We would like to acknowledge the assistance of the Department of Comparative Medicine staff and the Molecular and Histopathology core at Penn State Milton S. Hershey Medical Center. Funding sources: NIH NIAMS 1RO1AR071968-01A1 (F.K.), ANRF Arthritis Research Grant (F.K.).

Twelve-week-old male C57BL/6 mice were purchased from Jax Labs. All mice were housed in groups of 3&#8211;5 mice per micro-isolator cage in a room with a 12 h light/dark schedule. All animal procedures were performed according to the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Pennsylvania State University.
1. Post-traumatic osteoarthritis (PTOA) surgical model
<ol>
	<li>Anesthetize mice using a ketamine (100 mg/kg)/xylazine (10 mg/kg) combination via intraperitoneal injection, administer buprenorphine (1 mg/kg) via intraperitoneal injection for pain relief, and shave the hair over the knee. Check for the lack of a pedal reflex before starting the surgery.</li>
	<li>Perform destabilization of the medial meniscus (DMM) surgery as previously described22,23. 
	NOTE: Please refer to Glasson et al. and Singh et al. references for more detailed surgical protocol information22,23.
	<ol>
		<li>Make a 3 mm longitudinal incision from the distal patella to the proximal tibial plateau of the right knee joint. Use a suture to displace the patellar tendon.</li>
		<li>Open the joint capsule medial to the tendon and move the infrapatellar fat pad to visualize the intercondylar region, medial meniscus, and meniscotibial ligament23.</li>
		<li>Transect the medial meniscotibial ligament to complete DMM and destabilize the joint22,23. Close the incision site with staples or sutures.</li>
	</ol>
	</li>
	<li>Perform sham surgeries following a similar procedure, but without transection of the medial meniscotibial ligament. 
	NOTE: Mice were randomized to receive either DMM or sham surgery. According to the previously published literature, mice develop significant PTOA 12 weeks after the DMM surgery22,23,24.</li>
</ol>
2. Mouse euthanasia and sample collection
<ol>
	<li>Mouse euthanasia
	<ol>
		<li>Twelve weeks after DMM, anesthetize mice using a ketamine (100 mg/kg)/xylazine (10 mg/kg) combination via intraperitoneal injection.</li>
		<li>Make a midline incision through the skin along the thorax and retract the rib cage to expose the heart for perfusion fixation27. 
		NOTE: Refer to the Gage et al. reference for additional information on the protocol for proper rodent fixation as well as the video depiction of the proper equipment assembly and procedures27.</li>
		<li>Set up the perfusion apparatus according to the manufacturer&#8217;s protocol.</li>
		<li>Euthanize the mouse by perfusing heparinized saline through the left ventricle until blood is cleared out and the liver becomes pale. Perfuse the mouse with 10% neutral buffered formalin until complete mouse fixation is achieved. 
		NOTE: A successfully perfused mouse will become stiff. The average perfusion time using the automated pump system is 5&#8211;7 min.</li>
	</ol>
	</li>
	<li>Sample collection and preparation
	<ol>
		<li>Isolate the knees by cutting the bone at mid-femur and mid-tibia and dissect the surrounding muscle tissue.</li>
		<li>Continue the fixation by storing knee joints in 10% neutral buffered formalin at room temperature for 24 h, then at 4 &#730;C for 6 days.</li>
		<li>Decalcify the bone by storing the sample in EDTA decalcification buffer for 1 week at room temperature with mild shaking28. Change the decalcification buffer solution every 3 days. 
		NOTE: Decalcification buffer was prepared by dissolving 140 g of ultrapure EDTA tetrasodium in a solution of 850 mL of distilled water plus 15 mL of glacial acetic acid. The buffer was pH equilibrated to 7.6 by dropwise addition of glacial acetic acid to the solution. Upon reaching pH = 7.6, the buffer was brought to 1 L total volume with distilled water. Decalcification buffer was prepared fresh and used within 1 week of preparation.</li>
		<li>Wash the knees with 50% ethanol, then 70% ethanol for 15 min each, then process for embedding. 
