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skin should be approximately 15&#176;; then, the direction of the needle should be changed, and the needle should be withdrawn altogether.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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</td>
			<td>S-10-20</td>
			<td>Store product in the dark at 4&#176;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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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

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JoVE Journal

Medicine

995 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

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations.
Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are Gi/o and Gq coupled receptors, respectively3,4,5. M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum3,6,7. M2/Gi/o signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels5,8,9,10. These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle11. In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called "excitation-contraction coupling")4,7. More detailed reviews on signaling pathways controlling airway constriction can be found4,12. The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine13,14, both well-known ASM modulators in humans and other species5,15.
Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K+, Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

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 ...

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

Osteoarthritis Pain Model Induced by Intra-Articular Injection of Mono-Iodoacetate in Rats

The current animal models of osteoarthritis (OA) can be divided into spontaneous models and induced models, both of which aim to simulate the pathophysiological changes of human OA. However, as the main symptom in the late stage of OA, pain affects the patients&#39; daily life, and there are not many available models. The mono-iodoacetate (MIA)-induced model is the most widely used OA pain model, mainly used in rodents. MIA is an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, which causes chondrocyte death, cartilage degeneration, osteophyte, and measurable changes in animal behavior. Besides, expression changes of matrix metalloproteinase (MMP) and pro-inflammatory cytokines (IL1 &#946; and TNF &#945;) can be detected in the MIA-induced model. Those changes are consistent with OA pathophysiological conditions in humans, indicating that MIA can induce a measurable and successful OA pain model. This study aims to describe the methodology of intra-articular injection of MIA in rats and discuss the resulting pain-related behaviors and histopathological changes.

This study describes the method of intra-articular injection of mono-iodoacetate in rats and discusses the resulting pain-related behaviors and histopathological changes, which provide references for future applications.

The current animal models of osteoarthritis (OA) can be divided into spontaneous models and induced models, both of which aim to simulate the ...

Sleeve Gastrectomy in Mice using Surgical Clips

The number of people who are overweight and obese is continually increasing both in the adult and adolescent populations. This coincides with the increased universal phenomenon of type 2 diabetes (T2D) and other metabolic problems. Bariatric surgery, such as SG, is currently one of the most effective and commonly used long-term treatment for obesity and T2D, but the association between them is not completely explored yet. The mechanisms underlying the outcomes seen after bariatric surgery in humans can be investigated based on preclinical animal studies. The SG reduces body weight, glucose levels and many metabolic parameters, and is easy to perform with a low incidence of complications. The goal of this work is to provide a simple method and an uncomplicated preclinical model of bariatric surgery in animals for researchers.

The prevalence of diabetes and obesity is continuously increasing worldwide. The mechanisms between diabetes, obesity and their associated mortality and co-morbidities need to be further investigated. Here, we present a protocol for sleeve gastrectomy (SG) in animals as an uncomplicated preclinical model of bariatric surgery.

The number of people who are overweight and obese is continually increasing both in the adult and adolescent populations. This coincides with the ...

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, ...

Electrocardiogram Recordings in Anesthetized Mice using Lead II

The electrocardiogram is a valuable tool for evaluating the cardiac conduction system. Animal research has helped generate novel genetic and pharmacological information regarding the electrocardiogram. However, making electrocardiogram measurements in small animals in vivo, such as mice, has been challenging. To this end, we used an electrocardiogram recording method in anesthetized mice with many advantages: it is a technically simple procedure, is inexpensive, has short measuring time, and is affordable, even in young mice. Despite the limitations with using anesthesia, comparisons between control and experimental groups can be performed with enhanced sensitivity. We treated mice with agonists and antagonists of the autonomic nervous system to determine the validity of this protocol and compared our results with previous reports. Our ECG protocol detected increased heart rates and QTc intervals on treatment with atropine, decreased heart rates and QTc intervals after carbachol treatment, and higher heart rates and QTc intervals with isoprenaline but did not note any change in ECG parameters on administration of propranolol. These results are supported by previous reports, confirming the reliability of this ECG protocol. Thus, this method can be used as a screening approach to making ECG measurements that otherwise would not be attempted due to high cost and technical difficulties.

We present an ECG protocol that is technically easy, inexpensive, fast, and affordable in small mice, and can be performed with enhanced sensitivity. We suggest this method as a screening approach for studying pharmacological agents, genetic modifications, and disease models in mice.

The electrocardiogram is a valuable tool for evaluating the cardiac conduction system. Animal research has helped generate novel genetic and ...

Synovial Fluid Analysis to Identify Osteoarthritis

Synovial fluid (SF) analysis is important in diagnosing osteoarthritis (OA). Macroscopic and microscopic features, including total and differential white blood cell (WBC) count, help define the non-inflammatory nature of SF, which is a hallmark of OA. In patients with OA, WBC in SF samples usually does not exceed 2000 cells per microliter, and the percentage of inflammatory cells, such as neutrophils, is very low or absent. Calcium crystals are frequent in SF collected from OA patients. Although their role in the pathogenesis of OA remains unclear, they have been associated with a mild inflammatory process and a more severe disease progression. Recently, calcium crystals have been described in both the early and late stages of OA, indicating that they may play a vital role in diagnosing different clinical subsets of OA and pharmacological treatment. The overall goal of SF analysis in OA is two-fold: to ascertain the non-inflammatory degree of SF and to highlight the presence of calcium crystals.

Synovial fluid analysis under transmitted, polarized, and compensated light microscopy is used to evaluate the inflammatory or non-inflammatory nature of a sample through simple steps. It is particularly useful in osteoarthritis to detect calcium crystals and identify a more severe subset of osteoarthritis.

Synovial fluid (SF) analysis is important in diagnosing osteoarthritis (OA). Macroscopic and microscopic features, including total and differential white ...

Ameliorating Osteoarthritis in Mice Using Silver Nanoparticles

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In vitro Measurements of Tracheal Constriction Using Mice

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Osteoarthritis Pain Model Induced by Intra-Articular Injection of Mono-Iodoacetate in Rats

Sleeve Gastrectomy in Mice using Surgical Clips

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

Electrocardiogram Recordings in Anesthetized Mice using Lead II

Synovial Fluid Analysis to Identify Osteoarthritis