doi:10.1016/j.bbrc.2003.08.006
Copyright © 2003 Elsevier Inc. All rights reserved.
HBO suppresses Nogo-A, Ng-R, or RhoA expression in the cerebral cortex after global ischemia
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Changman Zhou, Yun Li, Anil Nanda and John H. Zhang
, 
Department of Neurosurgery, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA, USA
Received 1 July 2003.
Available online 26 August 2003.
Abstract
Nogo-A, a myelin-associated neurite outgrowth inhibitory protein, binds with the Ng-R receptor to activate RhoA intracellular signals and inhibit the plasticity after CNS injury. We evaluated the effect of hyperbaric oxygen (HBO) on the expression of Nogo-A, Ng-R, and RhoA after transient global ischemia in a rat 2 vessel occlusion global ischemic model. Male SD rats (n=78) were randomly divided into 13 groups: 1 sham group, 6 groups of global ischemia, and 6 groups of HBO treatment after global ischemia. HBO (3ATA) was applied for 2 hr at 1 hr after global ischemia. Rats were sacrificed at 6, 12, 24, 48, and 96 hr and 7days. Global ischemia (10 min) produced a marked increase of Nogo-A/B, Nogo-A, Ng-R, and RhoA expression. Immunohistochemistry showed increased Nogo-A/B and Nogo-A located in the myelin sheath of ischemic brain cortex. Ng-R expressed on the surface of neurons and their processes, and RhoA expressed inside the cytoplasm of neurons in ischemic brain. HBO significantly reduced neurological injury, decreased the levels of Nogo-A, Ng-R, and RhoA in ischemic injured cortex (p<0.05).
Author Keywords: Global ischemia; Hyperbaric oxygen; Nogo-A; Ng-R; Rat; RhoA
Fig. 1. Morphology and Nissl staining. Normal cortex is shown at two different magnifications (A1, A2). Most neuronal cells in the cortex are lost 7 days after global ischemia (B1, B2). One single HBO treatment preserved some neuronal cells 7 days after global ischemia (C1, C2).
Fig. 2. Immunohistochemistry staining demonstrated Nogo-A/B and Nogo-A expression in the cortex of normal, global ischemia (48 h), and HBO treatment animals. The expression of Nogo-A/B was localized in nucleus of neurons in normal tissue (A1, A2), and the expression of Nogo-A was in the cytoplasm of oligodendrocytes (B1, B2). Forty-eight hours after global ischemia, Nogo-A/B (C1, C2) and Nogo-A (D1, D2) were stained on the myelin sheath. HBO treatment reduced the expression of Nogo-A/B and Nogo-A on the myelin sheath but did not reduce the basal expression of Nogo-A/B in the nucleus (E1, E2). Some Nogo-A expression remained on the myelin sheath after HBO treatment, even though the expression level was reduced (F1, F2). Scale bars: A1, B1, C1, D1, E1, and F1=200 μm; A2, B2, C2, D2, E2, and F2=20 μm. Window in E2 shows the Nogo-A positive myelin at the beginning of axon. Scale bar=4 μm.
Fig. 3. Western blot analysis shows the expression of Nogo-A/B in the cortex from rats after 10 min ischemia with and without HBO. Representative bands from 6 h to 7 days after global ischemia, as well as corresponding β-actin bands, are shown on the top panel. The lower panel shows the summary of Nogo-A/B expression from 4 to 6 animals in each group. The expression of Nogo-A/B was significantly increased in the ischemic cortex of the 12, 24, and 48 h groups. HBO reduced the increases of Nogo-A/B expression. The “#” indicates
p<0.05 control vs. global ischemia groups. The “*” indicates
p<0.05 HBO vs. global ischemia groups (ANOVA).
Fig. 4. Western blot analysis shows the expression of Nogo-A in the cortex from rats after 10 min ischemia with and without HBO. Representative bands from 6 h to 7 days after global ischemia, as well as corresponding β-actin bands, are shown on the top panel. The lower panel shows the summary of Nogo-A expression from 4 to 6 animals in each group. The expression of Nogo-A was significantly increased in the ischemic cortex of the 24 and 48 h groups. HBO reduced the increases of Nogo-A expression. The “#” indicates
p<0.05 control vs. global ischemia groups. The “*” indicates
p<0.05 HBO vs. global ischemia groups (ANOVA).
Fig. 5. Immunohistochemistry staining demonstrated Ng-R and RhoA expression in the cortex of normal, global ischemia (48 h), and HBO treatment animals. The expression of Ng-R was localized on the membrane of neurons in the normal tissue (A1, A2). The expression of Ng-R increased after global ischemia (C1, C2) and HBO reduced the expression of Ng-R (E1, E2). Some slight expression of RhoA was localized in the cytoplasm in neurons in the normal tissue (B1, B2). The expression was markedly increased after global ischemia (D1, D2), and HBO reduced the expression of RhoA (F1, F2). Scale bars: A1, B1, C1, D1, E1, and F1=200 μm; A2, B2, C2, D2, E2, and F2=20 μm.
Fig. 6. Western blot analysis shows the expression of Ng-R in the cortex from rats after 10 min ischemia with or without HBO. Representative bands from 6 h to 7 days after global ischemia as well as corresponding β-actin bands are shown on the top panel. The lower panel shows the summary of Ng-R expression from 4 to 6 animals in each group. The expression of Ng-R was significantly increased from 6 h and lasted for 7 days in the ischemic cortex. HBO reduced the increases of Ng-R expression. The “#” indicates
p<0.05 control vs. global ischemia groups. The “*” indicates
p<0.05 HBO vs. global ischemia groups (ANOVA).
Fig. 7. Western blot analysis shows the expression of RhoA in the cortex from rats after 10 min ischemia with and without HBO. Representative bands from 6 h to 7 days after global ischemia, as well as corresponding β-actin bands, are shown on the top panel. The lower panel shows the summary of RhoA expression from 4 to 6 animals in each group. The expression of RhoA was significantly increased at 6 h and lasted for 7 days in the ischemic cortex. HBO reduced the increases of RhoA expression. The “#” indicates
p<0.05 control vs. global ischemia groups. The “*” indicates
p<0.05 HBO vs. global ischemia groups (ANOVA).
Table 1. Ratio of neurons in counted area in the cortex
#GI vs Control, p<0.001; *HBO vs. GI, p<0.001 (ANOVA).
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