This study was supported by the Zhejiang Natural Science Foundation (grant number LQ20H270009), the Natural Science Foundation of China (grant numbers 82074464 and 82104890), the Zhejiang Traditional Chinese Medical Science Foundation (grant numbers 2020ZA039, 2020ZA096, and 2022ZB137) and the Medical Health Science and Technology Project of Zhejiang Provincial Health Commission (grant number 2016KYA196).

The animal experiments were approved by the Medical Standards and Ethics Committee of Zhejiang University of Traditional Chinese Medicine, which conforms to the China legislation on the use and care of laboratory animals. In the present study, 6 week old male Sprague-Dawley (SD) rats weighing 150-180 g were used. The animals were obtained from a commercial source (see the Table of Materials).
1. Establishment of a full-thickness cartilage defects model in rats
<ol>
	<li>After 1 week of acclimatization to the new environment, randomly and equally divide the rats into two groups (n = 8 rats/group). The rats in the sham group will have the sham surgery, while the rats in the model group will have the experimental surgery involving drilling holes in the femoral trochlear groove. 
	NOTE: Each cage must be covered with sterile corncob paddings (see the Table of Materials) to protect the rats&#39; toes.</li>
	<li>Anesthetize the rats by an intraperitoneal injection (i.p.) of pentobarbital sodium (40 mg/kg). Then, gently press the toes of the rats to confirm adequate anesthesia. Use a vet ointment on the rats&#39; eyes to prevent dryness while under anesthesia. 
	NOTE: The animal surgery needs to be performed in a dedicated operating room using autoclaved surgical instruments. The operators must wear clean laboratory coats, facemasks, head covers, and sterile gloves during the surgery.&#160;Place sterile pads over the surgical area and sterilize all equipment before use. Provide thermal support throughout the procedure.</li>
	<li>Place the rat on the operating table in a supine position, shave the left and right hind limbs, and clean the knee joint area with surgical soap, followed by alternating antiseptic povidone-iodine solution and alcohol three times under sterile conditions. Place a sterile drape over the rat, and expose the disinfected knee joint only.</li>
	<li>Make a 1 cm incision with a scalpel blade (number 11) in the middle of the rat knee joint from top to bottom, and cut the joint capsule and quadriceps femoris tendon along the medial edge of the patella after the superficial dissection. 
	NOTE: The quadriceps femoris tendon is attached to the patella and to the femoral condyle during the flexion of the knee joint13. The groove seen in the joint capsule is the femoral trochlear groove, and the distal femoral condyle forms the medial and lateral condyles.</li>
	<li>Turn the patella to the outside, and flex the tibia and fibula at a 90&#176; angle to fully expose the trochlea of the femoral condyle. Use a 1.6 mm diameter circular drill bit (see the Table of Materials) vertical to the cartilage surface at 4,000 rpm for 10 s to make one full-thickness cartilage defect in the femoral trochlear groove with a depth of 0.1 mm. 
	NOTE: Use saline intermittently to minimize thermal trauma to the surrounding bone tissue during the drilling procedure.</li>
	<li>Wipe the surgical site with cotton balls soaked in a 0.9% saline solution, replace the patella, keep the knee in an extension position, and suture the incision layer by layer with nonabsorbable 4-0 sutures (see Table of Materials).
	<ol>
		<li>Place the animals on heating pads with sternal recumbency, monitor them until they wake up, and then return them to their cages. Inject buprenorphine (0.05 mg/kg) subcutaneously every 8 h three times after the operation for pain relief.</li>
	</ol>
	</li>
	<li>Test the pain-related behavior of all the rats at 3 days, 10 days, and 17 days after the surgery, as described in section 2.</li>
</ol>
2. Mechanical withdrawal threshold (MWT)
NOTE: The MWT of the bilateral posterior plantar of rats was measured by the classical von Frey filament pain measurement method14.
<ol>
	<li>Place the rat in a single plastic chamber (17 cm x 11 cm x 13 cm) on a wire mesh platform (see the Table of Materials), and place the wire mesh base 50 cm above a table. Measure the MWT after 30 min of adaptation.</li>
	<li>Press the von Frey filament (see the Table of Materials) perpendicularly on the plantar surface of each rat&#39;s hind paw, and bend the brush for about 2 s, avoiding the thickest part of the center of the hind paw.</li>
