Mechanisms of nitric oxide in spinal cord injury

INTRODUCTION

Spinal cord injury (SCI) is a pathological condition that results from damage to the spinal cord and associated tissues, leading to profound consequences.1 It may result in deficiency of motor, sensory, and autonomic functions.2 The prevalence of SCI is higher in males, and its peak incidence occurs within a few minutes after the occurrence of the injury. Following SCI, rapid swelling of the spinal cord occurs, leading to complete occupation of the spinal canal diameter at the level of injury. When cord swelling surpasses venous blood pressure, secondary ischemia is observed. Consequently, autoregulation of blood flow ceases, and neurogenic shock ensues in the spinal cord, which causes systemic hypotension that further exacerbates ischemia. Furthermore, release of toxic chemicals and transfer of electrolytes from damaged nerve membranes can trigger cascading reactions that result in secondary damage, which significantly amplifies the initial mechanical trauma by affecting or killing neighboring cells.3

Functioning as a gaseous chemical messenger, nitric oxide (NO) serves as a prominent endogenous molecule involved in various physiological processes within the central nervous system.4 NO plays a pivotal role in the regulation of interneuron communication, cerebral blood flow, memory formation, synaptic plasticity, intracellular signal transmission, and neurotransmitter release.5 NO is generated through the stereospecific oxidation of the guanidino group of L-arginine by a family of enzymes known as NO synthases (NOS). NOS has been classified into three isoforms based on their location: NOS-I, also known as neuronal or brain NOS (nNOS); NOS-II, also known as inducible NOS (iNOS); and NOS-III, also known as endothelial NOS (eNOS). nNOS and eNOS are expressed constitutively as constitutive NOS, whereas iNOS is induced in response to inflammatory processes.6,7 NO in the human brain has several key effects. First, it serves as a pivotal signaling molecule that triggers the activation of cyclic guanosine 3′,5′-monophosphate (cGMP), thereby inducing vasodilation in vascular smooth muscles. Second, NO mediates the impact of glutamate on N-methyl-D-aspartate receptors to finely regulate both blood flow and pressure, with its functionality being reliant on eNOS within cerebral and coronary vessels.8 Third, short-lived reactive species, such as dinitrogen trioxide, nitroxyl, and peroxynitrite (ONOO⁻/ONOOH), are generated to serve as sources of free radicals for oxidation–reduction reactions, thereby facilitating the direct oxidation of lipids, proteins, and DNA.9 Furthermore, as a pivotal mediator of diverse inflammatory responses, it exerts regulatory control over the intricate network of inflammation.10

After SCI occurs, the elevation of NO levels leads to the overexpression of iNOS. NO can induce neuronal loss and oxidative damage by reacting with reactive oxygen species (ROS). Furthermore, excessive NO production may result in protein nitrosylation, lipid peroxidation, and functional impairment of associated proteins. The generation of NO, peroxynitrite, and nitrated proteins rapidly increases following acute SCI. Depending on their quantity and microenvironmental conditions, these molecules play a crucial role in both destructive and reparative processes that ensue after acute SCI.11 However, other constitutive forms of NOS, such as eNOS and nNOS, may produce NO, which is neuroprotective in the case of SCI.12 In this study, we investigated the potential mechanisms underlying NO pathways following SCI, with a focus on pathological and biological reactions. Furthermore, this article provides an overview of the recent drugs and methods used to regulate NO in animal SCI models. A comprehensive understanding of these potential mechanisms can offer valuable guidance and assistance for future treatments of SCI. This paper searched PubMed for articles related to NO and SCI in the past 30 years, mainly in the past 3–5 years.

NITRIC OXIDE SYNTHESIS AFTER SPINAL CORD INJURY BASED ON THE TIME COURSE

Although constitutive NOS is constitutively expressed in the central nervous system to maintain cellular homeostasis by producing NO, iNOS is predominantly expressed in various cell types under pathological conditions, such as inflammation, immune response, and trauma. The induction of iNOS requires the presence of several inflammatory cytokines, including interferon-γ, tumor necrosis factor-alpha (TNF-α), and interleukin (IL)-1β.13 However, interpreting reports on changes in NO levels after SCI and their pathophysiological consequences poses challenges. Cherian et al.14 devised a method for categorizing the immediate- (< 30 minutes) and late-stage (> 6 hours) changes following SCI for the analysis of constitutive NOS and iNOS. Thirty minutes after SCI, the level of NO in the injured spinal cord is approximately three-fold higher than that in the uninjured spinal cord. During this time frame, significant metabolic and hemodynamic abnormalities manifest within the spinal cord. SCI triggers widespread depolarization, leading to a substantial increase in the extracellular concentration of potassium and excitatory toxic amino acids, while intracellular calcium accumulates.15 The activity of nNOS exhibits a similar pattern to the concentration of NO in the early stages, with an increase observed at 5 minutes after injury and subsequent return to the baseline level after 1 hour, followed by sustained increases for several consecutive days. The activation of iNOS in the later stages elucidates the delayed NO surge occurring within 24 hours to a few days following injury.15 In contrast, the expression of iNOS mRNA is specifically observed in the injured tissue within 2 hours after SCI, with its levels gradually increasing over consecutive days. The subsequent activation of iNOS during the later stages elucidates the delayed NO surge occurring between 24 hours and a few days following injury.16

NITRIC OXIDE/CYCLIC GUANOSINE 3',5'-MONOPHOSPHATE PATHWAY RELATED TO THE PATHOLOGY OF SPINAL CORD INJURY

The involvement of activated soluble guanylyl cyclases, the generation of cGMP, and the activity of cGMP-dependent protein kinases have been demonstrated to play a pivotal role in the NO signal transduction pathway within the central nervous system.17,18 Morris demonstrated that the superficial layers of the dorsal horn in the spinal cord of neonatal rats represent the primary site of the NO/cGMP pathway (Figure 1).19

Elevated cGMP levels in the dorsal horn of the spinal cord appear to be associated with nociceptive sensitization induced by the activation of cGMP-dependent protein kinase Iα.20 Other factors may also impact NO-mediated cGMP formation in SCI. The elevated levels of glutamate generated at the site of spinal cord lesions and excessive accumulation of intracellular Ca²⁺ can stimulate NOS activity,21 creating suitable conditions for NO synthesis and causing adverse effects on the white matter. The extreme production of NO and superoxide may stimulate the production of nitrite peroxide (ONOO⁻), which in turn may be decomposed, thus obtaining nitrogen dioxide and a compound with similar hydroxyl reactivity, leading to the oxidation of proteins, DNA, and membranes, damaging lipid peroxidation. Furthermore, free fatty acids released from SCI, particularly arachidonic acid,22 may lead to the overexpression of NOS, accompanied by an increase in cGMP in cells.23

The NO/cGMP pathway also plays a complex and pivotal role in pain modulation following SCI. SCI-induced neuronal cell discharge triggers the generation of NO, which diffuses to satellite glial cells. This, in turn, stimulates the synthesis of cGMP within satellite glial cells, leading to their activation and subsequent alterations that contribute to neuronal hyperexcitability and nociceptive signaling.24 Sensitization of peripheral pain is initiated by the release of inflammatory mediators from the injured tissues. In the context of inflammatory pain, NO can be derived from resident cells or newly migrated cells, such as neutrophils and monocytes, with neutrophil-derived NO potentially contributing to the induction of inflammatory pain and tissue damage. However, Spiller proposed that the activation of the soluble guanylyl cyclase/cGMP/protein kinase G pathway could lead to the activation of the ATP-sensitive potassium channel (K_ATP). This activation results in intracellular potassium expulsion, leading to hyperpolarization and directly blocking acute and persistent hyperalgesia. Several studies have demonstrated that the NO/cGMP pathway influences the analgesic effects of certain drugs used for neuropathic pain treatment,25,26,27 such as tramadol, clonidine gabapentin, and indomethacin.28 Furthermore, NO activates guanylate cyclase and enhances the synthesis of cGMP, which plays a crucial role in the regulation of muscle relaxation and synaptic transmission. These findings provide valuable insights into enhancing motor function and alleviating bladder spasm following SCI.

FUNCTIONS OF ENDOTHELIAL NITRIC OXIDE SYNTHASE THROUGH THE SER-1177 AND THR-495 PHOSPHORYLATION PATHWAY AFTER SPINAL CORD INJURY

Osuka et al.29 demonstrated that severe SCI leads to the degradation of eNOS and nNOS, whereas mild SCI results in the phosphorylation of eNOS at Ser-1177 for 1–2 days followed by a subsequent decrease in its levels. Phosphorylation and activation of eNOS at Ser-1177, predominantly expressed in the endothelial cells of microvessels within the gray matter, were indicated by an increase in the intensity of nicotinamide adenine dinucleotide phosphate-dependent flavotransferase immunoreactivity within the intact gray matter endothelium 1 day after SCI.30 eNOS expression was significantly elevated 1 and 14 days after spinal cord transection injury, as reported by Varziri et al.31 The phosphorylation of eNOS at Ser-1177 and Thr-495 has been demonstrated to be regulated by multiple protein kinases, including cyclic adenosine monophosphate-dependent kinase and adenosine monophosphate-activated protein kinase. Notably, phosphorylation at Ser-1177 plays a crucial role in activating eNOS.32 In contrast, phosphorylation at Thr-495 inhibits eNOS activity. The phosphorylation of eNOS is a determinant outcome after cerebral ischemia.33 NO from eNOS has neuroprotective effects and can induce dilation of surrounding blood vessels after cerebral ischemia.34 After brain injury, it plays a crucial role in safeguarding the cerebral blood flow. The phosphorylation of eNOS also contributes to the inhibition of platelet aggregation in penumbral microvessels by acting on N-methyl-D-aspartate receptors.35

The infiltration of leukocytes within the parenchyma36 and proliferation of smooth muscle cells.37 Meanwhile, Estévez et al.38 reported that NO produced by NOS can greatly inhibit apoptotic neuronal death. In the subacute stage following SCI, eNOS is generally activated, increasing blood flow, and shows potential involvement in protective and reparative responses via phosphorylation pathways.

RESPONSE OF INFLAMMATION AND INDUCIBLE NITRIC OXIDE SYNTHASE ACTIVATION

The characteristics of inflammatory responses following SCI encompass infiltration of inflammatory cells and release of inflammatory mediators, which enhance blood vessel permeability, resulting in edema and disruption of the blood-brain barrier.39 The inflammatory response following SCI primarily arises from the infiltration of neutrophils at the injury site. Neutrophils can release various substances, including ROS, free radicals of reactive nitrogen species, cytokines, chemokines, and diverse enzymes.40 The activity and accumulation of neutrophils are regulated by various cytokines, including TNF-α, IL-1, and IL-6, which are maintained through the intermediate transcriptional process known as nuclear factor kappa B (NF-κB). NF-κB is typically inactivated in the cytoplasm and is associated with inhibitor of NF-κB (IκB) as an endogenous inhibitor protein. In response to inflammatory stimulation and certain physical factors (e.g., depolarization, bleeding, hyperglycemia, and shear stress) commonly observed in SCI, NF-κB is activated. These biochemical and physical factors may induce the activation of IκB kinase, leading to the phosphorylation of IκB kinase that subsequently triggers the activation of NF-κB.41 NF-κB functions as a transcriptional regulator for the synthesis of iNOS.42 Consequently, iNOS generates substantial amounts of NO, leading to cellular damage. Following SCI, the infiltration of leukocytes increases ROS and NO levels. Central nervous system cells exposed to NO and ROS undergo oxidative stress induced by ROS and nitrogen-responsive species, which can result in pathological alterations in various biological substrates, such as lipid peroxidation. In summary, iNOS is activated within inflamed tissues10 and actively participates in the inflammatory response, and the use of iNOS inhibitors can effectively impede this reaction.

