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Ann Thorac Surg 2006;82:1480-1488
© 2006 The Society of Thoracic Surgeons

Original Articles: Cardiovascular

Biologically Variable Bypass Reduces Enzymuria After Deep Hypothermic Circulatory Arrest

Rohit K. Singal, MDa,e, Leanne M. Docking, MDb,e, Linda G. Girling, BSb,e, M. Ruth Graham, MDb,e, Peter W. Nickerson, MDc,e, Bruce M. McManus, PhD, MDd,e, Alexander B. Magil, MDd,e, Elizabeth K.-Y. Walker, BSd,e, R. Keith Warrian, MDa,e, Mary S. Cheang, MMathf,e, W. Alan C. Mutch, MDb,e,*

a Department of Surgery, University of Manitoba, Winnipeg, Manitoba
b Department of Anesthesiology, University of Manitoba, Winnipeg, Manitoba
c Department of Internal Medicine, University of Manitoba, Winnipeg, Manitoba
d James Hogg iCAPTURE Centre for Pulmonary and Cardiovascular Research, University of British Columbia, Vancouver, British Columbia
e Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia
f Community Health Sciences, University of Manitoba, Winnipeg, Manitoba, Canada

Accepted for publication May 3, 2006.

* Address correspondence to Dr Mutch, Department of Anesthesiology, University of Manitoba, A-504 Chown Bldg, 744 Bannatyne Ave, Winnipeg, MB R3C 0W3, Canada (Email: amutch{at}cc.umanitoba.ca).


Dr Mutch discloses that he has a financial relationship with Biovar Life Support, Inc.

 

   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
BACKGROUND: Renal injury is common after open-heart surgery. Cardiopulmonary bypass contributes to the problem. We compared conventional nonpulsatile perfusion (NP) to biologically variable perfusion (BVP), which uses a computer controller to restore physiological beat-to-beat variability to roller pump flow. We hypothesized BVP would decrease renal injury after deep hypothermic circulatory arrest.

METHODS: Pigs were randomly assigned to either BVP (n = 9) or NP (n = 9), cooled, arrested at 18°C (1 hour), reperfused, and rewarmed and maintained normothermic (3 hours). Additional pigs had NP for a similar time as above, but without circulatory arrest (n = 3), or were sham-treated without bypass (n = 3). Hemodynamics, acid-base status, temperature, and urine volumes were measured. Urinary enzyme markers of tubular injury were compared post-hoc for gamma glutamyl transpeptidase, alkaline phosphatase, and glutathione S-transferase and by urine proteomics using mass spectrometry.

RESULTS: Urine output at 1 hour after arrest was 250 ± 129 mL with BVP versus 114 ± 66 mL with NP (p < 0.02). All three renal enzyme markers were higher with NP after arrest compared with BVP. In animals on bypass without arrest or those sham-treated, no elevations were seen in renal enzymes. Urine proteomics revealed abnormal proteins, persisting longer with NP. Biologically variable perfusion decreased cooling to 21.0 ± 9.0 minutes versus 31.7 ± 7.5 minutes (p < 0.002), and decreased rewarming to 22.1 ± 3.9 minutes versus 31.2 ± 5.1 minutes (p < 0.002).

CONCLUSIONS: Biologically variable perfusion improved urine output, decreased enzymuria, and attenuated mass spectrometry urine protein signal with more rapid temperature changes. This strategy could potentially shorten bypass duration and may decrease renal tubular injury with deep hypothermic circulatory arrest.

Renal injury remains a common problem after open-heart surgery [1, 2]. The mortality rate is markedly elevated, especially if renal replacement therapy is required. A large retrospective review indicated an odds ratio for death of nearly 50 after cardiac surgery if dialysis was required postoperatively [3]. Intensive care utilization is also much higher for patients with renal dysfunction after bypass [4], and such morbidity derails the desired "fast track" paradigm [5]. Despite advances and increasing utilization of off-pump surgery [6], for the foreseeable future, the conventional on-pump technique will remain the most common for open-heart surgery—and the only approach for many procedures independent of coronary artery revascularization. Thus, improving the conduct and safety of on-pump bypass should be a high priority.

