Funding provided by National Institutes of Health (NIH) grant R01-HL088424; Veterans Affairs merit award to PPY.

1. Sponge Preparation
<ol>
	<li>Hydrate sponges by stirring overnight in 0.9% (w/v) aqueous sodium chloride solution.</li>
	<li>Sterilize hydrated sponges by autoclaving them. For 75 mL, autoclave for 25 minutes at 121 &deg;C.</li>
</ol>
Note: Loading sponges with a treatment as described below is optional.
<ol start="3">
	<li>In a sterile hood with sterile instruments, pick up one sponge from the solution using a tweezers. Squeeze the sponge with the tweezers and use a vacuum tip to remove as much solution as possible from the sponge. Place the wrung out sponge in a tissue culture dish, being careful to leave space between sponges. Multiple well plates can be used to separate different treatments.</li>
	<li>Pipette the treatment solution directly onto the center of a sponge. Press down gently on the sponge with the pipette tip after the solution has been placed on the sponge; this helps the sponge absorb the solution. Note: The 6mm diameter by 2.75 mm thick sponges that we employ are able to hold a maximum of 25 &mu;L.</li>
	<li>Once all sponges are loaded, the plate they are in can be stored on ice until they are implanted in the mouse.</li>
</ol>
2. Surgical Procedure
<ol>
	<li>Appropriately anesthetize animal and administer pre-emptive analgesic. The entire procedure will last approximately 20 minutes per animal. For a 30 g mouse 1.5 liters per minute of oxygen with 1.5% Isoflurane is used to induce and maintain anesthesia. When animal is no longer responsive to the pinch reflex test, place in nose cone in supine position. Use an electric shaver to shave a 1 in. by 1 in. area on the lower ventral side below the rib cage. Wipe clean of cut hair.</li>
	<li>Sterilize the shaved area by cleaning with betadine followed by 70% alcohol. Repeat this betadine-alcohol cleansing three times.</li>
	<li>While holding the skin taught, use the scalpel to make a vertical incision 1.5-2 times the diameter of the sponges to be inserted, being cautious not to cut through the body cavity.</li>
	<li>Use a tweezers to hold the edge of the skin and slowly begin separating the skin from the muscle on both sides of the incision using Metzembaum scissors. The pocket should go across the width of the mouse and from the rib cage to the hind legs.</li>
	<li>Use a tweezers to pick up the first sponge to be placed in the animal by pinching it from the sides. Transfer the sponge to a straight tweezers so that it is held from the top and bottom.</li>
	<li>Use tweezers to hold the edge of the skin and lift it so that the sponge can be inserted. Slowly insert the sponge trying to avoid touching the skin or the muscle underneath as the sponge will be difficult to move once it has touched tissue. Repeat for all sponges.</li>
	<li>Once all sponges have been inserted, place two or three sutures to close the incision. Use a pair of tweezers to hold both sides of the incision together and thread the suture through both layers of skin. Tighten the first throw just enough to close the skin edges. Finish the square knot with one more throw from the other direction, this time pulling the knot tight. Then tie two more square knots to finish the suture (Figure 2). Place additional sutures to close the entire incision. Other wound bonding methods may be used such as dermabond, wound clips, or staples.</li>
	<li>Remove animal from anesthesia system and monitor while consciousness is resumed. Animal should quickly begin ambulating and return to pre-op mobility. Assure that they are able to sufficiently reach food and water. Some moistened food may be placed in the bottom of the cage to assist in recovery. Continue to monitor animal daily for signs of distress or discomfort, and check the incision site for infection, inflammation, and dehiscence. Take appropriate action if any complications to recovery are found.</li>
</ol>
3. Optional Injection
Note: Once the sponges have been implanted into the animal they can be injected with a solution of cells, drugs, growth factors etc. This allows for analysis of the effects of the injected substance during varied delivery times and administration regimes. If injections are done a control should always be used.
<ol>
	<li>To inject the sponge with a desired solution, first determine the amount of the substance to administer to the mouse. The substance needs to be concentrated so that the injection volume is less than half the total volume the sponge can hold.</li>
	<li>Prepare a syringe for the injection (for mice we use a 28 G needle).</li>
	<li>Place the animal to receive the injections under anesthesia.</li>
	<li>When the animal no longer responds to the pinch test the injection can be given.</li>
	<li>To give the injection push the needle at a 45&deg; angle thorough the skin at the center of the sponge. Then push the needle into the sponge only going half way through its thickness. The goal is to inject into the exact center of the sponge.</li>
	<li>To check to make sure the needle is in the sponge, slowly lift the needle vertically. If the needle is in the sponge, the sponge should move up with the needle.</li>
	<li>Slowly inject into the sponge and remove the needle. If there are multiple sponges requiring injections it is possible that some of the injected fluid will be pushed out of the previous sites of injection.</li>
</ol>
4. Sponge Removal
Note: During the sponge removal, handle the sponge with extra caution. Avoid penetrating the sponge with the surgical instruments and prevent excessive bleeding into the sponge by avoiding the major arteries around the surrounding tissue.
<ol>
	<li>After the animal is euthanized, remove the sponge by holding the skin using a tweezers and making an incision close the sponge.</li>
	<li>Carefully separate the sponges from the surrounding capsule and avoid harvesting extra tissue surrounding the sponge. For assays such as drug delivery or morphometry, less accuracy is required for excision.</li>
</ol>
5. Representative Results
Removed sponges can be stored under different condition depending on the type of analysis that will be performed. For sectioning, the retrieved sponges can be embedded in a medium for freezing such as Optimal Cutting Temperature (O.C.T.) compound or placed in 10% formalin for embedding and sectioning in paraffin blocks (Figure 3A and B). Best results for sectioning are obtained when the sponge is cut in half across the diameter and embedded cut surface down (Figure 4A and B). Sections of the sponge should be taken so that entire width of the sponge is present. Figure 3A shows two sponges, the left sponge was embedded/sectioned incorrectly and the right sponge was processed correctly.
For analysis, sponges can be placed in an Eppendorf tube and stored at -20 &deg;C until ready for processing. For RNA and quantitative real time PCR analysis sponges should be placed in an Eppendorf tube, flash frozen, and stored at -80 &deg;C. In addition, an RNA preservative, such as RNAlater from QIAGEN, can be used to stabilize the RNA for safe storage at higher temperatures. Wound fluid can be collected by squeezing the sponges over a tube, and pooling together the fluid from multiple sponges to obtain a representative sample. Live cells, such as fibroblast, macrophages and lymphocytes, can also be extracted from the sponges. After removal from the animal, sponges can be place in an appropriate media for the desired type of cells. Sponges can be processed to release the cells by physical (i.e. mincing) or enzymatic (i.e. collagenase) methods.
<img alt="Figure 1." src="/files/ftp_upload/3885/3885fig1.jpg" /> 
Figure 1. Dehydrated PVA sponge disks in three different sizes.
<img alt="Figure 2." src="/files/ftp_upload/3885/3885fig2.jpg" /> 
Figure 2. Image of mouse wound-healing model showing position of lateral wound and four PVA sponges.
<img alt="Figure 3." src="/files/ftp_upload/3885/3885fig3.jpg" /> 
Figure 3. (A) H &amp; E staining at 4x and (B) Trichrome staining at 10x of sectioned paraffin embedded sponges.
<img alt="Figure 4." src="/files/ftp_upload/3885/3885fig4.jpg" /> 
Figure 4. (A) Image of sponge cut in half along the diameter. (B) PVA sponge half, embedded cut side down in OCT.