		NOTE: Fluid transfer processing was performed by the Molecular and Histopathology Core at Penn State Milton S. Hershey Medical Center. Consult the institution&#8217;s histology core for recommendations on optimal sample processing.</li>
		<li>Embed the mouse knees in paraffin with the medial aspect of the joint facing the bottom surface of the paraffin block. Apply slight pressure to the tibial side of the joint during embedding to ensure tibial and femoral surfaces are as close to the same plane as possible.</li>
	</ol>
	</li>
</ol>
3. Microtome sectioning and slide selection
<ol>
	<li>Microtome sectioning
	<ol>
		<li>Use the plane adjustment knobs on the block holder of the microtome to ensure proper facing of the sample block.</li>
		<li>Face the paraffin block so that the distal aspect of the femur, proximal region of the tibia, and anterior and posterior horns of the medial meniscus appear in the same plane for section collection (Supplementary Figure 1A). Begin collecting sections on slides when the anterior and posterior horns of the medial meniscus begin to separate from one another.</li>
		<li>Ensure that the tibia, femur, and menisci appear in same plane by comparing structure sizes. The tibia and femur should be of comparable size, with both meniscal horns similar in size and shape (Supplementary Figure 1A). If a structure appears too large in comparison to its counterpart, this indicates that additional facing is needed. Importantly, confirm that the joint space remains intact.</li>
		<li>Take sagittal sections of the paraffin-embedded mouse knees through the medial joint compartment in 5 &#956;m sections and collect the sections on slides. Collect approximately 30 sections per paraffin block.</li>
	</ol>
	</li>
	<li>Slide selection
	<ol>
		<li>Select three representative sections 50 &#956;m apart for each sample starting from the fifth section (i.e., slides 5, 15, and 25). Selected slides will be used for subsequent staining, OARSI scoring, and histomorphometric analysis. 
		NOTE: From a block with 30 total sections, select slides from the beginning (slides 5&#8211;10), middle (slides 15&#8211;20), and end (slides 25&#8211;30) of the set in order to clearly represent the entire sample. Select slides from similar levels of depth between samples.</li>
	</ol>
	</li>
</ol>
4. Hematoxylin, Safranin Orange, and Fast Green staining
<ol>
	<li>Deparaffinize the selected slides with three changes of xylene. Hydrate the slides with two changes of 100% ethanol, two changes of 95% ethanol, and one change of 70% ethanol.</li>
	<li>Stain the slides with hematoxylin for 7 min then wash with running water for 10 min. Stain with Fast Green for 3 min and rinse in 1.0% glacial acetic acid for 10 s. Stain with Safranin-O for 5 min and quickly rinse by dipping in 0.5% glacial acetic acid.</li>
	<li>Rinse slides in two changes of distilled water and let air dry.</li>
	<li>Clear slides in three changes of xylene for 5 min each then mount with xylene-based mounting media and a coverslip. 
	NOTE: Hematoxylin serves as the nuclear stain for identifying areas of bone marrow. Safranin-O is used to stain cartilage, proteoglycans, mucin, and mast cell granules. Fast Green is used as a counterstain for bone.</li>
</ol>
5. Slide imaging
<ol>
	<li>Image the Safranin-O and Fast Green stained slides using an available camera attached to a microscope and computer-based imaging software.</li>
	<li>Open the imaging software (see Table of Materials) and arrange the knee joint region of interest (ROI) in the center of the imaging window. If using a rotational microscope slide stage, align the slide so that the tibial surface spans the width of the horizontal axis of the imaging region.</li>
	<li>Set the white balance of the camera before beginning to image a set of samples (Supplementary Figure 2). Image the joint compartment at both 4x and 10x magnification, ensuring that both the tibial and femoral articular surface and tibial subchondral bone are included within each of the image ROIs (Supplementary Figure 1A, B). Save images for each of the three selected levels to be used for OARSI scoring. 
