The role of nitric oxide and hydrogen sulfide in spinal cord injury: an updated review

Abstract

Medical gases play an important role in the pathophysiology of human diseases and have received extensive attention for their role in neuroprotection. Common pathological mechanisms of spinal cord injury include excitotoxicity, inflammation, cell death, glial scarring, blood-spinal cord barrier disruption, and ischemia/reperfusion injury. Nitric oxide and hydrogen sulfide are important gaseous signaling molecules in living organisms; their pathological role in spinal cord injury models has received more attention in recent years. This study reviews the possible mechanisms of spinal cord injury and the role of nitric oxide and hydrogen sulfide in spinal cord injury.

INTRODUCTION

Spinal cord injury (SCI) is defined as motor, sensory, and autonomic dysfunction in the injured segment caused by a variety of factors; it has a high rate of death and disability. Over the past few decades, the global prevalence of SCI has risen among individuals, estimated to range from 250,000 to 500,000 people each year.1,2 SCI not only brings serious financial burden but also psychological pressure to patients.3 There is a lack of effective methods to treat SCI, and exploring treatment modalities for SCI was important.4

Neuroprotective properties have been observed with certain medicinal gases, common gases including hydrogen sulfide (H₂S), nitric oxide (NO), volatile and non-volatile anesthetics also evidence suggests they are potential neuroprotective agents.5 According to research, gas transmitters, also known as biogaseous gas molecules, have significant impacts on the pathological conditions of mammals.6 For instance, drinking hydrogen-rich water has been found to effectively reduce hepatic oxidative stress, apoptosis, inflammation, and hepatocarcinogenesis, making it a potential treatment for alcoholic hepatitis.7 Supplementation of H₂S in rectal cancer avoids the loss of resistance to 5-fluorouracil induced by cystine-glutamate antiporter interference, that suggesting a functional role for H₂S in the maintenance of chemoresistance.8 Gas molecules play an important role in various pathophysiological states, which are of great significance for clinical treatment.

Over the past few years, research on the increasing attention given to the involvement of gas molecules in SCI has been growing, even though their mechanisms of action are not identical. An in-depth exploration of the correlation between medical gas and SCI has the potential to expand the horizon, inform SCI treatment approaches, open new avenues for therapeutic interventions, and reduce the pain and economic burden of SCI patients. In this article, We will review the pathomechanisms of SCI and the research progress on the role of NO and H₂S in SCI. We searched PubMed for nearly 20 years of research primarily by the keywords “spinal cord injury and pathology” or “spinal cord injury and nitric oxide” or “spinal cord injury and hydrogen sulfide.”

PHYSIOLOGICAL AND PATHOLOGICAL MECHANISMS OF SPINAL CORD INJURY

The first stage of SCI is mechanical damage caused by temporary or permanent compression and crushing.9,10 The second stage primarily involves the destruction and death of related tissues in the spinal cord, which includes complex pathophysiological mechanisms.11,12,13 The mechanism of SCI is intricate, and understanding the molecular mechanism of SCI is crucial to its treatment. Here, we will discuss the related mechanisms after SCI (Figure 1).

Figure 1.

Pathological manifestations of spinal cord injury.

Ischemia/reperfusion injury of the spinal cord

Ischemia often occurs at the injury site following SCI. Ischemia/reperfusion (I/R) injury is one of the pathological changes recognized and studied by many researchers. Neurons in the spinal cord have a higher basal metabolism and are more susceptible to damage leading to cell death during ischemia. Large intracellular glutamate efflux after neuronal apoptosis, resulting in glutamate amino acid excitotoxicity.14,15,16,17 Restoration of blood flow after I/R in SCI stimulates the expression of adhesion molecules and chemokines, leading to changes in the immune-inflammatory microenvironment by activating immune cells such as microglia, astrocytes, and macrophages.18 Cytotoxic, ionic, and vasogenic factors contribute to spinal cord ischemia-induced edema.1 Progress has been made in alleviating spinal cord I/R injury, but it remains a challenge in clinical treatment.19 I/R injuries often have serious consequences, including motor and sensory dysfunction and autonomic dysfunction.20

