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

Neurobehavioral dysfunctions associated with traumatic brain injury (TBI) are the consequences of oxidative injury in the neurovascular unit that results in a damaging progression. These pathological events include continuous production of reactive oxidizing species and inflammation leading to disruption of the blood brain barrier (BBB), altered tissue homeostasis, axon/myelin loss, and cell death [1]. Previously, we reported the efficacy of S-nitrosoglutathione (GSNO) in TBI (short-term) [2], stroke [3, 4] and spinal cord injury (SCI) [5]. In this report, we investigate the mechanisms of GSNO's action and test whether GSNO stimulates neurorepair processes in a clinically relevant two-week long TBI study.

GSNO, a modulator of cellular redox, is a physiological metabolite produced by the reaction of nitric oxide (NO) with glutathione (GSH) [6]. It is an efficient nitrosylating agent, and the mechanism of nitrosylation modulates protein functioning in health and disease [7, 8]. Moreover, nitrosylation, like phosphorylation, functions as a signaling pathway and plays a major role in regulating several physiological and pathological processes. Under physiological conditions, GSNO and S-nitrosothiols are present in blood and brain [9‐12]. The concentration of GSNO in adult rat brain tissue is estimated to be 6-8 μM, which is ~0.3 to 0.7% of the tissue GSH level [10]. A study on GSNO metabolism and its membrane crossing ability has been reported [13]. Using an in vitro BBB model, we have also reported that significant levels of GSNO cross the cellular membrane [3].

Pharmacologically, GSNO has been shown to protect the central nervous system (CNS) against excitotoxicity, inflammation, and reactive oxygen species (ROS) in a variety of injury conditions [14]. GSNO invokes its anti-inflammatory effects on post-injury events mainly through the down regulation of the expression of NF-κB, adhesion molecules, cytokines and inducible NOS (iNOS) [2, 3, 15‐17]. It exerts its neuroprotective effects via reducing the neuronal apoptotic cell death and inhibiting the activity of caspase-3 [2, 3]. GSNO shows its antioxidant action through the modulation of redox [18], such as increasing glutathione (GSH) [4] and decreasing peroxynitrite levels [19‐21]. In acute CNS injury animal models, GSNO protects BBB, decreases edema, and reduces the expression of ICAM-1, ED1, and MMP-9 [2]. Furthermore, GSNO inhibits platelet activation, reduces embolization in humans [22‐26], and inhibits inflammatory events in endothelial [17] and T cells [27]. Nitrosylation's protective effect in heart disease has also been observed [22, 28, 29]. Deficient nitrosylation seems to be a general mechanism of disease pathogenesis [30]. However, the potential of GSNO to stimulate the neurorepair process in human or animal brain trauma models, such as TBI, has not been investigated.

To investigate the GSNO-mediated mechanisms of neurorepair, we used a rat controlled cortical impact (CCI) model because approximately 40% of all TBIs are contusions [31]. The rat model of TBI using the focal CCI technique is recognized as physiologically relevant to human TBI [32]. It reproduces many of the features of human brain injuries including inflammation, BBB disruption, neuron loss, motor deficit, and compromised memory [33].

Presently, effective therapy for TBI patients is not available, mainly because of the limited understanding of the crosstalk between the multiple pathways involved in acute and chronic phases of the injury [1]. Delineation of these mechanisms is complicated due, in part, to excessive production of reactive oxygen/nitrogen species (ROS/RNS), not only in the brain cells but also in the endothelial cells. The involvement of ROS/RNS is supported by the observed reduced damage and improved brain functions afforded by free radical trapping agents and antioxidants in TBI [1, 34‐37] and stroke [38, 39]. One of the highly deleterious ROS/RNS is peroxynitrite, a reaction product of a diffusion limited, instantaneous reaction between superoxide and NO. Peroxynitrite is documented to be highly injurious, and its reduction yields neuroprotection following experimental TBI and ischemia reperfusion (IR) injury [35, 40‐42]. Hence, 3-morpholino sydnonimine (SIN-1), a peroxynitrite forming agent, is deleterious in animal models of TBI [43] and stroke [4]. Treatment with GSNO has been shown to decrease the levels of peroxynitrite following brain injury [4, 20]. GSNO also reduces inflammation, protects neurons from apoptotic cell death, and improves neurobehavioral functions following TBI [2], SCI [5], and IR [3, 4]. However, the mechanisms through which GSNO reduces peroxynitrite, stimulates the neurorepair process, and improves neurobehavioral function are not clear.

