The authors thank Sara Minemyer and Alexander Comerci for the linguistic revision.

All animal protocols are approved by the Institutional Animal Care and Use Committee and adhered to the National Institutes of Health (NIH) Guidelines. Instruments used for any surgical procedure were thoroughly sterilized.
1. Initial preparation
<ol>
	<li>Before injecting cancer cells into the mouse spleen, autoclave and sterilize all instruments to be used during the procedure.</li>
	<li>Sterilize and/or autoclave a heating pad, surgical gloves, gauze, pairs of scissors, small clamps, vessel dilator, surgical forceps, and a needle holder.</li>
	<li>Prepare post-operative analgesic (0.1 mg/kg of buprenorphine) to be administered after splenectomy and every 12 h for 2 days.</li>
</ol>
2. Cell culture
<ol>
	<li>Ensure that cancer cells are free from mycoplasma contamination by using a mycoplasma ELISA kit.</li>
	<li>Prepare a 500 mL solution of Dulbecco&#39;s Modified Eagle medium (DMEM) culturing medium at 4 &#176;C for the culture of murine colorectal cancer cells (MC38). The culturing media should be supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL of penicillin, 100 &#181;g/mL of streptomycin, 15 mM of HEPES and 200 mM of L-glutamine.</li>
	<li>Culture cancer cells in a DNAse- and RNAse-free flask (75 cm2). Incubate the cell culture in a cell/tissue humidified incubator containing 5% CO2. Maintain temperature at 37 &#176;C.</li>
	<li>Once the proliferating cells reach 90&#8211;100% confluency, aspirate the old media, wash cells with 1x phosphate-buffered saline (PBS) and then treat them with 1x trypsin (0.25%) to detach cells from the flask.</li>
	<li>Collect cells in a 15 mL conical tube and centrifuge for 5 min at 700 x g.</li>
	<li>Aspirate the media and wash with 1x PBS twice by repeated centrifugation.</li>
	<li>Proceed to confirm cell viability by staining cells with trypan blue stain (0.4%).</li>
	<li>Resuspend cells to a concentration of 1 x 106 cells/ 100 &#181;L in 1x PBS. Pipet cells thoroughly to avoid any clumps. Keep cancer cells on ice prior to injection.</li>
</ol>
3. Injecting tumor cells
<ol>
	<li>Anesthetize 8&#8211;12-week-old male (C57BL6) mice by administering ketamine (150 mg/kg) and xylazine (12 mg/kg) intraperitoneally using a 1 mL 25 G (0.5 mm x 16 mm) needle.</li>
	<li>Shave the abdominal skin of the mice using clippers to avoid any postoperative infections.</li>
	<li>Place mice on the magnetic fixator retraction system. Confirm that the mice are completely under the effect of anesthesia by pinching a toe or the tail.</li>
	<li>Add saline drops into the eyes to avoid dryness during the procedure.</li>
	<li>Scrub povidone-iodine solution (7.5%) to the shaved abdominal wall to disinfect the skin before making a surgical incision.</li>
	<li>Initially lift the skin with the toothed forceps and make a midline incision with the help of surgical scissors. Then, lift the abdominal muscle and peritoneum to create a midline incision of approximately 3 cm length (midabdominal to xiphoid process to expose the abdominal contents. Take caution not to extend the incision beyond the xiphoid process to avoid extensive bleeding.</li>
	<li>Place the hemostat at both sides of the incision and under the xiphoid process. Extend the abdomen by pulling the tail downward and taping it. Use the 6-inch sterile cotton tip applicator to separate and expose the spleen from the pancreatic fat tissue.</li>
	<li>Before injection into the spleen, vortex the cancer cells to avoid any cell clumps.</li>
