidence that the local hyperemic response can be attributed to adenosine release, as it was prevented by the adenosine receptor antagonist theophylline [9] and the {alpha}1-adrenoceptor antagonist prazosin [20]. In the above-mentioned porcine model, Grund et al. [12] injected 15-µm microspheres and thereby induced a hyperemic response, which again was antagonized by theophylline, and is therefore attributable to adenosine release. In our model, we used similar-sized microspheres, suggesting that adenosine was also the mediator for the hyperemic response in our experiments.

Unlike the response to 10-µm microspheres, resting Bv and F was not preserved with injection of 100-µm microspheres. In contrast to our findings in pigs, injection of larger microspheres in canine myocardium also showed an initial increase and subsequent decrease in hyperemic response [9]. This difference is likely attributable to a differences in microvascular architecture between species with only sparse collateralization in porcine myocardium. When the microvasculature was maximally dilated before injection of microspheres, Bv and F decreased immediately and progressively with repetitive injections of microspheres, irrespective of microsphere size, both in porcine and canine myocardium.

Our findings support the concept of mobilization of a functional arteriolar and capillary Bv and F reserve in response to microspheres-injection. The increase in Bv and F after injection of low amounts of 10-µm microspheres at resting conditions may be explained by the increase in F and Bv within neighboring terminal arterioles and capillaries that is greater than the loss of Bv and F due to the embolized vessels. Such an excess mobilization of microvascular functional reserve may be required to compensate for the greater intercapillary distance between the recruited effective capillary exchange surface area and the tissue previously perfused by the embolized microvessel. Whether such compensation occurs due to an increase in the average volume of and flow through constantly perfused capillaries and connecting channels within a well-mixed capillary compartment [21] or due to flow through more capillaries, as suggested by the classical concept of capillary recruitment [22,23], or due to a combination of both, cannot be discriminated by our method. When microspheres are injected during adenosine infusion, the already mobilized microvascular functional reserve cannot counteract the loss of microvessels so that Bv and F decrease even after injection of small amounts of small microspheres. When large diameter microspheres embolize, then a large contiguous volume of myocardium is deprived of flow. As the functional capillary reserve can now only be mobilized at the surface of the embolized perfusion territory, this limited increase in capillary Bv and F cannot overcome the loss of capillaries at the center of the embolized perfusion territory.

The increase in resting coronary flow after injection of small microspheres does not, however, prevent myocardial ischemia and leads to persistent impairment in inotropic and coronary flow reserve [7,24]. We found an invariant transit time despite microspheres-induced reduction in perfused microvessels, i.e. an increase in blood velocity through the remaining functionally perfused microvascular volume. This may contribute to a ‘perfusion-contraction-mismatch’ [10] by reducing the time available for nutrient exchange at the capillary level. Our findings are consistent with the previously reported microspheres-induced decrease in arterio–venous O2 difference and lactate extraction [9] and may, in part, explain microembolization-related regional contractile dysfunction despite (pseudo-)normal regional flow. Accordingly, coronary microembolization at amounts that preserves coronary flow and that does not cause necrosis, results in reduced rather than increased tolerance against myocardial infarction in pigs [11], although adenosine is a major trigger of ischemic preconditioning [25].

4.2 Microvascular branching patterns and microvascular pattern of injury
The number of 10- and 100-µm microspheres that were injected to obtain a similar reduction in Bv and F is consistent with anticipated tree-like coronary branching patterns: Bv and F values after injection of maximum (i.e. total) amounts of approximately 108 10-µm and approximately 105 100-µm microspheres during adenosine infusion were statistically indistinguishable from baseline values and between groups. Thus, the impact of every one 100-µm microsphere was equivalent to the effect of about 1000 10-µm microspheres. Coronary artery daughter-branch diameters (Dd) are related to mother-branch diameters (Dm) by approximately Dm=Dd·21/3, for a symmetrical bifurcation branching geometry [26], so that sequential branch diameters downstream to a 100-µm arteriole are 100·2–1/3µm, 100·2–2/3µm, ..., 100·2–n/3µm, where n is the order of distal branchings. Accordingly, a 100 µm vessel is related to a 10-µm vessel by ten orders. Assuming a strictly bifurcational branching geometry, the number of 10-µm branches, i.e. capillaries, perfused by one 100-µm vessel is 210{approx}103. Thus, every 100-µm vessel is estimated to feed 103 10-µm vessels, which is approximately the difference in the number of 10- and 100-µm microspheres that were injected to reduce maximal Bv and F to values comparable to resting conditions before microspheres injections. If the embolized myocardium adjacent to the nonembolized myocardium results in local extravasation of blood into the embolized myocardium, then we would expect the 10-µm embolization to result in 10002/3, i.e. up to 100 times, more hemorrhage than would occur for the 100-µm embolic shut-off of blood flow to the same total number of capillaries. Further, while 10-µm microvessels may have induced rupture and leakage of capillaries resulting in predominantly hemorrhagic necrosis, occlusion of 100-µm microvessels may render dependant myocardium ischemic and eventually necrotic but prevents further in-flow of blood into this ischemic area resulting in nonhemorrhagic infarction.

These theoretical considerations on porcine coronary microvasculature, assuming tree-like branching patterns, are thus consistent with our findings in Bv and F-dynamics. In addition, the tree-like structure with only few collaterals in porcine myocardium may contribute to the observed differences in the patterns of injury from different-sized microspheres.

4.3 Methodological considerations
Due to the repetitive injections of microspheres, postmortem data were only obtained at the end of the study after injection of highest numbers of microspheres, which precluded the evaluation of a time course of the pattern and degree of injury. Our data show that the pattern of injury differs among different-sized microspheres. However, whether small numbers of microspheres induce the same different pattern of injury, whether and at what stage the associated changes in Bv and F are reversible and whether the changes respond to treatment requires further investigation.

This study provides insight into the mechanical contribution of the occlusion of different-sized microvessels to embolization-related phenomena, which may also play a role in clinical microembolization [12]. However, application of our findings to clinical microembolization warrants a caveat. Polymer microspheres result in mechanical plugging of microvessels but they do not have the biochemical attributes of clinical microemboli that frequently include platelets, endothelial cells, plaque debris with cholesterol crystals, etc. and which vary substantially in size [1]. Further, the relative importance of mechanical versus biochemical and inflammatory aspects and the potential interactions are not addressed in this study.


   5. Conclusion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
References
 
Coronary microembolization is followed by characteristic myocardial Bv, F and TT dynamics. The present study shows that microspheres-induced changes in Bv and F depend on the flow condition at the time of embolization, the size of embolized microvessels and the number of microspheres injected and hence the fraction of embolized microvessels within the perfusion territory. Irrespective of the size of microemboli and in contrast to nonembolized myocardium, TT remains unchanged when intramyocardial Bv decreases. Further, different-sized microemboli not only prompt functionally different responses but also induce characteristically different patterns of microvascular injury. Microvascular functional volume reserve mechanisms thus may play a key role accompanying flow preservation and TT-invariance following coronary microembolization.

Time for primary review 22 days.


   Acknowledgements
 
This study was funded in part by research grant HL-43025, National Institutes of Health, Bethesda, MD, USA.

The authors are indebted to Julie M. Patterson, who made the illustrations.


   Notes
 
1 Present address. Department of Cardiology, University Clinic Essen, Hufelandstrasse 55, 45122 Essen, Germany. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 

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