This work is supported by grants from the Sock it to Sarcoma! Foundation, the Australian and New Zealand Sarcoma Association, the Children&#39;s Leukemia &#38; Cancer Research Foundation and Perpetual Philanthropy. W.J.L is supported by a Simon Lee Fellowship and a research fellowship from the National Health and Medical Research Council, and the Cancer Council WA.

Animals used in these experiments were obtained from the Animal Resource Centre (Perth, Western Australia). Animals were maintained under standard pathogen-free conditions at the Harry Perkins Institute of Medical Research Bioresources North Facility (Perth, Western Australia). All experiments were carried out following the protocol as approved by the Harry Perkins Institute of Medical Research Animal Ethics Committee. BALB/c mice of 8-12 weeks of age were used in these experiments. The WEHI 164 fibrosarcoma cell line was obtained from CellBank Australia (Westmead, NSW).
1. Inoculation of cells
<ol>
	<li>Preparation of cells and animals
	<ol>
		<li>Ensure that the cell line is maintained in the recommended media. For example, maintain WEHI 164 cell line in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 2 mM L-glutamine, 10% fetal bovine serum, 20 mM HEPES, 0.05 mM 2-mercaptoethanol, 100 U/mL penicillin, and 100 &#181;g/mL streptomycin. 
		NOTE: Passage cells at least 3 and up to 5 times after being removed from cryogenic storage. To ensure an optimum cell viability, cells should be split when they are between 70-80% confluent. Tumor cell lines should be tested for mycoplasma, as infection can alter the cell growth and influence the immune response in vivo.</li>
		<li>One day before inoculation, shave mice on the lower right flank using clippers. 
		NOTE: Female BALB/c mice, aged between 8-12 weeks, of normal weight (16 -22 grams) were used in this experiment.</li>
		<li>On the day of inoculation, harvest WEHI 164 cells when 70-80% confluent by trypsinization.
		<ol>
			<li>Aspirate the culture medium from the tissue culture flasks and then add sterile phosphate buffered solution (1x PBS), to remove remaining traces of fetal bovine serum (FBS).</li>
			<li>Aspirate the PBS from the tissue culture flasks. Add 3 mL of 0.05% trypsin (for a T75 flask) and then swirl the flask so that the whole surface of flask with cells is covered by trypsin.</li>
			<li>Incubate the flask at 37 &#176;C, 5% CO2 incubator for 3 min. Check cells periodically, by tapping on the sides of the flask to see if cells have dislodged.</li>
			<li>Remove flasks from cell culture incubator and add 5 mL of media supplemented with FBS to neutralize the trypsin. 
			NOTE: Do not leave cells in trypsin longer than necessary, as this can damage cells and lead to low cell viability.</li>
			<li>Pipet suspension multiple times to obtain a single cell suspension. Transfer cell suspension to a conical centrifuge tube.</li>
			<li>Pellet cells by spinning at 350 x g for 3 min.</li>
		</ol>
		</li>
		<li>Wash the cells three times in 1x PBS.
		<ol>
			<li>Resuspend cells in 50 mL of sterile 1x PBS and wash cells by pipetting cell suspension up and down. Pellet cells by spinning at 350 x g for 3 min.</li>
			<li>Aspirate the supernatant and resuspend cells in 15 mL of sterile 1x PBS. Wash cells by pipetting cell suspension up and down. Pellet cells by spinning at 350 x g for 3 min.</li>
			<li>Aspirate the supernatant and resuspend cells in exactly 10 mL of sterile 1x PBS. Wash cells as in step 1.1.4.2 and transfer a small amount (approximately 100 &#181;L) of cell suspension to an centrifuge tube for counting. Pellet cells by spinning at 350 x g for 3 min.</li>
		</ol>
		</li>
		<li>Determine the cell number using the Trypan blue exclusion method by either using a hemocytometer or an automated cell counter. Resuspend cells in sterile 1x PBS at a concentration of 5 x 106 cells/mL. Keep cell suspension on ice. 
		NOTE: The viability of tumor cells should be equal or above 80 % to ensure reproducible tumor growth.</li>
	</ol>
	</li>
	<li>Subcutaneous inoculation
	<ol>
		<li>Mix the cell suspension thoroughly and fill a syringe with a 26 G needle with 100 &#181;L of cell suspension (5 x 105 cells) in sterile 1x PBS. Repeat mixing of cells before loading the next syringe. 
		NOTE: Keep cells on ice throughout the procedure to maintain viability.</li>
		<li>Restrain the mouse appropriately, ensuring access to the lower-right flank. Inoculate the mouse subcutaneously on the shaved lower-right flank. 
		NOTE: Make sure the inoculation is not in the peritoneum by lifting the needle slightly, which should be visible under the skin. A bubble-like lump should form under the skin following inoculation.</li>
		<li>Monitor mice as required by the applicable ethics approval and perform surgical resection when the tumours have grown to a size of about 50 mm2.</li>
	</ol>
	</li>
</ol>
2. Partial surgical resection of the tumor
NOTE: This protocol requires TWO researchers; one for surgical procedures (SURGEON), and another for mouse monitoring (ASSISTANT).
<ol>
	<li>Surgery setup
	<ol>
		<li>On day 12 post inoculation, when tumors have reached a size of approximately 50 mm2, dose mice with 100 &#181;L (0.1 mg/kg) of buprenorphine s.c. in the scruff of the neck, 30 minutes prior to surgery.</li>
		<li>Set up the surgical area with a heat pad covered with bench coat and set up a nose cone for anaesthesia. Sterilize surgical tools prior to use, and between each animal using a heat bead sterilizer, allowing tools to cool before use.&#160;Have the following surgical equipment clean and within easy reach: chlorhexidine, swab, gauze, eye gel, two curved forceps, scissors, clip applicator, clip remover, clip refills (Figure 2A, 2B).</li>
		<li>Warm the heating chamber to 37 &#176;C and set up another heat pad for recovery (Figure 2C).&#160;Place sterilized tools on a sterile surface such as autoclaved pads.</li>
	</ol>
	</li>
	<li>Anesthesia
	<ol>
		<li>Place the mouse in the induction chamber and anesthetize the mouse with 4% isoflurane (4% in 100% oxygen at a flow rate of 1 L/min) until the breathing rate slows to approximately 60 breaths per minute (1 per second) (this usually takes &#60;1 min). 
		NOTE: Do not leave the mouse in the chamber for too long as that may lead to asphyxiation and death. Only have one mouse under anesthesia at a time.</li>
		<li>Transfer the mouse onto the heat pad on the surgery table, place the mouse with its nose in the nose cone and maintain the anesthetic state with 3-4% isoflurane in 100% oxygen at a flow rate of 0.5 L/min. Monitor the breathing rate to ensure that the depth of anesthesia is maintained. 
		NOTE: The ASSISTANT must monitor the breathing of the mouse throughout the surgery to ensure the correct level of anesthesia is maintained. Lower the anesthetic concentration if breathing becomes too slow or increase the concentration if the depth of anesthesia is too shallow. If the mouse begins gasping, remove mouse from the nose cone, decrease the anesthetic concentration, and wait until breathing normalises before placing on the nose cone again.</li>
		<li>Perform a &#34;pinch test&#34; and &#34;corneal reflex test&#34;14 to ensure that the mouse is fully anesthetized before commencing surgery. 
		NOTE: Movement of any part of the mouse is an indication that the mouse is not fully anesthetized. The animal should immediately be given additional anesthetic by increasing the anesthetic concentration.</li>
		<li>Cover the mouse&#39;s eyes with a small amount of ophthalmic gel to avoid eye dryness.</li>
	</ol>
	</li>
	<li>Surgical procedure (SURGEON)
	<ol>
		<li>Swab the surgical area 3 times with alcoholic chlorhexidine. Using forceps and a pair of scissors, make a 1 cm straight incision along the dorsal side, 3 mm away from the tumor (Figure 3A, 3B). 
		NOTE: Standardizing the incision to 1 cm in every mouse (using a ruler) allows for even assessment of wound healing between mice. Locating the incision 3 mm away from the tumor allows for subsequent intratumoral adjuvant therapy without leakage from the wound.</li>
		<li>Using tweezers, pull away the facia and subcutaneous fatty tissue between the tumor and peritoneum. The subcutaneous tumor is normally attached to the skin-side.</li>
		<li>Open the wound by gently holding the skin on the tumor bearing side using tweezers, and &#34;invert&#34; the tumor so that it is visible outside (Figure 3C, 3D). 
		NOTE: The section of tumor to be debulked should be closest to the opening, to have enough skin to close the wound. Be careful not to cut the skin when removing the tumor.</li>
		<li>Using a pair of scissors, cut away the tumor capsule from the half to remove, starting from the base of the tumor closest to the opening.</li>
		<li>For 50% debulk surgery, cut across the middle of the tumor. Using curved forceps, scoop up the section of the tumor to be removed (50%); scoop up any remnants from the debulked area.</li>
		<li>For 75% debulk, perform a 50% tumor debulk as in part 2.3.5 above. Then cut in half the remaining 50% of tumor and scoop up 25% of the tumor, using curved forceps as described above.</li>
	</ol>
	</li>
	<li>Closing the surgical site
	<ol>
		<li>Place the remaining tumor back underneath the skin, and using forceps, pull the skin flaps together and line up the skin along the wound.</li>
		<li>Hold the skin together 5 mm from the edge of the wound, and use surgical clips to close the wound, starting on the side closest to the forceps. Apply as many clips as needed to ensure no underlying tissue is exposed. Generally, three to four clips are applied with 2 mm gaps between clips. 
		NOTE: If any clips is not well applied, remove it using a clip remover and replace with new clips.</li>
	</ol>
	</li>
	<li>Recovery of mice (ASSISTANT)
	<ol>
		<li>Allow the mice to recover by putting them into the warm (37 &#176;C) heating chamber.</li>
		<li>Place the mouse&#39;s cage on the heat pad. Monitor the mice in the heating chamber until they have recovered from the anesthetic (awake and walking) and then put the mice back into the cage. Leave the cage on the heat pad for a further 10 minutes, until the mice have become more active.</li>
		<li>Give the mice wet and soft food. Monitor the mice 1 hour after surgery for recovery and ensure clips remain in place.&#160;Ensure the cage is half on/half off the heat pad to allow animals to self-regulate temperature while unattended.</li>
		<li>Dose mice with 0.1 mg/kg buprenorphine (100 &#181;L subcutaneously in the scruff of the neck), 6-8 hours after surgery (at the end of the day). Monitor mice early the following morning, and dose mice again with 0.1 mg/kg buprenorphine (100 &#181;L subcutaneously in the scruff of the neck). Give more wet food as needed.</li>
		<li>Monitor mice daily for the next seven days. Clips may be removed after seven days using the clip remover.</li>
	</ol>
	</li>
	<li>Adjuvant or neoadjuvant treatment
	<ol>
		<li>Treat mice peri-operatively with (neo)adjuvant therapy at any given time, depending on the treatment of interest.</li>
		<li>For example, treat mice with one dose of 100 &#181;g of anti-CTLA-4 intraperitoneally (i.p.) on day 15 after inoculation, or with three doses of 200 &#181;g anti-PD-1 i.p. on day 15, 17 and 19 after inoculation.</li>
	</ol>
	</li>
	<li>Experimental controls
	<ol>
		<li>When using this model to assess the effects of inflammation/wound healing, consider using the following control groups: 1) No-surgery control (treatments can still be administered intratumorally); 2) Sham surgery control: A surgical incision is made in the skin; the tumor is manipulated and exposed, but no tumor tissue is removed; the wound is closed with clips.</li>
	</ol>
	</li>
</ol>

<ol>
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R., Moraco, N., Singer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+learned+from+the+study+of+10,000+patients+with+soft+tissue+sarcoma.">Lessons learned from the study of 10,000 patients with soft tissue sarcoma.</a> Annals of Surgery. 260 (3), 416-421 (2014).</li><li>Smith, H. 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No disclosures.

We provide a protocol for a mouse model of incomplete surgical resection of soft tissue sarcoma to test peri-operative therapies. We also standardized the surgical incision to allow assessment of wound healing between mice following treatment.
Tumor placement is an important part of this protocol. We have opted for a subcutaneous tumor model to allow easy surgical access to the tumor site and administration of local therapies with minimal burden on the mice. It is also important to ensure that...

Surgery remains the main treatment option for many solid tumors1, including soft tissue sarcoma2,3. Despite improvements in cancer surgery techniques, and combinations with (neo)adjuvant therapies, there is still a high risk of cancer relapse and metastasis following primary tumor resection4,5. In soft tissue sarcoma, relapses occur particularly locoregionally, at the site of surgery, resulting in increased morbidity and mortality. In the clinical setting, it can be difficult to obtain wide enough margins (e.g., due to anatomical constraints), resulting in incomplete resection and subsequent tumor recurrence6. Surgical stress and the subsequent process of wound healing are known to create an immunosuppressive tumor microenvironment favorable for tumor recurrence7,8. Therefore, the discovery and development of new therapies for soft tissue sarcoma, particularly immunotherapies, should ideally take the surgical wound healing response into account.
Most preclinical studies for adjuvant therapies are initially carried out using subcutaneous syngeneic or xenotransplant mouse models, without incorporating the surgical stress and wound healing response9,10. Therefore, we developed a syngeneic subcutaneous mouse soft tissue sarcoma model incorporating incomplete surgical resection. WEHI 164 fibrosarcoma cells are inoculated subcutaneously, and once tumors are established, we remove 50-75% of the tumor bulk (Figure 1A-E). Tumors consistently re-grow from the remaining tumor. This model allows for testing adjuvant therapies while considering the effect of surgical stress and wound healing. Similar surgical models of incomplete resection have been used in a number of studies by several groups and found to be reproducible and effective11,12,13. Here, we provide a detailed description of this protocol.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>26 gauge 0.5 mL insulin syringe</td>
			<td>Becton Dickinson, Australia</td>
			<td>326769</td>
			<td>None</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Life Technologies Australia Pty Ltd</td>
			<td>21985023</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Anaestetic gas machine</td>
			<td>Darvall Vet, Australia</td>
			<td>SKU: 2848</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Anti-CTLA-4</td>
			<td>BioXcell, USA</td>
			<td>BE0164</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Anti-PD-1</td>
			<td>BioXcell, USA</td>
			<td>BP0273</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Buprenorphine Hydrochloride Injection, 0.3mg/mL</td>
			<td>RB healthcare UK Limited, UK</td>
			<td>55175</td>
			<td>Prescription order</td>
		</tr>
		<tr>
			<td>Chlorhexidine Surgical Scrub 4%</td>
			<td>Perigo Australia, Australia</td>
			<td>CHL01449F(scrub</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Fetal Bovine serum</td>
			<td>CellSera, Australia</td>
			<td>AU-FBS-PG</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Forceps Fine 10.5 cm</td>
			<td>Surgical house, Western Australia</td>
			<td>CC74110</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Forceps Fine 12 cm Serrated</td>
			<td>Surgical house, Western Australia</td>
			<td>CC74212</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Forceps Halsted 14 cm</td>
			<td>Surgical house, Western Australia</td>
			<td>CD01114</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Heating chamber</td>
			<td>Datesand Ltd, UK</td>
			<td>Mini-Thermacage</td>
			<td>None</td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Life Technologies Australia Pty Ltd</td>
			<td>15630080</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein Animal Health, Australia</td>
			<td>SKU: 29405</td>
			<td>Prescription order</td>
		</tr>
		<tr>
			<td>Lubricating Eye Ointment</td>
			<td>Alcon</td>
			<td>n/a</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin 1000X</td>
			<td>Life Technologies Australia Pty Ltd</td>
			<td>15140122</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Solution 10x</td>
			<td>Life Technologies Australia Pty Ltd</td>
			<td>70013-032</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Reflex 7mm Clips</td>
			<td>Able scientific, Australia</td>
			<td>AS59038</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Reflex 7mm Wound Clip Applicator</td>
			<td>Able scientific, Australia</td>
			<td>AS59036</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Reflex Wound Clip Remover</td>
			<td>Able scientific, Australia</td>
			<td>AS59037</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Rodent Qube Anesthesia Breathing Circuit</td>
			<td>Darvall Vet, Australia</td>
			<td>#7885</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640 Medium + L-glutamine</td>
			<td>Life Technologies Australia Pty Ltd</td>
			<td>21870092</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Scissors Iris STR 11 cm</td>
			<td>Surgical house, Western Australia</td>
			<td>KF3211</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Scissors Iris STR 9 cm</td>
			<td>Surgical house, Western Australia</td>
			<td>JH4209</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Small Induction Chamber</td>
			<td>Darvall Vet, Australia</td>
			<td>SKU: 9630</td>
			<td>None</td>
		</tr>
		<tr>
			<td>TrypLE express 1x</td>
			<td>Life Technologies Australia Pty Ltd</td>
			<td>12604-021</td>
			<td>None</td>
		</tr>
	</tbody>
</table>

a mouse model of incompletely resected soft tissue sarcoma for testing (neo)adjuvant therapies

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

Tumor growth to a size of 50 mm2 is an ideal size for partial debulk. The incomplete surgical resection of 50 mm2 tumors results in 100% (n=5) reproducible regrowth of the tumors in the absence of adjuvant immunotherapy (Figure 4A). We next used the model to test adjuvant immunotherapies using antibodies against checkpoint molecules Cytotoxic T Lymphocyte Associated Protein 4 (CTLA-4) and Programmed Death Receptor 1 (PD-...

Watch this Scientific Journal Video about A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing Neoadjuvant Therapies at JoVE.com

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

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

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

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

a-mouse-model-incompletely-resected-soft-tissue-sarcoma-for-testing

Research

JoVE Journal

Medicine

9.4K Views.  University of Western Australia. In this protocol, we describe a mouse model of incomplete surgical resection of soft tissue sarcoma for testing (neo)adjuvant therapies.

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

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

Surgical Technique for Spinal Cord Delivery of Therapies: Demonstration of Procedure in Gottingen Minipigs

This is a compact visual description of a combination of surgical technique and device for the delivery of (gene and cell) therapies into the spinal cord. While the technique is demonstrated in the animal, the procedure is FDA-approved and currently being used for stem cell transplantation into the spinal cords of patients with ALS. While the FDA has recognized proof-of-principle data on therapeutic efficacy in highly characterized rodent models, the use of large animals is considered critical for validating the combination of a surgical procedure, a device, and the safety of a final therapy for human use. The size, anatomy, and general vulnerability of the spine and spinal cord of the swine are recognized to better model the human. Moreover, the surgical process of exposing and manipulating the spinal cord as well as closing the wound in the pig is virtually indistinguishable from the human. We believe that the healthy pig model represents a critical first step in the study of procedural safety.

Short visual description of the surgical technique and device used for the delivery of (gene and cell) therapies into the spinal cord. The technique is demonstrated in the animal but is entirely translatable and currently being used for human application.

This is a  compact visual description of a combination of surgical technique and device  for the  delivery of (gene and cell) therapies into the spinal ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies have suggested that nerves may also modulate skin disorders including atopic dermatitis, psoriasis and cancer. Here, we describe protocols for testing the requirement for nerves in maintaining a cutaneous mechanosensory organ, the touch dome (TD). Specifically, we discuss methods for genetically labeling, harvesting and visualizing TDs by whole-mount staining, and for performing unilateral surgical denervation on mouse dorsal back skin. Together, these approaches can be used to directly compare TD morphology and gene expression in denervated as well as sham-operated skin from the same animal. These methods can also be readily adapted to examine the requirement for nerves in mouse models of skin pathology. Finally, the ability to repeatedly sample the skin provides an opportunity to monitor disease progression at different stages and times after initiation.

This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount &#946;-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies ...