This work was funded by the Natural Science Foundation of Guangdong Province (Number: 2019A1515010209) and the Science and Technology Project of Guangzhou City, China (Number: 202102010164).

All animal work was approved by the Animal Ethical and Welfare Committee (AEWC) of Guangzhou Forevergen Medical Laboratory Animal Center (2018-0186).
1. Establishment of the KOA mouse model
<ol>
	<li>Maintain BALB/c mice (18-24 g; 12-14 weeks old) in an environment with 70% humidity and at 26 &#176;C with a light/dark cycle of 12 h. For this experiment, the animals were maintained in the Guangzhou Forevergen Medical Laboratory Animal Center.</li>
	<li>Use type II collagen to establish a KOA mouse model as described previously21. Perform the intra-articular injection as described below.
	<ol>
		<li>Apply 2% sodium pentobarbital (40 mg/kg) for anesthesia and buprenorphine (0.05 mg/kg, subcutaneous injection) for analgesia. Then, fix the mouse limbs with the tape, remove the hair with the razor, and disinfect with an alternating scrub of 0.1% iodophor and alcohol three times.</li>
		<li>Wear sterile gloves and use sterile scissors to sequentially expose the skin, subcutaneous tissue, and infrapatellar ligament. Keep the incision area below 0.5 cm. 
		NOTE: A heating blanket was used to maintain the body temperature of the mice during the operation.</li>
		<li>Use a 1 mL insulin syringe to inject 10 U of 30 mg/kg (0.4 mg/mL) type II collagenase into the joint cavity (beneath the infrapatellar ligament)22. 
		&#8203;NOTE: The angle between the needle and the skin should be approximately 15&#176;; then, the direction of the needle should be changed, and the needle should be withdrawn altogether.</li>
		<li>After the injection, suture the subcutaneous tissue first, and then the skin. Sterilize the suture area with 0.1% iodophor. Place the mice into the individually ventilated cages (IVCs) separately after they wake up from the anesthesia.</li>
	</ol>
	</li>
</ol>
2. Synthesis of silver nanoparticles
NOTE: The preparation of silver nanoparticles has been described previously in detail19. The whole formulation process is carried out on ice. After preparation, the mixture is stored at 4 &#176;C; otherwise, the mixture easily solidifies at room temperature.
<ol>
	<li>Add a total of 400 &#181;L of collagen type I (4 mg/mL) to a 1.5 mL microcentrifuge tube, and place on ice.</li>
	<li>Add a total of 200 &#181;L of phosphate-buffered saline (PBS) to the above collagen, mix the solution well, and place it on ice.</li>
	<li>Finally, add 400 &#181;L of silver nanoparticles to the above solution, and then mix sufficiently. The final concentration of the nanoparticle solution is 1 mM. 
	NOTE: The mean diameter of the silver nanoparticles ranges from 5 nm to 15 nm23. This was confirmed by electron microscopy.</li>
</ol>
3. Silver nanoparticle treatment of type II collagenase-induced KOA mice
<ol>
	<li>Remove the type II collagenase-induced KOA mice from their cages 1 week later, and inject them with silver nanoparticles. Inject the silver nanoparticles once per week, and collect the specimens 30 days later.</li>
	<li>Inject a total of 2% sodium pentobarbital (dose: 2 mL/kg) for anesthesia via an intraperitoneal injection, and then fix, prepare the skin, and sterilize as described in steps 1.2.</li>
	<li>Wear sterile gloves and use sterile scissors to sequentially expose the skin, subcutaneous tissue, and knee ligaments.</li>
	<li>Use a 1 mL insulin syringe, and enter the joint cavity at a 15&#176; angle with the needle. Slowly inject approximately 20 &#181;L of the silver nanoparticle collagen mixture, and slowly withdraw the needle24.</li>
	<li>Suture the subcutaneous tissue and skin in turn, and sterilize. Place the mice into individually ventilated cages (IVC) separately after they wake up from the anesthesia.</li>
	<li>Perform this injection of the silver nanoparticle collagen mixture (20 &#181;L) four times at a frequency of once per week. 
	&#8203;NOTE: The mice treated with the silver nanoparticle collagen mixture should be kept in individual cages. Mice fighting may occur when they are maintained together, and this would affect the experimental results. During the injection, there will be a feeling of tension when the needle reaches the joint cavity, and swelling occurs in the knee joint after the injection. The combination of these two methods allows the researcher to ensure that the drug has been successfully injected into the knee joint.</li>
</ol>
4. Collection of the knee joint and synovial tissue
<ol>
	<li>Sacrifice the mice with carbon dioxide asphyxiation or any other protocol approved by the relevant animal ethics committee.</li>
	<li>Sterilize and dissect the skin and subcutaneous tissue sequentially, and fully expose the knee joint.</li>
	<li>Harvest the knee joints, including the femur and tibia, and remove the musculature tissues.</li>
	<li>Collect the knee joint tissues, including the femur, tibia, and surrounding soft tissues (ligament and capsula), in 10% formalin for preservation and fixation.</li>
</ol>
5. Hematoxylin-eosin staining
<ol>
	<li>After overnight fixation, paraffin-embed the sections, and use a microtome to cut the paraffin-embedded tissue into 0.4 &#181;m thickness. Use the prepared sections for further staining (hematoxylin-eosin staining, Safranin O/Fast Green, and immunohistochemical (IHC) staining ).</li>
	<li>Deparaffinize the sections twice with xylene, soak in 100%, 95%, 80%, and 70% ethanol in sequence for 5 min each, and rehydrate.</li>
	<li>Stain the sections with hematoxylin (0.1 g/100 mL) for 5 min, and then directly place in 1% HCl for 10 s and eosin (0.5 g/100 mL) for 1 min.</li>
	<li>Observe the histopathological changes of the synovium under a microscope.</li>
</ol>
6. Safranin O/Fast Green
<ol>
	<li>Embed the tissue in paraffin, and prepare the histologic sections as described in step 5.1.</li>
	<li>Deparaffinize the sections twice with xylene, and rehydrate them with an ethanol series (such as 100%, 95%, 80%, and 70% ethanol in distilled water, each for 5 min).</li>
	<li>Stain the prepared sections with the hematoxylin, and wash them with PBS three times for 2 min each.</li>
	<li>Differentiate the sections with hydrochloric acid alcohol, and wash them with PBS three times for 2 min each.</li>
	<li>Immerse the sections in 0.02% Fast Green staining solution for 5-10 min, followed by 0.1% Safranin O staining for 1-2 min.</li>
	<li>Differentiate the sections with 1% acetic acid, followed by PBS washing.</li>
	<li>Detect and analyze fibrocartilage formation in the sections.</li>
</ol>
7. Immunohistochemical (IHC) staining
<ol>
	<li>Embedded the tissue in paraffin, and prepare the histologic sections as described in step 5.1.</li>
	<li>Deparaffinize the sections twice with xylene, and rehydrate them with an ethanol series (such as 100%, 95%, 80%, and 70% ethanol in distilled water, each for 5 min). Immerse the sections in Tris-EDTA buffer (10 mM Tris base, 1 mM EDTA solution; pH 9.0), and heat in a microwave oven at 95 &#176;C for 10 min to perform antigen retrieval.</li>
	<li>Expose the sections to 3% hydrogen peroxide solution for 10 min to remove endogenous peroxidase.</li>
	<li>Treat the sections with 5% goat serum to block nonspecific binding.</li>
	<li>Add the diluted primary antibodies (1:1,000 dilution) against CD177, and incubate overnight at 4&#176;C. Then, wash the sections with PBS three times.</li>
	<li>Soak the sections with PBS, and incubate the sections with the appropriate secondary antibody (HRP-conjugated polymer anti-rabbit system) for 30 min at room temperature.</li>
	<li>Perform the visualization of IHC staining by using 3,3&#39;-diaminobenzidine (DAB) as a chromogen.</li>
	<li>View the sections under a microscope, and analyze the acquired images.</li>
</ol>

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The authors have no conflicts of interest to disclose.

Silver nanoparticles exhibit anti-inflammatory, antibacterial, antioxidant, and immunomodulatory effects, meaning they could protect cells and tissues from damage by reducing the production of reactive oxygen species26. Some researchers are concerned about the toxicity of silver nanoparticles27. The toxicity of silver nanoparticles is directly related to the presence of free silver ions. Due to the nanoscale size of silver nanoparticles, they could easily interfere with biomolecules, cells, and human organs15,28,29. Several studies have reported that silver nanoparticles could induce oxidative stress and impair mitochondrial function in human cells30. In addition, Ag can be detected in human organs, especially in the liver and spleen, after the use of large quantities of silver nanoparticles. Researchers have also reported that silver nanoparticles have the ability to cross the blood-brain barrier via transsynaptic transport and accumulate in the brain31. A systematic report of the biotoxicity of silver nanoparticles has not beenconducted, although some researchers acknowledge the safety of silver nanoparticles32.
In this study, we prepared a silver nanoparticle collagen mixture. Indeed, the duration period of silver nanoparticles in human tissues is brief, but the duration period of silver nanoparticles can be prolonged when applied with a collagen mixture; this not only reduces the trauma but also the dose of the drugs. Considering the toxicity of silver nanoparticles, the dose of silver nanoparticles applied in this study was 30 mg/kg, in line with previous research33.
A few vital considerations of the experimental operation are as follows. Type II collagenase should be stored at &#8722;20 &#176;C after preparation to prevent degradation due to enzymatic cleavage. The preparation of the silver nanoparticle collagen mixture must be carried out on the ice continuously at room temperature because the silver nanoparticle collagen mixture becomes a semisolid gel rapidly and then cannot be used for injection. The solution should be stored at 4 &#176;C after preparation. A 1 mL insulin syringe with a smaller needle should be chosen for intra-articular administration, and this could effectively prevent the leakage of the injected drugs. The needle should be inserted at an angle of 15&#176; to inject the silver nanoparticle collagen mixture. When the needle isnonresistant, this indicates that the needle has reached the knee joint cavity. After injecting, the angle of the injection should be changed, and the needle should be withdrawn slowly to avoid leakage of the injected drug.
In this study, silver nanoparticles effectively improved the symptoms of type II collagenase-induced KOA in mice, demonstrating the anti-inflammatory effect of silver nanoparticles. Several studies have reported the presence of apoptosis in cells incubated in vitro with silver nanoparticles34,35,36. The reduction in synovial hyperplasia could have been caused by the silver nanoparticles due to their involvement in the impairment of mitochondrial function, or these outcomes may have been mediated by reactive oxygen species. Vascular hyperplasia was observed in the synovium of mice in the KOA model group. It was possible that chemokines drove neutrophils from the blood vessels to the synovial tissue during this process and that the burst of inflammation caused the cells to consume more oxygen, thus leading to vascular hyperplasia. Hence, further experiments are required to prove the reliability of this hypothesis. This study provides theoretical benefits for research into the treatment of clinical KOA. In future studies, we aim to combine the anterior cruciate ligament (ACL) method along with the chemically induced KOA model method to observe the effect of silver nanoparticles. The experimental results show that silver nanoparticles can significantly decrease the infiltration of inflammatory cells in the synovium in KOA mice, but the mechanisms of this effect still need further study, which might unravel the pathogenesis of KOA.

Knee osteoarthritis (KOA) is one of the most frequent forms of osteoarthritis and involves a complex disease process in the entire synovial joint1. As the world population is gradually aging, the incidence of KOA is increasing substantially. Consistent pain in the knee joint commonly prompts patients with KOA to seek medical treatment. The etiology of the pain in KOA may be related to the inflammatory response, synovial hyperplasia, and cartilage degeneration2. The synovium tissues are composed of two types of cells: synovial fibroblasts and macrophages3,4,5. Synovial fibroblasts produce synovial fluid. Synovial macrophages are normally dormant and are activated by the inflammatory response. Initial inflammation of the synovium causes knee joint pain6.
The synovium tissue inflammatory immune response plays a crucial part in the pathogenesis of KOA. Previous studies have confirmed that there are inflammatory responses in the synovium tissues in KOA, known as synovitis, and the synovitis degree of KOA is closely related to the inflammatory cell infiltration of the synovium tissues7,8,9. Synovitis is an inflammatory reaction of the synovium, and its pathological characteristics are the proliferation of synovial cells, new vessel formation, and the infiltration of inflammatory cells5,10,11.
The goal of KOA treatment is to relieve the inflammatory reaction of the synovium and delay the progression of the disease. Currently, the major clinical drugs for treating KOA are nonsteroidal anti-inflammatory drugs (NSAIDs); however, they exhibit significant side effects, such as nephrotoxicity12,13. Intra-articular glucocorticoid injections are another option for treating KOA; however, the glucocorticoid spreads quickly and could be rapidly metabolized by the joint effusion. Meanwhile, diabetic patients with underlying hyperglycemia should be cautious about ongoing steroid injections14. In summary, there is no available drug therapeutic strategy for KOA. Therefore, the exploration of new drugs for treating KOA is extremely urgent.
The size of silver nanoparticles is less than 100 nm. Due to their prominent anti-inflammatory, antibacterial, and antioxidant effects, they have been widely utilized invarious aspects of healthcare and medicine, such as wound healing and burn injury15,16. They are also used in targeted drug delivery, medical imaging, and molecular diagnosis17. Silver (Ag) has greater anti-inflammatory and anti-bacterial action than other metal nanoparticles, such as copper (Cu), zinc (Zn), and iron (Fe)15. Silver nanoparticles, a new type of nanomaterial, have broad-spectrum and potent antimicrobial properties. A previous study found that in burn injury and peritonitismouse models18,19, silver nanoparticles could effectively inhibit the production of inflammatory factors and promote wound healing. A previous study also demonstrated that silver nanoparticles improved the healing of diabetic wounds by promoting the synthesis of growth factors and collagen deposition20.
Based on the anti-inflammatory effects of silver nanoparticles, we aimed to use silver nanoparticles to treat type II collagen-induced KOA in mice. The results suggested that the number of inflammatory infiltration cells of the synovial joint in mice was significantly reduced with this treatment. The results also suggested that silver nanoparticles could significantly relieve the symptoms of KOA in mice. Therefore, the application of silver nanoparticles may support the development of new treatment options for clinical KOA.

<table><tbody><tr><td>1 mL insulin syringe</td><td>BD</td><td>305932</td><td>None</td></tr><tr><td>CD177 Polyclonal Antibody</td><td>ThermoFisher Scientific</td><td>PA5-98759</td><td>None</td></tr><tr><td>Chloral hydrate</td><td>Sigma-Aldrich</td><td>302-17-0</td><td>None</td></tr><tr><td>DAB</td><td>MCE</td><td>HY-15912</td><td>None</td></tr><tr><td>Eosin</td><td>Beyotime Biotechnology</td><td>C0109</td><td>None</td></tr><tr><td>Formalin</td><td>Sigma-Aldrich</td><td>HT501128</td><td>None</td></tr><tr><td>Hematoxylin</td><td>Beyotime Biotechnology</td><td>C0107</td><td>None</td></tr><tr><td>Light Microscopy</td><td>Leica</td><td>DM500</td><td>None</td></tr><tr><td>Silver nanoparticle</td><td>Wolcacvi&amp;nbsp;</td><td>S-10-20</td><td>Store product in the dark at 4&amp;deg;C</td></tr><tr><td>Safranine O-Fast Green FCF Cartilage Stain Kit</td><td>Solarbio</td><td>90-15-3</td><td>None</td></tr><tr><td>Type II collagen</td><td>Sigma-Aldrich</td><td>C6885-500mg</td><td>None</td></tr></tbody></table>

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.

