EVALUATION OF THE ANTI-INFLAMMATORY EFFICACY OF TEST FORMULATIONS IN MOUSE MODEL OF AIRWAY HYPERRESPONSIVENESS AND AIRWAY INFLAMMATION INDUCED BY DIESEL EXHAUST PARTICLES

 EVALUATION OF THE ANTI-INFLAMMATORY EFFICACY OF TEST FORMULATIONS IN MOUSE MODEL OF AIRWAY HYPERRESPONSIVENESS AND AIRWAY INFLAMMATION INDUCED BY DIESEL EXHAUST PARTICLES

1.0  TEST SYSTEM DETAILS:

Species                   : Mus musculus (Mouse)

Age                        : 6-8 weeks

Sex                        : Male/Female

No. of animals        : 8 /Group

Total animals         : 48

 

2.0   ALLOCATION OF GROUPS:

 

Group No.	Group Description	Disease-Induction agent administered	Treatment administered	Dose Volume and Route
G1	Normal Control	40µL of 0.01% Tween 80 prepared in normal saline on days 1, 4, and 7, administered oro-pharyngeally	0.5% MC, q.d.	10 ml/kg, p.o.
G2	Disease Control	40µL of diesel exhaust particles (20µg) suspended in 0.01% Tween 80 (prepared in normal saline) on days 1, 4 and 7, administered oro-pharyngeally	0.5% MC, b.i.d.
G3	Reference Control		Roflumilast-10 mg/kg, p.o., q.d.
G4	Treated orally with a low dose of TF		TF -X1 mg/kg, in 0.5% MC., q.d.
G5	Treated orally with intermediate dose of TF		TF -X2 mg/kg, in 0.5% MC., q.d.
G6	Treated orally with high dose of TF		TF -X3 mg/kg, in 0.5% MC., q.d.

 

Abbreviations: MC-Methyl Cellulose, p.o.-per os. q.d.: quaque die; bid: bis in die. X1, X2, and X3, are defined as the incremental doses of the Test formulations.

3.0  METHOD:

·         After completion of quarantine, healthy animals will be selected for the study. Subsequently, they will be randomized based on body weight and allocated into 6, different groups consisting of 8 animals each.

·         Post-randomization, animals will be acclimatized for 5 days in an experimental room earmarked for the experiment.

·         For the induction of airway hyperresponsiveness and airway inflammation, animals allocated to groups G2-G6 be transiently anaesthetized with isoflurane and administered 40µL of diesel exhaust particles (20µg) suspended in 0.01% Tween 80 (prepared in normal saline) by oro-pharyngeal route on day 1, 4 and 7. The animals assigned to the normal control group (G1) will be administered 40µL of 0.01% Tween 80 (prepared in normal saline) on day 1, 4 and 7.

·         Treatments to be administered:

Ø Animals of Group G1 and G2, designated as Normal control and disease-control respectively, will be administered 0.5% MC, p.o., q.d.

Ø Animals of group G3 will be treated with reference drug, Roflumilast, orally at the dose of 10 mg/kg, q.d.

Ø Animals of group G4-G6 will be treated with HF-1 orally at different dose levels ranging from 100-1000 mg/kg, q.d.  

·         On day 8, animals will be subjected to lung function measurement, wherein, the bronchial hyperresponsiveness of the animals to methacholine will be measured. For this procedure, the animals will be anaesthetized with thiopentone (50 mg/kg, i.p.) and then spontaneous respiration will be abolished by administration of succinylcholine (10 mg/kg, i.p.). The animals will be connected to a small animal ventilator (flexiVent-FX instrument) and their lung function will be measured. After completion of the procedure, the animals will be euthanized by overdose of thiopentone (150 mg/kg, i.p.). Subsequently, bronchoalveolar lavage fluid (BALF) will be collected and after that lung tissue will be harvested. The left lung will be fixed in 10% neutral buffered formalin for histopathological assessments and the right lung will be stored at -80°C for biochemical and molecular evaluations.

 

4.0  PARAMETERS TO BE EVALUATED:

·         Body weight: Twice a week

·         Lung function measurement: Total respiratory system and Newtonian resistance, dynamic lung compliance, tissue elastance and impedance.

·         Total and differential leukocyte counts in bronchoalveolar lavage fluid.

·         Cytokines and chemokines in the bronchoalveolar lavage fluid: TNF-α, IFN-γ, IL-1β, IL-6, IL-17, KC, IL-8, MIP-1α and MCP-1.

·         Oxidative stress parameters in lungs: Contents malondialdehyde, oxidized and reduced glutathione along with their ratio, total nitrite content, activities of myeloperoxidase, superoxide dismutase and catalase.

·         Quantitative real-time PCR: Expression of pro-inflammatory cytokines and chemokines, Nrf2, HO-1 and NQO-1

·         Histology of the lungs subsequent to hematoxylin and eosin staining.

 

5.0  REFERENCES:

1.        Nemmar, A., Al-Salam, S., Yuvaraju, P., Beegam, S. & Ali, B. H. Emodin mitigates diesel exhaust particles-induced increase in airway resistance, inflammation and oxidative stress in mice. Respir. Physiol. Neurobiol. 215, 51–57 (2015).

2.        Lee, Y. S. et al. Opuntia ficus-indica Alleviates Particulate Matter 10 Plus Diesel Exhaust Particles (PM10D)—Induced Airway Inflammation by Suppressing the Expression of Inflammatory Cytokines and Chemokines. Plants 11, (2022).

3.        Meldrum, K. et al. Diesel exhaust particle and dust mite-induced airway inflammation is modified by cerium dioxide nanoparticles. Environ. Toxicol. Pharmacol. 73, (2020).

END OF DOCUMENT

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