Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

Summary

The gastrointestinal tract is one of the most sensitive organs to injury upon radiotherapeutic cancer treatments. It is simultaneously an organ system with one of the highest regenerative capacities following such insults. The presented protocol describes an efficient method to study the regenerative capacity of the intestinal epithelium.

Abstract

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the active proliferation of intestinal stem cells located at the bottom of intestinal crypts. However, during events of acute intestinal injury, these active intestinal stem cells undergo cell death. Gamma irradiation is a widely used colorectal cancer treatment, which, while therapeutically efficacious, has the side effect of depleting the active stem cell pool. Indeed, patients frequently experience gastrointestinal radiation syndrome while undergoing radiotherapy, in part due to active stem cell depletion. The loss of active intestinal stem cells in intestinal crypts activates a pool of typically quiescent reserve intestinal stem cells and induces dedifferentiation of secretory and enterocyte precursor cells. If not for these cells, the intestinal epithelium would lack the ability to recover from radiotherapy and other such major tissue insults. New advances in lineage-tracing technologies allow tracking of the activation, differentiation, and migration of cells during regeneration and have been successfully employed for studying this in the gut. This study aims to depict a method for the analysis of cells within the mouse intestinal epithelium following radiation injury.

Introduction

The human intestinal epithelium would cover approximately the surface of half a badminton court if placed completely flat1. Instead, this single cell layer separating humans from the contents of their guts is compacted into a series of finger-like projections, villi, and indentations, crypts that maximize the surface area of the intestines. The cells of the epithelium differentiate along a crypt-villus axis. The villus primarily consists of nutrient-absorbing enterocytes, mucus-secreting goblet cells, and the hormone-producing enteroendocrine cells, while the crypts primarily consist of defensin-producing Paneth cells, active and reserve stem c....

Protocol

All mice were housed in the Division of Laboratory Animal Resources (DLAR) at Stony Brook University. The Stony Brook University Institutional Animal Care and Use Committee (IACUC) approved all studies and procedures involving animal subjects. Experiments involving animal subjects were conducted strictly in accordance with the approved animal handling protocol (IACUC #245094).

NOTE: Mouse strains B6;129-Bmi1tm1(cre/ERT)Mrc/J (Bmi1-CreER) and B6.129X1-

Representative Results

The use of 12 Gy total-body irradiation (TBI) in combination with murine genetic lineage tracing allows for a thorough analysis of the consequences of radiation injury in the gut. To start, Bmi1-CreER;Rosa26eYFP mice received a single tamoxifen injection, which induces enhanced yellow fluorescent protein (EYFP) expression within a Bmi1+ reserve stem cell population. Two days subsequent to the tamoxifen injection, the mice underwent irradiation or sham irradiation. Three hour.......

Discussion

This protocol describes a robust and reproducible radiation injury model. It allows for the precise analysis of the changes in the intestinal epithelium over the course of 7 days post injury. Importantly, the selected time points reflect crucial stages of injury and are characterized by distinct alterations to the intestine (injury, apoptosis, regeneration, and normalization phases)60. This model of irradiation has been established and carefully assessed, demonstrating a suitable manifestation of .......

Acknowledgements

The authors wish to acknowledge the Stony Brook Cancer Center Histology Research Core for expert assistance with tissue specimen preparation and the Division of Laboratory Animal Resources at Stony Brook University for assistance with animal care and handling. This work was supported by grants from the National Institutes of Health DK124342 awarded to Agnieszka B. Bialkowska and DK052230 to Dr. Vincent W. Yang.

....

Materials

NameCompanyCatalog NumberComments
1 mL syringeBD309659-
16G Reusable Small Animal Feeding Needles: StraightVWR20068-630-
27G x 1/2" needleBD305109-
28G x 1/2" Monoject 1mL insulin syringeCovidien1188128012-
5-Ethynyl-2′-deoxyuridine (EdU)Santa Cruz Biotechnologysc284628A10 mg/mL in sterile DMSO:water (1:4 v/v), aliquot and store in -20°C
Azer Scientific 10% Neutral Buffered FormalinFisher Scientific22-026-213-
B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/JThe Jackson LaboratoryStrain #:006148
B6;129-Bmi1tm1(cre/ERT)Mrc/JThe Jackson LaboratoryStrain #:010531
Bovine Serum Albumin Fraction V, heat shockMillipore-Sigma3116956001
Chicken anti-GFPAvesGFP-1020
Click-IT plus EdU Alexa Fluor 555 imaging kit, InvitrogenThermo Fisher ScientificC10638-
Corn oilMillipore-SigmaC8267-
Decloaking ChamberBiocare MedicalDC2012-
Dimethyl sulfoxide (DMSO)Fisher BioReagentsBP231-100light sensitive
DNase-free proteinase KInvitrogenC10618Hdiluted 25x in DPBS
Donkey anti-chicken AF647Jackson ImmunoResearch703-605-155
DPBSFisher Scientific21-031-CV-
EosinFisher ScientificS176
Fluorescence Microscope Nikon Eclipse 90i Bright and fluoerescent light, with objectives: 10X, 20XNikon
Fluoromount Aqueous Mounting MediumMillipore-SigmaF4680-25ML
Gamma Cell 40 ExactorBest Theratronics Ltd.-0.759 Gy min-1
Goat anti-rabbit AF488Jackson ImmunoResearch111-545-144
Hematoxylin Solution, Gill No. 3Millipore-SigmaGHS332
HM 325 Rotary Microtome from Thermo ScientificFisher Scientific23-900-668
Hoechst 33258, Pentahydrate (bis-Benzimide)Thermo Fisher ScientificH3569dilution 1:1000
Hydrogen Peroxide Solution, ACS, 29-32%, Spectrum ChemicalFisher Scientific18-603-252-
In Situ Cell Death Detection Kit, Fluorescein (Roche)Millipore-Sigma11684795910
Liquid Blocker Super PAP PEN, MiniFisher ScientificDAI-PAP-S-M
Lithium Carbonate (Powder/Certified ACS), Fisher ChemicalFisher ScientificL119-5000.5g/1L dH2O
Luer-Lok Syringe sterile, single use, 10 mLVWR89215-218-
MethanolVWRBDH1135-4LP
Pharmco Products Ethyl alcohol, 200 PROOFFisher ScientificNC1675398-
Pharmco-Aaper 281000ACSCSLT Acetic Acid ACS GradeCapitol ScientificAAP-281000ACSCSLT-
Rabbit anti-Ki67BioCare MedicalCRM325
Richard-Allan Scientific Cytoseal XYL Mounting MediumFisher Scientific22-050-262
Scientific Industries Incubator-Genie for baking slides at 65 degreeFisher Scientific50-728-103
Sodium Citrate DihydrateFisher ScientificS279-500
Stainless Steel Dissecting KitVWR25640-002
Superfrost Plus micro slides [size: 25 x 75 x 1 mm]VWR 48311-703
TamoxifenMillipore-SigmaT564830 mg/mL in sterile corn oil, preferably fresh or short-sterm storage in -20°C, light sensitive
Tissue-Tek 24-Slide Holders with Detachable HandleSakura4465
Tissue-Tek Accu-Edge Low Profile BladesSakura4689
Tissue-Tek Manual Slide Staining SetSakura4451
Tissue-Tek Staining Dish, Green with LidSakura4456
Tissue-Tek Staining Dish, White with LidSakura4457
Tween 20Millipore-SigmaP7949
Unisette Processing CassettesVWR87002-292-
VWR Micro Cover GlassesVWR48393-081
XyleneFisher ScientificX5P-1GAL

References

  1. Helander, H. F., Fandriks, L. Surface area of the digestive tract - Revisited. Scandinavian Journal of Gastroenterology. 49 (6), 681-689 (2014).
  2. vander Flier, L. G., Clevers, H. Stem cells, self-renewal, and differentiation in the....

This article has been published

Video Coming Soon

We use cookies to enhance your experience on our website.

By continuing to use our website or clicking “Continue”, you are agreeing to accept our cookies.

Learn More