EVALUATION
OF THE ANTI-ASTHMATIC POTENTIAL OF TEST FORMULATIONS IN MOUSE MODEL OF HOUSE
DUST MITE (HDM)-INDUCED AIRWAY HYPERRESPONSIVENESS AND AIRWAY INFLAMMATION
1.0 TEST SYSTEM DETAILS:
Species :Mus musculus(Mouse)
Strain :BALB/c
Age :8-10 weeks
Body Weight :22- 26g
Sex : Male/ Female
No. of animals : 8 animals /Group
Total animals : 56
Study duration : 60 days
2.0 ALLOCATION OF GROUPS:
|
Groups |
Treatment |
Dose; ROA |
No. of Animals |
|
G1 |
Normal
Control |
0.5%
MC, p.o. |
8 |
|
G2 |
Disease
Control |
0.5%
MC, p.o. |
8 |
|
G3 |
Reference
Control – Dexamethasone |
1
mpk, p.o. |
8 |
|
G4 |
Test Formulation 1 |
X1
mpk; p.o. |
8 |
|
G5 |
Test Formulation 1 |
X2
mpk; p.o. |
8 |
|
G6 |
Test Formulation 1 |
X3
mpk; p.o. |
8 |
|
G7 |
Test Formulation 1 |
X4
mpk; p.o. |
8 |
No extra animals
will be taken; X1, X2, X3, and X4 are defined as the incremental doses of the Test
formulations 1 respectively. MC-Methyl Cellulose; ROA-route of administration;
mpk-mg/kg, p.o.-per os
3.0 METHOD:
· Mice weighing 22 - 26 g will be procured and after quarantine they will be weighed. Subsequently, they will be randomly allocated into 7 different groups consisting of 8 animals each based on their body weights.
Development
of allergic airway hyperresponsiveness and airway inflammation:
Animals of the Group G2-G7 will be administered HDM (25μg protein dissolved in20μL saline) for 5 consecutive days per week for 7 weeks by intranasal administration; Group 1 will be administered 20 µL saline by intranasal route.
Animals of Group G1 will serve as Normal control and will receive 0.5% MC, p.o.
Animals of Group G2 will serve as disease control group will receive 0.5% MC, p.o.
Animals of Group G3 will serve as reference control and will be treated with dexamethasone – 1 mg/kg, p.o.
Animals of Group G4 to G7 will be treated with test compounds at respective dose levels as mentioned in the table (Refer section 2.0).
Measurement of Airway Hyperresponsiveness:
On day 36, animals will be anesthetized with ketamine and xylazine.
After animals are anaesthetized, tracheostomy will be performed and a cannula will be inserted in the trachea. Subsequently, the tracheal cannula will be connected to flexiVent instrument equipped with a small animal ventilator (Emka-Scireq) for the measurement of in vivo respiratory mechanics.
Succinylcholine will be administered to animals, intraperitoneally prior to pulmonary function testing to prevent respiratory drive artifacts.
Baseline measurements of respiratory mechanics will be assessed prior to challenge with increasing concentration of aerosolized methacholine/serotonin.
Finally, the total respiratory resistance (Rrs) and central airways Newtonian resistance (RN) will be determined.
Collection of blood, bronchoalveolar lavage fluid (BALF) and harvesting the lungs:
After measurement of airway hyperresponsiveness, blood will be collected from the retro-orbital sinus for biochemical evaluation and further the animals will be euthanized with overdose of thiopentone. Subsequently, BALF will be collected for enumeration of total and differential leukocyte counts. Finally, the lungs will be harvested. The left lung will be processed for histopathological evaluation whereas the right lung will be stored at -80°C for evaluation of biochemical and molecular parameters.
4.0 END POINT
PARAMETERS:
·
Measurement of Airway Hyperresponsiveness
·
Enumeration of total and differential
leukocyte counts in BALF
·
Quantification of Cytokines in BALF and Lung
·
qPCR in lungs
·
Quantification of HDM-Specific IgE in Serum
·
Quantification of total collagen content in
Lung
·
Flow Cytometric Analysis of Lung Cell Subsets
·
Histology and Immunohistochemistryof
lungs
5.0 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.
END THE DOCUMENT
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