EVALUATION OF TEST FORMULATION FOR ACUTE LUNGS INJURY (ALI) USING LIPOPOLYSACCHARIDE IN EXPERIMENTAL MICE

                                                                      STUDY PROTOCOL

EVALUATION OF TEST FORMULATION FOR ACUTE LUNGS INJURY (ALI) USING LIPOPOLYSACCHARIDE IN EXPERIMENTAL MICE

 

1.0 INTRODUCTION:

Acute Lung Injury (ALI) is an inflammatory disease of lungs, characterized by the disruption of alveolar endothelial and epithelial barriers, neutrophilic infiltration at pulmonary sites together with non-cardiogenic edema. It is most often seen as part of a systemic inflammatory process. The inflammatory process is vital in the development of ALI. The injury to the alveolar epithelium and endothelium, lung edema, and infiltration of neutrophils is the main pathological changes. Gram-negative bacterial infections are the main cause of ALI, and lipopolysaccharide (LPS), which is the main component of the cell wall of Gram-negative bacteria, is the major stimulus for the release of inflammatory mediators. LPS can also activate the host receptor TLR4 and trigger an inflammatory response, resulting in ALI.

 

2.0 TEST SYSTEM DETAILS:

Species             : Mus musculus (Mouse)

Strain                : C57BL/6; BALB/c; DBA/1; Swiss albino

Age                   : 6-8 Weeks

Body Wight        : 25-30 g

Sex                   : Male or Female

No. of animals    : 8 /Group

Total animal       : 56 + 5 Extra = 61

3.0  ALLOCATION OF GROUPS:

Groups	Treatment	Dose; ROA	No. of Animals
G1	Normal Control	0.9% Normal saline /Na-CMC	8
G2	Disease Control	0.9% Normal saline/Na-CMC	8
G3	Reference Drug- Dexamethasone	5 mpk; i.p daily	8
G4	Test Formulation-1	X mpk; p.o.	8
G5	Test Formulation-1	XX mpk; p.o.	8
G6	Test Formulation-1	X mpk; p.o.	8
G7	Test Formulation-1	XX mpk; p.o.	8

# 5 extra animals will be taken extra due to variability in the disease development and possibilities of animal mortalities

 

4.0 METHOD:

· Animals will be acclimatized for 7 days prior to main experiment and maintained at all standard condition as per CPCSEA guidelines.

· Animals will be randomly divided into five groups containing eight animals in each.

· Acute lung injury (ALI) will be induced by intranasal administration of 20 μg of LPS in 50 μl saline using micropipette.

· Animals of Group G1 served as normal control and received 0.25% Na-CMC (50μl saline intranasal without LPS) at 10ml/kg p.o. dose volume.

· Group G2 served as disease control (50μl LPS intranasal) received 0.25% Na-CMC at 10ml/kg p.o. dose volume.

· Group G3 treated with dexamethasone (DEXA + LPS) at the dose of 1mg/kg i.p.

· Group G4 to G7 animals will be treated with test compound (Test formulation + LPS) at different dose levels.

· After 24 h of intranasal LPS instillation, serum will be separated from blood and stored at −20 °C.

· Bronchoalveolar fluid (BALF) will be collected by trachea cannulation and flushing the lungs with 1 ml of sterile phosphate-buffered saline (PBS) three times.

· BALF will be centrifuged at 3000 rpm at 4 °C for 15 min, and supernatant will be collected and stored (−80 °C) for further analysis.

· Cell pellet will be washed three times with PBS and used for total cell count and differential cell count.

· The lungs will be removed and stored at −80 °C for biochemical analysis.

· Right lung lobes will be fixed for 24 hr in 10 % neutral buffered formalin (NBF) for histopathology

5.0 END POINT PARAMETER(S):

·       Clinical Observation

·       Body weight

·       Lung wet/dry ratio

·       Inflammatory cell count in BALF

·       Pulmonary myeloperoxidase activity analysis

·       Inflammatory cytokine assays

·       Histopathology of  lungs

 

6.0  REFERENCE(S):

6.1 Asha Kumari, Namitosh Tyagi, D Dash, and Rashmi Singh. Intranasal Curcumin Ameliorates Lipopolysaccharide-Induced Acute Lung Injury in Mice. Inflammation. DOI: 10.1007/s10753-014-0076-y.

6.2 Roderick J. Szarka , Nandi Wang  , Lyle Gordon , P.N. Nation , Richard H. Smith. A murine model of pulmonary damage induced by lipopolysaccharide via intranasal instillation. Journal of Immunological Methods 202 1997 49–57.

6.3 Xiaoyu Hu, Yuan Tian, Shihui Qu, Yongguo Cao, Shumin Li, Wenlong Zhang, Zecai Zhang, Naisheng.   Protective effect of TM6 on LPS-induced acute lung injury in mice. Scientific Report 572 (2017).

                       END OF DOCUMENT

You may like to read theses links:   

1. List of All SOPs and Documents for In-vivo Laboratory

2. List of All SOPs and Documents for Study Protocol

3. List of Pharmacological Study Models with details about disease Inducing agent, Standard or Reference Drug and related parameters.

4.List of OECD Guidelines or Toxicological Studies

5.List of Pharmacopeial Test or Quality Control Test or Batch Testing Parameters (In-vivo)

6.List of 32 Respiratory Pre-Clinical Study Models and Related Details

SHARE

Owner

Hi. I’m Writer of Researchsop.com. ’ ’ Please share these SOPs to all concern pharma people for their development. I like to fullfill the need of curious people. These things inspire me to make things looks better.

• 
• 
• 
• 
•