The authors wish to thank Edith Janssen for the critical review of the manuscript and Navin Rao and Jennifer Towne for their support of the publication of this manuscript.

All animal experiments were conducted in accordance within the policies of the Institutional Animal Care and Use Committee (IACUC) of Janssen R&#38;D.
1. Cryogenic Freezer Mill-based Pulverization Method
<ol>
	<li>At termination of the CIA study, sacrifice mice with 1.5% isoflurane followed by cervical dislocation.</li>
	<li>On the day of tissue processing, pre-chill all instruments (spatulas, grinding vials, etc.) on dry ice for a minimum of 10 min. Wear thermal gloves to avoid freeze damage.</li>
	<li>Cut hind paws with a scissor at the fur line as depicted in Figure 1A.</li>
	<li>Transfer each paw into one 1.5 mL microcentrifuge tube, snap freeze in liquid nitrogen immediately, and store frozen paws at -80 &#176;C. Do not keep samples frozen for more than one week.</li>
	<li>Fill freezer mill with liquid nitrogen as shown in Figure 1B and let it equilibrate for at least 10 min.</li>
	<li>Keep unprocessed sample on dry ice and avoid freeze-thaw cycles.</li>
	<li>Transfer one hind paw into pre-chilled large polycarbonate grinding vial with a bottom steel plug, insert the pre-chilled steel impactor and close the polycarbonate grinding vial with the pre-chilled top steel plug (Figure 1C).</li>
	<li>Transfer large polycarbonate grinding vials with the samples into the freezer mill pre-filled with liquid and close the lid with the rubber clasp found at the front of the instrument.</li>
	<li>Let samples cool in liquid nitrogen for 1 minute.</li>
	<li>Set the freezer mill to the 1 minute program with 10 cycles per second and press the start button. Wait for the freezer mill to complete its cycle. 
	NOTE: The machine makes a drumming sound as the steel impactor moves back and forth. Use ear plugs to avoid hearing loss.</li>
	<li>Open the lid of the freezer mill, take out the large polycarbonate grinding vial and place it in an extractor as depicted in Figure 1D. Remove the top steel plug by placing downward pressure on the black handle until the steel plug slides out of the polycarbonate tube.</li>
	<li>Transfer the opened large polycarbonate grinding vial onto dry ice. Remove the steel impactor with pre-chilled forceps.</li>
	<li>Transfer the frozen powder into pre-chilled 50 mL conical tube.</li>
	<li>Weight 30-50 mg of frozen powder into a tared frozen 1.5 mL microcentrifuge tube for RNA extraction and 100-200 mg of frozen powder for protein extraction. 
	NOTE: The frozen powder should look like white or light pink fine sand as depicted in Figure 1E.</li>
	<li>Store pulverized samples at -80 &#176;C and proceed to RNA and protein isolations within 24 h.</li>
</ol>
2. RNA Extraction
<ol>
	<li>Add 10 &#181;L of &#946;-mercaptoethanol (&#946;-ME) per 1 mL of RLT buffer.</li>
	<li>Calculate the volume of RLT Buffer corresponding to a ratio of 23.3 mL/mg of tissue and add the appropriate volume of RLT Buffer with &#946;-ME to the tube with frozen powder. This ratio was empirically determined to be ideal for mouse paws.</li>
	<li>Vortex the sample at 3,000 RPM for 10 s.</li>
	<li>Mix well by pipetting up-and-down 10 times vigorously with a 1 mL pipettor.</li>
	<li>Vortex the sample again at 3,000 RPM for 20 s.</li>
	<li>Centrifuge the tube at 13,000 x g for 2 min.</li>
	<li>Transfer 700 &#181;L of the supernatants to a fresh tube and add 700 &#181;L of 70% ethanol. 
	NOTE: If &#60;700 &#181;L is in the tube, it is acceptable to proceed, still adding equivalent volume of 70% ethanol.</li>
	<li>Load 700 &#181;L of the sample onto a RNA purification column.</li>
	<li>Centrifuge the tube at &#8805;10,000 x g for 30 s.</li>
	<li>Discard the flow-through and load the remaining part of the sample to the RNeasy column.</li>
	<li>Centrifuge the tube at &#8805;10,000 x g for 30 s.</li>
	<li>Discard the flow through and wash the column by adding 700 &#181;L of RW1 buffer with centrifugation of &#8805;10,000 x g for 30 s.&#160;If performing option DNASE digestion, 350 &#181;L of RW1 is added prior to DNASE treatment. &#160;And an additional 350 &#181;L of RW1 is added following DNASE treatment.</li>
	<li>Optional) Perform a DNase digestion step follow manufacturer&#39;s instructions.</li>
	<li>Wash the tube twice by adding 500 &#181;L of RPE buffer followed by centrifugation at &#8805;10,000 x g for 30 s. Discard flow through each time.</li>
	<li>Dry the column by centrifugation at &#8805;10,000 x g for 2 min.</li>
	<li>Elute RNA by adding 50 &#181;L water with centrifugation at &#8805;10,000 x g for 2 min. Collect flow through and transfer into a new 1.5 mL tube.</li>
	<li>Determine the quantity of RNA using the researcher&#39;s method of choice and store at -80 &#176;C before further analysis.</li>
</ol>
3. Protein Extraction
<ol>
	<li>Dilute the 10x cell lysis stock solution in 1x cell lysis stock solution using cell culture grade water.</li>
	<li>Reconstitute the Protease Inhibitor Cocktail Set I with 1 mL of water to make a 100x protease inhibitor stock.</li>
	<li>Add 100 &#181;L of protease inhibitor to 9.9 mL of 1x Cell lysis to make a 1x final stock solution.</li>
	<li>Add 4 &#181;L of ice cold 1x Cell Lysis Buffer per mg tissue powder (e.g., 800 &#181;L of lysis buffer to 200 mg of tissue powder).</li>
	<li>Add one 5 mm stainless steel bead to the tube.</li>
	<li>Vortex the tube at 3,000 RPM for 60 s. Transfer the tube to wet ice and continue to the next sample.</li>
	<li>Place all sample tubes in a box and shake the centrifuge box on a rocker at 1,000 RPM, 4 &#176;C for 1 h.&#160; 
	NOTE:&#160;Researchers may find that placing the box vertical can facilitate better mixing with the bead.</li>
	<li>Centrifuge the tubes at 10,000 x g and 4 &#176;C for 15 min.</li>
	<li>Transfer the supernatants into a fresh tube and avoid the fat layer on the top.</li>
	<li>Determine the total protein concentration. 10 to 30-fold dilutions of protein lysate are ideal for a BCA test.</li>
	<li>Aliquot the sample into 1.5 mL microcentrifuge tubes and store at -80 &#176;C before further analysis.</li>
</ol>

