Name: an in vivo mouse model of total intravenous anesthesia during cancer resection surgery
Uploaded: 2021-06-08T14:30:00
Description: Watch this Scientific Journal Video about An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery at JoVE.com

The authors wish to thank members of the Cancer Neural-Immune Laboratory and Dr. Cameron Nowell at Monash Institute of Pharmaceutical Sciences, Monash University, Parkville. This work was supported by grants from National Health and Medical Research Council 1147498, the National Breast Cancer Foundation IIRS-20-025, the Australian and New Zealand College of Anaesthetists (ANZCA), Perpetual and CTC for Cancer Therapeutics.

All animal studies were undertaken under the approval of the Institutional Animal Care and Use Committee at Monash University. In this study, female Balb/c mice aged 6-8 weeks were used.
1. Prepare cancer cells
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	<li>Culture tumor cells in medium. Culture 66cl4 murine mammary cancer cells in alpha-MEM containing 10% FBS and 200 mM glutamine. Cells should text negative for mycoplasma. OPTIONAL:&#160;Use cells that are stably transduced to express firefly luciferase for bioluminescence ima...

<ol>
	<li>Sullivan, R., 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=Global+cancer+surgery:+delivering+safe,+affordable,+and+timely+cancer+surgery.">Global cancer surgery: delivering safe, affordable, and timely cancer surgery.</a> Lancet Oncology. 16 (11), 1193-1224 (2015).</li><li>Lim, J. A., 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+effect+of+propofol+and+sevoflurane+on+cancer+cell,+natural+killer+cell,+and+cytotoxic+T+lymphocyte+function+in+patients+undergoing+breast+cancer+surgery:+an+in+vitro+analysis.">The effect of propofol and sevoflurane on cancer cell, natural killer cell, and cytotoxic T lymphocyte function in patients undergoing breast cancer surgery: an in vitro analysis.</a> BMC Cancer. 18 (1), 159 (2018).</li><li>Pandit, J. J., 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=5th+National+Audit+Project+(NAP5)+on+accidental+awareness+during+general+anesthesia:+protocol,+methods,+and+analysis+of+data.">5th National Audit Project (NAP5) on accidental awareness during general anesthesia: protocol, methods, and analysis of data.</a> British Journal of Anaesthesia. 113 (4), 540-548 (2014).</li><li>Yap, A., Lopez-Olivo, M. A., Dubowitz, J., Hiller, J., Riedel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthetic+technique+and+cancer+outcomes:+a+meta-analysis+of+total+intravenous+versus+volatile+anesthesia.">Anesthetic technique and cancer outcomes: a meta-analysis of total intravenous versus volatile anesthesia.</a> Canadian Journal of Anesthesia. 66 (5), 546-561 (2019).</li><li>Makito, K., Matsui, H., Fushimi, K., Yasunaga, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volatile+versus+total+intravenous+anesthesia+for+cancer+prognosis+in+patients+having+digestive+cancer+surgery.">Volatile versus total intravenous anesthesia for cancer prognosis in patients having digestive cancer surgery.</a> Anesthesiology. 133 (4), 764-773 (2020).</li><li>Oh, T. K., Kim, H. H., Jeon, Y. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrospective+analysis+of+1-year+mortality+after+gastric+cancer+surgery:+Total+intravenous+anesthesia+versus+volatile+anesthesia.">Retrospective analysis of 1-year mortality after gastric cancer surgery: Total intravenous anesthesia versus volatile anesthesia.</a> Acta Anaesthesiologica Scandinavica. 63 (9), 1169-1177 (2019).</li><li>Lai, H. C., 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=Propofol-based+total+intravenous+anesthesia+is+associated+with+better+survival+than+desflurane+anesthesia+in+hepatectomy+for+hepatocellular+carcinoma:+a+retrospective+cohort+study.">Propofol-based total intravenous anesthesia is associated with better survival than desflurane anesthesia in hepatectomy for hepatocellular carcinoma: a retrospective cohort study.</a> British Journal of Anaesthesia. 123 (2), 151-160 (2019).</li><li>Hong, B., 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=Anesthetics+and+long-term+survival+after+cancer+surgery-total+intravenous+versus+volatile+anesthesia:+a+retrospective+study.">Anesthetics and long-term survival after cancer surgery-total intravenous versus volatile anesthesia: a retrospective study.</a> BMC Anesthesiology. 19 (1), 233 (2019).</li><li>Flecknell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Special+Techniques.">Special Techniques.</a> Laboratory Animal Anaesthesia. Fourth edition. , (2015).</li><li>Cicero, L., Fazzotta, S., Palumbo, V. D., Cassata, G., Lo Monte, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+protocols+in+laboratory+animals+used+for+scientific+purposes.">Anesthesia protocols in laboratory animals used for scientific purposes.</a> Acta Biomedica. 89 (3), 337-342 (2018).</li><li>Sloan, E. K., 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+sympathetic+nervous+system+induces+a+metastatic+switch+in+primary+breast+cancer.">The sympathetic nervous system induces a metastatic switch in primary breast cancer.</a> Cancer Research. 70 (18), 7042-7052 (2010).</li><li>Al-Hashimi, M., Scott, S. W. M., Thompson, J. P., Lambert, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opioids+and+immune+modulation:+more+questions+than+answers.">Opioids and immune modulation: more questions than answers.</a> British Journal of Anaesthesia. 111 (1), 80-88 (2013).</li><li>DeMarco, G. J., Nunamaker, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Review+of+the+effects+of+pain+and+analgesia+on+immune+system+function+and+inflammation:+relevance+for+preclinical+studies.">A Review of the effects of pain and analgesia on immune system function and inflammation: relevance for preclinical studies.</a> Comparative Medicine. 69 (6), 520-534 (2019).</li><li>Hiller, J. G., Perry, N. J., Poulogiannis, G., Riedel, B., Sloan, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perioperative+events+influence+cancer+recurrence+risk+after+surgery.">Perioperative events influence cancer recurrence risk after surgery.</a> Nature Reviews Clinical Oncology. 15 (4), 205-218 (2018).</li></ol>

This study reports on a protocol for administering total intravenous anesthesia (TIVA) with propofol in a mouse model of breast cancer that replicates key aspects of clinical practice for TIVA in patients requiring cancer surgery. The protocol allows investigation of both short-term and long-term clinically relevant outcomes after cancer surgery in a mouse model of cancer progression, including measurement of cytokine levels and cancer recurrence (Figure 2, and Figure 3<...

More than 60% of patients with cancer receive anesthesia for surgical resection1. Currently, there are no specific clinical guidelines that determine the choice of anesthesia used in cancer patients. Surveys of anesthesiologists indicate a preference for volatile-based anesthesia, including during cancer surgery2,3. However, there is a growing body of evidence that the use of propofol-based total intravenous anesthesia (TIVA) during cancer surgery may associate with improved postoperative outcomes (progression-free survival, overall survival) when compared to volatile anesthesia4. Subsequent clinical studies continue to report contradicting results5,6,7,8. These findings support the need for preclinical studies to better understand the mechanistic effects of different anesthetic agents on cancer-related outcomes.
However, in in vivo studies that model cancer surgery, anesthesia is frequently an incidental part of the procedure. The rationale for the choice of anesthesia is&#160;often not the focus of the experimental design, and its impact on cancer-related endpoints may not be evaluated. For example, in vivo studies that require maintenance of anesthesia for cancer surgery most commonly use inhaled volatile anesthesia9. Where propofol has been used in in vivo studies, it has been delivered by single bolus dosing with intraperitoneal delivery, which does not replicate clinical onco-anesthetic protocols10. That approach of propofol administration induces light anesthesia that is suitable for rapid procedures. However, it does not allow maintenance of anesthesia that is required for cancer resection surgery which may be protracted. Furthermore, the absorption kinetics of intraperitoneal delivery is distinct to clinical methods of administration.
A model of propofol-based TIVA for cancer resection surgery was developed to address this need. A protocol for sustained maintenance of anesthesia with titration of the anesthetic agent to allow response to the surgical stimulus was developed to replicate key aspects of anesthetic delivery to patients having cancer surgery. The resulting protocol is used with a mouse model of cancer to provide TIVA during cancer resection surgery. The effect on short-term and long-term cancer-related outcomes is evaluated.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% saline</td>
			<td>Fresnius Kabi</td>
			<td>AUST R 197198</td>
			<td></td>
		</tr>
		<tr>
			<td>Artery forceps</td>
			<td>Proscitech</td>
			<td>TS1322-140</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Temgesic</td>
			<td>TEMG I</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated surgical mat</td>
			<td>Custom</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic needle (30 G, 1 mL insulin syringe)</td>
			<td>Terumo</td>
			<td>NN3013R</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina</td>
			<td>PerkinElmer</td>
			<td>126274</td>
			<td></td>
		</tr>
		<tr>
			<td>Luciferin</td>
			<td>Promega</td>
			<td>P1041/2/3</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyurethane catheter</td>
			<td>Intramedic</td>
			<td>427401</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone Iodine</td>
			<td>Betadine</td>
			<td>AUST R 29562</td>
			<td></td>
		</tr>
		<tr>
			<td>Propofol Lipuro, 2%</td>
			<td>Braun</td>
			<td>3521490</td>
			<td></td>
		</tr>
		<tr>
			<td>Sevoflurane</td>
			<td>Baxter</td>
			<td>ANZ2L9117</td>
			<td></td>
		</tr>
		<tr>
			<td>Sevoflurane vaporiser</td>
			<td>Vetquip</td>
			<td>VQ1334</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gauze</td>
			<td>Multigate Medical Products</td>
			<td>11-600A</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Proscitech</td>
			<td>TS1044</td>
			<td></td>
		</tr>
		<tr>
			<td>Sutures, 5-0 nylon</td>
			<td>Dynek</td>
			<td>V504</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>70-4500</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes (1 mL)</td>
			<td>Terumo</td>
			<td>SS+01T</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vivo mouse model of total intravenous anesthesia during cancer resection surgery

Anesthesia is a routine component of cancer care that is used for diagnostic and therapeutic procedures. The anesthetic technique has recently been implicated in impacting long-term cancer outcomes, possibly through modulation of adrenergic-inflammatory responses that impact cancer cell behavior and immune cell function. Emerging evidence suggests that propofol-based total intravenous anesthesia (TIVA) may be beneficial for long-term cancer outcomes when compared to inhaled volatile anesthesia. However, the available clinical findings are inconsistent. Preclinical studies that identify the underlying mechanisms involved are critically needed to guide the design of clinical studies that will expedite insight. Most preclinical models of anesthesia have been extrapolated from the use of anesthesia in in vivo research and are not optimally designed to study the impact of anesthesia itself as the primary endpoint. This paper describes a method for delivering propofol-TIVA anesthesia in a mouse model of breast cancer resection that replicates key aspects of clinical delivery in cancer patients. The model can be used to study mechanisms of action of anesthesia on cancer outcomes in diverse cancer types and can be extrapolated to other non-cancer areas of preclinical anesthesia research.

This method describes a model of total intravenous anesthesia (TIVA) with propofol during cancer resection surgery in mice. Propofol is delivered in this mouse model through an intravenous catheter using a syringe pump (Figure 1A,B) to replicate delivery of TIVA in the clinical setting of anesthesia for cancer surgery. Use of the syringe pump minimizes exposure to volatile anesthesia by allowing rapid conversion from initial induction by inhalational anesthesia to intravenou...

Watch this Scientific Journal Video about An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery at JoVE.com

An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery

This paper describes a method for modeling total intravenous anesthesia (TIVA) during cancer resection surgery in mice. The goal is to replicate key features of anesthesia delivery to patients with cancer. The method allows investigation of how anesthetic technique affects cancer recurrence after resection surgery.

An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery

Anesthesia is a routine component of cancer care that is used for diagnostic and therapeutic procedures. The anesthetic technique has recently been ...

This novel protocol describes surgical anesthesia in mice using propofol anesthesia. The method enables research into propofol anesthesia in mouse models of disease. The protocol allows the evaluation of total intravenous anesthesia and avoids the use of volatile anesthesia.The protocol can be used to explore how propofol anesthesia impacts disease-related outcomes. For example, here we evaluate short and long-term cancer outcomes after surgery to remove the tumor. To begin, culture 66 CL4 Mirai mammary cancer cells that are stably transduced to express firefly luciferase in alpha MEM medium containing 10%FBS and 200 millimolar glutamine.Incubate the cells at 37 degrees Celsius but 5%carbon dioxide. Subculture the cells when the cells reach approximately 80%confluency. When the cells are in a logarithmic growth phase with two milliliters of drips in EDTA solution, lift the adherent cells and count the cells using a hemocytometer.Then dilute the cells in PBS and keep the diluted cell suspension on ice until the mouse is ready for injection. Once the mouse is completely anesthetized, wipe the fourth left mammary fat pad area with a single-use alcohol swab to prepare the injection site. Fill a 25 microliter Hamilton syringe attached to a sterile 27 gauge hypodermic needle with cancer cell suspension.Use forceps to lift and secure the skin. Inject one times 10 to the fifth cells and 20 microliters of PBS into the fourth left mammary fat pad at approximately one millimeter from the nipple. If the cells are tagged with luciferase, inject 100 microliters of D-luciferin in the lateral tail vein of the anesthetized mouse using a 0.5 milliliter insulin syringe with a 30 gauge hypodermic needle.Two minutes after the injection place the mouse in a bioluminescence imaging system with the mammary fat pad facing up and capture the image for 10 seconds. After imaging, place the mouse in the clean cage and allow it to recover from anesthesia. Monitor the growth of the primary tumor growth using a caliper.Calculate the tumor volume and resect the tumor when the primary tumor reaches 80 to 90 cubic millimeters volume. Set up the automated syringe pump with a 30 gauge one milliliter insulin syringe containing two percent propofol formulation. Induce anesthesia in an induction chamber with three percent sevoflurane or isoflurane.Then transfer the anesthetized mouse to a heating pad at 37 degrees Celsius and maintain the anesthesia with two to three percent sevoflurane or isoflurane using a nose cone. Adjust the delivery of sevoflurane as required during intravenous cannulation to maintain a stable depth of anesthesia demonstrated by loss of pedal reflex and respiratory rate less than 100 breaths per minute. Apply aqueous lubricant to the eyes to prevent drying of the cornea, and then inject 0.05 milligram per kilogram of buprenorphine subcutaneously.To deliver the propofol based total intravenous anesthesia, cannulate the lateral tail vein using a sterile 30 gauge hypodermic needle attached to a sterile polyurethane catheter. Confirm correct placement by blood flashback into the catheter. Administer two percent propofol as an initial bolus of 27 milligrams per kilogram over one minute to begin total intravenous anesthesia.Cease the sevoflurane administration. Maintain propofol infusion at a rate of 2.2 to 4.0 milligram per kilogram per minute for retaining stable anesthesia depth during the surgery. Shave the abdomen region and disinfect the surgical area with povidone-iodine solution.Make a one centimeter incision inferior to the tumor in the left fourth mammary fat pad. Use forceps and scissors to dissect the tumor and the left inguinal lymph node. If luciferase-tagged tumor cells are used inject d-luciferin in the lateral tail vein as demonstrated and capture the images for 60 seconds to obtain clear margins.If a residual tumor is identified, resect additional tissue from the mammary fat pad. Once hemostasis is achieved at the surgical site, close the skin using 5-0 nylon sutures. Wipe the wound with povidone-iodine solution.Upon completion of the surgery, stop the anesthesia and place the mouse in a clean cage on a heating pad to allow it to recover from anesthesia. Monitor the mouse every 15 minutes until the mouse returns to normal alertness. Then monitor and provide analgesia every 12 hours for 48 hours after the surgery.For primary tumor recurrence or distant recurrence assessment, monitor the mice once per week, commencing the week following surgery using a bioluminescence imaging system. Bioluminescence imaging was used to identify the distant tumor recurrence in the lungs after resection of the primary tumor. This model can be used to assess events that occurred during the perioperative period.24 hours after cancer surgery under propofol, circulating plasma cytokines were evaluated. Practices required to rapidly cannulate the lateral tail vein and minimize sevoflurane exposure duration. The sevoflurane should gradually be down-titrated as the propofol bolus is administered to prevent over-sedation.This method may be used to investigate the impact of propofol anesthesia in mouse models of cardiac surgery or critical illness, such as sepsis.

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Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle screws after spinal surgery, which is a serious concern. To address this problem, diverse types of pedicle screws have been examined to identify those with good fixation strength and osseointegration in spine bone. The porcine spine is a good alternative for the human spine in the evaluation of pedicle screws due to the anatomical size, mechanical characteristics, and cost. Although several studies have reported that pedicle screws are efficient in the porcine model, no study has described detailed protocols for the evaluation of a pedicle screw using the porcine model. Here, we describe a detailed method for evaluating transpedicular screws using an in vivo porcine lumbar spine model. The technical details for anesthesia, spine surgery, and harvest provided here will facilitate with the evaluation of the transpedicular screw fixation model.

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Here, we introduce a method to evaluate transpedicular screws by using an in vivo porcine lumbar spine model.

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Noninvasive Determination of Vortex Formation Time Using Transesophageal Echocardiography During Cardiac Surgery

Trans-mitral blood flow produces a three-dimensional rotational body of fluid, known as a vortex ring, that enhances the efficiency of left ventricular (LV) filling compared with a continuous linear jet. Vortex ring development is most often quantified with vortex formation time (VFT), a dimensionless parameter based on fluid ejection from a rigid tube. Our group is interested in factors that affect LV filling efficiency during cardiac surgery. In this report, we describe how to use standard two-dimensional (2D) and Doppler transesophageal echocardiography (TEE) to noninvasively derive the variables needed to calculate VFT. We calculate atrial filling fraction (&#946;) from velocity-time integrals of trans-mitral early LV filling and atrial systole blood flow velocity waveforms measured in the mid-esophageal four-chamber TEE view. Stroke volume (SV) is calculated as the product of the diameter of the LV outflow track measured in the mid-esophageal long axis TEE view and the velocity-time integral of blood flow through the outflow track determined in the deep transgastric view using pulse-wave Doppler. Finally, mitral valve diameter (D) is determined as the average of major and minor axis lengths measured in orthogonal mid-esophageal bicommissural and long axis imaging planes, respectively. VFT is then calculated as 4 &#215; (1-&#946;) &#215; SV/(&#960;D3). We have used this technique to analyze VFT in several groups of patients with differing cardiac abnormalities. We discuss our application of this technique and its potential limitations and also review our results to date. Noninvasive measurement of VFT using TEE is straightforward in anesthetized patients undergoing cardiac surgery. The technique may allow cardiac anesthesiologists and surgeons to assess the impact of pathological conditions and surgical interventions on LV filling efficiency in real time.

We describe a protocol to measure vortex formation time, an index of left ventricular filling efficiency, using standard transesophageal echocardiography techniques in patients undergoing cardiac surgery. We apply this technique to analyze vortex formation time in several groups of patients with differing cardiac pathologies.

Trans-mitral blood flow produces a three-dimensional rotational body of fluid, known as a vortex ring, that enhances the efficiency of left ventricular ...