Amelioration of spinal cord injury in rats by blocking peroxynitrite/calpain activity

Spinal cord injury (SCI) results in locomotor deficits and pain due to the production of noxious metabolites which are held responsible for profound neurodegeneration [1, 2]. SCI is a major medical and socio-economic problem, and the rate of SCI is increasing every year [3]. The incidence of SCI is highest among young adults due to motor vehicle accidents, violence and sports accidents [4]. Other than critical care management, no current FDA-approved drug therapy exists for traumatic SCI [1]. Several pharmacological therapies, including methylprednisolone, have been evaluated time and again in SCI [2] without clinical success. SCI is divided into two distinct types of injury: primary and secondary. Primary (immediate phase after SCI) injury includes physical damage as a direct result of the traumatic event. It cannot be reversed. Secondary injury follows the initial physical insult, resulting from mechanistic crosstalk between and among several deleterious pathways, including redox and excitotoxicity [1]. Secondary injury is therefore amenable to reversal and treatment. A critical examination of injury mechanisms shows a disturbed nitric oxide (NO) metabolome [5, 6]. We hypothesize this metabolome to be responsible for the production of neuronal nitric oxide synthase (nNOS)-dependent deleterious peroxynitrite. As a consequence, much less NO is available for S-nitrosoglutathione (GSNO) biosynthesis and thus GSNO-mediated regulation of enzymatic activities is lost. Reduced NO bioavailability and the consequent decrease in GSNO levels are associated with chronic neurovascular injuries, and exogenous GSNO supplementation is reported to ameliorate CNS injuries [7–11]. Therefore, the objective of this study was to investigate the efficacy of GSNO for functional recovery and its role in regulation of the nNOS/calpain system in a rat model of contusion SCI.

GSNO is an endogenous molecule of the human body, produced mainly in NOS expressing cells by the reaction of NO with glutathione (GSH) in the presence of oxygen [12]. GSNO’s biosynthesis is also influenced by altered redox [13]. It is present in the brain and other organs [14]. GSNO reductase (GSNOR) is the major GSNO-degrading enzyme and thus GSNOR knock out mice store GSNO in excess [15]. GSNOR degrades GSNO to ammonia and oxidized glutathione (GSSG) without releasing free NO [16], indicating that the NO moiety of GSNO is not recycled by the enzymatic activity of GSNOR. GSNO is directly involved in cell signaling via S-nitrosylation of target proteins, including calpains, NF-κB, STAT3, neuronal NOS (nNOS) [8, 9, 17–21]. Several studies showing the efficacy of GSNO in human diseases have been listed by Hornyak et al. [22]. None of the studies reported significant side effects in humans associated with the use of exogenous GSNO. In animal studies, GSNO protects against cardiac ischemic injury [23], indicating the therapeutic potential of GSNO-mediated S-nitrosylation mechanism [9, 24]. S-nitrosylation of PTEN (a lipid phosphatase) has been shown to inhibit its activity, leading to the activation of Akt and thus the stimulation of neurorepair process in an animal model of stroke [25]. The Akt activation has been shown to be associated with stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), which, in turn, induces the expression vascular endothelial growth factor (VEGF) leading to therapeutic angiogenesis/neurogenesis and consequent recovery of function [26].

In spite of the significant role of GSNO in cellular functions, neither GSNO nor its S-nitrosylation mechanism has been investigated for anti-neurodegenerative efficacy in SCI. Decreased synthesis of GSNO due to reduced levels of either GSH [19] or NO [27] or both in SCI, combined with increased degradation of GSNO by inflammation-induced enzyme activity of GSNO reductase (GSNOR) [24], will likely contribute to the reduced levels of GSNO in SCI. Deficient S-nitrosylation is considered to be a general neurodegenerative mechanism [28–30]. Via S-nitrosylation, GSNO protects against neurodegeneration by targeting multiple signaling pathways, including anti-inflammatory, anti-oxidant and vascular effects [9, 31–33]. GSNO also stimulates production of neurotrophic factors [11, 34] and induces neuroregeneration [35]. On the other hand, peroxynitrite is formed by an instantaneous diffusion limited reaction between NO and superoxide under oxidative conditions. This reaction not only reduces NO bioavailability but also increases peroxynitrite-mediated tissue/cell damage. Peroxynitrite causes a sustained activation of calpains [36], leading to neurodegeneration and functional deficits [10, 21]. In SCI, the observed increased 3-nitrotyrosine (3NT) levels, a peroxynitrite adduct of tyrosine residue, in the injured cord [37, 38] suggest its pathological role in SCI. We observed that GSNO treatment of SCI decreased the levels of peroxynitrite via inhibition of nNOS activation, which paralleled with decreased calpain activity and improved Basso Beattie and Bresnahan (BBB) locomotor rating scale scores as well as the threshold for mechanical allodynia out to 2 weeks post-injury.

This is a preliminary mechanism-based study showing that SCI-induced functional deficits (Fig. 1), and neuropathic pain (Fig. 2) paralleled aberrant activation of nNOS, increased levels of peroxynitrite (Fig. 3) and high activity of calpains (Fig. 4) in a 2-week rat model of contusion SCI. The study further shows the therapeutic efficacy of GSNO. It improved functional deficits (Fig. 1) and increased the pain threshold (Fig. 2) by inhibiting the activities of both nNOS (Fig. 3) and calpains (Fig. 4) and reducing the levels of injurious peroxynitrite (Fig. 3).

Locomotor function deficits and pain are two major consequences intrinsic to SCI [49]. Deleterious metabolites, formed by the aberrant activities of otherwise regulatory enzymes such as nNOS, are primarily responsible for producing potent oxidizing/neurodegenerating agents, such as peroxynitrite, in neurons. Excessive accumulation of neuronal peroxynitrite is implicated in neuronal cell death and subsequent neurodegeneration [55]. In fact, scavenging peroxynitrite using peroxynitrite decomposition catalysts such as FeTPPS has been reported to ameliorate SCI [56], supporting this direct deleterious role of peroxynitrite. Inhibition of nNOS activity following SCI [57] has also been shown to provide neuroprotection, and nNOS KO mice show improved recovery after SCI [58], indicating a deleterious role of nNOS activity in SCI. An nNOS-based therapy for SCI therefore offers a logical approach. Reversible down regulation of nNOS activity, such as via-S-nitrosylation, is preferred because it maintains the required physiological activity of nNOS. The roles of other NOS enzymes (inducible and endothelial) in the chronic phase pathology, such as in neurodegeneration and pain, is not clear [59]. Peroxynitrite produced in neurons is a product of an instantaneous reaction between nNOS-derived NO and superoxide. Because NO is used for the formation of peroxynitrite (3-NT), the biosynthesis of GSNO, a product of a slow reaction between NO and GSH, and GSNO-mediated regulatory mechanisms are derailed. Because deleterious nNOS activity is down regulated by a GSNO-mediated S-nitrosylation mechanism [60], reduced NO/GSNO levels contribute to nNOS-dependent neurodegeneration and pain SCI pathology. Such a derailed NO/GSNO metabolism in SCI may also be responsible for functional deficits. Therefore, we tested the hypothesis that GSNO reduces the levels of peroxynitrite, inhibits the activity of nNOS, and improves behavioral function and cellular plasticity in young adult male rats.

