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Animal models in neonatal resuscitation research: What can they teach us?
Seminars in Fetal and Neonatal Medicine xxx (xxxx) xxx–xxx
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Seminars in Fetal and Neonatal Medicine journal homepage: www.elsevier.com/locate/siny
Animal models in neonatal resuscitation research: What can they teach us? Stuart B. Hoopera,b,∗, Arjan B. te Pasc, Graeme R. Polglasea,b, Myra Wyckoffd a
The Ritchie Centre, Hudson Institute for Medical Research, Melbourne, Australia Department of Obstetrics and Gynaecology, Monash University, Melbourne, Australia Division of Neonatology, Department of Paediatrics, Leiden University Medical Centre, Leiden, the Netherlands d Department of Pediatrics, Neonatal and Perinatal Medicine, University of Texas, South Western Medical Center, Dallas, TX, USA b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Fetal-to-neonatal transition Animal models Sheep Rabbits Pigs Rodents
Animal models have made and continue to make important contributions to neonatal medicine. For example, studies in fetal sheep have taught us much about the physiology of the fetal-to-neonatal transition. However, whereas animal models allow multiple factors to be investigated in a logical and systematic manner, no animal model is perfect for humans and so we need to understand the fundamental differences in physiology between the species in question and humans. Although most physiological systems are well conserved between species, some small differences exist and so wherever possible the knowledge generated from preclinical studies in animals should be tested in clinical trials. However, with the rise of evidence-based medicine the distinction between scientific knowledge generation and evidence gathering has been confused and the two have been lumped together. This misunderstands the contribution that scientific knowledge can provide. Science should be used to guide the gathering of evidence by informing the design of clinical trials, thereby increasing their likelihood of success. While scientific knowledge is not evidence, in the absence of evidence it is likely to be the best option for guiding clinical practice.
1. A historical perspective Traditionally, animal models have been used to understand the science of normal and pathophysiological states in humans and have given rise to much of our current understanding of normal human biology and the mechanisms of disease. Indeed, in the context of neonatal resuscitation, scientific research has greatly contributed to improving neonatal care and identifying infants that require assistance at birth. For instance, Dawes' pioneering studies on the fetal heart rate responses to birth asphyxia in lambs and primates provided the basic understanding for heart rate monitoring before and after birth [1]. Liggins' demonstration of improved survival and respiratory function following antenatal glucocorticoid administration in preterm lambs [2] has had remarkable success in reducing morbidity and mortality in preterm infants. Robertson's pioneering studies on surfactant in preterm rabbits [3–5] underpins the routine use of surfactant therapy for respiratory distress in preterm infants. All of these studies have had a profound impact on modern neonatal medical practice and the contribution of scientific studies in animal models to neonatal resuscitation research will likely continue into the future. In more recent years, several commentaries have raised concerns about the relevance of scientific studies, their validity, rigour and the
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contribution they make to improving health care in humans [6]. These commentaries argue that scientific studies are no longer relevant to modern human medicine and can be replaced by epidemiological studies and/or randomized controlled (RCT) trials and meta-analyses. Indeed, scientific studies in animal models have been relegated to the bottom of the evidence pyramid for changing clinical practice, even below expert opinion. However, this characterization reflects a lack of understanding for how scientific studies in animals generate the knowledge that informs and underpin the clinical studies that change clinical practice. Indeed, Liggins' scientific study in sheep [2] provided the knowledge that prompted the subsequent RCT on antenatal glucocorticoids [7]. As a result, that one RCT provided sufficient evidence to change clinical practice, leading to the routine use of antenatal corticosteroids in women at risk of delivering preterm. Thus, while it is important to question how science can contribute to ongoing improvements in clinical care, perhaps we should also question whether RCTs are underpinned by sufficient scientific knowledge. Ideally, they should form a continuum and we need to consider whether a lack of scientific knowledge explains why many RCTs fail to provide a definitive answer to the medical issues they seek to address. Indeed, a lack of high quality evidence from RCTs in neonatal resuscitation was highlighted by the most recent guidelines (2015) from the International
Corresponding author. The Ritchie Centre, Hudson Institute for Medical Research, 27–31 Wright St, Clayton, VIC, 3168, Australia. E-mail address: [email protected] (S.B. Hooper).
https://doi.org/10.1016/j.siny.2018.07.002
1744-165X/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Hooper, S.B., Seminars in Fetal and Neonatal Medicine (2018), https://doi.org/10.1016/j.siny.2018.07.002
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3. Differences between animal and human studies
Liaison Committee on Resuscitation [8].
Scientific studies in animal models have different priorities than human studies and RCTs. While the design of both human and animal studies is closely scrutinized by ethics committees, the oversight responsibilities of these committees are different. In animal studies, the hypothesis being tested has the highest priority, whereas in human studies the health and well-being of the patient takes precedence. Clearly the latter is a vital tenet of ethical studies in humans, but can significantly limit the capacity to rigorously test some hypotheses. This underpins the need for testing some hypotheses in animals. For instance, whereas birth asphyxia occurs naturally in humans, it varies markedly in severity between individuals. As such, studies aimed at determining the effect of different treatments on specific outcomes are difficult, particularly when the treatments are multifactorial. However, as birth asphyxia can be artificially induced in animals to produce a consistent level of severity [15,16], a systematic analysis for how differing treatments affect specific outcomes is far more feasible. At the very least, these types of animal studies can identify the treatments that are likely to be most effective, which can then be tested in a more focused human study. The ethical governance for the use of animals in research also requires several criteria to be met [17,18]. For example: (i) the minimum number of animals must be used which is still compatible with answering the question under investigation; (ii) the scientific question must be worthy of investigation; (iii) the experimental design must not be limited by factors that undermine its capacity to answer the question being investigated. Indeed, if animals are to be used for the sake of medical science, it is vital that answering the scientific question takes precedence, because otherwise there is no point in doing the study and using the animals. The ethical requirement to minimize animal numbers [17,18] can introduce limitations in animal studies. For instance, to account for the reduced statistical power associated with small numbers, it is important to control for as many variables as possible and to ensure that the expected treatment effect is sufficiently large to detect differences between groups. However, it is not always possible to anticipate all of the variables that can impact on the factor under investigation. As a result, the addition of unexpected variability reduces the power of the analysis and markedly reduces the capacity to identify differences, particularly when the numbers are low. Nevertheless, reducing animal numbers is one of the primary tenets for the ethical use of animals in research and is universally accepted by most scientific researchers [17,18]. The suggestion that scientific research is flawed because animal studies are rarely repeated, thereby precluding a meta-analysis [6], fails to understand this primary tenet. Indeed, repeating the same or similar animal studies in different laboratories just to fill the requirements for a meta-analysis is unethical and is contrary to every code of conduct specifying the ethical use of animals in medical research [17,18]. It also fails to understand the difference between the generation of fundamental knowledge and the provision of evidence that can change clinical practice.