Subgroups of patients especially at risk of end-organ damage and acute renal failure, secondary to ischemia-reperfusion, are those needing deep hypothermic circulatory arrest (DHCA). Improved approaches to perfusion, while on bypass, may be of particular benefit here.

We have previously shown that introducing biologically variable pulsation (BVP) to the roller pump output signal improves cerebral oxygenation [7, 8] and diastolic cardiac performance [9], suggesting brain and myocardial benefit from a variable bypass approach. In this study, using a porcine model of DHCA, we examined if a variable bypass approach could be renoprotective. Early markers of renal injury were compared when bypass was conducted using BVP or by conventional nonpulsatile perfusion (NP) before and after a period of DHCA. Early injury was assessed by way of enzyme markers in urine—so-called "enzymuria" [10]—urine proteomics, and histological analysis. Control experiments were also undertaken to examine renal tubular markers after NP without DHCA or in sham-treated animals. Our hypothesis was that renal tubular injury, as assessed by enzymuria, could be attenuated by using BVP as the bypass strategy, before and after DHCA.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Experimental Protocols
Eighteen female pigs weighing 30 to 40 kg were randomized to undergo either BVP or NP in an otherwise identical protocol of anesthesia, cannulation, cooling with CPB, DHCA, resumed CPB, and sacrifice (Fig 1). As controls, an additional 3 animals were placed on NP using the above protocol without undergoing DHCA; a further 3 animals were sham-treated, undergoing surgical intervention but without CPB. All animals were treated according to the guidelines of the Canadian Council on Animal Care as approved by the Committee for Animal Experimentation at the University of Manitoba, Winnipeg, Canada.


Figure 1
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  		Fig 1. Experimental protocol for the two groups of animals undergoing deep hypothermic circulatory arrest (DHCA). Animals underwent cooling with cardiopulmonary bypass (CPB), DHCA at 18°C (nasopharyngeal) for 1 hour, resumption of CPB with rewarming to 37°C (nasopharyngeal). Hemodynamic measurements, blood gases, and blood and urine samples were obtained at time periods denoted by circles. (Temp = temperature.)

 
The pigs were sedated with intramuscular ketamine 12 mg/kg, atropine 0.6 mg/kg, and midazolam 0.6 mg/kg followed by 4% isoflurane in oxygen by nose cone before endotracheal intubation and mechanical ventilation. Anesthesia was maintained with isoflurane 2% in oxygen. Pancuronium bromide was administered by continuous intravenous infusion at 10 mg/h for muscle relaxation. The bladder was catheterized. The right femoral artery and external jugular vein were cannulated. Rectal and nasopharyngeal temperature probes were inserted. Central venous pressure, mean arterial blood pressure, arterial blood gases, hemoglobin, serum and urinary electrolytes, liver enzymes and lactate, urine output, and temperature (rectal and nasopharyngeal) were monitored throughout. All hemodynamic data were recorded continuously on a Gould 2600 oscillograph (Gould, Cleveland, Ohio) and to an analog-to-digital data acquisition system (Advanced Codas; Dataq Instruments, Akron, Ohio). Arterial blood gases were analyzed using a Radiometer ABL500 (Copenhagen, NV, Denmark), and hemoglobin and mixed venous oxygen saturation were measured using a Radiometer OSM3 set for porcine blood.

After sternotomy, the aorta and right atrium were cannulated (6.5 mm [Stockert GmbH, Freiburg, Germany] and 32F DLP single-stage cannula [DLP, Walker, Michigan], respectively) to initiate CPB after administration of 20,000 units of intravenous heparin. No animals received antifibrinolytics. Baseline blood and urine samples were measured. Cardiopulmonary bypass was initiated with a prime of 1 L lactated Ringer's solution, 500 mL Pentaspan, and 5,000 units heparin. Extracorporeal perfusion was maintained using a membrane oxygenator (Cobe Optima 4700), alpha-stat pH management, and an in-line arterial filter (Medtronic Affinity 38 µm; Medtronic, Minneapolis, MN). Heparinization was maintained with 5,000 units intravenously every hour. Mannitol, 0.5 g · kg-1 · h-1, was administered by infusion, throughout the experiment. During CPB, 200 mL lactated Ringer's was added if the volume in the venous reservoir fell below 200 mL; these volumes were recorded. Once stable on CPB, mechanical ventilation was terminated and potassium chloride, 40 mEq, was injected into the left ventricle. An aortic cross-clamp was applied proximal to the aortic cannula. In animals undergoing DHCA, cooling to 18°C (nasopharyngeal) was undertaken. The time to reach this goal temperature was recorded, and CPB was maintained at goal temperature until 1 hour had elapsed from initiation of extracorporeal support. The circulation was arrested by termination of pump flow for 1 hour. Pump flow was then resumed and the animal rewarmed. The time taken to rewarm to 37°C nasopharyngeal was recorded. Perfusion with either mode was then continued for a total of 3 hours (refer to Fig 1). During both cooling and rewarming, the temperature gradient between the water bath and the animal was 8°C or less by minute-by-minute adjusting of the heat exchanger water bath temperature by the perfusionist.

