Figure 1.
Experimental Design.
Table 1.
Behavior and activity scores.
Table 2.
PCR primer sequences.
Figure 2.
Schematic representation of the areas taken for assay.
Figure 3.
Brain Injury Detection: (A) Water content results: Alterations in brain water content in the control group (n = 10), SAH group (n = 10), SAH+vehicle group (n = 10), and SAH+tBHQ group (n = 10).
The brain water content was significantly higher 48 h after SAH than at other times. TBHQ treatment was found to markedly reduce brain water content significantly. No difference in brain water content was detected between SAH and SAH+vehicle groups. (B)Evans blue results: Alterations in Evans blue extravasation in the control group (n = 10), SAH group (n = 10), SAH+vehicle group (n = 10), and SAH+tBHQ group (n = 10). SAH was found to induce a marked increase in BBB extravasation in rat brains relative to untreated controls. After administration of tBHQ, the Evans blue extravasation was significantly less pronounced than in the SAH+vehicle group. (C)Clinical behavior results: Effects of tBHQ administration on functional outcomes in the rats in the control group (n = 10), SAH group (n = 10), SAH+vehicle group (n = 10), and SAH+tBHQ group (n = 10). *P<0.05 and **P<0.01 vs. control group, #P<0.05 and ##P<0.01 vs. SAH+vehicle group, vs. P>0.05 vs SAH group.
Figure 4.
Fluoro-jade B (FJB) staining (400X).
Representative images demonstrating a lack of FJB positive neurons in the cortex of a saline-injected rats and a number of cells positively stained for FJB in cortex of rats in SAH and SAH+vehicle groups. In SAH+tBHQ group, the number of necrotic cells stained by FJB was decreased remarkably.
Figure 5.
Representative cortical sections from SAH animals showing TUNEL-positive cells co-localized with the nuclear marker DAPI in the SAH animal. Control group rats showing few TUNEL apoptotic cells; SAH group rats showing strong TUNEL staining; SAH + vehicle group rats still showing more TUNEL apoptotic cells; SAH + tBHQ group rats showing less TUNEL apoptotic cells than SAH or SAH + vehicle group.
Figure 6.
Administration of tBHQ significantly decreased the necrotic index and apoptotic index in rat brain following SAH.
**P<0.01 vs. control group, ##P<0.01 vs. SAH + vehicle group, nsP>0.05 vs. SAH group.
Figure 7.
Upper: Representative images of MWM trials of the rats of four groups; Bottom: Spatial learning and memory in the MWM.
Escape latency and swimming distance over 16 trials (A, C) and averaged for each day (B, D) over days 2–5. The SAH group exhibited significantly longer escape latency and swimming distance (A, C, *P<0.01 repeated ANOVA) over the 16 trials than control groups did. The tBHQ group exhibited significantly shorter escape latency and swimming distance (A, C, #P<0.01 repeated ANOVA) over the 16 trials than the vehicle group. The averaged data showed a similar increase in escape latency (B, *P<0.01 one-way ANOVA) in SAH animals on the fifth day relative to controls. In the tBHQ group, the averaged data exhibited significantly shorter escape latency (B, #P<0.01 one-way ANOVA) and swimming distance (D, #P<0.05 one-way ANOVA) on the 4th and 5th days than the vehicle group. On day 5, the relative improvement in escape latency from the previous training day was significantly lower in the SAH group than among controls (E, *P<0.01). The tBHQ group was higher on day 4 than the vehicle group (E, & P<0.05). The control group exhibited significantly more time saved on day 6 than in the SAH group on the working memory task (matching-to-place task), here indicated by the difference between the time required in latency to find the platform on the second (test) trial and that required to find the platform on the first (sample) trial (F, *P<0.01). There was no significant difference between the tBHQ group and the vehicle group (values are means±SD, n = 10 per group).
Figure 8.
Top: Representative autoradiogram of Keap1, Nrf2, and HO-1 expression in the brain after SAH.
