Name: micro-computed tomography analysis of the knee in aged dunkin-hartley guinea pigs after intra articular injection
Uploaded: 2024-08-02T13:00:00
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 m...

<ol>
	<li>Callahan, L. F., Cleveland, R. J., Allen, K. D., Golightly, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Racial/Ethnic,+socioeconomic,+and+geographic+disparities+in+the+epidemiology+of+knee+and+hip+osteoarthritis.">Racial/Ethnic, socioeconomic, and geographic disparities in the epidemiology of knee and hip osteoarthritis.</a> Rheum Dis Clin North Am. 47 (1), 1-20 (2021).</li><li>Mandl, L. 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S., Blanchet, T. J., Morris, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+surgical+destabilization+of+the+medial+meniscus+(DMM)+model+of+osteoarthritis+in+the+129/SvEv+mouse.">The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse.</a> Osteoarthritis Cartilage. 15 (9), 1061-1069 (2007).</li><li>Kuyinu, E. L., Narayanan, G., Nair, L. S., Laurencin, C. 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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+platelet-rich+plasma+injections+versus+single+platelet-rich+plasma+injection+in+early+osteoarthritis+of+the+knee:+An+experimental+study+in+a+guinea+pig+model+of+early+knee+osteoarthritis.">Multiple platelet-rich plasma injections versus single platelet-rich plasma injection in early osteoarthritis of the knee: An experimental study in a guinea pig model of early knee osteoarthritis.</a> Am J Sports Med. 47 (10), 2300-2307 (2019).</li><li>Patel, S., Mishra, N. P., Chouhan, D. K., Nahar, U., Dhillon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondroprotective+effects+of+multiple+PRP+injections+in+osteoarthritis+by+apoptosis+regulation+and+increased+aggrecan+synthesis-+Immunohistochemistry+based+Guinea+pig+study.">Chondroprotective effects of multiple PRP injections in osteoarthritis by apoptosis regulation and increased aggrecan synthesis- Immunohistochemistry based Guinea pig study.</a> J Clin Orthop Trauma. 25, 101762 (2022).</li><li>Cheng, J., Abdi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complications+of+joint,+tendon,+and+muscle+injections.">Complications of joint, tendon, and muscle injections.</a> Tech Reg Anesth Pain Manag. 11 (3), 141-147 (2007).</li><li>Wang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+central+role+for+complement+in+osteoarthritis.">Identification of a central role for complement in osteoarthritis.</a> Nat Med. 17 (12), 1674-1679 (2011).</li><li>Santangelo, K. 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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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#956;-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>&#956;-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&#160;</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...

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 ...

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.

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Medicine

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