KF and KO are co first authors along with KS

This work was supported in part by the AHPBA Fellowship (K.S.), NIH K23 CA148964-01 (L.Z.), Johns Hopkins School of Medicine Clinical Scientist Award (L.Z.), Viragh Foundation and the Skip Viragh Pancreatic Cancer Center at Johns Hopkins (E.M.J. and L.Z.), the National Pancreas Foundation (L.Z.), the Lefkofsky Family Foundation (L.Z.), the NCI SPORE in Gastrointestinal Cancers P50 CA062924 (E.M.J., L.Z.), the Lustgarten Foundation (L.Z.), and the Sol Goldman Pancreatic Cancer Center grants (K.S., B.H.E., L.Z.).

All animal experiments conformed to the guidelines of the Animal Care and Use Committee of the Johns Hopkins University, and animals were maintained in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Care (AAALAC).&#160;The surgical procedure is performed under sterile conditions in an operating room that undergoes a minimum of fifteen air exchanges per hour. All instruments are sterilized by autoclaving prior to the procedure and again with 70% ethanol in between each mouse during the procedure. All mice not expected to survive the surgical procedure are euthanized under CO2 for 3 min&#160;followed by cervical dislocation while still under anesthesia.
1. Cell Preparation
<ol>
	<li>Resuspend cultured Panc02 cells in phosphate buffered saline at 2 x107 cells/ml, and keep on ice.</li>
</ol>
2. Mouse Preparation
<ol>
	<li>Administer Ketamine (100 mg/kg) mixed with Xylazine (10 mg/kg) intraperitoneally to 8-12 week old C57BL/6 female mice in divided doses.</li>
	<li>Check to see that the toe pinch withdrawal reflex is abolished to ensure that the mouse is fully anesthetized.</li>
	<li>Administer additional doses of Ketamine (100 mg/kg) mixed with Xylazine (10 mg/kg) as needed to fully anesthetize the mice.</li>
	<li>Apply Puralube Vet Ointment to the eyes of the mice to prevent drying while the mice are under anesthesia.</li>
	<li>Shave the surgical area of the mice to avoid contamination of the wound.</li>
	<li>Prep the left subcostal area of the incision with 70% ethanol and then iodine.</li>
	<li>Place a surgical drape around the abdomen to maintain sterility.</li>
</ol>
3. Laparotomy
<ol>
	<li>Make a left subcostal incision in line with the left ear through the skin and through the peritoneum using a scalpel.</li>
	<li>Express the spleen through the incision by applying simultaneous digital pressure along the cranial and caudal aspects of the incision.</li>
	<li>Locate the splenic blood vessels at the inferior end of the spleen.</li>
	<li>Divide the spleen by placing two Horizon medium size ligating clips in the center of the spleen being careful to avoid damaging the splenic blood vessels at the splenic poles.</li>
	<li>Place the upper pole of the spleen back into the peritoneum to avoid contamination.</li>
</ol>
4. Tumor Injection
<ol>
	<li>Draw up 150 &#956;l of phosphate buffered saline into a 26 G&#160;x 5/8&#8221; syringe.</li>
	<li>Draw up 100 &#956;l&#160;of Panc02 (2 x106) cells into the same syringe maintaining the syringe in an upright position at all times during the injection.</li>
	<li>Dip the needle in a 70% ethanol solution.&#160;The needle is dipped in 70% ethanol to ensure sterility.</li>
	<li>Inject the cells slowly into the exposed hemispleen being sure the syringe is kept upright at all times. The bevel of the syringe should be observed at all times through the splenic capsule to avoid injecting the cells under the spleen. A whitening of the spleen and blood vessels should be observed upon injection.&#160;</li>
	<li>Elevate the spleen and apply one medium Horizon clip under the spleen to occlude the splenic blood vessels.</li>
	<li>Apply one small Horizon clip to the most distal aspect of the pancreas and splenic vessels.</li>
	<li>Ligate the pancreas and splenic vessels from the hemispleen by cutting above the Horizon clips.</li>
	<li>Place the mouse on its&#8217; side, and irrigate the incision with distilled water.</li>
</ol>
5. Abdominal Closure and Recovery from Anesthesia
<ol>
	<li>Close the peritoneum with a 4-0 running stitch.</li>
	<li>Apply two to three skin clips to close the skin incision.</li>
	<li>Administer 0.1 mg/kg buprenorphine or 5-10&#160;mg/kg Carprofen subcutaneously to alleviate post surgical pain.</li>
	<li>Place the mouse on a heating pad for recovery until mobile and the mouse demonstrates regular breathing patterns. Animals are only returned to the company of other animals after fully recovering from the surgical procedure.</li>
</ol>
6. Necropsy Examination and Harvesting of the Liver
<ol>
	<li>Between 30 to 60 days following the surgery, mice will begin to show clinical symptoms of metastasis and will require euthanasia. Signs of metastatic disease requiring humane euthanasia include enlarged abdomens and the development of ascites. Euthanize mice by inhalation of CO2.</li>
	<li>Following inhalation, perform cervical dislocation.</li>
	<li>After the animal has been determined to be non responsive, make an incision through the peritoneum to expose the abdomen using scissors.</li>
	<li>Using forceps and scissors, gently cut the liver out of the abdomen.</li>
	<li>Place the liver in OCT compound, and flash freeze using liquid nitrogen. Store the liver at -80&#176;C. Alternatively, the liver can be fixed in 16% neutral buffered formalin at RT&#160;for 24 hr and paraffin&#160;embedded.</li>
</ol>