2. Benefits and limitations of animal models Many examples demonstrate the contribution that animal research makes to neonatal resuscitation research, however, it is important to understand both the benefits and limitations of animal research in the context of improving health care in humans. For instance, whereas the basic underlying physiology is well conserved between species, all species are different and so no species is a perfect model for humans. This has led some to consider that no information derived from animal studies is relevant to humans, but this is clearly incorrect. For example, in some aspects sheep are clearly very different from humans as they have four legs and are ruminants with four stomachs. However, it is well established that their respiratory and circulatory systems as well as the underlying control systems are very similar, with few if any differences. For example, studies describing the normal transitional physiology in lambs described and explained the rapid (within minutes) onset of bidirectional shunting through the ductus arteriosus after birth [9]. As this is caused by changes in vascular resistances (decrease in pulmonary vascular resistance and increase in systemic vascular resistance caused by cord clamping), the laws of physics dictate that the same changes must also occur in humans. Subsequent observations in humans showed that normal healthy infants undergo these same transitional changes within minutes of birth [10,11]. Similarly, while it is not surprising that preterm lambs and infants require similar inflation pressures to achieve the same tidal volumes (corrected for body weight) at birth, preterm rabbits also require these same pressures to achieve the same tidal volumes despite being only 20–30 g in weight [12]. These examples highlight the need to understand the physiology of the animal under consideration and how they differ from humans when choosing an animal model for medical research. For instance, whereas sheep may be an appropriate model for studying respiratory and cardiovascular physiology in humans, their gastrointestinal physiology is very different and so are likely to have limited value in simulating pathophysiological states in humans. Furthermore, in view of the potential for unknown subtle differences to occur between species, it is important to recognize that responses in an animal model may be different from the responses in humans. Thus, any new potential treatments or therapies derived from animal studies should be trialled, wherever possible, in humans before they are adopted into widespread clinical use. However, this recognition should not devalue the information gained from animal-based research and instead perhaps we should change the way in which we view and utilize this information. With regard to neonatal resuscitation research it is also important to understand that the physiology of the fetus and newborn immediately after birth are very different from a newborn hours to days later. Indeed, as newborn infants immediately after birth have liquid-filled lungs and a fetal circulation that has not transitioned into the newborn phenotype, newborn resuscitation research using animal models should involve an animal in transition. For example, liquid-filled airways radically alter lung mechanics, resulting in airway resistances that are up to 100-fold higher than moments later when the lung is air-filled [13]. Furthermore, as lung aeration follows an exponential function that is very variable with different time constants and end points (degree of lung aeration) [14], particularly in very preterm newborns, it is not possible to accurately predict the state and homogeneity of lung aeration without imaging it. Similarly, the newborn circulation undergoes a major re-organization after birth, with the closure of shunts and separation of the two circulations (systemic and pulmonary), which is triggered by lung aeration [14]. As this process has such a profound influence on circulatory function and cannot be replicated in a fully transitioned animal model, an animal model undergoing this transitional process is most appropriate for investigations of the physiology/ pathophysiology involved.
4. What contribution can animal research make to resuscitation guidelines? Information gained from animal studies is commonly referred to as “weak evidence” for treatment recommendations in clinical practice guidelines and is usually raised when clinical evidence is absent. This wording is potentially misleading and contributes to a misunderstanding of the value that information from animal research can provide. Perhaps it should not be viewed as “evidence”, but rather as “knowledge” that informs the provision of evidence through appropriately designed clinical trials. In this context, Francis Bacon's quote “knowledge is power” is quite apt and perhaps the evidence pyramid should be inverted to reflect the concept that meta-analyses are not the 2
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The latter is currently a hotly debated topic and may have important implications for how infants are managed at birth in the future. Sheep have also been used to detail the effects of antenatal glucocorticoid (single and multiple doses) on the development of a variety of fetal organ systems as well as detailing the benefits to respiratory function after birth [26–28]. Studies in sheep have also been responsible for identifying the role of antenatal factors, such as intrauterine inflammation and fetal growth restriction, in markedly modifying the newborn's physiology and explaining the increased risk of morbidity and mortality [29–32]. They have also been used to investigate approaches that most effectively aerate the lung and ventilate preterm infants, while minimizing the risk of lung injury leading to bronchopulmonary dysplasia [33–37]. Whereas sheep have many advantages and similarities to humans, they do differ from humans in some important ways. Depending upon the breed of sheep, gestation length varies between 142 and 150 days, but within a breed, gestation length is very consistent ( ± 1 day). Single pregnancies are common in maiden ewes, but in multiparous ewes, multiple fetuses (twins) are more common. Sheep have a cotyledonary placenta and a relatively quiescent uterus; while this is quite different from humans, it does confer many advantages from an experimental perspective. For instance, incising the uterus and conducting sterile fetal surgery under general anesthesia is relatively simple and straightforward in sheep. Catheters, EMG leads, and flow probes can be implanted in the anesthetized fetus before the amniotic sac, uterus and abdomen are closed and the ewe and fetus allowed to recover from the anesthesia. As the uterus is very quiescent, there is little to no risk of triggering premature labour, unless there is an intrauterine infection, allowing the physiology of the fetus to be monitored for weeks after surgery. In addition, catheters and flow probes can be implanted days or immediately before delivery, allowing physiological recordings to be obtained before, during, and after transition. However, this paradigm precludes vaginal delivery and requires delivery by cesarean section either under general or spinal anesthesia. As normal healthy lambs are required to be mobile shortly after birth, so that they can feed from their mothers, organ development is in general more precocious than in humans to meet the higher levels of activity required of lambs. Indeed, the lungs are slightly more mature in sheep at birth than in humans, containing millions of alveoli, large sections of fused epithelial/endothelial cell basement membranes, and a fully differentiated epithelium [38]. Interestingly, whereas anatomically the lung of a premature lamb at ∼127 days of gestation appears quite mature, having millions of alveoli and a fully differentiated alveolar epithelium [38], functionally it is similar to that of a 26–28-week premature infant [33]. The latter has a lung with no alveoli, as it is only at the early saccular stage of development and a partially differentiated distal airway epithelium.