Excitotoxicity of SCI

The vital role in the advancement of central nervous system disorders is attributed to excitotoxicity induced by glutamate.21,22 Glutamate receptor activation after SCI substantially elevates glutamate levels, resulting in continuous excito-toxicity and cell death.23 Excessive glutamate release and impaired uptake following traumatic SCI cause neurotoxic concentrations around the injury site.24,25,26 Consequently, the neuronal ion pump is compromised, allowing an influx of Ca²⁺ into the neuron and inflicting direct and indirect spinal cord damage. Intracellular calcium overload triggers cell death via calpain activation, reactive oxygen species production, and mitochondrial impairment.27 Additionally, calcium overload stimulates protein kinases and phospholipases, contributing to calpain-related protein degradation and oxidative harm.28,29 Numerous medications targeting excitotoxicity have demonstrated strong effects in SCI animal models. In conclusion, excitotoxicity initiates a series of pathophysiological alterations and cascades after SCI, encompassing mitochondrial dysfunction, cytoskeleton disruption, free radical generation, axonal degeneration, glutamate release, and eventually apoptotic or necrotic cascade reactions within the pathway.30

Free radical of SCI

A larger number of studies indicate that early pathological changes in SCI include overproduction of reactive oxygen species and reactive nitrogen species. The generation of reactive oxygen specie and reactive nitrogen species during SCI mainly results from increased intracellular calcium levels, arachidonic acid metabolism, and activation of inducible nitric oxide synthase. The central nervous system is particularly vulnerable to damage caused by free radicals, and the excessive production of reactive oxygen specie and reactive nitrogen species leads to oxidative and nitrative harm to lipids, proteins, and nucleic acids. Free radicals not only disrupt neuronal cell membranes but also damage the cytoskeleton and organelles. Oxidative stress damage exacerbates mitochondrial dysfunction, intracellular calcium overload, activates proteases and leads to cytoskeletal proteolysis.13,31 Excessive free radicals can also lead to DNA damage and cellular aging.32,33

Glial scar formation after SCI

Glial scarring or gliosis, is an astrocyte-driven cellular response triggered under pathological conditions. Activated monocytes-macrophages, fibroblasts, and extracellular matrix proteins become part of the core of the lesion.34 Following SCI, astrocytes undergo proliferation, hypertrophy, and gradual migration along the periphery of extensively damaged tissue, intertwining around the core of the lesion and forming the primary component of the glial scar.35,36,37 The glial scar hinders axon regeneration by creating a physical obstacle that prevents axons from crossing the lesion area.38 The principal chemical constituents of glial scars are chondroitin sulfate proteoglycans. These components are predominantly expressed by reactive astrocytes and can significantly restrict axonal regeneration after SCI, as well as sprouting and remyelination.39,40,41 Protein tyrosine phosphatases are receptors for chondroitin sulfate proteoglycan and inhibit axonal regeneration when bound to chondroitin sulfate proteoglycan.42,43 Nonetheless, a membrane-permeable peptidomimetic that blocks this receptor not only reduces inhibition of axonal regeneration but also enhances the restoration of motor and urinary system functionality.42

But there is also recent evidence from different studies that glial scarring promotes axonal growth in the early stages of injury. Glial scarring protects normal tissues from neurotoxicity and excitotoxicity by restricting inflammatory factors and toxic substances to areas of damaged tissue.1 The intricate mechanisms of glial scarring in SCI have increasingly garnered attention, necessitating further research to comprehensively and profoundly comprehend its pros and cons.

Neuroinflammation and SCI

During traumatic SCI, peripheral inflammatory cells infiltrate and activate central inflammatory cells, initiating cascade reactions and releasing pro-inflammatory factors (such as interferon-gamma, tumor necrosis factor alpha, interleukin-1, interleukin-6, interleukin-8 and interleukin-12).44 In some cases, these inflammatory cells play a favorable role in SCI, but as the injury progresses, the continuous production of inflammatory factors can have effects on the body. Neutrophils, macrophages, and lymphocytes migrate to the injured spinal cord. The site, where microglia are also activated. Neutrophils migrate to the injured area rapidly, peaking after 24 hours, and gradually decreasing after 1 week.45 They engulf foreign cells and remove debris while also activating various inflammatory cells and microglia in tissues to accelerate neuronal destruction, which is considered detrimental. Rossignol et al.46 suggest that altering the external conditions can control the secondary inflammatory response in a direction beneficial to spinal cord repair; otherwise, the inflammatory response will accelerate cell necrosis and hinder axonal growth. The effect of neutrophils in SCI has been studied more recently, and some studies have said that it can play a role in tissue repair by reducing inflammation. For example, the release of secretory leukocyte protease inhibitors reduces inflammation and promotes axonal regeneration.47 Microglia are mainly polarized to the M1 phenotype after SCI, and polarized M1 amoeboid cells change with the release of neurotoxic substances, leading to neuronal damage. M2-polarized microglia undergo phenotypic changes that enable them to promote nerve regeneration, especially axon extension, after central nervous system injury.48

THE ROLE OF NITRIC OXIDE IN SPINAL CORD INJURY

NO is a widespread and important biomolecule involved in many physiological processes in mammalian life activities. Its production in the body is catalyzed by NO synthase under specific conditions. There are three types of NO synthases: endothelial NO synthase, neurogenic NO synthase (nNOS), and inducible NO synthase (iNOS). nNOS is mainly found in the central and peripheral nervous systems, endothelial NO synthase is expressed in cerebral vascular endothelial cells, and iNOS is expressed during inflammation.49 This subsection provides a review of the mechanisms of action of NO and NOS in SCI (Table 1).50,51,52,53,54,55,56

Table 1.

Main experimental studies of NO and NOS in SCI rat models

Study	Model	Animals/cells	Conclusion
Li et al.50	SCI	Rats	iNOS is involved in neuronal apoptosis after spinal cord ischemia-reperfusion injury via dephosphorylation of phospho-Bad (a proapoptotic Bcl-2 family protein).
Hamada et al.51	SCI	Rats	NO produced by iNOS may be neurotoxic in the subacute phase after SCI.
Estévez et al.52	SCI	Neuronal cells	Peroxynitrite, a strong oxidant formed by NO and superoxide, induces apoptosis of motor neurons.
Wang et al.53	Spinal root- avulsion	Rats	Upregulation of the nNOS protein prevents motor neuron degeneration in a radicular avulsion injury.
Xu et al.54	SCI	Rats	iNOS-induced NO, through nitration of tyrosine residues, damages cell membranes and organelles.
Kiziltepe et al.55	SCI	Rats	The neuroprotection of resveratrol may be mediated by its antioxidant and NO properties.
Chen et al.56	SCI	Rats	NO generated by nNO attenuates spinal cord injury transmission and neuropathic pain

Note: iNOS: Inducible NO synthase; nNOS: neurogenic NO synthase; NO: nitric oxide; SCI: spinal cord injury.

Many studies have found the role of NO in central nervous system diseases, but its potential role and mechanism in SCI are not very clear.50,51,57 As mentioned above, oxidative damage is one of the important pathological changes in secondary SCI. Currently, in a post-SCI study in rats, it was found that after treatment with NOS inhibitor, the movement disorders of the hindlimbs of the rats with SCI were significantly increased, and the concentration of thiobarbituric acid reactive substances and the level of myeloperoxidase activity was also significantly increased, indicating that the formation of NO by constitutive NOS has an effect on the cell damage after SCI. It was also observed that iNOS-induced NO might exhibit neurotoxic properties during the subacute phase following SCI.51

Physiological levels of NO are not highly toxic, but elevated concentrations can lead to neurological damage. Nitrite formed by the reaction of NO with O₂ is highly oxidizing and can oxidize lipids, DNA and proteins, ultimately inducing apoptosis in neuronal cells.52 In a study involving spinal radicular avulsion injury in adult male rats, inhibiting c-jun phosphorylation prevented nNOS levels in the spinal cord from dropping below baseline levels and partially alleviated motor neuron death post-injury,53 suggesting that nNOS-derived NO may mitigate neuronal damage after SCI, although the underlying mechanism remains unclear.

