Figure 1.
Effect of shear stress on primary cultured cortical neurons.
A) Histograms show that the cell viability decreases significantly after shear stress of 5, 10, 20 dyn/cm2 for 1 h, 4 h, 8 h, 12 h and 24 h respectively by MTT assay. B) Photomicrographs and C) histograms show that as shear stress levels increased (from 5, 10 dyn/cm2 to 20 dyn/cm2) TUNEL positive cells increased significantly by TUNEL assay. *p<0.05, **p<0.01 and ***p<0.001 were considered to be statistically significant.
Figure 2.
Influence of galanin on shear stress-induced neurotoxicity in primary cultured cortical neurons.
A) Histograms show that pretreatment with galanin (Gal) at 1 µM, 100 nM, and 10 nM for 24 h counteracts the shear stress-induced decrease in cell viability, assessed by MTT assay at 10 dyn/cm2 and 20 dyn/cm2. Cell viability does not change after shear stress of 5 dyn/cm2 and there is no marked influence of 100 nM galanin alone on the cell viability. B) Photomicrographs and C) histograms show that pretreatment with 100 nM of galanin for 24 h reduces shear stress-induced nerve injury assessed by TUNEL assay. *p<0.05, **p<0.01 and ***p<0.001 were considered to be statistically significant (Scale bars: B, 50 µm).
Figure 3.
Involvement of GalR2 in the protective effects of galanin against shear stress-induced neurotoxicity.
A) Galantide (M15), a galanin receptor antagonist, blocks the protective effects of galanin after shear stress of 10 dyn/cm2 for 12 h. B) Gal2-11, a galanin receptor 2/3 agonist, and pretreatments of 1 µM, 100 nM, 10 nM of the galanin receptor 2/3 antagonist Gal2-11 show significant protective effects against shear stress-induced nerve injury tested by MTT assay. *p<0.05, **p<0.01 and ***p<0.001 were considered to be statistically significant.
Figure 4.
Expressions of mRNAs for GalR1, R2 and R3 after shear stress of 5 dyn/cm2 for 12 h.
Quantitative real time PCR results show that GalR1 (Fig. 4A)and GalR2 (Fig. 4B) did not change significantly, and there was no GalR3 expression in the cortical neurons with galanin or galantide treatment after shear stress of 5 dyn/cm2 for 12 h. The data are presented as mean ± SD (n = 3).
Figure 5.
Expressions of mRNAs for GalR1, R2 and R3 after shear stress of 10 dyn/cm2 for 12 h.
(A) GalR1 mRNA levels in cultured cortical neurons after shear stress of 10 dyn/cm2 for 12 h with galanin or galantide treatment was assessed by real time PCR assay. (B) GalR2 mRNA levels in cultured cortical neurons after shear stress of 10 dyn/cm2 for 12 h with galanin or galantide treatment was assessed by real time PCR assay. GalR3 is not expressed in our system. The data are presented as mean ± SD (n = 3). *p<0.05, **p<0.01 and ***p<0.001 were considered to be statistically significant.
Figure 6.
Effect of galanin on the excitatory postsynaptic currents (EPSCs) after shear stress.
A) EPSC of cultured cortical neurons after shear stress of 5 dyn/cm2 and 10 dyn/cm2 by patch clamp assay. B) Shear stress of 5 dyn/cm2 for 12 h can not cause a significant change in amplitude. But shear stress of 10 dyn/cm2 for 12 h cause significantly decrease the amplitude, and pretreatment of galanin cause a significant increase in amplitude. C) Frequency of cortical neurons after galanin treatment. Shear stress of 5 dyn/cm2 for 12 h can not cause a marked decrease in frequency, but shear stress of 10 dyn/cm2 for 12 h induced a significant decrease in frequency, while galanin increased the frequency of cortical neurons after shear stress with 10 dyn/cm2. *p<0.05, **p<0.01 and ***p<0.001 were considered to be statistically significant.
Figure 7.
Effect of galanin on the cell apoptosis in the primary cortical neurons.
A) The effect of galanin on Bax in cortical neurons after shear stress was assessed by immunoprecipitation assay. There was no significant decrease in Bax after shear stress of 5 dyn/cm2, but there was a significant increase in active Bax after shear stress of 10 dyn/cm2; galanin inhibits the increase of active Bax. B) The expression of Bcl-2 and p53 were tested by galanin treatment. Shear stress of 10 dyn/cm2 for 12 h decreased Bcl-2 and increased p53 expression, while galanin may inhibit the effect of shear stress. There were no significant changes in p53 and Bcl-2 after shear stress of 5 dyn/cm2 for 12 h. *p<0.05, **p<0.01 and ***p<0.001 were considered to be statistically significant.