Name: PET/CT With 68Ga -NOTA-FAP-2286 for Imaging of Tendon Injuries in Rat Achilles Tendon Injury Models (Video) | JoVE Journal
Uploaded: 2025-06-06T12:50:00.000+00:00
Duration: 4 min 5 s
Description: Learn FAP-targeted PET/CT imaging using [68Ga]-NOTA-FAP-2286 for detecting tendon injuries in rat Achilles tendon models. This protocol enables precise localization and sensitive assessment of tendon damage.

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5 minutes

The study was funded Zhejiang&#160;Medical and Health Science and Technology&#160;Program (grant No.2023KY694), Zhejiang&#160;Natural Science Foundation (grant number: LTG23H180014).

All animal experiments were performed in accordance with the ethical standards for animal experimentation by the First Affiliated Hospital of Zhejiang University School of Medicine. (Reference Number: 20241008). 68Ga is a radioactive element that emits positrons from decay, which quickly combine with the surrounding electrons to release gamma rays. Gamma rays can penetrate skin, posing a risk of radiation damage to the body. All experimental personnel must undergo radiation safety and protection training before conducting related experiments. During the experiments, it is necessary to wear radiation dosimeters, shielding instruments, and other protective equipment. The radioactive waste produced in the experiment needs to be appropriately disposed of after the experiment.
1. Preparation and modeling process of animal models
<ol>
	<li>Preparation
	<ol>
		<li>Obtain male Sprague-Dawley (SD) rats (6-8 weeks old, ~250 g ,n = 8). Acclimate them under standard laboratory conditions with ad libitum access to food and water&#160;for 7 days. Use them to establish the models of Achilles tendon injury and rupture.</li>
		<li>Randomly divide the rats into two groups: group one: Achilles tendon injury model (n = 4); group two: Achilles tendon injury model (n = 4).</li>
	</ol>
	</li>
	<li>Modeling process
	<ol>
		<li>Prepare the instruments: scalpel, hemostatic forceps, standard forceps, isoflurane, oxygen, alcohol swabs, iodophor, fixation plate, and synthetic monofilament 4-0 sutures.</li>
		<li>Anesthetize the rat in an induction chamber with an isoflurane&#8211;oxygen mixture (1:1), using 2&#8211;3% isoflurane delivered in oxygen at a flow rate of 1.0&#8211;1.5 L/min, followed by maintenance anesthesia with 1&#8211;2% isoflurane at 0.5&#8211;1.0 L/min. Secure the unconscious rat onto a fixation board and maintain anesthesia using a gas mask delivering a continuous supply of isoflurane-oxygen.</li>
		<li>Apply depilatory cream evenly to the right limb, wait for 3 min, and remove hair and residual cream with a gauze. Sterilize the instruments by an autoclave or hot bead sterilization. Disinfect the surgical area 3x with alternating rounds of chlorhexidine/iodine-based scrub and alcohol.</li>
		<li>Establish the Achilles tendon injury model.
		<ol>
			<li>Use a scalpel to make a longitudinal incision to expose the Achilles tendon.</li>
			<li>Compress the tendon using hemostatic forceps until it is flattened.</li>
			<li>Close the incision with synthetic monofilament 4-0 sutures.</li>
		</ol>
		</li>
		<li>Establish the model of Achilles tendon rupture.
		<ol>
			<li>Follow steps 1.2.4.1 and 1.2.4.2 to expose the Achilies tendon.</li>
			<li>Grasp the Achilles tendon using hemostatic forceps and create a full-thickness midline incision using surgical scissors (Figure 1).</li>
			<li>Suture the severed tendon and close the skin incision.</li>
		</ol>
		</li>
		<li>Monitor the rats for signs of distress and ensure proper wound healing. Provide post-operative analgesia: Meloxicam: 1&#8211;2 mg/kg SC or PO q24h for 2&#8211;3 days. Perform PET imaging 1 week post surgery.</li>
	</ol>
	</li>
</ol>
2. Synthesis of 68 Ga-NOTA-FAP2286
NOTE: 68Ga (half-life: 68 min) is obtained from a 67Ge/68Ga generator. Confirm prior to animal administration that the synthesized 68Ga-NOTA-FAP2286 is at an appropriate pH, is sterile, and meets basic quality requirements suitable for animal use.
<ol>
	<li>Prepare 50 &#181;g of the precursor, dissolve it in 800 &#181;L of anhydrous acetonitrile, and add 3 mL of 0.1 M sodium acetate buffer solution to adjust the pH to ~4. Elute the 67Ge/68Ga generator (5 mL/3 min; make sure there are no air bubbles in the syringe).</li>
	<li>Draw 5 mL of 0.1 M hydrochloric acid using a silica gel-headed syringe and inject the hydrochloric into the 67Ge/68Ga generator to replace the 68Ga. Elute 68Ga (&#8764;17 mci) into a 10 mL glass vial 
	NOTE: Avoid contact of the hydrochloric acid with metal.</li>
	<li>Mix the dissolved precursor (NOTA-FAP-2286, CAS Registry Number: 2583823-71-4) and eluted 68Ga and place them in a heater at 35 &#176;C for 20 min (the labeling reaction is shown in Figure 2).</li>
	<li>To purify the product, preactivate the C18 column with 10 mL of ethanol and 10 mL of water. Place the product on the C18 column and rinse it with 1 mL of 50% ethanol to obtain the final product.</li>
	<li>Evaporate the ethanol. Heat the final product bottle at 80 &#176;C for ~20 min to remove excess ethanol from the solution by evaporation.</li>
	<li>Perform a quality control check using HPLC: Mobile phase: A (0.1% H3PO4 Aqueous solution), 72%-52%, 0-20 min; B (CH3CN), 28%-48%, 0-20 min; Flow rate: 3 mL/min.</li>
</ol>
3. Small animal PET imaging
<ol>
	<li>Secure the rat in a restrainer. Administer 500 &#181;ci of 68GA-NOTA-FAP2286 via the tail vein into both groups&#39; rats.</li>
	<li>Induce anesthesia using isoflurane in an induction chamber.</li>
	<li>Position the fully anesthetized rat in the scanning field with the Achilles tendon centred in the field of view, and maintain anesthesia during scanning using isoflurane (Figure 3).</li>
	<li>Perform small animal PET scanning and obtain Imaging results.
	<ol>
		<li>Turn on the computer connected to the small animal PET system. Click on CT Acquisition to initiate the preheating process of the CT system.</li>
		<li>Once the preheating is complete, select the scanning protocol and open the corresponding scanning workflow (the scan duration is preset to 10 min).</li>
		<li>Click on Scout View to confirm the positioning of the Achilles tendon in the center of the scanning field. If the position is correct, click Start Workflow to perform the PET and CT scans.
		<ol>
			<li>Use the following parameters : NCT bed view:0-36.2-76.4 mm, CT scan time:60 s/one bed. PET: Acquire by time: 600 s; 511 KeV Photopeak Energy Level; 350 Kev Lower-Level Discrimination; 650 KeV Upper-Level Discrimination; 3.432 ns Timing Window.</li>
		</ol>
		</li>
		<li>After the scan is completed, click on Reconstruction to reconstruct the scan files and obtain the imaging results. Use the Research Workplace for postprocessing of the images.</li>
	</ol>
	</li>
</ol>

<ol><li>de Morree, A., Rando, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+adult+stem+cell+quiescence+and+its+functions+in+the+maintenance+of+tissue+integrity%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">Regulation of adult stem cell quiescence and its functions in the maintenance of tissue integrity.</a> Nat Rev Mol Cell Biol. 24 (5), 334-354 (2023).</li>
<li>Kratochwil, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((68GaFAPI+PET%2FCT%3A+Tracer+uptake+in+28+different+kinds+of+cancer%5BTitle%5D)+AND+%22J+Nucl+Med%22%5BJournal%5D)">68GaFAPI PET/CT: Tracer uptake in 28 different kinds of cancer.</a> J Nucl Med. 60 (6), 801-805 (2019).</li>
<li>AndarawisPuri, N., Flatow, E. L., Soslowsky, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tendon+basic+science%3A+Development%2C+repair%2C+regeneration%2C+and+healing%5BTitle%5D)+AND+%22J+Orthop+Res%22%5BJournal%5D)">Tendon basic science: Development, repair, regeneration, and healing.</a> J Orthop Res. 33 (6), 780-784 (2015).</li>
<li>Weber, S., Chahal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Management+of+rotator+cuff+injuries%5BTitle%5D)+AND+%22J+Am+Acad+Orthop+Surg%22%5BJournal%5D)">Management of rotator cuff injuries.</a> J Am Acad Orthop Surg. 28 (5), e193-e201 (2020).</li>
<li>Maffulli, N., Longo, U. G., Maffulli, G. D., Khanna, A., Denaro, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Achilles+tendon+ruptures+in+elite+athletes%5BTitle%5D)+AND+%22Foot+Ankle+Int%22%5BJournal%5D)">Achilles tendon ruptures in elite athletes.</a> Foot Ankle Int. 32 (1), 9-15 (2011).</li>
<li>Figueroa, D., Figueroa, F., Calvo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patellar+tendinopathy%3A+Diagnosis+and+treatment%5BTitle%5D)+AND+%22J+Am+Acad+Orthop+Surg%22%5BJournal%5D)">Patellar tendinopathy: Diagnosis and treatment.</a> J Am Acad Orthop Surg. 24 (12), e184-e192 (2016).</li>
<li>Cutts, S., Gangoo, S., Modi, N., Pasapula, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tennis+elbow%3A+A+clinical+review+article%5BTitle%5D)+AND+%22J+Orthop%22%5BJournal%5D)">Tennis elbow: A clinical review article.</a> J Orthop. 17, 203-207 (2020).</li>
<li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibroblast+activation+protein-targeted+PET%2FCT+with+Al+18+FNODAFAPI04+for+in+vivo+imaging+of+tendon+healing+in+rat+Achilles+tendon+injury+models%5BTitle%5D)+AND+%22Am+J+Sports+Med%22%5BJournal%5D)">Fibroblast activation protein-targeted PET/CT with Al 18 FNODAFAPI04 for in vivo imaging of tendon healing in rat Achilles tendon injury models.</a> Am J Sports Med. 51 (14), 3790-3801 (2023).</li>
<li>Lindner, T., Giesel, F. L., Kratochwil, C., Serfling, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Radioligands+targeting+fibroblast+activation+protein+%28FAP%29%5BTitle%5D)+AND+%22Cancers+%28Basel%29%22%5BJournal%5D)">Radioligands targeting fibroblast activation protein (FAP).</a> Cancers (Basel). 13 (22), 5744(2021).</li>
<li>Chakravarty, R., Song, W., Chakraborty, S., Cai, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibroblast+activation+protein+%28FAP%29targeted+radionuclide+therapy%3A+which+ligand+is+the+best%5BTitle%5D)+AND+%22Eur+J+Nucl+Med+Mol+Imaging%22%5BJournal%5D)">Fibroblast activation protein (FAP)targeted radionuclide therapy: which ligand is the best.</a> Eur J Nucl Med Mol Imaging. 50 (10), 2935-2939 (2023).</li>
<li>Hotta, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nononcologic+incidental+uptake+on+FAPI+PET%2FCT+imaging%5BTitle%5D)+AND+%22Br+J+Radiol%22%5BJournal%5D)">Nononcologic incidental uptake on FAPI PET/CT imaging.</a> Br J Radiol. 96 (1142), 20220463(2023).</li>
<li>Bughda, R., Dimou, P., D'Souza, R. R., Klampatsa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibroblast+activation+protein+%28FAP%29targeted+CART+cells%3A+Launching+an+attack+on+tumor+stroma%5BTitle%5D)+AND+%22Immunotargets+Ther%22%5BJournal%5D)">Fibroblast activation protein (FAP)targeted CART cells: Launching an attack on tumor stroma.</a> Immunotargets Ther. 10, 313-323 (2021).</li>
</ol>

The authors have no conflicts of interest to disclose.

The images show remarkable differences between the right and left sides. The small animal PET scans clearly illustrate the differences between the normal and injured/ruptured Achilles tendons. These differences can be attributed to the accumulation of FAPs on the injured/ruptured side leading to increased uptake of imaging agent. Imaging experiments along with measurements of SUVmax and SUVmean clearly demonstrate the contrast between the injured/ruptured Achilles tendon and the normal Achilles tend...

Fibroblasts are ubiquitously distributed across nearly all tissues and typically reside in a quiescent state. Upon disruption of tissue integrity, fibroblasts become activated and migrate to the injury site, orchestrating the repair process1. Once the repair is complete, fibroblasts generally revert to their quiescent state; however, in conditions of chronic inflammation or fibrosis, they may remain persistently activated. FAP is a transmembrane protein expressed on the surfaces of activated fibroblasts. Recent studies have demonstrated a highly promising non-invasive method for tracking FAP to identify various important tumor entities, including breast, lung, and colorectal cancers2. This approach could be explored further for its potential in diagnosing tendon injuries.
Tendon injuries constitute a significant musculoskeletal issue, comprising approximately 30% of all musculoskeletal-related consultations in general medical practice3. These injuries are prevalent across various age groups and demographics, with a notably higher incidence among individuals aged 30 years or older, occupational groups engaged in repetitive motions, and athletes. Notable examples include rotator cuff injuries, Achilles tendon ruptures, patellar tendinopathy, and tennis elbow, each exhibiting distinct incidence rates and affected populations4,5,6,7. Diagnostic modalities, such as B-ultrasound and MRI, are commonly employed for assessing tendon injuries. However, given the characteristic healing process of tendons involving fibroblast accumulation around the wound surface, a study was conducted to investigate the use of Al[18F]-NOTA-FAPI-04 for Imaging tendon Injury models8. The findings support the hypothesis that PET-CT imaging with FAPI may serve as an effective method for monitoring tendon healing progress and evaluating injury severity.
[68Ga]-NOTAFAP-2286 exbibits the advantage of specifically targeting FAP in the tumor microenvironment with a prolonged retention time. It is currently used for tumor imaging. To our knowledge, PET/CT with [68Ga]-NOTA-FAP-2286 has not been used for imaging tendon injuries. Therefore, we conducted this study to explore the application of PET/CT with [68Ga]-NOTA-FAP-2286 in imaging tendon injuries.

<table><tbody><tr><td>&lt;sup&gt;68&lt;/sup&gt;Ga Generator</td><td>Eckert&amp;amp;Ziegier</td><td>10 84-470</td><td></td></tr><tr><td>C18 Cartridges</td><td>Waters</td><td>WAT020515</td><td></td></tr><tr><td>Chromatographic column</td><td>Waters</td><td>BEH C18 OBD Prep Column 5 &amp;mu;m,10 mm x 250 mm</td><td></td></tr><tr><td>HPLC system</td><td>Agilent</td><td>DE63062140</td><td></td></tr><tr><td>Radioactivity detector</td><td>BIOSCAN,INC,WASHINGTON,D,C</td><td>B-FC-3200 PR 253212</td><td></td></tr><tr><td>Small animal PET&amp;nbsp;</td><td>Siemens</td><td>SZ_200</td><td></td></tr></tbody></table>

pet/ct with [68ga]-nota-fap-2286 for imaging of tendon injuries in rat achilles tendon injury models

PET/CT With [68Ga]-NOTA-FAP-2286 for Imaging of Tendon Injuries in Rat Achilles Tendon Injury Models

Tendon injuries, particularly those affecting the Achilles tendon, are common sports-related conditions that significantly impact patients&#39; quality of life. Although existing diagnostic methods such as MRI and ultrasound are widely used, they may occasionally fail to provide accurate pathological information. This study aimed to investigate the feasibility of using [68Ga]-NOTA-FAP-2286 positron emission tomography/computed tomography (PET/CT) imaging for assessing an experimental rat model of Achilles tendon injury. The application of [68Ga]-NOTA-FAP-2286 PET/CT imaging demonstrated high sensitivity in detecting and precisely localizing tendon injuries, offering more comprehensive pathological insights compared to conventional imaging techniques. Fibroblast activation protein (FAP) ,a marker for activated fibroblasts, plays a crucial role in the tendon repair process. Tracer uptake started on the injured/ruptured side from the first week post modeling and peaked during the second week. These results show that FAP expression, activated by the Achilles tendon injury and rupture, reached its highest level during the second week of the repair process. By leveraging the specificity of the [68Ga]-NOTA-FAP-2286 tracer, this novel imaging approach accurately visualizes the extent and progression of tendon injuries in the animal model.

We successfully synthesized [68Ga]-NOTA-FAP-2286 with a yield of more than 70% (Decay corrected), achieving a radiochemical purity exceeding 95%. The HPLC profile is shown in Figure 4. Tendon injuries models were established surgically, and [68Ga]-NOTA-FAP-2286 was administered intravenously, followed by successful small-animal PET imaging. The results are presented in the Figure 5, Figu...

Here, we present a protocol to utilize [68Ga]-NOTAFAP-2286 PET/CT imaging to exhibit high sensitivity in detecting and precisely localizing tendon injuries.

Tendon injuries, particularly those affecting the Achilles tendon, are common sports-related conditions that significantly impact patients&#39; quality of ...

Our research investigates the potential of FAP targeted PET-CT imaging using NOTA-FAP-2286 for accurate detection and evaluation of tendon injuries. The latest advances in my research, it's the use of FAP-targeted PET-CT for precise imaging of tendon injuries. Current experimental challenges include optimizing tracers specificity, minimizing background signals, and correlating imaging findings with historic physiology.To begin, secure an anesthetized rat onto a fixation board. With a scalpel, make a longitudinal incision to expose the Achilles tendon. Compress the tendon using hemostatic forceps until it is flattened.Now close the incision with sutures. To establish the Achilles tendon rupture model, grasp the Achilles tendon with hemostatic forceps. Use surgical scissors to create a full thickness midline incision in the tendon, then suture the severed tendon and close the skin incision.For PET imaging, secure the rat in a restrainer. Administer 500 microcurie of GA-68-NOTA-FAP-2286 via the tail vein to both groups of rats. Position the fully anesthetized rat in the scanning field with the Achilles tendon aligned at the center.Turn on the computer connected to the small animal PET system. Now click on CT Acquisition to begin the preheating of the computed tomography system. Once the preheating is complete, select the scanning protocol, then open the corresponding scanning workflow, which has a preset scan duration of 10 minutes.Click on Scout View to verify the Achilles tendon is at the center of the scanning field. If the positioning is accurate, click Start Workflow to initiate the PET and CT scans. Set the scan parameters NCT bed view from zero to 36.2 to 76.4 millimeters, CT scan time of 150 seconds per bed, and PET acquire by time set to 600 seconds.Then set the photo peak energy level to 511 kiloelectron volt, lower level discrimination to 350 kilo electron volt, upper level discrimination to 650 kilo electron volt, and timing window to 3.432 nanoseconds. GA-68-NOTA-FAP-2286 showed a high radiochemical purity exceeding 95%Tracer uptake began at the site of Achilles tendon rupture or injury, 30 minutes post-injection, and intensified significantly over 90 minutes, showing higher signals compared to the surrounding muscle and the normal tendon. Computed tomography images failed to visually differentiate between injured ruptured and normal Achilles tendons, showing similar grayscale appearances across all samples.Maximum intensity projection imaging demonstrated that GA-68-NOTA-FAP-2286 uptake on the injury or rupture side increased from the first week and peaked at the second week, indicating elevated fibroblast activation protein expression during tendon repair.

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Learn FAP-targeted PET/CT imaging using [68Ga]-NOTA-FAP-2286 for detecting tendon injuries in rat Achilles tendon models. This protocol enables precise localization and sensitive assessment of tendon damage.

PET/CT With [⁶⁸Ga]-NOTA-FAP-2286 for Imaging of Tendon Injuries in Rat Achilles Tendon Injury Models

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