E Editorial
Pushing the Standards Forward: In-Depth Monitoring of Physiological Parameters in Anesthetized Neonatal Mice Laszlo Vutskits, MD, PhD,* and Piyush Patel, MD†
T
he translational value of experimental data obtained in laboratory animals is an important point of dissent between basic and clinical sciences.1–3 This is particularly true in the context of studies of anesthetic neuroprotection in experimental models of cerebral ischemia. Whether and to what extent general anesthetics can protect the brain during hypoxic-ischemic insults is an old but still unanswered question. In the past few decades, many anesthetic agents have been shown to have protective effects in a variety of adult experimental models including both global and focal, transient and permanent brain ischemia. None of these data have so far been conclusively translated into human practice, a situation that is ubiquitously shared with all other fields of neuroprotection research. The reasons for this discrepancy between animal and human trials have been extensively discussed, and the targeted outcomes of experimental approaches used in animal works have been identified as one potential factor underlying this failure.4 Indeed, many experimental studies focused primarily on short-term histopathological parameters, and only few of them evaluated long-term functional outcome. To improve the translational value of preclinical studies in stroke research, a panel of academicians and industry representatives has proposed guidelines, the Stroke Treatment and Academic Roundtable (STAIR) criteria, on how to conduct and report animal experiments on the field of ischemia-neuroprotection research.5 Key recommendations of the STAIR panel are the importance of morphologic and functional outcomes as well as appropriate physiologic monitoring of the experimental animals. The latter is based on investigations that have repeatedly revealed that minor changes in physiologic parameters, such as blood pressure, profoundly affect the extent of cerebral From the *Department of Anesthesiology, Pharmacology and Intensive Care, University Hospitals of Geneva, Geneva, Switzerland; and †Department of Anesthesiology, University of California San Diego School of Medicine, San Diego, California. Accepted for publication July 21, 2014 Funding: None. The authors declare no conflicts of interest. Reprints will not be available from the authors. Address correspondence to Laszlo Vutskits, MD, PhD, Department of Anesthesiology, Pharmacology and Intensive Care, University Hospital of Geneva, 4, rue Gabrielle-Perret-Gentil, 1205 Geneva, Switzerland. Address e-mail to [email protected]. Copyright © 2014 International Anesthesia Research Society DOI: 10.1213/ANE.0000000000000440
November 2014 • Volume 119 • Number 5
injury. In fact, a major concern is that transient hypotension attendant with anesthesia might preclude demonstration of potential anesthetic neuroprotection. In experimental studies in neonatal rodent pups, the small size of pups raises important technical challenges and limitations for investigators with respect to physiologic monitoring. This issue becomes especially relevant when these animals are exposed to general anesthetics, inducing not only loss of consciousness but also potentially significant respiratory, hemodynamic, and metabolic changes. These anesthesia-related pathophysiological alterations in systemic homeostasis can have a major impact on several organ systems and, therefore, may stand as important confounding factors precluding appropriate data interpretation in a wide range of experimental settings where young animals undergo general anesthesia.6,7 In an attempt to limit the magnitude of this problem, recent guidelines on experimental research reporting advocate for detailed description of animal monitoring modalities.5,8,9 Also, presentation of data from blood gas analyses performed at the end of the anesthesia procedure is increasingly found in experimental reports on anesthetized animals. While these efforts in laboratory research are clearly of utmost importance, we are still far from the clinical situation where providing general anesthesia is accompanied by continuous monitoring of an increasing number of physiological parameters, including, among others, blood pressure, heart rate, and oxygen saturation. In this issue of Anesthesia & Analgesia, Lin et al.10 performed experiments in which they raise monitoring of physiological parameters in anesthetized neonatal rodents to a new level. The principal goal of their study was to determine whether a combination of mild hypothermia and sevoflurane anesthesia can confer neuroprotective effects against cerebral hypoxia-ischemia in neonatal mice. To this aim, they employed a model of cerebral ischemia comprised of unilateral common carotid artery ligation followed by a subsequent 1-hour exposure to a hypoxic mixture containing 10% oxygen in 10-day-old mice. This preclinical model results in well-characterized brain injury and is widely employed for translational studies in neonatal encephalopathy research.11 Since anesthesia exposure in spontaneously breathing pups under these experimental conditions is fatal,12 the authors performed endotracheal intubation and mechanical ventilation to www.anesthesia-analgesia.org 1029