<ol>
	<li>Firestein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunologic+mechanisms+in+the+pathogenesis+of+rheumatoid+arthritis.">Immunologic mechanisms in the pathogenesis of rheumatoid arthritis.</a> Journal of Clinical Rheumatology. 11, S39-S44 (2005).</li><li>McInnes, I. B., Schett, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenesis+of+rheumatoid+arthritis.">The pathogenesis of rheumatoid arthritis.</a> New England Journal of Medicine. 365, 2205-2219 (2011).</li><li>Ahlqvist, E., Hultqvist, M., Holmdahl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+value+of+animal+models+in+predicting+genetic+susceptibility+to+complex+diseases+such+as+rheumatoid+arthritis.">The value of animal models in predicting genetic susceptibility to complex diseases such as rheumatoid arthritis.</a> Arthritis Research and Therapy. 11, 226 (2009).</li><li>David, C. S., Taneja, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+major+histocompatibility+complex+genes+in+murine+collagen-induced+arthritis:+a+model+for+human+rheumatoid+arthritis.">Role of major histocompatibility complex genes in murine collagen-induced arthritis: a model for human rheumatoid arthritis.</a> American Journal of the Medical Sciences. 327, 180-187 (2004).</li><li>Tarkowski, A., Holmdahl, R., Klareskog, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheumatoid+factors+in+mice.">Rheumatoid factors in mice.</a> Monographs in Allergy. 26, 214-229 (1989).</li><li>Schurgers, E., Billiau, A., Matthys, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen-induced+arthritis+as+an+animal+model+for+rheumatoid+arthritis:+focus+on+interferon-gamma.">Collagen-induced arthritis as an animal model for rheumatoid arthritis: focus on interferon-gamma.</a> Journal of Interferon &amp; Cytokine Research. 31, 917-926 (2011).</li><li>Joosten, L. A., Helsen, M. M., van de Loo, F. A., van den Berg, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticytokine+treatment+of+established+type+II+collagen-induced+arthritis+in+DBA/1+mice.+A+comparative+study+using+anti-TNF+alpha+anti-IL-1+alpha/beta,+and+IL-1Ra.">Anticytokine treatment of established type II collagen-induced arthritis in DBA/1 mice. A comparative study using anti-TNF alpha anti-IL-1 alpha/beta, and IL-1Ra.</a> Arthritis &amp; Rheumatology. 39, 797-809 (1996).</li><li>Asquith, D. L., Miller, A. M., McInnes, I. B., Liew, F. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+rheumatoid+arthritis.">Animal models of rheumatoid arthritis.</a> European Journal of Immunology. 39, 2040-2044 (2009).</li><li>Williams, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen-induced+arthritis+as+a+model+for+rheumatoid+arthritis.">Collagen-induced arthritis as a model for rheumatoid arthritis.</a> Methods in Molecular Medicine. 98, 207-216 (2004).</li><li>Shen, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+Blockade+of+TNF-alpha+and+IL-17A+Alleviates+Progression+of+Collagen-Induced+Arthritis+without+Causing+Serious+Infections+in+Mice.">Combined Blockade of TNF-alpha and IL-17A Alleviates Progression of Collagen-Induced Arthritis without Causing Serious Infections in Mice.</a> Journal of Immunology. 202, 2017-2026 (2019).</li></ol>

The authors BJ, SH, ML, RM, ML, TO and FS are employees of Janssen Research &#38; Development Inc and are shareholders in the parent company, Johnson &amp; Johnson, Inc.

Although there is strong scientific rationale to evaluate molecular pathways in synovial tissues, many reports on immune profiling of murine CIA model were focused on peripheral blood, while protein and RNA analysis data of inflamed paws are rather limited. There are several possible reasons for this bias: murine ankle joints are no larger than ~2 cm; the affected areas consist of skin, bone and connective tissues which are often difficult to homogenize using traditional methods like tissue grinders, pestle and mortar, silica bead disruption, trituration or enzymatic digestion. These methods typically result in incomplete homogenization, low protein or RNA yield, or inconsistent quality due to proteolytic and nucleic acid degradation. The robust method described herein provides a detailed procedure to generate a homogeneous pulverized frozen powder to enable downstream proteomic and transcriptional profiling efforts to establish molecular signatures of disease from a single uniform sample source.
Several critical steps should be considered when performing this method. Firstly, murine paws should be cut at the fur line during tissue harvest and large amounts of hair should be avoided. Excessive hair can clog columns during RNA extraction and interfere with protein extraction. Secondly, tubes, forceps and spatulas should be kept on dry ice throughout the procedure because elevated temperature in tissue powders will quickly turn the material into amorphous &#34;mud&#34; and the integrity of protein and RNA will be compromised. Thirdly, the amount of tissue powder used for protein or RNA extraction should be carefully optimized. More tissue powder may not always provide higher yield but can cause additional problems in downstream processing including clogging RNA isolation columns, altering the DNase/DNA ratio (if this step is employed) to prevent complete DNA degradation. Finally, normalization of protein and RNA input should be an integral part of the workflow and data analysis. Variability in tissue load can significantly mask changes in relative protein and RNA levels. In addition to total protein or total RNA, expression of housekeeping genes can also be considered as anchors for normalization.
Through extensive trial and error process, we have identified some potential frequent issues for first-time users of this method. There can be low yield of RNA. This is often a result of overloading the column with too much tissue extract. The amount of the tissue per column and ratio of RLT/powder are crucial for efficient RNA isolations. We have found that some commercial 96 well extraction kits may not work for this process as the samples will not spin through the column at an equivalent rate, thus compromising these samples in downstream washing and elution steps. There can be high variability in protein concentration in BCA analysis and cytokine concentrations in Luminex analysis. If there are no issues with the BCA or the Luminex process, it is typically caused by fat or other contaminants in the protein extract. It is important to avoid the fat layer at the top or insoluble matter at the bottom of the microcentrifuge tubes when harvesting for protein extraction. If needed, repeat the spin and collect supernatants for downstream analysis.
As this method involves a specialized cryogenic freezer mill and large quantities of dry ice and liquid nitrogen, additional safety measures should be considered. Proper personal protection equipment including safety glasses, lab coats and cryogenic gloves should always be worn to prevent potential freeze burns and injury due to explosion. Generation of aerosols should also be minimized and using of vented balance enclosures is advised. Finally, similar to any other protocols handling animal tissues, care should be taken to properly dispose of all unused tissue samples, while reusable tubes need to be decontaminated prior to cleaning.
While the method has many advantages over conventional methods, it still harbors some limitations. The transcriptome profile from a murine whole paw significantly overlaps with transcriptome profile from human synovial biopsy (data not shown). Additional mRNA from muscle, skin and bone marrow could dilute the signals from synovial tissue. An alternative solution is to collect murine synovial tissue through laser microdissection, which can be performed on OCT embedded mouse joints. However, low throughput and the relatively small quantity of tissue limits the broad application for laser microdissection. Additionally, even with significant automation, this method is still quite labor intensive. It takes at least 8 h to process 30-60 samples with the help of 3-4 investigators. Finally, this method requires large amounts of liquid nitrogen (~10-15 L for processing 30-60 samples).
In the preceding method description, the evaluation of proteomic and transcriptional endpoint analyses was demonstrated. However, additional endpoint, such as lipidomic, metabolomics and small RNA profiling could be of interest to the wider arthritis research community.

