Perfluorocarbons are hydrophobic synthetic molecules that can dissolve large quantities of gases.1 In an emulsified state, their potential as oxygen carriers (i.e., hemoglobin replacement compounds) has been investigated extensively since the late 1970s. Perfluorocarbons show a linear dissociation curve between Po2 and oxygen content2 that is independent of pH, 2,3-DPG, temperature, or other physiologic factors. As a result, elevated arterial oxygen partial pressures are very effective in improving oxygen transport capacity of perfluorocarbon emulsions in blood.3,4 In addition, the small size of the perfluorocarbon particles (<0.2 μm in diameter) enables them to enter the microvasculature not accessible to erythrocytes and hence improve local oxygen delivery. Particularly relevant to cardiopulmonary bypass (CPB) is the fact that otherwise relatively insoluble gases, such as nitrogen, are considerably more soluble in perfluorocarbons than in blood. This characteristic might allow for the use of perfluorocarbons to absorb systemic air emboli that are routinely generated during cardiac surgery.5
Despite advances in perioperative care, CPB equipment, and surgical techniques, cerebral injury remains a significant source of morbidity and mortality after cardiac surgery.6 Emboli, both gaseous and particulate, have been implicated in the etiology of adverse cerebral outcomes.7 Gaseous emboli can result from entrainment of air from the operative field, generation in the CPB apparatus from cavitation, and from manipulations (such as perfusionist injections) on the venous site of the CPB circuit.8 Experimental studies in pigs have demonstrated a significant improvement in the incidence and severity of neurologic injury after perfluorocarbon administration.9 Furthermore, pefluorocarbons have been demonstrated to increase oxygen delivery to organs at risk for injury during cardiac surgery. Several experiments have shown increased myocardial,10 gastrointestinal,11 and systemic oxygenation12 after administration of perfluorocarbons. In addition, we have previously demonstrated that perfluorocarbons administration may be useful in reducing the volume of gaseous bubbles present during CPB.13
In several experiments, we have performed CPB in rats demonstrating the utility of this model to study neurocognitive injury as well as other organ dysfunction.14–17 In the current experiment, our goal was to assess neurocognitive outcome, cytokine release, and organ histology after administration of the perfluorocarbon, perfluoro-tert-butylcyclohexane (PTBCH), in an experimental rat CPB model. We hypothesized that PTBCH administration would improve postoperative neurocognitive outcome in this animal CPB model by improving oxygen delivery and attenuate ischemia/reperfusion-induced inflammation.
The study was approved by the Duke University Animal Care and Use Committee and all procedures met the National Institutes of Health guidelines for animal care.18 Male 350–375 g Sprague-Dawley rats (Charles River Labs, Wilmington, MA) were housed 3 per cage with a 12-h light-dark cycle. Food and water were available ad libitum.
Anesthesia, Surgical Preparation, and CPB
Fasted rats were anesthetized with 5% isoflurane in 50% O2 in a plastic induction box. After orotracheal intubation with a 14G cannula (Insyte BD Medical, Sandy, UT), the animals were mechanically ventilated to a maximum airway pressure of 20 mmH2O (Harvard Model 687, Harvard Apparatus, Holliston, MA) maintaining a normal arterial carbon dioxide tension. During subsequent surgical preparation, anesthesia was maintained with 1.5%–2.0% isoflurane. A needle thermistor was inserted in the left temporal muscle adjacent to the skull to measure pericranial temperature. Body temperature was kept at 37°C throughout the procedure with both forced-air and surface heating systems.
Surgical preparation consisted of cannulating the tail artery with a 20-G catheter (Insyte BD Medical, Sandy, UT), which served as the arterial inflow cannula for the CPB circuit. Porcine heparin 150 IU and fentanyl 5 μg were administered after placement of this first catheter. Arterial blood pressure was monitored via the superficial caudal epigastric artery, which was cannulated with polyethylene tubing (PE-10 Intramedic Tubing, Becton-Dickinson, Sparks, MD). A multiorifice 4.5 French catheter (modified Desilets-Hoffman catheter; Cook, Bloomington, IN) was advanced through the right external jugular vein into the right heart to serve as a conduit for the venous outflow. Repeat injections of 150 IU of porcine heparin and 5 μg of fentanyl were administered just before the start of CPB. In addition, 0.2 mg of pancuronium was administered. Similar anesthetic regimens without pancuronium had previously been demonstrated in this model to prevent withdrawal responses to painful stimuli. Muscle relaxation was used to prevent spontaneous ventilation that would frequently interfere with venous return.
