EPIDEMIOLOGICAL studies reveal striking differences among children, adults, and elderly in risk factors, susceptibility, course, and outcome of the acute respiratory distress syndrome (ARDS).^1–10  Although ARDS is a major contributor to mortality in all age groups, the incidence, morbidity, and mortality tend to gradually increase with age,^4,5,11  which seems partially independent of comorbidity.^9,12–14  These findings suggest potential age-dependent differences in the pathophysiology of ARDS.

In the acute phase of ARDS, the innate immune response causes inappropriate accumulation and activation of blood leukocytes, excessive production of inflammatory mediators, and uncontrolled coagulation.^15,16  Interestingly, not only the innate immune response but also intrinsic properties of the lung are known to change during the process of maturation and aging.^17–27  Although newborns have a relatively impaired immune response to bacteria,^24,27  elderly have a persistent low-grade innate immune activation generating a constitutive proinflammatory environment (termed inflammaging).^22,28  Aging is also associated with a gradual deterioration of the immune system (termed immunosenescence).^22,28  Although the interplay between these age- dependent immunological and morphological changes correlate with the clinical patterns of disease,^18,23,24,26  the underlying molecular mechanisms are poorly understood.

Of the numerous animal models used to elucidate the pathophysiology and treatment of ARDS,^29,30  most of them used adolescent or young–adult animals. However, it is increasingly clear that age impacts the susceptibility for and severity of ARDS.³¹ 

Accordingly, we hypothesized that maturation and aging affect the pulmonary responses to injury in an age-dependent manner, which is associated with increased lung injury and augmented inflammatory responses with increasing age. This review aims to investigate age-related differences in pulmonary responses to injury in animal models of lung injury, discuss the potential underlying mechanisms, and outline the possible implications for research and clinical management.

This study followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines.³²  A search was conducted in PubMed and EMBASE (up to June 2014) using variants of the following search terms: “lung injury,” “maturation or aging,” and “animal.” The full electronic search strategy is presented in appendix 1.

L.R.S. and R.M.W. independently conducted the literature searches and assessed the eligibility of the identified publications. We included studies using standard in vivo animal models of lung injury caused by an acute pulmonary insult,^29,30  which compared at least two age groups. Studies included for data extraction were restricted to original full-text articles published in English. Excluded were studies that did not report at least one of the primary outcomes of interest (as described in the Data Extraction and Risk of Bias Assessment) or focused on surfactant-related infant respiratory distress syndrome.

L.R.S. and R.M.W. independently extracted data from all eligible studies. The primary outcome measures included the presence of increased lung permeability (wet-to-dry ratio, protein leakage, or pulmonary capillary filtration coefficient), lung injury (changes in respiratory compliance and lung injury score by histopathology), pulmonary inflammation (cell influx and levels of inflammatory mediators), oxidative stress (levels and activity of oxidants and antioxidants, including myeloperoxidase activity), and mortality. All these outcome measures are key features in the pathophysiology of ARDS.^15,16,29  In addition, information on potential underlying molecular mechanisms was extracted from the included studies.

The included studies were stratified by age groups that correspond to the developmental stages of the human lung (table 1).^35–38  First, mice aged 2 to 10 months and rats aged 3 to 12 months represented the (young-) adult reference group. Second, mice or rats aged 2 months or younger and 3 months were classified as infant/juveniles; this age group included lung developmental stages ranging from early alveolarization to lung growth. Third, mice or rats aged 10 months or older and 12 months were classified as middle aged/elderly.

We used a list of 10 items based on a recent publication evaluating a quality assessment tool for animal studies³⁹  to identify potential selection bias, performance bias, detection bias, and other bias. Because of the lack of a validated tool for quality assessment of animal studies, this checklist was only used to investigate the risk of bias and not to exclude studies from the data analysis due to poor quality.