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease with persistent symmetric synovitis in joints and extra articular involvement of organs such as the skin, heart, lungs, and eyes1. Although the systemic manifestations of the immune response are evident in human patients, one of the hallmarks of RA pathology is infiltration of immune cells in synovial tissue and proliferation of synovial fibroblast cells2.
Similar to human RA, mouse collagen induced arthritis (CIA) model elicits strong tissue inflammation with active immune responses in synovial tissues and systemic compartments. The susceptibility of different mouse strains to CIA model links to Major Histocompatibility Complex (MHC) haplotype and antigen specific T cell and B cell interactions3,4. In addition, many pathogenic pathways in human RA, including autoantibody production, immune complex deposition, myeloid cell activation, polyarticular manifestations and pannus formation with synovial immune infiltration, are also evident in this model5,6. Investigators have employed this well-established CIA model to investigate effects of anti-inflammatory cytokine treatments7. Many biologics approved for autoimmune or inflammatory diseases, such as anti-TNF&#945; and anti-IL-6, are found to be efficacious in the CIA model8,9.
Profiling the interactions of the immune system in synovial tissue is crucial to elucidate molecular mechanisms associated with the pathogenesis of RA. In the human clinical setting, a common practice is to perform needle synovial biopsies under the guidance of ultrasound imaging. In the preclinical settings, the smaller architecture of the murine joints makes biopsy procedures much more difficult if not impossible. Recently, we demonstrated the utilization of the murine CIA model to evaluate combinations of drugs to impact disparate end-points and resolve disease in a combinatorial approach10. A cryogenic freezer mill-based pulverization method was employed to process inflamed murine paws into homogeneous fine powders and established downstream processes to extract RNA and proteins. This method protects RNA and protein from enzymatic and chemical degrative processes and enables us to apply multiple analytical methods to a single homogenized sample source.

