Mini-endoscopic evaluation of the colon in live mice provides key experimental support for investigating intestinal manifestations of acute graph-versus-host disease, GvHD, upon allogenic, hematopoietic stem cell transplantation. The non-invasive assessment of acute GvHD-related colitis can function as a working substitute for gold standard histopathology, thereby rendering repeated animal sacrifices to obtain tissue specimens unnecessary. Demonstrating the procedure will be Vera Buchele, a post-doc from my laboratory.
One day after total-body irradiation of the recipient BALB/c mice, place a CD-45.1 live 5B6 SJL donor mouse in the prone position on a clean working sheath, and use one 13-cm curved and serrated tip of a simkin forceps to lift the fur at one Achilles heel. Using hardened 8.5-cm fine scissors with straight tips, incise the skin between the forceps and one heel, elongating the incision cranially to facilitate removal of the skin and fur from the hind limbs. When the skin has been removed from the hind limbs, cut through each hip, ankle, and knee joint.
Store the thigh and shank of every hind limb in a single 92-mm Petri dish filled with PVS on ice and carefully remove as much muscle as possible from each bone, transferring each cleaned femur and tibia into a 50-mL tube of sterile RPMI 1640 medium on wet ice. To isolate the bone marrow, transfer one bone at a time into a new 92-mL Petri dish filled with fresh RPMI 1640 medium, and use a scalpel to cut the ends off of each bone. Next, use a 1-mL syringe equipped with a 26-gauge needle to flush the bones with 1 mL of fresh medium at a time into a 50-mL collection tube containing 5 mL of medium.
When all the bones have been flushed, gently pipette the crumbly bone marrow pieces up and down several times to generate a single-cell suspension before filtering the cells through a 40-micrometer mesh-screen cell strainer into a new 50-mL collection tube. Pellet the cells by centrifugation and re-suspend the cells in 5 mL of ammonium chloride potassium lysis buffer for a three-minute incubation at room temperature. At the end of the incubation, stop the reaction with 10 mL of PVS, and centrifuge the cells again.
Re-suspend the pellet in 2 mL of fresh PVS for counting, and set aside a six times 10 to the sixth cell aliquot for downstream flow cytometric analysis. Next, use a commercially available cell purification kit to magnetically deplete the CD90.2-positive T cells from the bone marrow single-cell suspension, according to the manufacturer's instructions, and set aside a one times 10 to the sixth cell aliquot of the T-cell-depleted eluate for downstream flow cytometric analysis of the cell purity and composition before and after separation. Then, use a 1-mL syringe equipped with a 30-gauge needle to inject five times 10 to the fifth T-cell-depleted bone marrow cells in 100 microliters of PVS intravenously into the retrobulbar space of the venous sinus of each irradiated recipient animal.
The next day, place a spleen harvested from a donor animal onto a 40-micrometer mesh strainer in a 50-mL collection tube, and use a syringe plunger to press the spleen tissue through the strainer into the tube. Wash the strainer and plunger with PVS to collect all the splenocytes, and pellet the cells by centrifugation. After red blood cell lysis is demonstrated, re-suspend the white blood cells for counting, and set aside a six times 10 to the sixth cell aliquot for downstream flow cytometric analysis.
For a total CD3-positive T-cell isolation from total splenocytes, use a commercially available cell purification kit according to the manufacturer's instructions, and set aside a one times 10 to the sixth CD3-positive T-cell aliquot from the positive cell fraction for downstream flow cytometric analysis of the cell purity and composition before and after separation. Then, inject seven times 10 to the fifth alloreactive CD3-positive T cells in 100 microliters of PVS intravenously into the retrobulbar space of each recipient mouse to induce GvHD. To score GvHD-related lesions by mini-endoscopy of the distal colorectal region, lift the tail of a recipient animal just above the tail root with one hand, and use the other hand to carefully insert the endoscope into the rectum via the anus.
While the air stream inflates the colorectal lumen, slowly and carefully move the endoscope forward in the aboral direction. To avoid colonic bowel injuries, keep the endoscope in the middle area of the lumen using the live video screen to aid in this positioning. Start recording the video stream and scoring the inflammation, evaluating the GvHD-related inflammation of the colon by scoring the parameters as illustrated.
When a scoring spot has been located, move the endoscope gently and slightly back and forth to assess the different parameters. To assess the parameter translucency and stool consistency, position the endoscope at a wider distance in relation to the colonic wall. To assess the granularity and vascularity of the lesion, position the endoscope in the close proximity to the colonic wall, and carefully apply well-dosed tension to the colonic wall with the tip of the endoscope.
Upon completion of the scoring process, stop the recording. Compared to controls, allogenic hematopoieic stem cell transplantation as demonstrated produces a reproducibly robust induction of a systemic GvHD phenotype, with progressively increasing clinical GvHD scores in recipient animals. These mini-endoscopically assessable criteria were specifically adapted from previously reported syngeneic colitis in the context of allo-response-driven colitis scoring parameters for the precise description, scoring, and grading of intestinal GvHD-disease-associated lesions in the distal colon.
Indeed, the mini-endoscopically based grading system enables an easy discrimination of donor-lymphocyte-receiving mice with severe signs of intestinal inflammation from control mice that are essentially devoid of GvHD. Invalidation of the mini-endoscopically based grading system histopathological studies confirmed that the colon of mice severely affected by GvHD-related inflammation display similarly strong histopathological signs of inflammation. In contrast, colon tissues of control mice displayed no to, at most, mild histopathological signs of inflammation, in agreement with the virtual absence of mini-endoscopically detectable signs of colitis.
Furthermore, correlation studies between mini-endoscopically and histopathologically assessed colitis activity and systemic GvHD scores demonstrate that mini-endoscopically determined some scores of less than or equal to three reliably predict the absence of mid-to higher-grade intestinal GvHD-associated colonic inflammation scores obtained from histopathological grading. In addition, the severity of the endoscopically assessed intestinal GvHD shows a correlation with systemic GvHD disease activity. To standardize the results of mini-endoscopic evaluation of colonic inflammation and to ensure comparability across different mice and experiments over time, a defined anatomic site should be selected for scoring.
In addition to scoring colonic inflammation, the built-in working channel within the mini-endoscopic setup allows the retrieval of view-guided tissue biopsies applicable to various downstream analyses, such as standard histopathology. Due to the non-invasive nature of the mini-endoscopy, acute GvHD-related colitis can be repeatedly assessed within the same animal at virtually any time, thereby yielding fascinating insights into intestinal GvHD.