The KOA mouse model was induced using type II collagenase. Starting 1 week after model induction, the prepared silver nanoparticle collagen mixture was injected into the joint cavity once per week for 4 weeks (Figure 1). The weights of the mice in each group were observed and recorded daily. The results showed that the average body weight of the KOA mice was significantly lower than that of the mice in the normal control group. However, the average body weight of the mice in the type II collagenase + AgNPs group was higher compared to the KOA mice, although this difference was not statistically significant (Figure 2). After 30 days, the synovial tissues of the knee joints were collected from the mice and subjected to pathological examination. The hyperplasia, vascular proliferation, inflammatory infiltration of the synovium, and cartilage damage were analyzed5,10,11. The results showed that the synovial thickness of the mice in the KOA group was significantly higher compared with the normal control group. In the group treated with the silver nanoparticle collagen mixture, the synovial membrane thickness was reduced compared with the KOA group (Figure 3). There was vascular hyperplasia in the synovium of the KOA mice compared to the normal control group, and vascular hyperplasia was significantly reduced in the synovium of mice treated with the silver nanoparticle collagen mixture (Figure 4). The results of the Safranin-O staining showed that the cartilage matrix of KOA mice was destroyed, while the mice treated with the silver nanoparticle collagen mixture showed a significantly better cartilage matrix (Figure 5). The morphological feature scores in each group were assessed as described previously22. The results were as follows: 0 &#177; 0 for the saline group, 7 &#177; 0.63 for the type II collagen group, and 4.2 &#177; 1.17 for the type II collagenase + AgNPs group (Figure 6). CD177 is a major neutrophil marker25. CD177 is expressed in 40%-60% of neutrophils under normal conditions. However, the expression of CD177 in neutrophils increases significantly during acute inflammation. The results of the IHC staining demonstrated that the infiltrated neutrophils in the synovial region were significantly reduced in the group treated with AgNPs compared with the KOA group (Figure 7), which suggests that treatment with AgNPs could improve the symptoms of KOA.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65111/65111fig01.jpg" /> 
Figure 1: Injection location. (A) Representative images of the type II collagenase injection. (B) Representative images after the type II collagenase injection. (C) Representative images of the silver nanoparticle collagen mixture injection in the KOA mouse model. (D) Representative images after the silver nanoparticle collagen mixture injection in the KOA model mice. The red dotted line represents the line parallel to the mouse knee ligaments. The black arrow represents the angle between the insulin syringe needle and the skin. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65111/65111fig01large.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/65111/65111fig02.jpg" /> 
Figure 2: Body weight changes of the mice in each group. This panel shows the average weight of the mice in each group at different time points; the x-axis indicates the number of days after the injection of type II collagenase, and the y-axis indicates the fold-change in body weight. Saline group (n = 7), type II collagenase group (n = 5), type II collagenase + AgNPs group (n = 5). *p &#60; 0.05. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65111/65111fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65111/65111fig03.jpg" /> 
Figure 3: Hematoxylin-eosin (H&#38;E) staining representing synovial hyperplasia. The synovial tissues in each group of mice were collected, fixed, sectioned, and stained with H&#38;E at 30 days after surgery. The double arrows represent the detected synovial thickness. Scale bar = 0.1 mm. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65111/65111fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65111/65111fig04.jpg" /> 
Figure 4: Representative image of perisynovial vascular hyperplasia. The arrows indicate the vessels. Scale bar = 0.05 mm. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65111/65111fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65111/65111fig05.jpg" /> 
Figure 5: Safranin-O staining of the knee joint in each group of mice. Scale bar = 0.2 mm. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65111/65111fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65111/65111fig06.jpg" /> 
Figure 6: Morphological feature scores in each group.&#160;The synovial tissue was used to measure the morphological feature score for the mice in each group. Five tissue sections in each group were selected to analyze the degree of hyperplasia/enlargement of the synovial lining cell layer, the degree of neutrophil infiltration in the synovial tissue, and the degree of activation of the synovial stroma (Table 1). The average value was used as the final score. **p &#60; 0.01 and ***p &#60; 0.001 with a Student&#39;s t-test for each cohort compared to the untreated KOA group. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65111/65111fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65111/65111fig07.jpg" /> 
Figure 7: Immunohistochemical staining for a neutrophil marker in the synovial tissue in each group of mice. Immunohistochemical staining was used to detect the expression of the neutrophil marker CD177 in the synovial tissue of the mice in each group. The arrows indicate neutrophils. Scale bar = 100 &#181;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65111/65111fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Scoring morphological features. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65111/Table 1.xls" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Ameliorating Osteoarthritis in Mice Using Silver Nanoparticles at JoVE.com

Ameliorating Osteoarthritis in Mice Using Silver Nanoparticles

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 ...

ameliorating-osteoarthritis-in-mice-using-silver-nanoparticles

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

1.0K Views.  The Third Affiliated Hospital of Sun Yat-sen University. 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.

Video: Ameliorating Osteoarthritis in Mice Using Silver Nanoparticles

Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis

Destabilization of medial meniscus (DMM) model is an important tool for studying the pathophysiological roles of numerous arthritis associated molecules in the pathogenesis of osteoarthritis (OA) in vivo. However, the detailed, especially the visualized protocol for establishing this complicated model in mice, is not available. Herein we took advantage of wildtype and progranulin (PGRN)-/- mice as examples to introduce a protocol for inducing DMM model in mice, and compared the onset of OA following establishment of this surgically induced model. The operations performed on mice were either sham operation, which just opened joint capsule, or DMM operation, which cut the menisco-tibial ligament and caused destabilization of medial meniscus. Osteoarthritis severity was evaluated using histological assay (e.g. Safranin&#160;O staining), expressions of OA-associated genes, degradation of cartilage extracellular matrix molecules, and osteophyte formation. DMM operation successfully induced OA initiation and progression in both wildtype and PGRN-/- mice, and loss of PGNR growth factor led to a more severe OA phenotype in this surgically induced model.

We describe a protocol for the destabilization of the medial meniscus (DMM) model in mice, an effective tool for osteoarthritis (OA) research. In addition, we have demonstrated that deficiency of progranulincan exaggerate OA development and progression by using this model, indicating that progranulin plays a protective role in the pathogenesis of OA.

Destabilization of medial meniscus (DMM) model is an important tool for studying the pathophysiological roles of numerous arthritis associated molecules ...

Bioengineering

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 ...

A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections may be one possible cause. The typical animal model used for studying BA is established by inoculating a neonatal mouse with a rhesus rotavirus. Silver nanoparticles have been shown to exert antibacterial and antiviral effects; their function in the BA mouse model is evaluated in this study. Currently, in BA animal experiments, the methods used to improve the symptoms of BA mice are generally symptomatic treatments given via food or other drugs. The aim of this study is to demonstrate a new method for ameliorating BA syndrome in mice by the intraperitoneal injection of silver nanoparticles and to provide detailed methods for preparing the silver nanoparticle gel formulation. This method is simple and widely applicable and can be used to research the mechanism of BA, as well as in clinical treatments. Based on the BA mouse model, when the mice exhibit jaundice, the prepared silver nanoparticle gel is injected intraperitoneally to the surface of the lower liver. The survival status is observed, and biochemical indicators and liver histopathology are examined. This method allows a more intuitive understanding of both the establishment of the BA model and novel BA treatments.

This article describes in detail a method based on silver nanoparticles for ameliorating biliary atresia syndrome in an experimental biliary atresia mouse model. A solid understanding of the reagent preparation process and the neonatal mouse injection technique will help familiarize researchers with the method used in neonatal mouse model studies.

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections ...

Immunology and Infection

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.

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 ...

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 ...

Software-Assisted Quantitative Measurement of Osteoarthritic Subchondral Bone Thickness

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

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

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

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 ...

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 ...

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. ...