In conclusion, the phosphorylation-mediated involvement of eNOS in SCI may contribute to protective and reparative responses. Extracorporeal shock wave therapy has been reported to enhance angiogenesis and growth factor expression by activating eNOS and vascular endothelial growth factor, thereby promoting revascularization following SCI.43 The same objective can be accomplished through future drug development. The involvement of NO and cGMP in peripheral nociception following SCI remains contentious. Their protective effects during the initial hours after inflammation and their cytotoxic effects during the subsequent progression of inflammatory pain are widely acknowledged, contingent on varying concentrations of NO produced, dosage of NOS modulators, and the location and stage of the pathological process. Extensive clinical trials are required to establish the optimal timing and concentration for usage; iNOS plays a pivotal role in the inflammatory response after SCI, offering insights into the development of novel anti-inflammatory drugs.28

SPINAL CORD INJURY THERAPY

The pathophysiology of SCI encompasses primary injury and a cascade of secondary events. The inflammatory response plays a pivotal role in controlling neuronal damage and regeneration after SCI, making it a crucial component of secondary injury. Therefore, attenuating the early inflammatory response may limit tissue lesions and functional deficits. In terms of treatment modalities, medical intervention is probably the most expeditious approach for reducing the degree of spinal cord damage. Despite numerous studies conducted on this subject, no consensus protocol has been established. Methylprednisolone (MP) remains the sole U.S. Food and Drug Administration-approved drug for SCI treatment, with administration within 8 hours being recommended. However, confidence in the validity of the results has diminished following the adoption of a high-dose MP regimen based on the findings of the National Acute Spinal Cord Injury Study II and III. Note that MP is ineffective in patients with spinal cord rupture, whereas those with mild injuries can recover spontaneously without requiring MP treatment.44 However, caution must be exercised because high-dose MP may lead to pulmonary and gastrointestinal complications; elderly patients are particularly susceptible to respiratory complications and infection. Another steroid agent, dexamethasone, may offer benefits in cases that involves metastatic spinal cord compression by reducing inflammation and preventing further nerve damage; however, significant controversy exists regarding the therapeutic dosage.45

Recently, research on the inhibition of NO production to reduce injury after SCI has emerged as a prominent area of investigation, offering potential avenues for SCI treatment and inflammatory response mitigation. This field encompasses various approaches, including Chinese herbal medicine, clinical drugs, exosomes, hormones, and nerve electrical stimulation. Some interventions act by inhibiting specific targets, such as mitogen-activated protein kinases. Furthermore, these studies have identified novel pathways associated with the pathogenesis of SCI, such as IL-6 receptor/Janus kinase-signal transducer and activator of transcription and miR-146a-5p/G-protein-coupled receptor 17.46,47 Most importantly, many interventions facilitate body recovery and enhance motor function by attenuating NO-mediated oxidative stress.

LIMITATION

This paper has many shortcomings. First, due to time, resource, and knowledge limitations, investigators may not be able to cover all relevant literature, which may result in some important studies or ideas being missed in the review. Second, only PubMed articles were searched, and no other biomedical databases were involved. Third, the effect of NO on the related pathways and SCI was not discussed in terms of molecular structure.

CONCLUSION

Taken together, there is still a long way to go before existing basic research can be translated into clinical application. Although improvement in muscle or nerve function after SCI has been observed in animals, safety cannot be guaranteed, and validation of clinical trials is lacking. The clinical treatment direction of SCI is currently focused on the early anti-inflammatory stage and the later rehabilitation stage. The effectiveness of some methods has been fully verified; however, there is still a large gap in the recovery of normal function. It is gratifying that the safety of some new drugs or methods has been verified in humans. In the future, it will be necessary to expand the number of research objects based on safety to promote the recovery of neurological function in patients after SCI.

In this study, we conducted a comprehensive literature review to investigate the pivotal role of NO in SCI and elucidated its potential mechanisms, including temporal dynamics. Further investigations are warranted to unravel the neuroprotective and cytotoxic effects of distinct subtypes of NOS and other mediators involved in the modulation of the NO cascade. By adopting this approach, novel therapeutic targets can be identified and subsequently translated into clinical applications for mitigating secondary damage following SCI while enhancing functional recovery. The complete clinical significance of this article lies in successfully translating basic research findings into practical interventions, such as the development of NO inhibitors specifically tailored for treating SCI.