Fig 1.
Research design and in vitro model.
(A) Workflow of the in vitro stretch injury study. (B) Diagram illustrating the mechanism by which the 50 msec, 50 psi stretch injury is delivered onto the cells. The airtight plug is fitted into the well and delivers the burst of nitrogen gas. Figure adapted from Cohen et al. Prog Brain Res. (2007).161:143–69.
Fig 2.
Characterization of the immortalized H19-7 hippocampal cell line.
(A) H19-7 cells shown in bright field and stained positive with a neuronal marker Anti-MAP2 and DAPI. (B) Stretch injury levels were assessed using propidium iodide (PrI, red) and Hoechst 3342 (blue) staining. Unstretched H19-7 hippocampal neurons showed few PrI-stained cells. Increasing numbers of PrI-positive cells were detected in H19-7 cells subjected to a moderate injury level of 50 msec, 30 psi of pressure (~25% cell injury) and a more severe injury level of 50 msec, 50 psi (~57% cell injury). (C) The cell counts for PrI staining corresponded to increased levels of lactate dehydrogenase (LDH), a marker of tissue damage, released from stretch-injured cells.
Fig 3.
Characterization of the neuroprotective effects of clovanemagnolol (CM) in vivo and in vitro.
(A) Stereological counting of dying/injured Fluoro-Jade-positive (FJ+) pyramidal hippocampal neurons in TBI and TBI+CM rats showed that CM treatment significantly reduced neuronal injury in the CA1/2 (*p<0.02) and CA3 (**p<0.04) regions. (B) Quantitative real-time RT-PCR analysis of microRNA expression in the CA1-CA3 hippocampal subregions showed that CM treatment appeared to restore expression of miR-212 and miR-9 to naïve control levels. Results did not reach statistical significance. (C) Immunofluorescent staining of relative microglial activation (anti-CD11b) between TBI and TBI + CM-treated rats. The images show the hippocampal formation and surrounding regions. (D) Gene expression data from the RT profiler Neurogenesis PCR arrays (n = 4/group) were analyzed using bioinformatic software (Qlucore Omics Explorer). Principal component and hierarchical clustering heatmap analysis of significantly expressed genes (p = 0.004, q = 0.03) in the hippocampus of sham control, TBI and TBI+CM rats shows that eight genes can clearly discriminate the three groups from each other. Two genes (Gpi and Nrp2) that are restored to sham control levels by CM are associated with pro-survival roles in the brain. (E) H19-7 cells treated with 10nM CM 48 hours pre-injury were stretched at 50 msec, 30psi. A significant increase in cell viability of the drug treated stretched cells compared with stretched cells was shown by an MTS assay 24 hours after injury (#p<0.05). All error bars in A, B and E are SEM, standard error of the mean.
Fig 4.
Characterization of the pro-survival effects of vinaxanthone (VX) in vitro.
(A) H19 cells pre-treated with 8.675 uM of vinaxanthone 48 hrs prior to stretch injury were stretched (50 msec, 50 psi) and 4 and 24 hours later, media from each well was collected for LDH assays (n = 6). LDH release was elevated in stretch wells compared to control (*p < 0.001). Stretched + drug cells released less LDH compared to stretch alone wells (#p < 0.001). Error bars = SEM, standard error of the mean (B) Principal component analysis (PCA) and hierarchical clustering heatmap of microRNA (miRNA) expression in H19-7 cells showing that only two differentially expressed miRNAs distinguish stretch-injured from uninjured control cells. (C) PCA and hierarchical clustering heatmap of miRNA expression in VX-treated control and untreated H19-7 cells show that differential expression of only six miRNAs, whose target genes are known to be essential for critical brain functions and neuronal survival, clearly identify the VX-treated from untreated control cells. (D) PCA and hierarchical clustering heatmap of miRNA expression in stretch injured neurons showed that VX alters expression of miRNAs implicated in modulation of hippocampal spatial memory (miR-324-5p) and proliferation and migration of Schwann cells (miR-451-5p). (E) PCA and hierarchical clustering heatmap analysis of miRNA expression at a lower stringency p value cutoff showed that VX upregulated miR-702-5p which is known to inhibit apoptosis-related genes.
Fig 5.
Characterization of the pro-survival effects of pterostilbene (PT) in vitro.
(A) PT significantly increased cell viability of unstretched control H19-7 cells (*p<0.02). Error bar = SEM, standard error of the mean (B) PCA and hierarchical clustering heatmap analysis of miRNA expression in control cells showed that PT treatment downregulated three miRNAs which are known to target the pro-survival genes Bdnf and Bcl-2. (C) Although PT treatment had no apparent effects on the viability of stretch injured H19-7 cells as measured by the MTS assay, PCA and hierarchical clustering heatmap analysis revealed that PT downregulated two miRNAs (miR-29c-5p, miR-324-5p) whose altered expression could serve as a molecular marker of PT-induced pro-survival effects.