Secondary tissue damage following SCI might be attributed to inflammatory mediators. In SCI and other pathological conditions affecting the CNS, excess NO produced by activation of iNOS by nuclear factor-kappaB transcription factor promotes an inflammatory response with cytotoxic effects, leading to neuronal apoptosis.54,58 Cell membrane lipid peroxidation and loss of membrane function can result in apoptosis.57 However, research has demonstrated that during SCI, resveratrol can upregulate NO, inhibit membrane lipid peroxidation, scavenge oxygen free radicals, inhibit platelet aggregation, and protect multiple organs from I/R injury.55

Pain hypersensitivity induced by spinal nerve injury has gained increasing attention in recent times. Spinal N-methyl-D-aspartate receptors are closely related to chronic pain caused by peripheral nerve injury. Studies have shown that endogenous NO inhibits the activity of spinal cord N-methyl-D-aspartate receptors through S-nitrosylation, and the NO generated by nNOS can reduce spinal cord nociceptive transmission and neuropathic pain caused by nerve injury.56 It is not entirely clear which type of NOS is responsible for the pathological increase in NO production in SCI. The initial maximum increase in NO may be caused by nNOS, while the second wave of NO production may be caused by iNOS. Some researchers have found that NO produced by iNOS can have neurotoxic effects.51

At present, there is no unified conclusion on the changes of different NOS after SCI and the effect and mechanism of NO produced on neuroprotection and cytotoxicity to convince everyone. Analysis of the temporal distribution and cellular origin of the activation of different synthase isoforms to reduce NO-induced damage by inhibiting specific synthases is also the focus of our research.59 A lot of research is still needed to discover their specific potential effects, and it is possible to find New therapeutic targets to improve functional recovery after SCI in patients in the clinic.

THE ROLE OF HYDROGEN SULFIDE IN SPINAL CORD INJURY

H₂S smells like rotten eggs and has been considered a toxic gas since its discovery. Studies have found that H₂S is widely present in cardiovascular, nervous, gastrointestinal, respiratory, renal, liver, and reproductive systems, and plays a role in the body’s pathological and physiological processes.60 This subsection provides a review of the mechanisms of action of H₂S in SCI (Table 2).61,62,63 H₂S is synthesized primarily by cysteine β-synthase and cysteine γ-lyase. Cysteine β-synthase is mainly found in the central nervous system and is currently considered to be H₂S synthase and plays an important role in central nervous system pathophysiology.64,65 H₂S is a gas endogenous signaling molecule that can better pass through various biological barriers, regulate the expression of different types of microRNAs, and participate in the regulation of different signaling pathways in physiological and pathological processes. In the rat SCI model, H₂S was found to be neuroprotective, ameliorating symptoms and reducing inflammatory factor secretion, neuronal apoptosis, endoplasmic reticulum, and oxidative stress in rats.61,62

Table 2.

Main experimental studies of H₂S in SCI

Study	Model	Animals/cells	Conclusion
Xu et al.61	SCI	Rats	Reduces inflammatory factors, apoptosis, and oxidative stress by activating factor-erythroid 2-related factor 2 protein.
Xie et al.62	SCIR	Rats	Inhibited autophagic cell death significantly by reducing oxidative stress via the AKT/mTOR pathway.
Liu et al.63	SCI	Rats	Protects the spinal cord by upregulating CasC7 expression in the SCI rat model.
Li et al.66	SCIR	Rats	Protects the spinal cord and induces autophagy through miR-30c.
Yang et al.67	SCI	Rats	Reduce bone loss caused by SCI and improve motor function of the extremities.
Kakino et al.68	Transient spinal cord ischemia	Mice	Interacts with NO to exert anti-apoptosis and improve delayed paraplegia.
Wang et al.69 Campolo et al.70	SCI	Rats	Anti-oxidative stress and anti-inflammatory effects exert neuroprotection.

Note: CasC7: Cancer susceptibility candidate 7; H₂S: hydrogen sulfide; mTOR: mammalian target of rapamycin; NO: nitric oxide; SCI: spinal cord injury; SCIR: spinal cord ischemia/reperfusion.

As mentioned earlier, I/R injury after SCI is an important cause of tissue necrosis and apoptosis, and neurons cannot be regenerated.71 Therefore, the occurrence of cell necrosis after injury will cause serious consequences, and minimizing spinal cord I/R injury has become a research hotspot of SCI and has received more and more attention. Liu et al.63 found that NaHS treatment in a rat spinal cord ischemia model can reduce spinal cord neuron apoptosis and infarct size, which is mainly related to NaSH downregulating miR-30. Autophagy is a process of degradation and recycling of intracellular cytoplasm, long-lived proteins, and organelles. Numerous studies have found that autophagy has two sides in the pathological process, which can play a cytoprotective role and also can lead to cell death.72,73,74 Xie et al.62 found that autophagy promoted neuronal cell death after spinal cord I/R, and H₂S reduced I/R through the Akt/mammalian target of the rapamycin pathway in animal experiments. Interestingly, Li et al.66 found that H₂S increased cellular autophagy to protect the spinal cord from I/R injury by inhibiting miR-30c expression and upregulating the expression of autophagy-related proteins. This may be related to the duration of H₂S action in vitro, the route of administration, and the protective mechanism of H₂S in spinal cord I/R injury needs further study.

Both inflammation and oxidative stress are key factors in SCI and secondary injury. Glutathione is a reducing agent in the body, scavenging oxygen free radicals, hydrogen peroxide, and lipid peroxides in the central nervous system. H₂S promotes glutathione synthesis and protects neurons from oxidative stress damage.75 Nuclear factor-e2-related factor 2 (Nrf2) is a widely distributed protein in tissues and organs, acting as a crucial regulator of cellular redox responses.76 The activation of Nrf2 enhances the antioxidative stress capacity of neuronal cells, thus providing neuroprotective functions. Research has demonstrated that in a mouse SCI model, NaHS treatment significantly ameliorated the neurological recovery of injured mice, potentially due to the activation of Nrf2 signaling molecules, increased nuclear translocation of Nrf2, and subsequent activation of downstream target genes that play roles in SCI, including anti-inflammatory, antioxidant, and neuroprotective effects.61 SCI leads to the disruption of the blood-spinal cord barrier. A study by Wang et al.69 discovered that NaHS further safeguarded the blood-spinal cord barrier integrity by mitigating inflammatory responses and oxidative stress in a rat SCI model, ultimately enhancing functional recovery after SCI. In another study comparing ATB-346 (an H₂S-releasing naproxen derivative), naproxen, and a control group, ATB-346 accelerated motor function improvement in a mouse SCI model while significantly reducing inflammation.70 These experimental results support the hypothesis that H2S can reduce oxidative stress and anti-inflammatory effects in SCI. In addition, H₂S can also reduce bone loss under the lesion caused by SCI in rats, the bone density of the femur and tibia is significantly increased, and the motor function of the limbs is improved.67

Kakinohana et al.68 found that sulfide prevented spinal ventral horn motor neuron degeneration and delayed paraplegia in mice in an nNOS-dependent manner in a model of spinal cord ischemia. This study found that the protective effect of H₂S on delayed paraplegia after SCI was mediated by the anti-apoptotic effect of sulfide, which may be achieved through the hyper-sulfation of p65. This study also suggests that p65 hyper-sulfation may promote s-nitrosylation of p65 through nNOS-derived NO hours after reperfusion. The study discovered a new mechanism of action of H₂S, which provides the possibility for new H₂S-based therapeutic methods and broadens the therapeutic window of H₂S.

The research on H₂S and other sulfides is still not perfect, and most of them are at the level of experimental animals in the research of SCI. H₂S has a pungent odor and can be challenging to control inhalation in animal studies, which affects the reliability of experimental results and even causes animal death.77 Therefore, in most experiments, the experimenter adopts a safer and more reliable NaHS intravenous injection method to improve the accuracy of experimental data. However, other studies have shown that the therapeutic index of sulfide is very low and the therapeutic window is very short,78,79 which may be one of the reasons why sulfide-based clinical treatments have so far failed to achieve breakthroughs. Currently, different doses, different modes of administration, or different carriers need to be studied to fully study the effect of H₂S in animal models.

FUTURE OUTLOOK

Through a large number of studies on animals of SCI, it has been observed that NO and H₂S are crucial signaling molecules involved in the regulation of disease signaling pathways and play different mechanisms of action in the process of SCI. Its mechanism of action includes improving spinal cord I/R, reducing apoptosis, reducing inflammation, anti-oxidation, and improving angiogenesis and autophagy. Although some studies have found that their effects are not the same in different studies, this may be related to differences in the mode of administration and dosage, mode of action, and type of disease. The limitations of the current studies are that the role of NO and H₂S in patients is less studied; the lack of drugs targeting NO and H₂S is one of the great challenges for future research. In conclusion, medical gases, including NO and H₂S, play an important role in SCI. We believe that medical gases will become a milestone change in the treatment of SCI.