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Ann Thorac Surg 2000;69:84-89
© 2000 The Society of Thoracic Surgeons

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Original Articles

LV-powered coronary sinus retroperfusion reduces infarct size in acutely ischemic pigs

^a Division of Cardiac Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA

Address reprint requests to Dr Byrne, Division of Cardiac Surgery, Brigham and Women’s Hospital, 75 Francis St, Boston, MA 02115
e-mail: jgbyrne{at}bics.bwh.harvard.edu

	Abstract

Top Abstract Introduction Material and methods Results Comment References

 
Background. We developed a prosthetic left ventricle (LV) to coronary sinus (CS) shunt (LVCSS) that is autoregulating and provides LV-powered retrograde perfusion of the coronary sinus.

Methods. Each of 20 Yorkshire pigs underwent 1 hour of left anterior descending diagonal artery occlusion followed by 3 hours of reperfusion. The controls (n = 5) did not have shunt treatment. The LVCSS group (n = 9) underwent shunt treatment during the ischemic period. The LVCSS with partial coronary sinus occlusion (PCSO) group (LVCSS+PCSO, n = 6) underwent shunt treatment and PCSO during the ischemic period. Vital staining and planimetry techniques were used to determine the area at risk for infarction and the area of necrosis.

Results. The area at risk was not significantly different among groups. The area of necrosis was decreased by 53% in the LVCSS group and by 73% in the LVCSS+PCSO group when compared to controls (p < 0.01 among all groups).

Conclusions. The LVCSS reduces infarct size in pigs after acute coronary artery occlusion. The addition of PCSO to LVCSS further improves myocardial salvage.

	Introduction

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Most current therapies for coronary artery disease focus on restoring blood flow through the arterial system. However, the coronary venous system is an alternative route by which ischemic myocardium can receive its blood supply. Beck [1, 2] introduced his two-stage operation for venous revascularization of the heart in 1948. A venous bypass graft connecting the aorta to the coronary sinus (CS) was followed in 2 weeks by complete occlusion of the CS. Patients undergoing this procedure often experienced dramatic relief of anginal symptoms, but the procedure was not well accepted secondary to a high mortality rate, need for two technically difficult operations, and poor patient selection [3]. With the advent of coronary artery bypass grafting (CABG), interest in venous revascularization of the heart declined. Recently, however, several CS interventions have shown promising clinical and experimental results.

Pressure-controlled intermittent coronary sinus occlusion (PICSO), synchronized retrograde perfusion (SRP), and simplified retroperfusion (SR), have all been shown to decrease infarct size in pigs after acute coronary artery ligation [4]. The PICSO technique involves balloon occlusion of the CS, whereas the other techniques actively pump arterial (SRP) or venous (SR) blood into the CS. Also, PICSO has been shown to improve ischemic myocardial pH and wall motion in animals [5]. The SRP technique improves regional myocardial blood flow (RMBF) and regional wall motion in acutely ischemic animals [6], and has been used clinically for myocardial protection during high-risk angioplasty [7]. A major limiting factor is that each method requires an external pump or timing device.

We have developed an autoregulating, left ventricle (LV)–powered, left ventricle to coronary sinus shunt (LVCSS, Circulation Inc, San Francisco, CA) that provides a graded systolic retroperfusion of the CS. Previous experiments performed in our laboratory demonstrated a reduction in infarct size after acute coronary artery ligation in pigs while using the LVCSS [8]. We sought to demonstrate additional benefits with partial coronary sinus occlusion (PCSO), because PCSO alone has been shown to reduce infarct size in acutely ischemic animals [9].