The CPB circuit consisted of a 4-mL Plexiglas® venous reservoir, a roller pump (Masterflex; Cole-Parmer Instrument CO, Vernon Hills, IL) and a custom-designed small-volume oxygenator. The 4 mL priming volume oxygenator was comprised of 2 Plexiglas® shells (12.8 cm × 12.8 cm × 2.7 cm) that carry a sterile, disposable three layer hollow-fiber membrane providing a gas exchange surface area of 558 cm2.19,20 To prevent excessive heat loss, the oxygenator had an integrated heat exchanger. An in-line flow probe (2N806 probe and T208 flowmeter, Transonics Systems, Ithaca, NY) was used to continuously measure CPB flow. The entire circuit was primed with 10 mL of 6% hetastarch (Hextend®; Hospira, Lake Forest, IL). All parts were connected through single-use silicone tubing. During CPB, ventilation was discontinued and 0.5% to 1% isoflurane was administered through the oxygenator. CPB was performed for 60 min with a flow rate of 150 mL · kg−1 · min−1 adjusted to maximize flow and to maintain an optimal venous reservoir blood level. Mean arterial blood pressure (MAP) was maintained between 45 and 60 mm Hg, with the use of intermittent phenylephrine administered as necessary. At 30 min of CPB, a repeat dose of pancuronium (0.2 mg) was added to the bypass circuit. After 60 min of CPB, ventilation was reinitiated and CPB was discontinued and the venous cannula removed. The animals were ventilated for a further 120 min, after which the remaining cannulae were removed and the wounds closed. The rats recovered in a warmed and oxygen-enriched environment for at least 12 h before returning to their original cages.
After discontinuation of CPB, the remaining blood volume in the circuit was collected, 0.9% saline added, and the resulting solution centrifuged at 2000 rpm for 5 min. The supernatant was discarded and the resulting red blood cell suspension was reinfused into the animal.
Blood gas analysis was performed before the start of CPB, at 30 min and 60 min of CPB, then at 1 and 2 h post-CPB, using an IL-GEM Premier 3000 blood gas analyzer (Global Medical Instrumentation, Ramsey, MI).
The perfluorocarbon used was F-tert-butylcyclohexane, also known as PTBCH. It is a perfluorocarbon that contains only carbon and fluorine atoms (C10F20). It cannot exhibit cis-trans isomerism and thus exists as a single configuration. It is marketed as Oxycyte® (Synthetic Blood International, Costa Mesa, CA).
The dose used in this study (3 mL/kg) was chosen based on information available regarding its use in other animal studies. In a previous study describing cerebral ischemia in rats, a dose of 10 mL/kg was administered without serious side effects.21 Safety in doses up to four times the dose we used was described in safety studies in rats by the manufacturer (data on file with manufacturer). In addition, the dose we used, albeit lower than that used in rats before, was also based on perfluorocarbons being used in clinical CPB studies.22
Immediately after insertion of all the CPB cannulae, 28 animals were randomized into one of two groups: a group exposed to 60 min of CPB with administration of 3 mL/kg IV PTBCH into the CPB circuit prime (PTBCH CPB group), and a control group exposed to 60 min CPB with 3 mL/kg of saline added to the CPB prime (control CPB group).
Subsequently, in a separate series of experiments, 12 animals (5 controls, 7 PTBCH) underwent the same protocol as described above but were euthanized 4 h after CPB to obtain tissues for water content determination and histological analysis. In addition, these animals provided the 4-h post-CPB cytokine sample.
