Name: standardized histomorphometric evaluation of osteoarthritis in a surgical mouse model
Uploaded: 2020-05-06T13:00:00
Description: Watch this Scientific Journal Video about Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model at JoVE.com

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 usi...

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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