Pain is one of the major issues in SCI for obvious reasons but also because it impairs recovery after SCI [61]. Both inflammatory and neuropathic pain (caused by a lesion or disease of somatosensory function) are present in the majority of SCI patients [50, 62]. Due to the lack of mechanistic understanding of pain, satisfactory pain-management therapy of SCI is not yet available. We observed that the GSNO treatment significantly increased the pain threshold and reduced calpain activity after 2 weeks of SCI (Fig. 2), indicating that GSNO possesses an analgesic property in addition to improving functional deficits (Fig. 1). Furthermore, significant increases in tissue peroxynitrite levels (Fig. 3) correlated well with calpain-mediated cytoskeleton degradation (Fig. 4), indicating peroxynitrite’s contribution to neurodegeneration. Recently, we have shown that the activity of calpains is upregulated by peroxynitrite whereas GSNO, via S-nitrosylation, inhibits the activity of calpains in TBI [8], indicating a similar role of peroxynitrite versus GSNO in this SCI study. Peroxynitrite originating from nNOS [63] and NMDA receptor activity [64] is also recognized among the prominent causes of neuropathic pain following nerve injury. GSNO, likely via S-nitrosylation, down regulates the activity of nNOS, thus reducing the levels of peroxynitrite and its associated pain. Decreased levels of peroxynitrite in brains and improved neurological functions have also been shown after GSNO treatment in rat models of stroke and TBI [34, 51], indicating that the mechanism of S-nitrosylation invokes anti-neurodegeneration and anti-pain activities in CNS trauma. These observations establish the therapeutic potential of GSNO-mediated mechanisms in simultaneously treating neurodegeneration and neuropathic pain following SCI.

Inflammation is another significant component of SCI, contributing to neurodegeneration and pain. Interestingly, GSNO-mediated mechanisms are also shown to down regulate the expression of pro-inflammatory cytokines and NF-κB [18, 39], as well as the activation of STAT3 [20]. These actions contribute to the reduction of inflammation-mediated neurodegeneration and pain. As an alternative mechanism to alleviate pain in SCI, IL-10 has been shown to be a potent anti-neuropathic pain molecule [65–67], and GSNO-mediated mechanisms are reported to upregulate the levels of IL-10 [68] as well as to reduce pain in a rat model of cauda equine compression [44]. We add one caveat that excessive accumulation of GSNO, as observed in GSNOR knock out mice, creates altered redox pathology, leading to sensitization to pain [15] and thus rendering GSNOR knock out mice ‘not suitable’ for pain related studies. A critical balance of NO/GSNO versus peroxynitrite is requisite to maintain the homeostasis of the NO metabolome, ameliorating SCI pathology. Low dose exogenous supplementation of GSNO seems to be an ideal approach to improve pain threshold and to provide neuroprotection. One advantage of using GSNO supplementation is that GSNO-mediated regulatory mechanisms are reversible, and thus the physiological levels of activity of targeted enzymes can be maintained. The improved BBB score (Fig. 1) and increased pain threshold (Fig. 2) reported here support the efficacy of GSNO in ameliorating SCI and provide a rationale to further investigate GSNO therapy in SCI. Limitations the most affected population from SCI is young adult males. Therefore, we used young adult male animals in this preliminary study; however, the exclusion of female rats demands further testing in these populations. We are also aware that a 2-week SCI study is relatively short to sufficiently characterize the injury course. Moreover, biochemical studies of the early acute phase are needed. Therefore, in follow-up study, the efficacy of GSNO therapy and the cause-and-effect relationship between GSNO and peroxynitrite/calpain system will be investigated for both acute and longer chronic periods of time, using both male and female young adult animals.

Under SCI pathology, superoxide reacts with NO and this reaction produces a large amount of injurious peroxynitrite. Increased levels of peroxynitrite cause an upregulation of calpain activity and thus neuronal cytoskeleton degradation. Peroxynitrite is also recognized as a mediator of pain. Under such pathological conditions, S-nitrosylation-mediated biological regulation (inhibition) of the activity of nNOS and calpain is lost due to its reduced bioavailability, and thus levels of NO/GSNO. Replenishment of exogenous GSNO was found to inhibit the activation of nNOS, thus blocking the production of peroxynitrite and reducing the activity of calpains, leading to improved locomotor function and decreased mechanical allodynia acutely post-injury in SCI animals. Furthermore GSNO’s administration to humans is not associated with adverse effects [22]. Therefore, testing the efficacy of exogenous GSNO in humans may lead to an SCI therapy of clinical relevance.