<table><tbody><tr><td>5 mm stainless steel bead</td><td>Qiagen</td><td>69989</td><td></td></tr><tr><td>beta-mercaptoethanol</td><td>Sigma</td><td>M6250</td><td>Sample reducing agent that inhibits RNASE enzymes</td></tr><tr><td>Bioanalyzer Kit</td><td>Agilent</td><td>5067-1511</td><td>RNA qualification kit</td></tr><tr><td>b-mercaptoethanol</td><td>Sigma</td><td>M6250</td><td></td></tr><tr><td>Cell Culture Grade Water</td><td>Corning</td><td>25-055-CI</td><td>Water</td></tr><tr><td>Cell lysis stock solution</td><td>Cell Signaling</td><td>9803</td><td></td></tr><tr><td>Eppendorf Tube</td><td>Eppendorf</td><td>22363204</td><td>Microfuge tubes</td></tr><tr><td>Eppendorf tube centrifuge box</td><td>Nalgene</td><td>5055</td><td>Box for holding eppendorf tubes in horizontal tube arrangement</td></tr><tr><td>Everlast 247 Variable Speed Rocker</td><td>Benchmark Scientific</td><td>BR5000</td><td></td></tr><tr><td>Freezer Mill</td><td>Spex Sample Prep</td><td>6875</td><td>Freezer/Mill for processing paws into pulverized powder</td></tr><tr><td>Grinding Vial</td><td>Spex Sample Prep</td><td>6801</td><td>Polycarbonate vial for processing paws into pulverized powder</td></tr><tr><td>Pierce BCA kit</td><td>Pierce</td><td>23225</td><td>Kit for Total Protein Quantification</td></tr><tr><td>Protease Inhibitor Cocktail set 1</td><td>Calbiochem</td><td>539131</td><td>Protease Inhibitors</td></tr><tr><td>Protein BCA Kit</td><td>Pierce</td><td>23225</td><td></td></tr><tr><td>Quantigene Kit</td><td>Thermofisher</td><td>QP1013</td><td>bDNA analysis Kit</td></tr><tr><td>Refrigerated microcentrifuge</td><td>Eppendorf</td><td>5417R</td><td>Centrifugation</td></tr><tr><td>RLT Buffer</td><td>Qiagen</td><td>79216</td><td>RNA extraction buffer</td></tr><tr><td>RNeasy mini kit</td><td>Qiagen</td><td>74104</td><td>including RNeasy column, RLT Buffer and RW1 Buffer</td></tr><tr><td>Shaker</td><td>Benchmark Scientific</td><td>BR5000</td><td>Rocker/Shaker</td></tr><tr><td>Spatula</td><td>VWR</td><td>10806-412</td><td>Spatula for powder transfer</td></tr><tr><td>Stainless Steel Bead</td><td>Qiagen</td><td>69989</td><td>Bead for mixing during protein extraction</td></tr><tr><td>Tube Extractor</td><td>Spex Sample Prep</td><td>6884</td><td>Extractor for removing the top of grinding vial</td></tr><tr><td>Vortexer</td><td>VWR</td><td>10153-838</td><td>Sample mixing</td></tr></tbody></table>

a cryo-pulverization protocol for processing mouse paws to evaluate molecular pathways of tissue inflammation in a collagen induced arthritis model

Profiling molecular changes in local tissues is crucial to understand the mechanism(s) of action of therapeutic candidates in vivo. In the field of arthritis research, many studies are focused on inflamed joints that are composed of a complex mixture of bone, cartilage, muscle, stromal cells and immune cells. Here, we established a reliable and robust mechanical method to disrupt inflamed mouse paws into homogeneous pulverized samples in a cryogenically controlled environment. Protein and RNA lysates were processed to enable proteomic and transcriptional endpoints and molecular characterization of relevant disease pathways in local tissue.

Here, we show a representative gel image visualization of RNA extracted from front paws of CIA mice in Figure 2A. The 28S rRNA and the 18S rRNA band indicate all samples have sufficient amount of intact RNA. Next, we show a representative scatter plot of total protein concentrations based on protein BCA analysis in Figure 2B. Total protein concentrations from na&#239;ve mice, CIA mice or CIA mice under various treatments are comparable across groups. To determine the concentrations of inflammatory cytokines and chemokines in the protein extract, Luminex analyses were conducted. We show a representative scatter plot of the concentrations of normalized cytokines and chemokines (pg/mg total protein) in Figure 2C. Compared to na&#239;ve mice, several cytokines are elevated in CIA mice and treatment with anti-IL17A antibody significantly inhibits production of several cytokines. To evaluate transcriptome changes in CIA and treatment-related effects, microarray analysis was performed. We show a representative heatmap plot of genes significantly increased in CIA mice compared to na&#239;ve mice in Figure 2D.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60229/60229fig1.jpg" /> 
Figure 1: Equipment needed to pulverize murine paws. (A) Inflamed or uninflamed paws from CIA or na&#239;ve mice. (B) Liquid nitrogen Dewar used to fill the stainless-steel bath of the freezer mill. (C) Assembled freezer mill tube set. (D) Freezer mill tube opener. (E) Pulverized tissue from murine paws. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60229/60229fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60229/60229fig2.jpg" /> 
Figure 2: Representative data on analysis of protein and RNA from pulverized murine paws. (A) Gel image visualization of RNA integrity. (B) Total protein concentrations from different groups. (C) Chemokine and cytokine expression (Luminex) from different groups. (D) Hierarchical clustering heatmap of microarray analysis from different treatment groups. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60229/60229fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Cryo-pulverization Protocol for Processing Mouse Paws to Evaluate Molecular Pathways of Tissue Inflammation in a Collagen Induced Arthritis Model at JoVE.co

A Cryo-pulverization Protocol for Processing Mouse Paws to Evaluate Molecular Pathways of Tissue Inflammation in a Collagen Induced Arthritis Model

A cryogenic pulverization method to process murine paws using a liquid nitrogen freezer mill was developed to improve the yield and quality of RNA or protein extracted from the tissues and enable the analysis of molecular profiles associated with inflammatory responses.