E Editorial keep these animals alive while receiving sevoflurane/ mild hypothermia. Histopathological and functional outcome in this experimental group was then compared to a cohort of spontaneously breathing hypoxic-ischemic mice pups, as well as to control animals breathing room air. These experiments revealed that, compared to mouse pups subjected to hypoxia-ischemia, the mildly hypothermic and sevoflurane-anesthetized pups had significantly less cerebral injury and better behavioral and cognitive function. To this end, in separate cohorts of mice pups, the authors conducted detailed measurements of physiological parameters that included arterial blood gas sampling, continuous heart rate, and blood pressure monitoring during the entire duration of the hypoxic insult. Cerebral tissue oximetry was performed to ensure comparable severity of hypoxic-ischemic injury in experimental groups. Given physiologic parity among groups, the authors could conclude with a fairly high level of certainty that mild hypothermia, in combination with sevoflurane anesthesia, conferred both short-term and long-term neuroprotection and preserved memory function in a model of hypoxic-ischemic neonatal encephalopathy. An important limitation of the work, though, is that the protocol called for the study of a combination of sevoflurane and hypothermia. That the impact of sevoflurane alone, with maintenance of normothermia, was not evaluated is perplexing. Therefore, the work is relevant to a combination of sevoflurane and hypothermia only, and no conclusions about the putative neuroprotective effects of sevoflurane anesthesia per se can be drawn. This work deserves particular attention because (1) it provides the first demonstration that anesthetics might protect the immature brain in the context of neonatal ischemiahypoxia; (2) it employs detailed and invasive monitoring modalities to compare physiological variables between experimental groups; and (3) it presents proof of principle data showing that endotracheal intubation and mechanical ventilation are feasible even in rodent pups with very small body weight. Each of these 3 issues gives us thoughtprovoking new information that will definitely foster future avenues of research in neonatal animals with experimental approaches of high quality not only in the field of anesthesia-neuroprotection but also far beyond. The demonstration that experimental approaches to investigate these highly relevant issues are feasible clearly allows establishing a preclinical research agenda in this direction. The feasibility of monitoring physiological variables in newborn laboratory rodents of very small size is often considered a major limiting factor to extrapolate findings from these animal studies to human neonatal care. Accordingly, reporting of such data, other than blood gas parameters, is most often missing from research publications on neonatal rodents. This is indeed an important flaw in complex experimental procedures since even transient alterations in cardiac output and oxygen delivery can have a significant impact on morpho-functional outcome measures in these studies. Therefore, a very important aspect of the study by Lin et al. is that it clearly demonstrates that there is a place for invasive continuous monitoring of arterial blood pressure and even of cerebral oxygen saturations in experiments on
1030 www.anesthesia-analgesia.org
neonatal rodents. In this respect, it is important to note that sevoflurane-hypothermia protection was possible when the animals were mechanically ventilated. It is, of course, not possible, at least at the current stage of biomedical technology, to perform both invasive physiological recordings, morphological analysis, and neurocognitive testing in the very same small newborn rodent pup. However, an acceptable alternative is to use parallel sets of surrogate animals. These subjects, matched for sex and litters, share a very similar genetic and epigenetic background and, hence, are expected to respond rather homogeneously to any given standardized experimental paradigm. A major current criticism in this research area is that while human neonates are most often co-exposed to anesthesia and surgery in the perioperative period, only anesthesia exposure is applied in spontaneously breathing newborn rodent models. Endotracheal intubation followed by controlled ventilation permits achieving a surgical depth of anesthesia exposure without incurring the complications of respiratory depression. This approach will then allow us to determine the impact of both surgery and anesthesia exposure on the developing brain. The work, therefore, has relevance for studies not only of hypoxia-ischemia but also of anesthetic neurotoxicity in the developing brain. The experiments presented by Lin et al. provide an outstanding example of high-quality physiological monitoring and data reporting in newborn animals. They clearly push the standards of rodent preclinical neonatal anesthesia models forward and are important steps in our neverending effort to improve the translational value of animal models. This is particularly important in a socioeconomic context where the benefit of preclinical animal experimentation in biomedical research is continuously questioned. E DISCLOSURES
Name: Laszlo Vutskits, MD, PhD. Contribution: This author wrote the manuscript (i.e., editorial). Attestation: Laszlo Vutskits approved this manuscript. Name: Piyush Patel, MD. Contribution: This author wrote the manuscript (i.e., editorial). Attestation: Piyush Patel approved this manuscript. This manuscript was handled by: Gregory J. Crosby, MD. REFERENCES 1. Todd MM. Anesthetic neurotoxicity: the collision between laboratory neuroscience and clinical medicine. Anesthesiology 2004;101:272–3 2. Pound P, Ebrahim S, Sandercock P, Bracken MB, Roberts I; Reviewing Animal Trials Systematically (RATS) Group. Where is the evidence that animal research benefits humans? BMJ 2004;328:514–7 3. Pound P, Bracken MB. Is animal research sufficiently evidence based to be a cornerstone of biomedical research? BMJ 2014;348:g3387 4. STAIR. Recommendations for standards regarding preclinical neuroprotective and restorative drug development. Stroke 1999;30:2752–8 5. Fisher M, Feuerstein G, Howells DW, Hurn PD, Kent TA, Savitz SI, Lo EH; STAIR Group. Update of the stroke therapy academic industry roundtable preclinical recommendations. Stroke 2009;40:2244–50 6. Anand KJ, Soriano SG. Anesthetic agents and the immature brain: are these toxic or therapeutic? Anesthesiology 2004;101:527–30
anesthesia & analgesia
Neonatal Anesthesia Monitoring
7. Soriano SG, Anand KJ, Rovnaghi CR, Hickey PR. Of mice and men: should we extrapolate rodent experimental data to the care of human neonates? Anesthesiology 2005;102:866–8 8. Hooijmans CR, Leenaars M, Ritskes-Hoitinga M. A gold standard publication checklist to improve the quality of animal studies, to fully integrate the Three Rs, and to make systematic reviews more feasible. Altern Lab Anim 2010;38:167–82 9. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol 2010;8:e1000412
November 2014 • Volume 119 • Number 5
10. Lin EP, Miles L, Hughes EA, McCann JC, Vorhees CV, McAuliffe JJ, Loepke AW. A combination of mild hypothermia and sevoflurane affords long-term protection in a modified neonatal mouse model of cerebral hypoxia-ischemia. Anesth Analg 2014;119:1158–73 11. Patel SD, Pierce L, Ciardiello AJ, Vannucci SJ. Neonatal encephalopathy: pre-clinical studies in neuroprotection. Biochem Soc Trans 2014;42:564–8 12. Loepke AW, McCann JC, Kurth CD, McAuliffe JJ. The physiologic effects of isoflurane anesthesia in neonatal mice. Anesth Analg 2006;102:75–80
www.anesthesia-analgesia.org 1031
Pushing the Standards Forward: In-Depth Monitoring of Physiological Parameters in Anesthetized Neonatal Mice Laszlo Vutskits, MD, PhD,* and Piyush Patel, MD†
T
he translational value of experimental data obtained in laboratory animals is an important point of dissent between basic and clinical sciences.1–3 This is particularly true in the context of studies of anesthetic neuroprotection in experimental models of cerebral ischemia. Whether and to what extent general anesthetics can protect the brain during hypoxic-ischemic insults is an old but still unanswered question. In the past few decades, many anesthetic agents have been shown to have protective effects in a variety of adult experimental models including both global and focal, transient and permanent brain ischemia. None of these data have so far been conclusively translated into human practice, a situation that is ubiquitously shared with all other fields of neuroprotection research. The reasons for this discrepancy between animal and human trials have been extensively discussed, and the targeted outcomes of experimental approaches used in animal works have been identified as one potential factor underlying this failure.4 Indeed, many experimental studies focused primarily on short-term histopathological parameters, and only few of them evaluated long-term functional outcome. To improve the translational value of preclinical studies in stroke research, a panel of academicians and industry representatives has proposed guidelines, the Stroke Treatment and Academic Roundtable (STAIR) criteria, on how to conduct and report animal experiments on the field of ischemia-neuroprotection research.5 Key recommendations of the STAIR panel are the importance of morphologic and functional outcomes as well as appropriate physiologic monitoring of the experimental animals. The latter is based on investigations that have repeatedly revealed that minor changes in physiologic parameters, such as blood pressure, profoundly affect the extent of cerebral From the *Department of Anesthesiology, Pharmacology and Intensive Care, University Hospitals of Geneva, Geneva, Switzerland; and †Department of Anesthesiology, University of California San Diego School of Medicine, San Diego, California. Accepted for publication July 21, 2014 Funding: None. The authors declare no conflicts of interest. Reprints will not be available from the authors. Address correspondence to Laszlo Vutskits, MD, PhD, Department of Anesthesiology, Pharmacology and Intensive Care, University Hospital of Geneva, 4, rue Gabrielle-Perret-Gentil, 1205 Geneva, Switzerland. Address e-mail to [email protected]. Copyright © 2014 International Anesthesia Research Society DOI: 10.1213/ANE.0000000000000440
