Due to its high rate of mortality and morbidity, ischemic stroke is a devastating public health concern that also results in high socioeconomic burden (1–4). The effective treatments for acute ischemic stroke (AIS) are intravenous thrombolysis and mechanical thrombectomy. Due to the narrow time window for intravenous alteplase [tissue plasminogen activator (tPA)] of 4.5 h, the majority of patients are not eligible for this treatment. In addition, two-thirds of stroke patients still suffer from varying degrees of disability (5, 6) even after intravenous thrombolysis. Endovascular therapy (within 6 h of stroke and up to 24 h) has shown great benefit in improving functional outcomes in AIS patients with large vessel occluded (LVO) in the anterior circulation. However, only 46% of the patients achieve functional independence at 90 days with a 15.3% mortality rate (7). In the EXTEND trial (8), patients benefited from tPA between 4.5 and 9.0 h after the onset of stroke. This study suggested that the “tissue window” has advantages over the traditional “time window” in screening patients. Thus, exploring fast and effective neuroprotection strategies to save ischemic penumbra and to lengthen the “tissue window” may be the key in the treatment of AIS.

Ischemic stroke-induced brain damage results from the interaction of complex pathophysiological processes such as excitotoxicity, oxidative stress, inflammation, and apoptosis (9). NO has a central role in hypoxic signaling, and its physiologic and therapeutic levels exert potent cytoprotection after ischemia and reperfusion in various tissues including the brain (10). NO derived from endothelial nitric oxide synthase (eNOS) plays a critical role in the regulation of cerebral microvascular tone, the protection of the blood–brain barrier, the reduction of oxidative stress, and the alleviation of procoagulant stimulation (11–13). Various animal studies have reliably demonstrated a loss of cytoprotection when subjects were treated with NO scavengers or when NOS was inhibited or knocked out (14–16). NO donors are a heterogeneous group of drugs whose common feature is the ability to release NO or an NO-related species in vitro or in vivo independently of endogenous sources (17). NO donors have been implicated in improving cancer therapy, hypertension, and peripheral artery disease (17). Several preclinical studies suggest that NO donors could safely reduce infarct size, increase cerebral blood flow, and improve functional outcome in AIS in both transient and permanent stroke models (18). The neuroprotective effect of NO donors has been previously demonstrated to work at many different levels by several mechanisms including that of altering the cellular oxidative status, inhibiting monocyte activity, and diminishing primary hemostasis (19).

GTN, a Food and Drug Administration-approved vasodilator, is an example of a drug that functions as a NO donor. Transdermal GTN had been found to lower blood pressure (BP), have no deleterious effects on platelet function, and exert no changes in the middle cerebral artery blood flow velocity or regional cerebral blood flow in AIS patients (20–23). A recent larger sample size randomized controlled trial (RCT) to determine the efficacy of transdermal GTN for the management of high BP in AIS (ENOS) also revealed the potential to reduce BP without finding a functional improvement following AIS if administered within 48 h (24). However, this study indicated that administration of GTN within 6 h improved functional outcomes. The RIGHT-2 trial (25) focused on the safety and efficacy of transdermal GTN given within 4 h of onset of AIS assessed in the prehospital environment in the UK. This trial did not show that prehospital treatment with transdermal GTN improved functional outcomes in patients with presumed stroke. Importantly, the neuroprotective effect of NO donors after ischemia–reperfusion injury (IRI) has yet to be reported consistently with many factors contributing to this including dose, location, source, and environment (26). Further protocols have been proposed to evaluate the use of transdermal GTN in acute stroke treatment (26). In light of these parameters and owing to the short half-life of GTN, GTN administration as a patch on the arm or chest may not reach an effective concentration in the cerebrovascular system. As such, a continuous intravenous administration of GTN may be a rapid and effective way to maximize any benefit of this drug (26). Furthermore, 24-h continuous GTN administration can cause tolerance resulting in subtherapeutic levels. This knowledge hints that a better outcome may be possible if an “intermittent” therapy is used (27–29). At present, no study has been reported on the safety and efficacy of intravenous GTN as an adjuvant neuroprotective strategy for AIS. We have consequently designed this single-center, prospective RCT to evaluate the safety, feasibility, and preliminary efficacy of intravenous administration of GTN after AIS.

Over the past few decades, over 1,000 neuroprotective methods have been examined for adjunct neuroprotective administration in the setting of AIS (41). However, most neuroprotectants have only demonstrated benefit in animal stroke models without successful clinical transformation (42, 43). The cause of this failure was not immediately clear. One general conclusion would be that there is a low likelihood of success in targeting one pathway or one selective mechanism within a rather heterogeneous, nongenetically determined condition operative over many years (44). Ischemic stroke-induced brain injury results from the interaction of complex pathophysiological processes such as excitotoxicity, oxidative stress, inflammation, and apoptosis (9, 45–47). The post-ischemic cascade is a complex, multipathway, multifactorial process involving a variety of pathological mechanisms; therefore, multiple targets of neuroprotection drugs may be more effective.

