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 border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</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></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Isoflurane (e.g. Forene 100% v/v)</td>
 <td>Abott</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>cutane antiseptic (e.g. Octeniderm) </td>
 <td>Sch&uuml;lke</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Povidone Iodine (e.g. Betaisodona)</td>
 <td>Mundipharma</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>ophthalmic ointment (e.g. Bepanthen)</td>
 <td>Bayer</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Buprenorphin (e.g. Temgesic)</td>
 <td>RB Pharmaceuticals</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>HTK perfusion solution (e.g. CUSTODIOL HTK)</td>
 <td>Dr. Franz K&ouml;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&ouml;mel</td>
 <td>14739</td>
 <td></td>
 </tr>
 <tr>
 <td>Canula (e.g. Microlance 3, 27G &frac34;)</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...

Watch this Scientific Journal Video about Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET at JoVE.com

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

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

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 ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

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 ...

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). Impaired uptake and metabolism of glucose in multiple organ systems due to insulin resistance is a hallmark of type 2 diabetes. The ability of this technique to extract an image-derived input function from the vena cava using an iterative deconvolution method eliminates the requirement of the collection of arterial blood samples. Fitting of tissue and vena cava time activity curves to a two-tissue, three compartment model permits the estimation of kinetic micro-parameters related to the 18F-FDG uptake from the plasma to the intracellular space, the rate of transport from intracellular space to plasma and the rate of 18F-FDG phosphorylation. This methodology allows for multiple measures of glucose uptake and metabolism kinetics in the context of longitudinal studies and also provides insights into the efficacy of therapeutic interventions.

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This study describes a protocol that uses 18F-FDG and positron emission tomography/computed tomography (PET/CT) imaging, together with kinetic modelling, to quantify the in vivo, real-time uptake of 18F-FDG into tissues.

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

Semi-quantitative Assessment Using [18F]FDG Tracer in Patients with Severe Brain Injury

Patients with severe traumatic brain injury (sTBI) have difficulty knowing whether they are accurately expressing their thoughts and emotions because of disorders of consciousness, disrupted higher brain function, and verbal disturbances. As a consequence of an insufficient ability to communicate, objective evaluations are needed from family members, medical staff, and caregivers. One such evaluation is the assessment of functioning brain areas. Recently, multimodal brain imaging has been used to explore the function of damaged brain areas. [18F]-fluorodeoxyglucose positron emission tomography-computed tomography ([18F]FDG-PET/CT) is a successful tool for examining brain function. However, the assessment of brain glucose metabolism based on [18F]FDG-PET/CT is not standardized and depends on several varying parameters, as well as the patient&#39;s condition. Here, we describe a series of semiquantitative assessment protocols for a region-of-interest (ROI) image analysis using self-produced [18F]FDG tracers in patients with sTBI. The protocol focuses on screening the participants, preparing the [18F]FDG tracer in the hot lab, scheduling the acquisition of [18F]FDG-PET/CT brain images, and measuring glucose metabolism using the ROI analysis from a targeted brain area.

Semi-quantitative Assessment Using [18F]FDG Tracer in Patients with Severe Brain Injury

[18F]-fluorodeoxyglucose (FDG) positron emission tomography-computed tomography is useful for studying glucose metabolism related to brain function. Here, we present a protocol for an [18F]FDG tracer set-up and semiquantitative assessment of the region-of-interest analysis for targeted brain areas associated with clinical manifestations in patients with severe traumatic brain injury.

Patients with severe traumatic brain injury (sTBI) have difficulty knowing whether they are accurately expressing their thoughts and emotions because of ...

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...