	Material and methods

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Preparation
All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85-23, revised 1985). Yorkshire pigs (n = 22, mean weight 39.3 kg) were premedicated with ketamine (10 mg/kg IM) and masked with 7% isoflurane/6L O₂ while a tracheostomy was performed. Positive-pressure ventilation was initiated and anesthesia was maintained with 2% isoflurane/6L O₂. Central venous access was established and animals received MgSO₄ (2 g), bretylium tosylate (10 mg/kg) and lidocaine (100 mg). A 16-gauge catheter was placed in the femoral artery for serial blood gas and hematocrit measurements. A median sternotomy was performed and the heart was suspended in a pericardial cradle. Millar pressure-transducing catheters (Millar Instruments, Houston, TX) were placed in the left carotid artery and left ventricle. An electromagnetic flow probe (Carolina Medical Electronics, King, NC) was placed around the ascending aorta to measure aortic flow. The two largest diagonal branches of the left anterior descending coronary artery (LAD) were encircled with silicone elastomer vessel loops in preparation for coronary artery occlusion. One set of 5-MHz hemispherical piezoelectric ultrasonic crystals (Triton Technology Inc, San Diego, CA) were placed approximately 1 cm apart in the center of the distribution of the diagonal arteries and a second set was placed on the posterior free wall. A sonomicrometer (Triton Technology Inc, San Diego, CA) was used to transduce crystal signals. Heparin (2.5 mg/kg) was given to all animals to provide systemic anticoagulation. Animals that experienced ventricular fibrillation were excluded (n = 2), and the remaining animals (n = 20) were randomized to control, LVCSS, and LVCSS+PCSO as described below.

Control
In the control group (n = 5), a Millar (Millar Instruments) catheter was inserted directly into the CS. Animals underwent 60 minutes temporary occlusion of the diagonal arteries, followed by release of the snares and 3 hours reperfusion.

LVCSS
In the LVCSS group (n = 9, Fig 1), two pledgeted U stitches were placed in the apex of the LV and a polyurethane catheter was inserted. A silicone catheter was inserted approximately 2 cm into the CS through a purse-string suture in the right atrium. The two segments were connected by an in-line ultrasonic blood flowmeter (Transonic Systems Inc, Ithaca, NY) so that flow through the LVCSS could be measured. The LVCSS internal diameter was 2 mm and the external diameter was 3 mm. A Millar (Millar Instruments) catheter was placed through a side port to measure CS pressure. The LVCSS was clamped to record baseline data. The LAD diagonal arteries were then temporarily occluded for 60 minutes. We initiated LVCSS retroperfusion 5 minutes after arterial occlusion by releasing the clamp. At the end of the ischemic period the catheter was again clamped and the arteries were released. The animals then underwent 3 hours reperfusion.

View larger version (36K): [in this window] [in a new window]   Fig 1. Left ventricle to coronary sinus shunt (LVCSS). (CS = coronary sinus; LV = left ventricle; RV = right ventricle; SVC = superior vena cava.)

Measurements
Measurements were performed at baseline, during ischemia (5, 30, and 60 minutes), and during reperfusion (1, 2, and 3 hours). LabVIEW software (National Instruments, Austin, TX) was used to collect and analyze the data. Mean heart rate (HR), arterial blood pressure (MAP), and CS pressure (CSP) were calculated. The LV end diastolic pressure (LVEDP), cardiac output (CO), cardiac index (CI) and the maximal rate of rise in LV pressure (dP/dt max) were determined. Using subtended crystal length, percent segmental shortening (%SS) was calculated by the following equation: 100 x (diastolic length - systolic length) / diastolic length.

The LV stroke work index (LVSWI) was calculated as 1333 x (MAP - LVEDP) x CO / HR x (LV_wt), where LV_wt is the weight of the left ventricle [10].

Histologic staining
After reperfusion, animals underwent repeat ligation of the two LAD diagonal arteries. The ascending aorta was occluded and phthalo-blue dye was injected into the left ventricle. The hearts were arrested with an intracardiac injection of 80 meq KCl and excised. The right ventricle, atria and valvular tissue were resected. The LV was sliced, parallel to the atrioventricular groove, into several 5- to 10-mm slices and incubated for 30 minutes at 37°C in triphenyltetrazolium chloride (TTC, Sigma Chemical Co, St. Louis, MO). Myocardial tissue containing dehydrogenase enzymes is stained brick red by TTC, but areas of necrotic tissue depleted of these enzymes are not stained and appear as white or pale yellow [11]. A glass plate was placed over the slices and they were traced onto acetate sheets. The slices were weighed and planimetry techniques were used to determine the area at risk (Ar) and area of necrosis (An). The Ar was expressed as the ratio of the area unstained by phthalo-blue dye to the total area of all slices; the An, or the area unstained by TTC, was expressed as a ratio of the Ar [12].

Blood flow determination
Using an established technique, regional myocardial blood flow (RMBF) was determined in ischemic and nonischemic zones of the LVCSS+PCSO group with radioactive microspheres [13]. A 16-gauge catheter was placed in the left atrium for the injection of 15 µm radioactive microspheres (NEN, Boston, MA). The catheter in the left femoral artery was used for reference blood sample withdrawal. Microsphere suspensions were sonicated for 15 minutes and shaken for 2 minutes prior to injection. Injections were performed over 20 seconds with approximately 9 million microspheres per isotope in a volume of 6 mL. Reference blood sample withdrawal was performed at a rate of 9.8 mL/min with a withdrawal pump (Harvard, South Natick, MA). Withdrawal was initiated 10 seconds prior to injection and continued 2 minutes after its completion. ¹⁴¹Ce was injected at baseline, ¹¹³Sn immediately after LAD diagonal artery occlusion, and ⁴⁶Sc 25 minutes after opening the shunt (30 minutes ischemia). Two transverse slices containing the center of the Ar were used to determine RMBF. The Ar was divided into six samples and defined as the ischemic zone. The nonischemic zone was defined as the posterior LV wall, and four samples were taken from each slice. Each sample was weighed and counted with a gamma counter (Packard Instruments Co, Downers Grove, IL) along with the reference blood flow samples. Pure microsphere samples were counted so that spectral distribution stripping calculations could be performed [14]. Corrected counts obtained for each heart sample were multiplied by the known reference withdrawal rate and divided by the corrected reference blood sample counts to determine sample flow expressed as mL/min/g.

Statistical analysis
All results are reported as mean ± standard deviation. A Student’s t test was used to compare groups. Significance was assigned at p less than 0.05.

	Results

Top Abstract Introduction Material and methods Results Comment References

 
Hemodynamics
Mean shunt flow (mL/min) was 93 ± 35 in the LVCSS group and 111 ± 47 in the LVCSS+PCSO group (p = NS). Coronary sinus pressure (mm Hg) was 6.5 ± 4.9 in the control groups and 4.2 ± 2.2 in the LVCSS group (Fig 2, p = NS); CSP was elevated in the LVCSS+PCSO group (15 ± 2.7, p < 0.01 versus other groups). There was no difference at any time among the groups in MAP, LVEDP, CI, LVSWI, and dP/dt max (Table 1).

View larger version (10K): [in this window] [in a new window]   Fig 2. Mean coronary sinus pressure. *p < 0.01 versus other groups. (LVCSS = left ventricle to coronary sinus shunt; LVCSS/PCSO = left ventricle to coronary sinus shunt with partial coronary sinus occlusion.)

View larger version (23K): [in this window] [in a new window]   Fig 3. (A) Ischemic zone segmental shortening. No significant difference among groups at any time point. (B) Nonischemic zone segmental shortening. No significant difference among groups at any time point. (LVCSS = left ventricle to coronary sinus shunt; LVCSS/PCSO = left ventricle to coronary sinus shunt with partial coronary sinus occlusion; PreO = preocclusion; PostO = postocclusion; 30min = 30 minutes occlusion; 1hrR = 1 hour reperfusion.)

