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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Research

JoVE Journal

Medicine

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample3-5. One method which circumvents these issues is micro-Computed Tomography (&mu;CT). &mu;CT imaging can scan at resolutions &lt;10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)6-11. However, imaging vessels by ex vivo &mu;CT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by &mu;CT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for &mu;CT imaging.
Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) 12-14, or ex vivo via a Langendorff perfusion system 15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network &#x2013; arteries, capillaries, and veins alike.

Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (&mu;CT) imaging.

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Cochlear Implantation in the Guinea Pig

Cochlear implants are highly efficient devices that can restore hearing in subjects with profound hearing loss. Due to improved speech perception outcomes, candidacy criteria have been expanded over the last few decades. This includes patients with substantial residual hearing that benefit from electrical and acoustical stimulation of the same ear, which makes hearing preservation during cochlear implantation an important issue. Electrode impedances and the related issue of energy consumption is another major research field, as progress in this area could pave the way for fully implantable auditory prostheses. To address these issues in a systematic way, adequate animal models are essential. Therefore, the goal of this protocol is to provide an animal model of cochlear implantation, which can be used to address various research questions. Due to its large tympanic bulla, which allows easy surgical access to the inner ear, as well as its hearing range which is relatively similar to the hearing range of humans, the guinea pig is a commonly used species in auditory research. Cochlear implantation in the guinea pig is performed via a retroauricular approach. Through the bullostomy a cochleostomy is drilled and the cochlear implant electrode is inserted into the scala tympani. This electrode can then be used for electrical stimulation, determination of electrode impedances and the measurement of compound action potentials of the auditory nerve. In addition to these applications, cochlear implant electrodes can also be used as drug delivery devices, if a topical delivery of pharmaceutical agents to the cells or fluids of the inner ear is intended.

The goal of this protocol is to provide an animal model of cochlear implantation, which can be used to address a multitude of research questions. Potential applications include the evaluation of pharmaceutical interventions or electrical stimulation for beneficial effects on hearing thresholds or electrode impedances.

Cochlear implants are highly efficient devices that can restore hearing in subjects with profound hearing loss. Due to improved speech perception ...

Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit patients. Despite treatment advances over the last decades, ARDS patients still suffer mortality rates between 35 and 40%. There is still a need for further research to improve the outcome of patients suffering from ARDS. One problem is that no single animal model can mimic the complex pathomechanism of the acute respiratory distress syndrome, but several models exist to study different parts of it. Oleic acid injection (OAI)-induced lung injury is a well-established model for studying ventilation strategies, lung mechanics and ventilation/perfusion distribution in animals. OAI leads to severely impaired gas exchange, deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The disadvantage of this model is the controversial mechanistic relevance of this model and the necessity for central venous access, which is challenging especially in smaller animal models. In summary, OAI-induced lung injury leads to reproducible results in small and large animals and hence represents a well-suited model for studying ARDS. Nevertheless, further research is necessary to find a model that mimics all parts of ARDS and lacks the problems associated with the different models existing today.

In this article, we present a protocol to induce acute lung injury in pigs by central-venous injection of oleic acid. This is an established animal model for studying the acute respiratory distress syndrome (ARDS).

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit ...

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 ...

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 ...

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 &#181;m) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Here, we present a protocol to obtain high resolution micro computed tomography images of healthy and pathological large mammalian whole hearts with collagen-selective contrast enhancement.

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...