Profiling molecular changes in local tissues is crucial to understand the mechanism(s) of action of therapeutic candidates in vivo. In the field of ...

a-cryo-pulverization-protocol-for-processing-mouse-paws-to-evaluate

Nanyang Technological University

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JoVE Journal

Immunology and Infection

8.2K Views.  Janssen Research & Development, Inc.. A cryogenic pulverization method to process murine paws using a liquid nitrogen freezer mill was developed to improve the yield and quality of RNA or protein extracted from the tissues and enable the analysis of molecular profiles associated with inflammatory responses.

Video: A Cryo-pulverization Protocol for Processing Mouse Paws to Evaluate Molecular Pathways of Tissue Inflammation in a Collagen Induced Arthritis Model

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Neuroscience

Spontaneous and Evoked Measures of Pain in Murine Models of Monoarticular Knee Pain

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are necessary and useful for studying the mechanisms of pain and for evaluating efficacy of arthritis therapies. Measuring pain in animal models of arthritis is challenging. We have developed methods for measuring evoked and spontaneous pain in three models of murine arthritis. We quantitate the evoked pain responses of mice subjected to firm palpation of a painful knee. We also evaluate spontaneous pain by the proportion of weight and the amount of time placed on each of their 4 limbs after induction of arthritis pain in one knee. Joint pain in these mouse models produces a significant increase in evoked pain responses and an alteration in weight bearing. Since mice are quadrupeds, they offload the painful limb to the contralateral limb, to the forelimbs, or some combination. These methods are simple, require minimal equipment, and are reproducible and sensitive for detecting pain. They are useful for studying both disease-modifying arthritis treatments and analgesics in mice.

We have developed an evoked measure of arthritis pain and coupled it with a standardized method for measuring spontaneous pain in different murine models of chemically induced arthritis. These measures are sensitive and reproducible for different types of joint pain.

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are ...

Medicine

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.

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, ...

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

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

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

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

Destabilization of the Medial Meniscus and Cartilage Scratch Murine Model of Accelerated Osteoarthritis

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are used to mimic many aspects of the disease. The present protocol describes the destabilization and cartilage scratch model (DCS) of post-traumatic osteoarthritis. Based on the widely used destabilization of the medial meniscus (DMM) model, DCS introduces three scratches on the cartilage surface. The current article highlights the steps to destabilize the knee by transecting the medial meniscotibial ligament followed by three intentional superficial scratches on the articular cartilage. The possible analysis methods by dynamic weight-bearing, microcomputed tomography, and histology are also demonstrated. While the DCS model is not recommended for studies that focus on the effect of osteoarthritis on the cartilage, it enables the study of osteoarthritis development in a shorter time window, with special focus on (1) osteophyte formation, (2) osteoarthritic and injury pain, and (3) the effect of cartilage damage in the whole joint.

The present protocol describes the controlled microblade scratches on the surface of the articular cartilage after destabilizing the mouse knee by cutting the medial miniscotibial ligament. This animal model presents an accelerated form of osteoarthritis (OA) suitable for studying osteophyte formation, osteosclerosis, and early-stage pain.

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are ...

Ameliorating Osteoarthritis in Mice Using Silver Nanoparticles

Knee osteoarthritis (KOA) is one of the most commonly encountered degenerative diseases of the joints in people over 45 years of age. Currently, there are not any effective therapeutics for KOA,and the only end-point strategy is total knee arthroplasty (TKA); therefore, KOA is associated with economic burdens and societal costs. The immune inflammatory response is involved in the occurrence and development of KOA. We previously established a mouse model of KOA using type II collagen. Hyperplasia of the synovial tissue was present in the model, alongside a large number of infiltrated inflammatory cells. Silver nanoparticles have substantial anti-inflammatory effects and have been widely used in tumor therapy and surgical drug delivery. Therefore, we evaluated the therapeutic effects of silver nanoparticles in a collagenase II-induced KOA model. The experimental results showed that silver nanoparticles significantly reduced synovial hyperplasia and the infiltration of neutrophils in the synovial tissue. Hence, this work demonstrates the identification of a novel strategy for OA and provides a theoretical basis for preventing the progress of KOA.

Presented here is a protocol for using silver nanoparticles to effectively ameliorate the acute symptoms of type II collagenase-induced osteoarthritis mice, including synovial inflammation, synovial hyperplasia, vascular hyperplasia, etc.

Knee osteoarthritis (KOA) is one of the most commonly encountered degenerative diseases of the joints in people over 45 years of age. Currently, there are ...

Standardized Tuina Manipulation for Knee Osteoarthritis in Rats

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

Author Spotlight: Understanding the Mechanism of Tuina Manipulation in Rats 

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...

Impact of Non-Invasive ACL Injury and Surgery on Protease Activity in Mice

Non-Invasive Compression-Induced Anterior Cruciate Ligament (ACL) Injury and In Vivo Imaging of Protease Activity in Mice

Traumatic joint injuries such as anterior cruciate ligament (ACL) rupture or meniscus tears commonly lead to post-traumatic osteoarthritis (PTOA) within 10-20 years following injury. Understanding the early biological processes initiated by joint injuries (e.g., inflammation, matrix metalloproteinases (MMPs), cathepsin proteases, bone resorption) is crucial for understanding the etiology of PTOA. However, there are few options for in vivo measurement of these biological processes, and the early biological responses may be confounded if invasive surgical techniques or injections are used to initiate OA. In our studies of PTOA, we have used commercially available near-infrared protease activatable probes combined with fluorescence reflectance imaging (FRI) to quantify protease activity in vivo following non-invasive compression-induced ACL injury in mice. This non-invasive ACL injury method closely recapitulates clinically relevant injury conditions and is completely aseptic since it does not involve disrupting the skin or the joint capsule. The combination of these injury and imaging methods allows us to study the time course of protease activity at multiple time points following a traumatic joint injury.

Author Spotlight: Investigating Early Events and Long-Term Effects of ACL Injuries for Osteoarthritis Progression

Non-invasive ACL injury is a reliable and clinically relevant method for initiating post-traumatic osteoarthritis (PTOA) in mice. This injury method also allows for in vivo quantification of protease activity in the joint at early time points post-injury using protease-activatable near infrared probes and fluorescence reflectance imaging.

Traumatic joint injuries such as anterior cruciate ligament (ACL) rupture or meniscus tears commonly lead to post-traumatic osteoarthritis (PTOA) within ...

Detecting Migration and Infiltration of Neutrophils in Mice

Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. Neutrophils can be recruited to inflammation sites via the guidance of cytokines and chemokines. Overwhelming infiltration of neutrophils can lead to indiscriminate tissue damage, such as in rheumatoid arthritis (RA). Neutrophils isolated from peritoneal exudate respond to a defined chemoattractant, N-formyl-Met-Leu-Phe (fMLP), in vitro in Transwell or Zigmond chamber assays. The air pouch experiment can be used to evaluate the chemotaxis of neutrophils towards lipopolysaccharide (LPS) in vivo. The adjuvant-induced arthritis (AA) mouse model is frequently used in RA research, and immunohistochemical staining of joint sections with anti-myeloperoxidase (MPO) or anti-neutrophil elastase (NE) antibodies is a well-established method to measure neutrophil infiltration. These methods can be used to discover promising therapies targeting neutrophil migration.

Here, we present three methods to assess neutrophil migration and infiltration both in vivo and in vitro. These methods can be used to discover promising therapeutics targeting neutrophil migration.

Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. ...

Biology

Unveiling Inflammatory Arthritis&#58; Exploring Functions of Synovial Cells

Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central roles in the pathogenesis of rheumatoid arthritis. It is important to understand the functions of both cell populations to reveal the mechanisms underlying pathological progression and remission in inflammatory arthritis. In general, in vitro experimental conditions should mimic the in vivo environment as much as possible. Primary tissue-derived cells have been used in experiments characterizing synovial fibroblasts in arthritis. In contrast, in experiments investigating the biological functions of macrophages in inflammatory arthritis, cell lines, bone marrow-derived macrophages, and blood monocyte-derived macrophages have been used. However, it is unclear whether such macrophages actually reflect the functions of tissue-resident macrophages. To obtain resident macrophages, previous protocols were modified to isolate and expand both primary macrophages and fibroblasts from synovial tissue in an inflammatory arthritis mouse model. These primary synovial cells may be useful for in vitro analysis of inflammatory arthritis.

Author Spotlight: Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue  

The present study provides a modified protocol to isolate synovial macrophages and fibroblasts from murine inflammatory arthritis tissue.

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central ...