Structural Assessment of Intestinal Grafts Preserved With Phospholipase A 2 Inhibition J. Denney, N. Ranjbar, and R.E. Sonnino P RESERVATION of bowel for intestinal transplanta- tion remains an ongoing challenge. Our laboratory has been investigating the role of secretory phospholipase A 2 (sPLA 2 ) in the ischemia and reperfusion injury of intestinal grafts, as well as the effectiveness of a PLA 2 inhibitor (PX-13) in preventing the damage. 1 We hope that by inhibiting the effects of sPLA 2 on the graft and maintaining structural integrity intestinal graft preservation techniques may be improved. METHODS Grafts were harvested in a standard manner, 2 and preserved in University of Wisconsin (UW) solution alone for 24 hrs (group 1), UW + PX-13 for 24 hrs (group 2), UW solution alone for 48 hours (group 3), or UW + PX-13 for 48 hrs (group 4) (N = 5/group). All grafts were then studied for structural damage by light (LM) and transmission electron microscopy (TEM). LM samples were han- dled by standard histologic methods, with fixation in 10% formalin, dehydration in serial alcohol concentrations, embedding in paraf- fin, and staining with hematoxylin and eosin. TEM samples (1 to 3 mm) were fixed in cacodylate-buffered 3% glutaraldehyde for 24 hrs, rinsed in phosphate buffered sucrose, dehydrated in progres- sive concentrations of ethanol, heavy metal stained with osmium tetroxide, and embedded in resin. Ultrathin sections were obtained with an ultratome, doubly stained with uranyl acetate and lead citrate, and examined in a Philips 201 transmission electron microscope. All samples were blinded and evaluated in the TEM facility at the Medical College of Virginia. RESULTS LM In the absence of PX-13, most grafts preserved for 24 hrs (group 1) suffered some damage to the villi, 40% lost epithelial integrity, and scattered crypt injury was noted. After 48 hrs of ischemia (group 3), the findings were similar except for a greater degree of epithelial injury, evident in all samples, with only 33% having normal epithelium present. Structural preservation was improved in all grafts treated with PX-13. Crypts were intact after both 24 (group 2) and 48 hrs (group 4). A normal epithelial layer was evident in 100% of the samples examined at 24 hrs, while after 48 hours, some damage to the villi was found in up to 80% of samples. TEM The microvillous border of untreated grafts appeared well preserved at 24 hrs (group 1) but moderately irregular at 48 hours (group 3). Tight junctions between epithelial cells were intact at 24 hrs, but markedly widened at 48 hours. Mitochondrial cristae, usually intact at 24 hrs, were dis- rupted in at least 50% of the samples at 48 hrs, when significant swelling was present. Endothelial cells appeared remarkably intact. With PX-13 (groups 2 & 4), tissue edema in the grafts was conspicuously absent, and these grafts showed a trend toward lower weight than untreated con- trols. By TEM, the microvillous border, tight junctions, and mitochondrial morphology were well preserved at both time intervals. Mitchondrial swelling was not evident at either time period. CONCLUSIONS These findings suggest that PLA 2 inhibitors, such as PX-13, improve tissue morphology during cold storage of intestinal grafts of up to 48 hours. However, tissue protection is not complete, particularly after 48 hours of preservation. Addi- tional studies will be required to further improve the structural protection of preserved intestinal grafts. REFERENCES 1. Sonnino RE, Pigatt L, Schrama A, et al: Dig Dis Sci 42:972, 1997 2. Sonnino RE, Franson R, Pigatt LA, et al: J Invest Surg 9:313, 1996 From the Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia. Address reprint requests to Roberta E. Sonnino, MD, FACS, Division of Pediatric Surgery, PO Box 980015, Richmond, VA 23298. © 1998 by Elsevier Science Inc. 0041-1345/98/$19.00 655 Avenue of the Americas, New York, NY 10010 PII S0041-1345(98)00761-1 Transplantation Proceedings, 30, 2639 (1998) 2639