	NOTE: Setting the white balance of the camera will depend on the software and camera systems being used. Generally, navigate to the Camera Tab or Main Imaging Menu and scroll down to Set White Balance. Select Set White Balance and a small cursor or icon should appear (Supplementary Figure 2A). Using the cursor, select an area within the image/sample that is free of stain, such as the joint space, to set the white balance.</li>
</ol>
6. Osteoarthritis research society international (OARSI) scoring15
<ol>
	<li>Randomize the three selected Safranin-O and Fast Green stained slides from all samples.</li>
	<li>Perform OARSI scoring in a blinded manner with three observers. Briefly, display one image at a time in a randomized order and allow three observers to score each image, correlating to one of the three levels selected for each sample. 
	NOTE: Please refer to the OARSI scoring table for detailed descriptions of the grading scale15.</li>
	<li>Calculate the average score for each section using individual scores from each observer. Determine the average score per sample by taking the average score of the three representative sections.</li>
</ol>
7. Histomorphometric analysis
NOTE: Live images of the knee joint are viewed on a touchscreen monitor using a microscope camera, and a stylus is used to manually trace the ROIs. Built-in algorithms of the histomorphometry software quantify the specified parameters (see Protocol below) in the defined ROIs. Importantly, the same Safranin-O and Fast Green stained sections used in OARSI scoring are used for histomorphometric analysis.
<ol>
	<li>Software setup and measurement preparation
	<ol>
		<li>Turn on microscope, camera, computer, and touchscreen monitor. Click on the Software Icon to launch the software program. Place the slide on the microscope stage for viewing.</li>
		<li>Center the sample within the measurement window. Ensure that the measurement grid is set to the proper magnification to match the objective being used on the microscope (Supplementary Figure 3).</li>
		<li>Go to the File tab at the top of the screen and scroll down to Read Settings. Open the Settings window and select the appropriate settings file for the parameters to be measured.</li>
		<li>Select the parameter to be measured from the list on the right hand side of the screen (Supplementary Figure 3). Use the stylus or mouse cursor to trace images according to steps described below in order to measure specific tissue ROIs. When completed with each measurement, Right Click to end the measurement and continue to the next parameter. 
		NOTE: The list of parameters to be measured is created through a distinct preference file that is set up in consultation with the software company for the purpose of measuring the described ROIs. Please see discussion for further details regarding the complete setup.</li>
		<li>Complete all measurements and then navigate to the Summary Data tab at the bottom of the software screen (Supplementary Figure 3). Click on the screen window, hit the Insert key on the keyboard or copy and paste the data into a separate spreadsheet. 
		NOTE: Perform histomorphometric analysis in a randomized and blinded manner. After completing all parameter measurements for all samples, organize the data and average the measurements from the three slides from each sample.</li>
	</ol>
	</li>
	<li>Measurements of the total, calcified, and uncalcified cartilage areas 
	NOTE: Use a commercially available software (see Table of Materials) to perform these steps. Please see Supplementary Figure 1B,C for clarification on cartilage regions, junctions, and subchondral bone regions discussed throughout this section.
	<ol>
		<li>Draw a line across the superior edge of the tibial cartilage surface where the cartilage meets the joint space. Draw a second line at the chondro-osseous junction where the calcified cartilage meets the subchondral bone (Figure 1B). Use the software Area function to automatically obtain the total cartilage area measurement.</li>
		<li>Draw a line along the tidemark, a naturally occurring line separating the calcified and uncalcified regions of cartilage. Draw a second line at the chondro-osseous junction where the calcified cartilage meets the subchondral bone (Figure 1B). Use the software Area function to automatically obtain the calcified cartilage area measurement.</li>