Methodological diversity precluded combining quantitative data from the individual studies in a meta-analysis. Therefore, we present data on the primary outcomes by means of a qualitative comparison with the adult reference group.

The initial search yielded 2,840 publications and cross-referencing identified 28 additional publications; most of these were excluded for the reasons presented in figure 1. Finally, 51 publications were selected for data extraction, i.e., 22 studies on infant/juvenile animals and 29 studies on middle-aged/elderly animals. The models of lung injury that were used included ventilator-induced lung injury (VILI; 6 studies), hyperoxia (11 studies), intratracheal challenge with lipopolysaccharide (LPS; 10 studies), pneumonia models with bacteria (16 studies), and bleomycin (3 studies). In addition, five studies investigated two-hit models, combining LPS with high tidal volume mechanical ventilation, LPS with hyperoxia, or low tidal volume mechanical ventilation with hyperoxia. Of the included studies, two studies used genetically modified animals.^40,41  The effects found in these studies were similar compared with studies with nongenetically modified animals. Details on the included studies are presented in appendix 2. Overall, according to the quality checklist, there was a low risk of performance bias but a high risk of selection and detection bias (appendix 3).

A clear age-dependent difference in the severity of lung injury was found in the various animal models of lung injury (tables 2 and 3). Independent of the pulmonary challenge, the majority of studies assessing differences between infants/juveniles compared with adults showed less pulmonary edema,^{43,44,46,48–53,58}  decreased lung tissue damage on histology,^{43,44,46,48,49,51–54,58,59}  and a lower mortality^{44,45,48,49,51–55}  (table 2). In contrast, most studies investigating middle-aged/elderly animals showed more pulmonary edema,^{40,65–67,73,74,76,80–82,86,88}  increased lung tissue damage on histology,^{64–67,76,80–82,86,88–90} and a higher mortality compared with their adult reference group^{64,65,70,73,80,85–89}  (table 3). In addition, eight studies assessing lung compliance showed a more pronounced decrease in compliance with increasing age in VILI models^{42–44,47,64,65} or after administration of bleomycin.^89,90  Hyperoxia was the only pulmonary challenge in the middle aged/elderly, which was consistently associated with an increased tolerance.^68–71  However, this tolerance was attenuated in models that combined hyperoxia with a second hit, such as mechanical ventilation or LPS.^66,67,70 

Several studies assessed the baseline inflammatory status^{40,44,58,73,75,82,83,85,88}  (data not shown). These latter studies reported lower expression of inflammatory mediators and higher levels of antioxidants in infants/juveniles^44,58  and a more activated inflammatory milieu in the middle aged/elderly^{73,75,82,83,85,88}  when compared with adults. Data on the number of resident alveolar macrophages and neutrophils were conflicting.^{40,65,68,75,77,80,82,83} 

Given that each model produces different lung injury modifying the molecular mechanisms activated,²⁹  we present results on the pulmonary response to injury stratified by type of pulmonary insult (tables 4 and 5). In all age groups, cell influx was predominated by neutrophils. Independent of the model used, in most studies, the recruitment of neutrophils into the lung was lower in infants/juveniles^{44,46,51,52,54,56–58,60,61}  (table 4) and higher in the middle aged/elderly^{40,65–67,69,72–74,76–81,86,87,89}  (table 5), compared with adults. Some studies reported a delayed recruitment of neutrophils in both extremes of age: infants^52,54,57,58  and elderly.^{73,78,82,83,88}  In addition, macrophage function in the pneumonia models was impaired in these age groups and associated with a decreased clearance of bacteria (tables 4 and 5).^{60–63,77,80,81,85,86,88}

The pulmonary inflammatory mediator response in infants/juveniles was mainly studied in VILI and hyperoxia models, which showed decreased levels compared with adults^{43,44,46,53,54}  (table 4). Moreover, three hyperoxia models showed that the expression of antioxidants was increased in the infant/juvenile animals, which correlated with increased tolerance to hyperoxia.^48–50  The inflammatory mediator response in middle aged/elderly was dependent on the type of insult (table 5). VILI, LPS, and bleomycin all induced an increased proinflammatory response.^{41,65,67,74,76,89,90}  In contrast, no clear trend was seen in the pneumonia models^{80–83,85 –88} (table 5). Also, no correlation was found between the type of bacteria or time point of measurement and the cytochemokine and chemokine response. Although data were limited, an increased oxidative stress response was found in middle aged/elderly^66,67,72,80  when compared with adults.

Molecular mechanisms underlying the observed age-dependent differences in lung injury were investigated in several studies.^{40,41,46,49,50,55,58,59,68,73,76,81,84–86,89,90}  An age-dependent increase in susceptibility to lung injury was associated with differences in inflammatory and host defense signaling pathways, including altered gene expression,⁴⁶  transcription factors,^46,59,68,81  phosphorylation of intracellular signaling molecules,^76,81,84,86  and membrane sensing molecules^46,81,85,86  (table 6). In addition, age-related intrinsic dysregulation of proteostasis (protein homeostasis) induced a proinflammatory state that augmented lung injury.⁵⁹  In contrast, infants showed increased expression of genes encoding antioxidants resulting in decreased lung injury when compared with adults.^49,50,55  Finally, age-dependent differences were found in molecular pathways involved in the repair and remodeling phase of ARDS. Aged animals showed a lower expression of components of the vascular endothelial growth factor receptor pathway, known to play a protective role in ARDS.⁷³  In contrast, high age was associated with increased levels of components of the profibrotic response such as transforming growth factor and metalloproteinases.^41,58,89,90 Figure 2 provides a summary of the differences between infants/juveniles and middle-aged/elderly animals compared with the adult reference group.

The main finding of this systematic review is that increased age is associated with exaggerated pulmonary responses to injury. In vivo animal models of lung injury consistently show that age is an important independent host factor influencing fundamental pathophysiological mechanisms known to be involved in ARDS. This influence of age seems far more complex than merely a more pronounced proinflammatory or antiinflammatory response; age tends to affect multiple processes of the pulmonary response to injury (fig. 2).

The findings on preclinical studies are in line with epidemiological studies showing that the incidence of ARDS in children is lower than in adults, whereas the incidence and mortality are significantly higher in elderly.^1–10  The data from this review support the clinical findings that increased susceptibility to ARDS in elderly is not only due to comorbidity^9,12–14  but also due to age, which is an important independent determinant associated with severity of lung injury.

This review shows that, in different animal models of lung injury, increasing age is associated with increased endothelial–epithelial permeability, altered function of alveolar macrophages, increased influx of neutrophils, an exaggerated inflammatory mediator response, and increased oxidative stress (fig. 2). Studies addressing underlying molecular mechanisms of these age-dependent differences in the pulmonary response to injury are limited. However, they are in line with studies investigating the processes of maturation and aging in general. Prominent aging-associated alterations in the inflammatory response include dysfunctional immune cells, senescent cells that secrete proinflammatory cytokines, and the occurrence of a defective autophagy response.²²  Moreover, recent evidence indicates that neutrophils from humans of advanced age show untargeted tissue migration with increased primary granule release and neutrophil elastase activity leading to more tissue inflammation.⁹¹  This may in part explain the delayed but overwhelming recruitment and extensive alveolar damage found in elderly animals with lung injury. In addition, aging in general is associated with changes in intracellular signaling pathways involved in inflammation and cell integrity.²²  Increasing age is associated with overactivation of the nuclear factor-κB pathway.^22,59,68,81  Taken together, these alterations result in a proinflammatory state, failure to effectively clear pathogens, dysfunctional host cells, and impaired repair mechanisms making elderly prone to exaggerated responses to injury.

In the context of current evidence on maturation and aging, we speculate that age-dependent changes in morphology, cell integrity, and the innate immune response are important determinants of the severity of lung injury after a pulmonary challenge (fig. 3). Together with comorbidity and physiological reserves, these patient-related biological factors may ultimately determine the susceptibility of the patient to develop ARDS.

To the best of our knowledge, this is the first review to systematically investigate current knowledge on the effect of age on the pulmonary response to acute injury in preclinical models. Despite the clear association emerging between age and differences in the pulmonary response, some limitations of this study need to be addressed.

Systematic reviews are subject to publication bias of the studies showing no differences.⁹²  In addition, quality assessment showed a high risk of selection and detection bias in the included studies, which could overrate their conclusions. Only a minority of the studies reported the use of randomization (24%) and blinding (29%). Although in animal experiments, variation between groups is limited by genetic homogeneity and standardized (pre-) experimental conditions, lack of randomization and blinding can reduce the internal validity of an animal experiment,^93,94  implying that differences found between the experimental groups may not be attributed to the treatment under study.⁹⁵  Moreover, none of the included studies showed a power calculation. Sample size calculation is required to reduce the incidence of false-negative or false-positive outcomes between groups and to keep the number of animals used as low as possible in view of legal requirements and ethical/practical considerations.⁹⁵  These forms of potential bias have also been found in other reviews assessing the quality of animal models^95–97  and probably contribute to the large gap between preclinical and clinical research.^95–99  Use of recently published guidelines for reporting animal research, such as the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines¹⁰⁰  and STREAM (Studies of Translation, Ethics and Medicine) checklist,⁹⁷  have the potential to improve experimental design and reporting of animal studies, as such preventing these problems. Differential use of animal species, lung injury models, exposure time, doses, and variation in outcome measures makes it difficult to pool the data and may also account for some of the conflicting results.^29,30  Therefore, the current review focused only on the pulmonary processes and limited the search to models of lung injury induced by a pulmonary challenge. On the other hand, the consistent outcome in the various pulmonary insults studied underlines the importance of the influence of age. To summarize the current data, we decided to pool the data into three age groups; however, the delineation of these age groups is debatable. The infant/juvenile group includes many different maturational stages, whereas the elderly group includes studies with late-mature animals, corresponding with middle-aged adults. The heterogeneity in these age groups may have influenced the analysis; however, as we aimed to investigate a trend in increasing lung injury with increasing age, the precise classification of age may be less important. Finally, one could argue that the effects found in most of the studies is because of overdosing with increasing age, because most studies use body weight to titrate their pulmonary challenge. It is known that the ratio between lung volume and body weight in mice and rats declines with increasing age^19,101 ; therefore, dosing the pulmonary challenge based on lung weight or lung volume would be more accurate. However, because studies that used lung weight showed similar effects, it is unlikely that the associations found are exclusively because of overdosing or underdosing.

Our findings imply that results from animal models conducted in adolescent or young adult animals cannot be directly translated to patient populations of different age. To develop effective translational animal models of lung injury, appropriate age groups corresponding with the clinical patient population of interest should be used. In addition, unraveling the underlying mechanisms of age-dependent differences in ARDS could lead to more appropriate design of clinical trials for both children and elderly and may have potential therapeutic implications in the development of age-specific therapeutic targets. However, because the process of maturation and aging is continuous and dynamic, age groups do not necessarily have strict boundaries. Close collaboration between pediatric and adult intensive care physicians is important to further optimize treatment for the individual patient.

In conclusion, this systematic review shows that the pulmonary response to injury varies with age, which may have potential therapeutic implications. Although age-dependent changes in the innate immune response play an important role, the underlying molecular mechanisms are not well understood. In the future, well-designed animal and clinical studies, using appropriate age groups, could unravel these mechanisms and may provide new age-directed therapeutic targets for ARDS.

Supported by a research grant (PhD Scholarship) of the Academic Medical Center, Amsterdam, The Netherlands (to Dr. Schouten).

The authors declare no competing interests.