	<li>Use a 0.5 mL 28 G (0.36 mm x 13 mm) insulin syringe for injection. Avoid air bubbles.</li>
	<li>Slowly and carefully inject 100 &#181;L of cells into the tip of the spleen. Place a cotton tip and add gentle pressure to avoid backflow into the abdominal region. A successful injection can be observed by identifying the change in the color of the liver during the injection.</li>
	<li>Moisten a sterile gauze with 1x PBS and place it over the dissected area.</li>
	<li>Transfer the mice onto a heating pad for 15 mins to allow cancer cells to circulate within the system.</li>
	<li>To proceed with surgical ischemia and reperfusion injury, follow steps 5.3-5.10.&#160; 
	NOTE: This procedure is to study the effect of I/R induced establishment of metastatic foci.</li>
</ol>
4.&#160;Splenectomy
<ol>
	<li>To perform a splenectomy, use a hand-held cautery device. Carefully lift the spleen with smooth forceps and cauterize the splenic blood vessels to avoid excessive bleeding. Remove spleen by transecting the vessels at the cauterized section.</li>
	<li>Immediately following the procedure, close the incision in a double layer pattern by first suturing the muscle layer and then the skin. Use 4-0 polypropylene sutures for both the abdominal wall and the skin.</li>
	<li>Before repeating the procedure on another animal, disinfect all instruments by either spraying them with 70% isopropanol or inserting them into a bead bath.</li>
	<li>Place mice back into original cages and look for signs of distress and post-surgical pain.</li>
	<li>Inject post-operative analgesic (Buprenorphine 0.1 mg/kg) every 12 h for 2 days to avoid post-surgical pain.</li>
</ol>
5. Ischemia reperfusion Injury
<ol>
	<li>At 5 days after the first laparotomy, anesthetize mice by administering ketamine (150 mg/kg) and xylazine (12 mg/kg) intraperitoneally using a 1 mL 25 G (0.5 mm x 16 mm) needle. Follow steps 3.3&#8211;3.4.</li>
	<li>Scrub povidone-iodine solution (7.5%) on the shaved abdomen of the mouse to disinfect the skin and perform a midline laparotomy as described above in step 3.6.</li>
	<li>Using two moistened cotton tips, gently move the intestine from the cavity to expose the associated structures, including the portal vein. Dissect the liver hilum free of the surrounding tissue.</li>
	<li>Lift up the median and left lateral lobes against the diaphragm. Separate the quadrate lobe from the left lateral lobe by dissecting the liver hilum with the spring scissors using an operating microscope to allow clear visibility towards the portal triad structure.</li>
	<li>Place a small moist cotton swab between the median lobe and right lateral lobe to create sufficient space for clamping. Using the vessel dilator forceps, carefully pass the 10 cm thread (4.0 polypropylene suture) to lift the portal triad. Occlude all structures in the portal triad (hepatic artery, portal vein, and bile duct) to the left and median liver lobes by placing a microvascular clamp using a micro-serrefine clamp applicator with lock.</li>
	<li>If the lobes do not show significant blanching, readjust the clamp by removing and reapplying. 
	NOTE: If the immediate blanching of the liver does not occur even after readjusting the clamp, carefully consider whether or not to proceed with the I/R.</li>
	<li>Remove the small cotton swab placed between the median and right lateral lobes. Gently replace the intestine into the abdominal cavity. Cover the abdominal wall with a moist gauze (soaked with 1x PBS) and cover with a plastic wrap to minimize evaporative loss.</li>
