Figures & data
Figure 1 (A) Schematic of a connexin molecule showing the N-terminal region (NT), the membrane spanning domains (M1 to M4), the extracellular loops (EL1 and EL2), the intracellular loop (IL), and the C-terminal region (CT). (B) Schematic showing the mimetic peptide sequences (see ) and the amino acids of Cx43 to which they correspond. Amino acids that are part of the membrane-spanning domain are underlined and amino acids that are part of the extracellular loop are in italics.
TABLE 1 Sequences of connexin mimetic peptides
Figure 2 Culture of spinal cord segments leads to swelling which is reduced by connexin mimetic peptides. (A) Photograph showing the increase in swelling out of the ends of the dura (arrows) in ex vivo spinal cord segments after 24 h in culture (panel b), compared with segments fixed at time 0 (panel a). (B) Graph demonstrating the degree of swelling at 24 h, after treatment with connexin mimetic peptides (1 to 11) or vehicle. Peptide4 and peptide5 show a significant reduction in swelling; * p < 0.05; ** p < 0.01. (C) Photograph showing the difference in swelling in ex vivo spinal cord segments after 24 h culture with vehicle (panel a) and 500 μ M peptide5 (panel b).
Figure 3 Concentration-dependent reduction of spinal cord swelling after treatment with peptide5 for 24 h. Spinal cord swelling was measured after treatment with different concentrations of peptide5 and inactive control peptide (peptide8) for 24 h and expressed as a percentage of the swelling seen in vehicle treated cords. All concentrations of peptide5, 5 μ M and above show a significant reduction in swelling; *** p < 0.001.
Figure 4 Concentration-dependent reduction of spinal cord swelling after treatment with peptide5 for 4 days. Spinal cord swelling was measured after treatment with different concentrations of peptide5 and inactive control peptide (peptide8) for 4 days and expressed as a percentage of the swelling seen in vehicle-treated cords. Concentrations of peptide5 between 5 and 100 μ M show a significant reduction in swelling; * p < 0.05; *** p < 0.001.
Figure 5 Peptide5 reduces GFAP protein levels in ex vivo spinal cord segments after injury. (A) Western blot of GFAP protein levels in ex vivo spinal cord segments treated with 5 μ M peptide5 or control peptide (peptide8) for 24 h and cultured for up to 4 days, showing decreased GFAP protein levels in the presence of peptide5. (B) Immunohistochemical staining of corresponding sections from ex vivo spinal cord cultures treated as above. Scale bar = 100 μ m.
Figure 6 Peptide5 reduces Cx43 protein levels in ex vivo spinal cord segments after injury. Ex vivo spinal cord segments treated with 5 μ M peptide5 or control peptide (peptide8) for up to 24 h and cultured for up to 4 days. Western blot analysis shows the increase in Cx43 protein seen in control (peptide8)-treated cultures is reduced in peptide5-treated cultures.
Figure 7 Peptide5 prevents the reduction of NeuN protein levels in ex vivo spinal cord segments. (A) Western blot of NeuN protein levels in ex vivo spinal cord segments treated with 5 μ M peptide5 or control peptide (peptide8) for 24 h and cultured for up to 4 days, showing increased neuronal survival in the presence of peptide5. (B) Immunohistochemical staining of corresponding sections for NeuN at day 1 and day 4 of culture. Scale bar = 100 μ m.
Figure 8 Peptide5 prevents the reduction of SMI-32 protein levels in ex vivo spinal cord segments. (A) Western blot of SMI-32 protein levels in ex vivo spinal cord segments treated with 5 μ M peptide5 or control peptide (peptide8) for 24 h and cultured for up to 4 days, showing increased neuronal survival in the presence of peptide5. (B) Immunohistochemical staining of corresponding sections for SMI-32 at day 1 and day 4 of culture. Scale bar = 100 μ m.
Figure 9 Peptide5 prevents hemichannel opening. (A) Fluorescent images of NT2/D1 cells incubated with propidium iodide in low Ca2 + conditions. Both 5 and 500 μ M peptide5 reduce the uptake of propidium iodide to a similar degree as LaCl3 and carbenoxolone while peptide8 has no effect on dye uptake. (B) Graph demonstrating that the number of cells taking up dye is significantly reduced in the presence of peptide5, LaCl3 and carbenoxolone but not peptide 8; **p < 0.01.
Figure 10 High concentrations of peptide5 prevents gap junctional communication. (A) Fluorescent images of a cell settlement assay using NT2/D1 cells. A concentration of 500 μ M peptide5 reduces the transfer of calcein dye from loaded to unloaded cells to a similar degree as carbenoxolone-treated cells, whereas 5 μ M peptide5 and 500 μ M peptide8 has no effect on dye transfer. (B) Graph demonstrating the number of cells to which dye is transferred is significantly reduced in the presence of 500 μ M peptide5 and carbenoxolone but not 5 μ M peptide5 or 500 μ M peptide8; *p < 0.05.
