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Histological assessment of chondrocyte transplants
298
State-of-the-artlecture: From Stem Cells to Germ Cells Tumors and Back / Pathology - Research and Practice 200 (2004) 298
Aims: Activating of vasoactive mediators and inflammatory reactions are a major determinant of ischemia/reperfasion (I/R) injury after organ transplantation. We investigated the gene expression and synthesis of proinflammatbry cytokines (TNFct, IL-113, IL-6), prepro-endothelin-1 and endothelinA receptor (ETa-R) and their effects on morphologic changes after warm ischemia of the liver (LI) and pancreas transplantation (PTx) in a pig model. Methods: Two different models were used: 1. Total vascular exclusion of the liver for 2 h (n = 7). 2. PTx after 6 h of cold ischemia (n = 7). For measurement of mRNA-expression of prepro-ET-1, ET Areceptor, TNFct, IL-1[~ and IL-6 a real-time quantitative RT-PCR (TaqMan) was performed. Histologic assessment and immunohistochemical analysis (IHC) were graded semiqua.ntitatively. Plasma ET- 1 concentrations were determined by ELISA. Results: In both experiments RT-PCR lh after reperfusion revealed a significant upregulation of prepro -ET-1 and TNFct; ETA-receptor expression showed significant downregulation (p < 0.05). Expression of IL-113 and IL-6 was upregulated (p < 0.05). ET-1 plasma concentrations showed a significant increase. Histomorphologic analysis revealed interstitial edema, hemorrhage and leukocyte infiltration in both models. Immunostaining of ET-1 and ETA-receptor showed a significant increase lh after reperfusion (p < 0.05)~ Conclusion: Using quantitative RT-PCR a significant upregulation of mRNA-expression of ET-1 and proinflammatory cytokines was measured in both experimental models. It is responsible for initialisation of variable inflammatory cascades and a major determinant of postischemic microcirculatory disorders in liver and pancreas. Consequently blocking of ETA-receptors could be a promising approach for reducing ischemia/reperfusion injury.
162
HistologicalAssessmentof ChondrocyteTransplants
T. AIGNER 1~, M. BRITTBERG 2, P. BULLOUGH 2, A. HOLLANDER 2, E. HUNZIKER 2, R. KANDEL 2, S. NEHRER 2, K. PRITZKER 2, S. ROBERTS 2, E. STAUFFER2, P. MAINIL-VARLET2 1Institute of Pathology, University of Erlangen-Ntirnberg zHistology Endpoint Committee of the International Cartilage Repair Society (ICRS) Aims: Auto- and heterologous chondrocyte/cartilage transplantation is a very attractive upcoming therapeutic option for localized cartilage defects in the larger joints. So far, no accepted histological criteria for the determination of the outcome of such therapies are available. The aim of the Committee of the International Cartilage Repair Society was therefore the determination of histopathological criteria for evaluation of chondrocyte transplants. Methods: Conventional histology using hematoxylin-eosin stainings was performed on conventionally processed osteochondral biopsies obtained after chondrocyte transplantation. Results: Evaluation criteria and categories were identified and graded according to clinico-biological hypotheses: surface integrity, matrix appearance, cell distribution pattern, cellular viability, appearance of the subchondral bone, and cartilage mineralization pattern were named to be important parameters. All different categories axe supposed to be evaluated and scored by the observer at the same biopsy seperately. Basic requirements for sufficient bioptical specimens are a fullthickness biopsy from the transplantation area. Conclusions: This represents the first international consensus approach for the diagnosis and evaluation of cartilage repair tissue in particular after chondrocyte transplantation. It will be the basis for further testing and refining of generally accepted criteria and will help to evaluate outcome and prognosis of chondrocyte transplant approaches in the future.
: i ! J
16
.~
Clinical effects of androgen receptors
H. KLOCKER, Innsbruck
164
Genetic causes of male infertility
W. ENGEL, G6ttingen
16 5
From Testicular Biopsy to Human Embryo
D.~2EK Institute of Histology and Embryology, Medical School University of Zagreb Aims: The aim of the study was to investigate the role of a testicular biopsy in a diagnosis and therapy of infertile men with a nonobstructive azoospermia. Materials & methods: Overall, 70 testicular biopsies from infertile men were analysed. Samples were obtained by the "open testicular biopsy" method. Several pieces of the tissue (after dissection), were immediately immersed into the Sperm Prep (Medi-Cult) medium and fixative. As fixatives, 5.5% buffered glutaraldehyde and Bouin's fluid were used. Tissue samples transported in Sperm Prep medium were plunged into Sperm Freezing medium (MediCult) and were stored in the liquid nitrogen for potential in vitro fertilization procedures. The tissue fixed in glntaraldehyde and Bouin's fluid was processed for semi-thin sections & transmission electron microscopy and immunohistochemistry (IHC), respective-
ly. Results: Semi-thin sections from 8 infertile patients demonstrated regular testis structure and fully preserved spermatogenesis (control biopsies). In the remaining 62 cases, spermatogenesis was impaired and a variety of pathological changes could be seen: disorganization and desquamation of spermatogenic cells, spermatid or spermatocyte "stop", spermatogonia only, "Sertoli cells only" or tubular fibrosis. However, in 70% of cases (despite the above mentioned changes of seminiferous epithelium) foci of preserved spermatogenesis could be detected. IHC analysis showed increased presence of macrophages and mast cells in infertile group of patients as well as a decreased expression of testosterone. In 64% of infertile patients, a successful extraction of sperms from the biopsy could be performed. Conclusion: In azoospermic patients, histological analysis of testicular biopsy can be very useful in terms of diagnosis as well as therapy, i.e. further in vitro fertilization procedures.
166
From Stem Cells to Germ Cell Tumors and Back
I. DAMJANOV, Kansas City
State-of-the-artlecture: From Stem Cells to Germ Cells Tumors and Back / Pathology - Research and Practice 200 (2004) 298
Aims: Activating of vasoactive mediators and inflammatory reactions are a major determinant of ischemia/reperfasion (I/R) injury after organ transplantation. We investigated the gene expression and synthesis of proinflammatbry cytokines (TNFct, IL-113, IL-6), prepro-endothelin-1 and endothelinA receptor (ETa-R) and their effects on morphologic changes after warm ischemia of the liver (LI) and pancreas transplantation (PTx) in a pig model. Methods: Two different models were used: 1. Total vascular exclusion of the liver for 2 h (n = 7). 2. PTx after 6 h of cold ischemia (n = 7). For measurement of mRNA-expression of prepro-ET-1, ET Areceptor, TNFct, IL-1[~ and IL-6 a real-time quantitative RT-PCR (TaqMan) was performed. Histologic assessment and immunohistochemical analysis (IHC) were graded semiqua.ntitatively. Plasma ET- 1 concentrations were determined by ELISA. Results: In both experiments RT-PCR lh after reperfusion revealed a significant upregulation of prepro -ET-1 and TNFct; ETA-receptor expression showed significant downregulation (p < 0.05). Expression of IL-113 and IL-6 was upregulated (p < 0.05). ET-1 plasma concentrations showed a significant increase. Histomorphologic analysis revealed interstitial edema, hemorrhage and leukocyte infiltration in both models. Immunostaining of ET-1 and ETA-receptor showed a significant increase lh after reperfusion (p < 0.05)~ Conclusion: Using quantitative RT-PCR a significant upregulation of mRNA-expression of ET-1 and proinflammatory cytokines was measured in both experimental models. It is responsible for initialisation of variable inflammatory cascades and a major determinant of postischemic microcirculatory disorders in liver and pancreas. Consequently blocking of ETA-receptors could be a promising approach for reducing ischemia/reperfusion injury.
162
HistologicalAssessmentof ChondrocyteTransplants
T. AIGNER 1~, M. BRITTBERG 2, P. BULLOUGH 2, A. HOLLANDER 2, E. HUNZIKER 2, R. KANDEL 2, S. NEHRER 2, K. PRITZKER 2, S. ROBERTS 2, E. STAUFFER2, P. MAINIL-VARLET2 1Institute of Pathology, University of Erlangen-Ntirnberg zHistology Endpoint Committee of the International Cartilage Repair Society (ICRS) Aims: Auto- and heterologous chondrocyte/cartilage transplantation is a very attractive upcoming therapeutic option for localized cartilage defects in the larger joints. So far, no accepted histological criteria for the determination of the outcome of such therapies are available. The aim of the Committee of the International Cartilage Repair Society was therefore the determination of histopathological criteria for evaluation of chondrocyte transplants. Methods: Conventional histology using hematoxylin-eosin stainings was performed on conventionally processed osteochondral biopsies obtained after chondrocyte transplantation. Results: Evaluation criteria and categories were identified and graded according to clinico-biological hypotheses: surface integrity, matrix appearance, cell distribution pattern, cellular viability, appearance of the subchondral bone, and cartilage mineralization pattern were named to be important parameters. All different categories axe supposed to be evaluated and scored by the observer at the same biopsy seperately. Basic requirements for sufficient bioptical specimens are a fullthickness biopsy from the transplantation area. Conclusions: This represents the first international consensus approach for the diagnosis and evaluation of cartilage repair tissue in particular after chondrocyte transplantation. It will be the basis for further testing and refining of generally accepted criteria and will help to evaluate outcome and prognosis of chondrocyte transplant approaches in the future.
: i ! J
16
.~
Clinical effects of androgen receptors
H. KLOCKER, Innsbruck
164
Genetic causes of male infertility
W. ENGEL, G6ttingen
16 5
From Testicular Biopsy to Human Embryo
D.~2EK Institute of Histology and Embryology, Medical School University of Zagreb Aims: The aim of the study was to investigate the role of a testicular biopsy in a diagnosis and therapy of infertile men with a nonobstructive azoospermia. Materials & methods: Overall, 70 testicular biopsies from infertile men were analysed. Samples were obtained by the "open testicular biopsy" method. Several pieces of the tissue (after dissection), were immediately immersed into the Sperm Prep (Medi-Cult) medium and fixative. As fixatives, 5.5% buffered glutaraldehyde and Bouin's fluid were used. Tissue samples transported in Sperm Prep medium were plunged into Sperm Freezing medium (MediCult) and were stored in the liquid nitrogen for potential in vitro fertilization procedures. The tissue fixed in glntaraldehyde and Bouin's fluid was processed for semi-thin sections & transmission electron microscopy and immunohistochemistry (IHC), respective-
ly. Results: Semi-thin sections from 8 infertile patients demonstrated regular testis structure and fully preserved spermatogenesis (control biopsies). In the remaining 62 cases, spermatogenesis was impaired and a variety of pathological changes could be seen: disorganization and desquamation of spermatogenic cells, spermatid or spermatocyte "stop", spermatogonia only, "Sertoli cells only" or tubular fibrosis. However, in 70% of cases (despite the above mentioned changes of seminiferous epithelium) foci of preserved spermatogenesis could be detected. IHC analysis showed increased presence of macrophages and mast cells in infertile group of patients as well as a decreased expression of testosterone. In 64% of infertile patients, a successful extraction of sperms from the biopsy could be performed. Conclusion: In azoospermic patients, histological analysis of testicular biopsy can be very useful in terms of diagnosis as well as therapy, i.e. further in vitro fertilization procedures.
166
From Stem Cells to Germ Cell Tumors and Back
I. DAMJANOV, Kansas City