November 2014 • Volume 119 • Number 5
injury. In fact, a major concern is that transient hypotension attendant with anesthesia might preclude demonstration of potential anesthetic neuroprotection. In experimental studies in neonatal rodent pups, the small size of pups raises important technical challenges and limitations for investigators with respect to physiologic monitoring. This issue becomes especially relevant when these animals are exposed to general anesthetics, inducing not only loss of consciousness but also potentially significant respiratory, hemodynamic, and metabolic changes. These anesthesia-related pathophysiological alterations in systemic homeostasis can have a major impact on several organ systems and, therefore, may stand as important confounding factors precluding appropriate data interpretation in a wide range of experimental settings where young animals undergo general anesthesia.6,7 In an attempt to limit the magnitude of this problem, recent guidelines on experimental research reporting advocate for detailed description of animal monitoring modalities.5,8,9 Also, presentation of data from blood gas analyses performed at the end of the anesthesia procedure is increasingly found in experimental reports on anesthetized animals. While these efforts in laboratory research are clearly of utmost importance, we are still far from the clinical situation where providing general anesthesia is accompanied by continuous monitoring of an increasing number of physiological parameters, including, among others, blood pressure, heart rate, and oxygen saturation. In this issue of Anesthesia & Analgesia, Lin et al.10 performed experiments in which they raise monitoring of physiological parameters in anesthetized neonatal rodents to a new level. The principal goal of their study was to determine whether a combination of mild hypothermia and sevoflurane anesthesia can confer neuroprotective effects against cerebral hypoxia-ischemia in neonatal mice. To this aim, they employed a model of cerebral ischemia comprised of unilateral common carotid artery ligation followed by a subsequent 1-hour exposure to a hypoxic mixture containing 10% oxygen in 10-day-old mice. This preclinical model results in well-characterized brain injury and is widely employed for translational studies in neonatal encephalopathy research.11 Since anesthesia exposure in spontaneously breathing pups under these experimental conditions is fatal,12 the authors performed endotracheal intubation and mechanical ventilation to www.anesthesia-analgesia.org 1029
E Editorial keep these animals alive while receiving sevoflurane/ mild hypothermia. Histopathological and functional outcome in this experimental group was then compared to a cohort of spontaneously breathing hypoxic-ischemic mice pups, as well as to control animals breathing room air. These experiments revealed that, compared to mouse pups subjected to hypoxia-ischemia, the mildly hypothermic and sevoflurane-anesthetized pups had significantly less cerebral injury and better behavioral and cognitive function. To this end, in separate cohorts of mice pups, the authors conducted detailed measurements of physiological parameters that included arterial blood gas sampling, continuous heart rate, and blood pressure monitoring during the entire duration of the hypoxic insult. Cerebral tissue oximetry was performed to ensure comparable severity of hypoxic-ischemic injury in experimental groups. Given physiologic parity among groups, the authors could conclude with a fairly high level of certainty that mild hypothermia, in combination with sevoflurane anesthesia, conferred both short-term and long-term neuroprotection and preserved memory function in a model of hypoxic-ischemic neonatal encephalopathy. An important limitation of the work, though, is that the protocol called for the study of a combination of sevoflurane and hypothermia. That the impact of sevoflurane alone, with maintenance of normothermia, was not evaluated is perplexing. Therefore, the work is relevant to a combination of sevoflurane and hypothermia only, and no conclusions about the putative neuroprotective effects of sevoflurane anesthesia per se can be drawn. This work deserves particular attention because (1) it provides the first demonstration that anesthetics might protect the immature brain in the context of neonatal ischemiahypoxia; (2) it employs detailed and invasive monitoring modalities to compare physiological variables between experimental groups; and (3) it presents proof of principle data showing that endotracheal intubation and mechanical ventilation are feasible even in rodent pups with very small body weight. Each of these 3 issues gives us thoughtprovoking new information that will definitely foster future avenues of research in neonatal animals with experimental approaches of high quality not only in the field of anesthesia-neuroprotection but also far beyond. The demonstration that experimental approaches to investigate these highly relevant issues are feasible clearly allows establishing a