Blood samples (0.3 mL) for analysis of interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor (TNF)-α were collected from the tail artery for analysis immediately before bypass, 60 min after CPB, and at 2 and 4 h after CPB. All samples were immediately centrifuged (at 4°C) and the supernatant frozen and stored (at −80°C) for later batch analysis. The cytokines were analyzed by multiplexed sandwich enzyme-linked immunosorbent assay microtitre plate, according to the manufacturer’s instructions (Endogen®-Search Light ™, Woburn, MA). Results were expressed as pg/mL with the sensitivity for detection being 3.1 pg/mL for IL-β, 6.3 pg/mL for IL-6, 0.4 pg/mL for IL-10, and 3.1 pg/mL for TNF-α.
Beginning on the third postoperative day and continuing for 1 wk, surviving animals underwent neurocognitive testing in the Morris water maze (MWM).14 In brief, the MWM consisted of a 1.5-m diameter darkened pool filled with water (26 ± 1°C) with a fixed, submerged platform (submerged 1 cm) and various visual clues on the walls. The time to locate the hidden platform after placing the animal in the pool is measured and recorded as the latency. The animals were planned for daily testing with four trials per day.
Total Tissue Water Content
Immediately after killing the animals by decapitation, tissue samples (0.4–0.6 g) were collected from the brain, lung, heart, kidney, spleen, and liver. The samples were placed in preweighed open vials, reweighed immediately, and then placed in a drying chamber at 40°C. The vials were weighed daily until a stable weight was reached. Tissue water content was calculated as the tissue water weight recorded as g water/g wet weight × 100%.
The animals designated for histologic analysis were subjected to the same CPB procedures and randomization as described above. Four hours after CPB, they were decapitated and the tissues fixed in 3.7% phosphate-buffered neutral formaldehyde. The right kidney and right lung were dissected, and a cross-section of the heart was prepared by sectioning through the right and left ventricles approximately midway from the apex to the base of the heart. The organ samples were dehydrated with serial alcohols, embedded in paraffin, and 5 μm paraffin sections stained with hematoxylin and eosin. A pathologist (AP) blinded to group assignment evaluated the slides for any qualitative microscopic abnormalities in lungs and kidneys. Myocardial contraction band necrosis, a marker of cell death23 and associated with catecholamine excess,24 was quantified in the subendocardial and myocardial layers. The long and short axes of areas that exhibited hypereosinophilia and contraction bands were measured using a calibrated ocular micrometer and an estimated cross-sectional area calculated by multiplying the two diameters.
Physiologic variables were analyzed using a one-way analysis of variance. Tissue water content between the two groups was analyzed using an independent samples t-test. Cytokine data and areas of contraction band necrosis were analyzed using the Mann–Whitney U-test. All analyses were performed using SPSS 11.5 (SPSS, Chicago, IL).
The physiological variables in the two groups are presented in Table 1. During CPB, the pH was significantly lower in the PTBCH CPB group, but was still within normal physiological ranges (at 30 min CPB, 7.45 ± 0.04 control CPB group vs 7.38 ± 0.11 PTBCH CPB group; and at 60 min CPB, 7.44 ± 0.04 control CPB group vs 7.41 ± 0.04 PTBCH CPB group, P = 0.032 and P = 0.038, respectively). There was also a small difference in pericranial temperature at the end of CPB (36.7°C ± 0.2°C control CPB group vs 37.0°C ± 0.3°C PTBCH CPB group, P = 0.041). In addition, the PTBCH CPB group required more phenylephrine to maintain the targeted MAP (total dose in the PTBCH CPB Group 280 mcg vs 20 mcg in control CPB group, P = 0.03).
Of the 14 animals randomized into the PTBCH CPB group, four (29%) experienced significant hypotension and died during or shortly after the procedure. Of the 10 surviving animals, only one survived for 3 days while the other nine animals died within the first 24 postoperative hours. Of the 14 animals randomized to the control CPB group, two died perioperatively due to cannulation and/or equipment-related problems. Of the remaining 12 animals, one died on day 1 and one died on the second postoperative day; the remaining 10 animals survived through the duration of the experimental protocol. Because of the excessive mortality in the PTBCH CPB group, no comparisons between group neurocognitive performances in the MWM were possible.
At the end of the experiment, cytokines (IL-1β, IL-6, IL-10, and TNF-α) were analyzed in 12 control CPB and 10 PTBCH CPB animals with five animals in each group completing the 4-h time point cytokine measurements (Figs. 1–4). For all four cytokines, there was a significant difference between groups at the 2 and 4-h post-CPB time points. There were no significant differences at the baseline and 60 min CPB time points.
The tissue water content data are presented in Table 2. In the PTBCH CPB animals, the water content in kidneys and spleen was significantly lower than in the control CPB group (74.9% vs 76.1%, P = 0.01 and 73.8% vs 76.0%, P = 0.03, respectively). There were no differences in water content between the groups for the brain, heart, lung, and liver specimens.
Histologic sections of the lungs revealed only small areas of normal lung tissue. Most of the lung tissue had alveolar capillaries engorged with polymorphonuclear leukocytes and macrophages, along with lesser number of lymphocytes. There were no differences between the two groups. In the kidneys, the PTBCH CPB animals all demonstrated finely vacuolated tubular epithelial cells, while no histological abnormalities were seen in the control CPB group. Foci of hypereosinophilic myocytes with scattered contraction bands were seen in four of the five hearts in both the PTBCH and control CPB groups. However, the areas of contraction band necrosis were larger in the PTBCH CPB rats than in the control CPB rats (P = 0.034, Fig. 5). Some foci of contraction band necrosis, usually the larger foci, had a mild infiltrate of neutrophils and macrophages.
The aim of the present study was to investigate the effects of perfluorocarbon administration during CPB with a particular focus on its potential effects in modulating the inflammatory response to bypass as well any influence on neurocognitive outcome. The administration of PTBCH, a 60% perfluorocarbon emulsion, to the rats during CPB resulted in a significantly exaggerated inflammatory response, as demonstrated by increased serum cytokine levels and loss of vasomotor tone represented by higher vasopressor needs. Because of insufficient survival in the PTBCH CPB group, neurocognitive performance could not be assessed in the animals receiving PTBCH. Histologic examination revealed increased multifocal contraction band necrosis in the myocardium of the PTBCH CPB animals.
The degree to which the cytokines were increased in the PTBCH group was unexpected. Previous experiments in our rat model of CPB have demonstrated that after 60 min CPB, a predictable increase in cytokine levels occurs, with IL-1β, IL-6, and TNF-α normally reaching their peak levels approximately 2 h after cessation of CPB before slowly returning to baseline levels within a few hours.25 IL-10 usually has its peak at the end of CPB. As can be seen in Figures 1–4, the control CPB group replicates this expected pattern very closely. The PTBCH CPB group, however, demonstrated an exaggerated increase in IL-1β and IL-6 (up to 10-fold the otherwise expected increase) with the levels still increasing at 4-h post-CPB, suggesting the possibility of even higher peak levels occurring later. For IL-10, the levels in the PTBCH group were also increased when compared with the control group. For the TNF-α levels, they were also increased compared with the control group, but the groups demonstrated a similar pattern over time.
An inflammatory response, though not to this degree, has been previously demonstrated with perfluorocarbons. A clinical study in volunteers examining the effects of another perfluorocarbon (Oxygent, Alliance Pharmaceutical Corp., San Diego, CA) demonstrated that the infusion of this perfluorocarbon in the healthy volunteers lead to a dose-dependent increase in IL-6 at 8 h after infusion.26 Perfluorocarbons, particularly earlier generation compounds, have been identified as having marked febrile and flu-like responses, demonstrating their ability to activate the immune system.4 It is unclear as to the effects of later generation perfluorocarbons, such as the one we used, on the inflammatory response. Two earlier animal experimental studies9,12 used a second-generation perfluorocarbon emulsion in combination with CPB, but no inflammatory responses were measured and survival was not an end-point of those studies.9,27
Several processes are thought to play a role in cytokine release after CPB, including direct blood contact with the surface of the CPB circuit, surgical trauma, the occurrence of ischemia and reperfusion, and endotoxemia.27–29 Because in this study both groups were treated equally in regard to contact activation and surgical trauma, this can be excluded as a cause of additional inflammation in the PTBCH CPB group. Perfluorocarbons, due to their microscopic size and high oxygen-carrying capacities, especially during high partial pressures, have shown to improve tissue oxygen delivery.4 Therefore, the development of ischemia or the translocation of endotoxin across an ischemic gut wall seems unlikely. In the many studies investigating the inflammatory response to CPB, IL-6 is most consistently increased.28 We speculate that the large cytokine increase seen in this present study was due to a perfluorocarbon-specific amplication of the inflammatory response normally seen in CPB.
