Name: the influence of liver resection on intrahepatic tumor growth
Uploaded: 2016-04-09T14:00:00
Description: Watch this Scientific Journal Video about The Influence of Liver Resection on Intrahepatic Tumor Growth at JoVE.com

Special acknowledgements go to Dr. Benjamin Motsch for his assistance in technical questions. The authors would also like to acknowledge Dr. Marcus Forschner and Birk M&#252;ller for their multimedia support, Erica Magelky for her editorial expertise and Lisa Hornung, Dr. Roland Jurgons and Professor Stephan von H&#246;rsten (all from the Franz-Penzoldt-Center, University of Erlangen) for their professionalism in animal handling and care. We thank Professor Michael Neumaier at the Institute of Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, Germany for providing MC38 tumor cells.
The present work was performed in fulfillment of the requirements for obtaining the degree &#34;Dr. med.&#34; at the Friedrich-Alexander-University Erlangen-N&#252;rnberg (FAU).

The government of Middle Franconia in Bavaria, Germany, granted permission for the procedures described. Any similar experiments require prior authorization by the appropriate authorities.
Note: The following manual can be used for previously discussed groups A through C. Steps that have to be left out in groups A and B are marked accordingly.
1. Preparations
<ol>
	<li>Put on gloves, place the polystyrene pad underneath the microscope, and focus the lens on the area slightly above the pad.</li>
	<li>Split...

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+pathogens+of+laboratory+mice,+rats,+and+rabbits+and+their+effects+on+research.">Natural pathogens of laboratory mice, rats, and rabbits and their effects on research.</a> Clin. Microbiol. Rev. 11, 231-266 (1998).</li><li>FELASA working group on revision of guidelines for health monitoring of rodents and rabbits. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FELASA+recommendations+for+the+health+monitoring+of+mouse,+rat,+hamster,+guinea+pig+and+rabbit+colonies+in+breeding+and+experimental+units.">FELASA recommendations for the health monitoring of mouse, rat, hamster, guinea pig and rabbit colonies in breeding and experimental units.</a> Lab. 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Previous experiments performing surgery in rodents have been able to identify certain variables that could serve as sources for bias. In order to obtain reliable and valid results, consider the following precautions.
Routine pre-op fasting can lead to liver steatosis12, which may inhibit liver regeneration13,14. It is therefore not recommended. The highest mitotic activity of hepatocytes varies throughout the day15. If possible, conduct the procedures at a cert...

Colorectal cancer (CRC) accounts for nearly 9% of all malignant tumors. It is the third most common cancer, both in the U.S. and worldwide. Global mortality rates from CRC range from 300,000 to over 500,000 per year1. Twenty percent of patients suffer from liver metastases upon discovery of their colorectal tumor. Resectable metastases are normally treated by a partial liver resection2,3. Improved surgical techniques, new multimodal strategies and new definitions of resectable metastases render the therapy of a partial liver resection possible for an increasing number of patients4.
Recurrence of secondary liver malignancies, however, is a challenging clinical sequalae in modern gastrointestinal surgery. Patients with CRC who underwent resection of liver metastases have a 30 to 50% chance of developing a new tumor in their remnant liver5. Therefore, there is a need for further research on the mechanisms involved in recurrence of liver metastases.
A liver resection of about 70% is normally compensated within a few weeks by the remaining hepatic tissue. This regeneration involves multiple mechanisms, including cytokines like Interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-&#945;), hepatocyte growth factor (HGF), transforming growth factor beta (TGF-&#946;), vascular endothelial growth factor (VEGF), matrix metalloproteases (MMP-2 and MMP-9) and CXC-Chemokines6-11. These substances support hepatic regeneration and may also be responsible for the high recurrence rates of primary and secondary liver malignancies by inducing the growth of small tumor cell deposits in the remaining liver which are not detected by routine clinical imaging. This causality has not been proven so far.
The following hypothesis was established. After partial liver resection, the proliferation factors that are responsible for liver hypertrophy may also induce the growth of previously undiscovered tumor cells in the liver. A mouse model was designed which combined the techniques of liver resection and tumor induction. Thirty athymic nude-foxn1nu/nu mice were divided into three groups of ten animals each. Each of them was treated with either a laparotomy alone (Group A), a 30% liver resection (Group B) or a 70% liver resection (Group C). Animals in all groups subsequently received a tumor cell injection into a defined remaining part of the liver, to simulate dormant tumor cells. Animals where observed for two weeks and then evaluated for tumor growth and liver hypertrophy.
The objective was to create a model that could be used to search for the molecular and pathogenetic factors that may play a role in post-hepatectomy tumor formation. This method may be helpful in assessing: the origin of endocrine factors involved in liver regeneration; the responsible mechanisms for intrahepatic tumor growth after liver resection; and the liver resection volume necessary for intrahepatic tumor growth induction. The following method has only been performed on animals because they promise to contribute to the understanding of fundamental biological principles and to the development of knowledge that can be expected to benefit humans by improved treatment options. Due to the mechanisms involved in these matters, it had to be examined in vivo, as in vitro methods may not provide a realistic representation of the human pathology.
These investigations may lead to the discovery of relevant targets for prophylactic treatment options for decreasing tumor recurrence.

<table border="0" cellpadding="0" cellspacing="0" width="844">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Equipment</td>
			<td style="width:217px;"></td>
			<td style="width:143px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Operation Microscope</td>
			<td style="width:217px;">Zeiss</td>
			<td style="width:143px;">OPMI-1 FC S21</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Induction Cage (Plexiglas Box)</td>
			<td style="width:217px;">UNO BV, Netherlands</td>
			<td style="width:143px;">180000132</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Flowmeter + Connection Kit</td>
			<td style="width:217px;">UNO BV, Netherlands</td>
			<td style="width:143px;">180000008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">UNO Vaporizer Sigma Delta</td>
			<td style="width:217px;">UNO BV, Netherlands</td>
			<td style="width:143px;">180000002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Key Filler for Anesthetic</td>
			<td style="width:217px;">UNO BV, Netherlands</td>
			<td style="width:143px;">180000010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Activated Charcoal Filter Adsorber</td>
			<td style="width:217px;">UNO BV, Netherlands</td>
			<td style="width:143px;">180000140</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Gas Exhaust Unit</td>
			<td style="width:217px;">UNO BV, Netherlands</td>
			<td style="width:143px;">180000118</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Face Mask for mouse</td>
			<td style="width:217px;">UNO BV, Netherlands</td>
			<td style="width:143px;">180000065</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Vaporizer Stand</td>
			<td style="width:217px;">UNO BV, Netherlands</td>
			<td style="width:143px;">180000006</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Heat lamp</td>
			<td style="width:217px;">Physitemp Instruments</td>
			<td style="width:143px;">HL-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Styrofoam Pad</td>
			<td style="width:217px;">RAYHER</td>
			<td style="width:143px;">30074000&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Third Hand Tool</td>
			<td style="width:217px;">TOOLCRAFT&#160;</td>
			<td style="width:143px;">ZD-10F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Precision Scales</td>
			<td style="width:217px;">Kern</td>
			<td style="width:143px;">EW 220-3NM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Scales&#160;</td>
			<td style="width:217px;">Kern</td>
			<td style="width:143px;">EMB 500-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Sliding Caliper</td>
			<td style="width:217px;">MIB</td>
			<td style="width:143px;">MIB 82026100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Microdissection forceps</td>
			<td style="width:217px;">Braun/Aesculap</td>
			<td style="width:143px;">BD195R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Microdissection scissors</td>
			<td style="width:217px;">Braun/Aesculap</td>
			<td style="width:143px;">FD100R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Microdissection needle holder</td>
			<td style="width:217px;">Braun/Aesculap</td>
			<td>BM563R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Retractor</td>
			<td style="width:217px;">Fine Science Tools (F.S.T.)</td>
			<td style="width:143px;">No. 17001-0</td>
			<td>Type: Bowmann</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Clamp</td>
			<td style="width:217px;">Braun/Aesculap</td>
			<td style="width:143px;">BJ002R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Expendable Items</td>
			<td style="width:217px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:845px;">(NOTE: Quantities are per animal and procedure)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Foliodrape sterile cover (45x75cm)</td>
			<td style="width:217px;">Hartmann</td>
			<td style="width:143px;">2775001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Sterile Cotton Swabs (2x)</td>
			<td style="width:217px;">Hartmann</td>
			<td style="width:143px;">4700151</td>
			<td>Peha</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Sterile fluid (0,9%NaCl)</td>
			<td style="width:217px;">Braun</td>
			<td style="width:143px;">3570310</td>
			<td>PZN=04454809</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Disinfectant (Softasept - 250ml)</td>
			<td style="width:217px;">Braun</td>
			<td style="width:143px;">3887138</td>
			<td>PZN=0762008808505018</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">2 x 1ml syringe (Injekt-F )</td>
			<td style="width:217px;">Braun</td>
			<td>9166017V</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">26G canula (Sterican) - for Carprofen injection</td>
			<td style="width:217px;">Braun</td>
			<td>4665457</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">30G canula (Sterican)&#160; - for Tumor injection</td>
			<td style="width:217px;">Braun</td>
			<td>4656300</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Caprofen (=Rimadyl)</td>
			<td style="width:217px;">Pfizer</td>
			<td style="width:143px;">QM01AE91</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Metamizole (=Novaminsulfon)</td>
			<td style="width:217px;">Ratiopharm</td>
			<td style="width:143px;">16543.00.00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">4-0 Vicryl suture</td>
			<td style="width:217px;">Ethicon</td>
			<td style="width:143px;">J835G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">5-0 Prolene suture</td>
			<td style="width:217px;">Ethicon</td>
			<td style="width:143px;">8618G</td>
			<td></td>
		</tr>
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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.

In our specific experiment, we included 30 athymic nude-foxn1nu/nu mice. They received a median laparotomy, tumor cell injections of 500,000 MC38 tumor cells (dissolved in 50 &#181;l of saline), and were subsequently treated with either a 70% liver resection, a 30% liver resection, or no further intervention.
After 14 days, almost complete regeneration of the remaining liver following 30% or 70% liver resections (liver hypertrop...

Watch this Scientific Journal Video about The Influence of Liver Resection on Intrahepatic Tumor Growth at JoVE.com

The Influence of Liver Resection on Intrahepatic Tumor Growth

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 ...

The recurrence of primary and secondary liver malignoma after partial liver resection remains a common, yet unsolved, problem. The exact mechanisms of tumor formation in the remaining parts of the liver are not understood so far. The following mouse model studied the proliferation of secondary liver malignoma after partial liver resection.In mice, a liver resection of about 70%is normally compensated by liver hypertrophy of the remaining parts of the liver within two to three weeks. The endocrine factors, which play a role in the liver's regeneration, may also induce the growth of liver malignoma. This video introduces a method which can be used to investigate the relationship between liver hypertrophy after resection and tumor growth.The experiment tried to prove that a partial liver resection accelerates the growth of liver malignoma. The shown illustration outlines the murine liver anatomy. To achieve a liver volume reduction of 65 to 70%resection of the median and left lateral lobe is necessary.To induce a liver tumor, a predefined volume of cancerous cells is injected into the inferior right lobe. A Plexiglas box is used for anesthetic induction. The mouse is then positioned and the abdomen disinfected.To provide adequate analgesia, five milligrams per kilogram body weight of Carprofen are administered. The skin incision is made along the median line of the abdomen, from the area above the xiphoid down to the lower abdomen. The xiphoid is carefully dissected from the surrounding tissue, and the abdominal fascia opened, along the linea alba.A stay suture then fixes the now exposed xiphoid bone to a retainer above the animal's head, in order to expose the different liver parts. The retractor is introduced to spread the abdominal wall apart, and gain better access to the abdominal organs. A saline soaked cotton swab is used to shift the median lobe downward.The now exposed falciform ligament is dissected. Median lobe and left lateral lobe are shifted upward against the diaphragm, in order to dissect the thin membrane between the left lateral lobe and the caudate lobe. A size 4.0 ligature is placed at the base of the left lateral lobe, which is subsequently returned to its original position using only the saline soaked cotton swab.It is now tied up using multiple knots. A gradual change of the lobe's superficial color to a more cyanotic tone is a sign of adequately interrupted blood supply. After trimming the ligature threads, the left lateral lobe is resected by cutting along the slightly diagonal line along its base.Now a new ligature is put in place at the median lobe's base. The lobe is likewise repositioned by applying gentle pressure from above with a cotton swab. The median lobe is similarly tied up with multiple knots.With the previously described color change, again indicating effective disruption of blood flow. The ligature threads are trimmed, and the lobe is removed. Because the inferior vena cava runs through the dorsal portion of the lobe, it is prone to damage by the ligature, which may lead to liver necrosis.This part of the lobe must therefore remain in situ. The inferior right lobe is demonstrated by moving the intestinal loops to the left. A 13 gauge syringe containing the tumor cell suspension is carefully introduced into the lobe until the syringe's tip is approximately in the lobe's center part.Inject the tumor cells by slowly applying gentle pressure to the plunger over a period of about 30 to 45 seconds. Afterwards, the penetrated area must be compressed for at least three minutes to control bleeding. Retractor and stay suture are removed, and a single button suture is performed to close the fascia.It is highly recommended to use two lines of sutures, as some animals may try to remove the sutures and reopen the laparotomy after the procedure. The skin is therefore separately adapted with single button knots. To provide adequate recovery and analgesia, the mouse is placed into a warm environment and the animal's drinking water is mixed with 500 milligrams per kilogram body weight of Metamizole.In the experiment, 30 animals were divided into three groups. All groups were injected with tumor cells. Group A was treated only with a laparotomy.Animals in groups B and C underwent liver resection of 30 and 70%of the original liver volume, respectively. It was then found that a liver resection of 70%correlated significantly positive with a greater tumor volume and tumor weight. Immunohistochemistry also showed a greater proliferation rate of tumor cells in animals from the liver resection groups, when comparing them with tumor cells from the laparotomy group.This correlation confirms clinical observations, although further examinations are required in the human system.

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