preclinical research agenda in this direction. The feasibility of monitoring physiological variables in newborn laboratory rodents of very small size is often considered a major limiting factor to extrapolate findings from these animal studies to human neonatal care. Accordingly, reporting of such data, other than blood gas parameters, is most often missing from research publications on neonatal rodents. This is indeed an important flaw in complex experimental procedures since even transient alterations in cardiac output and oxygen delivery can have a significant impact on morpho-functional outcome measures in these studies. Therefore, a very important aspect of the study by Lin et al. is that it clearly demonstrates that there is a place for invasive continuous monitoring of arterial blood pressure and even of cerebral oxygen saturations in experiments on
1030 www.anesthesia-analgesia.org
neonatal rodents. In this respect, it is important to note that sevoflurane-hypothermia protection was possible when the animals were mechanically ventilated. It is, of course, not possible, at least at the current stage of biomedical technology, to perform both invasive physiological recordings, morphological analysis, and neurocognitive testing in the very same small newborn rodent pup. However, an acceptable alternative is to use parallel sets of surrogate animals. These subjects, matched for sex and litters, share a very similar genetic and epigenetic background and, hence, are expected to respond rather homogeneously to any given standardized experimental paradigm. A major current criticism in this research area is that while human neonates are most often co-exposed to anesthesia and surgery in the perioperative period, only anesthesia exposure is applied in spontaneously breathing newborn rodent models. Endotracheal intubation followed by controlled ventilation permits achieving a surgical depth of anesthesia exposure without incurring the complications of respiratory depression. This approach will then allow us to determine the impact of both surgery and anesthesia exposure on the developing brain. The work, therefore, has relevance for studies not only of hypoxia-ischemia but also of anesthetic neurotoxicity in the developing brain. The experiments presented by Lin et al. provide an outstanding example of high-quality physiological monitoring and data reporting in newborn animals. They clearly push the standards of rodent preclinical neonatal anesthesia models forward and are important steps in our neverending effort to improve the translational value of animal models. This is particularly important in a socioeconomic context where the benefit of preclinical animal experimentation in biomedical research is continuously questioned. E DISCLOSURES
Name: Laszlo Vutskits, MD, PhD. Contribution: This author wrote the manuscript (i.e., editorial). Attestation: Laszlo Vutskits approved this manuscript. Name: Piyush Patel, MD. Contribution: This author wrote the manuscript (i.e., editorial). Attestation: Piyush Patel approved this manuscript. This manuscript was handled by: Gregory J. Crosby, MD. REFERENCES 1. Todd MM. Anesthetic neurotoxicity: the collision between laboratory neuroscience and clinical medicine. Anesthesiology 2004;101:272–3 2. Pound P, Ebrahim S, Sandercock P, Bracken MB, Roberts I; Reviewing Animal Trials Systematically (RATS) Group. Where is the evidence that animal research benefits humans? BMJ 2004;328:514–7 3. Pound P, Bracken MB. Is animal research sufficiently evidence based to be a cornerstone of biomedical research? BMJ 2014;348:g3387 4. STAIR. Recommendations for standards regarding preclinical neuroprotective and restorative drug development. Stroke 1999;30:2752–8 5. Fisher M, Feuerstein G, Howells DW, Hurn PD, Kent TA, Savitz SI, Lo EH; STAIR Group. Update of the stroke therapy academic industry roundtable preclinical recommendations. Stroke 2009;40:2244–50 6. Anand KJ, Soriano SG. Anesthetic agents and the immature brain: are these toxic or therapeutic? Anesthesiology 2004;101:527–30
anesthesia & analgesia
Neonatal Anesthesia Monitoring
7. Soriano SG, Anand KJ, Rovnaghi CR, Hickey PR. Of mice and men: should we extrapolate rodent experimental data to the care of human neonates? Anesthesiology 2005;102:866–8 8. Hooijmans CR, Leenaars M, Ritskes-Hoitinga M. A gold standard publication checklist to improve the quality of animal studies, to fully integrate the Three Rs, and to make systematic reviews more feasible. Altern Lab Anim 2010;38:167–82 9. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol 2010;8:e1000412
November 2014 • Volume 119 • Number 5
10. Lin EP, Miles L, Hughes EA, McCann JC, Vorhees CV, McAuliffe JJ, Loepke AW. A combination of mild hypothermia and sevoflurane affords long-term protection in a modified neonatal mouse model of cerebral hypoxia-ischemia. Anesth Analg 2014;119:1158–73 11. Patel SD, Pierce L, Ciardiello AJ, Vannucci SJ. Neonatal encephalopathy: pre-clinical studies in neuroprotection. Biochem Soc Trans 2014;42:564–8 12. Loepke AW, McCann JC, Kurth CD, McAuliffe JJ. The physiologic effects of isoflurane anesthesia in neonatal mice. Anesth Analg 2006;102:75–80
www.anesthesia-analgesia.org 1031