Myocardial contraction band necrosis is a marker of cell death23 and also is associated with catecholamine excess.24 The myocardial contraction band necrosis as seen in the ventricular myocardium of PTBCH-treated animals could very well be a result of the generalized and significant myocardial hypoperfusion. However, it may also be a direct result of an endogenous catecholamine response to hypotension in combination with the catecholamine administration needed to maintain stable hemodynamics.24 We cannot completely exclude this possibility as we noted larger requirements for phenylephrine in the PTBCH CPB group to maintain a target MAP. Due to removal of the cannula 2 h after the end of CPB, MAP in the later postoperative period could not be determined. The other finding, vacuoles in the renal tubular epithelium, most likely reflects tubular reabsorption of PTBCH since such vacuoles are not a typical effect of renal ischemia. The pulmonary effect of 60 min CPB (capillary engorgement with leukocytes) was seen in the lungs of both groups suggesting that these did not contribute to the excessive mortality.
Tissue water analysis revealed that the kidney and spleen in the PTBCH CPB group had a lower water content compared with control animals. There are two possible explanations for this. First, there may have been less edema accumulation in these tissues. However, differences in edema were not observed in any of the other tissues. A more likely explanation is that accumulation of lipid containing PTBCH in these tissues resulted in overall net lower tissue water content. Emulsion particles of perfluorocarbons are cleared from the blood by the macrophages of the reticuloendothelial system in the spleen, which may have resulted in increased lipid content in this organ.30 The reason for an accumulation of lipid in the kidney is not as clear, but likely reflects tubular reabsorption of PTBCH manifesting as vacuolization of renal tubular epithelial cells.
There were several limitations to the present study. We did not administer PTBCH to a group of non-CPB rats. This would have allowed us to study the effects on cytokine release in the absence of bypass. Nonetheless, after the unexpected deaths in the PTBCH CPB group, we did inject two animals with the PTBCH emulsion and found them to be physiologically indistinguishable from other animals (data not shown). In addition, the manufacturer has administered this particular perfluorocarbon to several different animal species during the development phase of the drug and reported no clear adverse effects. Slight immune activation was seen as a consequence of macrophage clearance of emulsion particles but caused only some flu-like symptoms (data on file at Synthetic Blood International). The decision to determine IL-1β, IL-6, IL-10, and TNF-α was made because of their role in the inflammation process during CPB, which is thought to contribute to neurocognitive dysfunction after cardiac surgery.7,27 More extensive immunological evaluation of the effect of perfluorocarbons during CPB would have added information to this issue.
Several clinical studies of perfluorocarbon S administration in this perioperative setting have been undertaken. Hill et al.22 reported increased cerebral blood flow and total cerebral embolic load in cardiac surgical patients treated with a perfluorocarbon emulsion. Although with transcranial Doppler no differentiation between gaseous or particulate emboli could be made, this has potential significant clinical sequelae and may outweigh the potential benefits of absorption of gaseous emboli or enhanced tissue oxygen delivery distal to an atheromatous occlusion. In addition, a phase three trial in cardiac surgery was terminated prematurely due to an increased incidence of neurologic complications in the groups treated with the perfluorocarbon (Oxygent®) emulsion.22 Our experimental results corroborate these earlier safety concerns and suggest that additional information regarding the inflammatory response to perfluorocarbon administration during CPB is needed before advancing perfluorocarbon emulsions into the sphere of clinical CPB.
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