We are more than happy to resolve some of the misconceptions that may have been the primary motivation for Dr. Marx to write this letter. First of all, the two experimental studies under discussion were designed with the sole rationale to investigate the safety aspect of xenon when administered during cardiopulmonary bypass (CPB) in the presence of cerebral air emboli (CAE).1,2To achieve this goal, we first established an appropriate disease model that incorporates CAE into an existing rodent model of CPB.2,3Aiming to identify a suitable CAE volume that would allow for both a high degree of survival and a quantifiable cerebral injury, we selected a dose-escalating approach to investigate the effect of various volumes of CAE during CPB on survival and gross neurologic injury.2This study will be further referred to as the “dose-finding study.” Based on this work, we designed a second experiment (or “treatment study”) that analyzed the effect of xenon on the primary outcome cognitive performance following CPB combined with CAE.1 

We agree that the order of publication may have contributed to some of the confusion. However, knowing that the human study later published by Lockwood et al.  4was ongoing, this sequence was chosen to ensure timely information about relevant safety concerns for the use of xenon during CPB in the presence of CAE. The subsequent delay in publication of the dose-finding study was primarily due to a lengthy review process.

We appreciate the opportunity to further discuss any differences in terms of study design and primary endpoints of our two studies, because this will allow us to demonstrate that the findings are far from controversial. The primary endpoint of the dose-finding study was survival with an aspired survival rate of about 50%. Therefore, all animals euthanized due to severe neurologic damage were included in the study and were not replaced. There was no difference in survival in animals exposed to xenon versus  those exposed to nitrogen, but exposure to CPB was associated with lower survival rate compared with sham-operation.2Based on this dose-finding study, a CAE volume of 0.3 μl associated with a mortality rate of only 1% was used in the treatment study. As expected, none of the animals in the treatment study showed severe neurologic injury requiring euthanasia, but two animals were excluded from further data analysis and consequently replaced because of the development of cervical hematoma and inspiratory stridor, a condition due to postoperative bleeding rather than neurologic injury. In agreement with the dose-finding study, there was no difference between the xenon- and the nitrogen-treated animals in terms of survival rate in the treatment study.1 

In the two studies discussed, neurologic outcome was evaluated as both gross neurologic function, reflecting the integrity of the motor cortex, and cognitive performance, mirroring hippocampal injury. Of note, these two outcomes represent two very different executive functions assessed with two distinct batteries of tests: Gross neurologic outcome was assessed with assays of prehensile traction and beam performance and cognitive function with the modified hole board test.

Gross neurologic function in the dose-finding study was worse in animals exposed to CPB compared with sham-operated animals but was not different between xenon and nitrogen groups (original fig. 22) with identical findings in the treatment study (original fig. 11).

Cognitive function also was assessed in both studies. The modified hole board test was performed by two veterinarians, each acknowledged in the associated manuscripts. As mentioned by Dr. Marx, each veterinarian wrote a doctoral thesis in German. However, these theses will not be further discussed for several reasons: (a) They do not represent peer-reviewed publications; (b) they are written in German and are therefore not available to a broader audience; and (c) the results as represented in these theses are identical to those in the published manuscripts. Dr. Marx’s confusion about potential differences between the manuscripts and their respective theses is mainly due to the fact that he mistakenly assigned the theses to the publications, meaning the thesis associated with the dose- finding study was assigned to the treatment study and vice versa . However, the dose-finding study was designed to treat cognitive function as secondary outcome to explore the feasibility of cognitive assessment in the context of the combined model of CAE and CPB. As discussed in the manuscript, significant limitations need to be considered when interpreting the results: (a) Owing to the study design, only 26 out of 60 animals were available for cognitive testing; (b) surviving animals were subjected to different CAE volumes; and (c) the multiple logistic regression analysis was based on cognitive outcomes on postoperative day seven and not on the entire observation period.2Taken together, it does not come as a surprise that no differences in cognitive outcome were seen among treatment groups (original fig. 32). In fact, the dose-finding study did not even demonstrate a difference in cognitive function between the CPB and the sham-operated groups. In contrast, the treatment study was designed to treat cognitive outcome as the primary endpoint. In that study, 10 surviving animals per group (exposed to CPB and CAE with constant volumes) had their cognitive function assessed over 14 days with the modified hole board test. Statistical analyses were not restricted to one single day, but rather applied to the entire observation period as routinely done when analyzing learning tasks. Therefore, the treatment study appears to be sufficiently powered to evaluate cognitive outcome as primary endpoint while demonstrating a worse outcome for animals treated with xenon.

We agree with Dr. Marx that the results of our treatment study differ from at least one clinical and one experimental study demonstrating no adverse effects of xenon delivery during CPB.1,4,5However, this does not surprise us, because our study is the first to experimentally address the impact of xenon on neurocognitive and histologic outcome following CAE during CPB. Several other differences apply: Ma et al.  5did not integrate CAE into the rodent model of CPB, restricted the application of xenon to time on CPB, and used a different test to assess neurocognitive outcome. Although the recent small human study by Lockwood et al.  4concluded that xenon could safely be delivered to coronary artery bypass grafting patients while on CPB, that study did not include a detailed postoperative neurologic and neurocognitive assessment. Our results are not in disagreement with the results of the cited in vitro  studies, because they confirm the potential of xenon to expand air bubbles, albeit to a smaller extent compared with nitrous oxide.6,7 

We agree with Dr. Marx that blood pressure is an important factor potentially affecting bubble size and collateral perfusion of the brain, and therefore take issue with his statement that animals exposed to CPB were documented to have lower blood pressures while on CPB. As illustrated in table 1 of the treatment study, there is no difference in mean arterial blood pressure (MAP) between the two CPB groups, regardless of xenon.1Clinically, hypotension during CPB does not appear to impact cognitive or neurologic function after cardiac surgery,8,9and autoregulation remains intact within a wide normal range of MAP (50 to 100 mmHg) as long as pH and arterial carbon dioxide are kept constant.10,11In another clinical study investigating the effect of MAP on outcome, MAPs of 80–100 mmHg were assigned to the “high” MAP group whereas patients with MAPs of 50–60 mmHg during CPB were assigned to the “low” MAP group.12Therefore, Dr. Marx confuses the issue when considering MAP values of 70–80 mmHg during CPB in our study as “hypotensive.” In conclusion, blood pressure may present an important contributing factor to an adverse cerebral outcome following cardiac surgery, but is unlikely to be the reason for an adverse cognitive outcome in the CPB group treated with xenon, as MAPs were comparable to rats subjected to CPB and ventilated with nitrogen.1 

Although the kinetics and content of CAE were not studied directly, we believe that indirect information about xenon’s effect on cerebral air emboli was generated in our treatment study1; therefore, we speculate—but do not conclude—that potential neuroprotective effects of xenon may have been masked by the effects of xenon on CAE. Critical physiologic parameters such as MAP and arterial PCO2were controlled and any selection bias was avoided by selecting a suitable CAE volume based on the dose-finding study allowing for long-term survival and functional testing.2 

We strongly believe that interpretation of data obtained from any model must always take into consideration the limitation of the model itself. Even the most sophisticated animal models likely will fail to simulate the clinical situation completely. Models such as ours only offer insights into certain aspects of clinical problems, and therefore one needs to use caution when making an interpretation or comparison.

*Duke University Medical Center, Durham, North Carolina. b.mackensen@duke.edu

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