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 surgery as a control procedure on eight mice with the same background and age. 
	NOTE: Both sexes were used for the original project to meet the NIH requirement for consideration of sex as a biological variable, though examination of sex difference is not the scope of this protocol.</li>
	<li>Anesthetize animals by inhalation of Isoflurane. Check the depth of anesthesia by monitoring their respiratory rate/effort and lack of response to toe/tail pinch. Put animals in a supine position.</li>
	<li>Shave the skin in the knee area and clean the skin with Povidone-Iodine + alcohol skin scrub; three alternating cycles.</li>
	<li>Perform the DMM procedure on the right knee under a surgical microscope. Expose the knee joint through a medial parapatellar incision (1.2-1.5 cm in length) and incise the joint capsule. Keep the patella and the patellar tendon intact. After careful exposure of the medial meniscotibial ligament (MML) which anchors the medial meniscus to the tibial plateau, transect it with micro-surgical scissors to destabilize the medial meniscus.</li>
	<li>Perform sham surgery on the right knee as a control procedure, in which the MML was visualized but not transected.</li>
	<li>Close the joint capsule with 8-0 absorbable polyglactin sutures and skin incision with 7-0 non-absorbable sutures for both DMM and sham procedures to assure proper use of the knee once healing has occurred.</li>
	<li>Inject SR Buprenorphine (0.20-0.5 mg/kg) subcutaneously (SC) immediately before the surgical procedure for analgesia, which provides pain relief up to 72 h after a single injection. Monitor operated animals after surgery.</li>
	<li>Euthanize animals using a CO2 chamber at 2, 8, and 16 weeks post-surgery. After unconsciousness, confirm the death of the animals by a physical method (opening the chest cavity). These methods of euthanasia are consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association (AVMA).</li>
	<li>Harvest the knee joints for histological analyses at 2, 8, and 16 weeks after DMM surgery and at 2 and 16 weeks after Sham surgery to obtain mouse knee joints with different degrees of OA severity or SCB thickening.</li>
</ol>
2. Preparation of tissue sections and histologic images
<ol>
	<li>Fix mouse knee joint tissue samples in 2% paraformaldehyde, decalcify them in 25% formic acid, embed in paraffin, and section coronally to examine both the medial and lateral compartments.</li>
	<li>Cut knee specimens from the posterior side of the knee using a microtome and collect tissue sections that are 5-&#181;m-thick at 70-80 &#181;m intervals to obtain about 40 tissue slides across the entire knee joint. A micrometer-assisted estimation suggests that slide numbers 1-6 are from the far-posterior, 11-18 from mid-posterior, 23-30 from mid-anterior, and 35-40 from the far-anterior portion of the knee joint. Discard or collect intervening sections for additional stains.</li>
	<li>Perform Safranin-O and fast green stains according to the manufacturer&#39;s instructions to specifically identify cartilage cells and matrices on every five slides. Perform Hematoxylin-Eosin staining according to the manufacturer&#39;s instructions to examine the knee joints at the cellular and tissue levels as described previously19,20,21,22.</li>
	<li>Acquire histologic images with a microscope equipped with a digital camera. General histopathologic analysis and histologic OA grading were conducted as described previously15,19,20,21,22.</li>
</ol>
3. Quantitative measurement of osteoarthritic subchondral bone with ImageJ software
<ol>
	<li>Download the ImageJ software and open histologic images of interest.
	<ol>
		<li>Download the ImageJ bundled with Java 1.8.0_172 from&#160;https://imagej.nih.gov/ij/.</li>
		<li>Open the ImageJ program. Click the File tab on the Ribbon and click the Open option to open the histologic image.</li>
		<li>Find the file directory address, select the picture file, and click Open.</li>
	</ol>
	</li>
	<li>Calibrate ImageJ with the micrometer on the histologic images.
	<ol>
		<li>Use the straight-line tool to sketch one unit of length on the micrometer and click Analyze &#62; (then) Set scale. Set the Known distance and Pixel aspect ratio to 1 and click OK. ImageJ can convert the pixel length to the unit length on micrometer.</li>
		<li>Set the measured factor to area. Click Analyze &#62; Set Measurement and check Area and Limit to threshold box under new window. This step sets ImageJ to measure the parameter &#34;Area&#34; within selected &#34;Threshold&#34;.</li>
	</ol>
	</li>
	<li>Measure total subchondral bone (SCB) area of interest.
	<ol>
		<li>Define the SCB region of interest (ROI) as shown in the orange boxes of Figure 1A, which covers the SCB cortical plate and a portion of the underlying trabecular bone adjacent to the cortical plate in the medial femoral condyle (MFC) and medial tibial plateau (MTP) with specific dimensions for each ROI. Osteoarthritic SCB thickening usually occurs in these areas. Define the SCB ROI with the same shape and dimension in each MFC or MTP for all examined joints to ensure that the same size of the specific ROI was measured for all animals.</li>
		<li>Sketch the outline of the total SCB area of interest by using the Polygon selection tool under the main window of ImageJ. 
		NOTE: The selection tools give the system a threshold to limit the measured area.</li>
		<li>Measure the total SCB area: After the threshold is selected, click Analyze &#62; Measure. A &#34;Results&#34; window with area measurement will open.</li>
	</ol>
	</li>
	<li>Measure the bone substance area containing solid bone without bone marrow.
	<ol>
		<li>Click Edit &#62; Clear Outside to exclude the area outside the total SCB area. 
		NOTE: Only the total SCB area is visible after clicking Clear Outside option. The picture outside the total SCB area will turn black. This step allows observers to focus on the bone substance area within the area of interest.</li>
		<li>Click Image &#62; Adjust &#62; Color threshold to open the &#34;Threshold Color&#34; window. Click Original at the bottom of the &#34;Threshold Color&#34; window to restore the picture to the original status. Use selection tools in step 3.3.2 to draw a small box in the bone substance region. Click the Sample option at the bottom of the &#34;Threshold Color&#34; window to define the bone substance area. 
		NOTE: The &#34;Sample&#34; option in the &#34;Threshold Color&#34; window allows ImageJ to select all the same pixels on the total SCB area as the bone substance sample area. The selected bone substance area will turn red.</li>
		<li>Click Select at the bottom of the threshold color balance window to create an area measurement threshold. Click Analyze &#62; Measure at ImageJ main menu, and the bone substance area measurement result will show on the &#34;Results&#34; window.</li>
		<li>Save the data of the total SCB area and bone substance area.</li>
	</ol>
	</li>
	<li>Calculate the ratio of bone substance area (mm2) to total SCB area (mm2) of interest which represents the bone substance thickness (mm2/1.0 mm2) within the total SCB area.</li>
	<li>Measure the SCB thickness of histologic sections/images (as described in steps 3.1-3.5) of far-posterior, mid-posterior, mid-anterior, and far-anterior areas (as described in step 2.2) of DMM-induced OA to assess area-specific SCB thickness of 6 knee joints (Figure 1B). 
	&#8203;NOTE: This can validate the reliability of this quantitative measurement protocol because it is known that osteoarthritic SCB changes co-localize with cartilage lesions and that osteoarthritic cartilage damage with SCB thickening is more severe in the weight-bearing areas (mid-portion) of rodent knee joints14,15. Therefore, it is appropriate to use mid-sections for quantitative measurement of osteoarthritic SCB thickening.</li>
</ol>
4. Statistics
<ol>
	<li>Perform statistical analyses using data from quantitative measurement and visual grading of SCB thickness. Determine the inter- and intra-observer variability and reproducibility by Pearson&#39;s correlation coefficient analyses.</li>
	<li>Determine the significance of differences between study groups using Student&#39;s t-tests or one-way ANOVA, followed by a post-hoc test (Tukey) using spreadsheet software. Consider a p-value of less than 0.05 to be statistically significant.</li>
</ol>

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The authors declare no competing conflicts of interest.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Sigma-Aldrich</td>
			<td>GHS216</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>Sigma-Aldrich</td>
			<td>E4382</td>
			<td></td>
		</tr>
		<tr>
			<td>illustrator</td>
			<td>Adobe</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 ...

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2.8K Views.  University of Kansas Medical Center. 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. 

Video: Software-Assisted Quantitative Measurement of Osteoarthritic Subchondral Bone Thickness

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 ...

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment 1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded 2. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances 4, 5. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) 7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.
We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators 10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al 7. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14, 15. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16, 17. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18. This approach proved to be sufficiently sensitive and reproducible.

A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

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 ...

Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection may result in sustained inflammation, osteomyelitis, and eventual bone non-union. Establishment of a feasible, reproducible animal model is important to bone infection research and antibiotic treatment. As an in vivo model, the rabbit model is widely used in bone infection research. However, previous studies on rabbit bone infection models showed that the infection status was inconsistent, as the amount of bacteria was variable. This study presents an improved surgical method for inducing bone infection on a rabbit, by blocking the bacteria in the bone marrow. Then, multi-level evaluations can be carried out to verify the modelling method.
In general, debriding necrotic tissue and implantation of vancomycin-loaded calcium sulphate (VCS) are predominant in antibiotic treatment. Although calcium sulphate in VCS benefits osteocyte crawling and new bone growth, massive bone defects occur after debriding. Autogenous bone (AB) is an appealing strategy to overcome bone defects for the treatment of massive bone defects after debriding necrotic bone.
In this study, we used the tail bone as an autogenous bone implanted in the bone defect. Bone repair was measured using micro-computed-tomography (micro-CT) and histological analysis after animal sacrifice. As a result, in the VCS group, bone non-union was consistently obtained. In contrast, the bone defect areas in the VCS-AB group were decreased significantly. The present modeling method described a reproducible, feasible, stable method to prepare a bone infection model. The VCS-AB treatment resulted in lower bone non-union rates after antibiotic treatment. The improved bone infection model and the combination treatment of VCS and autogenous bone could be helpful in studying the underlying mechanisms in bone infection and bone regeneration pertinent to traumatology orthopedic applications.

This study presents an improved rabbit model infected with Staphylococcus aureus by blocking the same amount of bacteria in bone marrow. Vancomycin loaded calcium sulphate and autogenous bone are used for antibiotic and bone repair treatment. The protocol could be helpful for studying bone infection and regeneration.

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection ...

Using a Knee Arthrometer to Evaluate Tissue-specific Contributions to Knee Flexion Contracture in the Rat

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ROM is called a joint contracture and results in increased morbidity. Due to the difficulty of reversing established knee contractures, early detection is important, and hence, knowing risk factors for their development is essential. The rat represents a good model with which the effect of an intervention can be studied due to the similarity of rat knee anatomy to that of humans, the rat's ability to tolerate long durations of knee immobilization in flexion, and because mechanical data can be correlated with histologic and biochemical analysis of knee tissue.
Using an automated arthrometer, we demonstrate a validated, precise, reproducible, user-independent method of measuring the extension ROM of the rat knee joint at specific torques. This arthrometer can be used to determine the effects of interventions on knee joint ROM in the rat.

The goal of the protocol is to measure the extension range of motion of the rat knee. The effects of various diseases that increase the stiffness of the knee joint and the effectiveness of treatments can be quantified.

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ...

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.