Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.
Allergen challenge:
Anesthesia:
Intubation:
Intravenous line:
Airway resistance measurements:
Bronchoalveolar lavage:
TIMING:
Per mouse, the entire procedure should take no longer than 1 hour to accomplish: Step 3-4, 5-10 min.; Steps 5-21, 10 min.; Step 22, 20-30 min.; Steps 23-24, 10 min. With increased proficiency and by staggering subjects in the protocol, up to 3 mice/hour may be processed.
Representative Results:
Airway hyperreactivity in mice, as determined by measures of PC200 values, is a consequence of activation and recruitment to the lungs of T cells and secretion of the cytokine IL-135-7. Thus, airway hyperreactivity is not the inevitable consequence of airway challenge with allergen, but rather depends on an intact T cell immune compartment and the time required for T cell responses to develop in the setting of repeated allergen exposure. As shown in Fig. 2a, airway hyperreactivity, defined as PC200 values that are significantly lower compared to baseline values, developed after 5 allergen challenges with no further significant increase after the sixth challenge. For reasons that are not fully understood, airway reactivity decreased (PC200 values increased) after the first allergen challenge (Fig. 2a). Similar trends are apparent by comparing Ach dose response curves for the same mice (Fig. 2b). However, it is apparent here that full airway hyperreactivity develops abruptly after the fifth allergen challenge, such that mice become more than 30-fold more sensitive to Ach between the fourth and sixth challenges. Together, these findings indicate that the most reliable measurements of AHR are obtained after six allergen challenges (12 days); measurements at earlier timepoints are likely to yield highly variable data. Mice repeatedly challenged with vehicle intranasally (saline) do not develop airway hyperreactivity, and, at all doses of Ach given, RRS measurements do not significantly vary from baseline values (Fig. 3 and data not shown).
Prior to the emergence of robust AHR, the dominant cell type of the airway induced by allergen was the neutrophil (Fig. 4). Similar to the trend for AHR, however, eosinophilia gradually strengthened with repeated allergen challenge and the eosinophil became the numerically dominant cell type in BAL fluid after the sixth challenge, coincident with a marked decline in neutrophis numbers (Fig. 4). Macrophages initially increased in number with the first few allergen challenges and fluctuated in abundance thereafter. Lymphocyte abundance did not change significantly regardless of the number of allergen challenges and, paradoxically given their primary importance to the model, are typically the least numerous cell in the BAL fluid.
Airway resistance measurements in mice receiving neither allergen challenge nor BAL sampling did not vary over the 17 days of experimentation. Repeated BAL fluid sampling in the absence of airway physiology measurements or allergen challenge were also performed, and showed only an enhanced neutrophil and macrophage recruitment to the airways that did not persist beyond 5 days (data not shown). These findings demonstrate that the prominent neutrophilia observed in allergen challenged mice is largely the result of the procedure and not the antigen.
In control, PBS-challenged mice, airway resistance measurements also did not vary significantly over time. Enhanced macrophage and neutrophil, but not eosinophil, recruitment to BAL fluid was also seen in these mice, similar to those changes observed in mice receiving only repeated BAL fluid sampling (Fig. 4 b, d). Together, these data underscore the importance of the allergen, and not the different manipulations of the airway, to the induction of both allergic (eosinophilic) airway inflammation and AHR.
Similar results can be expected using intranasal allergens analogous to the proteinase that we have used here. However, many investigators use ovalbumin to induce allergic lung disease. After an appropriate period of intradermal or intraperitoneal priming (1-2 weeks) with ovalbumin precipitated in an aluminum salt, a robust asthma phenotype, including airway hyperreactivity, can be expected within 24 hours after a single intranasal challenge with soluble ovalbumin.
Figure 1. Photographic representation of a rodent plethysmograph, immediately prior to airway physiology measurement recording.
Figure 2. Airway resistance measurements. A) For statistical purposes, antilog PC200 values are reported. Note the large increase in the antilog PC200 after first challenge and subsequent decrease following further challenges. B) Respiratory system resistance (RRS): Note the steepness of the Ach-RRS dose-response curves after sixth and seventh challenges. Error bars represent SEM.
Figure 3. Representative real-time respiratory system resistance (RRS) tracings from a naïve (A) and 6X allergen challenged mouse (B) receiving consecutive IV doses of Ach. Dose values are presented in mg/kg units.
Figure 4. Differential immune cell counts in the bronchoalveolar lavage samples derived from the left lungs of mice treated with 7 consecutive intranasal challenges. Percent (%) abundance of immune cells in mice treated with allergen (A) or PBS (B). Total number of immune cells from mice treated with allergen (C) or PBS (D). Values represented as mean +/- sem.
The study of asthma, and various other airway obstructive diseases, constitutes an active and expanding field of biomedical research. An important component of asthma-related experimental research is the capacity to measure changes in airway size under varying conditions. Excessive airway narrowing in response to provocative challenge, a canonical feature of asthma and related lung diseases and a property of the airway termed airway hyperresponsiveness, is a major component of clinically significant attacks leading to shortness of breath and other symptoms, including death.
