Name: a mouse model of fatigue induced by peripheral irradiation
Uploaded: 2017-03-17T14:00:00
Description: Watch this Scientific Journal Video about A Mouse Model of Fatigue Induced by Peripheral Irradiation at JoVE.com

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</sup...

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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...

Watch this Scientific Journal Video about A Mouse Model of Fatigue Induced by Peripheral Irradiation at JoVE.com

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. ...

The overall goal of this procedure is to measure fatigue-like behavior induced by peripheral irradiation. This method can help answer key questions about fatigue and, in particular, fatigue related to irradiation. For example, it can be used to explore the different roles of the immune system and the central nervous system in fatigue after irradiation.The main advantage of this technique is that it specifically uses peripheral irradiation to affect essentially driven fatigue behavior. Begin by randomizing the mice into either sham irradiated control or irradiated groups. Then, identify five-week-old, male mice by tail tattoo, and assign each mouse to an individual, standard ventilated mouse cage.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. Next, introduce the mice to individual, voluntary wheel-running activity, or VWRA, cages, each equipped with a running wheel connected to an electronic counter for continuous recording. Initiate recording of VWRA through the computer software interface.Set recording intervals to one hour and the duration to at least five days. Finally, after five days, stop VWRA recording through the software interface, and return the mice to their standard cages without running wheels. After anesthetizing the mouse, confirm that the anesthesia was effective with a toe pinch.Use ointment on the eyes to prevent dryness. Next, transfer the anesthetized mouse into a lead shielding device. Arrange the mouse in the shielding so that only the lower abdominal and pelvic region is exposed.Then, use medical tape to secure the base of the mouse's tail in position within the shielding to ensure that the position does not change during irradiation. If the mouse is in the irradiation group, deliver 800 centigray at a dose rate of about 110 centigray per minute. If the mouse is in the sham irradiation control group, leave the mouse in the inactive irradiator for the equivalent time.After irradiation is complete, remove the mouse from the irradiator and shielding and return it to its original standard cage. Monitor the mouse continuously until it has regained sufficient consciousness to maintain sternal recumbency. After three successive days of irradiation, transfer the mice into their individual VWRA cages.Finally, to capture radiation-induced fatigue, record VWRA for 15 days. The effect was significant for the first seven days after radiation, with the lowest mean VWRA distance occurring on the third day after radiation. Here, the distribution of the change in VWRA from before to after irradiation is shown.While the majority of mice tested showed fatigue-like symptoms, there was a small number of mice that showed little change or even an increase in VWRA. While attempting this procedure, it's important to make sure the mice are completely anesthetized during irradiation since any movements may affect the irradiation targeting and dosage. After watching this video, you should have a good understanding of how to use peripherally targeted irradiation to induce fatigue-like behavior in mice.

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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 ...

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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.

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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 ...

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...