		<li>Calculate the uncalcified cartilage area by subtracting the calcified cartilage area from the total cartilage area (Figure 1B).</li>
	</ol>
	</li>
	<li>Determination of the chondrocyte number and phenotype
	<ol>
		<li>Create separate count functions within the settings in the histomorphometry software to distinguish matrix producing and matrix nonproducing chondrocytes (Figure 1B).</li>
		<li>Determine the number of matrices producing or not producing chondrocytes by using the stylus to make a small dot over each chondrocyte expressing the specified phenotype (Figure 1B). 
		NOTE: Count cells according to their phenotype as either matrix producing (i.e., strong Safranin-O staining) or matrix nonproducing (i.e., very faint or no Safranin-O staining).</li>
	</ol>
	</li>
	<li>Measurement of the tibial articular surface perimeter
	<ol>
		<li>Measure the absolute tibial articular surface perimeter by tracing a line across the surface of the tibial cartilage, carefully defining individual fibrillations (Figure 1C).</li>
		<li>Draw a second line along the tidemark to serve as an internal control for the plane of each section (Figure 1C).</li>
		<li>Calculate the tibial articular surface fibrillation index by dividing the tibial articular surface perimeter by the tidemark perimeter. 
		NOTE: Tidemark perimeters are generally equal across samples, so the difference in the articular surface perimeters between sham and DMM was generally small whether absolute perimeter or fibrillation index were used.</li>
	</ol>
	</li>
	<li>Measurement of the subchondral bone and marrow space areas
	<ol>
		<li>Measure the subchondral marrow space area using the Area function by outlining regions in the subchondral region stained with hematoxylin only (i.e., negative for Fast Green) to determine total marrow space area within the subchondral bone. Draw lines around the perimeter of these areas to determine the total area of all marrow space within the tibial subchondral bone (Figure 2B).</li>
		<li>Measure the total subchondral area using the area function by outlining the region between the chondro-osseous junction and the superior border of the growth plate, extending laterally to the regions of the anterior and posterior osteophytes (Figure 2B).</li>
		<li>Calculate the subchondral bone area by subtracting the subchondral bone marrow space area from the total subchondral area (Figure 2B,C).</li>
	</ol>
	</li>
	<li>Measurement of the anterior and posterior osteophyte areas
	<ol>
		<li>To measure the osteophyte area, trace the anterior and posterior osteophytes of the proximal tibia using the Area function (Figure 2B,E,F). 
		NOTE: Osteophytes are bony projections that form between the articular cartilage and the growth plate, anterior or posterior to the subchondral region. Osteophyte areas on the anterior and posterior sides of the tibia are determined by tracing the area anterior or posterior to the subchondral bone. The junction between the osteophytes and subchondral bone is clearly delineated by a line of cells extending from the articular cartilage to the growth plate. The junction between the osteophyte and synovium is defined by a transition between bony and fibrous tissue.</li>
	</ol>
	</li>
	<li>Measurement of the synovial thickness
	<ol>
		<li>Measure the anterior femoral synovial thickness using the Area function by drawing a line from the inner insertion point of the anterior synovium on the femur towards its attachment point on the meniscus (Figure 3B).</li>
		<li>Draw a second line from the outer insertion of the synovium on the femur towards its attachment to the meniscus (Figure 3B).</li>
		<li>Calculate the synovial thickness by dividing the total synovial area by the synovial perimeter.</li>
	</ol>
	</li>
</ol>
8. Statistical analysis
<ol>
	<li>Collect the measured parameters and average the results from the three representative sections for each sample. Analyze the data using an unpaired Student&#8217;s t-test to compare two groups or one-way ANOVA to compare three or more groups.</li>
	<li>Display the data as mean &#177; standard error of mean. 
	NOTE: Statistical significance was achieved at p &#8804; 0.05.</li>
</ol>

