STUDY PROTOCOL

EVALUATION OF TEST FORMULATION(S) FOR ANTI-COLITIS ACTIVITY USING 2,4,6- TRINITROBENZENE SULFONIC ACID INDUCED COLITIS IN EXPERIMENTAL ANIMAL

1.0 INTRODUCTION

Inflammatory bowel diseases (IBD) mainly comprised of Ulcerative Colitis and Crohn's Disease are complex and multifactorial disease with unknown etiology. For the past 20 years, to study human IBD mechanistically, number of murine models of colitis has been developed. These models are indispensable tools to decipher underlying mechanisms of IBD pathogenesis as well as to evaluate number potential therapeutics. Among various chemical induced colitis models, TNBS induced colitis model is widely used because of its simplicity and many similarities with human ulcerative colitis. This model has both advantages and disadvantages that need to be considered when employed. The current protocol aimed to extensively describe the TNBS-induced colitis model, focusing on its detailed protocol as well as factors that could affect TNBS-induced pathology.

2.0 TEST SYSTEM DETAILS:

Species : Mus musculus (Mouse)

Strain : Swiss albino or C57bl/6 or Balb/c

Age : 6-8 weeks

Body Wight : 20-25 g

Sex : Male or Female

No. of animals : 8 /Group

Total animals : 56 + 8 extra = 64

3.0 ALLOCATION OF GROUPS:

Groups	Treatment	Dose*	Animals
G1	Normal Control	0.25% Na-CMC	8
G2	TNBS Control	Normal saline or 0.25% Na-CMC	8
G3	Reference Drug- Dexamethasone	0.1mg/kg. i.p	8
G4	Test Formulation-1	X mpk	8
G5	Test Formulation-1	XX mpk	8
G6	Test Formulation-2	X mpk	8
G7	Test Formulation-2	XX mpk	8

*Route of drug administration will be decided at the time of experiment

4.0 METHOD:

· Healthy animals will be selected, randomized based on body weight and divided into 7 different groups consisting of 8+8 animals each.

· Induction of colitis by TNBS (Trinitrobenzenesulfonic acid induced)

· On day 0 (Induction day) the 24h fasted animals will be administered intracolonic with a single dose of TNBS at 25mg/animal in 50% v/v ethanol solution (0.5 mL/rat).

· The TNBS will be administration into the colon by an 8.5 cm long catheter with a 2.5 mm round Teflon tip under light anesthesia. The animals will be monitored several times daily until recovery.

· The Trendelenburg position will be used to avoid colonic reflux

· Treatment will be given as follows:

· Group G1 will be served as vehicle control.

· Group G2 will be treated as TNBS control and treated with 25mg/animal Trinitrobenzenesulfonic acid only.

· Animals of group G3 will be treated with standard drug dexamethasone at the dose of 0.1mg/kg i.p.

· Group G4 to G7 animals will be treated with ayurvedic formulation at different dose levels.

· Mice will be daily monitored for body weight, feed-water intake.

· All treatment will be started at day 0 and continued till the end of experiment day 5.

· However, the mice will be monitored daily for the stool consistency (0 = well-formed stool pellet, 2 = semi-formed stool, 4 = liquid stool that adhere to anal region) and hematochezia (0 = no blood, 2 = blood trace in the stool clearly visible, 4 = gross rectal bleeding).

5.0 END POINT PARAMETER(S):

· Colonic myeloperoxidase (MPO) assay

· Disease Activity Index (DAI)

· Colon length

· Biochemical markers - alkaline phosphatase (ALP), alanine aminotransferase (ALT), urea, and creatinine.

· Histopathology of colon

· Serum cytokine levels

· Cytokine/chemokine mRNA expression in the colon (TNF-α, IL-6 and IL-1β)

· Estimation of protein expression in the colon (TNF, IL-6, and COX-2)

6.0 REFERENCE(S):

6.1 Vanessa Mateus, João Rocha, Hélder Mota-Filipe, Bruno Sepodes, Rui Pinto. Hemin reduces inflammation associated with TNBS-induced colitis. Clinical and Experimental Gastroenterology 2018:11

6.2 Tianjing Liu, Yongyan Shi, Jie Du, XinGe, XuTeng, Lu Liu, EnboWang & Qun Zhao. Vitamin D treatment attenuates 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis but not oxazolone-induced colitis. Scientific Reports | 6:32889 | DOI: 10.1038/srep32889.

6.3 Magdy El-Salhy, Kazuo Umezawa, Odd Helge Gilja, Jan G. Hatlebakk, Doris Gundersen, and Trygve Hausken. Amelioration of Severe TNBS Induced Colitis by Novel AP-1 and NF-𝜅B Inhibitors in Rats. e Scientific World Journal Volume 2014, Article ID 813804, 8 pages http://dx.doi.org/10.1155/2014/813804

6.4 Soo Youn Oh, Kyung-Ah Cho, Jihee Lee Kang, Kwang Ho Kim, So-Youn Woo. Comparison of experimental mouse models of inflammatory bowel disease. November 27, 2013 https://doi.org/10.3892/ijmm.2013.1569 Pages: 333-340

                                         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.EVALUATION OF TEST FORMULATION(S) FOR ANTI-COLITIS ACTIVITY USING DEXTRAN SULFATE SODIUM INDUCED COLITIS IN EXPERIMENTAL ANIMAL

7.Important points for TNBS Induced Colitis Model in Mouse 

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