EVALUATION OF ANTI-ASTHMATIC POTENTIAL OF TEST FORMULATIONS IN MOUSE MODEL OF HOUSE DUST MITE (HDM) AND COMPLETE FREUND’S ADJUVANT (CFA)-INDUCED MIXED GRANULOCYTIC SEVERE AIRWAY INFLAMMATION.
1.0 TEST SYSTEM DETAILS:
Species :Mus Musculus (Mouse)
Strain :C57BL/6
Age :8-10 weeks
Body Weight :22- 26g
Sex : Male/ Female
No. of animals : 8 animals /Group
Total animals : 56
Study duration : 40 days
2.0 ALLOCATION OF GROUPS:
|
Groups |
Treatment |
Dose; ROA |
No. of Animals |
|
G1 |
Normal
Control |
0.5%
MC |
8 |
|
G2 |
Disease
Control |
0.5%
MC |
8 |
|
G3 |
Reference
Control – Dexamethasone |
2.5
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
formulation. 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 Mixed-Granulocytic
model of Severe Asthma:
·
Animals will be anesthetized with ketamine
and xylazine and injected under the skin of the back with 100 μL of CFA/HDM
emulsion(50 μL of Complete Freund’s Adjuvant supplemented
with 4 mg/ml of Mycobacterium tuberculosis extract and 25 μg of HDM protein
dissolved in 50 μL saline). Group 1 will be administered
saline subcutaneously.
·
Antigen challenges will be performed by intranasal inhalation of 10 μg
HDM in 20 μL of saline on days 19, 20, 21, and 22. Mice will be evaluated on
day 23.
·
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 treated with 2.5 mg/kg dexamethasone
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).
Assessment
of Pulmonary Responsiveness to Methacholine:
·
Responsiveness to inhaled methacholine will be assessed in
closed-chested mice.
· Animals
will be anesthetized
with ketamine and xylazine.
· After
the animals are anaesthetized,
tracheostomy will be performed followed by insertion of a cannula in the
trachea. The cannula will be connected to the flexiVent instrument, equipped
with a small animal ventilator for measurement of in vivo respiratory
mechanics.
·
Next, animals will be paralyzed with an intraperitoneal injection of succinylcholine.
·
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 and Measurement
of Specimens:
·
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
·
Cytokine and SAA Protein Analysis in BALF
·
Lung CD4+ and CD8+ T-Cell Analysis in Lung
·
Quantitative RT-PCRin
lung
·
Lung Histology and Inflammation Scoring
5.0 REFERENCES:
1.
Menson, Katherine E., et al.
"Therapeutic efficacy of IL-17A neutralization with corticosteroid
treatment in a model of antigen-driven mixed-granulocytic asthma." American
Journal of Physiology-Lung Cellular and Molecular Physiology 319.4
(2020): L693-L709.
2.
De Alba, Jorge, et al. "Double-stranded
RNA evokes exacerbation in a mouse model of corticosteroid refractory
asthma." Clinical Science 129.11
(2015): 973-987.
END THE DOCUMENT
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