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. 2011 Jul 6:8:78.
doi: 10.1186/1742-2094-8-78.

S-nitrosoglutathione reduces oxidative injury and promotes mechanisms of neurorepair following traumatic brain injury in rats

Mushfiquddin Khan  1 , Harutoshi Sakakima, Tajinder S Dhammu, Anandakumar Shunmugavel, Yeong-Bin Im, Anne G Gilg, Avtar K Singh, Inderjit Singh
Affiliations

S-nitrosoglutathione reduces oxidative injury and promotes mechanisms of neurorepair following traumatic brain injury in rats

Mushfiquddin Khan et al. J Neuroinflammation. .

Abstract

Background: Traumatic brain injury (TBI) induces primary and secondary damage in both the endothelium and the brain parenchyma, collectively termed the neurovascular unit. While neurons die quickly by necrosis, a vicious cycle of secondary injury in endothelial cells exacerbates the initial injury in the neurovascular unit following TBI. In activated endothelial cells, excessive superoxide reacts with nitric oxide (NO) to form peroxynitrite. Peroxynitrite has been implicated in blood brain barrier (BBB) leakage, altered metabolic function, and neurobehavioral impairment. S-nitrosoglutathione (GSNO), a nitrosylation-based signaling molecule, was reported not only to reduce brain levels of peroxynitrite and oxidative metabolites but also to improve neurological function in TBI, stroke, and spinal cord injury. Therefore, we investigated whether GSNO promotes the neurorepair process by reducing the levels of peroxynitrite and the degree of oxidative injury.

Methods: TBI was induced by controlled cortical impact (CCI) in adult male rats. GSNO or 3-Morpholino-sydnonimine (SIN-1) (50 μg/kg body weight) was administered orally two hours following CCI. The same dose was repeated daily until endpoints. GSNO-treated (GSNO group) or SIN-1-treated (SIN-1 group) injured animals were compared with vehicle-treated injured animals (TBI group) and vehicle-treated sham-operated animals (Sham group) in terms of peroxynitrite, NO, glutathione (GSH), lipid peroxidation, blood brain barrier (BBB) leakage, edema, inflammation, tissue structure, axon/myelin integrity, and neurotrophic factors.

Results: SIN-1 treatment of TBI increased whereas GSNO treatment decreased peroxynitrite, lipid peroxides/aldehydes, BBB leakage, inflammation and edema in a short-term treatment (4-48 hours). GSNO also reduced brain infarctions and enhanced the levels of NO and GSH. In a long-term treatment (14 days), GSNO protected axonal integrity, maintained myelin levels, promoted synaptic plasticity, and enhanced the expression of neurotrophic factors.

Conclusion: Our findings indicate the participation of peroxynitrite in the pathobiology of TBI. GSNO treatment of TBI not only reduces peroxynitrite but also protects the integrity of the neurovascular unit, indicating that GSNO blunts the deleterious effects of peroxynitrite. A long-term treatment of TBI with the same low dose of GSNO promotes synaptic plasticity and enhances the expression of neurotrophic factors. These results support that GSNO reduces the levels of oxidative metabolites, protects the neurovascular unit, and promotes neurorepair mechanisms in TBI.

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Figures

Figure 1
Figure 1
Effect of GSNO on the expression of 3-NT determined by dot blot and IHC at 4 h after TBI and colocalization of 3-NT with vWF. Animals were treated with GSNO (50 μg/kg body weight) at 2 h after TBI. Dot blot (A) and its densitometric analysis (B), performed at 4 h after TBI, show the accumulation of 3-NT in the traumatic penumbra region. The expression of 3-NT was significantly reduced in the GSNO group. Photomicrographs of IHC of 3-NT (C), determined at 4 h after TBI, show the enhanced expression of 3-NT in and around vessels. Treatment with GSNO reduced the TBI-mediated increased expression of 3-NT. Colocalization of 3-NT with an endothelial cell marker vWF as yellowish fluorescence (D) indicates that 3-NT was expressed in endothelial cells. Dot blots (A, B) and photomicrographs (C, D) are representative of n = 3 in each group. Densitometry results (B) are expressed as fold change, and data are presented mean ± SD. *p < 0.05 vs. Sham and GSNO.
Figure 2
Figure 2
Effect of GSNO on reduction of the expression of 3-NT at 14th day after TBI. Photomicrographs of IHC show enhanced reaction of 3-NT (red color) in the traumatic penumbra region of TBI compared to GSNO group. Sham brain does not show 3-NT-positive cells. Colocalization (yellowish, merged) of 3-NT (red color) with either neuronal marker NeuN (green) (B) or endothelial cell marker PECAM-1 (green) (C) indicates that both neurons and endothelial cells have significantly increased expression of 3-NT even after 14 days of TBI. Photomicrographs are representative of n = 3 in each group.
Figure 3
Figure 3
Effect of GSNO and SIN-1 on levels of 3-NT and NO in plasma at 4 h after TBI. Levels of 3-NT (A) and NO (B) were measured in plasma using colorimetric and fluorometric assay kits, respectively. While GSNO treatment of TBI decreased, the SIN-1treatment increased the levels of 3-NT (A). GSNO treatment also increased the levels of NO, whereas SIN-1 treatment did not alter the levels of NO in the injured animals. Results are expressed as nM for 3-NT and μmol/L for NO. Data are presented as mean ± SD of triplicate determinations from five different experiments. ***p < 0.001 vs. Control, Sham, GSNO, +p < 0.05 vs. TBI.
Figure 4
Figure 4
Effect of GSNO and SIN-1 on levels of TBARS in plasma and on the expression of 4-HNE and ferritin in the traumatic penumbra region at 4 h after TBI. Levels of TBARS were measured in plasma using TBARS assay kit. While GSNO treatment of TBI decreased, the SIN-1 treatment increased the levels of TBARS (A). TBARS results are expressed as nmol/ml plasma, and data are presented as mean ± SD of triplicate determinations from five different experiments. ***p < 0.001 vs. Control, Sham, GSNO, +p < 0.05 vs. TBI. The expression of 4-HNE (B) and ferritin (C) was determined using IHC. Photomicrographs show enhanced reaction of both 4-HNE and ferritin in the traumatic penumbra compared to GSNO group. Sham brain does not show significant number of 4-HNE or ferritin-positive cells. Photomicrographs are representative of n = 3 in each group.
Figure 5
Figure 5
Effect of GSNO and SIN-1 on BBB leakage and edema. Photographs showing Evan's blue (EB) extravasations in five coronal sections of brain starting at 4 h after TBI. Animals were sacrificed at 48 h, the brain was sectioned and photographed (A) and the intensity of EB (B) was determined by spectrofluorometric estimation. EB extravasations were not observed in sham brain, hence the photographs of sham brain are not shown. Edema (C) was measured at 24 h after TBI. While GSNO treatment of TBI decreased, SIN-1treatment increased both the Evan's blue extravasations and edema. Data are expressed as mean ± SD from five different experiments. ***p < 0.001 vs. TBI and SIN-1, *p < 0.05 vs. TBI.
Figure 6
Figure 6
Effect of GSNO and SIN-1on reduction of mRNA expression of ICAM-1 in brain at 24 h and 14th day after TBI. RNA was isolated from Sham, TBI, GSNO and SIN-1 treated traumatic penumbra region at 24 h (A) and 14th day (B) after TBI. Levels of ICAM-1 were determined by RT-PCR and normalized with β-actin. While GSNO treatment decreased, SIN-1 treatment increased ICAM-1 levels at both 24 h (A) and 14 days (B). Data are presented as mean ± SD (fold change) of duplicate determinations from three different experiments. ***p < 0.001 vs. Sham and GSNO, +++p < 0.001 vs. TBI.
Figure 7
Figure 7
Effect of GSNO on reduction of infarction after TBI. Representative TTC-stained brain section (#3 out of the six consecutive sections from cranial to caudate region) corresponding to the largest infarction (A) and infarct area (B) from each group. Brain sections (2 mm thick) were stained with TTC at 48 h after CCI to show the area of infarctions. The infarctions were not observed in the sham group. The area under the rectangle on GSNO section was used for IHC and histology studies. Data are presented as means ± SD from three different experiments. *** p < 0.001 vs. TBI.
Figure 8
Figure 8
Effect of GSNO and SIN-1 on levels of reduced glutathione (GSH) and total glutathione (TGSH; GSH+GSSG) in the traumatic penumbra at 24 h after TBI. Levels of GSH and GSSG were measured in plasma using a colorimetric assay kit. Results are expressed as nmol/mg wet tissue, and data are presented as mean ± SD of triplicate determinations from five different experiments. ***p < 0.001 vs. Control, Sham, GSNO, +p < 0.05 vs. TBI.
Figure 9
Figure 9
Effect of GSNO on reduction of inflammation, demyelination and axonal loss at 14th day after TBI. IHC photomicrographs of H&E staining (A) show enhanced inflammatory infiltration in the traumatic penumbra region of TBI group compared with GSNO group. Sham group does not show infiltration. LFB (B) and Bielschowsky silver (C) stainings show loss of myelin and axons respectively in the traumatic penumbra region. Treatment with GSNO reduced the loss of myelin and damage to axons. The sham brain does not show loss of myelin or axons. Photomicrographs are representative of n = 3 in each group.
Figure 10
Figure 10
Effect of GSNO on enhanced expression of synaptophysin, BDNF, and TrkB at 14th day after TBI. IHC photomicrographs show reduced expression of synaptophysin (A, red fluorescence), BDNF (B, brown diaminobenzidine staining), and TrkB (C, red fluorescence) in the traumatic penumbra region of the TBI group. Sham and GSNO groups show enhanced expression of synaptophysin, BDNF and TrkB compared to the TBI group. Photomicrographs are representative of n = 3 in each group.
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