NO is a ubiquitous molecule in the body, which plays a multitude of physiological actions such as a vasodilator, neurotransmitter, immunomodulator, and antagonist of platelets and leukocytes (48). In the brain, NO is mainly synthesized by various subtypes of NOSs: neuronal nitric oxide synthase (nNOS), eNOS, and inducible nitric oxide synthase (iNOS) (25). Previous studies demonstrate that organ injury can occur due to a reduction of NO, most commonly due to a reduction in eNOS activity during ischemia reperfusion injury (IRI) (49). In the setting of IRI, NO has been found to have various protective effects on inhibiting oxidative stress, leukocyte–endothelial adhesion, cytokine release, and apoptosis (50). NO can also reduce infarct size and inflammation after ischemic stroke and improve cerebral blood flow, cerebral metabolism, and nerve function in a preclinical study (51). A previous observational study found that patients with ischemic stroke had significantly lower plasma NO levels than matched normal volunteers, and the low NO levels were associated with more severe stroke and worse outcome assessed at discharge disposition (52). A meta-analysis showed a significant association between different eNOS gene polymorphisms and risk of ischemic stroke in the Asian population (52).

NO plays a pivotal role in preventing inflammation and attenuating oxidative stress after IRI, and, therefore, NO supplementation using NO donor drugs is a reasonable approach to minimizing the cerebral damage after ischemia. GTN is one of the most widely used exogenous NO donors in the clinic. Three studies (20–22) on transdermal GTN in acute stroke showed that GTN lowered peripheral and central BP, 24-h BP, pulse pressure, and augmentation index. However, RCTs ENOS (53) and RIGHT-2 (25) on transdermal GTN patch (5 mg) showed that there was no significant change in functional or secondary outcomes measured at day 90. In a prespecified subgroup analysis of participants within 6 h of stroke presentation in the ENOS trial, those who received GTN had a favorable improvement in functional outcomes, less death and disability, and improved cognition (25). Transdermal GTN is a simple method to the delivery of GTN; however, not all of the drug released from the patch reaches the systemic circulation (54). Compared with intravenous administration, about 75% of nitroglycerin is systemically bioavailable after patch administration (55). The reason for lost drug is related to retention at the application site, tissue binding, and breakdown (33). The development of an intravenous form of nitroglycerin has further enhanced the role of nitrates in the therapy of cardiovascular disorders. Intravenous form of nitroglycerin permits prompt initiation of therapy and rapid attainment of high systemic levels (56). Although there is concern that lowering BP may worsen outcomes in the context of carotid stenosis, an analysis of the ENOS trial demonstrated that transdermal GTN appeared safe in both ipsilateral and bilateral stenoses (25). Considering that both GTN and NO (57) have a very short half-life in the body, an intravenous form of nitroglycerin with rapid dose titration is both feasible and safe. This may be the best method in a clinical trial for stroke patients to receive targeted cerebral NO donors. Prior clinical trials on the continuous application of nitroglycerin patches showed that 24-h continued use of GTN results in developing tolerance in the majority of patients with stable angina and suggested that “intermittent” therapy may provide a more rational approach to therapy. With removal of the patch for 10–12 h in each 24-h period, this will provide a patch-free period, which may allow the reestablishment of sensitivity (27–29). The daily dose at 5 mg/day [0.4 mg/h (54)] was recommended in the two important RCTs ENOS and RIGHT-2 on transdermal GTN patch (25, 58). In order to rescue the ischemic penumbra that infarct completed within 48 h after stroke onset (59), GTN at a speed of GTN 0.4 mg/h for 12.5 h/day, which makes 5 mg/day in total, was used in the present study, for 2 days. We will determine whether intravenous GTN is safe and has the potential to improve functional outcomes in patients with AIS.

There are limitations to this study. First, this is a single-center, small-sample experiment, which may affect the generalizability of the interventions. Second, although the target dosage has been shown to be safe and reliable in other small-cohort experiments, the dose used in the present study may still need optimization. In addition, there is a concern that an intravenous administration of an equal dosage of GTN might have more interactions given better bioavailability.

Rapid Intravenous Glyceryl Trinitrate in Ischemic Damage (RIGID) is designed to identify the safety, feasibility, and possible efficacy of intravenous administration of GTN in AIS patients. The preliminary results will provide clues for the design of future clinical trials. Based on past basic research and previous clinical studies, we predict that intravenous administration of GTN is safe for patients with AIS. The current proposed study may suggest a neuroprotective role for GTN in AIS and, thus, warrants an RCT.

The studies involving human participants were reviewed and approved by the Ethics Committee of Beijing Luhe Hospital, Capital Medical University, Beijing, China. The patients/participants provided their written informed consent to participate in this study.

YD and XG conceptualized the study and contributed to the study design and implementation. LC made substantial contributions to the design, implementation, and writing of the protocol. GR and PP contributed to the design of the trial from their area of expertise. HD, JG, ZC, RX, and SJ contributed to the implementation of specific procedures, conceptualized the study and contributed to the study design and implementation. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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