	<li>Gradually increase the stimulus weight from the lowest 4 g until a positive response (paw withdrawal or paw licking) occurs. 
	NOTE: The interval between each stimulation should be more than 1 min. The MWT is defined as three positive responses in five stimulations; record the stimulus weight in grams.</li>
	<li>Calculate the mean values for the sham and model groups according to the recorded minimal stimulus weight in grams.</li>
</ol>
3. Histopathological and immunohistochemical analysis
<ol>
	<li>At 17 and 56 days after surgery, anesthetize the rats with a 40 mg/kg pentobarbital sodium (i.p.), and sacrifice all the rats by withdrawing blood from the heart. Isolate the knees by cutting the bone at the mid-femur and mid-tibia, and dissect the surrounding muscle tissue. Remove the knee joints for histological analysis.</li>
	<li>Fix the knee joints in 20 mL of 10% paraformaldehyde solution for 48 h at room temperature, and then decalcify them with 20 mL of 10% EDTA solution in an orbital shaker for 8 weeks at 4 &#176;C. Change the EDTA solution every day.</li>
	<li>Trim the knee joint to fit the size of the embedding box. Embed the dehydrated knee joints in 100% paraffin15.</li>
	<li>Place the paraffin-embedded knee joints on the holder of a microtome, adjust the angle, and trim the paraffin sample with a blade until the surface is flat.</li>
	<li>Set the thickness of the paraffin slices to 3 &#181;m, and flatten the slices in a water bath at 40 &#176;C.</li>
	<li>Stick the slices on glass slides, place them in a 45 &#176;C baking machine (see the Table of Materials) until they are dry, and store them at room temperature.</li>
	<li>Dewaxing and rehydration: Dewax the slices in an oven at 60 &#176;C for 4 h, and then place the slices successively in 100% xylene (three times), 100% ethanol (two times), 95% ethanol, 80% ethanol, and 75% ethanol for 5 min each time.</li>
	<li>Hematoxylin and eosin (H&#38;E) staining
	<ol>
		<li>Dewax, rehydrate, and wash the slices with double-distilled water for 2 min.</li>
		<li>Stain the slices with 0.5% hematoxylin (see Table of Materials) for 3 min, and wash the slices with double-distilled water until there is no hematoxylin residue on the slice surfaces.</li>
		<li>Immerse the slices in 1% hydrochloric acid alcohol for 3 s, and wash the slices with double-distilled water for 2 min.</li>
		<li>Soak the slices in 1% ammonia water for 10 s, and wash the slices with double-distilled water for 2 min.</li>
		<li>Stain the slices with eosin (see Table of Materials) for 1 min, and wash the slices with double-distilled water until there is no eosin residue on the slice surfaces.</li>
		<li>Immerse the slices in 95% ethanol, 100% ethanol, and 100% xylene (three times) successively for 1 min each time.</li>
		<li>Add a drop of neutral resin (see Table of Materials) to each slice, and seal it with a coverslip.</li>
	</ol>
	</li>
	<li>Safranin O/Fast Freen (SO) staining
	<ol>
		<li>Dewax, rehydrate, and wash the slices with double-distilled water for 2 min.</li>
		<li>Stain the slices with 0.05% Fast Green (see the Table of Materials) for 3 min, and wash the slices with double-distilled water until there is no Fast Green residue on the surface of the slices.</li>
		<li>Immerse the slices in 1% acetic acid solution for 10 s, and wash the slices with double-distilled water for 2 min.</li>
		<li>Stain the slices with 2.5% SO (see Table of Materials) for 2 min, and then wash the slices with double-distilled water until there is no SO residue on the slice surfaces.</li>
		<li>Immerse the slices in 95% ethanol, 100% ethanol, and 100% xylene (three times) successively for 1 min each time.</li>
		<li>Add a drop of neutral resin to each slice, and seal it with a coverslip.</li>
	</ol>
	</li>
	<li>Toluidine blue (TB) staining
	<ol>
		<li>Dewax, rehydrate, and wash the slices with double-distilled water for 2 min.</li>
		<li>Immerse the slices in 1% TB solution (see the Table of Materials) for 2 min, and wash the slices with double-distilled water until there is no toluidine blue residue on the surface of the slices.</li>
		<li>Immerse the slices in 95% ethanol, 100% ethanol, and 100% xylene (three times) successively for 1 min each time.</li>
		<li>Add a drop of neutral resin to each slice, and seal it with a coverslip.</li>
	</ol>
	</li>
	<li>Masson staining
	<ol>
		<li>Dewax, rehydrate, and wash the slices with double-distilled water for 2 min.</li>
