Name: micro-computed tomography analysis of the knee in aged dunkin-hartley guinea pigs after intra articular injection
Uploaded: 2024-08-02T13:00:00.000+00:00
Duration: 7 min 10 s
Description: The purpose of this protocol is to guide researchers in performing a palpation-guided technique of intra-articular knee injection in guinea pigs and ...

The research outlined in this manuscript was supported by South Carolina SmartState&#174; Endowed Chair in Drug Discovery Endowment funds (PMW),&#160;the MUSC Division of Laboratory Animal Resources, and the MUSC Drug Discovery Core. This publication was also supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Grant Numbers TL1 TR001451 &#38; UL1 TR001450, as well as the National Institute of Dental &#38; Craniofacial Research of the National Institutes of Health under Award Number R01DE029637.

All methods described here have been approved by the Institutional Animal Care and Use Committee of the Medical University of South Carolina. The study followed the principle of 3R.
1. Intra-articular injection preparations
<ol>
	<li>Allow Dunkin-Hartley guinea pigs to acclimate to facility for at least one week prior to starting the experiment. 
	NOTE: 5- (n=2), 9- (n=1), and 12- (n=2) month-old male guinea pigs were used. Males display an accelerated development and more severe phenotype of OA.</li>
	<li>Shave the knee area with an electric razor. 
	NOTE: Be careful of the nipples medially.</li>
	<li>Anesthetize guinea pig in an isoflurane chamber delivering 3-5% isoflurane in O2 mixture (flow rate 2.5 L/min) and then transfer the guinea pig to a nose cone connected to a non-rebreathing anesthesia circuit. Adjust isoflurane to maintain surgical plane of anesthesia during the injection, typically with an oxygen flow rate of 0.5-1 L/min and 1-3% isoflurane. 
	NOTE: Intra-articular injections cause mild, momentary pain. Animals are anesthetized during the procedure to prevent perception of painful stimuli and improve injection accuracy. In the presented study, the administration of analgesic agents, including nonsteroidal anti-inflammatory drugs, would interfere with OA progression21. Due to the momentary pain, provided anesthesia and potential of analgesics to confound the model, analgesics were not administered unless animals displayed side effects, including limping and signs of pain on joint palpation after injection. Investigators should consider the use of analgesics for routine injections. Analgesics are recommended when side effects occur. Analgesic regimens should be discussed with the institutional veterinarian and approved by the IACUC prior to initiating studies.</li>
	<li>Ensure guinea pig is at appropriate anesthesia depth by lack of toe pinch response.</li>
	<li>Place sterile ocular lubricant on both eyes to prevent desiccation and injury.</li>
	<li>Dilute betadine with sterile water to 10%.</li>
	<li>Dilute 200 proof ethanol with sterile water to 70% ethanol.</li>
	<li>Prepare solutions for injection, in a biosafety cabinet to maintain sterility. In the presented protocol, a sterile vehicle (1x phosphate buffered saline) was utilized to inject both knees. Solutions can be changed based on research objectives. 
	NOTE: Make sure to dilute fresh solutions for injection immediately before the injection session to ensure sterility. Any unused solutions should be discarded at the end of each injection session.</li>
	<li>Fill sterile one-time-use insulin syringes with solutions for injection. Take care to utilize the smallest volume achievable to prevent overloading the joint space with volume. In the present study, 50 &#181;L was used.</li>
	<li>Place guinea pig and nose cone on a clean surface with a heating pad for thermal support and padding under the head to elevate it slightly.</li>
	<li>Don surgical gown, hair net, sterile gloves, and mask while performing the injection procedure.</li>
	<li>Pour 10% betadine onto a cotton ball and wipe both knee areas.</li>
	<li>Pour 70% ethanol onto a cotton ball and wipe both knee areas.</li>
	<li>Repeat 1.12 and 1.13 two more times. 
	NOTE: For demonstration purposes, the corresponding video shows cleaning the knee and injection site once with 10% betadine and 70% ethanol. The injection site was subsequently cleaned using circular motions two more times, alternating these solutions. Three serial scrubs with alternating scrub solutions and alcohol are recommended to achieve aseptic technique.</li>
</ol>
2. Intra-articular injection
<ol>
	<li>Place the guinea pig in supine position for entirety of the procedure.</li>
	<li>Don a new pair of sterile gloves and palpate the knee joint. 
	NOTE: In the presented protocol and video, autoclaved nitrile gloves were utilized. Sterile gloves, including either autoclaved nitrile gloves or surgical gloves, should be utilized for aseptic technique.</li>
	<li>Manually flex the knee to 90&#176;.</li>
	<li>Move finger distal to the patella to locate the groove of the distal aspect of joint space by flexing and extending the hindlimb. 
	NOTE: The patella can be palpated in this position as a small, firm structure located directly over the joint space. The tibia can be felt as a bony structure distal to the patella. Once the location of the tibia and patella are determined, the joint, felt as a groove, is between them, distal to the patella and proximal to the tibia.</li>
	<li>Insert the insulin needle carefully on the midline distal to the patella within the joint space. The needle should be inserted 1-2 mm below the skin to enter the joint space. 
	NOTE: The largest access window for the joint space while the knee is flexed is on the anterior aspect of the limb on midline, directly distal to the patella. Injecting on midline in the anterior-to-posterior direction will aid in accurately injecting into the joint space without penetrating boney structures. Accurately injecting into the joint space can be achieved using a lateral-to-medial approach, although the access window is narrower especially when the knee is flexed.</li>
	<li>Inject 50 &#181;L of the solution into the joint slowly. Ensure that the needle inserts easily, and contents are injected without resistance. 
	NOTE: Make sure not to insert the needle too deep as it can cause joint or bone damage and result in unwanted inflammation and/or pain. If the groove corresponding to the joint space is not found, the needle could penetrate the femur, patella, or tibia. Therefore, it is beneficial to confidently palpate the groove corresponding to the joint space to prevent peri-articular injections or injuries associated with penetrating boney structures. If a bubble develops at the injection site under the skin, the injection was too shallow and the fluid has entered the subcutaneous space. Depending on the properties of the agent utilized, the drug may enter the joint space via diffusion, or another injection attempt may be needed.</li>
	<li>Once done discard the needle into sharps bin. 
	NOTE: For practice and training purposes, inject liquid containing a dye into the joint space of a cadaver in a similarly sized rodent or guinea pig. Then, dissect the joint to confirm the location of the injection.</li>
	<li>Massage the knee by flexing and extending the joint a few times promote diffusion of the drug within the joint space.</li>
	<li>Repeat steps 2.1-2.5 once on the contralateral limb with 1x PBS solution.</li>
</ol>
3. Recovery from intra-articular injection
<ol>
	<li>Turn off isoflurane and maintain 100% flow by oxygen until the animal regains consciousness.</li>
	<li>Place the animal on a heating pad for thermal support until ambulatory.</li>
	<li>Apply an ice pack to the knee for 30 s with a paper towel as a barrier to help decrease swelling from the injection.</li>
	<li>Assess animal gait when ambulatory prior to returning them to housing. 
	NOTE: If any gait abnormalities are noted, analgesics and supportive care may be warranted. It is advisable to assess their gait again several hours after recovery from anesthesia to ensure normal mobility.</li>
</ol>
4. Micro computed tomography (&#181;CT) scan
<ol>
	<li>For the tissue harvest, establish a surgical plane of anesthesia with 100% oxygen and 5% isoflurane mixture.</li>
	<li>Confirm a surgical plane of anesthesia with the lack of response to a toe-pinch stimulus. Humanely euthanize the animal via administration of &#8805; 150 mg/kg of pentobarbital intravenously according to institutional policies and approved animal use protocol. 
	NOTE: In the presented protocol, each of the five guinea pig received one injection in both knees. Animals were anesthetized and humanely euthanized one week after the injection.</li>
	<li>Harvest both hindlimbs by dissecting the skin away from the surrounding musculature.</li>
	<li>Next, disarticulate the hindlimb with Rongeurs at the mid-shaft of the femur and proximal to the ankle. 
	NOTE: The scanning bed and specimen holder used was unable to accommodate the entire hindlimb of an adult guinea pig. Large specimen holders are commercially available for larger specimen sizes.</li>
