Role of Airway Epithelial Injury in Murine Orthotopic Tracheal Allograft Rejection Elbert Kuo, MD, Ankit Bharat, MD, Jennifer Shih, BS, Tyler Street,

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Role of Airway Epithelial Injury in Murine Orthotopic Tracheal Allograft Rejection Elbert Kuo, MD, Ankit Bharat, MD, Jennifer Shih, BS, Tyler Street, Jenyi Norris, BS, Wei Liu, MD, William Parks, PhD, Michael Walter, MD, G. Alexander Patterson, MD, T. Mohanakumar, PhD The Annals of Thoracic Surgery Volume 82, Issue 4, Pages 1226-1233 (October 2006) DOI: 10.1016/j.athoracsur.2006.03.122 Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 (A) Orthotopic tracheal transplant model in which a 6-ring segment of tracheal is transplanted and secured with three 10-0 nylon sutures at both ends. Sections were taken at approximately 100, 250, and 400 vm (4-μm thickness), represented by the yellow lines. (B) Using Image Pro Express v 4.0, percent fibrosis was calculated by dividing the area between the basement membrane and the cartilage by the total area enclosed by the tracheal cartilage. (C) The ratio of the lamina propria to cartilage thickness was measured at 90, 180, and 270 degrees with the membranous portion of the trachea positioned at the bottom of the section. The Annals of Thoracic Surgery 2006 82, 1226-1233DOI: (10.1016/j.athoracsur.2006.03.122) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Hematoxylin and eosin staining of tracheas after transplantation. (A) Normal trachea. (B) Orthotopic isografts exhibit normal tracheal architecture with ciliated columnar epithelium. (C) Orthotopic allografts demonstrated a combination of cuboidal and ciliated columnar epithelial cells and a multicellular subbasement membrane infiltrate (I) at day 30 after transplantation. (D) Complete reepithelialization in orthotopic tracheal allografts by day 60 showing ciliated columnar epithelium and fibrosis (F). Arrows represent normal ciliated columnar epithelium and arrowheads represent denuded regenerating cuboidal epithelium. The Annals of Thoracic Surgery 2006 82, 1226-1233DOI: (10.1016/j.athoracsur.2006.03.122) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Orthotopic tracheal allografts (black bars) demonstrated increased (A) percent fibrosis and (B) thickness of lamina propria to cartilage ratio (LCR) at 30, 60, and 180 day time points compared with isografts (white bars). (C) The increase in fibrosis and LCR in allografts correlated with greater CD4+ and CD8+ lymphocytic infiltration (illustrated for day 30). (HPF = high-power field.) The Annals of Thoracic Surgery 2006 82, 1226-1233DOI: (10.1016/j.athoracsur.2006.03.122) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Allografts infected with a 5K dose of Sendai virus 15 days after transplantation showed delayed reepithelialization and increase in fibrosis (F). (A) There was incomplete reepithelialization and intramural fibrosis of tracheal allografts at day 60 along with fibrosis and cellular infiltration (I). (B) Infected isograft recipients at day 60 showed normal airway epithelium and minimal fibrosis. Arrows represent normal ciliated columnar epithelium and arrowheads represent denuded regenerating cuboidal epithelium. The Annals of Thoracic Surgery 2006 82, 1226-1233DOI: (10.1016/j.athoracsur.2006.03.122) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 Allografts infected with a 5K dose of Sendai virus 15 days after transplantation (black bars) showed increase in (A) fibrosis and (B) thickness of lamina propria to cartilage ratio (LCR) at day 60 compared with uninfected controls (white bars). For comparison, fibrosis in infected and uninfected isografts is illustrated. The Annals of Thoracic Surgery 2006 82, 1226-1233DOI: (10.1016/j.athoracsur.2006.03.122) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 6 The matrix metalloproteinase-7 knockout (MMP7-KO) recipients of orthotopic allografts demonstrated demonstrated areas of denuded epithelium at day 30 after transplantation. That correlated with high percent fibrosis (F) and a persistent subbasement membrane infiltrate (I). Arrows represent normal ciliated columnar epithelium and arrowheads represent denuded regenerating cuboidal epithelium. The Annals of Thoracic Surgery 2006 82, 1226-1233DOI: (10.1016/j.athoracsur.2006.03.122) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions