The authors would like to thank Michele Allen of the National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health (NIH) for generously sharing her expertise in murine phenotyping methods and for her ongoing technical assistance, as well as for Timothy Hunt of NHLBI for helping us develop the shielding device. This study is supported by the Division of Intramural Research of the National Institute of Nursing Research of the NIH, and part of the validation trial is supported by a grant from the Oncology Nursing Society Foundation.

Ethics Statement: This study was approved by the National Institutes of Health (NIH) Animal Care and Use Committee. All investigators taking part in animal handling and measurement of study outcomes were properly trained by the NIH Office of Animal Care and Use and the National Heart, Lung, and Blood Institute Murine Phenotyping Core. All aspects of animal testing, housing, and environmental conditions used in this study were in compliance with The Guide for the Care and Use of Laboratory Animals11.
1. Housing and Experimental Animals
NOTE: House male C57BL/6 mice (roughly five weeks old upon arrival) individually throughout the experiment and provide ad libitum access to food and water. All cages are kept on a 12:12 h light-dark cycle with the light phase beginning at 6 am and dark phase at 6 pm.
<ol>
	<li>Identify the mice and assign them to individual standard ventilated mouse cages. Allow 24 h after the identification procedure for recovery. 
	NOTE: Tail tattoo is recommended as a means of identification to eliminate the possibility that an ear tag could get caught in a running wheel. Tattoo a number on the tail of each mouse, with the number on the tail matching the number written on the mouse&#39;s cage.</li>
	<li>Allow mice to acclimate to their cages for at least three more days, handling each mouse gently for a period of three minutes per day.</li>
</ol>
2. Running Wheel Acclimation and Baseline
<ol>
	<li>Introduce the mice to individual VWRA cages, each equipped with a running wheel connected to an electronic counter for continuous recording. 
	NOTE: All wheel counters connect to a computer through a single USB interface (see Materials List). The computer software calculates the number of wheel rotations, distance traveled, and average speed across each designated time interval of the specified total duration. Once the recording stops, the data are automatically saved both as text and as spreadsheets.</li>
	<li>Initiate recording of VWRA through the computer software interface. Set recording intervals to one hour and the duration to at least five days. Continue recording VWRA for at least five days. 
	NOTE: At the end of step 2.2, all mice should achieve a relatively consistent amount of daily wheel running activity. If not, then identify and exclude any outliers.</li>
	<li>Stop VWRA recording through the software interface, and return the mice to their standard cages described in step 1.1 (cages without running wheels).</li>
	<li>Randomize mice into either sham-irradiated control or irradiated groups.</li>
</ol>
3. Irradiation
Note: Perform the following steps for all mice in both groups, once per day for three sequential days. Treat mice in the same order each day.
<ol>
	<li>Anesthetize each mouse by intraperitoneal injection of a ketamine (100 mg/kg) and xylazine (10 mg/kg) mixture.</li>
	<li>Confirm anesthesia with a toe pinch and use ointment on the eyes to prevent dryness while under anesthesia.</li>
	<li>Transfer the anesthetized mouse into a lead shielding device. Arrange the mouse in the shielding so that only the lower abdominal/pelvic region is exposed. 
	NOTE: The shielding is composed of two lead &#34;boxes,&#34; with a narrow open space in between that allows radiation exposure to a small, targeted region of the mouse (see Figure 1).</li>
	<li>Use medical tape to secure the base of the mouse&#39;s tail in position within the shielding. 
	NOTE: Step 3.4 is optional, but doing so can help ensure that the position of the mouse does not change during the following step.</li>
	<li>Transport the shielding device into the irradiator, ensuring that the animal position within the shielding is maintained.</li>
	<li>If the mouse is in the irradiation group, deliver 800 cGy at a dose rate of about 110 cGy/min. If the mouse is in the sham-irradiation control group, leave the mouse in the inactive irradiator for the equivalent time. 
	NOTE: Optimal irradiator settings will depend on the particular device. The dose rate of 110 cGy/min delivered from a 137Cesium source is the central dose rate of the irradiator used here. The exposure time was adjusted to reach the desired total dose of 800 cGy.</li>
	<li>Remove the mouse from the irradiator and shielding, and then return it to its original, standard cage mentioned in step 1.1.</li>
	<li>Continuously monitor the mouse until it has regained sufficient consciousness to maintain sternal recumbency.</li>
</ol>
4. Radiation-induced Fatigue Measurement
<ol>
	<li>On the day after completion of three successive days of irradiation, transfer the mice to their individual VWRA cages described in step 2.1.</li>
	<li>Record VWRA, as described in step 2.2, except here setting the recording duration to more than 15 days. At the end of the 15 days, manually stop VWRA recording through the software interface. 
	NOTE: The data from each recording period are automatically saved as spreadsheets, each of which includes the rotation, distance, and speed measurements for all animals (columns) and at all intervals (rows) throughout the duration of recording. At the end of the experiment, there are two spreadsheets generated by the recording software: one for the pre-irradiation VWRA, and one for the post-irradiation VWRA.</li>
</ol>

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	<li>Minton, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-related+fatigue+and+its+impact+on+functioning.">Cancer-related fatigue and its impact on functioning.</a> Cancer. 119, 2124-2130 (2013).</li><li>Bower, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-related+fatigue--mechanisms,+risk+factors,+and+treatments.">Cancer-related fatigue--mechanisms, risk factors, and treatments.</a> Nat Rev Clin Oncol. 11 (10), 597-609 (2014).</li><li>Saligan, L. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+cancer-related+fatigue:+a+review+of+the+literature.">The biology of cancer-related fatigue: a review of the literature.</a> Support Care Cancer. 23 (8), 2461-2478 (2015).</li><li>Norden, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+growth+increases+neuroinflammation,+fatigue+and+depressive-like+behavior+prior+to+alterations+in+muscle+function.">Tumor growth increases neuroinflammation, fatigue and depressive-like behavior prior to alterations in muscle function.</a> Brain Behav Immun. 43, 76-85 (2015).</li><li>Ray, M. A., Trammell, R. A., Verhulst, S., Ran, S., Toth, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+mouse+model+for+assessing+fatigue+during+chemotherapy.">Development of a mouse model for assessing fatigue during chemotherapy.</a> Comp Med. 61 (2), 119-130 (2011).</li><li>Zombeck, J. A., Fey, E. G., Lyng, G. D., Sonis, S. 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This research was supported by the Division of Intramural Research, National Institute of Nursing Research, National Institutes of Health.

We have described a protocol using targeted peripheral irradiation to induce a reduction of VWRA in mice without confounding morbidity or mortality. Importantly, a simple shielding device allows irradiation in this protocol to target a desired region consistently, mimicking the radiation treatments received by patients with pelvic cancer. In contrast to existing CRF models involving brain or total body irradiation8,9, this model explores how a peripherally-target...

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments1. The fatigue is neither proportional to recent activity nor alleviated by rest, and it is associated with a wide variety of disturbances related to mood, motivation, attention, and cognition2. The biological causes of CRF are unknown, though it has been shown in many cases to correlate with inflammation and cytokine levels, also in some cases with hemoglobin levels and the function of various hormone systems (see Saligan et al.3 for a review of biological studies of CRF).
Controlled studies using animal models are necessary to understand the behavior and biology associated with this complex condition. While tumor-related4 or chemotherapy-related5,6 fatigue has been studied in rodent models, the etiology of CRF may be treatment-specific. To investigate CRF related to radiation therapy, our group has recently developed a mouse model of irradiation-induced fatigue7. In contrast to existing CRF models involving brain or total body irradiation8,9, this model explores how a change in centrally-driven behavior, like fatigue, can be triggered by a peripherally-targeted irradiation procedure.
The procedure described here is designed to model radiation therapy administered to patients with pelvic cancer, using lead shielding to target the lower abdominal/pelvic region with irradiation. However, by modifying the lead shielding or its placement relative to experimental animals, this procedure could be adapted to model irradiation of other parts of the body. Voluntary wheel-running activity (VWRA) is used to measure fatigue-like behavior; because it&#39;s a voluntary and normal behavior10, it should allow the concurrent use of other behavioral and biological tests. We have found that peripheral irradiation is sufficient to reduce VWRA in mice without causing overt morbidity7. Future experiments with this model may help reveal effects of peripheral irradiation on immune and other biological signaling, as well as the downstream changes in the central nervous system that can produce deficits associated with CRF.

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a mouse model of fatigue induced by peripheral irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

Three batches of mice were run through the protocol described above. There were a total of 16 sham and 20 irradiated (2,400 cGy, 3 x 800 cGy/day) mice. After three consecutive days of irradiation, the irradiated group showed significantly reduced VWRA compared to sham (mixed repeated measures ANOVA: main effect of irradiation treatment, F1,13 = 19.233, p &#60; 0.001). The effect was significant for the first seven days after irradiation (simple main effects, p &#60; 0.05 with B...

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A Mouse Model of Fatigue Induced by Peripheral Irradiation

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

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7.8K Views.  National Institutes of Health. We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Video: A Mouse Model of Fatigue Induced by Peripheral Irradiation

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the gene of interest in hematopoietic cells and can be used to study the gene's role in normal and/or malignant hematopoiesis. This protocol provides a detailed description on how to perform transduction-transplantation using calreticulin (CALR) mutations recently identified in myeloproliferative neoplasm (MPN) as an example. In this protocol whole bone marrow cells from 5-flurouracil (5-FU) treated donor mice are transduced with a retrovirus encoding mutant CALR and transplanted into lethally irradiated syngeneic hosts. Donor cells expressing mutant CALR are marked with green fluorescent protein (GFP). Transplanted mice develop an MPN phenotype including elevated platelets in the peripheral blood, expansion of megakaryocytes in the bone marrow, and bone marrow fibrosis. We provide a step-by-step account of how to generate retrovirus, calculate viral titer, transduce whole bone marrow cells, and transplant into irradiated recipient mice.

This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation.

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without the ability to target a specific or well-delineated tumor volume. The delivery of radiation was achieved using fixed radiation sources or linear accelerators producing megavoltage (MV) X-rays. These devices are unable to achieve sub-millimeter precision required for small animals. Furthermore, the high doses delivered to healthy surrounding tissue hamper response assessment. To increase the translation between small animal studies and humans, our goal was to mimic the treatment of human glioblastoma in a rat model. To enable a more accurate irradiation in a preclinical setting, recently, precision image-guided small animal radiation research platforms were developed. Similar to human planning systems, treatment planning on these micro-irradiators is based on computed tomography (CT). However, low soft-tissue contrast on CT makes it very challenging to localize targets in certain tissues, such as the brain. Therefore, incorporating magnetic resonance imaging (MRI), which has excellent soft-tissue contrast compared to CT, would enable a more precise delineation of the target for irradiation. In the last decade also biological imaging techniques, such as positron emission tomography (PET) gained interest for radiation therapy treatment guidance. PET enables the visualization of e.g., glucose consumption, amino-acid transport, or hypoxia, present in the tumor. Targeting those highly proliferative or radio-resistant parts of the tumor with a higher dose could give a survival benefit. This hypothesis led to the introduction of the biological tumor volume (BTV), besides the conventional gross target volume (GTV), clinical target volume (CTV), and planned target volume (PTV).
At the preclinical imaging lab of Ghent University, a micro-irradiator, a small animal PET, and a 7 T small animal MRI are available. The goal was to incorporate MRI-guided irradiation and PET-guided sub-volume boosting in a glioblastoma rat model.

In the past, small animal irradiation was usually performed without the ability to target a well-delineated tumor volume. The goal was to mimic the treatment of human glioblastoma in rats. Using a small animal irradiation platform, we performed MRI-guided 3D conformal irradiation with PET-based sub-volume boosting in a preclinical setting.

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without ...

Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to objectively measure the severity of this condition. Here we describe an optimized method for assessing mitochondrial function of PBMCs collected from fatigued cancer patients. Using a compact extracellular flux system and sequential injection of respiratory inhibitors, we examined PBMC mitochondrial functional status by measuring basal mitochondrial respiration, spare respiratory capacity, and energy phenotype, which describes the preferred energy pathway to respond to stress. Fresh PBMCs are readily available in the clinical setting using standard phlebotomy. The entire assay described in this protocol can be completed in less than 4 hours without the involvement of complex biochemical techniques. Additionally, we describe a normalization method that is necessary for obtaining reproducible data. The simple procedure and normalization methods presented allow for repeated sample collection from the same patient and generation of reproducible data that can be compared between time points to evaluate potential treatment effects.

Our goal was to develop a practical protocol to evaluate mitochondrial dysfunction associated with fatigue in cancer patients. This innovative protocol is optimized for clinical use involving only standard phlebotomy and basic laboratory procedures.

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to ...

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 ...

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...