In this study, a novel method was developed to measure airway hyperresponsiveness and simultaneously sample inflammatory cells recruited to the airway in the mouse. We show that airway hyperresponsiveness is not immediately induced with the first, or even after the first several, of seven consecutive allergen challenges given over 15 days. Rather, airway hyperresponsiveness increases only gradually with progressive allergen challenge, becoming highly significant (relative to sham allergen challenged animals) only after 6 challenges (12 days). This marked alteration in airway physiology roughly paralleled the influx of lung inflammatory cells, marked most prominently by eosinophils, which first appear in the airway lavage fluid in large numbers (>106/ml) after the 5th allergen challenge.
These findings are consistent with the prior demonstration that T helper type 2 (Th2) cells mediate both airway hyperresponsiveness and eosinophilia6, but extend these observations by demonstrating the minimum time (12 days) required for these endpoints to become maximal with semi-continuous allergen exposure. The maximal degrees of airway hyperreactivity and airway eosinophilia reported here are likely the maximum possible with the mouse using this allergen as further allergen challenge failed to elicit greater responses (data not shown).
More importantly, these data demonstrate that airway hyperreactivity measurements and analysis of airway inflammatory cells can be rigorously performed repeatedly in the same cohort of mice. In addition to providing continuity to data collected serially, the described protocols allow for reduced experimental costs (fewer mice required for each study) and enhanced statistical power (repeated measures from the same animals as opposed to data from distinct groups). A strong theoretical foundation underlies the described physiologic method8, further adding confidence to the collected data. With dedicated effort, proficiency with the described techniques is readily achieved, providing a critically important tool set for investigating experimental allergic lung disease and other pulmonary disorders.
We thank Dr. W. Mintzer for the suggestion to perform fiberoptic orotracheal intubation. Supported by grants U19AI070973, R01AI057696, K02HL75243, and R01HL082487 from the National Institutes of Health.
Airway physiology measurement software (Rescomp) was custom prepared (Millenium Premier Group; 415-519-4371).
Data was analyzed using a PC workstation running Windows XP equipped with a Pentium III CPU (Intel, Inc. Santa Clara, CA) and a 17-pin analog to digital signal converter (National Instruments, #PC-LPM16).
A small animal airway physiology workstation was custom assembled (Millenium Premier Group) using commercially available pressure transducers (part #TRD5700 and TRD4510), preamp modules (part #MAX2270), chassis (part # MAX1320; all from Buxco, Inc. Wilmington, NC) and a customized small animal plethysmograph.
0.5mm external diameter fiber-optic thread, connected to light source (Cole Palmer Illuminator, 41722 series)
Ventilator (Harvard Apparatus Mouse Ventilator, #687)
10 mm, 27ga needle (BD Biosciences, cat. no. 309602)
Heat lamp
1 ml syringe (BD Biosciences, cat. no. 305109)
4 clamps (Pony 3200 spring clamp)
0.5 mm external wire for intubation guide
Hemacytometer
Superfrost/plus microscope slides (Fisher cat. no. 12-550-15)
Shandon Filter Cards (Thermo cat. no. 5991022)
Differential cell slide stain (Fisher cat. no. 122911)
Light microscope (Leica)
Cytospin 3 (Shandon)
20 ga, 1.25 inch ProtectIV intravenous catheters (Smith Medical)
0.5 mm polymer optical fiber (Edmund Optics # NT02-532).
Airway physiology measurement software (Rescomp) was custom prepared (Millenium Premier Group; 415-519-4371) and data were analyzed using a PC workstation running Windows XP equipped with a Pentium III CPU (Intel, Inc. Santa Clara, CA) and a 17-pin analog to digital signal converter (National Instruments, #PC-LPM16). Small animal airway physiology workstation was custom assembled (Millenium Premier Group) using commercially available pressure transducers (part #TRD5700 and TRD4510), preamp modules (part #MAX2270) and chassis (part # MAX1320; all from Buxco, Inc. Wilmington, NC) and a customized small animal plethysmograph. 0.5mm external diameter fiber-optic thread, connected to light source (Cole Palmer Illuminator, 41722 series); Ventilator (Harvard Apparatus Mouse Ventilator, #687); 10 mm, 27ga needle (BD Biosciences, cat. no. 309602); Heat lamp; 1 ml syringe (BD Biosciences, cat. no. 305109); 4 clamps (Pony 3200 spring clamp); 0.5 mm external wire for intubation guide); Hemacytometer; Superfrost/plus microscope slides (Fisher cat. no. 12-550-15); Shandon Filter Cards (Thermo cat. no. 5991022); Differential cell slide stain (Fisher cat. no. 122911); Light microscope (Leica); Cytospin 3 (Shandon); 20 ga, 1.25 inch ProtectIV intravenous catheters (Smith Medical); 0.5 mm polymer optical fiber (Edmund Optics # NT02-532).
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