		<li>Add Bouin solution (see the Table of Materials) dropwise to the slices, stain them at 37 &#176;C for 2 h, and wash them with double-distilled water until the yellow color on the surface of the slices disappears.</li>
		<li>Stain the slices with celestite blue (see the Table of Materials) for 3 min, and wash the slices with double-distilled water until there is no celestite blue residue on the slice surfaces.</li>
		<li>Stain the slices with hematoxylin for 3 min, and wash the slices with double-distilled water until there is no hematoxylin residue on the slice surfaces.</li>
		<li>Immerse the slices in acidic ethanol for 5 s, and wash the slices with double-distilled water for 2 min.</li>
		<li>Stain the slices with Ponceau fuchsin (see the Table of Materials) for 10 min, and wash the slices with double-distilled water until there is no Ponceau fuchsin residue on the slice surfaces.</li>
		<li>Immerse the slices in phosphomolybdic acid (see the Table of Materials) for 10 min, then immerse them in TB solution for 5 min, and wash the slices with double-distilled water until there is no TB residue on the surface of the slices.</li>
		<li>Immerse the slices in a weak acid solution for 2 min, and wash the slices with double-distilled water for 2 min.</li>
		<li>Immerse the slices in 95% ethanol, 100% ethanol,and 100% xylene (three times) successively for 1 min each time.</li>
		<li>Add a drop of neutral resin to each slice, and seal it with a coverslip.</li>
	</ol>
	</li>
	<li>Observe all the slices under a microscope in a double-blind setting to determine the degree of articular cartilage degeneration according to Mankin&#39;s scoring system16.</li>
	<li>Immunohistochemistry
	<ol>
		<li>Dewax and rehydrate the slices routinely, and wash the slices with PBS for 2 min.</li>
		<li>Immerse the slices in sodium citrate solution, and place the slices in an oven at 60 &#176;C for 4 h to repair the antigen. Wash the slices with PBS three times for 3 min each.</li>
		<li>Immerse the slices in a 0.3% Triton X-100 solution for 10 min, and wash the slices twice with PBS for 3 min each time.</li>
		<li>Block the endogenous peroxidase activity by adding a 3% H2O2 solution in methanol at room temperature for 30 min. Wash the slices twice with PBS for 3 min each time.</li>
		<li>Incubate the sections with 5% goat serum in PBS for 30 min at room temperature to block any non-specific binding. Wash the slices twice with PBS for 3 min each time.</li>
		<li>Add 100 &#181;L of PBS-diluted primary antibodies (anti-col1, 1:50; anti-col3, 1:50; anti-col2, 1:100; and anti-MMP13, 1:100; see the Table of Materials) to each slice, and incubate them overnight at 4 &#176;C. Wash the slices twice with PBS for 3 min each time.</li>
		<li>Incubate each slice with 100 &#181;L of PBS-diluted (1:100) secondary antibody (goat anti-rabbit or goat anti-mouse, see the Table of Materials) at room temperature for 20 min. Wash the slices twice with PBS for 3 min each time.</li>
		<li>Add 100 &#181;L of 3, 3 &#39;-diaminobenzidine (DAB, see the Table of Materials) working solution to each slice.</li>
		<li>Observe and record the appearance time of a brown color under a microscope; the chromogenic reaction turns the epitope sites brown17. Treat the rest of the samples with the same recorded reaction time.</li>
		<li>After the slices turn brown, wash the slices twice with double-distilled water for 3 min each time.</li>
		<li>Re-stain the slices with hematoxylin for 1 min, and wash the slices with double-distilled water for 2 min.</li>
		<li>Immerse the slices in hydrochloric acid for 3 s, and wash the slices with double-distilled water for 2 min.</li>
		<li>Soak the slices in 1% ammonia water for 10 s, and wash the slices with double-distilled water for 2 min.</li>
		<li>Immerse the slices in 95% ethanol, 100% ethanol, and 100% xylene (three times) successively for 1 min each time.</li>
		<li>Add a drop of neutral resin to each slice, and seal it with a coverslip.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This study describes an animal model for mimicking clinical cartilage defects by drilling holes in the femoral trochlear groove of rats (Supplementary Figure&#160;1). After cartilage injury, the excitability or responsiveness of peripheral nociceptors is enhanced, which can result in a decrease in the pain threshold and the enhancement of responsiveness to stimulation18. In preclinical studies, the modeling of cartilage defects in different species of animals has ...

Articular cartilage is a highly differentiated and dense tissue consisting of chondrocytes and extracellular matrix1. The surface layer of articular cartilage is a form of hyaline cartilage, which has a smooth surface, low friction, good strength and elasticity, and excellent mechanical stress tolerance2. The extracellular matrix comprises collagen proteoglycan and water, and type II collagen is the main structural component of the collagen, as it accounts for about 90% of the total collagen3. As no blood vessels or nerves exist in cartilage tissue, it lacks the ability to self-repair after injury4. Therefore, cartilage defects caused by trauma have always been an intractable joint disease in clinics; additionally, this joint disease tends to strike young people, and the global incidence is on the rise5,6. The knee joint is the most common site of cartilage defects, and defects here are accompanied by joint pain, joint dysfunction, and articular cartilage degeneration, eventually leading to knee osteoarthritis (kOA)7. Cartilage defects of the knee joint bring economic and physiological burdens to patients and seriously affect the patients' quality of life8. This disease poses a major and urgent clinical challenge with no imminent solutions. Currently, surgery is the mainstay of treatment for cartilage defects, but its long-term outcome remains unsatisfactory9.
Clinical cartilage defects eventually lead to kOA, and, thus, kOA animal models are commonly used for the pathological study of cartilage defects and drug development. The establishment of animal models is important for understanding the pathophysiological process of cartilage defect repair, which can be used to observe the cartilage regeneration and the alteration between fibrocartilage and hyaline cartilage10. However, commonly used kOA animal models, such as surgical models of anterior cruciate ligament transection (ACLT), destabilization of the medial meniscus (DMM), ovariectomy (OVX), and Hulth, usually need long-term modeling and only allow for pathological and pain evaluations, which poses limitations to the efficiency of drug development11. Besides the surgical models, chemical models, such as monoiodoacetate (MIA) and papain injection, also result in cartilage defects, but the degree of the defect cannot be well managed, and the conditions are far from the clinical reality11. Collision is another approach to model cartilage defects in larger animals, but this method depends on the use of specific instruments and is rarely applied12.
In summary, the existing kOA models are not ideal for studying the pathogenesis of cartilage defects or developing new drugs, and a specific and standardized model for cartilage defects is needed. This study established a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove in rats. Gross observation, pain behavior tests, and histopathological analysis were conducted for model evaluation. Unlike other animal models of kOA, this model has little effect on the rats' general condition. This modeling approach is accessible, can be well managed, and supports the understanding of progression from cartilage defects to kOA and the development of effective therapeutics. This model can also be used for testing therapies that prevent kOA by healing defects in pre-osteoarthritic joints.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3, 3 &#39;-diaminobenzidine &#160;</td>
			<td>Hangzhou Zhengbo Biotechnology Co., Ltd.</td>
			<td>ZLI-9019</td>
			<td>The dye for IHC staining</td>
		</tr>
		<tr>
			<td>Anti-Collagen III antibody</td>
			<td>Novus</td>
			<td>NB600-594</td>
			<td>Primary antibody for IHC</td>
		</tr>
		<tr>
			<td>Anti-Collagen II antibody</td>
			<td>Abcam (UK)</td>
			<td>34712</td>
			<td>Primary antibody for IHC</td>
		</tr>
		<tr>
			<td>Anti-Collagen I antibody</td>
			<td>Novus</td>
			<td>NB600-408</td>
			<td>Primary antibody for IHC</td>
		</tr>
		<tr>
			<td>Bouin solution</td>
			<td>Shanghai Yuanye Technology Co., Ltd.</td>
			<td>R20381</td>
			<td>The dye for Masson staining</td>
		</tr>
		<tr>
			<td>Celestite blue</td>
			<td>Shanghai Yuanye Technology Co., Ltd.</td>
			<td>R20381</td>
			<td>The dye for Masson staining</td>
		</tr>
		<tr>
			<td>Corncob paddings &#160;</td>
			<td>Xiaohe Technology Co., Ltd&#160;</td>
			<td></td>
			<td>Bedding for animal&#160;</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>Sigma-Aldrich</td>
			<td>861006</td>
			<td>The dye for HE staining</td>
		</tr>
		<tr>
			<td>Fast Green FCF</td>
			<td>Sigma-Aldrich</td>
			<td>F7252</td>
			<td>The dye for SO staining</td>
		</tr>
		<tr>
			<td>Goat anti-mouse antibody</td>
			<td>ZSGQ-BIO (Beijing, China)</td>
			<td>PV-9002</td>
			<td>Secondary antibody for IHC</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit antibody</td>
			<td>ZSGQ-BIO (Beijing, China)</td>
			<td>PV-9001</td>
			<td>Secondary antibody for IHC</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Sigma-Aldrich</td>
			<td>H3163</td>
			<td>The dye for HE staining</td>
		</tr>
		<tr>
			<td>Masson</td>
			<td>Shanghai Yuanye Technology Co., Ltd.</td>
			<td>R20381</td>
			<td>The dye for Masson staining</td>
		</tr>
		<tr>
			<td>Microdrill</td>
			<td>Rwd Life Science Co., Ltd</td>
			<td>78001</td>
			<td>Equipment for surgery</td>
		</tr>
		<tr>
			<td>MMP13</td>
			<td>Cell Signaling Technology, Inc. (Danvers, MA, USA)</td>
			<td>69926</td>
			<td>Primary antibody for IHC</td>
		</tr>
		<tr>
			<td>Modular tissue embedding center</td>
			<td>Thermo Fisher Scientific (USA)</td>
			<td>EC 350</td>
			<td>Produce paraffin blocks</td>
		</tr>
		<tr>
			<td>Neutral resin</td>
			<td>Hangzhou Zhengbo Biotechnology Co., Ltd.</td>
			<td>ZLI-9555</td>
			<td>Seal for IHC</td>
		</tr>
		<tr>
			<td>Nonabsorbable suture</td>
			<td>Hangzhou Huawei Medical Supplies Co.,Ltd.</td>
			<td>4-0</td>
			<td>Equipment for surgery</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium&#160;</td>
			<td>Hangzhou Zhengbo Biotechnology Co., Ltd.</td>
			<td>WBBTN5G</td>
			<td>Anesthetized animal</td>
		</tr>
		<tr>
			<td>phosphomolybdic acid&#160;</td>
			<td>Shanghai Yuanye Technology Co., Ltd.</td>
			<td>R20381</td>
			<td>The dye for Masson staining</td>
		</tr>
		<tr>
			<td>Ponceau fuchsin</td>
			<td>Shanghai Yuanye Technology Co., Ltd.</td>
			<td>R20381</td>
			<td>The dye for Masson staining</td>
		</tr>
		<tr>
			<td>Rotary and Sliding Microtomes</td>
			<td>Thermo Fisher Scientific (USA)</td>
			<td>HM325</td>
			<td>Precise paraffin sections</td>
		</tr>
		<tr>
			<td>Safranin-O</td>
			<td>Sigma-Aldrich</td>
			<td>S2255</td>
			<td>The dye for SO staining</td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Shanghai Lianhui Medical Supplies Co., Ltd.</td>
			<td>11</td>
			<td>Equipment for surgery</td>
		</tr>
		<tr>
			<td>Sodium citrate solution (20x)</td>
			<td>Hangzhou Haoke Biotechnology Co., Ltd.</td>
			<td>HK1222</td>
			<td>Antigen retrieval for IHC</td>
		</tr>
		<tr>
			<td>Sprague Dawley (SD) rats</td>
			<td>&#160;Shanghai Slake Experimental Animal Co., Ltd.</td>
			<td>SD</td>
			<td>Experimental animal</td>
		</tr>
		<tr>
			<td>Tissue-Tek VIP 5 Jr</td>
			<td>Sakura (Japan)</td>
			<td></td>
			<td>Vacuum Infiltration Processor</td>
		</tr>
		<tr>
			<td>Toluidine Blue</td>
			<td>Sigma-Aldrich</td>
			<td>89640</td>
			<td>The dye for TB staining</td>
		</tr>
		<tr>
			<td>Von Frey filament</td>
			<td>UGO Basile (Italy)&#160;</td>
			<td>37450-275</td>
			<td>Equipment for MWT assay</td>
		</tr>
		<tr>
			<td>Wire mesh platform&#160;</td>
			<td>Shanghai Yuyan Instruments Co.,Ltd.</td>
			<td></td>
			<td>Equipment for MWT assay</td>
		</tr>
	</tbody>
</table>

development and evaluation of a rat model of full-thickness cartilage defects

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired movement, and eventually, knee osteoarthritis (kOA). However, there is little effective treatment for cartilage defects or even kOA. Animal models are important for developing therapeutic drugs, but the existing models for cartilage defects are unsatisfactory. This work established a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats, and the subsequent pain behavior and histopathological changes were used as readout experiments. After surgery, the mechanical withdrawal threshold was decreased, chondrocytes at the injured site were lost, matrix metalloproteinase MMP13 expression was increased, and type II collagen expression decreased, consistent with the pathological changes observed in human cartilage defects. This methodology is easy and simple to perform and enables gross observation immediately after the injury. Furthermore, this model can successfully mimic clinical cartilage defects, thus providing a platform for studying the pathological process of cartilage defects and developing corresponding therapeutic drugs.

In this work, a rat model of FTCD was established by drilling holes in the femoral trochlear groove and detecting the subsequent pain behavior and histopathological changes. As shown in Figure 1, 3 days after modeling, compared with the sham group, the MWT of rats in the model group was significantly reduced, suggesting hyperalgesia caused by the FTCD. At 17 days after modeling, the mechanical withdrawal threshold of the rats in the model group remained at a low level, indicating that the pa...

Watch this Scientific Journal Video about Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects at JoVE.com

Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects

This protocol establishes a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats and measuring the subsequent pain behavior and histopathological changes.

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired ...

development-evaluation-rat-model-full-thickness-cartilage

Research

JoVE Journal

Medicine

1.5K Views.  Zhejiang Chinese Medical University. This protocol establishes a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats and measuring the subsequent pain behavior and histopathological changes.

Video: Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

A Multicenter MRI Protocol for the Evaluation and Quantification of Deep Vein Thrombosis

We evaluated a magnetic resonance venography (MRV) approach with gadofosveset to quantify total thrombus volume changes as the principal criterion for treatment efficacy&#160;in a multicenter randomized study comparing edoxaban monotherapy with a heparin/warfarin regimen for acute, symptomatic lower extremities deep vein thrombosis (DVT) treatment. We also used a direct thrombus imaging approach (DTHI, without the use of a contrast agent) to quantify fresh thrombus. We then sought to evaluate the reproducibility of the analysis methodology and applicability of using 3D magnetic resonance venography and direct thrombus imaging for the quantification of DVT in a multicenter trial setting. From 10 randomly selected subjects participating in the edoxaban Thrombus Reduction Imaging Study (eTRIS), total thrombus volume in the entire lower extremity deep venous system was quantified bilaterally. Subjects were imaged using 3D-T1W gradient echo sequences before (direct thrombus imaging, DTHI) and 5 min after injection of 0.03 mmol/kg of gadofosveset trisodium (magnetic resonance venography, MRV). The margins of the DVT on corresponding axial, curved multi-planar reformatted images were manually delineated by two observers to obtain volumetric measurements of the venous thrombi. MRV was used to compute total DVT volume, whereas DTHI was used to compute volume of fresh thrombus. Intra-class correlation (ICC) and Bland Altman analysis were performed to compare inter and intra-observer variability of the analysis. The ICC for inter and intra-observer variability was excellent (0.99 and 0.98, p &#60;0.001, respectively) with no bias on Bland-Altman analysis for MRV images. For DTHI images, the results were slightly lower (ICC = 0.88 and 0.95 respectively, p &#60;0.001), with bias for inter-observer results on Bland-Altman plots. This study showed feasibility of thrombus volume estimation in DVT using MRV with gadofosveset trisodium, with good intra- and inter-observer reproducibility in a multicenter setting.

The goal of this study is to use magnetic resonance venography with long-circulating gadolinium-based contrast agent and direct thrombus imaging for quantitative evaluation of DVT volume in a multicenter, clinical trial setting. Inter- and intra-observer variability assessments were conducted, and reproducibility of the protocol was determined.

We evaluated a magnetic resonance venography (MRV) approach with gadofosveset to quantify total thrombus volume changes as the principal criterion for ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained using laser Doppler flowmetry (LDF). This paper demonstrates a closed skull preparation that allows cerebral blood flow to be assessed without penetrating the skull or installing a chamber or cerebral window. To evaluate autoregulatory mechanisms, a model of controlled blood pressure reduction via graded hemorrhage can be utilized while simultaneously employing LDF. This enables the real time tracking of the relative changes in the blood flow in response to reductions in arterial blood pressure produced by the withdrawal of circulating blood volume. This paradigm is a valuable approach to study cerebral blood flow autoregulation during reductions in arterial blood pressure and, with minor modifications in the protocol, is also valuable as an experimental model of hemorrhagic shock. In addition to evaluating autoregulatory responses, LDF can be used to monitor the cortical blood flow when investigating metabolic, myogenic, endothelial, humoral, or neural mechanisms that regulate cerebral blood flow and the impact of various experimental interventions and pathological conditions on cerebral blood flow.

This article demonstrates the use of laser Doppler flowmetry to evaluate the ability of the cerebral circulation to autoregulate its blood flow during reductions in arterial blood pressure.

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained ...

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, ...

Development and Evaluation of 3D-Printed Cardiovascular Phantoms for Interventional Planning and Training

Catheter-based interventions are standard treatment options for cardiovascular pathologies. Therefore, patient-specific models could help training physicians&#39; wire-skills as well as improving planning of interventional procedures. The aim of this study was to develop a manufacturing process of patient-specific 3D-printed models for cardiovascular interventions.
To create a 3D-printed elastic phantom, different 3D-printing materials were compared to porcine biological tissues (i.e., aortic tissue) in terms of mechanical characteristics. A fitting material was selected based on comparative tensile tests and specific material thicknesses were defined. Anonymized contrast-enhanced CT-datasets were collected retrospectively. Patient-specific volumetric models were extracted from these datasets and subsequently 3D-printed. A pulsatile flow loop was constructed to simulate the intraluminal blood flow during interventions. Models&#39; suitability for clinical imaging was assessed by x-ray imaging, CT, 4D-MRI and (Doppler) ultrasonography. Contrast medium was used to enhance visibility in x-ray-based imaging. Different catheterization techniques were applied to evaluate the 3D-printed phantoms in physicians&#39; training as well as for pre-interventional therapy planning.
Printed models showed a high printing resolution (~30 &#181;m) and mechanical properties of the chosen material were comparable to physiological biomechanics. Physical and digital models showed high anatomical accuracy when compared to the underlying radiological dataset. Printed models were suitable for ultrasonic imaging as well as standard x-rays. Doppler ultrasonography and 4D-MRI displayed flow patterns and landmark characteristics (i.e., turbulence, wall shear stress) matching native data. In a catheter-based laboratory setting, patient-specific phantoms were easy to catheterize. Therapy planning and training of interventional procedures on challenging anatomies (e.g., congenital heart disease (CHD)) was possible.
Flexible patient-specific cardiovascular phantoms were 3D-printed, and the application of common clinical imaging techniques was possible. This new process is ideal as a training tool for catheter-based (electrophysiological) interventions and can be used in patient-specific therapy planning.

Here we present development of a mock circulation setup for multimodal therapy evaluation, pre-interventional planning, and physician-training on cardiovascular anatomies. With the application of patient-specific tomographic scans, this setup is ideal for therapeutic approaches, training, and education in individualized medicine.

Catheter-based interventions are standard treatment options for cardiovascular pathologies. Therefore, patient-specific models could help training ...

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 ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.