2Department of Environmental Health and Radiological Sciences, Colorado State University, Fort Collins, CO, USA
Quicktime (.m4v) video describing the construction and operation of the aerosol inhalation system.
The design for a simple, low-cost aerosol generation system for rodent inhalation studies is described here. This system is appropriate for low biohazard–level agents. In this study, two biosafety level 2 agents, Pasturella pneumotropica and Pseudomonas aeruginosa, were tested successfully. This system was also used to immunize mice and guinea pigs in ovalbumin-based models of pulmonary inflammation. This design is appropriate for studies with limited budgets and lower-level biosafety containment.
There are many well-made inhalation exposure systems on the market for treating rodents for inhalation studies, such as the one from Glas-Col, LLC (Catalog no. 099C A4212; Terre Haute, IN, USA). Many of these systems are used for biosafety level 3 agents like Mycobacterium tuberculosis and have been extensively characterized (1,2). However, these systems are costly ($25,000) and can be difficult to move, clean, and decontaminate. Here, we describe an inexpensive system with a total cost of less than $500, or $4500 with a biosafety cabinet. This system uses an air jet medical nebulizer to generate a high concentration aerosol with initial droplet diameters of 1–2 µm. Furthermore, this system utilizes a collection chamber for any potentially hazardous aerosol components and can be contained within a very small biological containment hood for biosafety level 2 agents.
The system is described in detail so that other investigators with limited resources can build their own systems. Initial tests for safety and decontamination procedures are also discussed. We started with the idea that a medical nebulizer could be used for rodent aerosol exposure studies, but were confronted with several problems related to biosafety and containment. Other groups had described the use of a medical nebulizer (3,4) and the design of aerosol systems (5) but we could not easily find suitable details to build our own. The best description actually came from the web site of a pet guinea pig owner (http://web.archive.org/web/20071023014537/http://home.houston.rr.com/lundquist/Purrie/Nebulizer/Nebulizer.htm) and we found that similar systems have apparently been used to treat birds and other small animals (www.petiatric.com/Medical-nebulizer.htm). We improved upon these designs to make sure that any aerosol would be safely contained and removed into a filter flask. Some systems did not prevent fur contamination (3,4), which we solved by using Decapicone bags (Braintree Scientific, Braintree, MA, USA). We also wanted a system that was easily decontaminated.
This system has been used in infection studies with Pasteurella pneumotropica and Pseudomonas aeruginosa. P. pneumotropica was used initially as a test organism because we planned to study this opportunistic pathogen. P. pneumotropica does not infect wild-type mice often, but can be more of a problem with immunocompromised strains of mice (6,7,8, http://www.criver.com/SiteCollectionDocuments/rm_rm_r_pasteurella_pneumotropica.pdf). P. pneumotropica was not a vigorous pathogen and was cleared easily in wild-type mice. Further studies were done with P. aeruginosa because it represents a more prototypical lung pathogen, had been used in a similar study with the Glas-Col aerosol system (9), and was available with a luciferase gene insertion for study of the infected lungs with a luminescence imager.
We have also done more conventional immunology studies with ovalbumin in Tg(DO11.10)10Dlo/J T cell receptor (TCR) transgenic mice, which can specifically respond to ovalbumin peptide (OVAp) amino acids 323–339 presented by Major Histocompatability Complex Class II H-2d, and is a commonly used model for T lymphocyte trafficking (3,10,11). Modifications were also done for preliminary studies in a guinea pig model of ovalbumin-mediated pulmonary inflammation (12) to show the utility of a larger version of this system.Materials and methods Materials
Materials for the nebulizer include a medical air-jet nebulizer (Part no. 9911–1, A Helping Hand HealthMed, Benicia, CA, USA); 15 mm × 22 mm adaptor pipe (Part no. 962-E, Hospitak Inc., Farmingdale, NY, USA); pump station (Catalog no. 13–875–240, Fisher Scientific, Pittsburgh, PA, USA); air regulator (Catalog no. EW-32460–48, Cole Parmer, Vernon Hills, IL, USA); Gladware 104-oz. Family Size disposable containers (for mouse studies; Glad, Oakland, CA, USA); Rubbermaid 2.4-gal food storage container (for guinea pig studies; Rubbermaid, Fairlawn, NJ, USA); pressure gauge (Part no. 1490; Ashcroft Inc., Stratford, CT, USA); 1-L polypropylene vacuum flask (Catalog no. DS4101-1000; Thermo Scientific, Barrington, IL, USA); pinch clamp (Catalog number S49103; Fisher Scientific); disposable 0.2-m air line HEPA filter (Cole Parmer); 0.25- and 0.125-in diameter Tygon tubing (Catalog nos. 13-310-271 and 13-310-192; Fisher Scientific); PVC duct tape (Catalog no. 40866-5VGA; Harbor Freight Tools, Camarillo, CA, USA); Scotch heavy-duty mounting tape (Catalog no. 110; St. Paul, MN, USA); barbed 0.25-in L and T connector tubes (Catalog nos. 48800 and 48780; Ace Hardware, Oak Brook, IL, USA); 0.24-in NPT brass barbed male fitting (Part no. 42777; Ace Hardware), female-female coupling (Part no. 43070; Ace Hardware); Teflon tape (Catalog no. 40973; Ace Hardware); O-ring set (Part no. 41018; Ace Hardware); plastic waterproof adhesive (Seal-All Adhesive, Eclectic Products, Inc., Pineville, LA, USA); circular cutting tools, round-edge knives or drill/Dremel tool (Catalog no. 8000-02; Dremel, Racine, WI, USA); and a Class I fume hood for biohazardous agents (Catalog no. 3970200; Labconco, Kansas City, MO, USA)