Recent osteoarthritis research has enhanced our understanding of the crosstalk between the different tissues within the joint and the role each tissue plays in disease initiation or progression8,9,10,35,36. Accordingly, it has become obvious that the assessment of OA should not be limited to analysis of the cartilage but should also include analysis of the sub...

One of the most prevalent joint disorders in the United States, OA is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life1,2,3. Articular cartilage is the specialized connective tissue of diarthrodial joints designed to minimize friction, facilitate movement, and endure joint compression4. Articular cartilage is composed of two primary components: chondrocytes and extracellular matrix. Chondrocytes are specialized, metabolically active cells that play a primary role in the development, maintenance, and repair of the extracellular matrix4. Chondrocyte hypertrophy (CH) is one of the principal pathological signs of OA development. It is characterized by increased cellular size, decreased proteoglycan production, and increased production of cartilage matrix-degrading enzymes that eventually lead to cartilage degeneration5,6,7. Further, pathological changes in the subchondral bone and synovium of the joint play an important role in OA development and progression8,9,10,11,12. To date, there are no existing curative therapies that inhibit cartilage degeneration1,2,3,13,14. Thus, there is extensive ongoing research that aims to understand OA pathology and discover novel therapeutic approaches that are able to slow down or even stop OA. Accordingly, there is an increasing need for a quantitative and reproducible approach that enables accurate evaluation of OA-associated pathological changes in the cartilage, synovium, and subchondral bone of the joint.
Currently, OA severity and progression are primarily assessed using the OARSI or Mankin scoring systems15. However, these scoring systems are only semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle changes that occur in the joint during disease or in response to genetic manipulation or a therapeutic intervention. There are sporadic reports in the literature describing histomorphometric analyses of the cartilage, synovium, or subchondral bone16,17,18,19,20,21. However, a detailed protocol for rigorous and reproducible histomorphometric analysis of all these joint components is still lacking, creating an unmet need in the field.
To study pathological changes in OA using histomorphometric analysis, we used a surgical OA mouse model to induce OA via destabilization of the medial meniscus (DMM). Among the established models of murine OA, DMM was selected for our study because it involves a less traumatic mechanism of injury22,23,24,25,26. In comparison to meniscal-ligamentous injury (MLI) or anterior cruciate ligament injury (ACLI) surgeries, DMM promotes a more gradual progression of OA, similar to OA development in humans22,24,25,26. Mice were euthanized twelve weeks after DMM surgery to evaluate changes in the articular cartilage, subchondral bone, and synovium.
The goal of this protocol is to establish a standardized, rigorous, and quantitative approach to evaluate joint changes that accompany OA.

<table>
	<tbody>
		<tr>
			<td>10% Buffered Formalin Phosphate</td>
			<td>Fisher Chemical</td>
			<td>SF100-20</td>
			<td>For sample fixation following harvest</td>
		</tr>
		<tr>
			<td>Acetic Acid, Glacial (Certified A.C.S.)</td>
			<td>Fisher Chemical</td>
			<td>A38S-212</td>
			<td>For Decalcification Buffer preparation and acetic acid solution preparation for staining</td>
		</tr>
		<tr>
			<td>Cintiq 27QHD Creative Pen Display</td>
			<td>Wacom</td>
			<td>https://www.wacom.com/en-es/products/pen-displays/cintiq-27-qhd-touch</td>
			<td>For histomorphometric analysis and imaging</td>
		</tr>
		<tr>
			<td>Cintiq Ergo stand</td>
			<td>Wacom</td>
			<td>https://www.wacom.com/en-es/products/pen-displays/cintiq-27-qhd-touch</td>
			<td>For histomorphometric analysis and imaging</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid, tetrasodium salt dihydrate, 99%</td>
			<td>Acros Organics</td>
			<td>AC446080010</td>
			<td>For Decalcification Buffer preparation</td>
		</tr>
		<tr>
			<td>Fast Green stain</td>
			<td>SIGMA Life Sciences</td>
			<td>F7258</td>
			<td>For sample staining</td>
		</tr>
		<tr>
			<td>Fisherbrand Superfrost Plus Microscope Slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
			<td>For sample section collection</td>
		</tr>
		<tr>
			<td>HistoPrep Xylene</td>
			<td>Fisherbrand</td>
			<td>HC-700-1GAL</td>
			<td>For sample deparrafinization and staining</td>
		</tr>
		<tr>
			<td>Histosette II Tissue Cassettes - Combination Lid and Base</td>
			<td>Fisher</td>
			<td>15-182-701A</td>
			<td>For sample processing and embedding</td>
		</tr>
		<tr>
			<td>HP Z440 Workstation</td>
			<td>HP</td>
			<td>Product number: Y5C77US#ABA</td>
			<td>For histomorphometric analysis and imaging</td>
		</tr>
		<tr>
			<td>Manual Rotary Microtome</td>
			<td>Leica</td>
			<td>RM 2235</td>
			<td>For sample sectioning</td>
		</tr>
		<tr>
			<td>Marking pens</td>
			<td>Leica</td>
			<td>3801880</td>
			<td>For sample labeling, cassettes and slides</td>
		</tr>
		<tr>
			<td>OLYMPUS BX53 Microscope</td>
			<td>OLYMPUS</td>
			<td>https://www.olympus-lifescience.com/en/microscopes/upright/bx53f2/</td>
			<td>For histomorphometric analysis and imaging</td>
		</tr>
		<tr>
			<td>OLYMPUS DP 73 Microscope Camera</td>
			<td>OLYMPUS</td>
			<td>https://www.olympus-lifescience.com/en/camera/color/dp73/</td>
			<td>For histomorphometric analysis and imaging (discontinued)</td>
		</tr>
		<tr>
			<td>ORION STAR A211 pH meter</td>
			<td>Thermo Scientific</td>
			<td>STARA2110</td>
			<td>For Decalcification Buffer preparation</td>
		</tr>
		<tr>
			<td>OsteoMeasure Software</td>
			<td>OsteoMetrics</td>
			<td>https://www.osteometrics.com/index.htm</td>
			<td>For histomorphometric measurement and analysis</td>
		</tr>
		<tr>
			<td>Perfusion Two Automated Pressure Perfusion system</td>
			<td>Leica</td>
			<td>Model # 39471005</td>
			<td>For mouse knee harvest</td>
		</tr>
		<tr>
			<td>PRISM 7 Software</td>
			<td>GraphPad</td>
			<td>Institutional Access Account</td>
			<td>Statistical Analysis</td>
		</tr>
		<tr>
			<td>Safranin-O stain</td>
			<td>SIGMA Life Sciences</td>
			<td>S8884</td>
			<td>For sample staining</td>
		</tr>
		<tr>
			<td>ThinkBoneStage - Rotating Microscope Stage</td>
			<td>Think Bone Consulting Inc. - OsteoMetrics (supplier)</td>
			<td>http://thinkboneconsulting.com/index_files/Slideholder.php</td>
			<td>For histomorphometric analysis and imaging</td>
		</tr>
		<tr>
			<td>Wacom Pro Pen Stylus</td>
			<td>Wacom</td>
			<td>https://www.wacom.com/en-es/products/pen-displays/cintiq-27-qhd-touch</td>
			<td>For histomorphometric analysis and imaging</td>
		</tr>
		<tr>
			<td>Weigerts Iron Hematoxylin A</td>
			<td>Fisher</td>
			<td>5029713</td>
			<td>For hematoxylin staining</td>
		</tr>
		<tr>
			<td>Weigerts Iron Hematoxylin B</td>
			<td>Fisher</td>
			<td>5029714</td>
			<td>For hematoxylin staining</td>
		</tr>
	</tbody>
</table>