<ol>
	<li>Efron, D. T., Barbul, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcutaneous+sponge+models.">Subcutaneous sponge models.</a> Methods Mol. Med. 78, 83-93 (2003).</li><li>Gottrup, F., Agren, M. S., Karlsmark, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+for+use+in+wound+healing+research:+a+survey+focusing+on+in+vitro+and+in+vivo+adult+soft+tissue.">Models for use in wound healing research: a survey focusing on in vitro and in vivo adult soft tissue.</a> Wound Repair. 8, 83-96 (2000).</li><li>Sprugel, K. H., McPherson, J. M., Clowes, A. W., Ross, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+growth+factors+in+vivo.+I.+Cell+ingrowth+into+porous+subcutaneous+chambers.">Effects of growth factors in vivo. I. Cell ingrowth into porous subcutaneous chambers.</a> Am. J. Pathol. 129, 601-613 (1987).</li><li>Alaish, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+polyvinyl+alcohol+sponge+and+expanded+polytetrafluoroethylene+subcutaneous+implants+as+models+to+evaluate+wound+healing+potential+in+human+beings.">Comparison of the polyvinyl alcohol sponge and expanded polytetrafluoroethylene subcutaneous implants as models to evaluate wound healing potential in human beings.</a> Wound Repair. 3, 292-298 (1995).</li><li>Davidson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+wound+repair.">Animal models for wound repair.</a> Arch. Dermatol. Res. 290, 1-11 (1998).</li><li>Alfaro, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=sFRP2+suppression+of+bone+morphogenic+protein+(BMP)+and+Wnt+signaling+mediates+mesenchymal+stem+cell+(MSC)+self-renewal+promoting+engraftment+and+myocardial+repair.">sFRP2 suppression of bone morphogenic protein (BMP) and Wnt signaling mediates mesenchymal stem cell (MSC) self-renewal promoting engraftment and myocardial repair.</a> J. Biol. Chem. 285, 35645-35653 (2010).</li><li>Andrade, S. P., Ferreira, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sponge+implant+model+of+angiogenesis.">The sponge implant model of angiogenesis.</a> Methods Mol. Biol. 467, 295-304 (2009).</li><li>Diegelmann, R. F., Lindblad, W. J., Cohen, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subcutaneous+implant+for+wound+healing+studies+in+humans.">A subcutaneous implant for wound healing studies in humans.</a> J. Surg. Res. 40, 229-237 (1986).</li><li>Efron, D. T., Most, D., Shi, H. P., Tantry, U. S., Barbul, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+of+studying+wound+healing.">A novel method of studying wound healing.</a> J. Surg. Res. 98, 16-20 (2001).</li><li>Lindblad, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+for+selecting+the+correct+animal+model+for+dermal+wound-healing+studies.">Considerations for selecting the correct animal model for dermal wound-healing studies.</a> J. Biomater. Sci. Polym. Ed. 19, 1087-1096 (2008).</li></ol>

We have nothing to disclose.

The sponge model of wound healing has multiple variations; it can be performed in many different species including humans and the sponges can also be implanted dorsally 1,2,8. For studies in humans and some other species, sponges may need to be placed in a porous silicone tubing to prevent the sponge from being encapsulated and to allow ease of removal 4,8. The location of the sponge implantation can be altered based on desired results and skill of technician. Dorsal implantation can allow the sponges to move under the skin, while ventral implants are more difficult due to probability of cutting through to the abdominal cavity. The sponges can be set up with a catheter so that drug delivery is constant and controlled, eliminating the need for multiple injections9. Smaller, tubular sponges can even be injected through a needle8. Sponges' composition is not limited to only polyvinyl alcohol. They can be prepared from many materials exclusively or in combination7. 
 It is crucial for sponge preparation and placement in the animal to be consistent and properly understood. If sponges are cut from sheets they must be of similar weight for comparability. If the sponge is to be loaded with cells or a treatment, the amount placed in each sponge must be exact. The healing process can vary based on the location of the placement of the sponge in the animal and healing will decrease as distance from the head increases 1,10.

<table border="1">
<tbody>
 <tr>
 <td>Material Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>PVA Sponge</td>
 <td>Medtronic</td>
 <td>CF120</td>
 <td>The size and porosity of the sponge depends on the experiment</td>
 </tr>
 <tr>
 <td>Scalpel blade size 15 </td>
 <td>BD Medial</td>
 <td>371115 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Metzembaum scissors</td>
 <td>Thermo Scientific</td>
 <td>79-211 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hemostat Forceps</td>
 <td>Fine Science Tools</td>
 <td>13004-14 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Needle holder</td>
 <td>Fine Science Tools</td>
 <td>12004-16 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>5-0 nylon suture</td>
 <td>Ethilon</td>
 <td>698</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>O.C.T. compound</td>
 <td>Tissue-Tek</td>
 <td>4583</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

the polyvinyl alcohol sponge model implantation

Wound healing is a complicated, multistep process involving many cell types, growth factors and compounds1-3. Because of this complexity, wound healing studies are most comprehensive when carried out in vivo. There are many in vivo models available to study acute wound healing, including incisional, excisional, dead space, and burns. Dead space models are artificial, porous implants which are used to study tissue formation and the effects of substances on the wound. Some of the commonly used dead space models include polyvinyl alcohol (PVA) sponges, steel wire mesh cylinders, expanded polytetrafluoroethylene (ePTFE) material, and the Cellstick1,2.
Each dead space model has its own limitations based on its material's composition and implantation methods. The steel wire mesh cylinder model has a lag phase of infiltration after implantation and requires a long amount of time before granulation tissue formation begins1. Later stages of wound healing are best analyzed using the ePTFE model1,4. The Cellstick is a cellulose sponge inside a silicon tube model which is typically used for studying human surgery wounds and wound fluid2. The PVA sponge is limited to acute studies because with time it begins to provoke a foreign body response which causes a giant cell reaction in the animal5. Unlike other materials, PVA sponges are easy to insert and remove, made of inert and non-biodegradable materials and yet are soft enough to be sectioned for histological analysis2,5.
In wound healing the PVA sponge is very useful for analyzing granulation tissue formation, collagen deposition, wound fluid composition, and the effects of substances on the healing process1,2,5. In addition to its use in studying a wide array of attributes of wound healing, the PVA sponge has also been used in many other types of studies. It has been utilized to investigate tumor angiogenesis, drug delivery and stem cell survival and engraftment1,2,6,7. With its great alterability, prior extensive use, and reproducible results, the PVA sponge is an ideal model for many studies1,2.
Here, we will describe the preparation, implantation and retrieval of PVA sponge disks (Figure 1) in a mouse model of wound healing.

Watch this Scientific Journal Video about The Polyvinyl Alcohol Sponge Model Implantation at JoVE.com

The Polyvinyl Alcohol Sponge Model Implantation

A useful tool to analyze the effects of drugs, growth factors, and/or manipulated cells in an animal model of wound repair is described. This technique utilizes the properties of a polyvinyl alcohol (PVA) sponge to deliver and contain the desired treatment and also provide a platform to be excised and analyzed.

Wound healing is a complicated, multistep process involving many cell types, growth factors and compounds1-3. Because of this complexity, wound healing ...

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19.7K Views.  Vanderbilt University School of Medicine. A useful tool to analyze the effects of drugs, growth factors, and/or manipulated cells in an animal model of wound repair is described. This technique utilizes the properties of a polyvinyl alcohol (PVA) sponge to deliver and contain the desired treatment and also provide a platform to be excised and analyzed.

Video: The Polyvinyl Alcohol Sponge Model Implantation

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

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Implantation of the Syncardia Total Artificial Heart

With advances in technology, the use of mechanical circulatory support devices for end stage heart failure has rapidly increased. The vast majority of such patients are generally well served by left ventricular assist devices (LVADs). However, a subset of patients with late stage biventricular failure or other significant anatomic lesions are not adequately treated by isolated left ventricular mechanical support. Examples of concomitant cardiac pathology that may be better treated by resection and TAH replacement includes: post infarction ventricular septal defect,&#160;aortic root aneurysm / dissection, cardiac allograft failure, massive ventricular thrombus, refractory malignant arrhythmias (independent of filling pressures), hypertrophic / restrictive cardiomyopathy, and complex congenital heart disease. Patients often present with cardiogenic shock and multi system organ dysfunction. Excision of both ventricles and orthotopic replacement with a total artificial heart (TAH) is an effective, albeit extreme, therapy for rapid restoration of blood flow and resuscitation. Perioperative management is focused on end organ resuscitation and physical rehabilitation. In addition to the usual concerns of infection, bleeding, and thromboembolism common to all mechanically supported patients, TAH patients face unique risks with regard to renal failure and anemia. Supplementation of the abrupt decrease in brain natriuretic peptide following ventriculectomy appears to have protective renal effects. Anemia following TAH implantation can be profound and persistent. Nonetheless, the anemia is generally well tolerated and transfusion are limited to avoid HLA sensitization. Until recently, TAH patients were confined as inpatients tethered to a 500 lb pneumatic console driver. Recent introduction of a backpack sized portable driver (currently under clinical trial) has enabled patients to be discharged home and even return to work. Despite the profound presentation of these sick patients, there is a 79-87% success in bridge to transplantation.

The purpose of this manuscript is to briefly review indications, management, and outcomes for the total artificial heart. Video operative techniques for device implantation are presented.

With advances in technology, the use of mechanical circulatory support devices for end stage heart failure has rapidly increased. The vast majority of ...

Implantation of Total Artificial Heart in Congenital Heart Disease

In patients with end-stage heart failure (HF), a total artificial heart (TAH) may be implanted as a bridge to cardiac transplant. However, in congenital heart disease (CHD), the malformed heart presents a challenge to TAH implantation. 
In the case presented here, a 17 year-old patient with congenital transposition of the great arteries (CCTGA) experienced progressively worsening HF due to his congenital condition. He was hospitalized multiple times and received an implantable cardioverter defibrillator (ICD). However, his condition soon deteriorated to end-stage HF with multisystem organ failure. 
Due to the patient's grave clinical condition and the presence of complex cardiac lesions, the decision was made to proceed with a TAH. The abnormal arrangement of the patient's ventricles and great arteries required modifications to the TAH during implantation.
With the TAH in place, the patient was able to return home and regain strength and physical well-being while awaiting a donor heart. He was successfully bridged to heart transplantation 5 months after receiving the device. This report highlights the TAH is feasible even in patients with structurally abnormal hearts, with technical modification.

This is a case report of a patient with congenitally corrected transposition of the great arteries (CCTGA) who received a total artificial heart (TAH) as a bridge to heart transplant. The TAH was successfully implanted with modifications to accommodate the patient's congenitally malformed heart.

In patients with end-stage heart failure (HF), a total artificial heart (TAH) may be implanted as a bridge to cardiac transplant. However, in congenital ...

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. ...

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 &#181;l whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.

The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 &#181;l&#160;blood sample is also described.

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Surgical Training for the Implantation of Neocortical Microelectrode Arrays Using a Formaldehyde-fixed Human Cadaver Model

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. Recent technological progress has enabled the fabrication of microelectrode arrays that allow recording the activity of multiple individual neurons in the neocortex of the human brain. These arrays have the potential to bring unique insight onto the neuronal correlates of cerebral function in health and disease. Furthermore, the identification and decoding of volitional neuronal activity opens the possibility to establish brain-computer interfaces, and thus might help restore lost neurological functions. The implantation of neocortical microelectrode arrays is an invasive procedure requiring a supra-centimetric craniotomy and the exposure of the cortical surface; thus, the procedure must be performed by an adequately trained neurosurgeon. In order to provide an opportunity for surgical training, we designed a procedure based on a human cadaver model. The use of a formaldehyde-fixed human cadaver bypasses the practical, ethical and financial difficulties of surgical practice on animals (especially non-human primates) while preserving the macroscopic structure of the head, skull, meninges and cerebral surface and allowing realistic, operating room-like positioning and instrumentation. Furthermore, the use of a human cadaver is closer to clinical daily practice than any non-human model. The major drawbacks of the cadaveric simulation are the absence of cerebral pulsation and of blood and cerebrospinal fluid circulation. We suggest that a formaldehyde-fixed human cadaver model is an adequate, practical and cost-effective approach to ensure proper surgical training before implanting microelectrode arrays in the living human neocortex.

We designed a procedure in which a formaldehyde-fixed human cadaver is used to assist neurosurgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain.

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. ...

Cochlear Implantation in the Guinea Pig

Cochlear implants are highly efficient devices that can restore hearing in subjects with profound hearing loss. Due to improved speech perception outcomes, candidacy criteria have been expanded over the last few decades. This includes patients with substantial residual hearing that benefit from electrical and acoustical stimulation of the same ear, which makes hearing preservation during cochlear implantation an important issue. Electrode impedances and the related issue of energy consumption is another major research field, as progress in this area could pave the way for fully implantable auditory prostheses. To address these issues in a systematic way, adequate animal models are essential. Therefore, the goal of this protocol is to provide an animal model of cochlear implantation, which can be used to address various research questions. Due to its large tympanic bulla, which allows easy surgical access to the inner ear, as well as its hearing range which is relatively similar to the hearing range of humans, the guinea pig is a commonly used species in auditory research. Cochlear implantation in the guinea pig is performed via a retroauricular approach. Through the bullostomy a cochleostomy is drilled and the cochlear implant electrode is inserted into the scala tympani. This electrode can then be used for electrical stimulation, determination of electrode impedances and the measurement of compound action potentials of the auditory nerve. In addition to these applications, cochlear implant electrodes can also be used as drug delivery devices, if a topical delivery of pharmaceutical agents to the cells or fluids of the inner ear is intended.

The goal of this protocol is to provide an animal model of cochlear implantation, which can be used to address a multitude of research questions. Potential applications include the evaluation of pharmaceutical interventions or electrical stimulation for beneficial effects on hearing thresholds or electrode impedances.

Cochlear implants are highly efficient devices that can restore hearing in subjects with profound hearing loss. Due to improved speech perception ...

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 ...

The Lower Body Positive Pressure Treadmill for Knee Osteoarthritis Rehabilitation

Here, based on a clinician&rsquo;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies.

Here, based on a clinician&#8217;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies.

Here, based on a clinician&rsquo;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in ...