Table 1.

Difficulties in modelling ARDS.

Problems	Factor	Problem in experimental trials	Solution
Endpoints	Endpoint in clinical trials is 28-day mortality	• Animals may die of shock and not of ARDS • In most studies, animals die within 72 h • Survivors of the initial phase recover completely	• Hemodynamic monitoring • Unknown • Unknown
	Do surrogate endpoints work?	• No accepted surrogate endpoint (i.e. hypoxemia seems not to work*)	• Unknown
	Relative vs. absolute endpoints	• Many experimental endpoints are relative (i.e. neutrophil fraction in BAL cells, Evans Blue, etc.) and provide little insight into the severity of lung injury	• Use of absolute endpoints such as P/F ratio, w/d ratio, or compliance
	Hyaline membranes	• Hyaline membranes are difficult to obtain in experimental animals4	• Unknown
ICU treatment	Fluid support	• Without fluid support hypodynamic shock is likely	• Animal ICU; cardiovascular monitoring
	Ventilation	• Mechanical ventilation is a critical risk factor	• Animal ICU; ventilation as second hit
	FiO2 > 30%	• Despite the general agreement on the important role of ROS in ARDS, the clinical reality where ICU patients are commonly ventilated at FiO2 > 30%,29 and the demonstrated increase in ROS formation during hyperoxic ventilation,38 most animal models are ventilated at an FiO2 of 21%	• Animal ICU; hyperoxia as second hit
Disease characteristics	Duration of disease	• Models > 72 h	• Large animal models
	MOF; animals die from shock and not from ARDS	• Contribution of shock and extrapulmonary organs is uncertain in many models	• Monitor extrapulmonary organs; cardiovascular monitoring
	Heterogeneity of patients, risk factors, sex, microbiome, age, co-morbidities	• Inbred animals may not be representative • Differences between mouse strains39–41 • Biological diversity (age, sex, co-morbidities, genetic heterogeneity),42–44 is commonly intentionally ruled out in animal studies	• Population heterogenization45 • Preclinical randomized controlled multicenter trials46 • Statistical adjustments47
	In pneumonia/sepsis there is a gradual/exponential bacterial growth with concomitant immune responses	• Rapid injection of high doses may produce untypical inflammation • Cytokine responses often higher than in patients which may explain why anti-cytokine therapies are more effective in animal models15	• Slow administration; multiple-hit models • Unknown
Lung properties	Lungs are strong and redundant	• Hypoxemia is difficult to induce in healthy animals	• Measure P/F ratio
	Pulmonary inflammation occurs easily	• Differentiate between benign and detrimental inflammation	• Measure degree of injury by absolute parameters
Other confounding factors	Ambient temperature	• Mice need 30℃, rats 28℃ for thermoneutrality48	• Animal ICU • Heated animal facilities
	Untrained immune system	• Humans have a trained immune system • Microbiome likely to modify the disease	• Unknown
	Standardization	• For example, i.t. administration,† cecal ligation, and puncture model‡	• Quality management
	Treatments	• Use of heparin in shock models problematic	• Avoid use of heparin

^*In the low tidal ARDSnet trial, oxygenation was worse in the low tidal volume group, which finally had a lower mortality.²⁴

^†It makes a huge difference where and how tracheal injections are placed. For instance, in our own hands in acid-induced lung injury, the outcome is very different if the same amount is deposited in the trachea as a drop (little effect), as a small streak of liquid (lung injury), or whether it is nebulized (little effect).

^‡In the CLP model, injury depends on the needle diameter and the rate of subsequent wound closure.⁴⁹

BAL, bronchoalveolar lavage; FiO₂, fraction of inspired oxygen; MOF, multiple organ failure; P/F ratio, ratio of arterial PO₂ over FiO₂; ROS, reactive oxygen species; SPF, specific-pathogen-free.