standardized histomorphometric evaluation of osteoarthritis in a surgical mouse model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

DMM-induced OA results in articular cartilage degeneration and chondrocyte loss 
DMM-induced OA resulted in an increased OARSI score compared to sham mice, distinctly characterized by surface erosion and cartilage loss (Figure 1A,D). The histomorphometry protocol detailed here detected several OA-associated changes, including a decrease in total cartilage area and in the uncalcified cartilage area (Figure 1A,B</stro...

Watch this Scientific Journal Video about Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model at JoVE.com

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

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11.8K Views.  Pennsylvania State College of Medicine. The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

Video: Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

The Lower Body Positive Pressure Treadmill for Knee Osteoarthritis Rehabilitation

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

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

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

Evaluation of Hepatic Glucose Production in a Polycystic Ovary Syndrome Mouse Model

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. Dysregulated glucose metabolism is an important manifestation of the disease and is the key to its pathogenesis. Therefore, studies involving evaluation of glucose metabolism in PCOS are of utmost importance. Very few studies have quantified hepatic glucose production directly in PCOS models using non-radioactive glucose tracers. In this study, we discuss step-by-step instructions for the quantification of the rate of hepatic glucose production in a PCOS mouse model by measuring M+2 enrichment of [6,6-2H2]glucose, a stable isotopic glucose tracer, via gas chromatography - mass spectrometry (GCMS). This procedure involves creation of stable isotopic glucose tracer solution, use of tail vein catheter placement and infusion of the glucose tracer in both fasting and glucose-rich states in the same mouse in tandem. The enrichment of [6,6-2H2]glucose is measured using pentaacetate derivative in GCMS. This technique can be applied to a wide variety of studies involving direct measurement of the rate of hepatic glucose production.

This study describes the direct measurement of hepatic glucose production in a polycystic ovary syndrome mouse model by using a stable isotopic glucose tracer via tail vein in both fasting and glucose-rich states in tandem.

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

Treating Knee Osteoarthritis with Tuina in a Rabbit Model

Tuina Intervention in Rabbit Model of Knee Osteoarthritis

Knee osteoarthritis (KOA) is mainly characterized by degenerative changes in the knee joint's cartilage and surrounding soft tissues. The efficacy of Tuina in treating KOA has been confirmed, but the underlying mechanism needs to be investigated. This study aims to establish a scientifically feasible KOA rabbit model treated with Tuina to reveal the underlying mechanisms. For this, 18, 6-month-old normal-grade male New Zealand rabbits were randomly divided into sham, model, and Tuina groups, with 6 rabbits in each group. The KOA model was established by injecting 4% papain solution into the knee joint cavity. The Tuina group was intervened with Tuina combined with the knee joint rotary correction method for 4 weeks. Only the standard grasping and fixation were performed in sham and model groups. At the end of the 1-week intervention, the knee joint range of motion (ROM) was observed, and cartilage hematoxylin-eosin (HE) staining was done. The study shows that Tuina could inhibit chondrocyte apoptosis, repair cartilage tissue, and restore knee joint ROM. In conclusion, this study demonstrates the scientific feasibility of Tuina treatment for KOA model rabbits, highlighting its potential application in the study of KOA and similar knee joint-related conditions.

Author Spotlight: Using a Rabbit Model to Explore the Efficacy of Tuina in Treating Knee Osteoarthritis 

The protocol describes a method for Tuina intervention in a rabbit model of knee osteoarthritis.

Knee osteoarthritis (KOA) is mainly characterized by degenerative changes in the knee joint's cartilage and surrounding soft tissues. The efficacy of ...

HE Staining and Micro-CT to Evaluate Murine Trabecular Bone Microarchitecture

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton is crucial for gaining insight into the pathophysiology of osteoporosis and improving its treatment. However, handling bone samples can be complex due to their hard and dense properties. Secondly, specialized software makes image processing and analysis difficult. In this protocol, we present a cost-effective and easy-to-use solution for trabecular bone microstructure analysis. Detailed steps and precautions are provided. Micro-CT is a non-destructive three-dimensional (3D) imaging technique that provides high-resolution images of trabecular bone structure. It allows for the objective and quantitative evaluation of bone quality, which is why it is widely regarded as the gold standard method for bone quality assessment. However, histomorphometry remains indispensable as it offers crucial cellular-level parameters, bridging the gap between two-dimensional (2D) and 3D assessments of bone specimens. As for the histologic techniques, we chose to decalcify the bone tissue and then perform traditional paraffin embedding. In summary, combining these two methods can provide more comprehensive and accurate information on bone microstructure.

Author Spotlight: An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model 

This protocol presents an economical and efficient method for quantitative evaluation of bone microarchitecture in a mouse model of osteoporosis by combining Hematoxylin-Eosin (HE) staining and micro-computed tomography (Micro-CT) techniques.

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton ...