This work was supported by the Deutsche Forschungsgemeinschaft (SFB 656, M&uuml;nster, Germany, projects C7 &amp; C6) and the IZKF M&uuml;nster (Core unit SmAP). The authors are grateful to Truc Van Le, Anne Kanzog, Ute Neugebauer, Wiebke Gottschlich and Roman Priebe for excellent technical assistance and to Daniel Burkert and Sven Fatum for producing radiotracers.

1. Donor Organ Recovery
<ol>
 <li>Set up the stereotactic microscope, record weight of the 8-10 week old rat (donor and recipient body weight should match).</li>
 <li>Anesthetize the donor rat (Lewis Brown Norway F1, LBN F1) using oxygen/isoflurane inhalation (isoflurane 4 %/2 L/min oxygen). Maintain anesthesia by lowering isoflurane to 2-2.5%.</li>
 <li>Place the anesthetized rat on the surgery pad. Fix the rat's extremities with adhesive tape onto the pad and apply ophthalmic ointment (Bepanthen, Bayer) to the rat's eyes.</li>
 <li>Shave and disinfect the abdomen of the rat and perform a ventral midline incision from the pubis to the caudal border of the liver. Expose the left kidney and its vessels by carefully moving the intestine to the right side. Place a thin sheet of gauze over the left kidney and moisten it with warmed isotonic saline solution to prevent desiccation.</li>
 <li>Carefully dissect the fat tissue with Dumont Angled Tip Forceps (Dumont SS-45 Forceps Inox Medical, Fine Science Tools, cat.no. 11203-25) enveloping the left ureter. Avoid any direct contact with the ureter, instead, mobilize it with sufficient surrounding fat tissue.</li>
 <li>Separate the left renal vein from the renal artery using Dumont Angled Tip forceps. Remove all fat and connective tissue from both vessels and cauterize the adrenal and testicular vessels.</li>
 <li>Dissect aorta and inferior vena cava (IVC) above and below its junction with the left renal artery and vein, respectively, and cauterize all outgoing arteries.</li>
 <li>Clamp the suprarenal aorta with a microsurgical clamp (Micro Serrefines, Fine Science Tools, cat.no. 18055-04) above the left renal artery, as near as possible to the superior mesenteric artery. Ligate the infrarenal aorta using surgical silk (5-0, V&ouml;mel, art no. 14739) approximately 5 mm below the left renal artery.</li>
 <li>Ligate the infrarenal IVC with surgical silk (5-0, V&ouml;mel), and clamp the suprarenal IVC with a microsurgical clamp (Micro Serrefines, Fine Science Tools, cat.no. 18055-03).</li>
 <li>Cut the renal vein as close as possible to the IVC using fine scissors (Iris Scissors - ToughCut Straight 11.5 cm, Fine Science Tools, cat.no. 14058-11).</li>
 <li>Slowly perfuse the kidney in situ with 2 ml ice-cold HTK perfusion solution (CUSTODIOL HTK, Dr. Franz K&ouml;hler Chemie) by inserting a canula (Microlance 3 27G &frac34;, BD, cat.no. 302200) in the infrarenal aorta. Confirm that the renal color changes (now olive-tinted).</li>
 <li>First, transect the suprarenal aorta, then the infrarenal aorta and finally the ureter as close as possible to the urinary bladder.</li>
 <li>Remove the kidney and its vascular supply together with the ureter and store in HTK perfusion solution on ice.</li>
 <li>Euthanize the donor rat by removing the vascular clamps and consecutive excision of the heart under anesthesia.</li>
</ol>
2. Recipient Preparation and Transplantation
<ol>
 <li>Set up the stereotactic microscope, record weight of the rat.</li>
 <li>Anesthetize the recipient rat (Lewis) using isoflurane 4%. Maintain anesthesia by lowering isoflurane to 2-2.5%.</li>
 <li>Place the anesthetized rat on the surgery pad. Fix the rat's extremities with adhesive tape onto the pad and apply ophthalmic ointment to the rat`s eyes. Monitor and control core body temperature using a rectal temperature sensor and a warming pad. During surgery repetitively control temperature, breathing and heart rate of the animal.</li>
 <li>Shave and disinfect the abdomen and perform a ventral midline incision from the pubis to the caudal border of the liver. Use a scalpel forcutting the skin in order to minimize skin trauma. Expose the left kidney and its vessels by moving the intestine to the right side.</li>
 <li>Carefully remove the renal capsule using Dumont Angled Tip forceps and cotton swabs.</li>
 <li>Occlude the left renal artery, vein and ureter near the renal hilum with two ligations using surgical thread (Mersilene 4-0, Ethicon, cat. no. EH6732H). Excise the greater part of the kidney, leaving only the renal hilum. Clean the cut surface with cotton swabs and check for bleeding. Add another ligature if necessary.</li>
 <li>Carefully dissect bluntly through the connective tissue covering the infrarenal aorta and vena cava on the caudal site with cotton swabs.</li>
 <li>Separate any visible nerve from the infrarenal aorta and vena cava using Dumont Angled Tip forceps.</li>
 <li>Carefully dissect through the connective tissue sheet between the infrarenal aorta and vena cava, forming two openings of 2-4 mm length: The first below the branching of the testicular vessels, and the second one above the branching of the common iliac vessels. Cauterize the iliolumbar arteries and veins in between.</li>
 <li>Draw a single surgical thread (Mersilene 0; Ethicon, cat. no.EH6665E) through each of the two openings. Using these threads pull the infrarenal aorta and vena cava gently to the right side of the animal to gain access to the vessels branching on the dorsal site. Cauterize any of these vessels between the two openings and remove the surgical threads thereafter.</li>
 <li>Next, occlude aorta and IVC by clockwise application of four microsurgical clamps (Micro Serrefines, Fine Science Tools, cat.no. 18055-04) beginning at the upper opening with the aorta and ending with the vena cava at the upper opening (Micro Serrefines, Fine Science Tools, cat.no. 18055-03).</li>
 <li>Afterwards carefully pierce the upper ending of the isolated part of the aorta with a needle (Microlance 3 27G &frac34;, BD, cat.no. 302200), insert the lower blade of a fine spring scissor (Vannas Spring Scissors - 3 mm Blades, Fine Science Tools, cat.no 15000-00) through the puncture opening and perform a short longitudinal incision. Flush the isolated aorta with ice-cold HTK perfusion solution to remove any remaining blood.</li>
 <li>Likewise, carefully pierce the lower ending of the isolated part of the vena cava with a needle, insert the lower blade of a fine spring scissor through the puncture opening and perform a longitudinal incision ending shortly below the aortic incision. Again, flush the vessel to remove any remaining blood.</li>
 <li>Remove the kidney graft from the storage solution. Place it below the former position of the left kidney, and ensure that it is correctly oriented.</li>
 <li>Check the orientation of the grafts renal artery and ensure that there are no twists. Then place a thin sheet of gauze over the graft and moisten it with ice-cold HTK perfusion solution to cool it, and to prevent desiccation.</li>
 <li>Fixate the opening of the suprarenal part of the aortic piece in which the renal artery of the graft opens into with surgical thread (Ethilon 9-0, Ethicon, cat. no. 2809G) - first at the upper, then at the lower end of the aortic incision. Close the incision clockwise with a continuous suture using surgical thread (Ethilon 9-0), Dumont Angled Tip forceps and a curved Castroviejo needle holder (Fine Science Tools, cat. no. 12061-01). Afterwards, cover the first suture with connective tissue through a counterclockwise second continuous suture. Now, only the lower part of the aortic piece of the graft remains to be closed. Regularly cool the graft with ice-cold HTK perfusion solution.</li>
 <li>Perfuse the graft with 1 ml of ice-cold HTK perfusion solution through a blunt needle inserted into the open end of the grafts aortic piece validating the tightness and integrity of the suture, as well as removing remaining blood in the graft. Afterwards, ligate the open end of the aortic piece with surgical silk (5-0, V&ouml;mel).</li>
 <li>Check the orientation of the grafts renal vein and ensure that there are no twists.</li>
 <li>Fixate the opening of the vein of the graft with surgical thread (Ethilon 9-0) - first at the upper, then at the lower end of the incision at the vena cava. Close the incision clockwise with a continuous suture using surgical thread (Ethilon 9-0), Dumont Angled Tip forceps and a curved Castroviejo needle holder. Regularly cool the graft with ice-cold HTK perfusion solution.</li>
 <li>Counterclockwise remove the microsurgical clamps, beginning with the uppermost vena cava clamp. After removing the last aortic clamp, carefully stop eventually mild bleeding with cotton swabs. After a few seconds the graft will turn rosy-tinted, and uretric contractions should appear.</li>
 <li>For the insertion of the grafts ureter into the recipient`s bladder carefully remove a small patch of the musculature at the top of the bladder using a Vannas Spring Scissor (Student Vannas Spring Scissor straight, Fine Science Tools, cat.no. 91500-09). Then, remove the surrounding tissue (mostly fat) from the graft ureter using Dumont Angled Tip forceps. Therefore, carefully grab the tip of the ureter and gently separate the surrounding fat, connective tissue and the embedded vessels from it by pulling them in the opposite direction. Fixate the uretric tip at the end of a surgical thread (Prolene 6-0, Ethicon, cat. no. 8697H) and perforate the previously prepared top of the bladder with the attached needle. Pull the ureter through the bladder and exit it at the base through a second perforation.</li>
 <li>Fixate the previously from the ureter separated tissue at the top of the bladder with surgical thread (Ethilon 9-0). Cut off the tip of the ureter with the attached surgical thread and pull the ureter back into the bladder by gently pushing from below. Afterwards, close the second bladder puncture with surgical thread (Ethilon 9-0).</li>
 <li>Close the abdominal wall and skin by two continuous but separate sutures using surgical suture thread (Mersilene 0). Disinfect the wound using Pividon Iodine and administer buprenorphine (0.1 mg per kg/BW/bid) s.c. for three days after surgery to control wound pain.</li>
</ol>
3. Imaging Allograft Rejection
<ol>
 <li>Anesthetize nonfasting rat using isoflurane 2-2.5%.</li>
 <li>30 MBq FDG (30 MBq FDG in 0.1 ml 0.9% NaCl) are administered i.v. via catheterizing a lateral tail vein using a 24 G catheter (Braun, Introcan, 4252500-01). Afterwards, purge the catheter with 0.9 ml 0.9% NaCl solution.</li>
 <li>Leave the rat in a restrainer without anesthesia until start of the scan and hydrate the animal by i.v. injection of 1 ml of 0.9% NaCl solution hourly.</li>
 <li>Re-anesthetize rat using isoflurane 2% immediately before the scan starts.</li>
 <li>Place anesthetized rat in a high-resolution multi-wire chamber-based animal PET camera quadHIDAC (Oxford Positron Systems Ltd, Oxford, UK). The spatial resolution of the PET scanner is 1.0 mm and is constant over the whole FOV (diameter, 165 mm; axial length, 280 mm). Monitor and control body temperature during the scan using a rectal temperature sensor and control anesthesia with a pulse oxymeter.</li>
 <li>Start dynamic acquisition for 60 min starting 180 min after FDG-injection to reduce tracer accumulation in the kidneys caused by renal excretion of FDG.</li>
 <li>Apply 5 MBq of 18F-fluoride i.v. and perform another PET scan immediately after 18F-fluoride injection for 60 min without moving the position of the rat in the scanner for identification of renal parenchyma and for calculation of 18F-clearance 5. Reconstruct images from both scans. Trace manually a volume of interest (VOI) on reconstructed images 2 min after 18F-fluoride injection (perfusion-phase) around renal cortex and transfer the VOI to the FDG images. Carefully exclude the renal pelvis from the VOI. Reconstruct images from both scans and manually trace a volume of interest (VOI) around renal cortex. List-mode data were reconstructed into images with a voxel size of 0.4 &times; 0.4 &times; 0.4 mm3. Carefully exclude the renal pelvis from the VOI.</li>
 <li>Compute FDG uptake by the ratio of total counts and volume and calculate the percentage of injected dose (%ID). We used MATLAB (Version R2011b, MathWorks, Natick, MA, USA) and tomographic imaging (TIM) Version 2.8 for image analysis.</li>
</ol>

<ol>
	<li>Wu, O., Levy, A. R., Briggs, A., Lewis, G., Jardine, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+rejection+and+chronic+nephropathy:+a+systematic+review+of+the+literature.">Acute rejection and chronic nephropathy: a systematic review of the literature.</a> Transplantation. 87, 1330-1339 (2009).</li><li>Miller, B. F., Gonzales, E., Wilchins, L. J., Nathan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+transplantation+in+the+rat.">Kidney transplantation in the rat.</a> Nature. 194, 309-310 (1962).</li><li>Reuter, S., Schn&#246;ckel, U., Schr&#246;ter, R., Schober, O., Pavenst&#228;dt, H., Sch&#228;fers, M., Gabri&#235;ls, G., Schlatter, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+imaging+of+acute+renal+allograft+rejection+in+rats+using+small+animal+F-FDG-PET.">Non-invasive imaging of acute renal allograft rejection in rats using small animal F-FDG-PET.</a> PLoS. One. 4, e5296 (2009).</li><li>Reuter, S., Schn&#246;ckel, U., Edemir, B., Schr&#246;ter, R., Kentrup, D., Pavenst&#228;dt, H., Schober, O., Schlatter, E., Gabri&#235;ls, G., Sch&#228;fers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+of+noninvasive+serial+assessment+of+acute+renal+allograft+rejection+by+18F-FDG+PET+to+monitor+treatment+efficiency.">Potential of noninvasive serial assessment of acute renal allograft rejection by 18F-FDG PET to monitor treatment efficiency.</a> J. Nucl. Med. 51, 1644-1652 (2010).</li><li>Schn&#246;ckel, U., Reuter, S., Stegger, L., Schlatter, E., Sch&#228;fers, K. P., Hermann, S., Schober, O., Gabri&#235;ls, G., Sch&#228;fers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+18F-fluoride+small+animal+PET+to+noninvasively+assess+renal+function+in+rats.">Dynamic 18F-fluoride small animal PET to noninvasively assess renal function in rats.</a> Eur. J. Nucl. Med. Mol. Imaging. 35, 2267-2274 (2008).</li><li>Grabner, A., Schn&#246;ckel, U., Kentrup, D., Sch&#228;fers, M., Reuter, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+Non-Invasive+Molecular+Imaging+of+Acute+Allograft+Rejection+by+Gamma+Scintigraphy+and+Positron+Emission+Tomography.">Strategies for Non-Invasive Molecular Imaging of Acute Allograft Rejection by Gamma Scintigraphy and Positron Emission Tomography.</a> Curr. Radiopharm. 4, 10-23 (2011).</li><li>Racusen, L. C., Solez, K., Colvin, R. B., Bonsib, S. M., Castro, M. C., Cavallo, T., Croker, B. P., Demetris, A. J., Drachenberg, C. B., Fogo, A. B., 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=The+Banff+97+working+classification+of+renal+allograft+pathology.">The Banff 97 working classification of renal allograft pathology.</a> Kidney Int. 55, 713-723 (1999).</li><li>Edemir, B., Reuter, S., Borgulya, R., Schr&#246;ter, R., Neugebauer, U., Gabri&#235;ls, G., Schlatter, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+rejection+modulates+gene+expression+in+the+collecting+duct.">Acute rejection modulates gene expression in the collecting duct.</a> J. Am. Soc. Nephrol. 19, 538-546 (2008).</li><li>Velic, A., Gabri&#235;ls, G., Hirsch, J. R., Schr&#246;ter, R., Edemir, B., Paasche, S., Schlatter, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+rejection+after+rat+renal+transplantation+leads+to+downregulation+of+Na++and+water+channels+in+the+collecting+duct.">Acute rejection after rat renal transplantation leads to downregulation of Na+ and water channels in the collecting duct.</a> Am. J. Transplant. 5, 1276-1285 (2005).</li><li>Reuter, S., Velic, A., Edemir, B., Schr&#246;ter, R., Pavenst&#228;dt, H., Gabri&#235;ls, G., Bleich, M., Schlatter, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+role+of+NHE-3+inhibition+in+rat+renal+transplantation+undergoing+acute+rejection.">Protective role of NHE-3 inhibition in rat renal transplantation undergoing acute rejection.</a> Pflugers Arch. 456, 1075-1084 (2008).</li></ol>

No conflicts of interest declared.

FDG-PET imaging is a new option for the diagnosis of acute rejection. Because of its non-invasive and specific nature, FDG-PET is advantages in comparison to classical diagnostics by core needle biopsy. In contrast to the limited sample size of a biopsy, FDG-PET analysis the whole graft. Moreover, one can apply it to patients on anticoagulant therapy, and one can perform PET measures repetitively e.g. to monitor treatment efficiency 4. In addition, we already have shown that two major different...

<table><tbody><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Mathieu Needle Holder - 14 cm</td><td>Fine Science Tools</td><td>12010-14</td><td></td></tr><tr><td>Castroviejo Micro Needle Holder - 9 cm</td><td>Fine Science Tools</td><td>12061-01</td><td></td></tr><tr><td>Surgical Scissors - Sharp_Blunt</td><td>Fine Science Tools</td><td>14001-12</td><td></td></tr><tr><td>Iris Scissors - ToughCut Straight 11.5 cm</td><td>Fine Science Tools</td><td>14058-11</td><td></td></tr><tr><td>Student Vannas Spring Scissors </td><td>Fine Science Tools</td><td>91500-09</td><td></td></tr><tr><td>Vannas Spring Scissors - 3 mm Blades</td><td>Fine Science Tools</td><td>15000-00</td><td></td></tr><tr><td>Student Tissue Forceps - 1x2 Teeth 12 cm</td><td>Fine Science Tools</td><td>91121-12</td><td></td></tr><tr><td>Dumont SS-45 Forceps - Inox Medical</td><td>Fine Science Tools</td><td>11203-25</td><td></td></tr><tr><td>Micro-Serrefine Clip Applicator with Lock</td><td>Fine Science Tools</td><td>18056-14</td><td></td></tr><tr><td>Micro-Serrefine 6 mm x 1 mm</td><td>Fine Science Tools</td><td>18055-03</td><td></td></tr><tr><td>Micro-Serrefine 4 mm x 0.75 mm</td><td>Fine Science Tools</td><td>18055-04</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>Isoflurane (&lt;em&gt;e.g.&lt;/em&gt; Forene 100% v/v)</td><td>Abott</td><td></td><td></td></tr><tr><td>cutane antiseptic (&lt;em&gt;e.g.&lt;/em&gt; Octeniderm) </td><td>Sch&amp;#252;lke</td><td></td><td></td></tr><tr><td>Povidone Iodine (&lt;em&gt;e.g.&lt;/em&gt; Betaisodona)</td><td>Mundipharma</td><td></td><td></td></tr><tr><td>ophthalmic ointment (&lt;em&gt;e.g.&lt;/em&gt; Bepanthen)</td><td>Bayer</td><td></td><td></td></tr><tr><td>Buprenorphin (&lt;em&gt;e.g.&lt;/em&gt; Temgesic)</td><td>RB Pharmaceuticals</td><td></td><td></td></tr><tr><td>HTK perfusion solution (&lt;em&gt;e.g.&lt;/em&gt; CUSTODIOL HTK)</td><td>Dr. Franz K&amp;#246;hler Chemie</td><td></td><td></td></tr><tr><td>surgical thread Mersilene 0</td><td>Ethicon</td><td>EH6665E</td><td></td></tr><tr><td>surgical thread Mersilene 4-0</td><td>Ethicon</td><td>EH6732H</td><td></td></tr><tr><td>surgical thread Prolene 6-0</td><td>Ethicon</td><td>8697H</td><td></td></tr><tr><td>surgical thread Ethilon 9-0</td><td>Ethicon</td><td>2809G</td><td></td></tr><tr><td>surgical silk 5-0</td><td>V&amp;#246;mel</td><td>14739</td><td></td></tr><tr><td>Canula (&lt;em&gt;e.g.&lt;/em&gt; Microlance 3, 27G &amp;#190;)</td><td>BD</td><td>302200</td><td></td></tr></tbody></table>

non-invasive imaging of acute allograft rejection after rat renal transplantation using 18f-fdg pet

The number of patients with end-stage renal disease, and the number of kidney allograft recipients continuously increases. Episodes of acute cellular allograft rejection (AR) are a negative prognostic factor for long-term allograft survival, and its timely diagnosis is crucial for allograft function 1. At present, AR can only be definitely diagnosed by core-needle biopsy, which, as an invasive method, bares significant risk of graft injury or even loss. Moreover, biopsies are not feasible in patients taking anticoagulant drugs and the limited sampling site of this technique may result in false negative results if the AR is focal or patchy. As a consequence, this gave rise to an ongoing search for new AR detection methods, which often has to be done in animals including the use of various transplantation models.
Since the early 60s rat renal transplantation is a well-established experimental method for the examination and analysis of AR 2. We herein present in addition small animal positron emission tomography (PET) using 18F-fluorodeoxyglucose (FDG) to assess AR in an allogeneic uninephrectomized rat renal transplantation model and propose graft FDG-PET imaging as a new option for a non-invasive, specific and early diagnosis of AR also for the human situation 3. Further, this method can be applied for follow-up to improve monitoring of transplant rejection 4.

Histology
During AR leukocytes, i.e. mainly T-lymphocytes are recruited into the transplant, whereas the severity of the rejection is reflected by the degree of inflammation. In the Periodic-Acid-Schiff (PAS) staining depicted here (Figure 1), the renal allograft shows significant histological signs of AR, namely glomerulitis, tubulitis, endothelialitis and graft infiltration (Figure 1, aTX POD4) (POD = postoperative day) while signs of rejection a...

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Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET | Medicine | Video

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

We herein present a rat renal transplantation model to non-invasively assess acute allograft rejection using positron emission tomography with 18F-fluorodeoxyglucose.

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

The number of patients with end-stage renal  disease, and the number of kidney allograft recipients continuously increases.  Episodes of acute cellular ...

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16.9K Views.  University of Münster. We herein present a rat renal transplantation model to non-invasively assess acute allograft rejection using positron emission tomography with 18F-fluorodeoxyglucose.

Video: Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

Machine Learning Algorithms for Early Detection of Bone Metastases in an Experimental Rat Model

Machine learning (ML) algorithms permit the integration of different features into a model to perform classification or regression tasks with an accuracy exceeding its constituents. This protocol describes the development of an ML algorithm to predict the growth of breast cancer bone macrometastases in a rat model before any abnormalities are observable with standard imaging methods. Such an algorithm can facilitate the detection of early metastatic disease (i.e., micrometastasis) that is regularly missed during staging examinations.
The applied metastasis model is site-specific, meaning that the rats develop metastases exclusively in their right hind leg. The model&#8217;s tumor-take rate is 60%&#8211;80%, with macrometastases becoming visible in magnetic resonance imaging (MRI) and positron emission tomography/computed tomography (PET/CT) in a subset of animals 30 days after induction, whereas a second subset of animals exhibit no tumor growth.
Starting from image examinations acquired at an earlier time point, this protocol describes the extraction of features that indicate tissue vascularization detected by MRI, glucose metabolism by PET/CT, and the subsequent determination of the most relevant features for the prediction of macrometastatic disease. These features are then fed into a model-averaged neural network (avNNet) to classify the animals into one of two groups: one that will develop metastases and the other that will not develop any tumors. The protocol also describes the calculation of standard diagnostic parameters, such as overall accuracy, sensitivity, specificity, negative/positive predictive values, likelihood ratios, and the development of a receiver operating characteristic. An advantage of the proposed protocol is its flexibility, as it can be easily adapted to train a plethora of different ML algorithms with adjustable combinations of an unlimited number of features. Moreover, it can be used to analyze different problems in oncology, infection, and inflammation.

This protocol was designed to train a machine learning algorithm to use a combination of imaging parameters derived from magnetic resonance imaging (MRI) and positron emission tomography/computed tomography (PET/CT) in a rat model of breast cancer bone metastases to detect early metastatic disease and predict subsequent progression to macrometastases.

Machine learning (ML) algorithms permit the integration of different features into a model to perform classification or regression tasks with an accuracy ...

Cancer Research

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1&#945; expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Preparing a 68Ga-labeled Arginine Glycine Aspartate (RGD)-peptide for Angiogenesis

The &#945;v&#946;3 integrin is a heterodimeric adhesion molecule involved in tumor cell migration and angiogenesis. The integrin is overexpressed in angiogenic tumor endothelial cells, where it typically has a low concentration. This specific expression of &#945;v&#946;3 makes it a valid biomarker for antiangiogenic and imaging drugs. As a functional imaging modality, positron emission tomography (PET) provides information about biochemical and physiological changes in vivo, due to its unique high sensitivity at the nanomolar scale. Hence, radiometal-based PET radiopharmaceuticals have received great attention for the non-invasive quantification of tumor angiogenesis. This paper provides a systemic protocol to prepare a new radiometal-labeled peptide for the evaluation of angiogenesis. This protocol contains information about radiochemical reliability, lipophilicity, cell uptake, serum stability, and pharmacokinetic properties. The 68Ga-RGD-peptide is one of the representative PET ligands toward &#945;v&#946;3 integrin. Here, we introduce a protocol to prepare a 68Ga-RGD-peptide and the evaluation of its biological efficacy.

Preparing a 68Ga-labeled Arginine Glycine Aspartate RGD-peptide for Angiogenesis

The &#945;v&#946;3 integrin is a type of adhesion protein that is highly expressed on activated endothelial cells undergoing angiogenesis. Thus, evaluating the integrity of the integrin is of great interest in oncology. Here, we introduce a method to prepare 68Ga-labeled radiopeptides and a method to assess its biological effectiveness.

The &#945;v&#946;3 integrin is a heterodimeric adhesion molecule involved in tumor cell migration and angiogenesis. The integrin is overexpressed in ...

A Modified Surgical Technique for Kidney Transplantation in Mice

Kidney transplantation in mice is a complicated and challenging surgery procedure. There are very few publications demonstrating the key steps of this operation. Therefore, this article introduces the technique and points out the surgical caveats associated with this operation. In addition, important modifications in comparison to the conventional procedure are demonstrated. Firstly, a patch of the abdominal aorta is cut and prepared so that the proximal bifurcations of the renal artery, including the ureteral artery are transected together with the donor kidney en bloc. This reduces the risk of a ureter necrosis and avoids the development of a urinary tract occlusion. Secondly, a new method of the vascular anastomosis is demonstrated that allows the operator to flexibly increase or decrease the size of the anastomosis after renal transplant reperfusion has already been initiated. This avoids the development of vessel strictures and intraabdominal bleeding. Thirdly, a technique that enables the anastomosis of the delicate donor ureter and the recipient bladder that does not cause a trauma is shown. Adopting this protocol can shorten the operation time and reduces the damage to the recipient's bladder, thereby significantly increasing the operation success rate for the recipient mice.

This protocol presents a new surgical technique of mouse kidney transplantation focusing on a modified arterial anastomosis strategy. A vascular suture technique including a simple and safer ureter-bladder anastomosis method is also presented. These modifications shorten the operation time and improve the success rate of the mouse kidney transplantation procedure.

Kidney transplantation in mice is a complicated and challenging surgery procedure. There are very few publications demonstrating the key steps of this ...

Immunology and Infection

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of renal replacement therapy. Over the past few decades, the rat kidney transplantation model has been used to study the immunological phenomena of rejection and tolerance. This model has become an indispensable tool to test new immunomodulatory pharmaceuticals and regimens prior to proceeding with expensive preclinical large animal studies.
This protocol provides a detailed overview of how to reliably perform orthotopic kidney transplantation in rats. This protocol includes three distinctive steps that increase the probability of success: perfusion of the donor kidney by flushing through the portal vein and the use of a cuff system to anastomose the renal veins and ureters, thereby decreasing cold and warm ischemia times. Using this technique, we have achieved survival rates beyond 6 months with normal serum creatinine in animals with syngeneic or tolerant kidney transplants. Depending on the aim of the study, this model can be modified by pre- or posttransplant treatments to study the acute, chronic, cellular, or antibody-mediated rejection. It is a reproducible, reliable, and cost-effective animal model to study different aspects of kidney transplantation.

The purpose of this manuscript and protocol is to explain and demonstrate in detail the surgical procedure of orthotopic kidney transplantation in rats. This method is simplified to achieve the correct perfusion of the donor kidney and shorten the reperfusion time by using the venous and ureteral cuff anastomosis technique.

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of ...

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand and face transplants and, more recently, abdominal wall, uterus, and urogenital transplants. Functional outcomes have exceeded initial expectations, and most recipients enjoy an improved quality of life. However, as clinical experience accumulates, chronic rejection and complications from the immunosuppression must be addressed. In many cases where grafts have failed, the causative pathology has been ischemic vasculopathy. <span style="font-size: 11pt; line-height: 115%; font-family: Arial, sans-serif; color: rgb(41, 43, 49); background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">The biological mechanisms of the acute and chronic rejection associated with VCA, especially ischemic vasculopathy, are important areas of research. However, due to the very small number of VCA patients, the evaluation of proposed mechanisms is better addressed in an experimental model. Multiple groups have used animal models to address some of the relevant unsolved questions in VCA rejection and vasculopathy. Several model designs involving a variety of species are described in the literature. Here we present a reproducible model of VCA heterotopic hindlimb osteomyocutaneous flap in the rat that can be utilized for translational VCA research. This model allows for the serial evaluation of the graft, including biopsies and different imaging modalities, while maintaining a low level of morbidity.

Vascularized composite allograft offers life-altering benefits to transplant recipients, but the biological causes of graft rejection and vasculopathy remain poorly understood. The rodent surgical model presented here offers a reproducible, clinically relevant model of transplantation, allowing researchers to evaluate rejection events and potential therapeutic strategies to prevent their occurrence.

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand ...

A Rat Orthotopic Renal Transplantation Model for Renal Allograft Rejection

Renal allograft rejection limits the long-term survival of patients after renal transplantation. Rat orthotopic renal transplantation is an essential model to investigate the mechanism of renal allograft rejection in pre-clinical studies and could aid in the development of novel approaches to improve the long-term survival of renal allografts. Donor kidney implantation in rat orthotopic renal transplantation is commonly performed by end-to-side anastomosis to recipients' aorta and inferior vena cava. In this model, the donor's kidney was implanted using end-to-end anastomosis to the recipients' renal artery and renal vein. The donor's ureter was anastomosed to the recipient's bladder in an end-to-side 'tunnel' method. This model contributes to better healing of ureter-bladder anastomosis and increases the recipients' survival by avoiding interference with blood supply and venous reflux of the lower body. This model can be used to investigate the mechanisms of acute and chronic immune and pathologic rejection of renal allografts. Here, the study describes the detailed protocols of this orthotopic renal transplantation between rats.

The rat orthotopic renal transplantation model contributes to investigating the mechanism of renal allograft rejection. The current model increases the recipients' survival without interference with blood supply and venous reflux of the lower body using an end-to-end anastomosis of kidney implantation and an end-to-side "tunnel" method of ureter-bladder anastomosis.

Renal allograft rejection limits the long-term survival of patients after renal transplantation. Rat orthotopic renal transplantation is an essential ...

High-Resolution Cardiac Positron Emission Tomography/Computed Tomography for Small Animals

Positron emission tomography (PET) and computed tomography (CT) are among the most employed diagnostic imaging techniques, and both serve in understanding cardiac function and metabolism. In preclinical research, dedicated scanners with high sensitivity and high spatio-temporal resolution are employed, designed to cope with the demanding technological requirements posed by the small heart size and very high heart rates of mice and rats. In this paper, a bimodal cardiac PET/CT imaging protocol for experimental mouse and/or rat models of cardiac diseases is described, from animal preparation and image acquisition and reconstruction to image processing and visualization.
In particular, the 18F-labeled fluorodeoxyglucose ([18F]FDG)-PET scan allows for the measurement and visualization of glucose metabolism in the different segments of the left ventricle (LV). Polar maps are convenient tools to display this information. The CT part consists of a time-resolved 3D reconstruction of the entire heart (4D-CT) using retrospective gating without electrocardiography (ECG) leads, allowing the morphofunctional evaluation of the LV and the subsequent quantification of the most important cardiac function parameters, such as ejection fraction (EF) and stroke volume (SV). Using an integrated PET/CT scanner, this protocol can be executed within the same anesthesia induction without the need to reposition the animal between different scanners. Hence, PET/CT can be seen as a comprehensive tool for the morphofunctional and metabolic evaluation of the heart in several small animal models of cardiac diseases.

Here, we present an experimental imaging protocol for the quantification of cardiac function and morphology using high-resolution positron emission tomography/computed tomography for small animals. Both mice and rats are considered, discussing the different requirements of computed tomography contrast agents for the two species.

Positron emission tomography (PET) and computed tomography (CT) are among the most employed diagnostic imaging techniques, and both serve in understanding ...

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using ¹⁸F-FDG PET

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