	<li>Place the tissues in neutral buffered formalin solution for 72 h for fixation before performing &#181;CT.</li>
	<li>Open &#181;CT scan software and place sample with formalin in a compatible container that will fit into the &#181;CT specimen folder while maintaining tissue in the field of vision.</li>
	<li>Calibrate &#181;CT machine for dark field and light field exposures according to manufacturer recommendations.</li>
	<li>Scan the sample with Al+Cu filter at 18 &#181;m. Use rotation step 0.7&#176; for 360&#176; with offset camera. 
	NOTE: The scan automatically saves.</li>
</ol>
5. Image processing for evaluating bone microarchitectural parameters
<ol>
	<li>Download and install &#181;CT reconstruction software for the reconstruction of &#181;CT images.</li>
	<li>Select the software folder and double click to open the software.</li>
	<li>Select one slice from the &#181;CT images by clicking on an image slice.</li>
	<li>Choose the reconstruction file destination. Select Browse and create a new folder named Recon. The selected file format should be BMP(8).</li>
	<li>Check Misalignment Compensation. 
	NOTE: Usually, the estimation is close to correct, but it can be manually adjusted to shift the overlapping images so that the right and left edges align as closely as possible.</li>
	<li>Under Settings, apply Smoothing, Beam Hardening, CS Rotation, and Ring Artifacts algorithms. 
	NOTE: It can be helpful to choose preview image to determine the clarity before reconstructing. The fine-tuning setting can also be helpful to determine which settings are the best.</li>
	<li>Select Start to begin processing the reconstruction.</li>
</ol>
6. Collection of microarchitectural data from reconstructed images
<ol>
	<li>Download and install Dataviewer.</li>
	<li>Select VOI and orient the sample to align vertically for easier analysis at a later time.</li>
	<li>Save edited VOI as a new folder.</li>
	<li>Download and install CTAnalyser for the bone property analysis of microarchitectural parameters. 
	NOTE: The free version of CTAnalyser is limited in functionality, so is recommended to obtain a full license.</li>
	<li>Split the analysis to subchondral plate and trabecular bone by saving them as separate range of images. 
	NOTE: Splitting the analysis is not necessary, but because subchondral plate and trabecular bone have different features, separate analyses can assist with robust measurements and comparison.</li>
	<li>Select the range of images to analyze, starting with subchondral plate by clicking on the image slice that you want to start with.</li>
	<li>Select the region of interest for each image to ensure that it is encompassing the bone by clicking on the region of interest tab.</li>
	<li>Select the Binary Selection tab. Adjust the histogram so that the background and the bone is completely separate.</li>
	<li>Select the Bone Mineral Density (BMD) tab. Save that data into a new analysis data folder.</li>
	<li>Select Custom Processing and go to Internal tab.</li>
	<li>First perform Thresholding and select Automatic Otsu, then Run.</li>
	<li>Then select Despeckle and choose Remove black speckles, then Run.</li>
	<li>Repeat Despeckle and choose Remove white speckles, then Run.</li>
	<li>Choose 3D analysis and select Basic values and Additional values.</li>
	<li>Repeat steps 6.2.2-6.4.5 to reset the image for trabecular bone analysis. 
	NOTE: Make sure the output file is in a new folder with the same file as the BMD data.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targets,+models+and+challenges+in+osteoarthritis+research.">Targets, models and challenges in osteoarthritis research.</a> Dis Model Mech. 8 (1), 17-30 (2015).</li><li>Vazquez-Portalatin, N., Breur, G. J., Panitch, A., Goergen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+ultrasound-guided+intra-articular+injections+in+guinea+pig+knees.">Accuracy of ultrasound-guided intra-articular injections in guinea pig knees.</a> Bone Joint Res. 4 (1), 1-5 (2015).</li><li>Nie, C., Wang, Z., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+depression+on+fracture+healing+and+osteoblast+differentiation+in+rats.">The effect of depression on fracture healing and osteoblast differentiation in rats.</a> Neuropsychiatr Dis Treat. 14, 1705-1713 (2018).</li><li>Jonsson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-CT+and+deep+learning:+Modern+techniques+and+applications+in+insect+morphology+and+neuroscience.">Micro-CT and deep learning: Modern techniques and applications in insect morphology and neuroscience.</a> Front Insect Sci. 3, (2023).</li></ol>

Despite recent advancements in symptomatic treatment of OA, there is a complete lack of therapeutic agents that prevent onset or delay progression of OA24. Currently, the only cure for severe OA is joint replacement, which is costly, invasive, and can result in patient morbidity and mortality25. As a result, there is a dire need for continued research with animal models of OA and the sustained development of novel therapeutics. Several animal models are available to study different components of OA, including spontaneous and chemically or surgically induced models. The injection of chemicals, like in the MIA mouse model, are vital for studying pain but do not allow investigators to follow the progression of OA development and intervene at desired timepoints throughout the course of the disease26. Surgical methods require invasive procedures and fail to faithfully mirror the initiation of OA in humans27. Dunkin-Hartley guinea pigs naturally develop OA, making them a widely available spontaneous model of OA28. This spontaneous model is ideal for translational research studying mechanistic OA development as it requires no interventions and features pathologic changes that mirror those in humans6. Previously published works have utilized intra-articular injections to test the effects of novel therapeutics on the progression of spontaneous OA in Dunkin-Hartley guinea pigs29,30,31.
While intra-articular injections in mice have been previously described, anatomical differences pose additional challenges to this procedure in guinea pigs7. Although intra-articular injections are integral to guinea pig OA studies, this procedure has yet to be systematically detailed. With the described methodology, we highlight key considerations for refining intra-articular injections in guinea pigs, thereby reducing the potential for procedure related complications. Common side effects following intra-articular injections in humans include pain, swelling, septic arthritis, and neuropathy and these should be considered as potential complications when performing injections in guinea pigs32. In our experience, side effects, including limping and pain on joint palpation, can occur when the needle is inserted too deeply into the joint space and/or contacted a boney structure during injection. Having cadavers to practice our injections on before performing them on live animals helped ensure more accurate injections. In refining our technique to ensure a shallow injection depth, we observed no side effects following intra-articular knee injections. Therefore, proper injection technique is paramount to ensuring animal welfare, particularly for studies requiring multiple injections over many months. Key steps in the procedure include using aseptic technique; sufficient training prior to performing survival procedures; achieving a surgical plane of anesthesia to prevent pain perception and movement upon injection; and ensuring appropriate injection depth, to avoid contact with bone distally and peri-articular injections which may require administering the compound again.
To systematically assess OA related changes, we describe &#181;CT analyses, including 3D image renditions, allowing for visualization, segmentation, labeling, and objective measurements of affected tissue. The methods described have been effective and efficient in systematically assessing OA joint and bone changes over time. Our &#181;CT results show that 12-month-old Dunkin-Hartley guinea pigs have more severe OA than 5-month-old guinea pigs, as indicated by increased bone mineral density and increased trabecular thickness. This demonstrated worsening of OA over time aligns with previously published results in this OA model, including &#181;CT analyses that assessed bone mineral density and trabecular thickness13. Previous studies, and the presented representative results, demonstrate and increase in BMD and trabecular thickness over time as measured by &#181;CT22,23. In addition, our histologic assessment of the knees in two-dimension supports the validity of the &#181;CT data. Histologically, signs of OA are more pronounced in the 9- and 12- month-old guinea pigs compared to the 5-month-old animals. Histologic changes associated with OA include proteoglycan loss, fissures, and hypocellularity.10 Each of these features can be seen in the older guinea pigs. Additionally, older guinea pigs have higher Modified Mankin scores, a widely utilized and validated method for assessing OA severity10. While our histologic analyses demonstrate expected OA changes, &#181;CT provides a more in-depth assessment of OA changes than histologic review as it scans an entire sample instead of one or few tissue sections18. Furthermore, it provides more objective measurements of the extent of bony lesions in both 2- and 3-dimensions18. Researchers should consult existing guidelines when scoring histologic OA changes, like the Osteoarthritis Research Society International histologic assessment guidelines10. Researchers should consider additional ancillary tests, including gait analyses, for in depth characterization of OA development34. While gait analyses can reveal functional changes over time, OA lesions develop well before the onset of lameness, making &#181;CT a more sensitive measure of OA related changes during early stages of disease.
While Dunkin-Hartley guinea pigs provide a robust model for studying OA progression and the effects of novel therapeutics, there are limitations to this model. Spontaneous models typically require a longer study period compared to the rapid development of changes following surgical or chemically induced OA35. This can result in increased cost and study duration. As previously described, onset of OA varies between animals of the same age, which may require assessing OA related changes serially36. Despite this variation, researchers can expect all Dunkin-Hartley guinea pigs to develop OA starting at 3 months, showing changes in subchondral and trabecular bone, followed by progressive OA changes as the animal ages10,19. Researchers may reduce the effect of inter-animal variability by applying treatments to one limb in each animal, allowing the contralateral limb to serve as an internal control. Although beyond the scope of the presented methodology, ultrasound guided injections may assist in further improving injection accuracy and has been utilized in guinea pigs37. The proposed study herein describes the feasibility of palpation-guided injection, without utilizing this imaging modality, a technique widely utilized in the field37. While &#181;CT is a powerful tool to analyze pathology and bone structure, the availability of software that supports extensive analytical features can introduce inter-user variability in analyses38. In the present study, the diameter of the &#181;CT scanning bed was 6.35 cm, thereby preventing imaging of the entire guinea pig or joint in situ. Larger bed attachments are commercially available that could facilitate whole animal imaging. Although the software is generally user friendly and offers several suggested settings, it may be beneficial to manually edit settings for higher resolution and accuracy. As automated techniques continue to evolve, including machine learning, these manual adjustments can be replaced with new, automatic software techniques to enhance standardization39. While the presented manuscript describes step-by-step instructions for the utilized &#181;CT and corresponding software, these same analyses can be performed with other brands and software; researchers should consult the corresponding user manuals to optimize and standardize image analyses when utilizing differing machines and/or software. To reduce variability, it is important to maintain the same settings across all samples. Subjectivity of image analyses can be further reduced by replicating analyses by multiple, blinded experimenters.
The primary goal of this protocol is to provide detailed guidance on performing a technically challenging procedure in guinea pigs and subsequent &#181;CT analyses of associated OA changes. Standardization of technique and data analyses will help minimize cost and increase efficiency.

Osteoarthritis (OA) impacts 32.5 million US adults. It is caused by progressive loss of articular cartilage, mild inflammation of the tissues in and around the joints, and formation of osteophytes and bone cysts1,2. Symptoms typically manifest in the later stages of the disease, with current treatments providing only palliative relief as well as having systemic side effects. The lack of disease-modifying drugs stems from a poor understanding of the underlying mechanisms of the disease3. As a result, there is a critical and ongoing medical need for improved agents to treat OA. 
Several animal models of OA are available that examine different components of the disease processes4. While several surgical models exist, including transection of the anterior cruciate ligament and destabilization of the medial meniscus, these are invasive and require a high level of technical skill5. Chemically induced models are comparatively less invasive procedures typically used to study OA pain mechanisms6. One such widely used mouse model involves OA induction by an intra-articular knee injection of monosodium iodoacetate (MIA). This model generates a reproducible, robust, and rapid pain-like phenotype that can be graded by altering MIA dosage7. Technical details of inducing this model have been previously described7. Translation of this technique to larger rodents, like guinea pigs, is difficult due to their anatomical differences. Some differences include increased musculature surrounding the adjacent bones and joint space in the guinea pig and an articulating fibula and tibia compared to distal fusion seen in mice8. Dunkin-Hartley guinea pigs, a widely available guinea pig strain, are an established OA animal model as they naturally develop this disease, thereby offering a robust model for investigating the effects of novel therapeutics administered by intra-articular injection on disease progression9. Dunkin-Hartley guinea pigs start developing OA at three months, with males displaying an accelerated development and more severe phenotype10. In guinea pigs, OA progresses with age, and at 12 months, associated pathology is apparent on imaging11. Spontaneous OA models, like the Dunkin-Hartley model, do not require any intervention to induce OA and thus recapitulate the development and progression of the disease phenotype in humans, thereby providing a powerful translational model10. Furthermore, the spontaneous development of OA allows for the internal control when novel therapeutics are administered unilaterally in a single knee of a given animal. This internal control minimizes the effects of inter-animal variabilities when analyzing data and may help reduce overall animal numbers. 
X-ray Micro Computed Tomography (&#181;CT) analysis is a powerful tool that allows for quantitative assessment of OA severity12. &#181;CT involves scanning multiple, high-resolution X-ray images, obtained from a rotating sample or rotating X-ray source and detector13. Then, three- dimensional (3D) volumetric data is reconstructed in the form of stacked image slices14. Because mineralized bone has excellent contrast on &#181;CT, this modality can be used to assess 3D features and perform quantitative analyses of changes associated with OA15,16,17. &#181;CT offers several advantages over more widely used tools, including histopathology and gait analyses. In contrast to histologic assessment of one or few sections of tissues, &#181;CT scans the entire joint and offers a more wholistic assessment of OA lesions18. While gait analysis can discern symptomatic changes in joint function over time, joint changes develop long before functional changes associated with OA. &#181;CT can provide a more sensitive measure of OA development prior to the onset of lameness. Two particularly relevant quantitative measurements include bone mineral density and trabecular thickness as both increase throughout the progression of OA19,20. It can be helpful to split the analysis into subchondral plate and trabecular bone, as they have different features, to achieve more robust measurements and comparisons.
The overall goal of this method is to help researchers successfully perform intra-articular injections on guinea pigs. The presented protocol utilized five-(n=2), nine- (n=1), and 12- (n=2) month-old intact, male Dunkin-Hartley guinea pigs; procedures can be extrapolated to other guinea pig strains and ages requiring intra-articular knee injections. In spontaneous models of OA, like the Dunkin-Hartley model, disease progression and response to serial treatment is often monitored over long periods of times, spanning weeks to months9. This extended protocol results in multiple intra-articular injections, and thus it is important to have proper injection technique to prevent adverse events, including pain, lameness, or infections, all of which can impact animal welfare and confound study results while necessitating additional animals on study. The presented protocol describes methodology of intra-articular injections in guinea pigs and subsequent analysis of &#181;CT data.

<table><tbody><tr><td>200 Proof Ethanol</td><td>Decon Laboratories</td><td>2701</td><td>sterilizing agent</td></tr><tr><td>3D.SUITE software</td><td>Bruker</td><td></td><td>&amp;mu;-CT analyzing software</td></tr><tr><td>Betadine Surgical Scrub</td><td>Avrio Health</td><td>67618-151-16</td><td>sterilizing agent</td></tr><tr><td>Insulin syringe with needle</td><td>Ulticare</td><td>91008</td><td>to perform injections</td></tr><tr><td>Isoflurane</td><td>Piramal</td><td>803249</td><td>anesthesize animal</td></tr><tr><td>Neutral Buffered Formalin</td><td>Fisher Scientific</td><td>23-427098</td><td>Fix tissue</td></tr><tr><td>Nrecon Software</td><td>Bruker</td><td></td><td>&amp;mu;-CT reconstruction software</td></tr><tr><td>Phosphate Buffered Saline</td><td>Cytiva</td><td>SH30258.01</td><td>control and diluting agent</td></tr><tr><td>SkyScan 1176</td><td>Bruker</td><td></td><td>to scan samples&amp;nbsp;</td></tr></tbody></table>

micro-computed tomography analysis of the knee in aged dunkin-hartley guinea pigs after intra articular injection

The purpose of this protocol is to guide researchers in performing a palpation-guided technique of intra-articular knee injection in guinea pigs and assessment using micro-computed tomography. Dunkin-Hartley guinea pigs are robust models for osteoarthritis research as they spontaneously develop osteoarthritis in their knees. Intra-articular drug delivery is a common method to study the effects of an investigational drug in vivo. In humans, therapeutic agents administered via intra-articular injection can offer pain relief and delay further progression of osteoarthritis. As with any species, the introduction of a needle into a joint space has the potential to cause injury, which can result in pain, lameness, or infection. Such adverse events can compromise animal welfare, confound study results, and necessitate additional animals to achieve study objectives. As such, it is imperative to develop proper injection techniques to prevent complications, especially in longitudinal studies that require multiple, repeated intra-articular injections. Using the presented methodology, five guinea pigs received bilateral knee injections under general anesthesia. Seven days after injection, animals were humanely euthanized for analysis of osteoarthritis severity. No adverse events occurred following anesthesia or knee injections, including limping, pain, or infection. X-ray micro-computed tomography analysis of the knee can detect pathologic changes associated with osteoarthritis. Micro-computed tomography data indicates osteoarthritis is more severe in older animals, as indicated by increased bone mineral density and trabecular thickness with age. These results are consistent with histologic changes and Modified Mankin scores, an established and widely used scoring system to assess arthritis severity in these same animals. This protocol can be utilized to refine intra-articular injections in guinea pigs.

Before performing intra-articular injections on live animals, the above protocol was practiced on three rat cadavers to ensure correct injection location. During the practice sessions, 50 &#181;L of 70% new methylene blue dye was injected into both knee joints using the methodology described above. This equates to six practice injections. After injections, the knee joint was dissected by incising through the cranial aspect of the joint space, distal to the patella and through the patellar ligament, to visualize the joint space and verify location of dye deposition. Because new methylene blue dye is a bright blue solution, the location of the dye deposition can be visualized grossly. Representative images from cadaver injections are included in Figure 1.&#160;Figure 1A demonstrates the presence of dye within the joint space, indicative of a correct location and technique of injection. During these practice sessions the knee joint was successfully injected at all six attempts (100% success rate). For the demonstration purposes, examples of incorrect injection location are included in Figure 1B-C. If the injection is administered in the subcutaneous space, fluid will accumulate causing a bleb under the skin as seen in Figure 1B. If the joint space is not injected, there will be no dye present within the knee joint, as seen in Figure 1C.
Each of the 5 guinea pigs underwent a single bilateral knee injection procedure with 1x phosphate buffered saline as part of a pilot experiment to ensure feasibility of injection and subsequent &#181;CT analyses of age-related OA changes. Animals were visually assessed at least once daily following the procedure for overall health and adverse events. None (0%) of these animals experienced adverse events associated with injection, including pain, lameness, or infection. Seven days post injection, animals were placed under a surgical plane of anesthesia and humanely euthanized for knee harvest and subsequent &#181;CT analyses.
As previously published, &#181;CT analysis can be utilized to assess knee changes in guinea pigs, including the ability to measure quantitative changes associated with OA severity over time22,23. In the presented study, &#181;CT was utilized to verify OA changes in guinea pigs of differing ages. These results can be used as baseline data for future studies assessing novel treatments intended to delay OA progression. The methodology described above will allow for standardization of &#181;CT analyses in future studies. The subchondral plate (Figure 2A) and trabecular bone (Figure 2B) have drastically different bone properties. As such, these regions are analyzed separately to yield a more robust result one week after the injection.
Bone mineral density (BMD) is greater in 12-month-old guinea pigs compared to 5- and 9- month olds (Figure 3). The mean subchondral plate BMD was 0.898 g/cm3, 0.952 g/cm3,&#160;and 0.588 g/cm3, in 12-, 9- and 5-month-old guinea pigs, respectively. This demonstrates 1.53 times increase in subchondral BMD for the 12-month old guinea pigs compared to 5-month old. The mean trabecular bone BMD was 0.825 g/cm3, 0.839 g/cm3, 0.427 g/cm3 in 12-, 9- and 5-month-old guinea pigs, respectively. This represents 1.93 times increase in trabecular BMD for the 12-month old guinea pigs compared to the 5-month old. Overall, BMD increases in Dunkin-Hartley guinea pigs as they age from 5 months to 12 months of age (Figure 3).
There were also differences in trabecular thickness between guinea pigs of various ages (Figure 4). For mean trabecular thickness, there is 1.38 times increase for the 12-month-olds (0.558 mm) compared to&#160;the 9-month old (0.403 mm), as well as a 2.48 times increase in the 12-month-old (0.558 mm) compared to the 5-month old (0.225 mm) guinea pigs.Therefore, the mean of the trabecular thickness is increased in Dunkin-Hartley guinea pigs as they age from 5- to 12-months.
Histologic changes and Modified Makin scores, a validated and widely utilized approach for assessing OA changes in 2D, support the &#181;CT findings (Figure 5). Modified Mankin scores increased as the guinea pigs age (Figure 5A). Histologically, signs of OA including proteoglycan loss, hypocellularity, and fissures, increase in prevalence as guinea pigs age from 5- to 12-months of age (Figure 5B-D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66053/66053fig01.jpg" /> 
Figure 1: Representative images of correct and incorrect injection locations. (A) Dissected rat knee with the presence of new methylene blue dye within the joint space. Scale included to the left of the knee for reference. (B) Knee demonstrating a shallow injection, resulting in a bleb forming in the subcutaneous space. (C) Knee from panel B dissected confirming a lack of new methylene blue dye within the joint space. Scale included to the right of the knee for reference. In all images, cranial is at the top of the image and caudal is at the bottom. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66053/66053fig01large.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/66053/66053fig02.jpg" /> 
Figure 2: Segmentation for subchondral plate versus trabecular bone measurement fields. The subchondral plate has different properties from the trabecular bone. Analyzing these regions separately allows for comparisons between different regions throughout the course of OA progression. Region A (red)&#160;depicts the coronal view for the region of the subchondral plate segment used for analysis. Region B (green)depicts the coronal view for the trabecular bone segment used for analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66053/66053fig02large.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/66053/66053fig03.jpg" /> 
Figure 3: Changes in Bone Mineral Density in aging Dunkin-Hartley guinea pigs. Bone mineral density (BMD) was assessed in guinea pigs at 5 (n=2), 9 (n=1), and 12 (n=2) months of age. Measurements were taken at the subchondral plate as well as the trabecular bone for two different measurements. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66053/66053fig03large.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/66053/66053fig04.jpg" /> 
Figure 4: Changes in Trabecular thickness in aging Dunkin-Hartley guinea pigs. Trabecular thickness measurements were taken from 5 (n=2), 9 (n=1), and 12 (n=2) month old guinea pigs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66053/66053fig04large.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/66053/66053fig05.jpg" /> 
Figure 5: Histology analysis of OA changes in Dunkin-Hartley guinea pigs. (A) Modified Mankin scores for histologic samples from 5- (n=2), 9- (n=1), and 12- (n=2) month old guinea pigs. The Modified Mankin score was calculated by adding together individual scores of cartilage structure, cellularity, tidemark, and osteophyte formation. (B) Representative histology image stained with toluidine blue from a 5-month-old guinea pig. (C) Representative histology image stained with toluidine blue from a 9-month-old guinea pig. black *= proteoglycan loss. D. Representative histology image stained with toluidine blue from a 12-month-old guinea pig. black *= fissures; white *= hypocellularity. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66053/66053fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Dunkin-Hartley guinea pigs are an established animal model for osteoarthritis research. Such studies may benefit from intra-articular injections for various reasons, including investigating novel agents or treating disease. We describe a methodology for intra-articular knee injections in Guinea pigs and subsequent micro-computed tomography analysis assessing arthritis-associated knee changes.

Micro-Computed Tomography Analysis of the Knee in Aged Dunkin-Hartley Guinea Pigs after Intra Articular Injection

The purpose of this protocol is to guide researchers in performing a palpation-guided technique of intra-articular knee injection in guinea pigs and ...

micro-computed-tomography-analysis-knee-aged-dunkin-hartley-guinea

Nanyang Technological University

Research

JoVE Journal

Medicine

258 Views.  Medical University of South Carolina. Artrose, of OA, is een degeneratieve gewrichtsaandoening die 32,5 miljoen Amerikaanse volwassenen treft. Vaak verschijnen artrosesymptomen pas nadat de ziekte ernstig is geworden. Het gebrek aan ziektemodificerende medicijnen komt voort uit een slecht begrip van de mechanismen die de ziekte initiëren en aandrijven.Als gevolg hiervan is er een kritieke en voortdurende medische behoefte aan verbeterde middelen voor de behandeling van artrose. Dunkin-Hartley-cavia's zijn een gevestigd ar...

Video: Microcomputertomografie-analyse van de knie bij oude Dunkin-Hartley-cavia's na intra-articulaire injectie

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are intensively and increasingly recruited in septic joints from the early phases of infection and the infiltration is supposed to regress once efficient removal of the pathogens is obtained. The ability to identify in vivo macrophage activity in an infected joint can therefore provide two main applications: early detection of acute synovitis and monitoring of therapy.
In vivo noninvasive detection of macrophages can be performed with magnetic resonance imaging using iron nanoparticles such as ultrasmall superparamagnetic iron oxide (USPIO). After intravascular or intraarticular administration, USPIO are specifically phagocytized by activated macrophages, and, due to their magnetic properties, induce signal changes in tissues presenting macrophage infiltration. A quantitative evaluation of the infiltrate is feasible, as the area with signal loss (number of dark pixels) observed on gradient echo MR images after particles injection is correlated with the amount of iron within the tissue and therefore reflects the number of USPIO-loaded cells.
We present here a protocol to perform macrophage imaging using USPIO-enhanced MR imaging in an animal model of septic arthritis, allowing an initial and longitudinal in vivo noninvasive evaluation of macrophages infiltration and an assessment of therapy action.

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

We demonstrate how to perform macrophage MR imaging using ultrasmall superparamagnetic contrast agent (USPIO) in septic arthritis, allowing an initial and longitudinal in vivo non-invasive evaluation of macrophages infiltration and an assessment of therapy efficacy.

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

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 ...

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, ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...

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 ...

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 ...

Establishment of the KOA Model in Rabbits Using the Modified Videman Method

Application of Acupotomy in a Knee Osteoarthritis Model in Rabbit

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of people with KOA. Among several pathogenic factors, the biomechanical imbalance of the knee joint is one of the main causes of KOA. Acupotomology believes that restoring the mechanical balance of the knee joint is the key to treating KOA. Clinical studies have shown that acupotomy can effectively reduce pain and improve knee mobility by reducing adhesion, contracture of soft tissues, and stress concentration points in muscles and tendons around the knee joint. 
In this protocol, we used the modified Videman method to establish a KOA model by immobilizing the left hindlimb in a straight position. We have outlined the method of operation and the precautions related to acupotomy in detail and evaluated the efficacy of acupotomy in conjunction with the theory of "Modulating Muscles and Tendons to Treat Bone Disorders" through the detection of the mechanical properties of quadriceps femoris and tendon, as well as cartilage mechanics and morphology. The results show that acupotomy has a protective effect on cartilage by adjusting the mechanical properties of the soft tissues around the knee joint, improving the cartilage stress environment, and delaying cartilage degeneration.

Author Spotlight: Investigating the Mechanism of Action of Acupotomy in Treating Knee Osteoarthritis

In this protocol, a knee osteoarthritis model was prepared using the modified Videman method, and the operation procedures and precautions of acupotomy are detailed. The effectiveness of acupotomy has been demonstrated by testing the mechanical properties of quadriceps femoris and tendon and the mechanical and morphological properties of cartilage.

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of ...

Micro-Computed Tomography Analysis of the Knee in Aged Dunkin-Hartley Guinea Pigs after Intra Articular Injection

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