View larger version (14K): [in this window] [in a new window]   Fig 4. (A) Area at risk. No significant difference among groups. (B) Area of necrosis. *p < 0.01 versus controls; p < 0.01 versus LVCSS. (LVCSS = left ventricle to coronary sinus shunt; LVCSS/PCSO = left ventricle to coronary sinus shunt with partial coronary sinus occlusion.)

View larger version (12K): [in this window] [in a new window]   Fig 5. (A) Ischemic zone regional myocardial blood flow. *p < 0.05 versus PreO, PostO vs LVCSS/PCSO not significantly different; (B) Nonischemic zone regional myocardial blood flow. No significant difference. (LVCSS/PCSO = left ventricle to coronary sinus shunt with partial coronary sinus occlusion; PostO = postocclusion; PreO = preocclusion.)

	Comment

Top Abstract Introduction Material and methods Results Comment References

Flow through the LVCSS is determined passively by resistance across the shunt. Catheter length and diameter were selected to give an approximate mean LV to CS flow of 100 mL/min; high enough to avoid LVCSS or CS clotting, and low enough to avoid decreasing CO or increasing CS pressure. As CS pressure is gradually increased with balloon inflation, shunt flow decreases, because increasing CSP decreases driving pressure (LV pressure - CSP). A slightly larger CS catheter was used in the LVCSS+PCSO group so that a similar flow rate could be obtained. Because complete coronary sinus occlusion is known to cause hypotension [15] and myocardial damage [2], PCSO was used.

In acutely ischemic pigs, LVCSS decreased infarct size and was enhanced by the addition of PCSO. Failure to improve RMBF leaves the mechanism of myocardial salvage largely unexplained. This is not surprising, however, because CS retroperfusate, regardless of flow rate, results in very little nutritive flow [4–6]. In fact, SR provided a 50% decrease in infarct size after perfusing the coronary sinus with venous blood at a flow rate of 7 mL/min [4]. Perhaps regional increases in coronary sinus back pressure or alterations in ischemia-reperfusion and endothelial cell injury play a role [4]. Ischemic myocardial washout is enhanced by SRP [16] and PICSO [17], possibly decreasing the local concentration of toxic ischemic metabolites such as lactic acid. Mechanisms such as these may play a role in LVCSS treatment. Furthermore, one problem with the microsphere technique is that it underestimates CS retroperfusate delivery. Approximately 10% of microspheres delivered by SRP are known to escape through venovenous and thebesian channels [18], and antegrade flow dislodges microspheres injected through the CS [19]. However, similar results regarding RMBF were reported in nonreperfused LVCSS-treated animals [8]. Another mechanism by which the LVCSS might work is by decreasing afterload. This is an unlikely mechanism, however, because shunt flow is typically only 4% CO. Furthermore, LVSWI, CI, and MAP are not significantly changed.

Another interesting finding is that regional function was not improved despite significant myocardial salvage. Postischemic myocardial stunning is likely responsible [10]. Other studies have demonstrated that, although CS interventions decrease infarct size, RMBF may not be enough to prevent postischemic dysfunction [4, 5]. A longer time course or placement of the crystals in the border zone might have been helpful to demonstrate improvements in regional function [10].

In summary, with LVCSS treatment, we demonstrated a 53% reduction in infarct size in an acutely ischemic pig model. This was further reduced to 73% when PCSO was added. Regional myocardial blood flow was not improved in the ischemic zone of LVCSS+PCSO-treated animals, suggesting that other mechanisms are responsible for myocardial salvage. The LVCSS is currently being evaluated for temporary myocardial support during off-pump CABG. Further studies are warranted to determine the mechanism of myocardial salvage and define potential uses of the LVCSS.

	Acknowledgments

 
We acknowledge the technical support of Paul Termin, DVM. This research was supported by the Cardiac Surgery Research Fund of Brigham and Women’s Hospital, Boston, MA.

	Footnotes

 
This work was supported in part by a grant from Circulation, Inc, San Francisco, California, the company that manufactured the reperfusion catheter.

	References

Top Abstract Introduction Material and methods Results Comment References

 

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