In TBI, reduced NO bioavailability in the neurovascular unit is recognized to be the consequence of its conversion to peroxynitrite. Once formed, peroxynitrite can 'uncouple' endothelial NOS (eNOS) via oxidation and thus depleting the eNOS substrate L-arginine, the cofactor tetrahydrobiopterin (BH4), or both [44, 45]. Excessive production of peroxynitrite can lead to endothelial cell activation and up regulation of ICAM-1 in such an oxidative microenvironment, thus compromising BBB integrity and contributing to edema [41]. Peroxynitrite is a highly reactive species for the nitration and inactivation of tyrosine in proteins in biological systems [46]. It also interacts with iron to form the most reactive hydroxyl radical [47]. Reducing iron or neutralizing ROS has been shown to be neuroprotective, indicating that iron and ROS play significant role in peroxynitrite-induced brain injury [48]. Antioxidant therapy down regulates neuroinflammation, stimulates the neurorepair process, and reduces neurological deficits following TBI [34, 49].

In this study, we demonstrated that post-injury administration of GSNO reduced the oxidative injury in the neurovascular unit caused by peroxynitrite and its metabolites. The injury was associated with BBB leakage, neuroinflammation, edema, depletion of GSH and NO, and reduced expression of neurotrophic factors. Increased BBB leakage and edema, and an enhanced mRNA expression of ICAM-1 in the SIN-1 group compared to the TBI group indicate the involvement of peroxynitrite in BBB pathology. These results are supported by the increased formation of peroxynitrite and lipid peroxide/aldehydes and decreased levels of NO and GSH in the SIN-1 group. Moreover, a long-term (2-week) treatment with GSNO not only inhibited the loss of myelin and axons, but also enhanced the expression of neurotrophic factors. These findings indicate that GSNO not only down regulates TBI-induced neurovascular exacerbations but also stimulates the mechanisms of neurorepair.

Following TBI, 3-NT (peroxynitrite) is formed early in and around the vessels (Figure 1) and in plasma (Figure 3A). The expression of 3-NT remains increased in neurons as well as in endothelial cells even two weeks after TBI (Figure 2). The increased 3-NT levels (Figures 1, 2, 3) correlated with the levels of lipid peroxidation products in both brain and plasma (Figure 4). While GSNO treatment of TBI animals significantly reduces the levels of 3-NT and lipid peroxidation products (Figures 1, 2, 3, 4), SIN-1 (a peroxynitrite forming agent) treatment significantly increases the levels of peroxynitrite (Figure 3A) and TBARS (Figure 4) in plasma compared to the TBI group. Furthermore, SIN-1 treatment of TBI increases BBB leakage (Figure 5A), edema (Figure 5C), and the expression of ICAM-1 (Figure 6), supporting the involvement of peroxynitrite in TBI-induced neurovascular pathology. Also, SIN-1 treatment of TBI remarkably decreases and GSNO significantly increases the levels of NO (Figure 3B) and GSH compared to TBI (Figure 8), indicating an association of antioxidant activity with GSNO. This antioxidant activity of GSNO might be responsible, at least in part, for its neurovascular protective ability. In the long-term two-week study, GSNO treatment of TBI stimulated the neurorepair mechanisms by blocking the loss of myelin/axons (Figure 9) and enhancing the expression of both synaptophysin and neurotrophic factors such as BDNF and its high affinity receptor TrkB (Figure 10).

The deleterious role of peroxynitrite in both IR and TBI is accepted because the scavenging of peroxynitrite has resulted in neurovascular protection [41, 42, 67, 71, 79]. However, reducing peroxynitrite for therapeutic purposes by GSNO has an advantage over reported metal-based synthetic scavengers [42, 80] because GSNO is an endogenous molecule of the human body and it is metabolized ultimately into beneficial -SNO/NO and GSH [18]. In the traumatized brain, NO bioavailability decreases early due to its conversion to peroxynitrite as shown in Figure 3. Once formed, peroxynitrite uncouples eNOS via oxidizing and depleting either the eNOS substrate L-arginine, the cofactor tetrahydrobiopterin, or both. Uncoupled eNOS produces more superoxide than NO, resulting in reduced NO levels and the formation of increasing amounts of peroxynitrite in the same compartment. This uncoupling of eNOS via peroxynitrite may lead to endothelial cell activation and enhanced expression of ICAM-1, thus compromising BBB integrity (Figure 5A-B) and causing a neuroinflammatory secondary injury (Figure 6) to the neurovascular unit [41, 70]. Inflammation-induced monocyte-endothelial cell interaction and up regulation of ICAM-1 have been reported to be inhibited by GSNO-mediated S-nitrosylation of NF-κB [17], indicating that GSNO invokes its anti-inflammatory effect through S-nitrosylation. eNOS is also reported to be dynamically regulated by S-nitrosylation at Cys¹⁰¹, resulting in inhibition of eNOS activity [81, 82]. Therefore, GSNO-mediated reduced levels of peroxynitrite formation (Figures 1, 2, 3) and the consequent BBB protection (Figure 5) may have resulted from the inhibition of aberrant eNOS by S-nitrosylation. The protective effect of S-nitrosylation signaling is well documented in the cardiovascular system [83]. A depletion of circulating nitroso/nitrosyl species in plasma has been observed in patients with endothelial dysfunction [84]. Deficient S-nitrosylation is considered a general mechanism in neurodegenerative diseases [27, 85‐87]. GSNO also inhibits platelet activation [22, 23] and protects BBB integrity and epithelial permeability [2, 88]. Like endothelial cells, neuronal cells also produced peroxynitrite (Figure 2B), which may induce cell death, thus compromising neurorepair. GSNO treatment of TBI also reduced these neuronal peroxynitrite levels, likely via nitrosylating and inhibiting neuronal NOS activity, thereby stimulating neurorepair mechanisms. Neuronal NOS activity has been shown to be down regulated by GSNO via nitrosylation of the NMDA receptor [89].