Blood and urine samples were collected before circulatory arrest, after 1 hour of reperfusion postcirculatory arrest, and every hour thereafter until the time of sacrifice. Serum and urine chemistries were assayed by the Manitoba Agriculture Veterinary Services Branch. Evidence of hemolysis in the serum was noted. Proteinuria was assessed by urinary protein/creatinine ratio, and urine mass spectrometry proteomics and enzymuria by the presence of specific enzyme markers of tubular injury.

At the end of the experiment, the animals that had undergone DHCA were sacrificed and the right kidney harvested. The kidney was perfused with iced saline until the venous effluent appeared clear. Sections of the kidney were snap frozen in liquid nitrogen and the remaining tissue was perfused with 1 L 5% formalin.

Animals undergoing NP without DHCA (normothermic bypass) and those sham-treated had total experimental duration the same as that in the DHCA experiments.

Computer-Controlled BVP
Once stable with pump flows of 3.0 to 3.5 L/min, mean arterial pressure 60 to 65 mm Hg, and PaO2 greater than 300 mm Hg, animals for DHCA were randomly assigned to either BVP or NP for the rest of the experiment. The technique and software for institution of variable bypass, modeled on the normal arterial pressure of a lightly anesthetized spontaneously breathing pig, has been previously described [7–9]. Briefly, the target minimal mean arterial pressure and maximum amount of computer-controlled modulation of the roller pump were set. A baseline mean arterial pressure of 65 mm Hg was chosen with a variation of 20 mm Hg. At any time, the roller pump revolutions per minute could be manually adjusted by rheostat control, although such adjustments were minimized. Figure 2 shows representative arterial pressure traces comparing NP and BVP.


Figure 2
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  		Fig 2. Representative femoral arterial pressure traces for pigs undergoing (1) conventional nonpulsatile perfusion (NP [gray trace]), and (2) biologically variable perfusion (BVP [black trace]). Time scales decrease by a factor of 10 with each trace from (A) to (C). Mean blood pressure was identical for both traces (65 mm Hg for the 150 s period). Long-term oscillations of approximately 75 s are evident with BVP in (A) as well as oscillations at 15/minute—reflective of encoded breathing rhythm. In (B), three complete respiratory oscillations are seen. Small rhythmic oscillations, best seen in (C) are reflective of the beat-to-beat variation with individual stroke volume and instantaneous heart rate (black trace); different from the monotonous rectified sine wave typical of nonpulsatile bypass (gray trace). (MAP = mean arterial pressure.)

 
Glutathione S-Transferase Measurement
The activity of urinary glutathione S-transferase (GST) in urine was measured using a spectrophotometric enzymatic assay for total GST activity (both cytosolic and microsomal [Cayman Chemical Company, Ann Arbor, Michigan]). This assay did not distinguish isomeric forms. Changes in absorbance levels were measured at 340 nm every 2 minutes for a total of 12 minutes. The increase in absorbance was plotted against time, and the resulting slope was converted into enzyme activity in units of nmol · min-1 · mL-1.

Gamma Glutamyl Transpeptidase and Alkaline Phosphatase Measurements
Gamma glutamyl transpeptidase (GGT) and alkaline phosphatase in U/L were quantified using reflectance spectrophotometry with a Vitros 700 analyzer (Ortho-Clinical Diagnostics, Rochester, New York).