pinnacle of evidence but rather the culmination of evidence that should start with knowledge and a basic understanding of the science. This is not a novel concept as there are few engineers who would consider building a large bridge without mathematically applying the scientific knowledge gained from studies on load distribution through substrates and structures, construction materials and design aerodynamics as well as knowledge of the local physical/environmental conditions, etc. While the ultimate goal is to support every treatment and therapy used in neonatal resuscitation with high-quality evidence from metaanalyses of good RCTs, currently this is an unrealistic expectation. This is primarily due to a loss of equipoise with some treatments and/or low incidence rates, making large trials with power to detect differences between treatments unfeasible. Thus, in the absence of evidence, what criteria should be used to decide on the best approach for recommendation and how should these criteria be weighted? In the absence of evidence, we consider that recommendations based on the best available scientific knowledge should be the primary criteria. Rather than referring to scientific knowledge gained from animal models simply as “weak evidence”, the knowledge should be assessed in terms of the quality of the scientific data and the weaknesses and strengths of the animal models used. In particular, the known differences and similarities between the animal species used and humans require careful consideration so that applicability of data can be appropriately assessed. Surely, if the science is sound and the animal model is similar to humans with few differences, basing treatment recommendations on the best available science is the most viable alternative when evidence from RCTs and meta-analyses are not available. In any event, the history of medicine is full of wonderful examples demonstrating that science should always be considered above expert opinion when deciding on treatment options. 5. Types of animal models Numerous animal models have been used to study the science underpinning the normal fetal-to-neonatal transition, antenatal conditions, and treatments that impact on this transition and on the best approaches for ventilating and resuscitating newborns immediately after birth. The most widely used species include sheep, rabbits, pigs, mice, and rats. Although some primates have been used, they are very costly relative to other species and require access to primate facilities that are not widely available. 5.1. Sheep Sheep are widely used in perinatal research and arguably have had the greatest impact on our understanding of perinatal medicine. The impact has probably been greatest in the discipline of maternal/fetal medicine, providing the foundations for fetal heart rate monitoring during labour [19], monitoring fetal breathing movements [20], fetal growth and other biophysical profiles, mechanisms of labour onset [21], antenatal glucocorticoid treatment for all women threatening preterm labour [2] as well as the transition to newborn life. Much of the information derived from fetal monitoring was adopted straight into clinical practice simply based on the findings, indicating the value that scientific knowledge can directly and safely add to clinical practice. In the context of neonatal resuscitation, research in sheep is almost entirely responsible for our understanding of the physiological changes that occur at birth. This includes our understanding of fetal lung physiology and how the airways are cleared of liquid at birth, allowing the lung to rapidly (within minutes) take over the role of gas exchange [22]. It also includes our understanding of fetal cardiovascular physiology and the role of vascular shunts, which is vastly more complex than the adult circulatory system [14,23,24]. Indeed, the transition of the circulatory system at birth is very complex and our scientific understanding of this process is still ongoing, particularly how umbilical cord clamping at birth interacts with the onset of air-breathing [25].
5.2. Pigs The need for cardiopulmonary resuscitation (CPR) in the delivery room is hard to predict, very rare if adequate ventilation is established, and is often performed under chaotic circumstances, making it very challenging to study clinically [39]. The neonatal piglet model has been used primarily to better understand various aspects of neonatal CPR following asphyxia-induced bradycardia and/or asystole. Strengths of the model are that the cardiac and pulmonary anatomy of the pig is very similar to that of the human. Newborn pigs are of similar size to newborn humans (2–4 kg) and although the chest is more keel-shaped (such that there is not a flat sternum directly above the heart as a landmark for cardiac compressions), due to its similar size, performing cardiac compressions on a piglet feels remarkably similar to the delivery room experience with CPR in newborn babies. Issues of compressor fatigue and ergonomic aspects of resuscitation can be explored in meaningful ways [40,41]. Another strength of the neonatal pig model of asphyxia-induced bradycardia and asystole is that it closely 3
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injury is much easier, as a cross-section of an entire lung can be placed on one slide.
mimics the biochemical profile of asphyxiated newborns who require CPR, with profound mixed metabolic and respiratory acidosis at the time of cardiovascular collapse. Important insights into neonatal cardiac compression techniques [41–45], prediction of return of spontaneous circulation [46], the effects of medications and volume [47–49], and both tissue and physiologic responses to resuscitation with various concentrations of oxygen [50–52] have come from studies of the pig model. One such example of cardiac compression technique is the pig studies by Schmölzer et al. that examined the method of performing cardiac compressions while maintaining a sustained inflation. This neonatal pig work suggests potential benefit with shorter time to return of spontaneous circulation compared to traditional interposed compressions and ventilations in a 3:1 ratio [40,44]. The initial exploratory science from the animal model led to a human pilot trial [53] and a larger trial is currently underway. The major limitation to the pig model is that the newborn pig transitions from fetal to newborn circulation in the first hours of life, such that even if animals are available on the first day of life, by the time the experimental surgical preparation is complete, the animal is unlikely to have an open duct, open foramen ovale, or very high pulmonary pressures such as would be present at birth in the human. The lack of these transitional shunts makes the neonatal pig model less ideal for study of delivery room resuscitation, although the information gained does translate better to resuscitations in the neonatal intensive care unit after neonatal transition has occurred.
5.4. Rodents Rodents, particularly mice, are by far the most frequently used animal models in biomedical research. The huge potential for genetic manipulation has unleashed an avalanche of new knowledge on genetic, epigenetic, and molecular regulation of developmental pathways and how these can influence the neonatal transition. Whereas it is impracticable to list all of the important discoveries attributed to this model, it is noteworthy to mention that rodents have been used extensively to demonstrate the adverse effect of hyperoxia on lung development and the role of hyperoxia in the development of bronchopulmonary dysplasia [59–64]. Gestation length is 19–21 days in mice and 21–23 days in rats, depending upon the strain. Rodent pups are considerably smaller and more immature at birth than rabbit pups and lambs, making it very difficult to make any physiological measurements or to ventilate them until they are least 4 days old. At this age they are well past the transitional stage and, as such, are not a particularly suitable model for neonatal resuscitation research. Furthermore, mice are born in the late canalicular stage of lung development, so all of the final two stages of lung development normally occurs postnatally, which is very different from humans. Structurally, the lungs are also quite different, with very large diameter proximal airways and truncated small airways to facilitate the very rapid flow of air into and out of the airways at a rate of 180 breaths per minute.
