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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 cysts^1,2. 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 (µCT) analysis is a powerful tool that allows for quantitative assessment of OA severity¹². µCT 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 µCT, this modality can be used to assess 3D features and perform quantitative analyses of changes associated with OA^15,16,17. µCT 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, µCT 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. µCT 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 OA^19,20. 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 µCT data.

Protokół

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

1. 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.
2. Shave the knee area with an electric razor.
   NOTE: Be careful of the nipples medially.
3. 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.
4. Ensure guinea pig is at appropriate anesthesia depth by lack of toe pinch response.
5. Place sterile ocular lubricant on both eyes to prevent desiccation and injury.
6. Dilute betadine with sterile water to 10%.
7. Dilute 200 proof ethanol with sterile water to 70% ethanol.
8. 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.
9. 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 µL was used.
10. 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.
11. Don surgical gown, hair net, sterile gloves, and mask while performing the injection procedure.
12. Pour 10% betadine onto a cotton ball and wipe both knee areas.
13. Pour 70% ethanol onto a cotton ball and wipe both knee areas.
14. 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.

2. Intra-articular injection

1. Place the guinea pig in supine position for entirety of the procedure.
2. 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.
3. Manually flex the knee to 90°.
4. 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.
5. 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.
6. Inject 50 µL 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.
7. 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.
8. Massage the knee by flexing and extending the joint a few times promote diffusion of the drug within the joint space.
9. Repeat steps 2.1-2.5 once on the contralateral limb with 1x PBS solution.

3. Recovery from intra-articular injection

1. Turn off isoflurane and maintain 100% flow by oxygen until the animal regains consciousness.
2. Place the animal on a heating pad for thermal support until ambulatory.
3. Apply an ice pack to the knee for 30 s with a paper towel as a barrier to help decrease swelling from the injection.
4. 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.

4. Micro computed tomography (µCT) scan

1. For the tissue harvest, establish a surgical plane of anesthesia with 100% oxygen and 5% isoflurane mixture.
2. Confirm a surgical plane of anesthesia with the lack of response to a toe-pinch stimulus. Humanely euthanize the animal via administration of ≥ 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.
3. Harvest both hindlimbs by dissecting the skin away from the surrounding musculature.
4. 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.
5. Place the tissues in neutral buffered formalin solution for 72 h for fixation before performing µCT.
6. Open µCT scan software and place sample with formalin in a compatible container that will fit into the µCT specimen folder while maintaining tissue in the field of vision.
7. Calibrate µCT machine for dark field and light field exposures according to manufacturer recommendations.
8. Scan the sample with Al+Cu filter at 18 µm. Use rotation step 0.7° for 360° with offset camera.
   NOTE: The scan automatically saves.

5. Image processing for evaluating bone microarchitectural parameters

1. Download and install µCT reconstruction software for the reconstruction of µCT images.
2. Select the software folder and double click to open the software.
3. Select one slice from the µCT images by clicking on an image slice.
4. Choose the reconstruction file destination. Select Browse and create a new folder named Recon. The selected file format should be BMP(8).
5. 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.
6. 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.
7. Select Start to begin processing the reconstruction.

6. Collection of microarchitectural data from reconstructed images

1. Download and install Dataviewer.
2. Select VOI and orient the sample to align vertically for easier analysis at a later time.
3. Save edited VOI as a new folder.
4. 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.
5. 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.
6. Select the range of images to analyze, starting with subchondral plate by clicking on the image slice that you want to start with.
7. Select the region of interest for each image to ensure that it is encompassing the bone by clicking on the region of interest tab.
8. Select the Binary Selection tab. Adjust the histogram so that the background and the bone is completely separate.
9. Select the Bone Mineral Density (BMD) tab. Save that data into a new analysis data folder.
10. Select Custom Processing and go to Internal tab.
11. First perform Thresholding and select Automatic Otsu, then Run.
12. Then select Despeckle and choose Remove black speckles, then Run.
13. Repeat Despeckle and choose Remove white speckles, then Run.
14. Choose 3D analysis and select Basic values and Additional values.
15. 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.

Wyniki

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 µL 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...

Dyskusje

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 d...

Materiały

Name	Company	Catalog Number	Comments
200 Proof Ethanol	Decon Laboratories	2701	sterilizing agent
3D.SUITE software	Bruker		μ-CT analyzing software
Betadine Surgical Scrub	Avrio Health	67618-151-16	sterilizing agent
Insulin syringe with needle	Ulticare	91008	to perform injections
Isoflurane	Piramal	803249	anesthesize animal
Neutral Buffered Formalin	Fisher Scientific	23-427098	Fix tissue
Nrecon Software	Bruker		μ-CT reconstruction software
Phosphate Buffered Saline	Cytiva	SH30258.01	control and diluting agent
SkyScan 1176	Bruker		to scan samples