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Pig in a Poke: Species Specificity in Modeling Lipid Resuscitation

Weinberg, Guy MD*,†; Rubinstein, Israel MD‡,§

doi: 10.1213/ANE.0b013e318240d8e7
Analgesia: Special Article
Free
SDC

Published ahead of print December 20, 2011 Supplemental Digital Content is available in the text.

From the *Department of Anesthesiology, University of Illinois College of Medicine, Chicago; Department of Anesthesiology, Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois; Department of Pulmonary, Critical Care, Sleep and Allergy Medicine, Department of Medicine, University of Illinois College of Medicine, Chicago; and §Department of Pulmonary and Critical Care Medicine, Medical Service, Jesse Brown VA Medical Center, Chicago, Illinois.

Funding: None.

Conflicts of Interest: See Disclosures at the end of the article.

Reprints will not be available from the authors.

Address correspondence to Guy Weinberg, MD, Department of Anesthesiology, University of Illinois, 1740 W. Taylor, Chicago, IL 60612. Address e-mail to guyw@uic.edu.

Accepted October 31, 2011

Published ahead of print December 20, 2011

We are convinced for several reasons that pigs are not an optimal model for studying IV lipid emulsion-based resuscitation. This is a major concern not only for the time, effort, and expense involved in such studies but also because of the potential for producing scientifically and clinically misleading data. Reports from highly respected research groups suggest that IV lipid might provide no benefit over saline infusion in treating bupivacaine overdose in pig models.1 This contrasts with the nearly uniform success in published intact rodent,2,3 canine,4 or isolated rat heart4 models of local anesthetic toxicity. One exception is a study by Candela et al.5 showing reversal of bupivacaine-induced electrocardiogram changes in piglets with infusion of either Ivelip® 20% (Baxter Belgium) or Medialipide® 20% (B. Braun, Germany). However, the effect of these formulations on recovery from bupivacaine-induced cardiac arrest was not tested.

The apparent problem with the porcine experimental model is not widely recognized nor has it been studied. Previously, reports of failed lipid resuscitation in pigs were ascribed to the introduction of other potential experimental confounders such as hypoxemia6 or high-dose pressor therapy,1 which by themselves either exacerbate local anesthetic toxicity or reduce efficacy of lipid infusion.7 However, the report of Litonius et al.8 in this issue of Anesthesia & Analgesia raises the possibility of another concern that might render the pig model itself specifically unsuitable for testing lipid-based resuscitation.

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SYSTEMIC CUTANEOUS MOTTLING HINTS AT A PROBLEM

Pigs are often chosen to model cardiovascular and metabolic interventions because of their size and anatomic and physiological similarities to humans. However, the first indication of a specific problem with the pig as a model for lipid resuscitation came in an observation from the same group that produced the paper in this issue.8 Niiya et al.9 reported in a study of lipid infusion in amiodarone overdose that every pig given lipid rapidly developed generalized, cutaneous mottling and turned red within minutes of the infusion, while none of the controls exhibited these changes. The degree of redness was variable but deepest in the 2 pigs that became significantly hypoxemic (SaO2 67% and 83%). They postulated that these adverse events were mediated by prostaglandin release and probably not specific to their choice of lipid, ClinOleic®. We propose an alternative explanation, that pigs might be sensitive (or hypersensitive) to the lipid emulsion itself.

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RELATED HYPERSENSITIVITIES

Pigs possess acute hypersensitivity to liposomal preparations such as those used as “nanomedicine” delivery vehicles.10 Descriptions of this phenomenon closely mimic that reported by Niiya et al. and have been studied for more than a decade.11 Szebeni et al. used pigs as a model of the nonimmunoglobulin E-mediated, acute hypersensitivity to liposomes loaded with doxorubicin and amphotericin, 2 commercially available formulations that are associated with anaphylactoid reactions in humans. Clinical symptoms in patients occur with a frequency between 2% and 30% and while generally mild, can include cutaneous changes, dyspnea, and chest pain, and occasionally result in cardiovascular instability. Szebeni et al.12 clearly established in pigs that this reaction is complement-mediated and gave it the moniker “complement activation-related pseudoallergy,” or CARPA. These liposomes contain strongly acidic, phosphate anions in the lipid bilayer that activate the complement cascade through the classical pathway. Subsequent release of the potent anaphylatoxins C5a and C3a leads to activation of immune cells, including pulmonary intravascular macrophages that are localized in the pulmonary microcirculation of pigs but not humans or rodents. Mast cell and basophil degranulation follow, with systemic release of histamine, platelet activating factor, thromboxane B2, prostaglandins, leukotrienes, and other cytokines and chemokines. This immediate response is highly reproducible in pigs, in which after even very small challenges, pulmonary hypertension and cutaneous flushing are constant features. Hypotension, arrhythmias, and low cardiac output can also occur. Intralipid® and ClinOleic® are both composed of negatively charged, predominantly spherical solid, lipid nanoparticles, 300 to 400 nm in diameter. The nanoparticles are similar in size, shape, and surface charge to the Doxil liposomes, suggesting that pigs are similarly predisposed to develop a CARPA-like reaction to lipid emulsions. Therefore, it is possible that lipid emulsion infusion activates the complement cascade in pigs, resulting in cutaneous, pulmonary, and cardiovascular effects similar to those reported by Niiya et al.9

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A SURVEY SUPPORTS OUR HYPOTHESIS

Consequently, we sent e-mails to the communicating authors of 16 papers identified by a PubMed search for peer-reviewed studies of lipid infusion in pigs from 1991 to 2011. We asked whether they had observed cutaneous flushing in pigs during lipid emulsion infusion. We received 12 replies, of which 3 were negative or the authors could not recall. However, 9 of 12 respondents described having seen generalized mottling of the skin with the pig turning red, purple, or even ashen during the experiment. In some instances, on the basis of experimental design, it was difficult to separate effects of lipid infusion from those of other drugs or the shock state induced by a drug, such as bupivacaine. Moreover, in some cases the respondent had been blinded to the treatment group and could not say with certainty that the effect covaried with lipid emulsion infusion. However, several investigators noted the flushing occurred only after infusion of lipid emulsion. One investigator confirmed that after opening their study key, the dark, ashen color noted in several pigs by a blinded investigator was specific to animals treated with lipid emulsion (Brent Bushey, Lt. Cmdr, USN. Personal communication, November 2010). These phenomena were apparently independent of sex, age, weight, breed, and origin of the pigs.

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POSSIBLE MECHANISMS INTERFERING WITH RESUSCITATION

If these investigators observed a CARPA-like phenomenon, it is critical to determine the mechanism(s) by which complement activation interferes with lipid resuscitation. Such interference could certainly result from pulmonary hypertension and other cardiovascular effects that reduce cardiac output and tissue perfusion. However, it is also possible that complement-mediated opsonization of the circulating lipid particles comprising Intralipid® and ClinOleic® could prevent either bupivacaine sequestration or metabolic utilization of their component fatty acids by the heart. Interestingly, the adverse response of pigs to IV lipid emulsion is also reminiscent of the “Red Man Syndrome,” a hypersensitivity reaction to bolus injection of vancomycin that is independent of both immunoglobulin E and complement.13 This phenomenon is elicited by systemic mast cell degranulation leading to release of histamine and other potent proinflammatory mediators. In this case, it is expected that histamine release could interfere with lipid resuscitation by contributing to cardiovascular instability. Given that pigs often exhibit idiosyncratic and unpredictable hemodynamic and metabolic responses to various stressors, it is plausible that complement activation and/or mast cell degranulation could induce or exacerbate such responses, particularly in the setting of cardiopulmonary instability, a major oxidative stress.

Unfortunately, published studies were not specifically designed to study the effects of lipid infusion alone in pigs. None tested either the CARPA or Red Man Syndrome hypothesis or identified the specific mechanism(s) of lipid resuscitation that is impaired. One of the scientists who confirmed the relationship of cutaneous flushing with lipid emulsion infusion has developed a protocol to study the phenomenon, and we look forward to his findings (John Capacchione, MD. Personal communication, May 2011).

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CLINICAL RELEVANCE

Fortunately, acute hypersensitivity reactions have not been described in any reported case of lipid resuscitation in humans. There is a strong precedent for species specificity of this sensitivity. Rats, for instance, are relatively resistant to CARPA and require doses 10- to 100-fold larger than pigs to invoke these symptoms. One proposed explanation for this difference is the site of processing the lipid particles. In rats and humans, hepatic and splenic Kupffer cells capture the opsonized circulating particles. However, in pigs, pulmonary intravascular macrophages remove these circulating particles and are consequently activated to release potent proinflammatory mediators.14,15 This contention is supported by the observation that a 30-minute Intralipid® 20% infusion in rats showed increased lipid content in the hepatocytes at all volumes infused.16 By contrast, microscopic evidence of lipid accumulation in the lung occurred at only the highest volumes of Intralipid® 20% infused (60 to 80 mL/kg). Notably, previous studies in humans have shown that infusion of Intralipid® 20% does not activate the complement cascade.17

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EXPERIMENTAL SEQUELAE

Clearly, the cardiovascular effects of rapid lipid emulsion infusion in pigs are problematic in terms of choosing the pig as a model of lipid resuscitation. Several questions remain to be answered with respect to the underlying pathogenesis and mechanism(s) of this acute CARPA-like response. As long as the nature of this phenomenon is uncertain, we maintain that the pig may not be a suitable model for studying lipid resuscitation. We recommend that any such study also report measures of complement activation in lipid-treated and appropriate control groups to exclude a CARPA-like phenomenon.

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DISCLOSURES

Name: Guy Weinberg, MD.

Contribution: This author helped prepare the manuscript.

Conflict of Interest: Dr. Weinberg holds US Patent #7261903 related to lipid resuscitation. Dr. Weinberg is a cofounder of ResQ Pharma, LLC.

Name: Israel Rubinstein, MD.

Contribution: This author helped prepare the manuscript.

Conflict of Interest: Dr. Rubinstein is a cofounder of ResQ Pharma, LLC.

This manuscript was handled by: Terese T. Horlocker, MD.

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