5.3. Rabbits The rabbit model has also made a huge impact on neonatal resuscitation research and was famously used by Robertson et al. to demonstrate the effect of exogenous surfactant administration in premature newborns [3–5]. Similarly, we have used the rabbit model in our phase-contrast X-ray imaging studies to demonstrate the roles of inspiration in airway liquid clearance [54,55], the benefits of positive end-expiratory pressures [12], and sustained inflations on lung aeration [13,56] in ventilated newborns and to characterize ventilation/perfusion relationships in the lung at birth [57,58]. Like all models, the rabbit model has similarities with and differences from humans. Rabbits frequently have litters of 4–12 pups and growth-restricted pups are common, which can add variability to the data if all pups from a litter are used. Gestation length is only 32 days, so fetal development is greatly compressed in time relative to humans. In the latter part of gestation, 1 day of development in a rabbit equates to ∼1–2 weeks of development in a human. As such, a 27-day-old rabbit fetus has a similar level of maturity as a 26–27-week human, with development rapidly progressing with each day. Even half a day difference can markedly alter the level of maturity, so care should be taken to avoid this influence on data variability. Nevertheless, at birth lung maturity is very similar to humans and from an anatomical and functional perspective, rabbits are arguably one of the most suitable animal models for humans to study transitional respiratory physiology. Like sheep, rabbits also tolerate recoverable fetal surgery, although success rates for surviving fetuses are less than in fetal sheep. As newborn rabbit pups are very small (50–60 g at term), the physiological measurements that can be performed are more limited compared with sheep and mostly require purpose-built equipment. For instance, preterm rabbits (∼25 g) can be intubated and have an intravenous catheter inserted, but to ventilate them in a manner relevant to preterm infants (e.g. in volume guarantee mode) requires the ability to measure and administer very small tidal volumes; to ventilate a 25 g pup at 7 mL/kg requires giving a tidal volume 0.175 mL. Nevertheless, their small size confers other advantages. For instance, their small size makes it quite easy to measure lung volume changes by plethysmography, which is ideal for measuring the temporal and spatial patterns of lung aeration in a lung that is structurally and functionally very similar to that of humans. Similarly, histological examination of the lung for
6. Conclusion Animal models have made and continue to make an important contribution to neonatal resuscitation research. However, ignorance of this contribution is widespread, as is the misconception that humans are different from animals and therefore the knowledge gained from animal studies is of no real value for clinical medicine. However, some of the most profound improvements in neonatal health care have come from knowledge generated by animal-based research that have informed the design of subsequent clinical trials. As such, the most direct and effective pathway leading to improvements in neonatal care is likely to be a continuum that commences with the generation of scientific knowledge and is followed by the collection of evidence though clinical trials. Indeed, animal models have served as a valuable source of information that has provided a large component of our understanding of normal and abnormal human biology. In the context of neonatal resuscitation, they have provided the foundation for our entire understanding of the fetal-to-neonatal transition at birth and for testing new or improved approaches for facilitating this process. Whereas it is important to recognize the limitations of each model and its applicability to human biology, it is also important to recognize that the primary physiological changes that occur at birth are extraordinarily well conserved across all mammalian species. Accordingly, this means that we can learn much from animal-based research, which can be used to design better clinical trials and ensure that these trials are asking the right questions in the right subpopulations of infants. 6.1. Practice points
• Animal models continue to make an important contribution to improving the practice of neonatal medicine. • Knowledge from animal studies should guide the design of clinical trials in order to increase the likelihood of trial success. • Although not the ideal, if the science is sound and the animal model
is similar to that of humans with few differences, basing treatment recommendations on the best available animal study is the most viable alternative when evidence from clinical studies is not
4
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6.2. Gaps in knowledge
• Much of neonatal resuscitation has limited clinical evidence. Studies
using the best available neonatal transitional animal models to better inform future clinical studies need to be done for a variety of delivery room interventions including: o umbilical cord management o ventilation techniques o advanced resuscitation techniques o resuscitation medications.
References [1] Dawes GS. Fetal and neonatal physiology. Chicago: Year Book Medical Publishers, Inc; 1968. [2] Liggins GC. Premature delivery of foetal lambs infused with glucocorticoids. J Endocrinol 1969;45:515–23. [3] Enhorning G, Robertson B. Lung expansion in the premature rabbit fetus after tracheal deposition of surfactant. Pediatrics 1972;50:58–66. [4] Enhorning G, Grossman G, Robertson B. Tracheal deposition of surfactant before first breath. Am Rev Respir Dis 1973;107:921–7. [5] Enhorning G, Grossmann G, Robertson B. Pharyngeal deposition of surfactant in the premature rabbit fetus. Biol Neonate 1973;22:126–32. [6] Pound P, Bracken MB. Is animal research sufficiently evidence based to be a cornerstone of biomedical research? BMJ 2014;348. g3387. [7] Liggins GC, Howie RN. A controlled trial of antepartum glucocorticoid treatment for prevention of the respiratory distress syndrome in premature infants. Pediatrics 1972;50:515–25. [8] Perlman JM, Wyllie J, Kattwinkel J, et al. Part 7: neonatal resuscitation: 2015 international consensus on cardiopulmonary resuscitation and emergency cardiovascular care science with treatment recommendations. Circulation 2015;132:S204–41. [9] Crossley KJ, Allison BJ, Polglase GR, Morley CJ, Davis PG, Hooper SB. Dynamic changes in the direction of blood flow through the ductus arteriosus at birth. J Physiol 2009;587:4695–704. [10] van Vonderen JJ, Roest AA, Siew ML, et al. Noninvasive measurements of hemodynamic transition directly after birth. Pediatr Res 2014;75:448–52. [11] van Vonderen JJ, Roest AA, Walther FJ, Blom NA, van Lith JM, Hooper SB, te Pas AB. The influence of crying on the ductus arteriosus shunt and left ventricular output at birth. Neonatology 2015;107:108–12. [12] Siew ML, Te Pas AB, Wallace MJ, et al. Positive end expiratory pressure enhances development of a functional residual capacity in preterm rabbits ventilated from birth. J Appl Physiol 2009;106:1487–93. [13] te Pas AB, Siew M, Wallace MJ, et al. Effect of sustained inflation length on establishing functional residual capacity at birth in ventilated premature rabbits. Pediatr Res 2009;66:295–300. [14] Hooper SB, Te Pas AB, Lang J, et al. Cardiovascular transition at birth: a physiological sequence. Pediatr Res 2015;77:608–14. [15] Klingenberg C, Sobotka KS, Ong T, et al. Effect of sustained inflation duration; resuscitation of near-term asphyxiated lambs. Arch Dis Child Fetal Neonatal Ed 2013;98(3):F222–7. [16] Sobotka KS, Morley C, Ong T, et al. Circulatory responses to asphyxia differ if the asphyxia occurs in utero or ex utero in near-term lambs. PLoS One 2014;9:e112264. [17] Festing S, Wilkinson R. The ethics of animal research. Talking point on the use of animals in scientific research. EMBO Rep 2007;8:526–30. [18] Ghasemi M, Dehpour AR. Ethical considerations in animal studies. J Med Ethics Hist Med 2009;2:12. [19] Westgate JA, Wibbens B, Bennet L, Wassink G, Parer JT, Gunn AJ. The intrapartum deceleration in center stage: a physiologic approach to the interpretation of fetal heart rate changes in