Results show that there was more expression of these proteins in the SAH groups and further up-regulated after tBHQ treatment. Lane 1, control; lane 2, SAH; lane 3, SAH+vehicle; lanes 4, SAH+tBHQ, respectively. Bottom: Quantitative analysis of the Western blot results shows that these protein levels in SAH groups are significantly higher than in control group and progressively induced by tBHQ. Bars represent the mean±SD (n = 10, each group). **P<0.01 and *P<0.05 between control animals vs. SAH animals; ##P<0.01 and #P<0.05 between SAH+vehicle animals vs. SAH+tBHQ animals; nsP >0.05 between SAH animals vs. SAH+vehicle animals.
Figure 9.
Nrf2 activity in the brain area surrounding the blood clot in control group (n = 10), SAH group (n = 10), SAH+vehicle group (n = 10), and SAH+tBHQ group (n = 10).
Top, EMSA autoradiography of NF-κB DNA binding activity. Lane 1, control; lane 2, SAH; lane 3, SAH+vehicle; lane 4, SAH+tBHQ, respectively. Bottom, Levels of Nrf2 DNA binding activity quantified by computer-assisted densitometric scanning and expressed as arbitrary densitometric units (ADU). Nrf2 binding activity measured by EMSA was significantly higher than in the control group after SAH. tBHQ rendered Nrf2 activation significantly higher in the SAH+tBHQ group than in the SAH+vehicle group. *P<0.05 between control animals vs. SAH animals; ##P<0.01 between SAH+vehicle animals vs. SAH+tBHQ animals; nsP>0.05 between SAH animals vs. SAH+vehicle animals.
Figure 10.
Immunohistochemical results: (A) Immunohistochemical study of Keap1, Nrf2, and HO-1 on brain samples.
Few Keap1-, Nrf2-, and HO-1-positive cells were observed in the control group, which indicates the constitutional activation of Keap1/Nrf2/HO-1 pathway in the normal cortex of rats. High numbers of Keap1-, Nrf2-, and HO-1-positive cells (arrows) were stained brown and observed in the brains of the rats in the subarachnoid hemorrhage (SAH) groups. Significant up-regulation of Keap1, Nrf2, and HO-1 immunoreactivity (arrows) was observed in the neurons and glia cells of SAH brains treated with tBHQ (400X). (B) Quantitative analysis showed relatively low concentrations of Keap1, Nrf2, and HO-1 in the control group. The concentrations of Keap1, Nrf2, and HO-1 expression were higher in the SAH and SAH+vehicle groups. After tBHQ therapy, all three proteins were progressively activated in the treatment group. Bars represent the mean ± SD (n = 10, each group). *P<0.05 compared with control group, #P<0.05 and ##P<0.01 compared with SAH+vehicle group.
Figure 11.
The mRNA expressions of HO-1, NQO1, and GST-α1 in the brains in control group (n = 10), SAH group (n = 10), SAH+vehicle group (n = 10), and SAH+tBHQ group (n = 10).
SAH was found to induce a marked increase in HO-1, NQO1, and GST-α1 mRNA expression in the rat SAH brains compared with control group. After tBHQ administration, the mRNA expressions of the downstream Keap1/Nrf2/ARE pathway related agents were significantly up-regulated relative to the SAH+vehicle group. ** P<0.01 and * P<0.05 vs. control group, ## P<0.01 and # P<0.05 vs. SAH+vehicle group, vs. P>0.05 vs. SAH group.
Figure 12.
Changes of NQO1 and GST-α1 enzymes activity in the brain as determined by biochemical tests in control group (n = 10), SAH group (n = 10), SAH+vehicle group (n = 10) and SAH+tBHQ group (n = 10).
SAH was found to induce the significant increases in the activity of NQO1 and GST-α1 in rat brain tissue. In the SAH+tBHQ group, the cortical activity of NQO1 and GST-α1 was markedly up-regulated relative to the SAH+vehicle group. *P<0.05 vs. control group; #P<0.05 vs. control group; vs. P>0.05 relative to the SAH group.
Figure 13.
Cerebral antioxidant status of the experimental group of animals (n = 10 for each group).
After tBHQ administration, the post-SAH reduced antioxidative status was ameliorated in this SAH model. Values are expressed as mean±SD. (*P<0.05 and *P<0.01 vs. Control group, nsP>0.05 vs. SAH group, #P<0.05 and ##P<0.01 vs. SAH+ vehicle group).