Time course and cellular localization of inducible nitric oxide synthases expression during cardiac allograft rejection Neil K Worrall, MD, Thomas P.

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Time course and cellular localization of inducible nitric oxide synthases expression during cardiac allograft rejection Neil K Worrall, MD, Thomas P Misko, PhD, Mitchell D Botney, MD, Patrick M Sullivan, MA, Jia-J Hui, MD, Gloria M Suau, MD, Pamela T Manning, PhD, T.Bruce Ferguson, MD The Annals of Thoracic Surgery Volume 67, Issue 3, Pages 716-722 (March 1999) DOI: 10.1016/S0003-4975(98)01346-0

Fig 1 Representative ribonuclease protection assay gel demonstrating inducible nitric oxide synthase (iNOS) mRNA expression in allograft hearts but not isograft or normal hearts. Grafted hearts were harvested on the indicated postoperative day and ribonuclease protection assay performed independently using rat iNOS and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; internal control) antisense probes. Lanes are as follows: 1, undigested probe; 2, probe hybridized with transfer RNA demonstrating that protection was specific for iNOS mRNA; 3, normal heart; 4 to 9, allograft hearts on postoperative days 3, 4, 5, 6, 8, and 10, respectively; 10 and 11, isograft hearts on days 5 and 8, respectively. The same results were obtained with duplicate samples in these animals and in two additional animals at each time point. The Annals of Thoracic Surgery 1999 67, 716-722DOI: (10.1016/S0003-4975(98)01346-0)

Fig 2 Inducible NOS (iNOS) enzyme activity present in allograft and isograft hearts harvested on the indicated postoperative day. Normal hearts produced 0.11 ± 0.11 pmol l-citrulline · mg protein−1 · min−1 (n = 7, P > 0.9 vs isografts and day 3 allograft). (Mean ± SEM; n = 4 to 8; ∗P < 0.0005 vs normal, isograft, and day 3 allograft; †P < 0.0005 vs normal and isograft and P = 0.002 vs day 3 allograft; and ‡P < 0.0005 vs normal, P = 0.002 vs isograft, and P = 0.02 vs day 3 allograft.) The Annals of Thoracic Surgery 1999 67, 716-722DOI: (10.1016/S0003-4975(98)01346-0)

Fig 3 Time course of increased in vivo nitric oxide production in allografts. Serum nitrite/nitrate levels were determined in allografts and isografts on the indicated postoperative day. Serum nitrite/nitrate levels in normals were 15 ± 1 μmol/L (n = 13, P > 0.2 vs isografts and day 3 allograft). (Mean ± SEM; n = 7 for isografts and n = 8 to 14 for allografts; ∗P = 0.0002 vs normal and isograft and P < 0.05 vs day 3 allograft; †P = 0.0002 vs normal, isograft, day 3 allograft, and P < 0.005 vs day 4 allograft; and ‡P = 0.0002 vs normal, P < 0.002 vs isograft and days 5 and 6 allograft, and P < 0.05 vs day 3 allograft.) The Annals of Thoracic Surgery 1999 67, 716-722DOI: (10.1016/S0003-4975(98)01346-0)

Fig 4 Representative darkfield and brightfield photomicrographs of in situ hybridization with rat iNOS antisense and sense mRNA probes demonstrating iNOS mRNA expression in infiltrating mononuclear inflammatory cells in allografts (oriented with the left ventricular cavity at top). (A) No iNOS expression in day 3 allograft. (B) iNOS expression in infiltrating mononuclear inflammatory cells in day 4 allograft but not in adjacent myocytes. (C) iNOS expression in interstitial space in day 6 allograft but not in myocytes. (D) iNOS expression in interstitial space in day 8 allograft. (E) No hybridization with the sense probe in the same day 8 allograft section. (F) iNOS mRNA was not detected in day 6 isografts. Similar results were obtained in 3 animals at each time. (Eosin; 100× except 1,000× for [B].) The Annals of Thoracic Surgery 1999 67, 716-722DOI: (10.1016/S0003-4975(98)01346-0)

Fig 5 Representative sections from allograft and isograft hearts stained with iNOS antisera. (A) Interstitial inflammatory cells expressed iNOS and appear to outline the myocytes, which did not stain for iNOS. (B) Perivascular infiltrating cells were positive for iNOS. iNOS was not present in endothelial cells or vascular smooth muscle cells. (C) Higher power demonstrating that iNOS-positive cells are mononuclear inflammatory cells. (D) There was no staining in the isograft heart, the normal heart (not shown), or with preimmune sera in any animal (not shown). Similar results were examined in 3 animals per group. (Mayer’s hematoxylin; 100× except 400× for [C].) The Annals of Thoracic Surgery 1999 67, 716-722DOI: (10.1016/S0003-4975(98)01346-0)