Intraoperative situs: the free peroneus brevis muscle flap, obliterates and closes the defect (flap anastomosis to the lateral tarsal artery).

<p>Intraoperative situs: the free peroneus brevis muscle flap, obliterates and closes the defect (flap anastomosis to the lateral tarsal artery).</p

Preoperative planning of the arterialized venous flap.

<p>Bypass is anastomosed to the popliteal artery proximally, and the medial plantar artery distally.</p

60 y. old patient with diabetes after reconstruction with a parascapular flap covering exposed bone and plantar surface.

<p>60 y. old patient with diabetes after reconstruction with a parascapular flap covering exposed bone and plantar surface.</p

55y.old patient with diabetes and peripheral arterial disease and defect of the heel and exposed bones.

<p>The patient required flap and bypass. A reversed greater saphenous bypass was planned including an arterialized venous flap. The bypass supplies the flap by arterial means. 2 separate veins are anastomosed to provide venous outflow.</p

Scratching Assay of primary glial cell cultures.

<p>Cells were incubated with 100 µM minocycline for 72 h after wound scratching. All values in (A) are given as mean distance % of the starting distance of the wound ± SD. Closure of the wound was delayed in minocycline incubated cells compared to the control after 24 and 72 h. Images in (B) show phase contrast images of cultures with control cells and minocycline treated cells after 0, 24, 48 and 72 h incubation time. Wound distances are marked with black <b>lines</b>. Minocycline delayed wound closure compared to the control. Bars illustrate 250 µm. Statistical analysis was done with a paired Student’s t-test. Significant differences are marked with * and demonstrate p<0.05. The number of samples was n = 17 for 24 and 72 h and n=14 for 48 h.</p

Treatment of organotypic spinal cord cultures with 100 µM minocycline.

<p>Comparison of organotypic spinal cord cultures incubated with minocycline starting at DIV 1 or DIV 4 and analyzed at DIV 7. (A) Number of surviving motor neurons (anti-pan-NF stained). Minocycline incubation decreased this number significantly compared to controls. (B) Number of anti-NeuN stained neurons. Minocycline incubation reduced this number significantly compared to controls. (C) Percentage of anti-pan-NF stained area (neurofilaments). Minocycline incubation from DIV 1 onwards increased the percentage of neurofilament staining. Minocycline incubation from DIV 4 onwards, however, did not alter it. (D) Percentage of DAPI stained area. Minocycline incubation significantly decreased the DAPI cell nuclei staining in cultures incubated from DIV 1 and DIV 4 onwards. (E) Percentage of anti-IBA-1 stained area (microglia). Minocycline incubation reduced microglia pattern significantly compared to the control. (F) Anti-IBA-1 fluorescence staining intensity. Minocycline incubation reduced the fluorescence intensity of microglia in accordance with the reduced microglia spreading. (G) Percentage of anti-GFAP stained area (astroglia). Minocycline incubation did not influence the percentage of astroglia staining significantly if cultures were incubated from DIV 1 onwards. Incubation starting at DIV 4, however, reduced the percentage of astroglia staining significantly compared to controls. (H) Anti-GFAP fluorescence staining intensity. Minocycline incubation influenced fluorescence intensity of astroglia in accordance with the changes of area. If treatment started at DIV 1, fluorescence intensity was enhanced, whereas under treatment started at DIV4 a reduction was found. For statistical analysis a paired Student’s t-test was used to compare control and +Mino for each incubation period. Numbers of samples are: number of motor neurons and percentage of anti-pan-NF area: DIV 1 n=26, DIV 4 n=19; number of anti-NeuN stained neurons n=6 (DIV 1, DIV 4); percentage stained area of anti-IBA-1, anti-GFAP and DAPI: DIV 1 n=7, DIV 4n=6 (1 animal = mean of up to 3 replicates). All values are means ± standard deviation (SD). Significant differences are marked with * and demonstrate p<0.05.</p

Western Blot analysis of primary glial cell cultures.

<p>Primary glial cell cultures were incubated for 72 h with 100 µM minocycline (+Mino). Control cells received medium without any supplement. Values in (A) are given as mean ratio of β-actin/GAPDH density ± SD and values in (C) are given as mean ratio of Cx43/GAPDH density ± SD. (<b>B</b>) shows representative images of β-actin bands of control and +Mino cells with corresponding GAPDH bands. (<b>D</b>) displays representative images of Cx43 bands of control and +Mino cells with corresponding GAPDH bands. Minocycline did not change the expression of β-actin but enhanced the expression of Cx43 compared to control cells. Statistical analysis was done with a paired Student’s t-test. A p-value ≤ 0.05 was considered to be statistically significant and is marked with *. The number of samples was n = 7.</p

Treatment of organotypic spinal cord cultures with 10 µM minocycline.

<p>Comparison of cultures incubated from DIV 1 or DIV 4 onwards and analyzed at DIV 7. (<b>A</b>) Number of surviving anti-pan-NF stained motor neurons. Minocycline incubation starting at DIV 1, but not at DIV 4, decreased the number of surviving motor neurons significantly. (<b>B</b>) <b>Percentage of anti-pan-NF stained area (neurofilaments). Minocycline incubation did not alter it</b>. (<b>C</b>) Percentage of anti-IBA-1 stained area (microglia). Minocycline incubation did not show a significant influence on microglia pattern. (<b>D</b>)Anti-IBA-1 fluorescence staining intensity. Early (long-term) minocycline incubation reduced the fluorescence intensity (activity) of microglia significantly. (<b>E</b>) Percentage of anti-GFAP stained area (astroglia). Minocycline incubation did not show an influence on astroglia pattern. (<b>F</b>) Anti-GFAP fluorescence staining intensity. Minocycline incubation did not show an influence on the fluorescence intensity (activity) of astroglia. (<b>G</b>) Percentage of DAPI stained area. Minocycline incubation increased the percentage area of DAPI cell nuclei staining in cultures incubated with minocycline from DIV 4, but not DIV 1 onwards. For statistical analysis a paired Student’s t-test was used to compare control and +Mino for each incubation period. Number of samples n=6 for each incubation time with minocycline (1 animal = mean of up to 3 replicates). All values are means ± SD. Significant differences are marked with * and demonstrate p<0.05.</p

Fluorescence images of spinal cord cultures treated with 10 µM minocycline.

<p>Fluorescent images of spinal cord cultures of control (A, D) and of slices incubated with 10 µM minocycline from DIV 1 (B, E) and from DIV 4 (C, F) onwards. Slices were co-stained for anti-pan-NF (green) and anti-IBA-1 (red) in (A, B, C) or for anti-pan-NF (green) and anti-GFAP (red) in (D, E, F) at DIV 7. Motor neuronal areas are magnified and marked with a box. Control cultures show various anti-pan-NF stained motor neurons, neurons, modest microglia activation (A) and formation of a glia cover (B). All cultures treated with minocycline show control-like staining patterns. No clear differences in the staining pattern are visible. Bars = 500 µm, bars in magnified area = 200 µm.</p

Cytotoxicity of minocycline.

<p>MTT assay of glial cell cultures incubated with increasing minocycline concentrations (25, 50, 75, 100 and 125 µM) and incubation times (24, 48, 72 h and 7 days). Cytotoxicity of minocycline increased with incubation time and concentration. Statistical analysis was done with a 2-way ANOVA. Significant differences to the control are marked with * and demonstrate p<0.05. The assay was done in triples (n = 3 preparations) with a mean of 6 replicates each.</p