According to WHO, at present, there are above 1.3 billion tobacco smokers globally. Cigarette smoke comprises more than 4000 harmful chemical composites, out of which approximately 200 constituents are highly toxic. It is commonly recognised that bidi or cigarette smoking is a highly vital risk factor for the advancement and evolution of chronic obstructive pulmonary disease (COPD) and the reason for roughly 80% of COPD cases. Bidi or cigarette smoking is connected with several respiratory ailments comprising emphysema, bronchitis and airway obstruction. Communally these respiratory disorders are represented as a chronic obstructive pulmonary disease (COPD). COPD, a word signifying two pulmonary diseases: chronic bronchitis and emphysema, is described by an airflow restriction that is not rescindable. Alterations in the lungs due to smoking embrace inflammation, epithelial impairment, and modernizing of the airways. Airway inflammation probably plays a serious responsibility in the beginning and development of cigarette smoke-induced airway disorder.
Cigarette
smoke extract (CSE) preparation
CSE is prepared by bubbling the cigarette smoke in a Phosphate-buffered solution (PBS) (pH 7.4). Considerable amounts of carbon monoxide, hydrogen cyanide, and hydrocarbons like ethylene and methane are found in conventional bidi smoke. Additionally, cotinine, 3-methylpyridine, α & β-amyrin have also existed at high levels in cigarette smoke. A cigarette smoke comprises 3 to 5 times the quantity of nicotine as an ordinary cigarette and causes users at risk for nicotine dependence. Bidi smoking enhances the risk for different types of cancers such as lung cancer, oral cancer, stomach cancer, and esophageal cancer.
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Animal model for CSE induced lung inflammation
CSE is administered in C57BL/6 mice by
intranasal route to induce respiratory inflammation. The smoke extract is prepared
daily by using a vacuum pump and instilled in mice that are anaesthetized by using
isoflurane. For best results, intranasally instil the CSE continuously
for 10 days. On day 11, perform airway hyperresponsiveness (AHR) under
anaesthesia by using a flexivent ventilator system. Before AHR, inject
succinylcholine intraperitoneally to paralyse the respiratory system and AHR is
measured at various concentrations of methacholine.
Cigarette smoke extract (CSE) preparation and administration
Cigarette smoke extract (CSE) is prepared by bubbling the cigarette smoke from 25 nos., research cigarette (Kentucky University) through 10 mL of sterile, pyrogen-free PBS (pH = 7.4). Smoke is drawn through the medium using a standard vacuum pump. One Bidi is burned for 6 minutes. CSE is made fresh before each treatment and administered within 30 minutes. Mice are anaesthetized by exposure to isoflurane in a jar until effect. Mice are then held in a vertical position and 50 uL of CSE will be placed on the edge of the nostril and inhaled by the mouse. Mice are exposed to CSE for 7 days. All procedure is conducted under a fume hood so that the experimenter and laboratory environment are not exposed to cigarette smoke.
Poly
(I: C) preparation and administration
Poly I: C is purchased from Sigma Aldrich (25mg Vial) and must be stored at -20 °C until its use. Required quantity of Poly I: C is weighed and mixed in the predetermined volume of pyrogen-free PBS to obtain a 1mg/mL stock concentration. After the preparation stock solution, a different aliquot is prepared in 200 uL PCR tubes. Aliquots are centrifuged for about 1 minute at 3000 RPM to settle down the mixture drop present at the cap & wall of the tube. The mixture is annealed for 10 minutes by using a thermocycler. The block temperature of the thermocycler should be 65-70 °C and the lid temperature should be 98°C. After 10 min annealing, aliquots should be placed for 1 hour at room temperature to ensure proper annealing. The instillation procedure of Poly (I: C) is the same as CSE instillation.
A stock concentration of 50mg/mL of methacholine is prepared in normal saline and 6 different dilutions (25, 12.5, 6.25, 3.12 and 1.56 mg/mL) of methacholine are prepared in normal saline. The stock concentration of Succinylcholine (0.75mg/mL) is prepared in normal saline and injected to animals at the dose level of 7.5mg/kg. MCH & SCH stocks and dilutions must be stored in the icebox to prevent degradation. Methacholine is inhaled in animals by using a computer-controlled small animal ventilator (Flexivent) and airway hyperresponsiveness (AHR) is recorded. Succinylcholine is injected into animals to cause respiratory paralysis.
Bronchoalveolar lavage fluid (BALF) collection and
harvesting of the lungs
After measurement of
pulmonary function testing, Mice are lavaged through an 18-gauge tracheal
cannula with 1 ml of cold Hank balanced salt solution (HBSS) and lungs are
harvested, the right lung is stored at -80°C for further biochemical &
molecular analysis, and the left lung is fixed in 10% paraformaldehyde
solution. The lavage fluid is centrifuged at 2800 x g for 10 minutes at 4°C to
obtain cell-free supernatants that are snap-frozen for further cytokines
analysis. Cell pellets are resuspended in cold HBSS solution for differential
leukocyte counts and mounted on slides by using Cytocentrifuge for Giemsa
staining.
Important endpoint parameters of the study
BALF Contents:
Total BAL cells, % macrophages, Total macrophages, PMN, total PMNs, BAL
neutrophils, BAL cytokines (TNF-α, IFNα-2a, IL-1β, IL-6, IL-8, CXCL10/IP-10,
CXCL1/KC, CCL3/MIP-1α, CCL5, TGF-β1).
Lung Tissue-
SOD, catalase, GSH, GPx, MDA, hydroxyproline, HDAC-2.
Gene expression/qRT/PCR - TNF-α, IL-1β, IFN-γ, IL-6, MMP-9 & MMP-12,
TIMP-1, NRF-2, NQO-1, HO-1, MUC5AC, MUC5B & TGF-β1.
Histopathological staining- H&E staining, Masson Trichome (MT) staining &
PAS Staining.
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