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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Research

JoVE Journal

Medicine

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A non-mammalian soil round worm, Caenorhabditis elegans, is a valuable model organism for toxicology studies due to its convenience and lack of animal ethics issues compared with mammalian animal systems. In this protocol, a detailed procedure of toxicological evaluation of chemicals in C. elegans is described. A clinical anticancer drug, etoposide, which targets human topoisomerase II and inhibits DNA replication of human cancer cells, was selected as a model testing chemical. Age-synchronized C. elegans eggs were exposed to either dimethyl sulfoxide (DMSO) or etoposide, and then the growth of C. elegans was monitored every day for 4 days by the stereo microscope observation. The total number of eggs laid from C. elegans treated with DMSO or etoposide was also counted by using the stereo microscope. Etoposide treatment significantly affected the growth and reproduction of C. elegans. By comparison of the total number of eggs laid from worms with different treatment periods of chemicals, it can be decided that the reproductive toxicity of chemicals on C. elegans reproduction is reversible or irreversible. These protocols may be helpful for both the development of various drugs and risk assessment of environmental toxicants.

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

A basic protocol to evaluate the toxicity of chemicals in a model animal, Caenorhabditis elegans, is described. The method is convenient and useful for the development of pharmaceuticals as well as for the risk assessment of various environmental pollutants.

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A ...

Transaxillary First Rib Resection for Treatment of the Thoracic Outlet Syndrome

Thoracic outlet syndrome (TOS) is a common disorder that causes a significant loss of productivity. The transaxillary first rib resection (TFRR) protocol has been used for the decompression of trapped neurovascular structures in the TOS. Among the other surgical procedures, the advantage of the TFRR is that it has the smallest rate of recurrence and better cosmetic outcomes. The disadvantage of TFRR is that it provides a narrow, and deep working corridor that makes obtaining vascular control challenging.

Here, we present a protocol of the transaxillary resection of the first rib for treatment of thoracic outlet syndrome caused by compression of the brachial plexus, subclavian vein and artery.

Thoracic outlet syndrome (TOS) is a common disorder that causes a significant loss of productivity. The transaxillary first rib resection (TFRR) protocol ...

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice.

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the ...

Modified Posterior Vertebral Column Resection for Patients with Thoracolumbar Kyphotic Deformity

Old compression vertebrae fracture or congenital kyphoscoliosis with abnormal vertebral body development and other diseases that invade the spine may cause severe thoracolumbar kyphotic deformity, often accompanied by intractable low back pain or compression of the spinal cord, leading to severe neurological symptoms or even paralysis. If conservative treatment cannot relieve the symptoms or correct the deformities, surgical treatment is usually needed. For severe kyphotic deformity, reconstruction of the physiological curvature and rigid fixation determine the prognosis of the patients. Osteotomy and orthopedics are the standard procedure for deformities with severe compression of the front and middle column, but the trauma to the patients is high, with a long operation time and massive blood loss. To avoid these disadvantages, we have developed a modified technique to remove the diseased vertebra unilaterally. In this technique, we use a modified trephine to resect the vertebral columns like in the pedicle screw technique by adding a locking instrument that can restrict the trephine to lower the risk of osteotomy and shorten the surgery time and blood loss.

We describe a modified technique for resecting the posterior vertebral column unilaterally based on a modified trephine for patients with thoracolumbar kyphotic deformity.

Old compression vertebrae fracture or congenital kyphoscoliosis with abnormal vertebral body development and other diseases that invade the spine may ...

An Apical Resection Model in the Adult Xenopus tropicalis Heart

It is known that in adult mammals, the heart has lost its regenerative capacity, making heart failure one of the leading causes of death worldwide. Previous research has demonstrated the regenerative ability of the heart of the adult Xenopus tropicalis, an anuran amphibian with a diploid genome and a close evolutionary relationship with mammals. Additionally, studies have shown that following ventricular apex resection, the heart can regenerate without scarring in X. tropicalis. Consequently, these previous results suggest that X. tropicalis is an appropriate alternative vertebrate model for the study of adult heart regeneration. A surgical model of cardiac regeneration in the adult X. tropicalis is presented herein. Briefly, the frogs were anesthetized and fixed; then, a small incision was made with iridectomy scissors, penetrating the skin and pericardium. Gentle pressure was applied to the ventricle, and the apex of the ventricle was then cut out with scissors. Cardiac injury and regeneration were confirmed by histology at 7-30 days post resection (dpr). This protocol established an apical resection model in adult X. tropicalis, which&#160;can be employed to elucidate the mechanisms of adult heart regeneration.

An Apical Resection Model in the Adult Xenopus tropicalis Heart

Xenopus tropicalis is an ideal model for regenerative research as many of its organs possess a remarkable regenerative capacity. Here, we present a method for constructing a heart injury model in X. tropicalis via apex resection.

It is known that in adult mammals, the heart has lost its regenerative capacity, making heart failure one of the leading causes of death worldwide. ...

Microscopic Cyst Resection for the Treatment of Patients Diagnosed with Epididymal Cyst

Epididymal cysts mostly occur in men aged 20-40 years old. Previous reports have covered concerns about postoperative complexes, including postoperative asoedema, hematoma, sustaining pain, and seminal tract obstruction in patients who have undertaken nonmicroscopic epididymal cyst resection or epididymal resection. Nonmicroscopic epididymal cyst surgery is suggested for patients with childbirth plans as a precaution. The treatment of male epididymal cysts via microtechnology is obviously a beneficial option; we took the lead in carrying out microscopic epididymal exploration and cyst resection surgery in China. From September 2017 to April 2021, 41 young and middle-aged male patients diagnosed with epididymal cysts underwent microtechnology treatment in a program titled &#34;microscopic epididymal exploration and cystectomy&#34;. The postoperative follow-up lasted for 3-50 months. The results confirmed that, as microscopic manipulation largely improved visualization of the subtle tissue structures of the epididymis, the cyst could be clearly dissected apart and completely removed intact under the microscope. Bleeding during the operation was significantly reduced (2-3 mL) and wound drainage was not required. According to follow-up data, microscopic treatment significantly reduced the incidence of postoperative scrotal hematoma, edema, and long-term postoperative pain, thereby promising a higher surgical success rate as well as recurrence prevention. Besides, preliminary experience and reflection suggest that microscopic epididymal exploration and cystectomy provide efficient preservation of the epididymal patency through refined treatment, while a better prognosis can be achieved. We recommend that surgery be carried out before the epididymal cyst develops to 0.8 cm in diameter, for fear that a larger epididymal cyst (&#62;0.9 cm in diameter) could cause the complete destruction of all tubules of the ipsilateral epididymis - a more severe case with damage to the testicular output network.

Here, we present a protocol to treat patients diagnosed with an epididymal cyst using microscopic cyst resection. Based on microscopic manipulation, a largely improved visualization of the subtle tissue structures of the epididymis was obtained, so the cyst could be clearly dissected apart and completely removed intact.

Epididymal cysts mostly occur in men aged 20-40 years old. Previous reports have covered concerns about postoperative complexes, including postoperative ...