Urine Proteomics
All urine was collected with the urometer in an ice bath. Decanted urine was immediately centrifuged at 500g for 7 minutes. The urine was frozen at –80°C until time of processing. Urine samples were thawed on ice, vortexed, and centrifuged for 5 minutes at 10,000g. Supernatant, 5 µL, was applied in duplicate to normal phase chips (ProteinChip NP20; Ciphergen, Fremont, California). One microliter (µL) 35% {alpha}-cyano-4-hydroxycinnamic acid (Ciphergen) was applied to each spot and air-dried. Chips were read using surface enhanced laser desorption/ionization time of flight mass spectrometry (ProteinChip Reader II; Ciphergen) in the positive ion mode with the following settings: laser intensity, 230; detector sensitivity, 6; detector voltage, 1700 V; 240 laser shots were collected per sample. Peak labeling was performed using the ProteinChip Software (Version 3.1) for peaks with a signal-to-noise ratio of 3 or greater displayed for a mass over charge (m/z) ratio from 2 to 80 kD.

Histology
The formalin-fixed, paraffin-embedded kidney tissue blocks were stained with periodic acid Schiff stain. The stained histologic sections were assessed in a blinded fashion by an experienced nephropathologist. Sections were stained with a TUNEL (terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labelling) method. The ApopTag peroxidase in situ apoptosis detection kit (Chemicon International, Temecula, CA) was used.

Statistics
Data are presented as mean ± SD unless otherwise noted. All continuous variables between groups were compared at baseline and at each time interval by a split-plot one-way analysis of variance for repeated measures. When significant group or group-by-time interactions (GxT) were found, least mean square matrices were generated and within- and between-group comparisons were made. Bonferroni's correction for repeated measures was applied to within- and between-group comparisons (p less than or equal to 0.05/n; n = number of comparisons). Nonparametric data were compared by {chi}2 analysis or Fisher's exact test; p less than or equal to 0.05 was considered significant. The SAS 9.1 software (SAS Institute, Cary, North Carolina) was utilized for statistical analysis.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
The temperature and hemodynamic data for animals subjected to DHCA are shown in Table 1. Nasopharyngeal temperature was not different between groups at any specified time period as planned by protocol. Mean arterial pressure, PaO2, and PaCO2 did not differ between groups. The mixed venous oxygen saturation during CPB was higher in the BVP group after DHCA compared with the NP group (p < 0.04, GxT). Arterial pH (pHa) was significantly lower in the NP group in the third hour after circulatory arrest; 7.34 ± 0.07 compared with 7.39 ± 0.08 in the BVP group (p < 0.04, between groups). Hemoglobin was greater in the NP group compared with the BVP group at all post-arrest periods (p < 0.03, GxT). The pulse pressure was 26.4 ± 4.5 mm Hg in the BVP group compared with 6.8 ± 1.7 mm Hg in the NP group (p < 0.0001, unpaired t test). With respect to hemolysis; none (5 of 9 animals), very slight (1 of 9 animals), and slight (3 of 9 animals) was seen in both groups at 3 hours after DHCA (not significant between groups, {chi}2 analysis).


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  		Table 1. Hemodynamic and Blood Gas Data With Deep Hypothermic Circulatory Arrest (DHCA)
 
Creatinine concentrations (serum and urine) and urine volumes for the two groups with DHCA are shown in Table 2. No difference in serum creatinine was seen between groups at any time period, and serum creatinine was not increased at end experiment. Dilutional effects were seen in urine creatinine concentration after institution of CPB and mannitol administration, but there were no differences between groups at any time. Urine volume also increased in both groups after initiating CPB and mannitol administration. In the first hour after DHCA, volume was greater in the BVP group (250 ± 129 mL compared with 114 ± 66 mL in the NP group; p < 0.02 between groups). In the NP group, fluid balance was positive at 1,204 ± 1,414 mL compared with 612 ± 866 mL in the BVP group (not significant between groups).


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  		Table 2. Creatinine Concentration and Urine Volume With Deep Hypothermic Circulatory Arrest (DHCA)
 
The hemoglobin concentration and serum activity of GGT, alkaline phosphatase, and GST are shown in Figure 3. A decrease in hemoglobin concentration is seen in the three groups of animals placed on CPB. Serum alkaline phosphatase activity correlated strongly with the changes in hemoglobin concentration, serum GGT activity less so, and serum GST activity between groups was not noticeably different despite the significant hemoglobin dilution in the bypass groups.


Figure 3
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  		Fig 3. Hourly measures of (A) hemogloblin concentration (g/L) and serum enzyme activity (U/L) for (B) gamma glutamyl transpeptidase (GGT), (C) alkaline phosphatase (AP), and (D) glutathione S-transferase (GST). *Difference between deep hypothermic circulatory arrest (DHCA) groups at time interval by least mean squares test. +Differences between sham-treated group and other three groups. The time scale references experiments with DHCA. For nonpulsatile perfusion (NP) without DHCA and sham-treated groups, the time intervals were similar. —{blacklozenge}— = BVP; Figure 3 = NP; Figure 3 = CPB-No DHCA; Figure 3 = Sham-Treated.

 
Urine protein/creatinine ratio, and enzymuria as assessed by GGT, alkaline phosphatase, and GST are shown in Figure 4 for the four experimental groups. Proteinuria was not detected in either the sham-treated or NP without DHCA animals. Protein/creatinine ratio was significantly greater in the NP group compared with the BVP group (p < 0.0001, GxT). Stable minimal activity of the three urinary enzymes were seen in the sham-treated group and the NP group without DHCA. In contrast, with DHCA, marked elevation in the urine activity of the three tubular enzymes was seen (Fig 4B through D), especially noticeable at 1 hour after DHCA, and decreasing but still elevated compared with the sham-treated and NP group without DHCA at 2 and 3 hours. In the NP group with DHCA, GGT and alkaline phosphatase, enzymuria was greater at all times after DHCA compared with the BVP group; for GST, enzymuria differences were only seen at 1 hour after DHCA. By 2 hours after DHCA, enzyme activity for the three enzymes in the BVP group did not differ from the activity in the sham-treated or NP without DHCA groups.


Figure 4
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  		Fig 4. Hourly measures of protein/creatinine ratio and "enzymuria" for the four groups for urine enzyme activity (U/L): (A) protein/creatinine ratio, (B) gamma glutamyl transpeptidase (GGT), (C) alkaline phosphatase (AP), and (D) total glutathione S-transferase (GST). *Significant difference between deep hypothermic circulatory arrest (DHCA) groups at time interval by least mean squares test. +Differences between nonpulsatile perfusion (NP) group and sham-treated and NP without DHCA group. Differences between biologically variable perfusion (BVP) group and sham-treated and NP without DHCA group. The time scale references experiments with DHCA. For NP without DHCA and sham-treated groups, the time intervals were similar. —{blacklozenge}— = BVP; Figure 4 = NP; Figure 4 = CPB-No DHCA; Figure 4 = Sham-Treated.

 
Abnormal urine proteins were confirmed by mass spectrometry. Before DHCA, few distinct peaks were observed corresponding to a protein mass of 7 kD or greater. In contrast, both groups with DHCA demonstrated the presence of new peaks at 8.5 and 9.8 kD after circulatory arrest (Fig 5). These peaks decreased in amplitude with reperfusion as time progressed; however, the decrease was visibly greater in the BVP group. By end of the experiment, 8 of 9 animals in the NP group had persistent peaks at 8.5 kD defined as a signal to noise ratio greater than 1, compared with only 2 of 9 animals in the BVP group (Fisher's exact test, p = 0.015). The 9.8 kD protein signal was present in 7 of 9 animals in the NP group and 2 of 9 in the BVP group (Fisher's exact test, p = 0.056).


Figure 5
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  		Fig 5. Representative urine protein spectra over time for one experiment in each group subjected to deep hypothermic circulatory arrest (DHCA). Spectra were generated using surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS). The time periods for measurement are shown between the two panels (refer to Fig 1 for time periods). Note, after DHCA there is more rapid attenuation of the protein peaks in the biologically variable perfusion (BVP) group. (CPB = cardiopulmonary bypass; NP = nonpulsatile perfusion.)