labor. Am J Obstet Gynecol 2007;197:236. e231–11. [20] Patrick J, Challis JR, Cross J, Olson DM, Lye SJ, Turliuk R. The relationship between fetal breathing movements and prostaglandin E2 during ACTH-induced labour in sheep. J Dev Physiol 1987;9:287–94. [21] Bassett JM, Thorburn GD. Foetal plasma corticosteroids and the initiation of parturition in sheep. J Endocrinol 1969;44:285–6. [22] Hooper SB, Te Pas AB, Kitchen MJ. Respiratory transition in the newborn: a threephase process. Arch Dis Child Fetal Neonatal Ed 2016;101:F266–71. [23] Rudolph AM. Distribution and regulation of blood flow in the fetal and neonatal lamb. Circ Res 1985;57:811–21. [24] Dawes GS, Mott JC, Widdicombe JG. The patency of the ductus arteriosus in newborn lambs and its physiological consequences. J Physiol 1955;128:361–83. [25] Hooper SB, Polglase GR, Te Pas AB. A physiological approach to the timing of umbilical cord clamping at birth. Arch Dis Child Fetal Neonatal Ed 2014;100(4):F355–60. [26] Dunlop SA, Archer MA, Quinlivan JA, Beazley LD, Newnham JP. Repeated prenatal corticosteroids delay myelination in the ovine central nervous system. J Matern Fetal Med 1997;6:309–13. [27] French NP, Hagan R, Evans SF, Godfrey M, Newnham JP. Repeated antenatal corticosteroids: size at birth and subsequent development. Am J Obstet Gynecol
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pattern of lung liquid clearance and lung aeration at birth. J Appl Physiol 2009;106:1888–95. te Pas AB, Siew M, Wallace MJ, et al. Establishing functional residual capacity at birth: the effect of sustained inflation and positive end expiratory pressure in a preterm rabbit model. Pediatr Res 2009;65:537–41. Lang JA, Pearson JT, Binder-Heschl C, et al. Increase in pulmonary blood flow at birth: role of oxygen and lung aeration. J Physiol 2016;594:1389–98. Lang JA, Pearson JT, te Pas AB, et al. Ventilation/perfusion mismatch during lung aeration at birth. J Appl Physiol 1985;2014(117):535–43. Han RN, et al. Changes in structure, mechanics, and insulin-like growth factor-related gene expression in the lungs of newborn rats exposed to air or 60% oxygen. Pediatr Res 1996;39:921–9. Bouch S, O'Reilly M, Harding R, Sozo F. Neonatal exposure to mild hyperoxia causes persistent increases in oxidative stress and immune cells in the lungs of mice
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without altering lung structure. Am J Physiol Lung Cell Mol Physiol 2015;309:L488–96. O'Reilly M, Harding R, Sozo F. Altered small airways in aged mice following neonatal exposure to hyperoxic gas. Neonatology 2014;105:39–45. Sozo F, Horvat JC, Essilfie AT, O'Reilly M, Hansbro PM, Harding R. Altered lung function at mid-adulthood in mice following neonatal exposure to hyperoxia. Respir Physiol Neurobiol 2015;218:21–7. Royce SG, Nold MF, Bui C, et al. Airway remodeling and hyperreactivity in a model of bronchopulmonary dysplasia and their modulation by IL-1 receptor antagonist. Am J Respir Cell Mol Biol 2016;55:858–68. Yi M, Jankov RP, Belcastro R, et al. Opposing effects of 60% oxygen and neutrophil influx on alveologenesis in the neonatal rat. Am J Respir Crit Care Med 2004;170:1188–96.
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Seminars in Fetal and Neonatal Medicine journal homepage: www.elsevier.com/locate/siny
Animal models in neonatal resuscitation research: What can they teach us? Stuart B. Hoopera,b,∗, Arjan B. te Pasc, Graeme R. Polglasea,b, Myra Wyckoffd a
The Ritchie Centre, Hudson Institute for Medical Research, Melbourne, Australia Department of Obstetrics and Gynaecology, Monash University, Melbourne, Australia Division of Neonatology, Department of Paediatrics, Leiden University Medical Centre, Leiden, the Netherlands d Department of Pediatrics, Neonatal and Perinatal Medicine, University of Texas, South Western Medical Center, Dallas, TX, USA b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Fetal-to-neonatal transition Animal models Sheep Rabbits Pigs Rodents
Animal models have made and continue to make important contributions to neonatal medicine. For example, studies in fetal sheep have taught us much about the physiology of the fetal-to-neonatal transition. However, whereas animal models allow multiple factors to be investigated in a logical and systematic manner, no animal model is perfect for humans and so we need to understand the fundamental differences in physiology between the species in question and humans. Although most physiological systems are well conserved between species, some small differences exist and so wherever possible the knowledge generated from preclinical studies in animals should be tested in clinical trials. However, with the rise of evidence-based medicine the distinction between scientific knowledge generation and evidence gathering has been confused and the two have been lumped together. This misunderstands the contribution that scientific knowledge can provide. Science should be used to guide the gathering of evidence by informing the design of clinical trials, thereby increasing their likelihood of success. While scientific knowledge is not evidence, in the absence of evidence it is likely to be the best option for guiding clinical practice.
1. A historical perspective Traditionally, animal models have been used to understand the science of normal and pathophysiological states in humans and have given rise to much of our current understanding of normal human biology and the mechanisms of disease. Indeed, in the context of neonatal resuscitation, scientific research has greatly contributed to improving neonatal care and identifying infants that require assistance at birth. For instance, Dawes' pioneering studies on the fetal heart rate responses to birth asphyxia in lambs and primates provided the basic understanding for heart rate monitoring before and after birth [1]. Liggins' demonstration of improved survival and respiratory function following antenatal glucocorticoid administration in preterm lambs [2] has had remarkable success in reducing morbidity and mortality in preterm infants. Robertson's pioneering studies on surfactant in preterm rabbits [3–5] underpins the routine use of surfactant therapy for respiratory distress in preterm infants. All of these studies have had a profound impact on modern neonatal medical practice and the contribution of scientific studies in animal models to neonatal resuscitation research will likely continue into the future. In more recent years, several commentaries have raised concerns about the relevance of scientific studies, their validity, rigour and the
∗
contribution they make to improving health care in humans [6]. These commentaries argue that scientific studies are no longer relevant to modern human medicine and can be replaced by epidemiological studies and/or randomized controlled (RCT) trials and meta-analyses. Indeed, scientific studies in animal models have been relegated to the bottom of the evidence pyramid for changing clinical practice, even below expert opinion. However, this characterization reflects a lack of understanding for how scientific studies in animals generate the knowledge that informs and underpin the clinical studies that change clinical practice. Indeed, Liggins' scientific study in sheep [2] provided the knowledge that prompted the subsequent RCT on antenatal glucocorticoids [7]. As a result, that one RCT provided sufficient evidence to change clinical practice, leading to the routine use of antenatal corticosteroids in women at risk of delivering preterm. Thus, while it is important to question how science can contribute to ongoing improvements in clinical care, perhaps we should also question whether RCTs are underpinned by sufficient scientific knowledge. Ideally, they should form a continuum and we need to consider whether a lack of scientific knowledge explains why many RCTs fail to provide a definitive answer to the medical issues they seek to address. Indeed, a lack of high quality evidence from RCTs in neonatal resuscitation was highlighted by the most recent guidelines (2015) from the International
Corresponding author. The Ritchie Centre, Hudson Institute for Medical Research, 27–31 Wright St, Clayton, VIC, 3168, Australia. E-mail address: [email protected] (S.B. Hooper).
https://doi.org/10.1016/j.siny.2018.07.002
1744-165X/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Hooper, S.B., Seminars in Fetal and Neonatal Medicine (2018), https://doi.org/10.1016/j.siny.2018.07.002
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3. Differences between animal and human studies
Liaison Committee on Resuscitation [8].
Scientific studies in animal models have different priorities than human studies and RCTs. While the design of both human and animal studies is closely scrutinized by ethics committees, the oversight responsibilities of these committees are different. In animal studies, the hypothesis being tested has the highest priority, whereas in human studies the health and well-being of the patient takes precedence. Clearly the latter is a vital tenet of ethical studies in humans, but can significantly limit the capacity to rigorously test some hypotheses. This underpins the need for testing some hypotheses in animals. For instance, whereas birth asphyxia occurs naturally in humans, it varies markedly in severity between individuals. As such, studies aimed at determining the effect of different treatments on specific outcomes are difficult, particularly when the treatments are multifactorial. However, as birth asphyxia can be artificially induced in animals to produce a consistent level of severity [15,16], a systematic analysis for how differing treatments affect specific outcomes is far more feasible. At the very least, these types of animal studies can identify the treatments that are likely to be most effective, which can then be tested in a more focused human study. The ethical governance for the use of animals in research also requires several criteria to be met [17,18]. For example: (i) the minimum number of animals must be used which is still compatible with answering the question under investigation; (ii) the scientific question must be worthy of investigation; (iii) the experimental design must not be limited by factors that undermine its capacity to answer the question being investigated. Indeed, if animals are to be used for the sake of medical science, it is vital that answering the scientific question takes precedence, because otherwise there is no point in doing the study and using the animals. The ethical requirement to minimize animal numbers [17,18] can introduce limitations in animal studies. For instance, to account for the reduced statistical power associated with small numbers, it is important to control for as many variables as possible and to ensure that the expected treatment effect is sufficiently large to detect differences between groups. However, it is not always possible to anticipate all of the variables that can impact on the factor under investigation. As a result, the addition of unexpected variability reduces the power of the analysis and markedly reduces the capacity to identify differences, particularly when the numbers are low. Nevertheless, reducing animal numbers is one of the primary tenets for the ethical use of animals in research and is universally accepted by most scientific researchers [17,18]. The suggestion that scientific research is flawed because animal studies are rarely repeated, thereby precluding a meta-analysis [6], fails to understand this primary tenet. Indeed, repeating the same or similar animal studies in different laboratories just to fill the requirements for a meta-analysis is unethical and is contrary to every code of conduct specifying the ethical use of animals in medical research [17,18]. It also fails to understand the difference between the generation of fundamental knowledge and the provision of evidence that can change clinical practice.
2. Benefits and limitations of animal models Many examples demonstrate the contribution that animal research makes to neonatal resuscitation research, however, it is important to understand both the benefits and limitations of animal research in the context of improving health care in humans. For instance, whereas the basic underlying physiology is well conserved between species, all species are different and so no species is a perfect model for humans. This has led some to consider that no information derived from animal studies is relevant to humans, but this is clearly incorrect. For example, in some aspects sheep are clearly very different from humans as they have four legs and are ruminants with four stomachs. However, it is well established that their respiratory and circulatory systems as well as the underlying control systems are very similar, with few if any differences. For example, studies describing the normal transitional physiology in lambs described and explained the rapid (within minutes) onset of bidirectional shunting through the ductus arteriosus after birth [9]. As this is caused by changes in vascular resistances (decrease in pulmonary vascular resistance and increase in systemic vascular resistance caused by cord clamping), the laws of physics dictate that the same changes must also occur in humans. Subsequent observations in humans showed that normal healthy infants undergo these same transitional changes within minutes of birth [10,11]. Similarly, while it is not surprising that preterm lambs and infants require similar inflation pressures to achieve the same tidal volumes (corrected for body weight) at birth, preterm rabbits also require these same pressures to achieve the same tidal volumes despite being only 20–30 g in weight [12]. These examples highlight the need to understand the physiology of the animal under consideration and how they differ from humans when choosing an animal model for medical research. For instance, whereas sheep may be an appropriate model for studying respiratory and cardiovascular physiology in humans, their gastrointestinal physiology is very different and so are likely to have limited value in simulating pathophysiological states in humans. Furthermore, in view of the potential for unknown subtle differences to occur between species, it is important to recognize that responses in an animal model may be different from the responses in humans. Thus, any new potential treatments or therapies derived from animal studies should be trialled, wherever possible, in humans before they are adopted into widespread clinical use. However, this recognition should not devalue the information gained from animal-based research and instead perhaps we should change the way in which we view and utilize this information. With regard to neonatal resuscitation research it is also important to understand that the physiology of the fetus and newborn immediately after birth are very different from a newborn hours to days later. Indeed, as newborn infants immediately after birth have liquid-filled lungs and a fetal circulation that has not transitioned into the newborn phenotype, newborn resuscitation research using animal models should involve an animal in transition. For example, liquid-filled airways radically alter lung mechanics, resulting in airway resistances that are up to 100-fold higher than moments later when the lung is air-filled [13]. Furthermore, as lung aeration follows an exponential function that is very variable with different time constants and end points (degree of lung aeration) [14], particularly in very preterm newborns, it is not possible to accurately predict the state and homogeneity of lung aeration without imaging it. Similarly, the newborn circulation undergoes a major re-organization after birth, with the closure of shunts and separation of the two circulations (systemic and pulmonary), which is triggered by lung aeration [14]. As this process has such a profound influence on circulatory function and cannot be replicated in a fully transitioned animal model, an animal model undergoing this transitional process is most appropriate for investigations of the physiology/ pathophysiology involved.
4. What contribution can animal research make to resuscitation guidelines? Information gained from animal studies is commonly referred to as “weak evidence” for treatment recommendations in clinical practice guidelines and is usually raised when clinical evidence is absent. This wording is potentially misleading and contributes to a misunderstanding of the value that information from animal research can provide. Perhaps it should not be viewed as “evidence”, but rather as “knowledge” that informs the provision of evidence through appropriately designed clinical trials. In this context, Francis Bacon's quote “knowledge is power” is quite apt and perhaps the evidence pyramid should be inverted to reflect the concept that meta-analyses are not the 2
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The latter is currently a hotly debated topic and may have important implications for how infants are managed at birth in the future. Sheep have also been used to detail the effects of antenatal glucocorticoid (single and multiple doses) on the development of a variety of fetal organ systems as well as detailing the benefits to respiratory function after birth [26–28]. Studies in sheep have also been responsible for identifying the role of antenatal factors, such as intrauterine inflammation and fetal growth restriction, in markedly modifying the newborn's physiology and explaining the increased risk of morbidity and mortality [29–32]. They have also been used to investigate approaches that most effectively aerate the lung and ventilate preterm infants, while minimizing the risk of lung injury leading to bronchopulmonary dysplasia [33–37]. Whereas sheep have many advantages and similarities to humans, they do differ from humans in some important ways. Depending upon the breed of sheep, gestation length varies between 142 and 150 days, but within a breed, gestation length is very consistent ( ± 1 day). Single pregnancies are common in maiden ewes, but in multiparous ewes, multiple fetuses (twins) are more common. Sheep have a cotyledonary placenta and a relatively quiescent uterus; while this is quite different from humans, it does confer many advantages from an experimental perspective. For instance, incising the uterus and conducting sterile fetal surgery under general anesthesia is relatively simple and straightforward in sheep. Catheters, EMG leads, and flow probes can be implanted in the anesthetized fetus before the amniotic sac, uterus and abdomen are closed and the ewe and fetus allowed to recover from the anesthesia. As the uterus is very quiescent, there is little to no risk of triggering premature labour, unless there is an intrauterine infection, allowing the physiology of the fetus to be monitored for weeks after surgery. In addition, catheters and flow probes can be implanted days or immediately before delivery, allowing physiological recordings to be obtained before, during, and after transition. However, this paradigm precludes vaginal delivery and requires delivery by cesarean section either under general or spinal anesthesia. As normal healthy lambs are required to be mobile shortly after birth, so that they can feed from their mothers, organ development is in general more precocious than in humans to meet the higher levels of activity required of lambs. Indeed, the lungs are slightly more mature in sheep at birth than in humans, containing millions of alveoli, large sections of fused epithelial/endothelial cell basement membranes, and a fully differentiated epithelium [38]. Interestingly, whereas anatomically the lung of a premature lamb at ∼127 days of gestation appears quite mature, having millions of alveoli and a fully differentiated alveolar epithelium [38], functionally it is similar to that of a 26–28-week premature infant [33]. The latter has a lung with no alveoli, as it is only at the early saccular stage of development and a partially differentiated distal airway epithelium.
pinnacle of evidence but rather the culmination of evidence that should start with knowledge and a basic understanding of the science. This is not a novel concept as there are few engineers who would consider building a large bridge without mathematically applying the scientific knowledge gained from studies on load distribution through substrates and structures, construction materials and design aerodynamics as well as knowledge of the local physical/environmental conditions, etc. While the ultimate goal is to support every treatment and therapy used in neonatal resuscitation with high-quality evidence from metaanalyses of good RCTs, currently this is an unrealistic expectation. This is primarily due to a loss of equipoise with some treatments and/or low incidence rates, making large trials with power to detect differences between treatments unfeasible. Thus, in the absence of evidence, what criteria should be used to decide on the best approach for recommendation and how should these criteria be weighted? In the absence of evidence, we consider that recommendations based on the best available scientific knowledge should be the primary criteria. Rather than referring to scientific knowledge gained from animal models simply as “weak evidence”, the knowledge should be assessed in terms of the quality of the scientific data and the weaknesses and strengths of the animal models used. In particular, the known differences and similarities between the animal species used and humans require careful consideration so that applicability of data can be appropriately assessed. Surely, if the science is sound and the animal model is similar to humans with few differences, basing treatment recommendations on the best available science is the most viable alternative when evidence from RCTs and meta-analyses are not available. In any event, the history of medicine is full of wonderful examples demonstrating that science should always be considered above expert opinion when deciding on treatment options. 5. Types of animal models Numerous animal models have been used to study the science underpinning the normal fetal-to-neonatal transition, antenatal conditions, and treatments that impact on this transition and on the best approaches for ventilating and resuscitating newborns immediately after birth. The most widely used species include sheep, rabbits, pigs, mice, and rats. Although some primates have been used, they are very costly relative to other species and require access to primate facilities that are not widely available. 5.1. Sheep Sheep are widely used in perinatal research and arguably have had the greatest impact on our understanding of perinatal medicine. The impact has probably been greatest in the discipline of maternal/fetal medicine, providing the foundations for fetal heart rate monitoring during labour [19], monitoring fetal breathing movements [20], fetal growth and other biophysical profiles, mechanisms of labour onset [21], antenatal glucocorticoid treatment for all women threatening preterm labour [2] as well as the transition to newborn life. Much of the information derived from fetal monitoring was adopted straight into clinical practice simply based on the findings, indicating the value that scientific knowledge can directly and safely add to clinical practice. In the context of neonatal resuscitation, research in sheep is almost entirely responsible for our understanding of the physiological changes that occur at birth. This includes our understanding of fetal lung physiology and how the airways are cleared of liquid at birth, allowing the lung to rapidly (within minutes) take over the role of gas exchange [22]. It also includes our understanding of fetal cardiovascular physiology and the role of vascular shunts, which is vastly more complex than the adult circulatory system [14,23,24]. Indeed, the transition of the circulatory system at birth is very complex and our scientific understanding of this process is still ongoing, particularly how umbilical cord clamping at birth interacts with the onset of air-breathing [25].