<ol>
	<li>American Cancer Society. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cancer Facts &amp; Figures. , (2013).</li><li>Clark, C. E., Beatty, G. L., Vonderheide, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosurveillance+of+pancreatic+adenocarcinoma:+insights+from+genetically+engineered+mouse+models+of+cancer.">Immunosurveillance of pancreatic adenocarcinoma: insights from genetically engineered mouse models of cancer.</a> Cancer letters. 279, 1-7 (2009).</li><li>Rhim, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EMT+and+dissemination+precede+pancreatic+tumor+formation.">EMT and dissemination precede pancreatic tumor formation.</a> Cell. 148, 349-361 (2012).</li><li>Frese, K. K., Tuveson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+mouse+cancer+models.">Maximizing mouse cancer models.</a> Nature reviews. Cancer. 7, 645-658 (2007).</li><li>Corbett, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+chemotherapeutic+response+of+two+transplantable+ductal+adenocarcinomas+of+the+pancreas+in+C57BL/6+mice.">Induction and chemotherapeutic response of two transplantable ductal adenocarcinomas of the pancreas in C57BL/6 mice.</a> Cancer research. 44, 717-726 (1984).</li><li>Leao, I. C., Ganesan, P., Armstrong, T. D., Jaffee, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+depletion+of+regulatory+T+cells+allows+the+recruitment+of+mesothelin-specific+CD8+T+cells+to+the+antitumor+immune+response+against+a+mesothelin-expressing+mouse+pancreatic+adenocarcinoma.">Effective depletion of regulatory T cells allows the recruitment of mesothelin-specific CD8 T cells to the antitumor immune response against a mesothelin-expressing mouse pancreatic adenocarcinoma.</a> Clinical and translational science. 1, 228-239 (2008).</li><li>Jain, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+effect+of+a+granulocyte-macrophage+colony-stimulating+factor-transduced+tumor+vaccine+and+systemic+interleukin-2+in+the+treatment+of+murine+colorectal+cancer+hepatic+metastases.">Synergistic effect of a granulocyte-macrophage colony-stimulating factor-transduced tumor vaccine and systemic interleukin-2 in the treatment of murine colorectal cancer hepatic metastases.</a> Annals of surgical oncology. 10, 810-820 (2003).</li><li>Yoshimura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+targeting+of+antitumor+immune+responses+with+engineered+live-attenuated+Listeria+monocytogenes.">Selective targeting of antitumor immune responses with engineered live-attenuated Listeria monocytogenes.</a> Cancer research. 66, 1096-1104 (2006).</li><li>Yoshimura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+attenuated+Listeria+monocytogenes+effectively+treats+hepatic+colorectal+cancer+metastases+and+is+strongly+enhanced+by+depletion+of+regulatory+T+cells.">Live attenuated Listeria monocytogenes effectively treats hepatic colorectal cancer metastases and is strongly enhanced by depletion of regulatory T cells.</a> Cancer research. 67, 10058-10066 (2007).</li><li>Olino, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-associated+antigen+expressing+Listeria+monocytogenes+induces+effective+primary+and+memory+T-cell+responses+against+hepatic+colorectal+cancer+metastases.">Tumor-associated antigen expressing Listeria monocytogenes induces effective primary and memory T-cell responses against hepatic colorectal cancer metastases.</a> Annals of surgical oncology. 19, 597-607 (2012).</li><li>Hingorani, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trp53R172H+and+KrasG12D+cooperate+to+promote+chromosomal+instability+and+widely+metastatic+pancreatic+ductal+adenocarcinoma+in+mice.">Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice.</a> Cancer cell. 7, 469-483 (2005).</li><li>Marion, A., Aoudi, W., Basarab, A., Delachartre, P., Vray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+flow+evaluation+in+high-frequency,+40+MHz+imaging:+a+comparative+study+of+four+vector+velocity+estimation+methods.">Blood flow evaluation in high-frequency, 40 MHz imaging: a comparative study of four vector velocity estimation methods.</a> Ultrasonics. 50, 683-690 (2010).</li><li>Zheng, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosine+23+phosphorylation-dependent+cell-surface+localization+of+annexin+A2+is+required+for+invasion+and+metastases+of+pancreatic+cancer.">Tyrosine 23 phosphorylation-dependent cell-surface localization of annexin A2 is required for invasion and metastases of pancreatic cancer.</a> PloS one. 6, e19390 (2011).</li></ol>

The authors have no relevant conflicts of interest to disclose.

This preclinical murine model of pancreatic cancer metastasis to the liver via a hemispleen injection technique is the first model described that mirrors many of the clinical and immunological conditions of patients with metastatic disease to the liver. This model offers several advantages over other murine models. First, unlike transgenic models of pancreatic cancer in which only 30% of the mice develop metastases to the liver and the timing of metastases formation is quite variable, this model offers the advantage of c...

Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer related deaths in the United States1. The five year survival for patients with metastatic PDA is 2-12%1. Although adjuvant and neoadjuvant chemotherapy for pancreatic cancer is more effective today than ever, there still remains a desperate need for novel therapies.
The development of novel therapeutics necessitates reliable preclinical animal models. Although subcutaneous tumor models are straightforward to use, drawbacks include the inability of these tumors to metastasize and mimic the tumor progression and microenvironments seen in human cancers. Genetically engineered models, such as the genetically engineered mouse containing Kras and p53 knock-in oncogenic mutations (the KPC model)2 and the KPC model with a YFP lineage tracer (the PKCY model)3 allow for the study of naturally occurring tumors in immunocompetent mice. Additionally, the tumor microenvironment is unaltered and more closely resembles that seen in human tumor progression. Unfortunately, the timing of tumor development is variable within these models, which makes it difficult to study experimental therapies. Additionally, backcrossing of these mice requires a significant amount of time and financial commitment, which is not always feasible. Finally, implanted xenograft PDA mouse models necessitate immunocompromised mice, which have altered or absent tumor microenvironments. As a result, the efficacy of experimental therapies demonstrated in these preclinical models are often not reproducible in human clinical trials4.
This hemisplenectomy mouse model utilizes Panc02 tumor cells, which are a highly tumorigenic, methylcholanthrene induced pancreatic tumor cell line derived from C57BL/6 mice5,6. Additionally, other murine PDA cell lines derived from the KPC mice can be used in this model. Schulick et al. have previously developed a hemisplenectomy technique and created a syngeneic mouse model of colon cancer metastases7. This hemisplenectomy model (Figure 1) offers consistent and equal tumor inoculation in immunocompetent mice lending itself to the investigation of novel therapeutic agents7-10.

<table border="0" cellpadding="0" cellspacing="0" width="713">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell culture media and components</td>
			<td style="width:197px;"></td>
			<td style="width:119px;"></td>
			<td style="width:181px;">will vary depending on cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:197px;">Gibco by Life Technologies</td>
			<td style="width:119px;">10010-023</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 mL centrifigure tubes</td>
			<td style="width:197px;">Corning</td>
			<td style="width:119px;">430052</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Curved Operating Scissor</td>
			<td style="width:197px;">Roboz Surgical Store</td>
			<td style="width:119px;">RS-6835</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micro dissecting Graefe Forceps</td>
			<td style="width:197px;">Roboz Surgical Store</td>
			<td style="width:119px;">RS-5130</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Needle holder (regular)</td>
			<td style="width:197px;">World Precision Instruments, Inc</td>
			<td style="width:119px;"></td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3-0 or 4-0 suture</td>
			<td style="width:197px;"></td>
			<td style="width:119px;"></td>
			<td style="width:181px;">courtesy of dept of surgery, Johns Hopkins</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Weck Horizon Open Ligating Clip Applier, small, angled</td>
			<td style="width:197px;">Teleflex</td>
			<td style="width:119px;">137085</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Weck Horizon Open Ligating Clip Applier, medium, angled</td>
			<td style="width:197px;">Teleflex</td>
			<td style="width:119px;">237115</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Weck Horizon medium ligating clips</td>
			<td style="width:197px;">Teleflex</td>
			<td style="width:119px;">002200</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Weck Horizon small ligating clips</td>
			<td style="width:197px;">Teleflex</td>
			<td style="width:119px;">001200</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syringe, 1 cc, tb syringe, 3/8&#34; slip tip</td>
			<td style="width:197px;">Becton Dickinson</td>
			<td style="width:119px;">309625</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syringe, 1 cc, tb syringe, 5/8&#34; slip tip</td>
			<td style="width:197px;">Becton Dickinson</td>
			<td style="width:119px;">309597</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">9 mm Autoclip Applier</td>
			<td style="width:197px;">Mikron</td>
			<td style="width:119px;">427630</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">9 mm Autoclip</td>
			<td style="width:197px;">Becton Dickinson</td>
			<td style="width:119px;">427631</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heating pad</td>
			<td style="width:197px;">Sunbeam</td>
			<td style="width:119px;">CAT93</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70% Ethanol</td>
			<td style="width:197px;"></td>
			<td style="width:119px;"></td>
			<td style="width:181px;">sold by numerous vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine Hydrochloride&#160;</td>
			<td style="width:197px;">Hospira</td>
			<td style="width:119px;">22395DD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine hydrochloride</td>
			<td style="width:197px;">Sigma</td>
			<td style="width:119px;">X1251</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Panc02 tumor cells injected into the hemispleen (Figure 1) form liver metastases in 100% of the mice, while lung and peritoneal metastases are not observed if the technique is performed correctly. This technique has been performed on over one hundred mice using Panc02 cells in multiple, independent experiments, and reproducibly, all untreated mice die with liver metastases between 30 and 60 days following the hemisplenectomy procedure (Figure 2). To obtain statistical significance betwee...

Watch this Scientific Journal Video about A Preclinical Murine Model of Hepatic Metastases at JoVE.com

A Preclinical Murine Model of Hepatic Metastases

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, ...

a-preclinical-murine-model-of-hepatic-metastases

Research

JoVE Journal

Medicine

28.4K 次观看.  The Johns Hopkins University School of Medicine. 通过hemispleen注射技术进行肝转移的临床前，小鼠模型。 

视频: A Preclinical Murine Model of Hepatic Metastases

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 ...

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

Percutaneous Hepatic Perfusion (PHP) with Melphalan as a Treatment for Unresectable Metastases Confined to the Liver

Unresectable liver metastases of colorectal cancer can be treated with systemic chemotherapy, aiming to limit the disease, extend survival or turn unresectable metastases into resectable ones. Some patients however, suffer from side effects or progression under systemic treatment. For patients with metastasized uveal melanoma there are no standard systemic therapy options. For patients without extrahepatic disease, isolated liver perfusion (IHP) may enable local disease control with limited systemic side effects. Previously, this was performed during open surgery with satisfying results, but morbidity and mortality related to the open procedure, prohibited a widespread application. Therefore, percutaneous hepatic perfusion (PHP) with simultaneous chemofiltration was developed. Besides decreasing morbidity and mortality, this procedure can be repeated, hopefully leading to a higher response rate and improved survival (by local control of disease). During PHP, catheters are placed in the proper hepatic artery, to infuse the chemotherapeutic agent, and in the inferior caval vein to aspirate the chemosaturated blood returning through the hepatic veins. The caval vein catheter is a double balloon catheter that prohibits leakage into the systemic circulation. The blood returning from the hepatic veins is aspirated through the catheter fenestrations and then perfused through an extra-corporeal filtration system. After filtration, the blood is returned to the patient by a third catheter in the right internal jugular vein. During PHP a high dose of melphalan is infused into the liver, which is toxic and would lead to life threatening complications when administered systemically. Because of the significant hemodynamic instability resulting from the combination of caval vein occlusion and chemofiltration, hemodynamic monitoring and hemodynamic support is of paramount importance during this complex procedure.

Percutaneous Hepatic Perfusion PHP with Melphalan as a Treatment for Unresectable Metastases Confined to the Liver

In this manuscript, we describe percutaneous isolated hepatic perfusion with simultaneous chemofiltration as treatment for unresectable liver metastases. This procedure is performed under general anaesthesia in the angiosuite by an experienced team, consisting of an interventional radiologist, a clinical perfusionist and anaesthesiologist.

Unresectable liver metastases of colorectal cancer can be treated with systemic chemotherapy, aiming to limit the disease, extend survival or turn ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

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

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

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

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...