Name: an in vivo mouse model of total intravenous anesthesia during cancer resection surgery
Uploaded: 2021-06-08T14:30:00.000+00:00
Duration: 6 min 40 s
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
<ol>
	<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 imaging to enable cancer recurrence monitoring after resection surgery11 (see step 4). 
	NOTE: The cell line and the medium mentioned above were used in this study.</li>
	<li>Grow cells at 37 &#176;C with 5% CO2. Passage cells aseptically in a hood&#160;at &#60;80% confluency. Use low-passage cells in the logarithmic growth phase for optimal in vivo results.</li>
	<li>Lift the adherent cells with 0.5 mg/mL of trypsin in PBS with 10 mM EDTA; 2 mL for a T75 flask. When lifted, add medium to inactivate trypsin, and wash the cells in PBS.</li>
	<li>Count the cells using a hemocytometer. Dilute the cells in PBS for injection. For 66cl4 mammary cancer cells, inject 1 x 105 cells in 20 &#956;L of PBS per mouse.</li>
	<li>Place the cells on ice prior to the injection.</li>
</ol>
2. Generate a mouse model of breast cancer
<ol>
	<li>Use 4% isoflurane to anesthetize the mouse in an induction chamber. Then, maintain anesthesia with 2%-3% isoflurane using a nose cone. Confirm proper anesthetization by lack of response to toe pinch.</li>
	<li>Prepare the injection site by wiping the fourth left mammary fatpad area using a single-use alcohol swab.</li>
	<li>Draw up tumor cells (see step 1.4) into a 25 &#956;L Hamilton syringe attached to a sterile 27 G hypodermic needle.</li>
	<li>Inject the cells into the fourth left mammary fatpad. Use forceps to secure and lift the skin. Inject approximately 1 mm from the nipple.</li>
	<li>OPTIONAL: If cells are tagged with luciferase, confirm successful injection of tumor cells by bioluminescence imaging. Inject 100 &#956;L of 150 mg/kg D-luciferin into the lateral tail vein of the anesthetized mice using a 0.5 mL insulin syringe with a 30 G hypodermic needle.</li>
	<li>OPTIONAL: Place the mouse in a bioluminescence imaging system with the mammary fatpad facing up. Wait for 2 min from the luciferin injection for optimal tissue uptake of luciferin, then image for 10 s.</li>
	<li>Place the mouse in a clean cage and allow it to recover from anesthesia.</li>
	<li>Continue to monitor animal welfare as per the institutional animal ethics guidelines.</li>
</ol>
3. Induce stable anesthesia with intravenous delivery of propofol
<ol>
	<li>Monitor growth of the primary tumor using caliper measurement and calculate the tumor volume using the equation: Volume (mm3) = (length x (width)2 &#247; 2).</li>
	<li>Perform tumor resection surgery on mice when the primary tumor reaches the required volume (here, 80-90 mm3).</li>
	<li>Set up an automated syringe pump with a 30 G 1 mL insulin syringe containing propofol formulation (2% Lipuro propofol) (Figure 1A).</li>
	<li>Induce anesthesia of the mouse in an induction chamber with 3% sevoflurane or isoflurane. 
	NOTE: Here, sevoflurane was used as this is the predominant volatile used clinically.</li>
	<li>Transfer the mouse to a 37 &#176;C heating pad for the duration of the surgery. Briefly maintain anesthesia with 2%-3% sevoflurane using a nose cone.</li>
	<li>Apply aqueous lubricant to the eyes to prevent drying.&#160;</li>
	<li>Inject 0.05 mg/kg of buprenorphine subcutaneously for analgesia.</li>
	<li>To prepare for surgery, shave the abdomen and prepare the skin for surgery with an iodine-povidone solution. Wipe the skin using an alcohol or sterile wipe.</li>
	<li>To deliver propofol-based TIVA, cannulate the lateral tail vein using a sterile 30 G hypodermic needle attached to a sterile polyurethane catheter. Confirm correct placement by blood flashback into the catheter (Figure 1B). Adjust the delivery of sevoflurane as required during intravenous cannulation to maintain a stable depth of anesthesia demonstrated by loss of corneal and pedal reflex, and respiratory rate &#60; 100 breaths per min.</li>
	<li>Commence propofol-TIVA by administering 2% propofol as an initial bolus of 27 mg/kg for over 1 min. Cease sevoflurane administration.</li>
	<li>Continue the infusion of propofol at a maintenance rate of 2.2-4.0 mg/kg/min to maintain a stable depth of anesthesia for the duration of the surgery (Figure 1C).</li>
</ol>
4. Resect the primary tumor
<ol>
	<li>Make a straight&#160;1 cm incision inferior to the tumor in the region of the left fourth mammary fatpad. Carefully resect the tumor and the left inguinal lymph node using dissection with blunt forceps.</li>
	<li>OPTIONAL: If using luciferase-tagged tumor cells, use bioluminescence imaging to confirm clear surgical margins. Inject 150 mg/kg D-luciferin into the lateral tail vein, wait for 2 min, and then image for 60 s using a bioluminescence imaging system. If a residual tumor is identified, resect additional tissue from the mammary fatpad and re-image to achieve clear margins.</li>
	<li>Ensure hemostasis at the surgical site and close the skin using 5-0 nylon sutures. Ensure that the fur does not enter the surgical site.</li>
	<li>At the conclusion of resection surgery, cease anesthesia. Place the mouse in a clean cage on a 37 &#176;C heating pad and allow it to recover from anesthesia.</li>
	<li>Monitor every 15 min after anesthesia until the mouse has returned to normal alertness. Then, monitor the mouse every 12 h for 48 h after surgery.</li>
	<li>Administer 0.05 mg/kg of buprenorphine subcutaneously every 12 h for 48 h after surgery.</li>
	<li>After 7-10 days, remove sutures using sterile curved stitch cutters under brief sevoflurane or isoflurane anesthesia.</li>
</ol>
5. Track cancer recurrence with in vivo imaging
<ol>
	<li>Use bioluminescence imaging to track cancer recurrence after resection surgery non-invasively. Use a bioluminescence imaging system to monitor the mice once per week for evidence of primary tumor recurrence or distant recurrence, commencing the week following surgery.</li>
	<li>Induce anesthesia of the mouse in an induction chamber with 4% isoflurane. Then, transfer the mouse to a 37 &#176;C heating pad for the duration of anesthesia and maintain anesthesia with 2%-4% isoflurane using a nose cone.</li>
	<li>Apply aqueous lubricant to the eyes to prevent drying.</li>
	<li>Inject D-luciferin 150 mg/kg into the lateral tail vein. Wait for 2 min, then measure bioluminescence over a 60 s exposure to detect recurrence of the primary tumor or distant metastasis.</li>
	<li>If the primary tumor recurs and becomes palpable, commence monitoring of tumor growth using caliper measurements.</li>
	<li>At the end of the experiment, humanely kill the mice according to the approved protocol. Here, CO2 was used, followed by cervical dislocation.</li>
</ol>