5.2. Pigs The need for cardiopulmonary resuscitation (CPR) in the delivery room is hard to predict, very rare if adequate ventilation is established, and is often performed under chaotic circumstances, making it very challenging to study clinically [39]. The neonatal piglet model has been used primarily to better understand various aspects of neonatal CPR following asphyxia-induced bradycardia and/or asystole. Strengths of the model are that the cardiac and pulmonary anatomy of the pig is very similar to that of the human. Newborn pigs are of similar size to newborn humans (2–4 kg) and although the chest is more keel-shaped (such that there is not a flat sternum directly above the heart as a landmark for cardiac compressions), due to its similar size, performing cardiac compressions on a piglet feels remarkably similar to the delivery room experience with CPR in newborn babies. Issues of compressor fatigue and ergonomic aspects of resuscitation can be explored in meaningful ways [40,41]. Another strength of the neonatal pig model of asphyxia-induced bradycardia and asystole is that it closely 3
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injury is much easier, as a cross-section of an entire lung can be placed on one slide.
mimics the biochemical profile of asphyxiated newborns who require CPR, with profound mixed metabolic and respiratory acidosis at the time of cardiovascular collapse. Important insights into neonatal cardiac compression techniques [41–45], prediction of return of spontaneous circulation [46], the effects of medications and volume [47–49], and both tissue and physiologic responses to resuscitation with various concentrations of oxygen [50–52] have come from studies of the pig model. One such example of cardiac compression technique is the pig studies by Schmölzer et al. that examined the method of performing cardiac compressions while maintaining a sustained inflation. This neonatal pig work suggests potential benefit with shorter time to return of spontaneous circulation compared to traditional interposed compressions and ventilations in a 3:1 ratio [40,44]. The initial exploratory science from the animal model led to a human pilot trial [53] and a larger trial is currently underway. The major limitation to the pig model is that the newborn pig transitions from fetal to newborn circulation in the first hours of life, such that even if animals are available on the first day of life, by the time the experimental surgical preparation is complete, the animal is unlikely to have an open duct, open foramen ovale, or very high pulmonary pressures such as would be present at birth in the human. The lack of these transitional shunts makes the neonatal pig model less ideal for study of delivery room resuscitation, although the information gained does translate better to resuscitations in the neonatal intensive care unit after neonatal transition has occurred.
5.4. Rodents Rodents, particularly mice, are by far the most frequently used animal models in biomedical research. The huge potential for genetic manipulation has unleashed an avalanche of new knowledge on genetic, epigenetic, and molecular regulation of developmental pathways and how these can influence the neonatal transition. Whereas it is impracticable to list all of the important discoveries attributed to this model, it is noteworthy to mention that rodents have been used extensively to demonstrate the adverse effect of hyperoxia on lung development and the role of hyperoxia in the development of bronchopulmonary dysplasia [59–64]. Gestation length is 19–21 days in mice and 21–23 days in rats, depending upon the strain. Rodent pups are considerably smaller and more immature at birth than rabbit pups and lambs, making it very difficult to make any physiological measurements or to ventilate them until they are least 4 days old. At this age they are well past the transitional stage and, as such, are not a particularly suitable model for neonatal resuscitation research. Furthermore, mice are born in the late canalicular stage of lung development, so all of the final two stages of lung development normally occurs postnatally, which is very different from humans. Structurally, the lungs are also quite different, with very large diameter proximal airways and truncated small airways to facilitate the very rapid flow of air into and out of the airways at a rate of 180 breaths per minute.
5.3. Rabbits The rabbit model has also made a huge impact on neonatal resuscitation research and was famously used by Robertson et al. to demonstrate the effect of exogenous surfactant administration in premature newborns [3–5]. Similarly, we have used the rabbit model in our phase-contrast X-ray imaging studies to demonstrate the roles of inspiration in airway liquid clearance [54,55], the benefits of positive end-expiratory pressures [12], and sustained inflations on lung aeration [13,56] in ventilated newborns and to characterize ventilation/perfusion relationships in the lung at birth [57,58]. Like all models, the rabbit model has similarities with and differences from humans. Rabbits frequently have litters of 4–12 pups and growth-restricted pups are common, which can add variability to the data if all pups from a litter are used. Gestation length is only 32 days, so fetal development is greatly compressed in time relative to humans. In the latter part of gestation, 1 day of development in a rabbit equates to ∼1–2 weeks of development in a human. As such, a 27-day-old rabbit fetus has a similar level of maturity as a 26–27-week human, with development rapidly progressing with each day. Even half a day difference can markedly alter the level of maturity, so care should be taken to avoid this influence on data variability. Nevertheless, at birth lung maturity is very similar to humans and from an anatomical and functional perspective, rabbits are arguably one of the most suitable animal models for humans to study transitional respiratory physiology. Like sheep, rabbits also tolerate recoverable fetal surgery, although success rates for surviving fetuses are less than in fetal sheep. As newborn rabbit pups are very small (50–60 g at term), the physiological measurements that can be performed are more limited compared with sheep and mostly require purpose-built equipment. For instance, preterm rabbits (∼25 g) can be intubated and have an intravenous catheter inserted, but to ventilate them in a manner relevant to preterm infants (e.g. in volume guarantee mode) requires the ability to measure and administer very small tidal volumes; to ventilate a 25 g pup at 7 mL/kg requires giving a tidal volume 0.175 mL. Nevertheless, their small size confers other advantages. For instance, their small size makes it quite easy to measure lung volume changes by plethysmography, which is ideal for measuring the temporal and spatial patterns of lung aeration in a lung that is structurally and functionally very similar to that of humans. Similarly, histological examination of the lung for
6. Conclusion Animal models have made and continue to make an important contribution to neonatal resuscitation research. However, ignorance of this contribution is widespread, as is the misconception that humans are different from animals and therefore the knowledge gained from animal studies is of no real value for clinical medicine. However, some of the most profound improvements in neonatal health care have come from knowledge generated by animal-based research that have informed the design of subsequent clinical trials. As such, the most direct and effective pathway leading to improvements in neonatal care is likely to be a continuum that commences with the generation of scientific knowledge and is followed by the collection of evidence though clinical trials. Indeed, animal models have served as a valuable source of information that has provided a large component of our understanding of normal and abnormal human biology. In the context of neonatal resuscitation, they have provided the foundation for our entire understanding of the fetal-to-neonatal transition at birth and for testing new or improved approaches for facilitating this process. Whereas it is important to recognize the limitations of each model and its applicability to human biology, it is also important to recognize that the primary physiological changes that occur at birth are extraordinarily well conserved across all mammalian species. Accordingly, this means that we can learn much from animal-based research, which can be used to design better clinical trials and ensure that these trials are asking the right questions in the right subpopulations of infants. 6.1. Practice points
• Animal models continue to make an important contribution to improving the practice of neonatal medicine. • Knowledge from animal studies should guide the design of clinical trials in order to increase the likelihood of trial success. • Although not the ideal, if the science is sound and the animal model
is similar to that of humans with few differences, basing treatment recommendations on the best available animal study is the most viable alternative when evidence from clinical studies is not
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6.2. Gaps in knowledge
• Much of neonatal resuscitation has limited clinical evidence. Studies
using the best available neonatal transitional animal models to better inform future clinical studies need to be done for a variety of delivery room interventions including: o umbilical cord management o ventilation techniques o advanced resuscitation techniques o resuscitation medications.
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