For the full assessment of lung mechanics without challenge, mice will be anesthetized, tracheotomised (end-point measurement) or intubated (repeated measurement) and attached to Scireq’s Flexivent FX and ventilated. We consecutively apply a series of different perturbations. The response from these perturbations is then analyzed by the software to calculate the lung mechanical parameters as described below. Measures are repeated until at least 4 valid measurements are obtained for every perturbation.

The measurements with tracheotomy give more accurate results, compared to the results obtained with intubation, because leakage of air can be much better controlled (ligature around the trachea) and shorter tubes (less resistance) can be used with a tracheotomy. However, as the trachea can’t be closed after tracheotomy, this measurement is an end-point measurement. Measurements with intubation can be performed more than once in the same mouse.

Deep-Inflation

During this perturbation lungs are inflated until the tracheal pressure reaches 30 cm H2O. Inspirational capacity, the volume of air which can enter the lungs, can be calculated from this pertubation. This parameter can be affected by any pathology which reduces the size of the airway lumen (e.g. airway wall thickening or edema) but also by parameters affecting the elasticity of the lungs (e.g. emphysema or fibrosis). Also the weight of the mouse has an influence on this parameter.

SnapShot

The SnapShot is a sinusoidal perturbation of the airway pressure. The response of this perturbation is fit to the very basic ‘single compartment model’ of the lungs and airways. In this model the lungs are represented as an elastic balloon, giving the compliance, while the airways are represented as a tube, which confers resistance to the air going in and out of the lungs. The following parameters are calculated from this model: C: Dynamic compliance: a measure for the elasticity of the lung tissue. This will be typically affected in diseases like emphysema or fibrosis. E: Dynamic elastance: The elastance is the inverse of the compliance (E = 1/C) and thus represents the same phenomenon as the dynamic compliance. R: Resistance: a measure for the resistance the air encounters to enter and exit the lungs, which is typically increased in cases of airway narrowing and bronchoconstriction, typical landmarks for asthma.

Quick-Prime-3 and Prime-8

Both perturbations are multi-frequency perturbations. The Quick-prime-3 takes 3 seconds while the prime-8 takes 8. As the prime-8 perturbation is longer it allows for a more accurate determination of the lung mechanics parameters. However in mice with diseased lungs and thus reduced oxygen exchange in the lungs this perturbation can be too long, causing a drop in oxygen in the lungs and consequent breathing reflexes of the animal. In these animals the Quick-prime-3 perturbation is a better choice as the oxygen levels will not drop as severely. The response to these perturbations is fit to the ‘constant phase’ model of the lungs. The advantage of this model is that one can make a difference between the resistance encountered in the central large airways and in the lung tissue (small airways and alveoli). This is interesting in disease where the central airway is not particularly affected while the small airways are, like in case of emphysema and fibrosis. The following parameters are calculated: Rn: Newtonian resistance: This parameter represents the resistance the air encounters in the central airways. Tissue damping (G): The tissue damping is a measure for the air resistance in the small airways and alveoli. Tissue elastance (H): The tissue elastance is closely related to the dynamic elastance calculated in the ‘single compartment model’ from the SnapShot perturbations and is also a measure for the elasticity of the lung and is thus influenced by the same disorders as the dynamic elastance as emphysema and fibrosis.

PV-Loops

For the pressure-volume (PV) loops the lungs are inflated in small steps of 4 cm H2O and then left in that ventilator position for 1 second before the next step until 30 cm H2O is reached after which they are deflated in the same small steps. The advantage is that, because of the slow maneuver, lungs can reach a state of quasi static equilibrium. The compliance measured this way is thus a better measure of the true compliance of the lung. However the question is if this measure is truly very relevant, as the lung will not reach this state of quasi static equilibrium in normal breathing. Due to its slow nature, PV-loops can also help to characterize lungs in fibrosis models, in which the response is usually slowed down so much that they can’t be characterized accurately with other perturbations which are relatively fast and dynamic. PV-loops can also be helpful for translational research as they are performed in humans under ventilation. We only use this perturbation to calculate the static compliance. Full analysis of these curves is not included in the standard phenotyping but can be performed upon user’s request or the curves can be sent to the customer so that they can analyze them themselves. The following parameter is obtained from PV-loops: Cst: Static compliance: As explained above this is a measure for the elasticity of the lung in a quasi-static equilibrium.

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As for the full assessment of lung-function without challenge, we can perform this measurement as end-point measurement, with tracheotomy, or as repeated measures, with intubation. The challenge is given by aerosol which is generated in-line with the ventilation.

The choice between end-point and repeated measures is more critical for these experiments, as administration of the challenge compound (typically methacholine), will cause increased pressure in the lungs during ventilation and during the perturbations. To maintain integrity of the lungs and insure that we have no pressure leakage from the tube we will have to stop the registration of the dose response curves once the pressure reaches 30 cm H2O when using intubation in a survival measurement. This should not be an issue for baseline measurements in which we do not expect the pressure to increase a lot, but could be a problem if airway hyper-reactivity is already established. In case of doubts, please feel free to contact us, so that we can establish the best strategy to suit your needs.

With the challenge one is usually interested in the peak response after each dose of methacholine. Therefore we can only use 1 type of short perturbation to measure the response, which means that the following options are available:

SnapShots

Is the shortest perturbation we can apply and thus the best choice to characterize the peak response. However the amplitude of the SnapShot is relatively high and we can’t limit the pressure during the perturbation. We can reduce the amplitude in the higher doses, to avoid reaching the 30 cm H2O pressure limit. However this will require an additional mouse to find the right amplitude. The parameters we can measure from these perturbations are the parameters from the ‘single compartment model’ R, E and C as described above.

Quick-Prime-3

This perturbation is slightly longer, but the maximal amplitude of the input signal is much lower, which means that we can go to higher doses of methacholine without reaching the 30 cm H2O lung integrity pressure limit. As we are using the prime wave we can measure the parameters from the ‘constant phase model’: Rn, G and H; but also the parameters from the ‘single compartment model’: R, E and C.

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Asthma Models

Severe OVA-Induced Asthma

This is model is one of the oldest models of asthma which gives rise to a severe form of allergic asthma in the mouse with a strong Th2 type response due to the use of alum during the sensitization phase. A schematic representation of the model is shown in figure 1. Considering the robustness of the model and the fact that it is one of the best characterized models of asthma, it is a good choice for many studies. Its weaknesses are the exaggerated Th2 response and the lack of extensive airway remodeling. Also, in some cases, the phenotype could be too strong to see effects of interventions on the model.

Mild OVA-Induced Asthma

This model, which is also driven by OVA as model allergen, gives rise to a much milder form of allergic asthma, with much milder Th2 inflammation and less severe development of airway hyper-reactivity. A schematic representation of the protocol is shown in figure 2. This model can be useful in cases that the severe model is too strong, due to high sensitivity of the used mouse strain for example. As for the severe model its Th2 response is still exaggerated and there is a lack of extensive airway remodeling.

Short House Dust Mite Model of Asthma

This model uses a natural aero-allergen which also causes asthma in humans and animals. It is generally milder than the OVA driven models but the phenotype more closely mimics the pathology of allergic asthma as observed in human asthma, with a much milder Th2 inflammatory component and a more extensive airway remodeling. The protocol is shown in Figure 3.

Long House Dust Mite Model of Asthma

This model induces a more severe asthma phenotype compared to the short house dust mite model. The airway remodeling is more extensive, so it is a good choice if airway remodeling is your area of interest. A schematic overview is shown in Figure 4.

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Emphysema Model

Elastase-Induced Model of Emphysema

The elastase model of emphysema is a fast and simple protocol to establish emphysema. It consists of just 1 oro-pharyngal instillation of elastase after which progressive emphysema is established. Despite the fact that it has a completely different etiology as the natural disease in humans it has shown to be a powerful tool to study the development of the disease once it is established. A mild form of emphysema can already be observed 3 days after the administration of elastase and will progress to GOLD stage 2 within 3 weeks of elastase treatment.

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Pulmonary Fibrosis Model

Bleomycin-Induced Model of Pulmonary Fibrosis

The bleomycin model of pulmonary fibrosis is one of the most used pulmonary fibrosis models in rodents. As for the emphysema model it consists of only 1 oro-pharyngal application of the compound to establish the disease. A significant fibrosis can be observed 3 weeks after administration of Bleomycin. In contrast to the emphysema model this model is not a true chronic and progressive model and some recovery from fibrosis is observed in the mouse after this 3 week period.

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Acute Lung Injury

LPS-Induced Model of Acute Lung Injury

Acute lung injury is a common problem in intensive care patients under ventilation, as the mechanical stress of the ventilation exacerbates mild pulmonary infections to a level in which they become a serious health issue for the patient. We are not equipped for infectious studies, but acute lung injury can be induced in mice by instillation of LPS. It will cause an acute lung injury like disease, which is strongest 24 h after LPS administration and resolves within 1 week.

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Histopathology

Tissues from subject to lung function assessments can be fixed, processed, embedded, sectioned and stained upon request.

Serum IgE

Serum IgE levels can be determined using ELISA.

Immune Cell Typing

A standard FACS procedure using 2 cocktails of antibodies allows differentiation between: Total T cells, αβ T cells, CD4+ αβ T cells, CD8+ αβ T cells, γδ T cells, NKT cells, NK cells, B cells, monocytes, granulocytes, eosinophils, CD4+ CD25+ regulatory T cells, CD4+ CD44hi CD62Llo T cells, CD4+ KLRG1+ T cells, CD8+ CD44hi CD62Llo T cells, CD8+ KLRG1+ T cells, KLRG1+ NK cells, IgD+ B cells, Ly6Chi I-A/I-Elo monocytes and Ly6Clo I-A/I-Elo monocytes.

Inflammatory Cytokines

Multiplex bead assay can be used to accurately measure the following inflammatory cytokines:

• Panel I: IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17a, Eotaxin, G-CSF, GM-CSF, IFNg, IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG, MIP-1a, MIP-1b, MIP-2, RANTES, TNFa, VEGF.
• Panel II: IL-16, IL-21, IL-22, IL-25/IL-17, IL-28B, EPO, Exodus-2, Fractalkine, MCP-5, MIP-3a, MIP-3b, TARC
• Panel III: IL-20, IL-23, Il-27, IL-33, MDC, TIMP-1

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Lung unit was upgraded with the support from OP RDE project CZ.02.1.01/0.0/0.0/18_046/0015861 CCP Infrastructure Upgrade II.