 
The BVP group demonstrated more rapid cooling and rewarming, with a mean cooling time of 21.0 ± 9.0 minutes compared with 31.7 ± 7.5 minutes in the NP group and a mean warming time of 22.1 ± 3.9 minutes compared with 31.2 ± 5.1 minutes in the NP group (p < 0.002, unpaired t test for both periods). The mean cumulative time saved for cooling and rewarming with BVP was 19.8 ± 11.2 minutes.

Qualitative assessment of the formalin-fixed, periodic acid Schiff, and TUNEL-stained tissues under light microscopy did not demonstrate noteworthy necrosis of the renal parenchyma in either group.

Serum aspartate aminotransferase was elevated in the BVP group during the experiment compared with the NP group (p < 0.012, GxT). Peak levels were 140 ± 46 U/L in the BVP group compared with 100 ± 13 U/L in the NP group. In contrast, serum levels of alanine aminotransferase (ALT), alkaline phosphatase, GGT, conjugated and unconjugated bilirubin, albumin, and lactate were not different between groups.


   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
The mechanisms of postoperative renal damage after CPB have not been fully elucidated. However, bypass-related factors contributing to renal dysfunction include contact activation from biomaterials [11], ischemia, and endotoxin translocation from the gut to the kidney [12]. The problem is compounded with DHCA, secondary to flow cessation, further contributing to vascular obstruction and congestion [12]. In addition, the kidney may be at special risk as a postbypass cardiorenal syndrome has been identified [13].

Early detection of acute renal impairment remains a clinical goal, especially for patients at significant risk of kidney damage. Increasingly, proteins in the urine have been studied for suitability as early markers of injury and to guide therapy for patients identified at risk after renal ischemia/reperfusion. In health, the kidney actively resorbs filtered protein. Proteinuria indicates impaired renal function [14]. Proteins detected in urine may represent a failure of resorption by the convoluted tubules or may represent enzymatic proteins sloughed after ischemic damage to the convoluted tubules themselves.

Westhuyzen and colleagues [10] have shown the value of measuring tubular enzymuria for early detection of acute renal dysfunction. Although numerous proteins have been touted to be specific early markers, we chose GGT, alkaline phosphatase, and GST as that group found excellent discriminating power for acute renal failure (an increase in plasma creatinine of 50% or more, or 0.15 mmol/L or more) with these three enzymes. The receiver operating characteristic curve area was 0.95 for GGT, 0.86 for alkaline phosphatase, and 0.89 to 0.93 for GST depending on the enzyme isoform. Herget-Rosenthal and colleagues [15] showed similar, but less robust findings for GGT and GST enzymuria in nonoliguric acute tubular necrosis. These authors note, however, that early peak loss of these enzymes may have been missed owing to a delay in urine collection after the initial renal insult. In our study, the enzyme markers were measured within 1 hour of DHCA.

In this experiment, significantly greater proteinuria was seen in the NP group after DHCA. As hypothesized, the three urine enzymes—GGT, alkaline phosphatase, and GST—were elevated after DHCA in both groups studied; however, BVP attenuated enzymuria compared with conventional NP bypass. The two control groups (NP without DHCA and the sham-treated animals) did not demonstrate changes in the concentration of urine enzymes over time. By 2 hours after DHCA, enzymuria did not differ between the two control groups and animals in the BVP group whereas significantly greater GGT and alkaline phosphatase enzymuria was noted out to 3 hours in the NP group. Serum activity of the three enzymes did not follow the same urine activity profiles seen after DHCA. The serum activity of the three enzymes did not differ significantly in the three groups undergoing CPB. The temporal difference between serum and urine enzyme activity strongly suggests renal tubular injury with DHCA, most marked in the NP group.

We also assessed proteinuria through mass spectrometry urine proteomics—previously used to identify characteristic protein patterns with renal transplant rejection [16]. In this experiment, the technique identified abnormal protein profiles in both groups after DHCA. The abnormal proteins persisted longer in the NP group.

Urine output may have been confounded by the use of mannitol. Preliminary studies indicated that a mannitol infusion was necessary to maintain urine output after DHCA. However, the administration protocol was the same for all four groups studied, suggesting that the observed, but modest differences between the two groups at 1 hour after DHCA were truly related to perfusion strategy.

Histologic examination of the kidneys after DHCA of this duration did not demonstrate any visible differences between these groups. That may reflect the relative insensitivity of histology in the detection of acute tubular injury in this setting of a 1-hour period of ischemia-reperfusion with protective hypothermia. The absence of distinct changes in cellular morphology was confirmed by an absence of TUNEL-staining for apoptosis. The use of enzyme-linked immunosorbant assay and other antibody-related techniques could help elucidate as yet unrecognized histologic differences. Such techniques are not readily available for the swine proteome.

A beneficial effect of BVP on whole body perfusion was implied by a significant decrease in both the cooling and warming times. The BVP group required roughly two thirds of the time to achieve target temperatures for cooling and rewarming compared with the NP group. The accelerated cooling and rewarming with BVP may have important clinical implications because duration of CPB is correlated to renal dysfunction [17]. These findings imply better microcirculatory perfusion with more homogeneous heat transfer. The mixed venous oxygen saturation provides further evidence for enhanced perfusion with BVP. Goldman and colleagues [18] have demonstrated greater venous desaturation as microcirculatory stasis increases with sepsis. As CPB can initiate a systemic inflammatory response syndrome, such microcirculatory derangements may occur with bypass. Indeed, Ince [19] has commented on such a microcirculatory problem with CPB [19]. The BVP group had higher mixed venous oxygen saturation than the NP group at the same mean perfusion pressure and mean pump flow during CPB. Finally, the NP group showed greater acidosis in the third hour after DHCA compared with the BVP group with no difference in PaCO2, indicating an increased metabolic acidosis with conventional perfusion in the final hour.

Biologically variable pulsation perfusion is a novel approach to address the limitations of nonphysiologic flow of traditional nonpulsatile or conventional pulsatile CPB. Biologically variable perfusion restores physiologic timing sequences to the roller pump signal—a restoration of biological variability, or noise. Normal physiology is associated with a specific variation in each of these signals; all have identifiable temporal characteristics [20]. With BVP, the "noisy" variation in heart rhythm, blood pressure, and respiratory rhythm are all restored by computer control of the roller pump output [7, 8].

Enhanced perfusion with BVP may be explained by the relationship between flow (Q) and pressure (P) that occurs after a period of circulatory arrest, as demonstrated in the coronary circulation when flow is restored with cardioplegia delivery [21]. This relationship has been modeled as Q = Pa, a convex curve, where "a" is a constant. We have previously shown that when the flow-pressure relationship is convex, as with cardioplegia delivery, enhanced flow occurs at the same mean driving pressure with the addition of biological variation or noise [9]. Additionally, we have compared BVP with monotonous pulsation and shown less jugular venous desaturation during rewarming from moderate hypothermia with BVP [8]. We have recently demonstrated, mathematically, how variation about a mean driving pressure can enhance output of a biologically variable life support device [22]. Such a mathematical proof can be generalized to account for enhanced output from an input that varies about a mean value for any smooth convex function.

The higher hemoglobin concentration in the NP group was not expected given, similar fluid resuscitation between groups. The differences may suggest greater third space losses in the NP group, although this is speculative. Post–experimental body weight and analysis of peritoneal fluid would be required to better understand these fluid shifts.

The finding of elevated aspartate aminotransferase in the BVP group may imply decreased hepatic perfusion or be an index of hemolysis. There were no significant differences in all other measured parameters of hepatic function that would corroborate liver injury. Furthermore, hemolysis in the serum was not different between the NP or BVP groups.

In conclusion, in an experimental model of DHCA, BVP decreased enzymuria, an index of renal tubular injury, while permitting more rapid whole body cooling and rewarming. Such improvements could potentially be of benefit to patients undergoing operative interventions requiring DHCA. To date, BVP remains to be tested clinically as an adjunct to on-pump bypass.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
The authors would like to recognize the technical expertise of Kevin Sangster for urinary proteomic results and Amrit Samra for the preparation of specimens for histologic examination. Biovar Life Support, Inc; the Industrial Research Assistance Program; the Canadian Institutes of Health Research; and the James S. McDonnell Foundation funded research for this article.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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