Fig 1: HPC regulates GLT-1 and the activity of GS against tGCI through regulation of Cx43 and c-Src. Generally, glutamates (Glu) can be synthesized inside the presynaptic terminal and then released into the synaptic cleft. Most Glu are transported into astrocytes by GLT-1 and shall be converted to glutamine (Gln) by GS and released into the extracellular space. Subsequently, Gln shall be uptaken into the presynaptic terminals and metabolized to Glu, the process of which is termed glutamate-glutamine cycle. Under physiological conditions, extracellular glutamate concentration shall be maintained at a proper level. However, under ischemic conditions, the decreased expression of Cx43 and /or the increased c-Src activity can decrease the expression of GLT-1 and reduce the activity of GS, and then disrupt the operation of glutamate-glutamine cycle, leading to the accumulation of extracellular glutamate. HPC can prevent the reduction of GLT-1 expression and maintain GS activity by regulating Cx43 and inactivating c-Src to remove the released glutamate from the extracellular space, eventually protecting neurons against tGCI. HPC, hypoxic preconditioning; tGCI, transient global cerebral ischemia; Glu, glutamate; Gln, glutamine; Cx43, connexin 43; c-Src, cellular-Src; p-c-Src, phosphorylated cellular-Src; GLT-1, glutamate transporter 1; GS, glutamine synthetase.
Fig 2: Effect of Gap26 or PP2 on the expression of GLT-1 and GS activity in CA1 of rats exposed to tGCI or HPC. The effect of Gap26 on GTL-1 expression (A) and GS activity (B) in CA1 at 4 h after reperfusion in tGCI and HPC group. (C) The effect of Gap26 on extracellular glutamate in CA1 at 24 h after reperfusion in tGCI and HPC group. The effect of PP2 on GLT-1 expression (D) and GS activity (E) in CA1 at 4 h after reperfusion in tGCI and HPC rats. (F) The effect of PP2 on extracellular glutamate at 24 h after reperfusion in tGCI and HPC rats. Each bar represents the mean ± S.D. *p < 0.05 vs. Sham group, #p < 0.05 vs. the same group with vehicle, and &p < 0.05 vs. HPC group with Gap26 (A–C) or tGCI group with PP2 (D–F; n = 3 in each group). Sham, sham-operated; tGCI, transient global cerebral ischemia; HPC, hypoxic preconditioning. GS, glutamine synthetase; GLT-1, glutamate transporter 1; Gap26, Cx43 mimetic peptide, PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo (3, 4-d) pyrimidine.
Fig 3: Cellular localization of Cx43 in CA1 in Sham group and tGCI group with or without hypoxia at 168 h of reperfusion. Representative images of triple fluorescent staining of Cx43 (green), NeuN (red) and DAPI (blue); Cx43 (green), GFAP (red) and DAPI (blue); Cx43 (green), Iba-1 (red) and DAPI (blue) in CA1. (A) The overlapped images showed that Cx43 prominently surrounded NeuN (a–d), and located slightly in GFAP-positive (e–h) and Iba-1-positive (i–l) cells in Sham group. (B) Cx43 mainly located in GFAP-positive (e–h) and Iba-1-positive (i–l) cells at 168 h after tGCI, and part of cells with NeuN (a–d). (C) Cx43 located mainly in GFAP-positive (e–h) and Iba-1-positive (i–l), and partly in NeuN-positive cells (a–d) in CA1 at 168 h after reperfusion of HPC group. Scale bar: 25 μm. (D–F) Quantitative analysis of Cx43 positive neurons, Cx43 positive astrocytes and Cx43 positive microglia in the CA1, respectively. Data are expressed as percentage of the number of Cx43-positive cells. Each bar represents the mean ± SD; *p < 0.05 vs. Sham group and #p < 0.05 vs. tGCI group (n = 4 in each group). Sham, sham-operated; tGCI, transient global cerebral ischemia; HPC, hypoxic preconditioning; Cx43, connexin 43; NeuN, neuronal nuclei; GFAP, glial fibrillary acidic protein; Iba-1, ionized calcium binding adaptor molecule 1; DAPI, 4’,6-diamidino-2-phenylindole.
Fig 4: HPC increases the Cx43 expression in CA1 after tGCI. (A) Immunohistochemistry for Cx43 in hippocampus after tGCI with or without HPC. Representative images show Cx43-immunopositive cells in Sham group (a,b), 4 h after reperfusion of tGCI group (c,d) and HPC group (e,f), and 168 h after reperfusion of tGCI group (g,h) and HPC group (i,j), respectively. Scale bar: (a,c,e,g,i) 250 μm; (b,d,f,h,j) 25 μm. (B) Quantitative analysis of optical density of Cx43 immunopositive cells in CA1 (n = 6 in each group). (C) Western blot analysis of Cx43 in CA1 of tGCI and HPC rats. The histogram presents the quantitative analyses of Cx43 levels (n = 3 in each group). Data are expressed as percentage of value of Sham animals. Each bar represents the mean ± SD *p < 0.05 vs. Sham animals and #p < 0.05 vs. tGCI group at the same time point. Sham: sham-operated; tGCI: transient global cerebral ischemia; HPC: hypoxic preconditioning; Cx43: connexin 43.
Fig 5: Inhibition of c-Src activity with PP2 alleviates neuronal damage in CA1 of tGCI rats with or without hypoxia. (A) Nissl staining, NeuN immunostaining and FJ-B staining from tGCI and HPC rats with or without PP2 treatment at 168 h after reperfusion. Boxes indicated that the magnified regions displayed in the right panel. Scale bar: (a,e,i,m,q): 250 µm; (b,c,f,g,j,k,n,o,r,s) 25 µm; (d,h,l,p,t) 75 µm. Quantitative analyses of survival cells (B), NeuN-positive cells (C), and FJ-B positive cells (D) in CA1. Each bar represents the mean ± SD *p < 0.05 vs. Sham group pretreated with PP2, #p < 0.05 vs. the same group with vehicle, and &p < 0.05 vs. tGCI group with PP2 injection (n = 6 in each group). The effect of Gap26 (10 µL and 20 µL, respectively) on Cx43 expression (E) and p-c-Src level (F) in CA1 at 4 h after reperfusion in tGCI and HPC group. The effect of PP2 (10 µL and 20 µL, respectively) on p-c-Src level (G) and Cx43 expression (H) in CA1 at 4 h after reperfusion in tGCI and HPC group. Data are expressed as the percentage of value of Sham group with vehicle. Each bar represents the mean ± SD *p < 0.05 vs. Sham group with vehicle, #p < 0.05 vs. the same group with vehicle, and &p < 0.05 vs. HPC group with Gap26 (n = 3 in each group; E,F) or tGCI group with PP2 (n = 3 in each group; G,H). Sham, sham-operated; tGCI, transient global cerebral ischemia; HPC, hypoxic preconditioning; p-c-Src, phosphorylated cellular-Src; Cx43, connexin 43; Gap26, Cx43 mimetic peptide, PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo (3, 4-d) pyrimidine; Nissl, cresyl violet; NeuN, neuronal nuclei; FJ-B: Fluoro-Jade B.
Supplier Page from Abcam for Anti-Connexin 43 / GJA1 antibody - C-terminal