Name: software-assisted quantitative measurement of osteoarthritic subchondral bone thickness
Uploaded: 2022-03-18T14:00:00
Description: Watch this Scientific Journal Video about Software-Assisted Quantitative Measurement of Osteoarthritic Subchondral Bone Thickness at JoVE.com

This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (NIH) under Award Number R01 AR059088, the Department of Defense (DoD) under Research Award Number W81XWH-12-1-0304, and the Mary and Paul Harrington Distinguished Professorship Endowment.

All animal procedures included in this protocol were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Kansas Medical Center, in compliance with all federal and state laws and regulations.
1. Creation of knee OA in mice
<ol>
	<li>Create a mouse model of knee OA by surgical destabilization of the medial meniscus (DMM) as described by Glasson et al.18 in 22 wild-type BALB/c mice at 10-11 weeks of age. Perform sham ...

<ol>
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N., Mueller-Gerbl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basic+science+of+the+subchondral+bone.">The basic science of the subchondral bone.</a> Knee Surgery, Sports, Traumatology, Arthrosclerosis. 18 (4), 419-433 (2010).</li><li>Milz, S., Putz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+morphology+of+the+subchondral+plate+of+the+tibial+plateau.">Quantitative morphology of the subchondral plate of the tibial plateau.</a> Journal of Anatomy. 185, 103-110 (1994).</li><li>Blalock, D., Miller, A., Tilley, M., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Joint+instability+and+osteoarthritis.">Joint instability and osteoarthritis.</a> Clinical Medicine Insights: Arthritis and Musculoskeleton Disorders. 8, 15-23 (2015).</li><li>Waung, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+X-ray+microradiography+for+high-throughput+phenotyping+of+osteoarthritis+in+mice.">Quantitative X-ray microradiography for high-throughput phenotyping of osteoarthritis in mice.</a> Osteoarthritis Cartilage. 22 (10), 1396-1400 (2014).</li><li>Botter, S. M., 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=Cartilage+damage+pattern+in+relation+to+subchondral+plate+thickness+in+a+collagenase-induced+model+of+osteoarthritis.">Cartilage damage pattern in relation to subchondral plate thickness in a collagenase-induced model of osteoarthritis.</a> Osteoarthritis Cartilage. 16 (4), 506-514 (2008).</li><li>Nalesso, G., 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=Calcium+calmodulin+kinase+II+activity+is+required+for+cartilage+homeostasis+in+osteoarthritis.">Calcium calmodulin kinase II activity is required for cartilage homeostasis in osteoarthritis.</a> Science Reports. 11 (1), 5682 (2021).</li><li>Ding, M., Christian Danielsen, C., Hvid, I. <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+hyaluronan+on+three-dimensional+microarchitecture+of+subchondral+bone+tissues+in+guinea+pig+primary+osteoarthrosis.">Effects of hyaluronan on three-dimensional microarchitecture of subchondral bone tissues in guinea pig primary osteoarthrosis.</a> Bone. 36 (3), 489-501 (2005).</li><li>Kraus, V. B., Huebner, J. L., DeGroot, J., Bendele, 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+OARSI+histopathology+initiative+-+recommendations+for+histological+assessments+of+osteoarthritis+in+the+guinea+pig.">The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the guinea pig.</a> Osteoarthritis Cartilage. 18, 35-52 (2010).