Administration of S-nitrosoglutathione after traumatic brain injury protects the neurovascular unit and reduces secondary injury in a rat model of controlled cortical impact - PubMed (original) (raw)

Administration of S-nitrosoglutathione after traumatic brain injury protects the neurovascular unit and reduces secondary injury in a rat model of controlled cortical impact

Mushfiquddin Khan et al. J Neuroinflammation. 2009.

Abstract

Background: Traumatic brain injury (TBI) is a major cause of preventable death and serious morbidity in young adults. This complex pathological condition is characterized by significant blood brain barrier (BBB) leakage that stems from cerebral ischemia, inflammation, and redox imbalances in the traumatic penumbra of the injured brain. Once trauma has occurred, combating these exacerbations is the keystone of an effective TBI therapy. Following other brain injuries, nitric oxide modulators such as S-nitrosoglutathione (GSNO) maintain not only redox balance but also inhibit the mechanisms of secondary injury. Therefore, we tested whether GSNO shows efficacy in a rat model of experimental TBI.

Methods: TBI was induced by controlled cortical impact (CCI) in adult male rats. GSNO (50 microg/kg body weight) was administered at two hours after CCI. GSNO-treated injured animals (CCI+GSNO group) were compared with vehicle-treated injured animals (CCI+VEH group) in terms of tissue morphology, BBB leakage, edema, inflammation, cell death, and neurological deficit.

Results: Treatment of the TBI animals with GSNO reduced BBB disruption as evidenced by decreased Evan's blue extravasation across brain, infiltration/activation of macrophages (ED1 positive cells), and reduced expression of ICAM-1 and MMP-9. The GSNO treatment also restored CCI-mediated reduced expression of BBB integrity proteins ZO-1 and occludin. GSNO-mediated improvements in tissue histology shown by reduction of lesion size and decreased loss of both myelin (measured by LFB staining) and neurons (assayed by TUNEL) further support the efficacy of GSNO therapy. GSNO-mediated reduced expression of iNOS in macrophages as well as decreased neuronal cell death may be responsible for the histological improvement and reduced exacerbations. In addition to these biochemical and histological improvements, GSNO-treated injured animals recovered neurobehavioral functions as evaluated by the rotarod task and neurological score measurements.

Conclusion: GSNO is a promising candidate to be evaluated in humans after brain trauma because it not only protects the traumatic penumbra from secondary injury and improves overall tissue structure but also maintains the integrity of BBB and reduces neurologic deficits following CCI in a rat model of experimental TBI.

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Figures

Figure 1

Figure 1

Effect of GSNO treatment on TBI-mediated tissue injury and on the expression of ICAM-1 and ED1 in ipsilateral rat brain after CCI. Animals were treated with GSNO (0.05 mg/kg) 2 h following CCI. Seven days after TBI, brains were removed, sectioned and stained with H&E (A) and LFB (C) and analyzed for lesion size (B) using morphometric measurement. GSNO reduced TBI-mediated tissue injury, decreased lesion size (B), and reduced the expression of ICAM-1 (D) and ED1 (E) (detected in traumatic penumbral area at 72 h following TBI). Photomicrographs are the representative of n = 3 in each group. Lesion size data are presented as mean ± SD (n = 3). ***p < 0.001 vs. CCI+VEH.

Figure 2

Figure 2

Effect of GSNO on TBI-mediated BBB disruption, edema and on the expression of ZO-1, occludin and MMP-9 in ipsilateral rat brain after CCI. Representative photographs showing Evan's blue extravasation at 48 h in 2 out of 6 coronal sections (A). Significant EB leakage was observed in CCI+VEH brain, and the leakage was reduced in the CCI+GSNO group. EB extravasation was not observed in Sham animals (see Table 1). A similar treatment with GSNO decreased the brain water content (edema) in ipsilateral brain (C) measured at 24 h after CCI. Treatment with GSNO also enhanced TBI-mediated reduced expression of ZO-1 and occludin in traumatic penumbral area from ipsilateral hemisphere measures at 24 h following TBI by Western blot (B). TBI-mediated increased expression of MMP-9 was also decreased following GSNO treatment (detected in traumatic penumbral area at 72 h following TBI) (D). Photomicrograph of Evan's blue is the representative of n = 5. Edema data are presented as mean ± SD (n = 5). Western blot and photomicrographs of MMP-9 immunohistochemistry are representative of n = 3 in each group. *p < 0.05 vs. CCI+VEH or Sham. ips, ipsilateral; conr, contralateral.

Figure 3

Figure 3

Effect of GSNO on the expression of iNOS present mainly in macrophages/microglia. Red fluorescence indicates induced higher expression of iNOS in the traumatic penumbral area of CCI+VEH brain than in the treated (CCI+GSNO) brain determined by immunohistochemistry at 72 h following CCI. Sham animal did not show any significant staining of iNOS (A). Colocalization of the expression of iNOS with ED1 as yellowish fluorescence (B) indicates that iNOS was mainly expressed in macrophages/microglia.

Figure 4

Figure 4

Effect of GSNO on TBI-mediated deficits in neurobehavioral function. A treatment at 2 h post-injury following CCI with GSNO improved motor function evaluated by rotarod task (A), decreased modified neurological severity score (mNSS) (B) evaluated on 6th day after CCI and reduced time to remove the stimuli from limbs (tactile strength test) (C). Data are presented as mean ± SD (n = 7). ***p < 0.001, **p < 0.01, *p < 0.05 vs. CCI+VEH.

Figure 5

Figure 5

Effect of GSNO on neuronal apoptotic cell death (TUNEL assay) at 72 h after CCI. Traumatic penumbral region in CCI+VEH brain had a significantly higher number of TUNEL positive cells compared to CCI+GSNO treated brain. Sham brain had no TUNEL positive cells (A). TUNEL positive cells were found to colocalize with neuronal marker NSE (B). Number of TUNEL positive cells (C) and number of stained positive cells (D) were counted in three different fields and averaged. Data are presented as mean ± SD TUNEL staining was carried out as described in Methods. Photomicrographs are the representative of n = 3 in each group. **p < 0.01 vs. Sham and CCI+GSNO.

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