The authors thank the dental technician Nicola Lanfranconi for producing the master reference model and the Alicona Company for their ongoing support with improving the scan software.

1. Specimen Preparation/Base
<ol>
	<li>Apply a flat base around the specimen. Place the specimen on the scanning table. Orientate the occlusal surface to the horizontal plane (Figure 1).</li>
</ol>
2. Software Analysis
<ol>
	<li>Start the program and then the Laboratory Measurement Module (Figure 2)</li>
	<li>Position the specimen at the center of the scanning table.</li>
	<li>Choose the correct magnification lens. For large objects, like full arch scans, use the 5X objective.</li>
	<li>Move the scan optic by using the 3D mouse until the surface of the specimen is displayed on the live view window (Figure 3).</li>
	<li>Use sensor control to set Exposure and Contrast to achieve optimal scanning parameters. For metal surfaces, use an exposure between 400 and 800 &#181;sec and a contrast between 0.3 and 0.8 (Figure 4).</li>
	<li>Check image quality by checking the button "Show Image Quality in Live Preview".</li>
	<li>Set the correct parameters in the section "Measurement Control". The measurement type is "3D Dataset"; ImageField Type is "General ImageField" (Figure 5).</li>
	<li>Click on "New ImageField".</li>
	<li>Define the scan volume. The software needs the bounding coordinates of the measure volume.
	<ol>
		<li>Move the specimen to the highest and lowest scan level and click on "Add Position" at both points. The "Z Range" value shows the actual height of the scan volume (Figure 6).</li>
		<li>Move the specimen to the X-Y boundings of the scan volume. Click on "Add Position" to define X-axis and Y-axis lengths. The dimension of the scan volume is shown at the "Information" tab and should exceed the dimensions of the specimen by 1 cm in the X-axis and Y-axis.</li>
	</ol>
	</li>
	<li>Check the "Points" number. The software is capable of scanning 100 million surface points in one "ImageField" scan. The actual number of points exceeds this limit. "Decimate" the measure point's number by a "Lateral Downsampling" of the point size.
	<ol>
		<li>Click on "Advanced Settings" (Figure 7).</li>
		<li>Move the "Lateral Downsampling" slider to the right until the "Points" number is reduced below 100 M (Figure 7). Downsampling reduces the lateral resolution of the surface points, resulting in larger pixel size of the scanned object. To ensure optimal scan results, the vertical resolution is increased.</li>
	</ol>
	</li>
	<li>Click on "Start Measurement". This will start the "Preview Mode". The software performs a prescan with the selected X- and Y-axis dimensions.</li>
	<li>After the prescan is completed, select the region of interest. This helps to reduce file size and scanning time (Figure 8).</li>
	<li>Select all parts of the prescan except the specimen and two measure fields around the specimen, containing the flat base (Figure 9).</li>
	<li>Click on "Start" to begin the scan.</li>
	<li>Control the scan in the "ImageViewer" using the mouse and press the left mouse button (Figure 10).</li>
	<li>Close the window and click on "View Pseudo Color Only".</li>
	<li>Click on "Settings" and "Pseudo Coloring", and choose "Repeatability".</li>
	<li>Set the "Max." value to 0.2 and click on "Apply Range".</li>
	<li>Control the repeatability, it should be equal for areas with the same incline and material. In particular, the base around the specimen should display a homogeneous repeatability (Figure 11).</li>
	<li>Click on "Database" and save the scan to the appropriate folder.</li>
	<li>Export the scan to different file formats, if needed. Click on "File/Export/3D Data As/&#8230;." The protocol can be paused at that point and continued later.</li>
</ol>
3. Difference Analysis
<ol>
	<li>Use the "3D-Editor" to cut away the base. This area will not be used for the difference analysis.</li>
	<li>To compare and analyze two scans, start the "Difference Measurement" software (Figure 12).</li>
	<li>Choose the second model to compare.</li>
	<li>Click on "Automatic Rough Alignment" to perform a first match of the models.</li>
	<li>Click on "Manual Adjustment" and align the models with rotating and moving until they are in the same orientation (Figure 13).</li>
	<li>Click on "Automatic Alignment" and "Apply" to start the best fit algorithm for the optimal model matching.</li>
	<li>Click on "Differences" to view the difference map of the two matched models.</li>
	<li>Select a proper color range to display the deviations (Figure 14). Save the visual deviation pattern as a screenshot for analysis.</li>
</ol>
Click on "Statistics" to display the statistical values of the differences. Choose 1 &#181;m class size and save histogram data to a text file for statistical comparison (Figure 15).

