Asthma and Study protocol of House dust mite (HDM) induced chronic allergic asthma in mice

 Introduction

Asthma is a varied disease illustrated by long-lasting airway inflammation, mucus overproduction, bronchial hyperreactivity, and airway restoration. Allergic asthma is a persistent inflammatory ailment of the leading airways illustrated by the existence of allergen-specific IgE, Th2 cytokine generation, eosinophilic airway inflammation, respiratory hyperactivity, mucus overproduction, and physical changes in the airways. Researchers have attempted to copy these characters of human allergic asthma in murine models. While the substitute allergen ovalbumin has been tremendously valued for unravelling causing mechanisms of the disease, murine asthma models depend currently on biologically appearing allergens, for example, HDM (House Dust Mite).

The House dust mite (HDM) Dermatophagoides pteronyssinus is a multifaceted arthropod that lives in human housings and nourishes on flecks of moult human skin. To digest skin, the HDM generates potent proteases in its gastrointestinal tract (GIT) that are evacuated in faecal pellets that humans breathe in. HDM faecal pellets comprise numerous allergens that belong to the chief triggers of allergic asthma globally.

 Experimental animals

Species	:	Mus musculus
Strain	:	Mouse
Sex	:	Male/Female
Age	:	7-8
Bodyweight range	:	20-25 g
No. of animals/group	:	08

Allocation of animals

Groups	Treatment	Dose & Regimen
G1	Normal Control	0.5% MC
G2	Disease Control	0.5% MC
G3	Reference Control- Dexamethasone	1 mg/kg
G4	Test compound dose-1	X1 mg/kg
G5	Test compound dose-1	X2 mg/kg
G6	Test compound dose-1	X3 mg/kg

The route of administration of the test compound will be decided at the time of experiment initiation.

Experimental procedure

Mice will be randomly assigned into 6 different groups consisting of 8 animals each based on their body weights. Further, the animals will be acclimatized to the experiment room for four days.

After acclimatization, mice of the Group G2-G6 will be administered house dust mite (HDM) (25μg protein dissolved in 20μL saline) for 5 repeated days per week for 5 weeks by intranasal administration; Group 1 will be administered 20 µL normal saline by the intranasal route. HDM is instilled to develop allergic airway hyperresponsiveness (AHR), airway inflammation and airway remodelling in mice.

Subsequently, Animals will be treated with a test compound with HDM instillation for 5 weeks.

Measurement of Airway Hyperresponsiveness (AHR)

On day 36, animals will be anaesthetized with 50 mg/kg thiopentone administered intraperitoneally.

After animals are anaesthetized, a cannula will be inserted in the trachea to perform a tracheostomy. Consequently, the tracheal cannula will be connected to a flexiVent instrument equipped with a small animal ventilator (Emka-Scireq) for the measurement of in-vivo respiratory mechanics and airway hyperresponsiveness (AHR). Before pulmonary function testing, Succinylcholine (7.5 mg/kg) will be administered intraperitoneally to prevent respiratory drive artifacts. Baseline measurements of respiratory mechanics will be evaluated before the challenge with increasing concentration of aerosolized methacholine/serotonin.

Finally, the total respiratory resistance (Rrs) and central airways Newtonian resistance (RN) will be determined.

Bronchoalveolar lavage fluid (BALF) and harvesting of the lungs

After measurement of airway hyperresponsiveness, BALF will be collected for enumeration of total and differential leukocyte counts. Further, a thread will be tied around the right bronchus and the left lung will be inflated with 10% neutral buffered saline followed by its excision and subsequent processing for histopathological evaluation. The right lung will be stored at -80°C for evaluation of biochemical and molecular parameters.

Endpoint parameters

•   Measurement of Airway Hyperresponsiveness

•   Enumeration of total and differential leukocyte counts in BALF

•   Quantification of Cytokines in BALF and Lung- – IL-4, IL-5, IL-13, IL- 17, IL-33, IL-10, KC, TNF-α, IP-10.

•   Oxidative stress parameters – SOD, CAT, GPX, MDA, GSH, GSSG, GSH/GSSG ratio.

•   EPO and nitrite levels in lung

•   qPCR in lungs– Nrf2, HO-1, NQO-1, MUC5AC, MUC5B.

•   Quantification of total collagen content in Lung

•   Histopathology of lungs- periodic acid- Schiff (PAS) stain for the evaluation of goblet cell metaplasia, Haematoxylin & Eosin (H&E) for evaluation of lung inflammation, Masson-Trichrome stain evaluation of subepithelial fibrosis.

References

1.  Johnson, Jill R., et al. "Continuous exposure to house dust mite elicits chronic airway inflammation and structural remodeling." American journal of respiratory and critical care medicine 169.3 (2004): 378-385.

2.     Yang, Zhimei, et al. "Roles of Bronchopulmonary C-fibers in airway Hyperresponsiveness and airway remodeling induced by house dust mite." Respiratory research 18.1 (2017): 199.

3.   Yao, Lu, et al. "Huangqi–Fangfeng protects against allergic airway remodeling through inhibiting epithelial–mesenchymal transition process in mice via regulating epithelial derived TGF-β1." Phytomedicine 64 (2019): 153076.

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