Lumbar spines were harvested from 4-year-old Merino cross adult ewes (Ovis aries) following the ethical guidelines of the Animal Care and Ethics Committee of the Surgical and Orthopaedic Research Laboratories. Following the institutionally approved method of humane euthanasia, the lumbar spines were harvested using a sharp dissection tool, first incising through the skin and subcutaneous tissues, followed by fascia and musculature prior to disarticulation at the thoracolumbar and lumbosacral junctions. A harvested sample is shown in Figure 1A.
1. Preparation for the initial bath
<ol>
	<li>Manually remove soft tissue (muscle, connective tissue, fat, etc.) using a sharp dissection tool (number 22 scalpel blade) from the bone sample before processing further. 
	NOTE: This step is optional; however, removing as much soft tissue as possible reduces the time the water bath takes to macerate the tissues. The specimen size (~20 cm x 10 cm x 8 cm) is also reduced; therefore, more samples can be fitted into the bath.</li>
	<li>Seal the sample in a heat-safe sealable plastic bag after removing the air. It is recommended to use a vacuum bag using a commercial vacuum-sealing device (see Table of Materials). 
	NOTE: No additives are required for the initial 24 h bath. If there is significant muscle covering all surfaces of the bone and if there is already minimal soft tissue and most of the bone surfaces of the sample are exposed, proceed to step 3.2 (Figure 1B).</li>
</ol>
2. Procedure for the initial bath
<ol>
	<li>Completely submerge the sealed sample in a 70 &#176;C water bath for 24 h.</li>
	<li>After 24 h, remove the bag from the bath, open the bag, and allow the sample to cool until handleable.</li>
</ol>
3. Preparation for subsequent baths
<ol>
	<li>Remove as much soft tissue from the bone as possible using a sharp scalpel and running water as needed.</li>
	<li>Disarticulate any joints using a sharp scalpel to expose the cartilaginous tissue.
	<ol>
		<li>Keep the disarticulated pieces in situ using material such as orthopedic wire or cable ties (see Table of Materials) to maintain the anatomical position.</li>
	</ol>
	</li>
	<li>Seal the sample in a vacuum bag along with 10 mL of dishwashing detergent (see Table of Materials) and 10 mL of tap water. 
	&#8203;NOTE: The volume of detergent depends on the strength, concentration, and size of the sample.</li>
</ol>
4. Procedure for subsequent baths
<ol>
	<li>Completely submerge the sealed sample in a 70 &#176;C water bath for 24 h.</li>
	<li>After 24 h, remove the bag from the bath, open the bag, and allow the sample to cool until handleable.
	<ol>
		<li>Avoid letting the sample cool completely, as this allows softened cartilage to harden and adhere to bone, becoming more difficult to remove. 
		&#8203;NOTE: The time required for sample processing may vary based on size and type, and repeated removal of subsequent baths may be unnecessary. Additionally, the sample can remain in the bath for extended periods, which may aid in the interim removal of tissues.</li>
	</ol>
	</li>
	<li>Remove as much soft tissue as possible using a sharp dissection tool (number 22 scalpel blade on a dedicated scalpel handle) and running water.</li>
	<li>Repeat step 4 as required until the bone is free of soft tissue material. In our experience, this was required to be repeated only once.</li>
</ol>
5. Completion of the procedure
<ol>
	<li>Wash the sample with liquid detergent and rinse thoroughly with water. 
	NOTE: Alcohol may be used to speed drying.</li>
	<li>Allow the sample to dry for approximately 48 h.</li>
</ol>

<ol>
	<li>Leary, O. P., 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=Three-dimensional+printed+anatomic+modeling+for+surgical+planning+and+real-time+operative+guidance+in+complex+primary+spinal+column+tumors:+single-center+experience+and+case+series.">Three-dimensional printed anatomic modeling for surgical planning and real-time operative guidance in complex primary spinal column tumors: single-center experience and case series.</a> World Neurosurgery. 145, 116-126 (2021).</li><li>Tack, P., Victor, J., Gemmel, P., Annemans, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-printing+techniques+in+a+medical+setting:+a+systematic+literature+review.">3D-printing techniques in a medical setting: a systematic literature review.</a> BioMedical Engineering OnLine. 15 (1), 115 (2016).</li><li>Ventola, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medical+applications+for+3D+printing:+current+and+projected+uses.">Medical applications for 3D printing: current and projected uses.</a> Pharmacy and Therapeutics. 39 (10), 704-711 (2014).</li><li>Wilcox, B., Mobbs, R. J., Wu, A. M., Phan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+review+of+3D+printing+in+spinal+surgery:+the+current+state+of+play.">Systematic review of 3D printing in spinal surgery: the current state of play.</a> Journal of Spinal Surgery. 3 (3), 433-443 (2017).</li><li>Mairs, S., Swift, B., Rutty, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detergent:+an+alternative+approach+to+traditional+bone+cleaning+methods+for+forensic+practice.">Detergent: an alternative approach to traditional bone cleaning methods for forensic practice.</a> The American Journal of Forensic Medicine and Pathology. 25 (4), 276-284 (2004).</li><li>Husch, C., Berner, M., Goldammer, H., Lichtscheidl-Schultz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+note:+A+novel+method+for+gentle+and+non-destructive+removal+of+flesh+from+bones.">Technical note: A novel method for gentle and non-destructive removal of flesh from bones.</a> Forensic Science International. 323, 110778 (2021).</li><li>Couse, T., Connor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+maceration+techniques+for+use+in+forensic+skeletal+preparations.">A comparison of maceration techniques for use in forensic skeletal preparations.</a> Journal of Forensic Investigation. 3, 1-6 (2015).</li><li>Mahon, T. J., Maboke, N., Myburgh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+different+detergents+in+skeletal+preparations.">The use of different detergents in skeletal preparations.</a> Forensic Science International. 327, 110967 (2021).</li><li>Hussain, M., Hussain, N., Zainab, H., Qaiser, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+preservation+techniques+to+enhance+veterinary+anatomy+teaching.">Skeletal preservation techniques to enhance veterinary anatomy teaching.</a> IJAVMS. 1, 21-23 (2007).</li><li>Simonsen, K. P., Rasmussen, A. R., Mathisen, P., Petersen, H., Borup, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fast+preparation+of+skeletal+materials+using+enzyme+maceration.">A fast preparation of skeletal materials using enzyme maceration.</a> Journal of Forensic Science. 56 (2), 480-484 (2011).</li><li>Burkhard, M., Furnstahl, P., Farshad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensionally+printed+vertebrae+with+different+bone+densities+for+surgical+training.">Three-dimensionally printed vertebrae with different bone densities for surgical training.</a> European Spine Journal. 28 (4), 798-806 (2019).</li><li>Rose, A. S., 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=Multi-material+3D+models+for+temporal+bone+surgical+simulation.">Multi-material 3D models for temporal bone surgical simulation.</a> The Annals of Otology, Rhinology, and Laryngology. 124 (7), 528-536 (2015).</li><li>Werz, S. M., Zeichner, S. J., Berg, B. I., Zeilhofer, H. F., Thieringer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+printed+surgical+simulation+models+as+educational+tool+by+maxillofacial+surgeons.">3D printed surgical simulation models as educational tool by maxillofacial surgeons.</a> European Journal of Dental Education. 22 (3), 500-505 (2018).</li></ol>

The authors have to disclose.

This technical note aims to describe a simple, safe, and low-cost method to produce an anatomical bone model for the benefit of veterinary and medical education and for use in anatomical education and surgical planning. 
Pilot testing found that a bath temperature of 70 &#176;C provided the fastest processing time without causing damage to the samples. Higher temperatures caused an extensive breakdown of collagen within the bone, resulting in brittle samples with a chalky texture. The hot bath...

Removing soft tissue and cleaning bones are required for forensics, medical and biological research, and veterinary and medical education. Most techniques have been developed for forensic purposes, minimizing damage to the bone to preserve as much detail as possible. This can provide an accurate, tangible bone model for preoperative surgical planning, as well as intraoperative decision-making to help minimize complications1,2,3. This is beneficial in surgery by reducing operation times and blood loss and improving communication between surgeons, compared to planning with 2D images4. The use of these models may also reduce the reliance on intraoperative imaging, such as fluoroscopy, which may reduce radiation exposure to personnel.
Skeletal bone from cadavers has historically been used for these models; however, technological advances have pushed toward the use of manufactured models and, more recently, three-dimensional (3D) printed models. Bone models rely on the availability of cadaveric samples and the efficiency of processing these samples into usable models. 3D printing has the advantage of creative freedom, allowing for anatomical and patient-specific models, especially when anatomical abnormalities or neoplasms are present, or if the hardware needs to be manufactured or augmented to fit the patient1. These samples are also able to be sterilized and manipulated by surgeons during a procedure. However, this freedom comes with a cost, as it requires computed tomography (CT) scans, the materials and equipment required can be expensive, and expertise is essential to create the models in the required software1,4. Additionally, these factors can limit the precision and quality of the model, and hence the surgical planning and success1. 3D printed models may not be the best choice for cases where there is no need for patient-specific anatomy and where there is an immediate requirement for the model.
Commonly applied methods for the removal of soft tissue from cadaveric bone include manual cleaning, bacterial maceration, chemical maceration, cooking, and insect maceration5,6. The success of these methods is generally based on the cost, time, labor, equipment, safety, and quality of the final product5,7. Manual cleaning requires the most labor and a significant amount of time, but involves minimal equipment5. Bacterial maceration consists of leaving the sample in a cold or warm water bath for long periods of time, often up to 3 weeks, allowing bacteria to decompose the tissue6. This creates unpleasant odors, requires additional equipment to treat the bacteria, and creates a biosecurity hazard for the user5,6. The use of dermestid beetles is very effective with minimal labor, but requires the acquisition of a colony and husbandry of the animals, and is not considered an economic investment if used infrequently6,7. Chemical maceration usually involves the use of enzymes such as trypsin, pepsin, and papain, or commercial detergents containing substances such as surfactants and enzymes5,8. Although this method provides faster results, the chemicals used, such as sodium hydroxide, ammonia, bleach, and gasoline, may represent a health and safety risk and produce noxious odors that require personal protective equipment (PPE) and a fume hood5,7,8,9. Finally, extended heating provides another minimally intensive method but may produce odors requiring ventilation10.
A simple, safe, and low-cost method for the preparation of anatomical bone models would provide a useful tool for surgeons, students, educators, and researchers. This article describes a novel method for preparing skeletal bone models that avoids unpleasant odors and noxious chemicals, and produces a detailed surgical model with minimal equipment and labor.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dimension Elite 3D printer</td>
			<td>Stratasys, Eden Prairie, MN, United States</td>
			<td></td>
			<td>3D printer for production of surgical bone models based on reconstructed CT scans</td>
		</tr>
		<tr>
			<td>Mimics Innovation Suite</td>
			<td>Materialise NV, Leuven, Belgium</td>
			<td>Suite 24</td>
			<td>Software to create 3D models from imaging scans</td>
		</tr>
		<tr>
			<td>Nylon cable ties</td>
			<td>4Cabling, Alexandria, NSW, Australia</td>
			<td>011.060.1042/011.060.1039</td>
			<td>Used to maintain connection between vertebral bodies</td>
		</tr>
		<tr>
			<td>Orthopaedic wire</td>
			<td>B Braun, Bella Vista, NSW, Australia</td>
			<td></td>
			<td>Used to maintain connection between vertebral bodies</td>
		</tr>
		<tr>
			<td>Support Cleaning Apparatus</td>
			<td>Phoenix Analysis and Design Technologies, Tempe, AZ, United States</td>
			<td>SCA-1200</td>
			<td>Hot water bath for immersion of the sealed sample.</td>
		</tr>
		<tr>
			<td>Ultra Strength Original Dishwashing Liquid</td>
			<td>Colgate-Palmolive, New York, NY, United States</td>
			<td></td>
			<td>Dishwashing liquid added to sealed bag with sample for cleaning of the bone model.</td>
		</tr>
		<tr>
			<td>Vacuum bags</td>
			<td>Pacfood PTY LTD</td>
			<td></td>
			<td>Heat safe, sealable plastic bags</td>
		</tr>
		<tr>
			<td>Vacuum Food sealer</td>
			<td>Tempoo (Aust) PTY LTD</td>
			<td></td>
			<td>Vacuum food sealer to seal vacuum bags prior to bath immersion</td>
		</tr>
	</tbody>
</table>

vacuum-sealed hot water bath immersion for the preparation of anatomical and surgical cadaveric bone models

Bone models serve many purposes, including improving anatomical understanding, preoperative surgical planning, and intraoperative referencing. Several techniques for the maceration of soft tissues have been described, mainly for forensic analysis. For clinical research and medical use, these methods have been superseded by three-dimensional (3D) printed models, which require substantial equipment and expertise, and are costly. Here, cadaveric sheep vertebral bone was cleaned by vacuum sealing the specimen with commercial dishwashing detergent, immersing in a hot water bath, and subsequently manually removing the soft tissue. This eliminated the disadvantages of the previously existing maceration methods, such as the existence of foul odors, usage of hazardous chemicals, substantial equipment, and high costs. The described technique produced clean, dry samples while maintaining anatomical detail and structure to accurately model the osseous structures that can be useful for preoperative planning and intraoperative referencing. The method is simple, low-cost, and effective for bone model preparation for education and surgical planning in veterinary and human medicine.

Following this protocol, clean and dry sheep lumbar vertebral column models were created for surgical planning and reference. Samples consisting of seven lumbar vertebrae were processed within 4 days using this method, with one initial bath to remove the bulk of the muscle and three subsequent baths. Completion of the baths was indicated by the ease at which cartilage and connective tissue were removed from the bone. This varied based on the type and location of cartilage; thin layers were easily removed after one or two...

Watch this Scientific Journal Video about Vacuum-Sealed Hot Water Bath Immersion for the Preparation of Anatomical and Surgical Cadaveric Bone Models at JoVE.com

Vacuum-Sealed Hot Water Bath Immersion for the Preparation of Anatomical and Surgical Cadaveric Bone Models

The present protocol describes the maceration and cleaning of cadaveric bone with a vacuum-sealed, hot water bath immersion technique. This is a low-cost, safe, and effective method to produce anatomical specimens for surgical planning and medical education as an alternative to three-dimensional (3D) printed models.

Bone models serve many purposes, including improving anatomical understanding, preoperative surgical planning, and intraoperative referencing. Several ...

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1.2K Views.  University of New South Wales (UNSW) Sydney. The present protocol describes the maceration and cleaning of cadaveric bone with a vacuum-sealed, hot water bath immersion technique. This is a low-cost, safe, and effective method to produce anatomical specimens for surgical planning and medical education as an alternative to three-dimensional (3D) printed models.

Video: Vacuum-Sealed Hot Water Bath Immersion for the Preparation of Anatomical and Surgical Cadaveric Bone Models

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Adjustable Stiffness, External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo. The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.

One constraint of preclinical research in the field of bone repair is the lack of experimental control over the local mechanical environment within a healing bone lesion. We report the design and use of an external fixator for bone repair with the ability to change fixator stiffness in vivo.

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices currently entering the market. In order to evaluate the safety of these devices, in preclinical studies, reliable techniques are needed. However, the hard metal components utilised in some retinal implants are not compatible with traditional histological processes, particularly in consideration for the delicate nature of the surrounding tissue. Here we describe techniques for assessing the health of the eye directly adjacent to a retinal implant secured epiretinally with a metal tack.
Retinal prostheses feature electrode arrays in contact with eye tissue. The most commonly used location for implantation is the epiretinal location (posterior chamber of the eye), where the implant is secured to the retina with a metal tack that penetrates all the layers of the eye. Previous methods have not been able to assess the proximal ocular tissue with the tack in situ, due to the inability of traditional histological techniques to cut metal objects. Consequently, it has been difficult to assess localized damage, if present, caused by tack insertion.
Therefore, we developed a technique for visualizing the tissue around a retinal tack and implant. We have modified an established technique, used for processing and visualizing hard bony tissue around a cochlear implant, for the soft delicate tissues of the eye. We orientated and embedded the fixed eye tissue, including the implant and retinal tack, in epoxy resin, to stabilise and protect the structure of the sample. Embedded samples were then ground, polished, stained, and imaged under various magnifications at incremental depths through the sample. This technique allowed the reliable assessment of eye tissue integrity and cytoarchitecture adjacent to the metal tack.

Here we describe histological techniques for visualising ocular tissue directly adjacent to a metal epiretinal tack and retinal prosthesis.

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices ...

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation of the animal for surgery, opening the thoracic body wall, exposing the desired rib from the surrounding intercostal muscles, excising the desired section of rib without inducing a pneumothorax, and closing the incisions. Compared to the bones of the appendicular skeleton, the ribs are highly accessible. In addition, no internal or external fixator is necessary since the adjacent ribs provide a natural fixation. The surgery uses commercially available supplies, is straightforward to learn, and well-tolerated by the animal. The procedure can be carried out with or without removing the surrounding periosteum, and therefore the contribution of the periosteum to repair can be assessed. Results indicate that if the periosteum is retained, robust repair occurs in 1 - 2 months. We expect that use of this protocol will stimulate research into rib repair and that the findings will facilitate the development of new ways to stimulate bone repair in other locations around the body.

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

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 ...

Proximal Cadaveric Femur Preparation for Fracture Strength Testing and Quantitative CT-based Finite Element Analysis

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength. Because ex vivo biological tissues are prone to lose their mechanical properties over time, specimen preparation for experimental testing must be performed carefully to obtain reliable results that represent in vivo conditions. For that reason, we designed a protocol and a set of fixtures to prepare the femoral specimens such that their mechanical properties experienced minimal changes. The femora were kept in a frozen state except during preparation steps and mechanical testing. The relevant clinical measures of total hip and femoral neck bone mineral density (BMD) were obtained with a clinical dual X-ray absorptiometry (DXA) bone densitometer, and the 3D geometry and distribution of bone mineral were obtained using CT with a calibration phantom for quantitative estimations based on the greyscale values. Any possible bone disease, fracture, or the presence of implants or artifacts affecting the bone structure, was ruled out with X-ray scans. For preparation, all bones were carefully cleaned of excess soft tissue, and were cut and potted at the internal rotation angle of interest. A cutting fixture allowed the distal end of the bone to be cut off leaving the proximal femur at a desired length. To allow positioning of the femoral neck at prescribed angles during later CT scanning and mechanical testing, the proximal femoral shafts were potted in polymethylmethacrylate (PMMA) using a fixture designed specifically for desired orientations. The data collected from our experiments were then used for validation of quantitative computed tomography (QCT)-based finite element analysis (FEA), as described in a different protocol. In this manuscript, we present the protocol for the precise bone preparation for mechanical testing and subsequent QCT/FEA modeling. The current protocol was successfully applied to prepare about 200 cadaveric femora over a 6-year time period.

We present a robust protocol on how to carefully preserve and prepare cadaveric femora for fracture testing and quantitative computed tomography imaging. The method provides precise control over input conditions for the purpose of determining relationships between bone mineral density, fracture strength, and defining finite element model geometry and properties.

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength.  Because ex vivo biological tissues are prone to ...

An Anatomical Study of Nerves at Risk During Minimally Invasive Hallux Valgus Surgery

The growing popularity of minimally invasive surgical (MIS) procedures makes it necessary that new anatomical references arise, to aid in tridimensional orientation and localization of structures that are not directly visible to the surgeon. This is especially critical for structures at risk like nerves or blood vessels. Optimization of the handling of cadaveric material and the combination of multiple techniques compensate for the limited availability of adequate specimens. The described protocol combines anatomical plane-by-plane dissection and sectional anatomy of fresh-frozen specimens to help localize relevant structures, such as nerves, arteries, veins and to correctly position the portals during MIS procedures. Depiction of these structures in anatomy textbooks can differ from what is encountered in the surgical field; and for this reason, new anatomical studies with a surgical orientation are needed. However, this is a complex, time-consuming technique requiring specific training. The anatomical references described with the so-called 'clock method' provide the surgeon with an easy and reproducible system to locate the path of the nerves at risk in Hallux Valgus MIS procedures. This model can be extrapolated to many other minimally invasive surgical procedures.

Minimally invasive surgical (MIS) procedures rely on anatomical references to localize structures not directly visible to the surgeon. This manuscript describes a combined method of plane-by-plane dissection and sectional anatomy of fresh-frozen specimens to locate the structures at risk during MIS procedures.

The growing popularity of minimally invasive surgical (MIS) procedures makes it necessary that new anatomical references arise, to aid in tridimensional ...