Volume 127, Issue 2, Pages 595-606 (August 2004) Biliverdin protects the functional integrity of a transplanted syngeneic small bowel  Atsunori Nakao, Leo E. Otterbein, Marcus Overhaus, Judit K. Sarady, Allan Tsung, Kei Kimizuka, Michael A. Nalesnik, Takashi Kaizu, Takashi Uchiyama, Fang Liu, Noriko Murase, Anthony J. Bauer, Fritz H. Bach  Gastroenterology  Volume 127, Issue 2, Pages 595-606 (August 2004) DOI: 10.1053/j.gastro.2004.05.059

Figure 1 In vitro contractile activity of control nonoperated and transplanted jejunal circular muscularis that did not undergo a period of preservation is shown. Jejunal circular muscle strips from control animals with saline and nonoperated controls receiving biliverdin treatment showed a dose-dependent increase in contractile area in response to bethanechol. This activity was significantly diminished in graft muscle taken 24 hours following transplantation without preservation. Significant improvement was measured in transplanted animals treated with biliverdin (P < 0.05 biliverdin vs. saline treated rats; n = 5 or 6 rats/group). Gastroenterology 2004 127, 595-606DOI: (10.1053/j.gastro.2004.05.059)

Figure 2 (A-D) Representative images of histochemically stained full-thickness muscularis whole mounts for the presence of MPO-positive PMN extravasated into muscularis of the transplanted grafts without preservation. (A) The muscularis whole mount from nonoperated animals treated with saline was virtually devoid of MPO positive leukocytes. (B) Biliverdin-treated control animals showed a slight increase in MPO-positive cells 24 hours after injection. (C) Transplanted graft muscularis from saline-treated rats showed the presence of numerous extravasated MPO-positive cells within the graft muscularis 24 hours after SITx (without preservation). (D) Transplanted graft muscularis from biliverdin-treated rats showed a significant decrease in the number of MPO-positive cells present in the tissue 24 hours after SITx (without preservation). (E) Histogram quantifying the number of extravasated PMN within muscularis externa from each group. Although SITx with saline resulted in significant cellular inflammatory response within the muscularis, biliverdin treatment significantly decreased the number of MPO positive cells, which extravasated in response to transplantation. Images are representative of 5 rats/group; ∗P < 0.02 vs. grafts from saline-treated rats. Gastroenterology 2004 127, 595-606DOI: (10.1053/j.gastro.2004.05.059)

Figure 3 RT-PCR analysis revealed a significant increase in mRNA expression present in the nonpreserved graft muscularis for IL-6 (A), IL-1β (B), TNF-α (C), and ICAM-1 (D) 4 hours after transplantation compared with nonoperated animals. In the animals treated with biliverdin, the mean expressions for IL-6, IL-1β, and ICAM-1 mRNA were significantly reduced. Expression of TNF-α mRNA showed no significant difference vs. controls. Results are mean ± SEM of 5 samples from 5 rats/group (∗P < 0.05 vs. saline-treated recipients). SITx without preservation results in significant up-regulation of iNOS (E) and COX-2 (F) expression in the graft muscularis vs. nonoperated animals, which are significantly reduced in those animals treated with biliverdin. Results are mean ± SEM of 5 samples from 5 rats/group. ∗P < 0.02 vs saline-treated recipients; BV, biliverdin. Gastroenterology 2004 127, 595-606DOI: (10.1053/j.gastro.2004.05.059)

Figure 4 RT-PCR analysis revealed a significant increase in mRNA expression present in the nonpreserved graft muscularis 4 hours after transplantation for the anti-inflammatory genes, IL-10 (A) and HO-1 (B) mRNA, as well as the antioxidant enzyme MnSOD (C). Results showed no significant difference in IL-10 and HO-1 mRNA in the saline vs. biliverdin-treated animals. However, induction of MnSOD mRNA was completely abrogated by biliverdin injection. Results are mean ± SEM of 5 samples from 5 rats/group. ∗P < 0.05 vs. saline-treated recipients; BV, biliverdin. Gastroenterology 2004 127, 595-606DOI: (10.1053/j.gastro.2004.05.059)

Figure 5 (A) Serosal microcirculatory blood flow of the intestine from nontransplanted rats ranged from 35 to 48 mL/min/100 g tissue. Blood flow in transplanted intestine following 6 hours cold preservation showed that blood flow decreased to below 15 mL/min/100 g when measured 3 hours after reperfusion. Biliverdin administration had no effect on microcirculatory blood flow measured at the same time point. Results are mean ± SEM of 4 measurements averaged from 4 rats/group. (B) A significant increase in gut permeability was observed in the 6-hour preserved transplanted intestine as measured by fluorescent dextran translocation. Biliverdin treatment maintained graft permeability, maintaining normal nonoperated control levels. Results are mean ± SEM of 4 rats/group. ∗P < 0.01 vs. saline-treated rats; solid bar, saline, shaded bar, biliverdin. Gastroenterology 2004 127, 595-606DOI: (10.1053/j.gastro.2004.05.059)

Figure 6 (A) Intestinal reductive capacity was assessed using a “total antioxidant power” kit. Biliverdin treatment for the nonoperated intestine increased reductive capacity in the intestine 3 hours after IP injection of biliverdin. Although reductive capacity was significantly impaired in the cold preserved (6 hours) grafts 1 hour after reperfusion, biliverdin treatment showed significant improvement of the capacity in the graft intestine. (B) Total MDA levels were determined in tissue grafts 1 hour after reperfusion. Note that biliverdin treatment resulted in reduced MDA. Results are mean ± SEM of 4 rats/group; ∗P < 0.05 vs. saline-treated recipients. (C) Effects of biliverdin in the presence of NF-κB inhibition. Biliverdin-induced inhibition in graft permeability is significantly less in the presence of SN50, a selective inhibitor of NF-κB. ∗P < 0.05 vs. biliverdin-treated rats (n = 6 rats/group. (D) Effects of SN50 on biliverdin (Bv)-induced NF-κB binding. Animals were treated with SN50 (SN) followed by Bv. NF-κB DNA binding evaluated by EMSA in tissue grafts 3 hours after injection of biliverdin. Note that SN50 prevented DNA binding of NF-κB. Blot is representative of 3 independent experiments from 3 to 6 rats/group. CC, cold competition; FP, free probe. Gastroenterology 2004 127, 595-606DOI: (10.1053/j.gastro.2004.05.059)

Figure 7 (A) Six hours of preservation prior to transplantation resulted in an overall animal survival rate in saline-treated control rats for 14 days of 57.1%. Significant prolongation of transplant recipient’s survival of 100% was achieved by biliverdin administration (∗P < 0.02 vs. saline-treated rats; BV, biliverdin). (B) The effects of biliverdin administration on transplant-induced intestinal injury were evaluated histopathologically using 6-hour cold preserved and transplanted grafts. Massive loss of intestinal epithelial cells, severe mucosal erosion, and hemorrhage were seen in the saline-treated, 6-hour cold-preserved grafts (3 hours after reperfusion). Biliverdin-treated grafts showed less severe erosion and hemorrhage. Loss of the intestinal epithelial cells was limited at the top of the villi. Images are representative of 4 rats/group. (H&E, original magnification ×200.) Gastroenterology 2004 127, 595-606DOI: (10.1053/j.gastro.2004.05.059)