</li><li>McNulty, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+histological+assessment+of+osteoarthritis+lesions+in+Mice.">A comprehensive histological assessment of osteoarthritis lesions in Mice.</a> Cartilage. 2 (4), 354-363 (2011).</li><li>Glasson, S. S., Chambers, M. G., Van Den Berg, W. B., Little, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+OARSI+histopathology+initiative+-+recommendations+for+histological+assessments+of+osteoarthritis+in+the+mouse.">The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the mouse.</a> Osteoarthritis Cartilage. 18, 17-23 (2010).</li><li>Pritzker, K. 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=Osteoarthritis+cartilage+histopathology:+grading+and+staging.">Osteoarthritis cartilage histopathology: grading and staging.</a> Osteoarthritis Cartilage. 14 (1), 13-29 (2006).</li><li>Mankin, H. J., Dorfman, H., Lippiello, L., Zarins, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+metabolic+abnormalities+in+articular+cartilage+from+osteo-arthritic+human+hips.+II.+Correlation+of+morphology+with+biochemical+and+metabolic+data.">Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data.</a> Journal of Bone and Joint Surgery American. 53 (3), 523-537 (1971).</li><li>Glasson, S. S., Blanchet, T. J., Morris, E. 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+surgical+destabilization+of+the+medial+meniscus+(DMM)+model+of+osteoarthritis+in+the+129/SvEv+mouse.">The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse.</a> Osteoarthritis Cartilage. 15 (9), 1061-1069 (2007).</li><li>Wang, J., 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=Transcription+factor+Nfat1+deficiency+causes+osteoarthritis+through+dysfunction+of+adult+articular+chondrocytes.">Transcription factor Nfat1 deficiency causes osteoarthritis through dysfunction of adult articular chondrocytes.</a> Journal of Pathology. 219 (2), 163-172 (2009).</li><li>Zhang, M., Lu, Q., Budden, T., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NFAT1+protects+articular+cartilage+against+osteoarthritic+degradation+by+directly+regulating+transcription+of+specific+anabolic+and+catabolic+genes.">NFAT1 protects articular cartilage against osteoarthritic degradation by directly regulating transcription of specific anabolic and catabolic genes.</a> Bone Joint Research. 8 (2), 90-100 (2019).</li><li>Zhang, M., 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=Epigenetically+mediated+spontaneous+reduction+of+NFAT1+expression+causes+imbalanced+metabolic+activities+of+articular+chondrocytes+in+aged+mice.">Epigenetically mediated spontaneous reduction of NFAT1 expression causes imbalanced metabolic activities of articular chondrocytes in aged mice.</a> Osteoarthritis Cartilage. 24 (7), 1274-1283 (2016).</li><li>Rodova, M., 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=Nfat1+regulates+adult+articular+chondrocyte+function+through+its+age-dependent+expression+mediated+by+epigenetic+histone+methylation.">Nfat1 regulates adult articular chondrocyte function through its age-dependent expression mediated by epigenetic histone methylation.</a> Journal of Bone and Mineral Research. 26 (8), 1974-1986 (2011).</li><li>Jackson, M. T., 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=Depletion+of+protease-activated+receptor+2+but+not+protease-activated+receptor+1+may+confer+protection+against+osteoarthritis+in+mice+through+extracartilaginous+mechanisms.">Depletion of protease-activated receptor 2 but not protease-activated receptor 1 may confer protection against osteoarthritis in mice through extracartilaginous mechanisms.</a> Arthritis and Rheumatology. 66 (12), 3337-3348 (2014).</li></ol>

Measuring SCB thickening is an important component of histologic assessments of OA severity. Most existing OA grading systems focus mainly on cartilage changes15,16,17. A widely used murine osteoarthritic SCB thickness grading method that categorizes SCB thickening as mild, moderate, and severe is largely subjective, and its reliability has not been fully validated15. The present study has developed and v...

Osteoarthritis (OA), characterized radiographically by joint space narrowing due to the loss of articular cartilage, osteophytes, and subchondral bone (SCB) sclerosis, is the most common form of arthritis1,2. Although the role of peri-articular bone in the etiology of OA is not fully understood, osteophyte formation and SCB sclerosis are generally thought to be the results of the disease process rather than causative factors, but changes in peri-articular bone architecture/shape and biology may contribute to the development and progression of OA3,4. The development of an accurate and easily executed OA grading system, including SCB measurement, is critical for comparative studies among research laboratories and in evaluating the efficacy of therapeutic agents designed to prevent or attenuate OA progression.
SCB is built with a thin dome-like bone plate and an underlying layer of trabecular bone. The SCB plate is the cortical lamella, lying parallel to and immediately under the calcified cartilage. Small branches of arterial and venous vessels, as well as nerves, penetrate through the channels in the SCB plate, communicating between the calcified cartilage and the trabecular bone. The subchondral trabecular bone contains blood vessels, sensory nerves, bone marrow and is more porous and metabolically active than the SCB plate. Therefore, SCB exerts shock-absorbing and supportive functions and is also important for cartilage nutrient supply and metabolism in normal joints5,6,7,8.
SCB thickening (in histology) and sclerosis (in radiography) are the major hallmarks of OA and key research areas of OA pathophysiology. Measuring SCB thickening is an important component of histologic assessments of OA severity. Previously reported digital microradiography for measuring rodent SCB mineral density9 as well as micro-computed tomography (micro-CT) based quantitative SCB measurement in rodent models of OA10,11,12,13 have improved our understanding of SCB structure and the role of SCB changes in OA pathophysiology. SCB area and thickness has also been quantified with histological slides using a sophisticated computer system with specific and expensive bone histomorphometry software14. Nevertheless, visual estimate-based semi-quantitative OA grading systems, including SCB thickening grading, are more widely used than micro-CT at the present time because the grading systems are easy to use, particularly for screening numerous histologic images. However, most existing OA grading systems focus mainly on cartilage changes15,16,17. A widely used osteoarthritic SCB thickness grading method that categorizes SCB thickening as mild, moderate, and severe is largely subjective, and its reliability has not been fully validated15. A reliable and easily executed step-by-step osteoarthritic SCB thickness measurement protocol is either not fully developed or un-standardized.
This study aimed to develop a reproducible, sensitive, and easily executed protocol to quantitatively measure the SCB thickness in a mouse model of OA. Our rigorous measurement tests and statistical analysis demonstrated that this ImageJ software-assisted quantitative measurement protocol could quantify the SCB thickness in both normal and osteoarthritic knee joints. The newly developed protocol is reproducible and more sensitive to mild SCB changes than the widely used visual grading systems. It can be used for detecting early osteoarthritic SCB changes and for assessing in vivo efficacy of OA treatments in concert with OA cartilage grading.

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			<td>Safranin-O</td>
			<td>Sigma-Aldrich</td>
			<td>S8884</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast green</td>
			<td>Sigma-Aldrich</td>
			<td>F7252</td>
			<td></td>
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		<tr>
			<td>Hematoxylin</td>
			<td>Sigma-Aldrich</td>
			<td>GHS216</td>
			<td></td>
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			<td>Eosin</td>
			<td>Sigma-Aldrich</td>
			<td>E4382</td>
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software-assisted quantitative measurement of osteoarthritic subchondral bone thickness

Subchondral bone thickening and sclerosis are the major hallmarks of osteoarthritis (OA), both in animal models and in humans. Currently, the severity of the histologic subchondral bone thickening is mostly determined by visual estimation based semi-quantitative grading systems. This article presents a reproducible and easily executed protocol to quantitatively measure subchondral bone thickness in a mouse model of knee OA induced by destabilization of the medial meniscus (DMM). This protocol utilized ImageJ software to quantify subchondral bone thickness on histologic images after defining a region of interest in the medial femoral condyle and the medical tibial plateau where subchondral bone thickening usually occurs in DMM-induced knee OA. Histologic images from knee joints with a sham procedure were used as controls. Statistical analysis indicated that the newly developed quantitative subchondral bone measurement system was highly reproducible with low intra- and inter-observer variabilities. The results suggest that the new protocol is more sensitive to subtle or mild subchondral bone thickening than the widely used visual grading systems. This protocol is suitable for detecting both early and progressing osteoarthritic subchondral bone changes and for assessing in vivo efficacy of OA treatments in concert with OA cartilage grading.

Reproducibility comparison between visual estimate grading and ImageJ-assisted quantitative measurement: 
SCB thickness in 48 regions of interest (ROI) (24 MFC and 24 MTP), defined from a mid-section of each knee from 24 knees/animals was scored by three independent individuals using the existing 0-3 visual scoring scheme as described in the literature15,23, where 0 = normal (no SCB thickening), 1 = mild, 2 = moderate, and 3 = severe SCB thi...

Watch this Scientific Journal Video about Software-Assisted Quantitative Measurement of Osteoarthritic Subchondral Bone Thickness at JoVE.com

Software-Assisted Quantitative Measurement of Osteoarthritic Subchondral Bone Thickness

This methodology article presents a software-assisted quantitative measurement protocol to quantify histologic subchondral bone thickness in murine osteoarthritic knee joints and normal knee joints as controls. This protocol is highly sensitive to subtle thickening and is suitable for detecting early osteoarthritic subchondral bone changes.

Subchondral bone thickening and sclerosis are the major hallmarks of osteoarthritis (OA), both in animal models and in humans. Currently, the severity of ...

Subchondral bone thickening, or sclerosis, is one of the hallmarks of osteoarthritis, both in animal models and in humans. Currently, the severity of histologic subchondral bone thickening is mostly determined by visual estimation based on semi-quantitative grading systems. This study aim to develop a reproducible and easily executed protocol to quantitatively measure subchondral bone thickness in a mouse model of knee OA induced by destabilization of the medial meniscus.We created a mouse model for knee osteoarthritis by surgical destabilization of the medial meniscus which is performed under surgical microscope and the sterile condition. The histological process of mouse knee joints include tissue fixation, decalcification, trimming, processing, embedding, sectioning, and staining, followed by microscopic imaging. The image on the screen shows mouse knee osteoarthritis with articular cartilage lesions, osteophyte formation, and subchondral bone thickening Quantitative measurement of osteoarthritic subchondral bone with ImageJ software.Download ImageJ from imagej.nih.gov/ij. Open ImageJ. Click the File tab and then click Open option to open the histologic image.Find the file directory address, select the picture file, and click Open. Use the straight line tool to sketch one unit of length, a micrometer, and click Analyze, then Set Scale. Set the known distance and the pixel aspect ratio to one and click Ok.ImageJ can convert the pixel length to the unit length, a micrometer.Set the measured factor to area. Click Analyze then Set Measurement and check Area and Limit to Threshold box under the new window. This step sets ImageJ to measure parameter area within selected threshold.Defined total subchondral bone area of interest as shown in radio which covers the subchondral cortical plate and a portion of the underlying trabecular bone adjacent to cortical plate. Sketch the outline of total subchondral bone area by using the selection tool under the main window of image. The selection tools gives the system a threshold to limit the measurement after threshold being selected.Click Analyze then Measure. A result window with area measurement will open. Click Edit then Clear Outside to exclude the area outside total subchondral bone area.Only total subchondral bone area is visible after clicking Clear Outside option. Click Image then Adjust then Color Threshold to open the threshold color window. Click Original at the bottom of threshold color window to restore picture to original status.Use selection tools to draw a small box in the bone substance region. Click Sample Option at the bottom of window to define the bone substance area. Selected bone substance area will turn to red.Click Select at the bottom of threshold color window to create area measurement threshold. Click Analyze then Measure in ImageJ main menu. The bone substance area measurement result will show on result window.Save the data of total subchondral bone area and bone substance area. The inter and intraobserver variability and the reproducibility were determined by Pearson's correlation coefficient analysis. The significance of difference between study groups were determined by using Student t-test or one-way ANOVA, followed by post-hoc test.This figure shows upper and lower panels. The upper panel shows photomicrographs of histologic images from Sham and DMM groups for visual estimate-based subchondral bone grading. The lower panels show photomicrographs of histologic images from Sham and DMM groups for ImageJ-assisted, quantitative subchondral bone measurement.The boxes outlined with a dotted yellow line made with Adobe Illustrator defines the total subchondral bone area of interest. The area of bone substance within the boxes is highlighted in orange. Remarkable subchondral bone thickening in the medial femoral condyle and medial tibial plateau can be quantified using ImageJ software.In this figure, interobserver variation tests indicate a high reproducibility between observers for both the first and second measurements of subchondral bone thickness in the medial tibial plateau and the medial femoral condyle regions of interest. In this figure, intraobserver variation tests indicate a high reproducibility between the first and second subchondral bone thickness measurements in the medial tibial plateau and medial femoral condyle regions of interest. Table 1 shows comparative analysis of reproducibility between the visual grading and ImageJ-assisted quantitative measurement of subchondral bone thickness.Correlation coefficient test suggests that the quantitative measurement was relatively more reproducible than the visual grading system. This figure shows comparative sensitivity analysis of visual grading and ImageJ-assisted quantitative measurement of subchondral bone thickness in the medial femoral condyle and the medial tibial plateau. The histologic images for visual estimate grading were divided into three groups.The quantitative subchondral bone thickness value from all three observers for the DMM images with a zero visual score were significantly higher than that of the Sham images with a zero visual score, indicating that the quantitative measurement is more sensitive than the visual grading to mild subchondral bone thickening. The newly developed protocol for quantitative measurement of osteoarthritic subchondral bone thickness is more sensitive to mild subchondral bone changes than the widely used visual grading systems. And this protocol can be used for detecting early osteoarthritic subchondral bone changes and for assessing in vivo efficacy of osteoarthritis treatments in concert with osteoarthritis cartilage grading.

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Research

JoVE Journal

Medicine

Quantitative Autonomic Testing

Disorders associated with dysfunction of autonomic nervous system are quite common yet frequently unrecognized. Quantitative autonomic testing can be invaluable tool for evaluation of these disorders, both in clinic and research. There are number of autonomic tests, however, only few were validated clinically or are quantitative. Here, fully quantitative and clinically validated protocol for testing of autonomic functions is presented. As a bare minimum the clinical autonomic laboratory should have a tilt table, ECG monitor, continuous noninvasive blood pressure monitor, respiratory monitor and a mean for evaluation of sudomotor domain. The software for recording and evaluation of autonomic tests is critical for correct evaluation of data. The presented protocol evaluates 3 major autonomic domains: cardiovagal, adrenergic and sudomotor. The tests include deep breathing, Valsalva maneuver, head-up tilt, and quantitative sudomotor axon test (QSART). The severity and distribution of dysautonomia is quantitated using Composite Autonomic Severity Scores (CASS). Detailed protocol is provided highlighting essential aspects of testing with emphasis on proper data acquisition, obtaining the relevant parameters and unbiased evaluation of autonomic signals. The normative data and CASS algorithm for interpretation of results are provided as well.

Standardized, comprehensive and fully quantitative testing of autonomic functions is described. The autonomic tests consist of evaluation of all three major autonomic domains including cardiovagal, adrenergic and sudomotor. The severity and distribution of dysautonomia is quantitated using Composite Autonomic Severity Scores.

Disorders associated with dysfunction of autonomic nervous system are quite common yet frequently unrecognized. Quantitative autonomic testing can be ...

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 ...

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing continuous remodeling with the competing activity of osteoblasts, which produce the new bone matrix, and osteoclasts, whose function is to eliminate mineralized bone. [18F]-NaF is a positron emission tomography (PET) radiotracer that enables visualization of bone metabolism. [18F]-NaF is chemically absorbed into hydroxyapatite in the bone matrix by osteoblasts and can thus noninvasively detect osteoblastic activity, which is occult to conventional imaging techniques. Kinetic modeling of dynamic [18F]-NaF-PET data provides detailed quantitative measures of bone metabolism. Conventional semi-quantitative PET data, which utilizes standardized uptake values (SUVs) as a measure of radiotracer activity, is referred to as a static technique due to its snapshot of tracer uptake in time.&#160; Kinetic modeling, however, utilizes dynamic image data where tracer levels are continuously acquired providing tracer uptake temporal resolution. From the kinetic modeling of dynamic data, quantitative values like blood flow and metabolic rate (i.e., potentially informative metrics of tracer dynamics) can be extracted, all with respect to the measured activity in the image data. When combined with dual modality PET-MRI, region-specific kinetic data can be correlated with anatomically registered high-resolution structural and pathologic information afforded by MRI. The goal of this methodological manuscript is to outline detailed techniques for performing and analyzing dynamic [18F]-NaF-PET-MRI data. The lumbar facet joint is a common site of degenerative arthritis disease and a common cause for axial low back pain.&#160; Recent studies suggest [18F]-NaF-PET may serve as a useful biomarker of painful facetogenic disease.&#160; The human lumbar facet joint will, therefore, be used as a prototypical region of interest for dynamic [18F]-NaF-PET-MRI analysis in this manuscript.

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts

Bone development and homeostasis is dependent upon the differentiation and activity of bone forming osteoblasts. Osteoblast differentiation is sequentially characterized by proliferation followed by protein synthesis and ultimately bone matrix secretion. Proliferation and protein synthesis require a constant supply of amino acids. Despite this, very little is known about amino acid consumption in osteoblasts. Here we describe a very sensitive protocol that is designed to measure amino acid consumption using radiolabeled amino acids. This method is optimized to quantify changes in amino acid uptake that are associated with osteoblast proliferation or differentiation, drug or growth factor treatments, or various genetic manipulations. Importantly, this method can be used interchangeably to quantify amino acid consumption in cultured cell lines or primary cells in vitro or in isolated bone shafts ex vivo. Finally, our method can be easily adapted to measure the transport of any of the amino acids as well as glucose and other radiolabeled nutrients.

This protocol presents a radiolabeled amino acid uptake assay, which is useful for evaluating amino acid consumption either in primary cells or in isolated bones.

Bone development and homeostasis is dependent upon the differentiation and activity of bone forming osteoblasts. Osteoblast differentiation is ...

Precision of In Vivo Quantitative Tooth Wear Measurement Using Intra-Oral Scans

Quantitative wear measurement is of increasing interest for measuring tooth wear progression. However, most research on quantitative wear measurement has focused on simulated wear or scanned gypsum casts. A 3D Wear Analysis (3DWA) protocol has been developed that analyzes tooth wear in vivo through intra-oral scanners available to dental clinicians. This study investigated the precision of the 3DWA protocol for measuring wear through maximum height loss (mm) and volume change (mm3). Observational prospective wear data from 55 patients were analyzed after 0-1-, 0-3-, and 0-5-year intervals to determine rates of wear, and convenience samples were chosen to test the protocol&#39;s precision on dentitions scanned twice in one sitting and its intra- and inter-rater precision on scans with 0-3- and 0-5-year intervals. Scans were made using intra-oral scanners (IOS) and superimposed using 3D measurement software. T-tests were performed to determine the structural and random error, and trimmed ranges were calculated to interpret the error. For protocol precision, the mean difference was 0.015 mm (-0.002; 0.032, p = 0.076) for height and -0.111 mm3 (-0.250; 0.023, p = 0.101) for volume. The duplicate measurement error was 0.062 mm for height and 0.268 mm3 for volume. The height measurements were precise enough to measure wear after intervals of 0-3 or 0-5 years; however, volume measurements were susceptible to procedural error and operator sensitivity. The 3DWA protocol is precise enough to adequately measure tooth height loss after intervals of a minimum of 3 years or in patients with severe wear progression, but it is not suited to measuring volumetric changes.

Precision of In Vivo Quantitative Tooth Wear Measurement Using Intra-Oral Scans

Quantitative wear measurement is a method of increasing importance in measuring tooth wear progression. We here describe a protocol, its precision, and its intra/inter-rater precision for the acquisition and superimposition of repeated in vivo scanned dentitions in patients with moderate to severe wear, reporting on both height and volume measurements.

Quantitative wear measurement is of increasing interest for measuring tooth wear progression. However, most research on quantitative wear measurement has ...

Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired movement, and eventually, knee osteoarthritis (kOA). However, there is little effective treatment for cartilage defects or even kOA. Animal models are important for developing therapeutic drugs, but the existing models for cartilage defects are unsatisfactory. This work established a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats, and the subsequent pain behavior and histopathological changes were used as readout experiments. After surgery, the mechanical withdrawal threshold was decreased, chondrocytes at the injured site were lost, matrix metalloproteinase MMP13 expression was increased, and type II collagen expression decreased, consistent with the pathological changes observed in human cartilage defects. This methodology is easy and simple to perform and enables gross observation immediately after the injury. Furthermore, this model can successfully mimic clinical cartilage defects, thus providing a platform for studying the pathological process of cartilage defects and developing corresponding therapeutic drugs.

This protocol establishes a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats and measuring the subsequent pain behavior and histopathological changes.

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired ...