Development of an experimental model of neutrophilic pulmonary response induction in mice

ORIGINAL ARTICLE

Development of an experimental model of neutrophilic pulmonary response induction in mice^*

Leonardo Araújo Pinto^I; Camila Camozzato^II; Monique Avozani^II; Denise Cantarelli Machado^III; Marcus Herbert Jones^IV; Renato Tetelbom Stein^IV; Paulo Márcio Condessa Pitrez^V (te sbpt)

^IResident Physician in Pediatric Pneumology

^IIScientific Initiation Sponsored

^IIIAssociate Professor, Internal Medicine do Department

^IVAssociate Professor, Pediatrics Department. Master in Pneumology

^VPhysician at the Pediatric Pneumology Team. Master in Pneumology. Specialist title by the Brazilian Society on Pneumology and Tisiology.

^{Correspondence}

ABSTRACT

BACKGROUND: Several lung diseases are characterized by a predominantly neutrophilic inflammation. A better understanding of the mechanisms of action of some drugs on the airway inflammation of such diseases may bring advances to the treatment.

OBJECTIVE: To develop a method to induce pulmonary neutrophilic response in mice, without active infection.

METHODS: Eight adult Swiss mice were used. The study group (n = 4) received an intranasal challenge with 1 x 10¹² CFU/ml of Pseudomonas aeruginosa (Psa), frozen to death. The control group (n = 4) received an intranasal challenge with saline solution. Two days after the intranasal challenge, a bron­choalveolar lavage (BAL) was performed with total cell and differential cellularity counts.

RESULTS: The total cell count was significantly higher in the group with Psa, as compared to the control group (median of 1.17 x 10⁶ and 0.08 x 10⁶, respectively, p = 0.029). In addition to this, an absolute predominance of neutrophils was found in the differential cellularity of the mice that had received the Psa challenge.

CONCLUSIONS: The model of inducing a neutrophilic pulmonary disease using frost-dead bacteria was successfully developed. This neutrophilic inflammatory response induction model in Swiss mice lungs may be an important tool for testing the anti-inflammatory effect of some antimicrobial drugs on the inflammation of the lower airways.

Key words: Animal experimentation. Lung disesases. Inflammation. Neutrophils. Pseudomonas.

Acronyms and abbreviations used in this study

Psa – Pseudomonas aeruginosa

PBS – Phosphate buffered saline

CUF – Colony Unit Former

SS – Saline solution

BAL – Bronchoalveolar lavage

TCC – Total cell count

Background

Several diseases or respiratory infections, such as cystic fibrosis, diffuse pan-bronchiolitis, or acute viral bronchiolitis may induce a predominantly neutrophilic pulmonary inflammatory response. ^(1-3) A better understanding of the pathophysiology and of the action mechanism of drugs used for these disorders may be beneficial for future therapy.

Some medications, such as macrolides, in addition to the antibacterial action may have an anti-inflammatory effect on pulmonary diseases, in addition to the antibacterial effect. ^{(2, 4, 5)} To investigate this type of medication it is important to develop a model of a neutrophilic pulmonary inflammation without infection. Recent studies with erythromycin, of the macrolides group, used to treat bacterial infections, disclosed that this agent may inhibit neutrophil chemotaxis - interleukin-8 is one of the most important substances involved in this process.^(5,6) Other studies showed that erythromycin delays the metabolism of corticosteroids, which might cause or heighten the anti-inflammatory effect of macrolides.⁽⁷⁾ Furthermore, the use of erythromycin for long periods (at least two months) was clinically effective in treating diffuse pan-bronchiolitis, a disease characterized by diffuse inflammation predominantly in the small bronchi.⁽⁸⁾ In studies of patients with cystic fibrosis, other macrolides, such as azythromycin, also had an anti-inflammatory effect.⁽⁹⁾

Protocols for induction of pulmonary infection with live Pseudomonas aeruginosas (Psa) in animal models are already well established. ^{(10, 11)} The use of bacterial lipopolysaccharide for pulmonary immune response stimulation is also a used experimental model. ^{(4, 12)} However no previous study was found on the efficacy of dead bacteria to induce a neutrophil pulmonary inflammatory response in animal models. In theory, this would, be a protocol technically easy to carry out at low costs. The objective of the present study is to develop a method to induce neutrophilic pulmonary response in Swiss mice without an active bacterial infection.

Material and methods

Animals

Eight adult (six to eight weeks old) male Swiss mice from the PUCRS vivarium were used. They were kept at Instituto de Pesquisas Biomédicas during the study period.

Protocol for pulmonary neutrophil induction with Pseudomonas aeruginosas

The Microbiology Department of the Hospital São Lucas of PUCRS provided the Psa sample in a culture plate. After being scraped from the plate, the material was diluted in a phosphate buffered saline solution (PBS), to a 1 x 10^13vUFC/mL concentration. All Psas were frozen to death (–20°C), to suppress the bacterial effect in this type of experiment. The Psa solution was diluted again to 1 x 10¹² UFC/ml, for the intranasal challenge.

The study group (n = 4) received an intranasal challenge with Psa (Day 0), for induction of neutrophilic inflammatory response in the lower airways. Eighty microlitres were intranasally injected under sedation. ^{(14, 15)} The control group (n = 4) received an intranasal challenge with saline solution (SS), at the same volume. Sedation was done to allow pulmonary aspiration of the Psa solution, due to loss of upper airways reflexes, and consisted of intraperitoneal administration of 0.1mL of a ketamine (0.4mL), xylazine (0.1mL) and SS (0.5mL) combination.

Bronchoalveolar lavage

Two days after intranasal challenge with Psa or SS, a bronchoalveolar lavage (BAL) was performed. All mice were put under anesthesia with the same ketamine and xylazine solution used at the intranasal challenge, at 0.2mL intraperitoneal dose. After anesthesia, a tracheostomy was performed, with trachea canulation and tube fixation, 1mL SS was instilled by means of a syringe. After a five second pause, the material was aspirated. This procedure was repeated three times with the same solution.

Total cell count and differential cytological test

The BAL was weighed and centrifuged (2,000rpm for two minutes). The precipitate was diluted in 1mL of PBS. Total cell count (TCC) and cell viability were performed based upon this solution, on all samples, with tripan blue staining, Neubauer chamber (Boeco, Germany).

For preparation of the slides for differential cytology, a 40ml solution was centrifuged (FANEM, São Paulo, Mod. 218), at 500 rpm, for five minutes. Slides were fixed with methylic alcohol and stained with May Grunwald Giemsa. Cells were analyzed according to their morphology. The cell types at light microscopy were expressed in percentage after count of 200 cells.

Statistical analysis

Values are described as mean and median, and the statistical difference was calculated by the Mann-Whitney test. Differences were considered significant with p < 0.05.

Ethics

The study was approved by the Ethics Committee for Animal Research of the Institution and was based on animal models research guidelines. ⁽¹³⁾

Results

TCC on BAL was significantly higher for the Psa group, as compared with the control group. Median was 1.17 x 10⁶ and 0.08 x 10⁶ for the Psa and control group, respectively (p = 0.029). Furthermore, an absolute neutrophil predominance was observed (mean: 82%) in differential cell count of the Psa mice group. The control group had no significant neutrophil count in the BAL (mean: 0%). The results of TCC and neutrophil percentage in BAL for both groups are shown in Figures 1 and 2, respectively. Viability of the studied samples in the group given Psa and in the control group was 80% and 85%, respectively.

Discussion

This experimental model permitted to produce a method for induction of a pulmonary neutrophil response in mice without active bacterial infection. There was a significant increase in the total cell count, and an absolute predominance of neutrophils in the Swiss mice bronchoalveolar lavage with pulmonary inflammation induced by bacteria.

There are several experimental models for induction of pulmonary inflammation. One of the best known is a model that induces eosinophil inflammation used for research in asthma. The method uses Balb/c mice, which are allergic to ovalbumin (OVA). The protein is applied for

allergic sensitization by means of intraperitoneal injections. The same protein is used for challenge through nebulization or intranasal. One or two days after challenge, BAL should be performed. This model is widely used for research of asthma. It permits to test different drugs and other factors that may influence pulmonary eosiniphil inflammation in asthma. Another widely known model uses inoculation of live bacteria. It is primarily employed for research in cystic fibrosis, and is very useful to assess the antimicrobial effect of many drugs.

The use of live P. aeruginosa to induce a neutrophilic pulmonary disease in mice had already been described. ^{(10, 11)} The fact that some antibiotics present an anti-inflammatory effect and the need to assess their effects and anti-inflammatory potency on some diseases render the model of this study a quite useful tool, to induce a neutrophilic inflammation with no active bacterial infection.

The objective of the present study was to develop a model compatible with research of pulmonary diseases with Europhilic inflammation, but with no active bacterial infection. Other models use virus or lipopolysaccharides (LPS) inoculation, but they are too expensive and complex for use in most Brazilian research laboratories.^(4,12) In this study a similar more simple and more accessible to Brazilian laboratories was reproduced. Since pathological bacteria usually do not withstand extreme temperature, freezing causes death of P. aeruginosa. This is a simple and low cost way to induce pulmonary inflammation in animal models. Additionally, the use of intranasal challenge simplifies induction when compared with intra-tracheal instillation models. ⁽⁴⁾ Models that use viruses require higher investments, considering that breeding and infection through viral methods are usually more complex. ^{(16, 17)}

Therefore, this model of neutrophilic inflammation may become an important tool for research in diseases such as cystic fibrosis, acute viral bronchiolitis, and obliterant bronchiolitis. In addition to macrolides antibiotics, corticosteroids, leucotrien antagonists, diuretics and immunossupressants are some examples of drugs that may be tested and compared by means of this experimental model.

This experimental model may be considered original, in terms of other already described ones because it describes an easy and accessible way of developing pulmonary neutrophil inflammation. It is a short term protocol, that doesn’t involve complex procedures, such as virus culture, tracheal inoculation through intubation or by using nebulization chambers on mice. The material used is of low cost and the pulmonary inflammation is intense.

In conclusion, a simple and low cost model was developed for induction in mice of neutrophilic pulmonary disease with dead bacteria. The method may be used on experimental pharmacology studies in the treatment of diseases such as acute bronchiolitis, cystic fibrosis, or obliterant bronchiolitis.

Received for publication on 07/04/03

Approved, after review, on 30/04/03

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*

This study was performed at Instituto de Pesquisas Biomédicas da Pontifícia Universidade Católica do Rio Grande do Sul-PUCRS, Pediatric Pneumology Team, Pediatrics Department, São Lucas Hospital. Sponsorship: CNPq.

Publication Dates

• Publication in this collection
  02 Dec 2003
• Date of issue
  Aug 2003

History

• Received
  07 Apr 2003
• Accepted
  30 Apr 2003