Figure 1.
NAMPT and NAD levels in rat brain after ischemia.
(A-C) NAMPT protein expression level was measured by using Western blot in the contralateral (A), boundary zone (B) and ischemic core (C). N=8. (D-F) NAMPT mRNA expression level was measured by using RT-PCR in the cortex of ischemic core. N=5. (G)NAMPT protein level in cerebral-spinal fluid was measured after 30 min MCAO and 24 h reperfusion. N=10. (H) NAD level was measured in rat brain after cerebral ischemia. N=4. *P<0.05, **P<0.01, ***P<0.001, compared with sham, One-way ANOVA.
Figure 2.
Distribution of NAMPT protein in rat brain after MCAO.
(A-C) Representative images of double immunofluorescent staining of NeuN (for neuron) and NAMPT (A), Iba-1 (for microglia) and NAMPT (B), GFAP (for astrocyte) and NAMPT (C) in rat brain after 30 min MCAO and 24 h reperfusion. Immunofluorescent staining was repeated on three rats. (D) Using Western blot (upper panel) and immunochemistry staining (lower panel), the i.v. injected His-tagged NAMPT was detected in the ischemic brain area after 30 min MCAO and 24 h reperfusion.
Figure 3.
Recombinant NAMPT protein aggravated MCAO-induced brain injury.
The rats were i.c.v. injected with 4 μg albumin, wild type NAMPT (WT) or H247A NAMPT (H247A) at 12 h interval for three times with the first injection 12 h before MCAO. (A) Representative brain sections stained with TTC. (B) Statistical analysis of the infarct volume. (C) Neurological deficit score evaluated before sacrifice. N=6, *P<0.05, **P<0.01, compared with NS, One-way ANOVA. NS represents normal saline.
Figure 4.
The effect of recombinant NAMPT protein on primary cultured neuron cell injury induced by OGD and reperfusion.
Wild type NAMPT (WT) or H247A NAMPT (H247A) was applied 30 min before OGD. (A) Using MTT assay, cell viability was determined after the treatment of NAMPT or combined treatment with NAMPT and OGD. (B) LDH in medium was determined after the treatment of NAMPT or after combined treatment with NAMPT and OGD. N=12. *P<0.05, **P<0.01, ***P<0.001, compared with relative control, One-way ANOVA.
Figure 5.
The effect of recombinant NAMPT protein on OGD-induced cell necrosis in neuron-glial mixed culture.
Wild type NAMPT (WT) or H247A NAMPT (H247A) protein was applied 30 min before OGD. (A and B) Representative images of triple immunofluorescence staining of NeuN (green, neuron specific marker), PI (red, for necrotic cells) and DAPI (blue, for all cells) under normal condition (A) or after OGD and recovery (B). (C) Percentage of necrotic cells in total cells (PI-positive cell number over DAPI-positive number). N=6. (D) Percentage of necrotic neurons in total neuron cells (PI/NeuN double positive cell number over NeuN-positive cell number). N=6. *P<0.05, **P<0.01, ***P<0.001, compared with the control of OGD, One-way ANOVA. ###P<0.001, compared with the control of control, One-way ANOVA.
Figure 6.
The effect of recombinant NAMPT on ischemic-induced TNF-α release in primary neuron-glial mixed culture.
Wild type NAMPT (WT) or H247A NAMPT (H247A) was applied 30 min before OGD. (A and B) TNF-α in the medium was determined at 24 h after NAMPT treatment (A) or after combined treatment with OGD (B). N=6. ***P<0.001, compared with E-S Glu (A) or E-S Glu-free (B), One-way ANOVA. (C and D) TNF-α in the medium was determined at 3, 6, 12 or 24 h after the treatment of NAMPT (C) or after combined treatment with NAMPT and OGD (D). N=5. *P<0.05, **P<0.01, ***P<0.001, compared with “0” of E-S Glu (C) or E-S Glu-free (D), One-way ANOVA. #P<0.05, ##P<0.01, ###P<0.001, compared with 0 h recovery in each group, One-way ANOVA. E-S is Earle’s solution; Glu is glucose.
Figure 7.
TNF-α neutralizing antibody protected neuronal injury induced by OGD and NAMPT in primary neuron-glial mixed culture.
NAMPT and TNF-α neutralizing antibody (5μg/ml, TNF-α Ab) was applied 30 min before OGD. (A) The percentage of necrotic cells in total cells determined by using PI and DAPI staining. (B) The percentage of necrotic neuron cells determined by using NeuN, PI and DAPI staining. N=5-6. *P<0.05, ***P<0.001, compared with no NAMPT no TNF-α Ab in control, One-way ANOVA. #P<0.05, ##P<0.01, ###P<0.001, compared with no NAMPT no TNF-α Ab in OGD, One-way ANOVA.