Degenerative Alterations in RVOT Samples – What to learn from Histopathological Analysis? Peivandi A.D. 1 , Seiler M. 1 , Asfour B. 2 , Sindermann J. 1 , Martens S. 1 , Osada, N. 1 , Malec E. 3 , Lück S. 3 1 Klinik für Herz- und Thorax-Chirurgie, Abteilung für Herzchirurgie, Universitätsklinikum Münster, Münster, Germany, 2 Deutsches Kinderherzzentrum, Kinderherz- und Thoraxchirurgie, Sankt Augustin, Germany, 3 Klinik für Herz- und Thorax-Chirurgie, Abteilung für Kinderherzchirurgie, Universitätsklinikum Münster, Münster, Germany Introduction: Re-obstruction of the RVOT is a common phenomenon in CHD-patients that lacks broader histopathological analysis and understanding. To examine the histological correlation of degenerative alterations , explanted specimen were processed and investigated. Methods: Human (homologous and autologous), bovine (pericardium and jugular vein conduits) and other explants (synthetic, equine or porcine) of 42 patients (male: n=27, 64.3%; female: n=15, 35.7%), who underwent re-operation of the RVOT at a median age of 12.5 years [1.9-57.0 years] (median 8.8 years [1.3-49.2years] after primary surgery), were analysed. Alizarin red S staining was used to analyse calcification degree. Nuclear fast red dye and Image J analysis enabled determination of nuclei per mm 2 . Immunohistochemistry, in detail expression of Tissue Inhibitor of Metalloproteinase 1 (TIMP 1) and Matrix Metalloproteinase 9 (MMP 9) was performed, then examined microscopically. Forschungslabor Herzchirurgie, Klinik für Herz- und Thorax-Chirurgie, Universitätsklinikum Münster, Münster, Germany E-Mail: [email protected]; www.klinikum.uni-muenster.de Macroscopic degeneration: Count of nuclei per mm 2 : 11,02 11,68 14,12 0 2 4 6 8 10 12 14 Human Bovine Other Average nuclei per mm 2 Picture 5: 10x Nuclear fast red staining displaying nuclei in a bovine explant of a nine-year-old. Nuclei were counted using ImageJ software. Degree of Calcification in different explants: Human 0 Human 1 Human 2 Human 3 Bovine 0 Bovine 1 Bovine 2 Bovine 3 Other 0 Other 1 Other 2 Other 3 Future Questions and Goals of Research: • What is the chemical composition of calcification encountered? • What role do inflammatory cells play in re- obstruction on a histological level? • How can neo-endothelialisation be characterised? • How are degenerative alterations linked to clinical data? Immunohistochemistry TIMP1 and MMP9 staining: Calcification in different types of explants – Alizarin red S staining: Picture 4: Massive calcification clod in a bovine explant Picture 3: Synthetic PTFE patch (GORETEX ® ) showing calcification inside and on the surface Picture 2: Calcified surface of an autologous pericardial patch . • Calcification was a major cause for early re-operation • Severe calcification was encountered in a range of different types of explants • Human explants showed a lower degree of calcification than bovine tissue and other materials Conclusion: • Degenerative changes were present in most of the analysed samples • Severe calcification was encountered in a range of different types of explants leading to early re-operation • Human explants were on average less calcified than bovine and other materials • Count of nuclei per mm 2 was lower in human and bovine samples • Balance of MMP 9 and TIMP 1 seems to be shifted towards MMP 9 Picture 6: 10x EvG staining displaying Contegra ® of a ten-year-old Picture 7: 10x MMP 9 staining displaying Contegra ® of a ten-year-old Picture 8: 10x TIMP 1 staining displaying Contegra ® of a ten-year-old Picture 1: Explanted Contegra ® Conduit (bovine jugular vein in pulmonary position) of a two-year-old showing severe calcification and valve stenosis. • Dark shades represent higher calcified samples in the different examined groups • All in all, 18 of 42 samples were extremely calcified
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Degenerative Alterations in RVOT Samples – What to learn from
Histopathological Analysis? Peivandi A.D.1, Seiler M.1, Asfour B.2, Sindermann J.1, Martens S.1, Osada, N.1, Malec E.3, Lück S.3 1Klinik für Herz- und Thorax-Chirurgie, Abteilung für Herzchirurgie, Universitätsklinikum Münster, Münster, Germany, 2Deutsches Kinderherzzentrum, Kinderherz- und
Thoraxchirurgie, Sankt Augustin, Germany, 3Klinik für Herz- und Thorax-Chirurgie, Abteilung für Kinderherzchirurgie, Universitätsklinikum Münster, Münster, Germany
Introduction: Re-obstruction of the RVOT is a common phenomenon in CHD-patients that lacks broader histopathological analysis and understanding. To examine the
histological correlation of degenerative alterations , explanted specimen were processed and investigated.
Methods: Human (homologous and autologous), bovine (pericardium and jugular vein conduits) and other explants (synthetic, equine or porcine) of 42 patients (male:
n=27, 64.3%; female: n=15, 35.7%), who underwent re-operation of the RVOT at a median age of 12.5 years [1.9-57.0 years] (median 8.8 years [1.3-49.2years] after
primary surgery), were analysed.
Alizarin red S staining was used to analyse calcification degree. Nuclear fast red dye and Image J analysis enabled determination of nuclei per mm2.
Immunohistochemistry, in detail expression of Tissue Inhibitor of Metalloproteinase 1 (TIMP 1) and Matrix Metalloproteinase 9 (MMP 9) was performed, then
examined microscopically.
Forschungslabor Herzchirurgie, Klinik für Herz- und Thorax-Chirurgie, Universitätsklinikum Münster, Münster, Germany
E-Mail: [email protected]; www.klinikum.uni-muenster.de
Macroscopic degeneration:
Count of nuclei per mm2:
11,02 11,68
14,12
0
2
4
6
8
10
12
14
Human Bovine Other
Average nuclei per mm2
Picture 5: 10x Nuclear
fast red staining
displaying nuclei in a
bovine explant of a
nine-year-old. Nuclei
were counted using
ImageJ software.
Degree of Calcification in different explants:
Human 0
Human 1
Human 2
Human 3
Bovine 0
Bovine 1
Bovine 2
Bovine 3
Other 0
Other 1
Other 2
Other 3
Future Questions and Goals of
Research:
• What is the chemical composition of
calcification encountered?
• What role do inflammatory cells play in re-
obstruction on a histological level?
• How can neo-endothelialisation be
characterised?
• How are degenerative alterations linked to
clinical data?
Immunohistochemistry TIMP1 and MMP9 staining:
Calcification in different types of explants – Alizarin red S staining:
Picture 4:
Massive calcification clod in a bovine
explant
Picture 3:
Synthetic PTFE patch (GORETEX ®)
showing calcification inside and on the
surface
Picture 2:
Calcified surface of an autologous
pericardial patch
.
• Calcification was
a major cause for
early re-operation
• Severe
calcification was
encountered in a
range of different
types of explants
• Human explants
showed a lower
degree of
calcification than
bovine tissue and
other materials
Conclusion: • Degenerative changes were present in most of the analysed samples
• Severe calcification was encountered in a range of different types of explants leading to early re-operation
• Human explants were on average less calcified than bovine and other materials
• Count of nuclei per mm2 was lower in human and bovine samples
• Balance of MMP 9 and TIMP 1 seems to be shifted towards MMP 9
Picture 6:
10x EvG staining displaying
Contegra ® of a ten-year-old
Picture 7:
10x MMP 9 staining displaying
Contegra ® of a ten-year-old
Picture 8:
10x TIMP 1 staining displaying
Contegra ® of a ten-year-old
Picture 1:
Explanted Contegra® Conduit (bovine jugular
vein in pulmonary position) of a two-year-old
showing severe calcification and valve stenosis.
• Dark shades represent
higher calcified
samples in the
different examined
groups
• All in all, 18 of 42
samples were
extremely calcified
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