Role of Rac1 GTPase in NADPH oxidase activation and cognitive impairment following cerebral ischemia in the rat - PubMed (original) (raw)
Role of Rac1 GTPase in NADPH oxidase activation and cognitive impairment following cerebral ischemia in the rat
Limor Raz et al. PLoS One. 2010.
Abstract
Background: Recent work by our laboratory and others has implicated NADPH oxidase as having an important role in reactive oxygen species (ROS) generation and neuronal damage following cerebral ischemia, although the mechanisms controlling NADPH oxidase in the brain remain poorly understood. The purpose of the current study was to examine the regulatory and functional role of the Rho GTPase, Rac1 in NADPH oxidase activation, ROS generation and neuronal cell death/cognitive dysfunction following global cerebral ischemia in the male rat.
Methodology/principal findings: Our studies revealed that NADPH oxidase activity and superoxide (O(2)(-)) production in the hippocampal CA1 region increased rapidly after cerebral ischemia to reach a peak at 3 h post-reperfusion, followed by a fall in levels by 24 h post-reperfusion. Administration of a Rac GTPase inhibitor (NSC23766) 15 min before cerebral ischemia significantly attenuated NADPH oxidase activation and O(2)(-) production at 3 h after stroke as compared to vehicle-treated controls. NSC23766 also attenuated "in situ" O(2)(-) production in the hippocampus after ischemia/reperfusion, as determined by fluorescent oxidized hydroethidine staining. Oxidative stress damage in the hippocampal CA1 after ischemia/reperfusion was also significantly attenuated by NSC23766 treatment, as evidenced by a marked attenuation of immunostaining for the oxidative stress damage markers, 4-HNE, 8-OHdG and H2AX at 24 h in the hippocampal CA1 region following cerebral ischemia. In addition, Morris Water maze testing revealed that Rac GTPase inhibition after ischemic injury significantly improved hippocampal-dependent memory and cognitive spatial abilities at 7-9 d post reperfusion as compared to vehicle-treated animals.
Conclusions/significance: The results of the study suggest that Rac1 GTPase has a critical role in mediating ischemia/reperfusion injury-induced NADPH oxidase activation, ROS generation and oxidative stress in the hippocampal CA1 region of the rat, and thus contributes significantly to neuronal degeneration and cognitive dysfunction following cerebral ischemia.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Rac GTPase Activation Contributes Significantly to Neuronal Cell Death in the Hippocampus CA1 Region Following Global Cerebral Ischemia (GCI).
(A) NSC23766 significantly attenuated cerebral ischemia-induced Rac1 activity at 3 h after ischemia. *Represents P<0.05 (four to 5 animals) vs. reperfusion 3 hour (R3h) and saline treatment groups. (B) Representative hippocampal CA1 sections from sham, saline–treated (ischemia) and NSC23766-treated (ischemia) male rats were labeled with NeuN (Blue), TUNEL (Red) and FJadeB (Green) staining at 7 d following GCI. Merged images represent CA1 neurons undergoing degeneration, indicated by white staining. (C) Quantification showed that NSC23766 was strongly neuroprotective of CA1 region. Data was obtained from five independent animals and a typical experiment is presented. Results are expressed as neuronal density with mean ± SE. * p<0.05 vs. sham control and NSC23766.
Figure 2. Temporal Expression of NADPH Oxidase Activity and Superoxide Production in the Hippocampal CA1 Region Following GCI.
Homogenates taken from the hippocampus CA1 at 30 min, 3 h, 6 h, 24 h after reperfusion were subjected to NADPH oxidase activity (A) and superoxide production (B) assays to assess temporal expression following ischemia-reperfusion. Data is expressed as optical density (OD) and represented as fold vs. sham from four to five animals. * p<0.05 vs. sham control.
Figure 3. Rac GTPase Activation is Critical for Enhanced NADPH Oxidase Activity and Superoxide Production in the Hippocampal CA1 Region Following GCI.
NSC23766 pre-treatment reduces NADPH oxidase activity (A) and superoxide production (B) at 3 h post-reperfusion in the hippocampus CA1 after GCI. Data for experiments (A) and (B) was measured as optical density (OD) and expressed as fold difference in comparison to sham from four to five animals. *p<0.05 vs. sham, # p<0.05 vs. saline control. HET staining measuring endogenous superoxide production in CA1 coronal brain sections obtained from four to five animals; magnification 40X (C).
Figure 4. Contributions of NADPH oxidase to Ischemic Neuronal Damage in Hippocampal CA1.
(A) Representative staining of coronal CA1 sections with NeuN (red) show neuroprotection of gp91ds-tat peptide at day 7 after reperfusion. a: Sham; b: Ischemic reperfusion; c: Scrambled gp91ds-tat control; d: gp91ds-tat (91ds-tat). (B) CA1 neuronal density was counted per 250 µm length of medial CA1 region from five to six rats in each group. *p<0.05 vs. reperfusion at 7 days (R7d) and Scr treatment groups. Scr: gp91ds-tat control.
Figure 5. Rac GTPase Activation is Critical for Enhanced Oxidative Damage in the Hippocampus CA1 Following GCI.
DAB staining of representative coronal CA1 sections show NSC23766 ability to attenuate staining for oxidative stress markers for lipid peroxidation (4-HNE), DNA damage (8-OHdG) and histone phosphorylation (p-H2A.X). (Four to five animals per treatment group, magnification used was 20X).
Figure 6. Effect of the Rac GTPase Inhibitor, NSC23766 Upon Cognitive Function Following GCI.
Latency trial (A) and probe trial (B) results in Morris water maze of non-ischemic control sham and NSC23766 animals showing no effect of NSC23766 in nonischemic control animals. Panel C shows no significant differences between sham and NSC23766-treated animals in swimming speed (cm/sec); Five animals per condition, mean ± SE. (D) Time (sec) spent finding the submerged platform at 7 d, 8 d and 9 d after ischemic injury. (E) Exploration time spent in the quadrant which initially contained the platform at 9 d following reperfusion. (F) Representative traces indicating the sample paths of the rats from the maze latency trials (a–d) and the swimming traces from probe trials (e–h) (a, e: sham; b, f: I/R; c, g: vehicle + I/R; d, h: NSC23766 + I/R). Data is expressed as mean ± SE from five different animals.*p<0.05 vs. sham, # p<0.05 vs. vehicle + I/R. I: ischemia; R: reperfusion.
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