Die BSE-Krise: Lernen unter Nichtwissensbedingungen

Die BSE-Krise: Lernen unter Nichtwissensbedingungen / S. Böschen ... - In: Handeln trotz Nichtwissen : vom Umgang mit Chaos und Risiko in Politik, Industrie und Wissenschaft / Stefan Böschen ... (Hg.). - Frankfurt/Main u.a. : Campus-Verl., 2004. - S. 99-12

<i>In vivo</i> transplantation of LPC and HPC in injured muscles.

<p>(A) Experimental plan: single GFP+LPC and HPC were transplanted in <i>Tibialis Anterior</i> 2 days after cardiotoxin injury and muscle sections were analysed 30 days after cell injection. (B and C) GFP+LPC and HPC cultured at 20% and 2% O₂ transplanted in damaged muscles. Haematoxylin and Eosin staining revealed a physiological muscle structure in both conditions 1 month post injection (upper row, bar = 100 µm). GFP+fibers were present in both groups with an increased presence in the LPC treated group in respect to HPC (staining for Laminin and GFP on transplanted TA sections, nuclei were counterstained with DAPI; lower row, bar = 100 µm). (D–G) Graphs of row numbers and percentages of GFP+fibers and GFP+centrally nucleated fibers in LPC and HPC 20% or 2% O₂ treated muscles. (D and E) On the left, number of total fibers (gray: GFP- fibers, black: GFP+fibers <i>per</i> field) in representative fields of transplanted muscle sections. On the right, average of the percentages of GFP+fibers. (F and G) On the left, number of centrally nucleated fibers (gray: GFP- fibers, black: GFP+fibers <i>per</i> field) in randomly selected representative fields of transplanted muscle sections. On the right, average of the percentages of GFP+and GFP- centrally nucleated fibers (LPC 20% O₂ n = 12, HPC 20% O₂ n = 10, LPC 2% O₂ n = 11, HPC 2% O₂ n = 8). TA injected with normoxia cultured LPC possessed one third more green fibers than HPC (D) while GFP+centrally nucleated fibers were found in the same proportion in both treated animal groups (F). For hypoxia cultured clones, muscles LPC injected possessed about twice the number of green fibers than HPC (E). Centrally nucleated fibers were found in the same proportion in both treated animal groups but the TA treated with clones cultured under 2% O₂ highlighted more centrally nucleated fibers than those transplanted with normoxia cultured clones (G) (no statistical differences between 20% and 2% O₂ results). (H) PCR analysis of transplanted TA with GFP+single clones. First lane: amplification of the <i>GFP</i> gene (307 bp). Second lane: amplification of genomic <i>TERT</i> (132 bp) to confirm the quality of the extracted DNA. For each PCR, the positive control (C+) for GFP amplification was the genomic DNA extracted from C57BL/6-(ACTB-EGFP)/J mice muscle (NTC = no template control). In all samples GFP protein was detected.</p

Marker expression in SC clones cultured under normoxia and hypoxia.

<p>Immunofluorescence analyses of LPC and HPC cultured for 5 days at 20% and 2% O₂ revealed differences in the expression of MyoD, Myf5 (only at 2% O₂) and CD34. Phase-contrast and corresponding immunostaining examples for the specific marker (in red) merged with DAPI (left, scale bar = 100 µm, insets with higher magnification). Graphs indicate the percentage of marker-positive cells <i>per</i> clone (right, mean±SEM, * p<0.05, ** p<0.01, *** p<0.001): α7integrin (A) and (B) (n = 50), MyoD (C) and (D) (n = 70), Myf5 (E) and (F) (n = 85), CD34 (G) and (H) (n = 50), CD31 (I) and (L) (n = 45). (M, N and O) <i>MyoD</i>, <i>Myf5</i> and <i>Hif-1α</i> gene expression quantification was performed using RealTime PCR. Graphs indicate the amount of <i>MyoD</i>, <i>Myf5</i> and <i>Hif-1α</i> expression respectively at single cell level (n = 4, mean±SEM, ANOVA analysis determined a significant difference among groups for <i>MyoD</i> and <i>Hif-1α</i> expression; Student t-test: * p<0.05, *** p<0.001).</p

Mouse satellite cell isolation, cloning and <i>in vitro</i> proliferation.

<p>(A) Experimental plan<b>.</b> For satellite cell (SC) isolation, hindlimb skeletal muscles of C57BL/6J mice were digested and single myofibers collected. SCs were mechanically disengaged (stripping passage) and cloned with the limited dilution method. For each experiment, cloned cells were randomly cultured in normoxic (20% O₂) or hypoxic (2% O₂) conditions with a proliferative medium. (B) After 5 days of culture, all the clones had a similar morphology (bar = 75 µm). (C) The number of clones generated in hypoxia was higher than in the normoxia conditions, expressed as the number of clones obtained starting from the same seeded cell number (n = 16, mean±SEM, * p<0.05). (D and H) Number of cells <i>per</i> clone distribution (example experiment) under normoxia or hypoxia conditions respectively (a model based cluster analysis with normal mixture modelling has been applied to identify the groups): cells distribute in 2 clusters easily separable with a cut-off value (light grey bar) in LPC and HPC. (E and I) Averages of the number of cells <i>per</i> clone both in LPC and HPC demonstrated that the proliferation increases in those clones cultured with 2% O₂ (total number of clones counted: 20% O₂ n = 600, 2% O₂ n = 700, mean±SEM, ** p<0.01, *** p<0.001). (F and L) Pie charts indicate LPC and HPC proportions calculated as percentage of the total number of clones (n = 16, mean±s.d., 20% O₂: LPC = 73%±8.1, HPC = 27%±8.1; 2% O₂: LPC = 69%±8.3, HPC = 31%±8.3; p<0.001). (G and M) ATP quantification: LPC show lower ATP content with respect to HPC, both in normoxia and hypoxia. ATP measurements uphold the higher number of cells both in LPC and HPC under hypoxia (n = 132, mean±SEM, *** p<0.001).</p

Spontaneous myogenic differentiation in LPC and HPC at day 5 of culture.

<p>(A) Spontaneous myotubes formation after 5 days of culture in proliferative medium was evaluated with desmin staining (in red). Immunofluorescence shows the appearance of single desmin+cells in LPC cultured in 20% O₂ and also some myotube formation in HPC at 20% O₂ and both in LPC and HPC cultured at 2% (bar = 100 µm, nuclei counterstained with DAPI). (B and C) Graphs indicate the number of myotubes <i>per</i> clone calculated for all the clone types (LPC 20% O₂ n = 390, HPC 20% O₂ n = 205, LPC 2% O₂ n = 425, HPC 2% O₂ n = 265; mean±SEM). Both LPC and HPC showed increased myotube formation when cultured at 2% O₂ (ANOVA analysis determined a statistical difference among the groups; Student t-test: p<0.05 for number of myotube/clone in hypoxia cultured clones with respect to both clone types cultured under normoxia).</p

<i>In vivo</i> engraftment potential of LPC and HPC single cells.

<p>(A) Graphs with the Engraftment Index average calculated as proportion between GFP+fibers percentage and number of GFP+transplanted cells (for raw data see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049860#pone.0049860.s005" target="_blank">Table S2</a>). In both normoxia and hypoxia conditions cells from LPC possess stronger myogenic potential (mean±SEM, p<0.01 with ANOVA, * p<0.05, ** p<0.01 with Student t-test). (B) Cartoon emphasizing on one hand the same myogenic potential at single cell level (dotted line) for LPC and HPC respectively when cultured at different conditions and, on the other (continuous line), the greater muscle regeneration ability of LPC cultured under 2% O₂. In this condition, LPC proliferation is enhanced and as a consequence the achievable myogenic engraftment is higher.</p

Quantification of extracellular matrix components in samples stored with the SCM protocol.

<p>Quantification of extracellular matrix components in samples stored with the SCM protocol.</p

Functional analysis of decellularised scaffolds.

<p>(A,B) Synchrotron analysis after 2 DET cycles confirmed extracellular matrix preservation in the lamina propria, submucosa and muscularis. (C) An intact basement membrane was detected across the entire scaffold segment. (D) Blood vessels were seen converging towards the scaffold in a spoked wheel manner after 10 days from placement on the CAM as confirmed by blinded quantification of the vessels compared to the negative control (**p< 0.01). (E) The maximum tensile stress at which the samples broke and the elasticity modulus remained comparable to fresh after decellularisation. (F) Characteristic stress-strain curves showing that by increasing the number of DET cycles the tensile stress at which the samples break remains the same as seen in E. DET = Detergent-Enzymatic Treatment, CAM = Chicken chorioallantoic membrane assay.</p

Decellularisation efficiency and scaffolds characterization.

<p>(A) Efficient cell removal after 2 DET cycles was evident by macroscopic appearance, H&E staining and DNA quantification. (B) Immunohistochemistry for extracellular matrix composition. Masson’s Trichrome and Picrosirius Red staining demonstrated collagen preservation in both submucosa and among muscle fibers (inset). Elastin Van Gieson staining showed elastic fibers in the submucosa, around blood vessels and surrounding muscle fascicles both in the fresh and DET tissues. Muscular elastin staining was reduced after 3 DET cycles (inset). Alcian Blue staining indicated glycosaminoglycan preservation (bar = 100μm). (C) Extracellular component quantification demonstrated a gradual decrease in collagen after the first and second DET cycle. Elastin decreased after 3 DET cycles. glycosaminoglycan were partially reduced by the first DET cycle. DET = Detergent-Enzymatic Treatment, MT = Masson’s Trichrome, PR = Picrosirius Red, EVG = Elastin Van Gieson, AB = Alcian Blue. *p<0.05; **p<0.01.</p

Macro- and microscopic appearance of stored decellularised scaffolds at 6 months.

<p>(A) Macroscopic appearances varied in the two protocols. (B) Scanning electron microscopy analysis. While SCM scaffolds demonstrated a good preservation of all oesophageal layers, in 4°C samples extracellular matrix was falling apart with signs of degradation. SCM = slow cooling medium, 4°C = 4°C in PBS.</p