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

Osteoarthritis, or OA, is a degenerative joint disease that affects 32.5 million US adults. Often, OA symptoms appear only after the disease has become severe. The lack of disease-modifying drugs stems from a poor understanding of the mechanisms initiating and driving the disease.

As a result, there's a critical and ongoing medical need for improved agents for the treatment of OA.Dunkin-Hartley guinea pigs are an established OA animal model, as they naturally develop this disease, offering a robust model for investigating the effects of novel therapeutics administered by intraarticular injection on disease progression. Because mineralized bone has excellent contrast on micro-CT, this modality can be used to assess 3D features and perform quantitative analyses of changes associated with OA.Two particularly relevant quantitative measurements include bone mineral density and trabecular thickness, as both increase throughout the progression of the disease. The described methodology of intraarticular injections in guinea pigs and subsequent analysis of micro-CT data will help to reduce study variability and increase efficiency of data analyses.

Shave the knee with an electric razor. Anesthetize the animal and confirm an adequate depth of anesthesia by lack of toe pinch response. Place sterile ocular lubricant in both eyes to prevent desiccation and injury.

Clean the injection site three times using a circular motion and alternate by using the diluted scrub and alcohol solutions. Sterile gloves should be utilized. This video includes the use of autoclave nitro gloves.

Aseptic technique should be utilized throughout the procedure. Flex the knee to 90 degrees, move finger distal to the patella to locate the groove of the distal aspect of joint space by flexing and extending the hind limb. The tibia can be felt as the bony structure distal to the patella.

Once the location of the tibia and patella are determined, the joint is between them. Insert the insulin needle carefully in the midline distal to the patella within the joint space. The needle should be inserted one to two millimeters below the skin.

Inject the volume slowly. Massage the knee by flexing and extending the joint a few times to ensure proper distribution of the drug. Place samples with formalin in a compatible container.

Calibrate the micro-CT machine for dark field and light field exposures, according to manufacturer recommendations. Scan sample with aluminum copper filter at 18 microns. Use rotation step 0.7 degrees for 360 degrees with offset camera.

Select one slice from the micro-CT images. Check misalignment compensation. Under settings, apply smoothing, beam hardening, CS rotation, and ring artifact.

Select Start to begin the reconstruction. Select VOI and orient the sample to align vertically for easier analysis later on. Save edited VOI as a new folder.

Select the range of images to analyze, starting with the subchondral plate. Splitting the analysis of subchondral plate in trabecular bone is recommended because they have different bony features. Select the region of interest for each image to ensure that it is encompassing the bone.

Select binary selection. Adjust the histogram so that the background in the bone is completely separate. Select the bone mineral density tab.

Save that data into a new analysis data folder. Select custom processing. First, perform thresholding and select Automatic then run.

Then select Despeckle and choose Remove black speckles. Repeat Despeckle, and choose Remove white speckles. Choose 3D analysis and select basic values and additional values.

A shows a dissected knee with a presence of methylene blue dye within the joint space, showing correct injection. B is a shallow injection resulting in a bleb forming in the subcutaneous space. C shows the lack of methylene blue dye within the joint space, showing incorrect injection.

It can be helpful to split up the analysis for the subchondral plate, which is shown in A and the trabecular bone, which is shown in B, as sometimes they have different parameters for different bone analysis. For bone mineral density, we see that there's an increase in mean of the 12 month old guinea pigs compared to the five month old guinea pigs, and this is true in the subchondral plate as well as the trabecular bone, which shows that bone mineral density increases over time. In addition for trabecular thickness, we see that there's an increase amount of thickness in millimeters between the 12 month old guinea pig and the nine month old guinea pig and the five month old guinea pig, which shows that trabecular thickness is increasing over time.

Modified Mankin scores increase as the guinea pigs age, as seen in figure A.Figures B through D show toluidine blue stains. B is a five month old guinea pig, C is a nine month old guinea pig, and D is a 12 month old guinea pig. The black asterisk in figure C shows proteoglycan loss, while the black asterisk in figure D shows fissures, and the white asterisk in figure D shows hypocellularity.

Currently the only cure for severe OA is replacement, which is costly, invasive, and can result in patient morbidity and mortality. As a result, there's a dire need for continuing to research with animal models of OA and the sustained development on novel therapeutics. Although intraarticular injections are integral to guinea pig OA studies, this procedure has yet to be systematically detailed.

Proper injection procedures paramount to ensuring animal welfare and reducing overall animal numbers, particularly for studies requiring multiple injections over many months. To systematically assess OA-related changes, we describe micro-CT analyses. Micro-CT provides a more in-depth assessment of OA changes than histologic review of the scans in an entire sample instead of one or few tissue sections.

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 OA.Furthermore, 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.