Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

This protocol exhibits highly comparable pathophysiology of Crohn's fibrosis in human and also discusses the rapamycin-mediated inhibitory effects on intestinal fibrosis. The main advantage of this protocol is that it describes intestinal fibrosis development in a very short amount of time varying from four to eight weeks, which give us the opportunity to study tissue repair and tissue regeneration. This protocol will be helpful to researchers who are in a rush to getting the mechanism of fibrosis, and help finding a better tech to predict intervention for Crohn's associated intestinal fibrosis.

To begin this procedure, shave adult mice around the neck area in order to pre-sensitize them to TNBS via dermal exposure. Then soak a coton swab with TNBS and apply it to the shaved area of the mouse's neck. Eight days post-sensitization, induce colitis by intrarectal administration once a week, for six weeks.

To do this, apply four milligrams of TNBS in 25%ethanol using a 100 microliter enema via a one milliliter syringe attached to a steel gavage needle. Give control mice 100 microliters of 25%ethanol only. After anesthetizing the mice, intraperitoneally inject either rapamycin at two milligrams per kilogram per day, or a vehicle, or both, every weekday for three to six weeks to both the control and TNBS-treated mice.

After this, use six-week TNBS post-treated mice intestines for analyzing the extracellular assays. First, open the colon longitudinally in ice cold HBSS and wash the colon in HBSS. Using sterile scissors, cut the colon into small pieces that are approximately five centimeters in HBSS.

Transfer the small colon tissue pieces to a 15 milliliter conical tube containing 10 milliliters of pre-digestion buffer. Shake the tube at 100 RPM in an incubator at 37 degrees Celsius for 20 minutes. Next, pass the suspension through a 40 micrometer cell strainer, and discard the attached colonic epithelium.

Collect the remaining tissue from the strainer and digest them further in digestion buffer containing collagenase Type IV and DNASE1 in 1X HBSS with 5%FBS for 20 minutes in an incubator at 37 degrees Celsius while shaking at 100 RPM. After this, vortex the digested tissues for approximately 20 seconds and pass it through a 40 micrometer cell strainer to obtain lamina propria fractions. Centrifuge the lamina propria fractions at 700 times G and four degrees Celsius to pellet down the cells.

Then, make 100 milliliters of both 30%and 70%density gradient media solutions. Resuspend the obtained cells in 10 milliliters of the 30%solution and overlay this on top of five milliliters of the 70%solution in a 15 milliliter tube. Centrifuge the gradient in a break free condition at 1, 000 times G and room temperature.

Collect the white ring phase containing the lamina propria lymphocytes, which will be between the 30%and 70%gradient layers. Wash the obtained cells by resuspending them in ice-cold HBSS and centrifuging at 500 times G and 10 degrees Celsius for 10 minutes. Then resuspend the cells in FACS buffer.

Prior to antibody staining, first block the cell surface of the lamina propria cells by incubating them with anti-mouse CD16 by 32FC blocker on ice for 15 minutes. Next incubate the cells with anti-CX3CR1 PE antibodies along with anti-PE microbeads on ice for 30 minutes to capture the bound cells, then wash the cells with FACS buffer. Pass the antibody and bead-bound cells through a magnetic activated cell sorting column in a magnetic field to remove the unbound cells.

Wash the cells three times with FACS buffer. After this, remove the column from the magnetic field and push the plunger in the column to yield the bead-bound cells. First, block the lamina propria cells with anti-mouse CD16 by 32FC blocker on ice.

Strain the cells by incubating with anti-CD64, CD11c, CD11b, CX3CR1, Ly6C and MHC Class II antibodies Then sort the cells using a FACS flow cytometer as outlined in the text protocol. Lyse the sorted cells for total RNA preparation and detect cytokine and fibrotic markers. Analyze mRNA expression of fibrotic markers and inflammatory cytokine analysis from isolated single cell suspensions from magnetic purification.

To begin cell surface staining and analysis, resuspend between 500, 000 and 1 million isolated lamina propria cells in 15 milliliters of FACS buffer. Incubate the cells with anti-CD16 by 32 antibodies at a dilution of 1 to 50 on ice for 10 minutes. After this, wash the cells with 500 microliters of ice cold FACS buffer to remove unbound antibodies.

Surface stain colonic single cell suspensions with fluorescent-labeled antibodies at a dilution of 1 to 100 on ice for 30 minutes. Wash the labeled cells twice to remove the unbound antibodies using 500 microliters of ice cold FACS buffer for each wash. Then analyze the labeled mononuclear cells by flow cytometer as outlined in the text protocol.

To begin intracellular cytokine staining and analysis, use a fixation permeabilization solution kit to fix and permeabilize the surface-stained cells according to the manufacturer's instructions. Gate the lamina propria cells as outlined in the text protocol to detect the intracellular level of IL-1 beta cytokine. Next, wash the cells by adding FACS buffer and centrifuging at 200 times G and four degrees Celsius for five minutes to remove excess fixative buffer.

Repeat this wash once. To determine the alpha smooth muscle actin level, first permeabilize cells with the fixation permeabilization solution kit. Incubate the cells with anti-alpha SMA Alexa Fluor 488 antibodies at a dilution of 1 to 1000 on ice for 30 minutes.

Then wash the cells by adding FACS buffer and centrifuging at 200 times G and four degrees Celsius for five minutes to remove excess fixative buffer. Repeat this wash once. Perform FACS analysis and gate the cells as outlined in the text protocol.

In this study the TNBS colitis mouse model is adopted to study and elucidate the underlying mechanisms of intestinal fibrosis. After six weeks of TNBS treatment, it can be seen that the colonic length shortened progressively over the course of the TNBS treatment from approximately five centimeters in the control group to approximately three centimeters in the TNBS group. To ensure the TNBS Crohn's disease model is comparable to the human Crohn's fibrosis model and is not an artifact related to the methodology, the fibrotic markers are analyzed at multiple levels in a detailed timecourse study.

Accumulation of alpha smooth muscle actin positive cells and collagen deposition within submucosal layers have been reported in most of the fibrosis incidences and is regarded as a hallmark for fibrotic events To compare TNBS fibrosis with Crohn's associated fibrosis, the expression of fibrosis markers and cytokines is analyzed in fresh tissue biopsy from the ileum of patients with active CD or under remission. Remarkably, marked induction is seen of the thickening of alpha smooth muscle actin positive layers and increased collagen deposition in active CD sections. Western blot analysis is performed to confirm the induction of alpha smooth muscle actin expression in active CD samples.

In addition, significant induction of fibrosis markers is detected by qPCR analysis. The effects of rapamycin, a pharmacological inhibitor of M4 activity are then evaluated to determine a mechanism to limit TNBS fibrosis. Mice are treated with both TNBS and rapamycin and the levels of alpha smooth muscle actin and collagen in the colon histology are analyzed.

It is important to remember to inoculate the same amount of TNBS to each mouse and to process isolated lamina propria cells as soon as possible to avoid cell loss as well as to maintain cell viability. Our protocol details on TNBS fibrosis and remission of fibrosis as well as the inhibitory factor of rapamycin in fibrosis formation. Other researcher may be interested in using this technique to find a better drug target for fibrosis and colon cancer.

Please remember that TNBS is a skin irritant and lab coats should be worn during handling to avoid skin contact.