<ol>
	<li>Wettstein, F., Sailer, I., Roos, M., H&#228;mmerle, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+study+of+the+internal+gaps+of+zirconia+and+metal+frameworks+for+fixed+partial+dentures.">Clinical study of the internal gaps of zirconia and metal frameworks for fixed partial dentures.</a> Eur. J. Oral Sci. 116 (3), 272-279 (2008).</li><li>Persson, A. S., Oden, A., Andersson, M., Sandborgh-Englund, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digitization+of+simulated+clinical+dental+impressions:+virtual+three-dimensional+analysis+of+exactness.">Digitization of simulated clinical dental impressions: virtual three-dimensional analysis of exactness.</a> Dent. Mater. 25 (7), 929-936 (2009).</li><li>Del'Acqua, M. A., Arioli-Filho, J. N., Compagnoni, M. A., Mollo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=de+A+Accuracy+of+impression+and+pouring+techniques+for+an+implant-supported+prosthesis.">de A Accuracy of impression and pouring techniques for an implant-supported prosthesis.</a> Int. J. Oral Maxillofac. Implants. 23 (2), 226-236 (2008).</li><li>Schneider, D., Grunder, U., Ender, A., H&#228;mmerle, C. H. F., Jung, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+gain+and+stability+of+peri-implant+tissue+following+bone+and+soft+tissue+augmentation:+1-year+results+from+a+prospective+cohort+study.">Volume gain and stability of peri-implant tissue following bone and soft tissue augmentation: 1-year results from a prospective cohort study.</a> Clinical Oral Implants Research. 22 (1), 28-37 (2011).</li><li>Windisch, S. I., Jung, R. E., Sailer, I., Studer, S. P., Ender, A., H&#228;mmerle, C. H. 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+new+optical+method+to+evaluate+three-dimensional+volume+changes+of+alveolar+contours:+a+methodological+in+vitro+study.">A new optical method to evaluate three-dimensional volume changes of alveolar contours: a methodological in vitro study.</a> Clinical Oral Implants Research. 18 (5), 545-551 (2007).</li><li>Caputi, S., Varvara, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimensional+accuracy+of+resultant+casts+made+by+a+monophase,+one-step+and+two-step,+and+a+novel+two-step+putty/light-body+impression+technique:+an+in+vitro.">Dimensional accuracy of resultant casts made by a monophase, one-step and two-step, and a novel two-step putty/light-body impression technique: an in vitro.</a> 99 (4), 274-281 (2008).</li><li>Hoyos, A., Soderholm, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+tray+rigidity+and+impression+technique+on+accuracy+of+polyvinyl+siloxane+impressions.">Influence of tray rigidity and impression technique on accuracy of polyvinyl siloxane impressions.</a> Int. J. Prosthodont. 24 (1), 49-54 (2011).</li><li>Luthardt, R. G., Kuhmstedt, P., Walter, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+computer-aided+evaluation+of+three-dimensional+changes+in+gypsum+materials.">A new method for the computer-aided evaluation of three-dimensional changes in gypsum materials.</a> Dent. Mater. 19 (1), 19-24 (2003).</li><li>Mehl, A., Ender, A., M&#246;rmann, W., Attin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+testing+of+a+new+intraoral+3D+camera.">Accuracy testing of a new intraoral 3D camera.</a> International Journal of Computerized Dentistry. 12 (1), 11-28 (2009).</li><li>Fickl, 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=Dimensional+changes+of+the+ridge+contour+after+socket+preservation+and+buccal+overbuilding:+an+animal+study.">Dimensional changes of the ridge contour after socket preservation and buccal overbuilding: an animal study.</a> J. Clin. Periodontol. 36 (5), 442-448 (2009).</li><li>Vlaar, S. T., vander Zel, 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=Accuracy+of+dental+digitizers.">Accuracy of dental digitizers.</a> Int. Dent. J. 56 (5), 301-309 (2006).</li><li>Ender, A., Mehl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+complete-arch+dental+impressions:+a+new+method+of+measuring+trueness+and+precision.">Accuracy of complete-arch dental impressions: a new method of measuring trueness and precision.</a> 109 (2), 121-128 (2013).</li><li>Ender, A., Mehl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+scanning+strategies+on+the+accuracy+of+digital+intraoral+scanning+systems.">Influence of scanning strategies on the accuracy of digital intraoral scanning systems.</a> Int. J. Comput. Dent. 16 (1), 11-21 (2013).</li><li>Ender, A., Mehl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full+arch+scans:+conventional+versus+digital+impressions--an+in-vitro+study.">Full arch scans: conventional versus digital impressions--an in-vitro study.</a> Int. J. Comput. Dent. 14 (1), 11-21 (2011).</li><li>Meer, W. J., Andriessen, F. S., Wismeijer, D., Ren, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+intra-oral+dental+scanners+in+the+digital+workflow+of+implantology.">Application of intra-oral dental scanners in the digital workflow of implantology.</a> PLoS One. 7 (8), (2012).</li></ol>

The authors declare that they have no competing financial interests or other conflicts of interest.

Accuracy is a basic demand in dental medicine. The reference scanner is capable of scanning small and large objects with high trueness and precision. With the optimal scanning method, even individual morphological detailed dental surfaces can be scanned with a high resolution and repeatability. With the different magnification levels of the scanner, macro and micro morphological structures can be acquired. It is possible to scan a variety of model materials.
The evaluation of the reference sca...

Accuracy is a major interest in many fields in dental medicine. Replacing dental hard tissue needs an exact fitting prosthesis to ensure proper function and prevent further destroying the remaining tooth structure1,2. Fixed partial dentures and total prosthesis are especially critical for exact fitting at supporting structures like prepared teeth or implants3. This is why a highly accurate reproduction is needed, especially in the field of dental impressions and dental laboratory workflow. However, other fields of dental treatment also benefit from a true and precise metrical outcome, to verify treatment success and evaluate new treatment strategies, e.g.&#160;soft and hard tissue augmentation, erosion and abrasion monitoring, periodontal treatments, and orthodontic treatments4,5. In many of these fields, current validation procedures are linear distance measurements with calipers or microscopes6,7. These methods are limited to only few measuring points and limited information of three-dimensional (3D) changes of the testing area. Newer measuring methods include the optical or radiographic capturing of the entire surface of the test object8,9. Here, the entire surface or volume is measured and displayed as a 3D object on the computer screen. Linear measurements are possible, as well as superimpositions of models from different scan times. With this superimposition, an evaluation of the surface changes at every scan point is possible. This enables the possibility of monitoring a specific area or displaying deformations in all three coordinate axes. Also, volumetric changes can be measured10. The limiting point with these new methods is the accuracy of the scanner, used to capture the test object. None of the changes within the accuracy of the reference scanner can be divided into changes of the test object or scan errors. Scan accuracy is often a value given by the manufacturer derived from scanning small, calibrated objects11. This minimal scan error is different when scanning large objects like a dental arch. Accuracy consists of trueness and precision. Trueness is the deviation of the scanned object from its real geometry. Precision is the deviation between repeated scans (ISO 5725-1). In this study, a new optical reference scanner, based on the focus variation scanning technique, was introduced to scan specimens from single tooth up to full arch models with highest accuracy. This reference scanner was used as a base for several studies, comparing dental impression accuracy from conventional and digital techniques12&#8211;14 and for actual projects concerning dental occlusion and abrasion of dental materials. The aim of this study was to provide basic information of the accuracy of the reference scanner and some possibilities to use this device in the field of dental research.

<table border="0" cellpadding="0" cellspacing="0" width="669">
	<tbody>
		<tr>
			<td>Reference model</td>
			<td></td>
			<td></td>
			<td>individual non-precious metal model, derived from a patient impression</td>
		</tr>
		<tr>
			<td>Araldit repair</td>
			<td>Huntsmen Advanced Material, Basel, Switzerland</td>
			<td></td>
			<td>used for making the base of the reference model</td>
		</tr>
		<tr>
			<td>CamBase</td>
			<td>Dentona, Dortmund, Germany</td>
			<td></td>
			<td>Type IV dental ston for pouring conventional impressions</td>
		</tr>
		<tr>
			<td>Identium</td>
			<td>Kettenbach, Eschenburg, Germany</td>
			<td></td>
			<td>Vinylsiloxanether impression material for conventional impression</td>
		</tr>
		<tr>
			<td>inEOS model holder</td>
			<td>Sirona Dental Systems, Bensheim, Germany</td>
			<td></td>
			<td>used for fixing stone models at the reference scanner</td>
		</tr>
		<tr>
			<td>Accutrans</td>
			<td>Coltene Whaledent, Altst&#228;tten, Switzerland</td>
			<td></td>
			<td>used for making the base of thestone models</td>
		</tr>
		<tr>
			<td>President putty</td>
			<td>Coltene Whaledent, Altst&#228;tten, Switzerland</td>
			<td></td>
			<td>mix with accutrans for betterstability of the base</td>
		</tr>
		<tr>
			<td>Alicona Infinite Focus</td>
			<td>Alicona Imaging, Graz, Austria</td>
			<td></td>
			<td>Reference scanner&#160;</td>
		</tr>
	</tbody>
</table>

accuracy in dental medicine, a new way to measure trueness and precision

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base for dental restorations. The current limitation of many reference scanners is the lack of accuracy scanning large objects like full dental arches, or the limited possibility to assess detailed tooth surfaces. A new reference scanner, based on focus variation scanning technique, was evaluated with regards to highest local and general accuracy. A specific scanning protocol was tested to scan original tooth surface from dental impressions. Also, different model materials were verified. The results showed a high scanning accuracy of the reference scanner with a mean deviation of 5.3 &#177; 1.1 &#181;m for trueness and 1.6 &#177; 0.6 &#181;m for precision in case of full arch scans. Current dental impression methods showed much higher deviations (trueness: 20.4 &#177; 2.2 &#181;m, precision: 12.5 &#177; 2.5 &#181;m) than the internal scanning accuracy of the reference scanner. Smaller objects like single tooth surface can be scanned with an even higher accuracy, enabling the system to assess erosive and abrasive tooth surface loss. The reference scanner can be used to measure differences for a lot of dental research fields. The different magnification levels combined with a high local and general accuracy can be used to assess changes of single teeth or restorations up to full arch changes.

Figure 16A shows the scan of a gypsum specimen. To verify optimal scan parameters, control the repeatability for the material. A section of the specimen is scanned with different contrast and brightness settings and the repeatability is checked after each scan to find the optimal scan settings. Metal surfaces show the best results with a contrast lower 1.0 and gypsum or resin materials with a higher contrast. Figure 16B shows the repeatability without optimal scanning parameters after th...

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Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Accuracy is a major demand in dental medicine. To verify accuracy, reference scanners are needed. This article presents a new reference scanner with an adjusted scanning method to acquire a broad variety of dental morphologies with high trueness and precision.

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base ...

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JoVE Journal

Medicine

13.3K visualizações.  University of Zürich.  Precisão é uma grande demanda na medicina dentária. Para verificar a exatidão, são necessários scanners de referência. Este artigo apresenta um novo scanner de referência com um método de varredura ajustado para adquirir uma ampla variedade de morfologias dentárias com alto rigor e precisão. 

Vídeo: Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic processes that prepare the alimentary canal for digestion. Steady progress is being made towards understanding the early biochemical and molecular events underlying taste transduction (for a review, Breslin and Spector, 20081). However, progress to date has largely resulted from animal models. Yet, since marked differences in receptor specificity and receptor density vary among species, human taste transduction will only be understood by using human taste tissue. Here we describe a biopsy technique to collect human fungiform papillae, visible as rounded pink anterior structures, about 0.5 mm in diameter that contain taste buds. These biopsied papillae are used for several purposes including the isolation of viable taste bud cells, in situ hybridization, immunohistochemistry and, through techniques of molecular biology, the identification of taste-specific novel proteins.

Technique to Collect Fungiform Taste Papillae from Human Tongue

Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on human tongue.

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic ...

Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.

We demonstrate the construction of alcohol vapor chambers using readily available materials that simultaneously house 6 mouse cages. We further describe their use in a mouse model of fetal alcohol exposure equivalent to all 3&#160;trimesters of human pregnancy. This paradigm exposes animals during gestation and postnatal days 1-12.

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is important to standardize protocols to ensure proper analyses and interpretations. In our previous studies, we have measured boldness by monitoring a series of behaviors (time near surface, latency in transitions, number of transitions, and darts) in a 1.5 L trapezoidal tank. Likewise, we quantified aggression by observing bites, lateral displays, darts, and time near an inclined mirror in a rectangular 19 L tank. By dividing a 76 L tank into thirds, we also examined shoaling preferences. The shoaling assay is a highly customizable assay and can be tailored for specific hypotheses. However, protocols for this assay also must be standardized, yet flexible enough for customization. In previous studies, end chambers were either empty, contained 5 or 10 zebrafish, or 5 pearl danios (D. albolineatus). In the following manuscript, we present a detailed protocol and representative data that accompany successful applications of the protocol, which will allow for replication of behavioral syndrome experiments.

This manuscript describes the setup, implementation, and analysis of boldness, aggression, and shoaling in zebrafish and testing for the presence of a behavioral syndrome. A standardized approach for behavioral quantification will allow for easier comparison across studies. Modifications to this protocol are possible as each assay can be run individually.

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Conducting Maximal and Submaximal Endurance Exercise Testing to Measure Physiological and Biological Responses to Acute Exercise in Humans

Regular physical activity has a positive effect on human health, but the mechanisms controlling these effects remain unclear. The physiologic and biologic responses to acute exercise are predominantly influenced by the duration and intensity of the exercise regimen. As exercise is increasingly thought of as a therapeutic treatment and/or diagnostic tool, it is important that standardizable methodologies be utilized to understand the variability and to increase the reproducibility of exercise outputs and measurements of responses to such regimens. To that end, we describe two different cycling exercise regimens that yield different physiologic outputs. In a maximal exercise test, exercise intensity is continually increased with a greater workload resulting in an increasing cardiopulmonary and metabolic response (heart rate, stroke volume, ventilation, oxygen consumption and carbon dioxide production). In contrast, during endurance exercise tests, the demand is increased from that at rest, but is raised to a fixed submaximal exercise intensity resulting in a cardiopulmonary and metabolic response that typically plateaus. Along with the protocols, we provide suggestions on measuring physiologic outputs that include, but are not limited to, heart rate, slow and forced vital capacity, gas exchange metrics, and blood pressure to enable the comparison of exercise outputs between studies. Biospecimens can then be sampled to assess cellular, protein, and/or gene expression responses. Overall, this approach can be easily adapted into both short- and long-term effects of two distinct exercise regimens.

To assess the influence of exercise intensity on physiologic and biologic responses, two different exercise testing protocols were utilized. Methods outlining exercise testing on a cycle ergometer as an incremental maximal oxygen consumption test and endurance, steady state submaximal endurance test are described.

Regular physical activity has a positive effect on human health, but the mechanisms controlling these effects remain unclear. The physiologic and biologic ...

Precision Implementation of Minimal Erythema Dose (MED) Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary to produce erythema (inflammatory reddening) on the surface of the skin. In this context, the MED is regarded as a key factor in determining starting doses for UV phototherapy for common skin conditions such as psoriasis and eczema. In research settings, MED testing also has potential to be a powerful tool for assessing within- and between-persons variation in inflammatory responses. However, MED testing has not been widely adopted for use in research settings, likely owing to a lack of published guidelines, which is a barrier to obtaining reproducible results from this assay. Also, protocols and equipment for establishing MED vary widely, making it difficult to compare results across laboratories. Here, we describe a precise and reproducible method to induce and measure superficial erythema using newly designed protocols and methods that can easily be adapted to other equipment and laboratory environments. The method described here includes detail on procedures that will allow extrapolation of a standardized dosage schedule to other equipment so that this protocol can be adapted to any UV radiation source.

Precision Implementation of Minimal Erythema Dose MED Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is used to establish dosage schedules for ultraviolet radiation phototherapy. It can assess individual variation in inflammatory response but lacks methodology for achieving reproducible results. Here, we present a precision implementation of MED and demonstrate its ability to capture individual variation in inflammatory response.

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Intraoperative Assessment of Resection Margins in Oral Cavity Cancer: This is the Way

The goal of head and neck oncological surgery is complete tumor resection with adequate resection margins while preserving acceptable function and appearance. For oral cavity squamous cell carcinoma (OCSCC), different studies showed that only 15%-26% of all resections are adequate. A major reason for the low number of adequate resections is the lack of information during surgery; the margin status is only available after the final histopathologic assessment, days after surgery.
The surgeons and pathologists at the Erasmus MC University Medical Center in Rotterdam started the implementation of specimen-driven intraoperative assessment of resection margins (IOARM) in 2013, which became the standard of care in 2015. This method enables the surgeon to turn an inadequate resection into an adequate resection by performing an additional resection during the initial surgery. Intraoperative assessment is supported by a relocation method procedure that allows accurate identification of inadequate margins (found on the specimen) in the wound bed.
The implementation of this protocol resulted in an improvement of adequate resections from 15%-40%. However, the specimen-driven IOARM is not widely adopted because grossing fresh tissue is counter-intuitive for pathologists. The fear exists that grossing fresh tissue will deteriorate the anatomical orientation, shape, and size of the specimen and therefore will affect the final histopathologic assessment. These possible negative effects are countered by the described protocol. Here, the protocol for specimen-driven IOARM is presented in detail, as performed at the institute.

The goal of this protocol is to provide a clear overview of specimen-driven intraoperative assessment of resection margins. It is encouraged to implement this protocol to improve patient care at other institutes.