	<li>Place the mouse on the heating pad and apply the clamp for a period of 60 min.</li>
	<li>Throughout the ischemic interval, seek evidence of ischemia injury by visualizing the pale blanching of the right medial and left medial and lateral lobes.</li>
	<li>Initiate reperfusion by removing the clamps after the 60 min period. 
	NOTE: Evidence of reperfusion can be observed by an immediate color change of the median and left lateral lobes.</li>
	<li>Immediately following reperfusion, close the incision with a double layer suture pattern by first suturing the muscle layer and then the skin. Use the 4-0 polypropylene suture with the help of a needle holder to close the abdominal wall and the skin.</li>
	<li>Before repeating the procedure on another animal, disinfect all instruments by either spraying them with 70% isopropanol or inserting them into a heated bead bath.</li>
	<li>Place mice back into original cages and look for signs of distress and post-surgical pain.</li>
	<li>Inject post-operative analgesic (0.1 mg/kg of buprenorphine) every 12 h for 2 days to avoid post-surgical pain.</li>
	<li>For liver I/R sham mice, perform laparotomy, hilum dissection and abdominal sutures. 
	NOTE: The role of surgical stress influencing the establishment of liver metastases can be investigated through two different experimental designs. The above protocol (Model-1) is used to establish micrometastatic liver disease and study the effect of liver I/R on their growth (Figure 1A). Alternatively, liver I/R and tumor injection can be performed concurrently (Model-2) to study the effect of I/R injury in the establishment of new metastatic foci (Figure 1B). To do this, inject cancer cells into the spleen as described above and allow them to circulate for 15 min. Perform liver I/R or sham surgery after the circulating period for 60 min. Perform lateral splenectomy 60 min later, and then close the laparotomy incision.</li>
</ol>
6. Assessment of operated mice
<ol>
	<li>During the surgical procedure ensure that the mice are under the influence of stage III anesthesia by performing palpebral and corneal reflex test. Additional anesthetic dose must be given upon the signs of reflexes.</li>
	<li>Provide post-operative analgesic (Buprenorphine 0.1mg/kg) right after the surgery and every 12h for 2 days to avoid post surgical pain.</li>
	<li>Allow mice 30&#8211;60 min of recovery time from anesthesia. Constantly monitor mice and do not leave them unattended until complete recovery.</li>
	<li>Look for distress signs such as hunched back, closed eyes, slow movement, and failure to groom. Treat accordingly until mice return to their normal activity.</li>
	<li>Supplimental care including fluids, heat, Yohimbine reversal agent for xylazine, soft paper towel bedding (to avoid aspiration) should be provided after the surgery to improve the recovery period.</li>
</ol>
7. Assessment of liver ischemia reperfusion injury
<ol>
	<li>Immediately after applying the clamp, make sure the pale blanching of the median and left lateral lobes occurs compared to the caudate and quadrate lobes.</li>
	<li>Assess liver ischemia injury by measuring serum alanine transaminase (sALT), serum aspartate transaminase (sAST) and serum lactate dehydrogenase (sLDH) levels. The blood can be drawn from the facial vein to extract serum 3&#8211;6 h after the initiation of reperfusion. Perform liver histology to analyze the percent tumor area within the ischemic lobe.</li>
</ol>

<ol><li>Riihimäki, M., Hemminki, A., Sundquist, J., Hemminki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patterns+of+metastasis+in+colon+and+rectal+cancer%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Patterns of metastasis in colon and rectal cancer.</a> Scientific Reports. 6 (1), 29765(2016).</li>
<li>Oki, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+advances+in+treatment+for+colorectal+liver+metastasis%5BTitle%5D)+AND+%22Annals+of+gastroenterological+surgery%22%5BJournal%5D)">Recent advances in treatment for colorectal liver metastasis.</a> Annals of gastroenterological surgery. 2 (3), 167-175 (2018).</li>
<li>Wolpin, B. M., Mayer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systemic+treatment+of+colorectal+cancer%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Systemic treatment of colorectal cancer.</a> Gastroenterology. 134 (5), 1296-1310 (2008).</li>
<li>van Golen, R. F., Reiniers, M. J., Olthof, P. B., van Gulik, T. M., Heger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sterile+inflammation+in+hepatic+ischemia%2Freperfusion+injury%3A+present+concepts+and+potential+therapeutics%5BTitle%5D)+AND+%22Journal+of+Gastroenterology+and+Hepatology%22%5BJournal%5D)">Sterile inflammation in hepatic ischemia/reperfusion injury: present concepts and potential therapeutics.</a> Journal of Gastroenterology and Hepatology. 28 (3), 394-400 (2013).</li>
<li>Demicheli, R., Retsky, M. W., Hrushesky, W. J. M., Baum, M., Gukas, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effects+of+surgery+on+tumor+growth%3A+a+century+of+investigations%5BTitle%5D)+AND+%22Annals+of+Oncology%22%5BJournal%5D)">The effects of surgery on tumor growth: a century of investigations.</a> Annals of Oncology. 19 (11), 1821-1828 (2008).</li>
<li>Tohme, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+Extracellular+Traps+Promote+the+Development+and+Progression+of+Liver+Metastases+after+Surgical+Stress%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Neutrophil Extracellular Traps Promote the Development and Progression of Liver Metastases after Surgical Stress.</a> Cancer Research. 76 (6), 1367-1380 (2016).</li>
<li>Tseng, W., Leong, X., Engleman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Orthotopic+mouse+model+of+colorectal+cancer%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Orthotopic mouse model of colorectal cancer.</a> Journal of Visualized Experiments. (10), 484(2007).</li>
<li>Elkin, M., Vlodavsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tail+vein+assay+of+cancer+metastasis%5BTitle%5D)+AND+%22Current+Protocols+in+Cell+Biology%22%5BJournal%5D)">Tail vein assay of cancer metastasis.</a> Current Protocols in Cell Biology. 19 (19.2), (2001).</li>
<li>Thalheimer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+intraportal+injection+model%3A+A+practical+animal+model+for+hepatic+metastases+and+tumor+cell+dissemination+in+human+colon+cancer%5BTitle%5D)+AND+%22BMC+Cancer%22%5BJournal%5D)">The intraportal injection model: A practical animal model for hepatic metastases and tumor cell dissemination in human colon cancer.</a> BMC Cancer. 9 (1), 29(2009).</li>
<li>Frampas, E., Maurel, C., Thedrez, P., Le Saëc, P. R. emaud-, Faivre-Chauvet, A., Barbet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+intraportal+injection+model+for+liver+metastasis%3A+Advantages+of+associated+bioluminescence+to+assess+tumor+growth+and+influences+on+tumor+uptake+of+radiolabeled+anti-carcinoembryonic+antigen+antibody%5BTitle%5D)+AND+%22Nuclear+Medicine+Communications%22%5BJournal%5D)">The intraportal injection model for liver metastasis: Advantages of associated bioluminescence to assess tumor growth and influences on tumor uptake of radiolabeled anti-carcinoembryonic antigen antibody.</a> Nuclear Medicine Communications. 32 (2), 147-154 (2011).</li>
<li>Goddard, E. T., Fischer, J., Schedin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Portal+Vein+Injection+Model+to+Study+Liver+Metastasis+of+Breast+Cancer%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer.</a> Journal of Visualized Experiments. (118), (2016).</li>
<li>Limani, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+portal+vein+injection+for+the+design+of+syngeneic+models+of+liver+malignancy%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Gastrointestinal+and+Liver+Physiology%22%5BJournal%5D)">Selective portal vein injection for the design of syngeneic models of liver malignancy.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 310 (9), G682-G688 (2016).</li>
</ol>

The authors disclose no conflicts of interest that pertain to this work.

The animal model described in this manuscript is based upon two major approaches. The first is to recognize the ability of cancer cells to localize and proliferate in the liver lobes. The second is to study the effect of hepatic ischemia reperfusion injury influencing the tumor growth and metastases. This model permits the relevant study of liver metastases in the absence of secondary metastases in an immunocompetent mouse. The model is useful in addressing the questions of metastatic efficiency, such as cell survival ex...

The liver is one of the most common sites for the development of metastatic disease1. Mortality is almost invariably attributable to complications associated with tumor growth in the liver. In patients with metastatic solid tumors in the liver, surgery remains a crucial intervention for disease control and a possible curative approach. However, the vast majority of patients ultimately present with recurrent disease, predominantly in the liver2,3. During hepatic surgery, intraoperative bleeding is common, often necessitating blood transfusion and different technical approaches for control of bleeding, including vascular clamping methods. However, such measures cause hepatic ischemia/reperfusion (I/R) to the liver tissue. The adverse effects of I/R on hepatocellular function have been well documented. The liver I/R insult ignites inflammatory cascades during the restoration of blood flow via inflammatory pathways4. Not only does liver I/R injury contribute to liver failure, but current evidence also shows that I/R injury stimulates tumor cell adhesion, and promotes the incidence of metastases formation and the growth of existing micrometastatic disease5. We have previously reported that surgical stress induces activation of immune cells which not only helps in the growth of the primary tumor, but also facilitates metastases by capturing cancer cells within the circulation6.
Here we describe in detail a technique to establish a liver metastasis mouse tumor model. In this model, we also present a method to induce hepatic ischemia reperfusion injury which acts as a surrogate to the surgical stress present clinically during hepatectomies. The combined methods of cancer injection and hepatic I/R can successfully interpret the development of CRLM in patients who have undergone primary tumor resection.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Lonza</td><td>12-614F</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Lonza</td><td>900-108</td><td></td></tr><tr><td>L-Glutamin</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>Penicilin</td><td>Fisher scientific</td><td>15-140-122</td><td></td></tr><tr><td>Stretomysin</td><td>Fisher scientific</td><td>15-140-122</td><td></td></tr><tr><td>HEPES</td><td>Fisher Scientific</td><td>SH3023701</td><td></td></tr><tr><td>Trypsin</td><td>Hyclone</td><td>sh30042.02</td><td></td></tr><tr><td>Cell culture Flask 75cm</td><td>5 Cells Star</td><td>658170</td><td></td></tr><tr><td>15ml PP Conical Tubes</td><td>BioExcell</td><td>41021037</td><td></td></tr><tr><td>Trypan Blue Stain</td><td>Giibco</td><td>15250-061</td><td></td></tr><tr><td>Gauze</td><td>Fisherbrand</td><td>1376152</td><td></td></tr><tr><td>Cautry</td><td>Bovie</td><td>AA01</td><td></td></tr><tr><td>Microvascular clamp</td><td>Finescience tools</td><td>18055-03</td><td></td></tr><tr><td>Micro-Serrefine clamp applicator with lock</td><td>Fine science toosl</td><td>FST-18056-14</td><td></td></tr><tr><td>Spring scissor</td><td>Fine science toosl</td><td>FST-15021-15</td><td></td></tr><tr><td>Vessel Dilator</td><td>Fine science toosl</td><td>FST-00276-13</td><td></td></tr><tr><td>Magnetic fixator Retraction system</td><td>Fine science toosl</td><td>FST-18200020</td><td></td></tr><tr><td>Micro-Adson Forceps</td><td>Fine science toosl</td><td>FST-11019-12</td><td></td></tr><tr><td>Micro-Adson Forceps</td><td>Fine science toosl</td><td>FST-11018-12</td><td></td></tr><tr><td>4-0 polypropylene suture</td><td>Ethicon</td><td>K881H</td><td></td></tr><tr><td>Needle holder</td><td>Harvard Apparatus</td><td>72-8826</td><td></td></tr><tr><td>Heating Pad</td><td>Fisher scientific</td><td>1443915</td><td></td></tr><tr><td>Clipper</td><td>Oster</td><td>559A</td><td></td></tr><tr><td>Povidone-Iodine solution</td><td>Medline</td><td>MDS093945</td><td></td></tr><tr><td>Syringe 1ml 25G</td><td>BD safety Glide</td><td>305903</td><td></td></tr><tr><td>Insulin syringe 0.5 ml</td><td>BD insulin Syringes</td><td>32946</td><td></td></tr><tr><td>Cotton -Tipped Applicator</td><td>Fisher Scientific</td><td>23-400-101</td><td></td></tr><tr><td>Surgical Microscope</td><td>Leica</td><td>LR92240</td><td></td></tr><tr><td>Mycoplasma Elisa Kit</td><td>Roche</td><td>11663925910</td><td></td></tr><tr><td>Ketamine</td><td>Putney</td><td>#056344</td><td></td></tr><tr><td>Xylazine</td><td>NADA</td><td>#139-236</td><td></td></tr><tr><td>ALT strip</td><td>Heska</td><td>15809554</td><td></td></tr><tr><td>AST strip</td><td>Heska</td><td>15809542</td><td></td></tr><tr><td>LDH strip</td><td>Heska</td><td>15809607</td><td></td></tr></tbody></table>

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.

All wildtype (C57BL6) mice (n = 20) were subjected to the liver metastases model using the protocol described above. All injected mice with or without ischemia reperfusion injury survived until the date of sacrifice. The schematic diagram Figure 1A of a cancer-injected liver illustrates the clamping of the portal triad (hepatic artery, portal vein, and bile duct) which induces a partial liver ischemic (70%) insult towards the median and left lateral lobes. An increase in the number of liver ...

Watch this Scientific Journal Video about Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury at JoVE.com

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

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 ...

murine-model-metastatic-liver-tumors-setting-ischemia-reperfusion

Research

JoVE Journal

Cancer Research

11.6K Views.  University of Pittsburgh. Experimenteel en klinisch bewijs toont aan dat lever ischemie reperfusie de vorming van gemetastaseerde foci kan verhogen door circulerende kankercellen en de groei van micrometastatische ziekte. Dit reproduceerbare murinemodel maakt de relevante studie mogelijk van zowel de vestiging van metastasen als de groei van reeds bestaande tumor in de lever. Leverchirurgie, bloeden, en septische shock zijn allemaal geassocieerd met lever ischemie reperfusie en kan invloed hebben op de groei van uitge...

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

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

Medicine

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

Murine Model of Intestinal Ischemia-reperfusion Injury

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

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

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

The Influence of Liver Resection on Intrahepatic Tumor Growth

The high incidence of tumor recurrence after resection of metastatic liver lesions remains an unsolved problem. Small tumor cell deposits, which are not detectable by routine clinical imaging, may be stimulated by hepatic regeneration factors after liver resection. It is not entirely clear, however, which factors are crucial for tumor recurrence.
The presented mouse model may be useful to explore the mechanisms that play a role in the development of recurrent malignant lesions after liver resection. The model combines the easy-to-perform and reproducible techniques of defined amounts of liver tissue removal and tumor induction (by injection) in mice. The animals were treated with either a single laparotomy, a 30% liver resection, or a 70% liver resection. All animals subsequently received a tumor cell injection into the remaining liver tissue. After two weeks of observation, the livers and tumors were evaluated for size and weight and examined by immunohistochemistry.
After a 70% liver resection, the tumor volume and weight were significantly increased compared to a laparotomy alone (p &#60;0.05). In addition, immunohistochemistry (Ki67) showed an increased tumor proliferation rate in the resection group (p &#60;0.05).
These findings demonstrate the influence of hepatic regeneration mechanisms on intrahepatic tumor growth. Combined with methods like histological workup or RNA analysis, the described mouse model could serve as foundation for a close examination of different factors involved in tumor growth and metastatic disease recurrence within the liver. A considerable number of variables like the length of postoperative observation, the cell line used for injection or the timing of injection and liver resection offer multiple angles when exploring a specific question in the context of post-hepatectomy metastases. The limitations of this procedure are the authorization to perform the procedure on animals, access to an appropriate animal testing facility and acquisition of certain equipment.

A high incidence of tumor recurrence after resection of liver metastases remains an unsolved problem. The illustrated mouse model may be useful to investigate the reasons for such recurrences. It combines a liver resection model with intrahepatic tumor cell injection for the first time.

The high incidence of tumor recurrence after resection of metastatic liver lesions remains an unsolved problem. Small tumor cell deposits, which are not ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

Developing Murine Acute Kidney Injury Model Using Bilateral Ischemia-Reperfusion

Bilateral Renal Ischemia-Reperfusion Model for Acute Kidney Injury in Mice

Acute kidney injury (AKI) is defined as a rapid decline in renal function, in which persistent kidney dysfunction gradually progresses to chronic kidney disease (CKD) due to the irreversible loss of nephrons and their maladaptive repair. In recent years, the incidence of AKI has been increasing concerning diverse etiologies, including volume depletion, sepsis, nephrotoxicity, muscle injury, and major trauma, in which ischemia-reperfusion injury (IRI)&#160;accounts for most episodes. Development of the IRI model in mice is induced by surgical clamping of the renal pedicles, which provides powerful and controllable tools for preclinical models of AKI. Importantly, the IRI model is deployed at different stages of the AKI development, especially in the processes of AKI to CKD. Despite the IRI model being widely practiced in many laboratories, a series of variables still influence the results of this model. Here, we describe the procedure of IRI model development to provide a repeatable and reliable method for researchers to explore the underlying pathogenesis in the development of AKI and the progression of AKI to CKD.

The ischemia-reperfusion injury (IRI) model can be used at different stages of acute kidney injury (AKI) development, especially during AKI to chronic kidney disease (CKD) progression. Here, we describe the procedure for IRI model development in mice via a trans-abdominal approach, clamping renal pedicles via a vascular clip to induce ischemic injury.

Acute kidney injury (AKI) is defined as a rapid decline in renal function, in which persistent kidney dysfunction gradually progresses to chronic kidney ...

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

Samenvatting

Meer video's verkennen