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.

The authors have no conflicts of interest.

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 ...

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 cells, and progenitor cells2,3,4,5. Furthermore, the bi-directional communication these cells have with the stromal and immune cells of the underlying mesenchymal compartment and the microbiota of the lumen generate a complex network of interactions that maintains gut homeostasis and is critical to recovery after injury6,7,8.
The intestinal epithelium is the most rapidly self-renewing tissue in the human body, with a turnover rate of 2-6 days9,10,11. During homeostasis, active stem cells at the base of intestinal crypts (crypt base columnar cells), marked by the expression of leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), rapidly divide and provide progenitor cells that differentiate into all other intestinal epithelial lineages. However, owing to their high mitotic rate, active stem cells and their immediate progenitors are particularly sensitive to gamma-radiation injury and undergo apoptosis following irradiation5,12,13,14. Upon their loss, reserve stem cells and non-stem cells (subpopulation of progenitors and some terminally differentiated cells) within intestinal crypts undergo activation and replenish the basal crypt compartment, which can then reconstitute cell populations of the villi and, thus, regenerate the intestinal epithelium15. Using lineage tracing techniques, multiple research groups have demonstrated that reserve (quiescent) stem cells are capable of supporting regeneration upon the loss of active stem cells13,16,17,18,19,20,21,22. These cells are characterized by the presence of polycomb complex protein 1 oncogene (Bmi1), mouse telomerase reverse transcriptase gene (mTert), Hop homeobox (Hopx), and leucine-rich repeat protein 1 gene (Lrig1). In addition, it has been shown that non-stem cells are capable of replenishing intestinal crypts upon injury23,24,25,26,27,28,29,30,31. In particular, it has been shown that progenitors of secretory cells and enterocytes undergo dedifferentiation upon injury, revert to stem-like cells, and support the regeneration of the intestinal epithelium. Recent studies have identified cells expressing multiple markers that possess the capacity of acquiring stem-like characteristics upon injury (such as DLL+, ATOH1+, PROX1+, MIST1+,&#160;DCLK1+)32,33,34,35,36. Surprisingly, Yu et al. showed that even mature Paneth cells (LYZ+) can contribute to intestinal regeneration37. Furthermore, in addition to causing apoptosis of intestinal epithelial cells and disrupting epithelial barrier function, irradiation results in dysbiosis of the gut flora, immune cell activation and the initiation of a pro-inflammatory response, and the activation of mesenchymal and stromal cells38,39.
Gamma radiation is a valuable therapeutic tool in cancer treatment, especially so for colorectal tumors40. However, irradiation significantly affects intestinal homeostasis by inducing damage to the cells, which leads to apoptosis. Radiation exposure causes multiple perturbations that slow down a patient&#39;s recovery and is marked by mucosal injury and inflammation in the acute phase and diarrhea, incontinence, bleeding, and abdominal pain long term. This panoply of manifestations is referred to as gastrointestinal radiation toxicity. Additionally, radiation-induced progression of transmural fibrosis and/or vascular sclerosis may only manifest years after the treatment38,41. Simultaneous to the injury itself, radiation induces a repair response in intestinal cells that activates signaling pathways responsible for initiating and orchestrating regeneration42. Radiation-induced small bowel disease can originate from pelvic or abdominal radiotherapy provided to other organs (such as cervix, prostate, pancreas, rectum)41,43,44,45,46. Intestinal irradiation injury is, thus, a significant clinical issue, and a better understanding of the resulting pathophysiology is likely to advance the development of interventions to alleviate the gastrointestinal complications associated with radiotherapy. There are other techniques that allow for investigating the regenerative purpose of the intestinal epithelium apart from radiation. Transgenic and chemical murine models to study inflammation and the regeneration thereafter have been developed47. Dextran sodium sulfate (DSS) induces inflammation in the intestine and leads to the development of characteristics similar to those of inflammatory bowel disease48. A combination of DSS treatment with the pro-carcinogenic compound azoxymethane (AOM) can result in the development of colitis-associated cancer48,49. Ischemia reperfusion-induced injury is another method employed to study the regenerative potential of the intestinal epithelium. This technique requires experience and surgical knowledge50. Furthermore, the aforementioned techniques cause different types of injury than radiation and may lead to the involvement of different mechanisms of regeneration. In addition, these models are time-consuming, while the radiation technique is fairly brief. Recently, in vitro methods utilizing enteroids and colonoids generated from the intestine and colon have been used in combination with radiation injury to study the mechanisms of intestinal regeneration51,52. However, these techniques do not fully recapitulate the organ they model53,54.
The protocol presented includes the description of a murine model of gamma-radiation injury in combination with a genetic model that, following tamoxifen treatment, permits tracing of lineages originating from the reserve stem cell population (Bmi1-CreER;Rosa26eYFP). This model utilizes a 12 Gy total-body irradiation, which induces significant enough intestinal injury to activate reserve stem cells while still allowing for the subsequent investigation of intestinal regenerative capability within 7 days of injury55.

<table><tbody><tr><td>1 mL syringe</td><td>BD</td><td>309659</td><td>-</td></tr><tr><td>16G Reusable Small Animal Feeding Needles: Straight</td><td>VWR</td><td>20068-630</td><td>-</td></tr><tr><td>27G x 1/2&quot; needle</td><td>BD</td><td>305109</td><td>-</td></tr><tr><td>28G x 1/2&quot; Monoject 1mL insulin syringe</td><td>Covidien</td><td>1188128012</td><td>-</td></tr><tr><td>5-Ethynyl-2&amp;prime;-deoxyuridine (EdU)</td><td>Santa Cruz Biotechnology</td><td>sc284628A</td><td>10 mg/mL in sterile DMSO:water (1:4 v/v), aliquot and store in -20&amp;deg;C</td></tr><tr><td>Azer Scientific&amp;nbsp;10% Neutral Buffered Formalin</td><td>Fisher Scientific</td><td>22-026-213</td><td>-</td></tr><tr><td>B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J</td><td>The Jackson Laboratory</td><td>Strain #:006148</td><td></td></tr><tr><td>B6;129-Bmi1tm1(cre/ERT)Mrc/J</td><td>The Jackson Laboratory</td><td>Strain #:010531</td><td></td></tr><tr><td>Bovine Serum Albumin Fraction V, heat shock</td><td>Millipore-Sigma</td><td>3116956001</td><td></td></tr><tr><td>Chicken anti-GFP</td><td>Aves</td><td>GFP-1020</td><td></td></tr><tr><td>Click-IT plus EdU Alexa Fluor 555 imaging kit, Invitrogen</td><td>Thermo Fisher Scientific</td><td>C10638</td><td>-</td></tr><tr><td>Corn oil</td><td>Millipore-Sigma</td><td>C8267</td><td>-</td></tr><tr><td>Decloaking Chamber</td><td>Biocare Medical</td><td>DC2012</td><td>-</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Fisher BioReagents</td><td>BP231-100</td><td>light sensitive</td></tr><tr><td>DNase-free proteinase K</td><td>Invitrogen</td><td>C10618H</td><td>diluted 25x in DPBS</td></tr><tr><td>Donkey anti-chicken AF647</td><td>Jackson ImmunoResearch</td><td>703-605-155</td><td></td></tr><tr><td>DPBS</td><td>Fisher Scientific</td><td>21-031-CV</td><td>-</td></tr><tr><td>Eosin</td><td>Fisher Scientific</td><td>S176</td><td></td></tr><tr><td>Fluorescence Microscope Nikon Eclipse 90i Bright and fluoerescent light, with objectives: 10X, 20X</td><td>Nikon</td><td></td><td></td></tr><tr><td>Fluoromount Aqueous Mounting Medium</td><td>Millipore-Sigma</td><td>F4680-25ML</td><td></td></tr><tr><td>Gamma Cell 40 Exactor</td><td>Best Theratronics Ltd.</td><td>-</td><td>0.759 Gy min&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Goat anti-rabbit AF488</td><td>Jackson ImmunoResearch</td><td>111-545-144</td><td></td></tr><tr><td>Hematoxylin Solution, Gill No. 3</td><td>Millipore-Sigma</td><td>GHS332</td><td></td></tr><tr><td>HM 325 Rotary Microtome from Thermo Scientific</td><td>Fisher Scientific</td><td>23-900-668</td><td></td></tr><tr><td>Hoechst 33258, Pentahydrate (bis-Benzimide)</td><td>Thermo Fisher Scientific</td><td>H3569</td><td>dilution 1:1000</td></tr><tr><td>Hydrogen Peroxide Solution, ACS, 29-32%, Spectrum Chemical</td><td>Fisher Scientific</td><td>18-603-252</td><td>-</td></tr><tr><td>In Situ Cell Death Detection Kit, Fluorescein (Roche)</td><td>Millipore-Sigma</td><td>11684795910</td><td></td></tr><tr><td>Liquid Blocker Super PAP PEN, Mini</td><td>Fisher Scientific</td><td>DAI-PAP-S-M</td><td></td></tr><tr><td>Lithium Carbonate (Powder/Certified ACS), Fisher Chemical</td><td>Fisher Scientific</td><td>L119-500</td><td>0.5g/1L dH2O</td></tr><tr><td>Luer-Lok Syringe sterile, single use, 10 mL</td><td>VWR</td><td>89215-218</td><td>-</td></tr><tr><td>Methanol</td><td>VWR</td><td>BDH1135-4LP</td><td></td></tr><tr><td>Pharmco Products&amp;nbsp;Ethyl alcohol, 200 PROOF</td><td>Fisher Scientific</td><td>NC1675398</td><td>-</td></tr><tr><td>Pharmco-Aaper 281000ACSCSLT Acetic Acid ACS Grade</td><td>Capitol Scientific</td><td>AAP-281000ACSCSLT</td><td>-</td></tr><tr><td>Rabbit anti-Ki67</td><td>BioCare Medical</td><td>CRM325</td><td></td></tr><tr><td>Richard-Allan Scientific Cytoseal XYL Mounting Medium</td><td>Fisher Scientific</td><td>22-050-262</td><td></td></tr><tr><td>Scientific Industries Incubator-Genie for baking slides at 65 degree</td><td>Fisher Scientific</td><td>50-728-103</td><td></td></tr><tr><td>Sodium Citrate Dihydrate</td><td>Fisher Scientific</td><td>S279-500</td><td></td></tr><tr><td>Stainless Steel Dissecting Kit</td><td>VWR</td><td>25640-002</td><td></td></tr><tr><td>Superfrost Plus micro slides [size: 25 x 75 x 1 mm]</td><td>VWR</td><td>&amp;nbsp;48311-703</td><td></td></tr><tr><td>Tamoxifen</td><td>Millipore-Sigma</td><td>T5648</td><td>30 mg/mL in sterile corn oil, preferably fresh or short-sterm storage in -20&amp;deg;C, light sensitive</td></tr><tr><td>Tissue-Tek 24-Slide Holders with Detachable Handle</td><td>Sakura</td><td>4465</td><td></td></tr><tr><td>Tissue-Tek&amp;nbsp;Accu-Edge&amp;nbsp;Low Profile Blades</td><td>Sakura</td><td>4689</td><td></td></tr><tr><td>Tissue-Tek&amp;nbsp;Manual Slide Staining Set</td><td>Sakura</td><td>4451</td><td></td></tr><tr><td>Tissue-Tek Staining Dish, Green with Lid</td><td>Sakura</td><td>4456</td><td></td></tr><tr><td>Tissue-Tek Staining Dish, White with Lid</td><td>Sakura</td><td>4457</td><td></td></tr><tr><td>Tween 20</td><td>Millipore-Sigma</td><td>P7949</td><td></td></tr><tr><td>Unisette Processing Cassettes</td><td>VWR</td><td>87002-292</td><td>-</td></tr><tr><td>VWR Micro Cover Glasses</td><td>VWR</td><td>48393-081</td><td></td></tr><tr><td>Xylene</td><td>Fisher Scientific</td><td>X5P-1GAL</td><td></td></tr></tbody></table>

intestinal epithelial regeneration in response to ionizing irradiation

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.

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&#160;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...

Watch this Scientific Journal Video about Intestinal Epithelial Regeneration in Response to Ionizing Irradiation at JoVE.com

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

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.

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the ...

intestinal-epithelial-regeneration-in-response-to-ionizing-irradiation

Research

JoVE Journal

Biology

2.1K Views.  Renaissance School of Medicine at Stony Brook University. Gammabestraling is een veel gebruikte behandeling voor darmkanker. En hoewel het therapeutisch effectief is, heeft het negatieve effecten op het maagdarmkanaal. Het gepresenteerde protocol beschrijft een efficiënte methode om de activering, differentiatie en migratie van cellen tijdens regeneratie na stralingsletsel te bestuderen.Dit protocol beschrijft een robuust en reproduceerbaar stralingsschademodel. Belangrijk is dat dit model het mogelijk maakt om het lot van darmcellen na lets...

Video: Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell proliferation and increased nutrient absorption, are critical in recovery, yet poorly understood. Understanding the molecular mechanism behind the adaptive responses is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Different approaches and models have been described throughout the literature, but a detailed descriptive way to essentially perform the procedures is needed to obtain reproducible data. Here, we describe a method to estimate important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis in mice. We demonstrate the detection of proliferating cells using a cell cycle specific marker, as well as using small intestinal weight, crypt depth, and villus height as endpoints. Some of the critical steps within the described method are the removal and weighing of the small intestine and the rather complex software system suggested for the measurement of this technique. These methods have the advantages that they are not time-consuming, and that they are cost-effective and easy to carry out and measure.

Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell ...

Cancer Research

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Medicine

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between colorectal tumor cells and the immune system. Colorectal carcinoma is among the most common type of cancer, typically treated by surgery, chemotherapy, and radiotherapy. Advantages of radiotherapy in targeting the tumor are well known. However, even limited exposures of healthy tissues are of great concern, particularly regarding the effects on the intestinal barrier and the immune system. The adopted setup allows to study the interplay between two cell populations in a condition more similar to the physiological one, when compared to normal cell cultures. For this purpose, we resort to different techniques and we used an in vitro co-culture model, based on Caco-2 cells differentiated as a monolayer and PBMC, sharing the same culture medium. This protocol has been developed to focus on both macroscopic effects, i.e. cell viability and Trans-Epithelial Electrical Resistance (TEER), and, through western blot, molecular alterations, i.e. the activation of inflammatory pathway in immune cells and the tight junction protein expression in Caco-2 cells. Initial evaluation of radiation effects on Caco-2 cell viability was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays, while TEER was measured at fixed time intervals through an ohmmeter specifically designed for co-culture systems. In this way, the effects due to radiation, the presence of Peripheral Blood Mononuclear Cells (PBMC), and eventually their synergistic effect, can be demonstrated. Through these complementary techniques, we observed a high radio-resistance of Caco-2 within the range of 2 - 10 Gy of X-rays and an increased Caco-2 monolayer permeability when PBMCs were added. In particular, PBMC presence was found to be associated with the variation in the tight junction scaffold proteins expression.

We present a protocol to investigate the crosstalk between X-ray-irradiated Caco-2 and peripheral blood mononuclear cells (PBMC). The protocol starts with Caco-2 irradiation and set-up of the co-culture with PBMC; subsequently, trans-epithelial electrical resistance is measured regularly over 48 h and western blot performed in both Caco-2 and PBMC.

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between ...

Immunology and Infection

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

Bioengineering

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the safety of stem cell transplantation. Although the transplantation of differentiated rather than undifferentiated cells is common practice, teratomas can still arise from the presence of residual undifferentiated stem cells at the time of transplant or from spontaneous mutations in differentiated cells. Because stem cell therapies are often delivered into anatomically sensitive sites, even small tumors can be clinically devastating, resulting in blindness, paralysis, cognitive abnormalities, and cardiovascular dysfunction. Surgical access to these sites may also be limited, leaving patients with few therapeutic options. Controlling stem cell misbehavior is, therefore, critical for the clinical translation of stem cell therapy.
External beam radiation offers an effective means of delivering targeted therapy to decrease the teratoma burden while minimizing injury to surrounding organs. Additionally, this method avoids genetic manipulation or viral transduction of stem cells-which are associated with additional clinical safety and efficacy concerns. Here, we describe a protocol to create pluripotent stem cell-derived teratomas in mice and to apply external beam radiation therapy to selectively ablate these tumors in vivo.

Research on treatment strategies for pluripotent stem cell-derived teratomas is important for the clinical translation of stem cell therapy. Here, we describe a protocol to, first, generate stem cell-derived teratomas in mice and, then, to selectively target and treat these tumors in vivo using a small-animal irradiator.

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the ...

Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with radiotherapy. Experimental approaches to understanding mechanisms of salivary gland dysfunction and restoration have focused on in vivo models, which are handicapped by an inability to systematically screen therapeutic candidates and efficiencies in transfection capability to manipulate specific genes. The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. We utilized immunofluorescent staining and confocal microscopy to determine when specific cell populations and markers are present during a 30-day culture period. In addition, cellular markers previously reported in in vivo radiation models are evaluated in cultures that are irradiated ex vivo. Moving forward, this method is an attractive platform for rapid ex vivo assessment of murine and human salivary gland tissue responses to therapeutic agents that improve salivary function.

Using three-dimensional organotypic cultures to visualize morphology and functional markers of salivary glands may provide novel insights into the mechanisms of tissue damage following radiation. Described here is a protocol to section, culture, irradiate, stain, and image 50&#8211;90 &#956;m thick salivary gland sections prior to and following exposure to ionizing radiation.

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with ...

Epithelial Effects of Intestinal Inflammation on Established Murine Colonoids

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.

Author Spotlight: Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids  

We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic ...

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

Samenvatting

Meer video's verkennen