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

                                                                      STUDY PROTOCOL

EVALUATION OF TEST FORMULATION(S) FOR ANTI-COLITIS ACTIVITY USING DEXTRAN SULFATE SODIUM 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, DSS 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 DSS-induced colitis model, focusing on its detailed protocol as well as factors that could affect DSS-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	DSS 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.

·       3.5% of DSS will be prepared in autoclaved drinking water by dissolving DSS powder until a clear solution is reached.

·       The water bottles will be filled  in the cages with approximately 100 ml for 2 days per cage with 4-5 mice however remaining water will be discarded after 2 days and replenish with freshly prepared DSS containing water.

·       Experimental colitis will be induced by administration of 3.5% of w/v DSS with drinking water for 5 days and later on normal drinking water for next 7 days.

·       Treatment will be given as follows:

·       Group G1 will be served as vehicle control.

·       Group G2 will be treated as DSS control and treated with 3.5% dextran sulfate sodium 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 Test formulation at different dose levels.

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

·       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

·       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 Manish Kumar Jeengar, Dinesh Thummuri, Mattias Magnusson, V. G. M. Naidu & Srinivas Uppugunduri. Uridine Ameliorates Dextran Sulfate Sodium (DSS)-Induced Colitis in Mice. Scientific Report, May 2017; DOI: 10.1038/s41598-017-04041-9; 7: 3924.

6.2 Benoit Chassaing, Jesse D. Aitken, Madhu Malleshappa, and Matam Vijay-Kumar. Dextran Sulfate Sodium (DSS)-Induced Colitis in Mice. Current Protocols in Immunology; February 2014; DOI: 10.1002/0471142735.im1525s104. y 15.25.1-15.25.14.

6.3 Das, S., Batra, S. K. and Rachagani, S. (2017). Mouse Model of Dextran Sodium Sulfate (DSS)-induced Colitis. Bio-protocol 7(16): e2515. DOI: 10.21769/BioProtoc.2515.

6.4 Mi-Rae Shin, Kyeong Jo Kim, Soo Hyun Kim, Su Ji Kim, Bu-Il Seo, Hyo-Jin An, and Seong-Soo Roh. Comparative Evaluation between Sulfasalazine Alone and in Combination with Herbal Medicine on DSS-Induced Ulcerative Colitis Mice. Bio Med Research International. Volume 2017, Article ID 6742652, 10 pages https://doi.org/10.1155/2017/6742652

6.5 Patel PP and Trivedi ND: Karanjin Ameliorate DSS Induced Colitis in C57bl/6 Mice. Int J Pharm Sci Res 2015; 6(11): 4866-74.doi: 10.13040/IJPSR.0975-8232.6 (11).4866-74.

6.6 HAIFENG ZHANG and WEICHANG CHEN. Interleukin 6 inhibition by triptolide prevents inflammation in a mouse model of ulcerative colitis. Experimental and therapeutic medicine ;DOI: 10.3892/etm.2017.4778; 14: 2271-2276, 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.EVALUATION OF AYURVEDIC FORMULATION(S) FOR ANTI-COLITIS ACTIVITY USING 2,4,6- TRINITROBENZENE SULFONIC ACID INDUCED COLITIS IN EXPERIMENTAL ANIMAL

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