Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, Emory University and Atlanta VA Medical Center
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Reddy, A. T., Lakshmi, S. P., Reddy, R. C. Murine Model of Allergen Induced Asthma. J. Vis. Exp. (63), e3771, doi:10.3791/3771 (2012).
Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.
I. Allergen Sensitization and Challenge (see Figure 1)
A. For Intratracheal Challenge
B. For Challenge by Nebulization
II. Determination of Airway Hyperresponsiveness to Methacholine
A. Noninvasive Measurement of Airway Hyperresponsiveness by Whole-body Plethysmography (WBP; Buxco Research Systems, Wilmington, NC)
B. Invasive Measurement of Airway Responsiveness by Computer-controlled Ventilator (flexiVent; SCIREQ Inc., Montreal, Canada)
III. Measurement of Cellular Infiltration into the Airspace
A. Perform Bronchoalveolar Lavage (BAL)
B. Count Cells and Determine Differentials
IV. Representative Results
Excessive airway constriction following provocative stimuli is a prominent feature of clinical asthma. We describe two methods for measuring such airway hyperresponsiveness to methacholine in OVA-sensitized and challenged mice: Whole-body plethysmography (Figure 2) and forced oscillation using the flexiVent system (Figure 3). Both methods demonstrate that OVA sensitization and challenge produces airway hyperresponsiveness in mice.
Eosinophil-rich airway inflammation is another prominent feature of both clinical asthma and allergic airway disease in mice. As shown in Figure 4, OVA sensitization and challenge greatly increases the total number of cells that can be recovered from the airways by BAL. The numbers of eosinophils and, to a lesser extent, neutrophils are especially increased.
Evidence indicates that allergic airway disease results from overproduction of IgE antibodies to sensitizing antigens. Sensitization and challenge with OVA using the protocols we describe increases IgE levels in both serum and BAL fluid of treated mice (Figure 5).
Figure 1. Experimental schema for OVA-induced allergic asthma. Mice were sensitized twice i.p. with 20 μg of OVA emulsified in 2 mg of aluminum hydroxide in 0.2 ml of sterile PBS, or 2 mg of aluminum hydroxide in 0.2 ml of sterile PBS alone, followed at the indicated time points by i.t. challenge with 0.1% OVA or sterile PBS solution or by daily exposure for 30 minutes to nebulized 1% OVA in PBS or PBS alone delivered via an ultrasonic nebulizer (Buxco). Twenty-four hours after the final OVA exposure, airway responsiveness was determined. Subsequently, BAL fluid, blood samples, lung cells, and tissues were collected for further analysis.
Figure 2. Assessment of allergen-induced airway hyperresponsiveness by a noninvasive method. Mice (n=4/group) were sensitized and challenged with OVA. Twenty-four hours following the last challenge, airway hyperresponsiveness to inhaled methacholine was determined using whole-body plethysmography as described in the protocol. Penh was determined and expressed as Penh ratio (average Penh over the 8-min time interval with methacholine divided by the average Penh over the 8-min interval with PBS). *, P < 0.05 vs. PBS.
Figure 3. Assessment of allergen-induced airway hyperresponsiveness by an invasive method (forced oscillation). Mice (n=4/group) were sensitized and challenged with OVA. Twenty-four hours following the last challenge, airway hyperresponsiveness to increasing concentrations of inhaled methacholine was determined by the forced oscillation (flexiVent) method as described in the protocol. A, B) Airway resistance; C) Lung elastance. *, P < 0.05 vs. PBS.
Figure 4. BAL fluid cell count. Mice (n=4/group) were sensitized and challenged with OVA. Twenty-four hours following the last challenge, (Top) BAL cells were collected and total cells were counted as described in the protocol. (Bottom) Cytospin slides were prepared and stained with Diff-Quick. Tot = total cells; Eos = eosinophils; Neu = neutrophils; Mac = macrophages; Lym = lymphocytes. *, P < 0.05 vs. PBS.
Figure 5. OVA-specific IgE. Mice (n=4/group) were sensitized and challenged with OVA. Twenty-four hours following the last challenge, IgE was measured in BAL fluid and in serum from the blood collected by cardiac puncture as described in the protocol. *, P < 0.05 vs. PBS.
Animal models of allergic airway disease provide important tools for studies relevant to clinical asthma. A number of different models, employing varying species and antigens, have been developed. The mouse, an attractive and frequently used laboratory species, also offers a number of advantages for models of allergic airway disease.9,10 Although such models do not mimic asthma in every respect,11 with aspects of chronic disease being particularly difficult to reproduce,12,13 we confirm here that many of the major features are reproduced. We also show that, as in human asthma, these features are associated with increases in antigen-specific IgE in both serum and BAL fluid. We present two murine models, both employing OVA as the antigen but utilizing different challenge techniques. Intratracheal administration is somewhat complex and time-consuming, but offers the advantage of delivering a known quantity of antigen directly to the lung. It is also possible that this method delivers the antigen more deeply into the lung than do alternatives. We describe an invasive method for intratracheal delivery, but antigen can also be delivered intratracheally via a tube or cannula inserted via the oral cavity. This method is described elsewhere in JoVE.14 Nebulization is simpler and more directly mimics the usual route of human exposure. Quantitation is more variable, however, and delivery to the lung may be less efficient.15 Indeed, it is likely that a sizable but unknown fraction of the dose is deposited in the upper airways. A third alternative, not described here, is intranasal delivery. Again, it is likely that a sizable fraction of the dose will be deposited in the upper airways.
Although our demonstration is performed using C57BL/6 mice, evidence suggests that certain other strains may give more robust results on specific endpoints. In comparisons of C57BL/6 with BALB/c16 or with both BALB/c and FVB/NJ17 mice, the C57BL/6 mice showed the smallest increase in AHR. On the other hand, C57BL/6 mice showed greater increases in eosinophilia in one study16 but not in the other.17 Cytokine elevations were both strain- and cytokine-specific. The preferred strain may therefore depend on the endpoint deemed most important.
Two main features of allergic airway disease are commonly chosen for assessment in order to determine the effects of experimental manipulations: AHR and extent of airway inflammation. AHR may be measured either by whole-body plethysmography (WBP) or by forced oscillation. In both cases, methacholine is used as the provocative challenge. WBP is a functional in vivo measurement that allows analysis of airway reactivity on conscious, freely moving or minimally restrained mice without invasive surgery and anesthesia. Airway responsiveness is expressed using the "enhanced pause" (Penh) as a parameter of altered airway function. Penh is an empirical parameter that reflects changes in the box flow waveform from both inspiration and expiration and combines it with the comparison of early and late expiratory box flow. WBP has several potential advantages compared to invasive means for measuring lung resistance, since it is technically less demanding and allows measurements of airway responsiveness to aerosolized stimulants. This method minimizes both effects of psychological stress and animal preparation time, making it ideal for repeated measurements (examination of the same animal at different time points) and measurement over long periods of time (>24 hr), during which a variety of aerosols can be administered in a controlled and repeatable manner. The results correlate strongly with those of the invasive methods, but it is faster and easier.
Two alternative noninvasive methods provide measurements that are said to be more directly related to airway constriction. In double-chamber plethysmography the mouse is forced to stick its head through a hole in the front of the thoracoabdominal chamber.18 A nasal chamber is then fitted to the front of the thoracoabdominal chamber and a head-hole is cut in the sealing latex film between the chambers. This hole is precisely sized to provide an air-tight seal between the chambers without restricting respiratory airflow. Plethysmographic measurements are then taken in both chambers and the delay between nasal and thoracoabdominal flows are used to calculate the specific airway resistance (sRaw). Head-out plethysmography is similar except that air flows freely in the nasal chamber and no plethysmographic measurements are made there.19 The primary calculated parameter is flow rate at midexpiratory phase (EF50). Both techniques, like WBP, are suitable for repeated measurements over a number of hours, permitting assessment of both early and late responses. However, both require the animals to be restrained, which can produce erratic results unless the animals are accustomed to the apparatus over the course of several days. It can also be difficult to assure an effective air-tight seal, which is essential in both cases. Furthermore, since both Penh and sRaw have been shown to correlate strongly with invasive measurements, and Penh and sRaw have been shown to correlate directly in guinea pigs,20 it can be questioned whether the additional information obtained justifies the increased experimental difficulty.
In the forced oscillation method, deeply anesthetized mice are tracheotomized and a mechanical ventilator is connected to the tube. Pressure-flow measurements as the ventilator inflates and deflates the lung then allow direct measurement of pulmonary resistance and dynamic compliance. This provides information about airway mechanics that is not available with WBP. Forced oscillation is technically more difficult, however, and is typically a terminal procedure. As experimental endpoints, the two procedures generally give similar results.19
The other prominent feature of clinical asthma commonly used to assess the effect of experimental manipulations in murine allergic airway disease is eosinophilic inflammation. Although other aspects of inflammation may be measured as well, this most commonly involves determination of the extent of inflammatory cell (especially eosinophil) infiltration into the airways, the lung, or both. The number and type of cells in the airways is determined in BAL fluid: First total cell number is determined by counting in a hemacytometer, and then cell type is determined by differential staining following cytospin. Although BAL fluid collection can in principle be performed in living mice, as it is in humans, it is more usually and conveniently performed following euthanasia. Eosinophil infiltration into the lungs is assessed by lung excision followed by standard histopathological techniques.
In some instances it may also be useful to assess the extent to which antigen sensitization and challenge stimulates goblet (mucus-producing) cell proliferation. This is easily accomplished by staining the lung sections with periodic acid-Schiff's, which is specific for mucus. Antigen-specific IgE may also be measured in BAL fluid and serum using readily available kits. IgE measurement may be important in settings where the degree of sensitization, as distinct from the end-organ response, is a crucial parameter. There are a wide variety of other measurements, including cytokines and T-cell responses, that are valuable adjuncts to the study of allergic airway disease in animal models that we have not described here. It is the wide variety of relevant responses that antigen sensitization and challenge can elicit that render such models valuable in study of this disease.
Experiments on animals were performed in accordance with the guidelines and regulations set forth by the Atlanta VAMC IACUC Committee under protocol #V010-10.
This work was supported by NIH Grant HL093196 (R.C.R.) and the Atlanta Research and Education Foundation (AREF).
|Pentobarbital sodium salt||Sigma-Aldrich||P3761|
|Whole body plethysmography (WBP) system||Buxco Research Systems||http://www.buxco.com|
|Light microscope||Leica Microsystems|
|Cytospin 4||Thermo Fisher Scientific, Inc.|
|Diff-Quick stain||Siemens AG||B4132-1A|