<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>

The authors declare no competing financial interests.

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). The methodology will be useful for the evaluation of the effects of TIVA on cancer-related outcomes and its comparison with other anesthetic techniques such as volatile anesthesia.
In contrast to existing protocols that deliver a single intraperitoneal bolus of propofol for sedation during minor interventions such as blood sampling delivery10, this protocol allows prolonged intravenous delivery of propofol for maintenance of anesthesia during major interventions such as surgery. Both the induction and maintenance dose of propofol was carefully titrated to optimize anesthetic depth suitable for major surgical interventions while minimizing mortality from hypotension or cardiac arrest. It was found that hypotension could be avoided by using a strict induction dose of 27 mg/kg administered over 60 s, with concurrent down-titration and cessation of inhaled sevoflurane. Maintenance was achieved using an infusion of propofol at 2.2-4.0 mg/kg/min. During major surgical interventions, such as cancer resection, titration within this range was important to respond to and obtund the magnitude of surgical stimulation. This replicates clinical practice and prevents either over-dosing anesthesia, which can result in hypotension or death, or under-dosing, which can result in emergence from anesthesia, movement, or surgical stress.
A limitation of the model is the brief use of volatile anesthesia to induce anesthesia prior to canulation. This approach was chosen because of the ease of cannulation after induction of anesthesia, allowing the rapid delivery of a therapeutic dose of propofol to continue anesthesia. In addition, mice were briefly anesthetized with volatile anesthesia for tumor cell inoculation, to remove suture, and for imaging. Sevoflurane was used for volatile anesthesia during resection surgery as it is often used in clinical practice. However, isoflurane is also used in clinical practice. Future studies may use a single agent for all episodes of inhalation anesthesia. With experience, less than 2 min elapsed from loss of righting reflex in response to volatile anesthesia exposure to commencement of propofol dosing. Nonetheless, for analyses that intend to compare propofol-based TIVA with inhalational volatile anesthesia techniques, interpretation may be confounded by even the brief use of volatile anesthesia.
An alternative approach to volatile anesthesia induction is to cannulate the lateral tail vein of an awake mouse. While not suitable for all situations, this may provide an alternative to induction by inhalation anesthesia. However, movement of the awake mouse may result in the cannula being dislodged, leading to failure of anesthesia induction. In addition, movement of the needle can result in extravasation of propofol from the vein, which puts the tail at risk of tissue necrosis. This has welfare implications for the mouse, and any associated adrenergic activation as a result of physiological stress may impact the validity of the observed results11.
An additional potential limitation is the use of buprenorphrine as a postoperative analgesic. Opioids may modulate postoperative inflammatory and immune responses12,13. Buprenorphine was chosen for analgesia as its effects on the immune response are less than for other opiates13. Nevertheless, future studies might consider the use of non-opioid analgesic agents.
Despite advances in oncological therapy, local and distant cancer recurrence can occur after surgery and remains a dominant cause of mortality in cancer patients. Many patients will be exposed to anesthesia, often multiple times, during diagnostic and therapeutic operative procedures. A growing body of evidence from in vivo and in vitro&#160;studies implicates anesthetic agents in modulating the perioperative response to surgery and impacting diverse aspects of tumor cell biology14. To better understand the impact of anesthetic agents on cancer progression, the model of intravenous propofol anesthesia developed here will be important in future mechanistic preclinical research. This model may be used to interrogate the mechanisms underlying the effects of anesthetic agents on immunomodulation, perioperative inflammatory response, and tumor cell growth and invasion. Furthermore, this model could be extrapolated for use in non-cancer surgery research where anesthetic agents may have effects on other systems, such as cardiac surgery, trauma research, or critical illness (e.g., sepsis) as propofol is a common sedation used in intensive care units.

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><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 intravenous delivery.
After stable anesthesia was achieved by propofol-based TIVA, the primary mammary tumor was resected. In vivo bioluminescence imaging was used to confirm complete resection of the primary tumor (Figure 2). Regular monitoring of mice by non-invasive in vivo bioluminescence imaging identified distant recurrence by luciferase-tagged tumor cells in the lung (Figure 2). This method is also suitable to track local recurrence in the mammary fatpad.
In addition to tracking long-term events such as recurrence, the model may be used to assess events that occur during the perioperative period. These early events may provide mechanistic insight into the effects of anesthesia and other surgical factors on cancer-related outcomes. Twenty-four hours after&#160;the cancer surgery under propofol, a multiplex enzyme-linked immunosorbent assay was used to quantify circulating plasma cytokines (Figure 3). Cytokines were evaluated in 7 mice; the appropriate group size will be influenced by the effect size of the endpoint of interest.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62747/62747fig01.jpg" /> 
Figure 1: Experimental set-up for propofol-based TIVA. (A) A syringe pump is used to ensure the controlled delivery of propofol from a 1 mL insulin syringe. (B) Propofol is delivered into the lateral tail vein by intravenous catheter, connected to a syringe pump, via a 30 G needle. The asterisk shows the needle insertion point. (C) Schematic illustrating the plasma concentration of propofol achieved by sequential administration of a bolus followed by a constant infusion using the syringe pump, compared to delivery by bolus or infusion only. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62747/62747fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62747/62747fig02.jpg" /> 
Figure 2: Cancer progression after surgical resection of the primary mammary tumor under propofol-based TIVA. Non-invasive bioluminescence imaging of luciferase-tagged tumor cells was used to track initial growth of the primary tumor growth, successful surgical resection, and subsequent distant recurrence to the lung. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62747/62747fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62747/62747fig03.jpg" /> 
Figure 3: Circulating cytokine levels measured after cancer resection under propofol-based TIVA. Multiplex enzyme-linked immunosorbent assay was used to quantify plasma cytokines 24 h after surgery. Each data point represents data from one mouse. Lines show mean and standard error (N = 4-7). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62747/62747fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

an-vivo-mouse-model-total-intravenous-anesthesia-during-cancer

Nanyang Technological University

Research

JoVE Journal

Medicine

2.3K Views.  Monash University. Dit nieuwe protocol beschrijft chirurgische anesthesie bij muizen met behulp van propofol-anesthesie. De methode maakt onderzoek mogelijk naar propofol-anesthesie in muismodellen van ziekten. Het protocol maakt de evaluatie van totale intraveneuze anesthesie mogelijk en vermijdt het gebruik van vluchtige anesthesie.Het protocol kan worden gebruikt om te onderzoeken hoe propofol-anesthesie ziektegerelateerde uitkomsten beïnvloedt. Hier evalueren we bijvoorbeeld de resultaten van kanker...

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

Mouse Complete Stasis Model of Inferior Vena Cava Thrombosis

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE). In the United States (U.S.), the high morbidity and mortality rates make VTE a serious health concern 1-2. After heart disease and stroke, VTE is the third most common vascular disease 3. In the U.S. alone, there is an estimated 900,000 people affected each year, with 300,000 deaths occurring annually 3. A reliable in vivo animal model to study the mechanisms of this disease is necessary.
The advantages of using the mouse complete stasis model of inferior vena cava thrombosis are several. The mouse model allows for the administration of very small volumes of limited availability test agents, reducing costs dramatically. Most promising is the potential for mice with gene knockouts that allow specific inflammatory and coagulation factor functions to be delineated. Current molecular assays allow for the quantitation of vein wall, thrombus, whole blood, and plasma for assays. However, a major concern involving this model is the operative size constraints and the friability of the vessels. Also, due to the small IVC sample weight (mean 0.005 grams) it is necessary to increase animal numbers for accurate statistical analysis for tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent (ELISA), zymography, vein wall and thrombus cellular analysis, and whole blood and plasma assays 4-8.
The major disadvantage with the stasis model is that the lack of blood flow inhibits the maximal effect of administered systemic therapeutic agents on the thrombus and vein wall.

The mouse complete stasis model of inferior vena cava thrombosis yields quantifiable amounts of vein wall tissue and thrombus. It has proven useful for evaluating interactions between the vein wall and the occlusive thrombus and in assessing the progression from acute to chronic inflammation.

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE).  In the United States (U.S.), the high morbidity and ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Cancer Research

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Implementation of Minimally Invasive Brain Tumor Resection in Rodents for High Viability Tissue Collection

The present protocol describes a standardized paradigm for rodent brain tumor resection and tissue preservation. In clinical practice, maximal tumor resection is the standard-of-care treatment for most brain tumors. However, most currently available preclinical brain tumor models either do not include resection, or utilize surgical resection models that are time-consuming and lead to significant postoperative morbidity, mortality, or experimental variability. In addition, performing resection in rodents can be daunting for several reasons, including a lack of clinically comparable surgical tools or protocols and the absence of an established platform for standardized tissue collection. This protocol highlights the use of a multi-functional, non-ablative resection device and an integrated tissue preservation system adapted from the clinical version of the device. The device applied in the present study combines tunable suction and a cylindrical blade at the aperture to precisely probe, cut, and suction tissue. The minimally invasive resection device performs its functions via the same burr hole used for the initial tumor implantation. This approach minimizes alterations to regional anatomy during biopsy or resection surgeries and reduces the risk of significant blood loss. These factors significantly reduced the operative time (&#60;2 min/animal), improved postoperative animal survival, lower variability in experimental groups, and result in high viability of resected tissues and cells for future analyses. This process is facilitated by a blade speed of ~1,400 cycles/min, which allows the harvesting of tissues into a sterile closed system that can be filled with a physiologic solution of choice. Given the emerging importance of studying and accurately modeling the impact of surgery, preservation and rigorous comparative analysis of regionalized tumor resection specimens, and intra-cavity-delivered therapeutics, this unique protocol will expand opportunities to explore unanswered questions about perioperative management and therapeutic discovery for brain tumor patients.

The present protocol describes a standardized resection of brain tumors in rodents through a minimally invasive approach with an integrated tissue preservation system. This technique has implications for accurately mirroring the standard of care in rodent and other animal models.

The present protocol describes a standardized paradigm for rodent brain tumor resection and tissue preservation. In clinical practice, maximal tumor ...

Optimized Venous Thrombosis Assay in a Mouse Cancer Model Using Vascular Clips

Venous Thrombosis Assay in a Mouse Model of Cancer

This methodology paper highlights the surgical nuances of a rodent model of venous thrombosis, specifically in the context of cancer-associated thrombosis (CAT). Deep venous thrombosis is a common complication in cancer survivors and can be potentially fatal. The current murine venous thrombosis models typically involve a complete or partial mechanical occlusion of the inferior vena cava (IVC) using a suture. This procedure induces a total or partial stasis of blood and endothelial damage, triggering thrombogenesis. The current models have limitations such as higher variability in clot weights, significant mortality rate, and prolonged learning curve. This report introduces surgical refinements using vascular clips to address some of these limitations. Using a syngeneic colon cancer xenograft mouse model, we employed customized vascular clips to ligate the infrarenal vena cava. These clips allow residual lip space similar to a 5-0 polypropylene suture after IVC ligations. Mice with the suture method served as controls. The vascular clip method resulted in a consistent reproducible partial vascular occlusion and greater clot weights with less variability than the suture method. The larger clot weights, greater clot mass, and clot to the IVC luminal surface area were expected due to the higher pressure profile of the vascular clips compared to a 6-0 polypropylene suture. The approach was validated by gray scale ultrasonography, which revealed consistently greater clot mass in the infrarenal vena cava with vascular clips compared to the suture method. These observations were further substantiated with the immunofluorescence staining. This study offers an improved method to generate a venous thrombosis model in mice, which can be employed to deepen the mechanistic understanding of CAT and in translational research such as drug discovery.

Author Spotlight: Advancing Cancer Associated Thrombosis Research in Rodent Models 

This article aims to present an optimized method for assessing venous thrombosis in a mouse cancer model, using vascular clips to achieve venous ligation. Optimization minimizes variability in thrombosis-related measurements and enhances relevance to human cancer-associated venous thrombosis.

This methodology paper highlights the surgical nuances of a rodent model of venous thrombosis, specifically in the context of cancer-associated thrombosis ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Immunology and Infection

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Neuroscience

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing (Neo)adjuvant Therapies

Surgery is often the first treatment for many solid tumors. However, local relapses frequently occur following primary tumor resection, despite adjuvant or neo-adjuvant therapies. This occurs when surgical margins are insufficiently tumor-free, resulting in residual cancer cells. From a biological and immunological perspective, surgery is not a null event; the wound healing environment is known to induce both pro- and anti-tumorigenic pathways. As a consequence, preclinical models for drug development aimed at preventing local relapse should incorporate surgical resection when testing new (neo)adjuvant therapies, to model the clinical settings in patients treated with surgery.
Here, we describe a mouse model of incomplete surgical resection of WEHI 164 soft tissue sarcoma that allows testing of (neo)adjuvant therapies in the setting of a wound healing response. In this model, 50% or 75% of the tumor is removed, leaving behind some cancer tissue in situ to model gross residual disease after surgery in the clinical setting. This model allows testing therapies in the context of surgery while also considering the wound healing response, which may affect the efficacy of (neo)adjuvant treatments. The incomplete surgical resection results in reproducible regrowth of the tumor in all mice in the absence of adjuvant therapy. Adjuvant treatment with checkpoint blockade results in reduced tumor regrowth. This model is thus appropriate for testing therapies in the context of debulking surgery and its associated wound healing response and can be extended to other types of solid cancer.

A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing Neoadjuvant Therapies

In this protocol, we describe a mouse model of incomplete surgical resection of soft tissue sarcoma for testing (neo)adjuvant therapies.

Surgery is often the first treatment for many solid tumors. However, local relapses frequently occur following primary tumor resection, despite adjuvant ...

Surgical Cisterna Magna Injection: A Method for Administering Tumor Cells Directly into Central Nervous System of Murine Model

Source: Law, V. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/62033/a-murine-ommaya-xenograft-model-to-study-direct-targeted-therapy">A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease.</a> J. Vis. Exp. (2021)
In this video, we describe the procedure to deliver human-derived tumor cells into the cisterna magna of a mouse&#8217;s brain. The orthotopic mouse model can be used to study metastatic spread of cancer to the cranial meninges.

Source: Law, V. et al. A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease. J. Vis. Exp. (2021)
In this video, we ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video