In vivo, the action of GSNO is modulated mainly by nitrosylation/transnitrosylation [7, 8, 18]. Via nitrosylation, GSNO exerts its anti-inflammatory action mainly through inhibition of NF-κB, TNF-α, and iNOS/neuronal NOS [3, 16, 90, 91], its antiapoptotic action through inhibition of caspase-3 activity [19], and its antioxidant activity through down regulation of peroxynitrite [20, 21] and up regulation of GSH [4]. Another target of GSNO might be the inhibition of superoxide production by nitrosylation of NADPH oxidase. Nitrosylation of p47phox of NADPH oxidase has been reported to suppress superoxide production in human endothelial cells [92]. Superoxide is implicated in the reduction of GSNO because NO has higher reactivity with superoxide than GSH, a substrate of GSNO biosynthesis. Reduced levels of GSNO may also result from its decomposition (denitrosylation) by the enhanced expression of GSNO reductase [8]. The expression of GSNO reductase is increased during inflammation [8]. GSNO reductase is the only known enzyme that degrades GSNO without releasing NO. Therefore, an exogenous supplementation of GSNO under neuroinflammatory conditions may reduce the oxidative burden (Figures 4, 8) and protect BBB integrity (Figure 5A-B), resulting in reduced edema (Figure 5C) and decreased brain damage (Figures 7, 9). Moreover, GSNO/hemoglobin homeostasis is required in the circulation to maintain GSNO-based S-nitrosylation mechanisms. Enhancing S-nitrosylation by ethyl nitrite, a nitrosylating agent of hemoglobin, has improved outcomes in mice with subarachnoid hemorrhage [93].

Our previous study on neurovascular protection by S-nitrosylating agents in TBI and stroke shows that GSNO down regulated the expression of inflammatory mediators ICAM-1, ED-1, iNOS, MMP-9, protected the BBB, and improved neurological functions [2, 3]. In this study, we observed that a long-term post-TBI treatment with GSNO not only improved tissue structure (Figures 7, 9), reduced the expression of ICAM-1 (Figure 6), and restored myelin/axon loss (Figure 9) but also increased synaptogenesis (increased expression of synaptophysin; Figure 10) and stimulated the expression of neurotrophic factors (enhanced expression of BDNF and TrkB; Figure 10). Neurorepair following brain injury is facilitated by synaptophysin and BDNF [94, 95]. Synaptophysin is a protein involved in the biogenesis of synaptic vesicles and budding. NO can influence plastic changes in specific brain regions and can change the expression of synaptophysin [95]. Because NO is converted to peroxynitrite in an oxidative environment such as that which occurs in TBI, it is difficult to dissect the effects on neurotrophins of NO/peroxynitrite vs. nitrosylation. However, our data showing enhanced expression of synaptophysin, BDNF and TrkB in the GSNO brain compared to the TBI (Figure 10) indicate that nitrosylation accelerates the neurorepair process. Preservation of myelin and axons in the GSNO group (Figure 9) further supports GSNO-mediated mechanisms of neurorecovery.

The therapeutic potential of GSNO is also supported by increased levels of GSH and TGSH in the GSNO brain following TBI (Figure 8). Higher levels of TGSH in the GSNO group compared to TBI and SIN-1 are likely due to increased GSSG, which may be converted back to GSH by GSH reductase. GSNO may also increase GSH indirectly via neutralizing ROS. A similar observation after GSNO treatment in our IR study [4] indicates that GSNO's infarct-reducing effect (Figure 7) may be dependent on its antioxidant activity.

Because clinically proven TBI therapy is not currently available and GSNO shows clinically relevant therapeutic potential in our animal model of TBI, a further investigation is warranted to test GSNO's efficacy in human TBI. GSNO occurs naturally in the human body and is safe, as its exogenous administration in humans for other indications was not associated with toxicity or side effects [24, 26, 96].

GSNO reduces peroxynitrite and neuroinflammation, increases NO and GSH, and protects the integrity of the BBB and the neurovascular unit following TBI. Furthermore, it stimulates the mechanisms of the neurorepair in the injured animals. Therefore, GSNO has therapeutic potential to be investigated in human TBI.