Arterial Glyceryl Trinitrate in Acute Ischemic Stroke After Thrombectomy for Neuroprotection (AGAIN): A Pilot Randomized Controlled Trial

Although endovascular therapy demonstrates robust clinical efficacy in acute ischemic stroke (AIS), not all stroke patients benefit from successful reperfusion. This study aimed to evaluate the safety, feasibility, and preliminary efficacy of intra-arterial administration of glyceryl trinitrate (GTN) after endovascular recanalization for neuroprotection. This is a prospective randomized controlled study. Eligible patients were randomized to receive 800 μg GTN or the same volume of normal saline through the catheter after recanalization. The primary outcome was symptomatic intracranial hemorrhage (ICH), while secondary outcomes included mortality, functional outcome, infarction volume, complications, and blood nitrate index (NOx). A total of 40 patients were enrolled and randomized with no participants being lost to follow-up. There was no significant difference in the proportion of sICH between GTN and control groups. Additionally, no significant difference was observed in mortality or rates of neurological deterioration and other complications. Favorable trends, while non-significant, were noted in both outcome and imaging for functional independence at 90 days and reduction in final infarct volume (75.0% vs 65.0%; 33.2 vs 38.9 ml) for the GTN group. Moreover, the concentration of blood NOx in the GTN group was significantly higher than in the control group at 2 h after GTN administration (26.2 vs 18.0 μmol/l, p < 0.05). The AGAIN study suggests intra-arterial administration of GTN post-endovascular therapy is safe and feasible and GTN successfully raised NOx levels over controls at 2 h. A multi-center randomized controlled trial with a larger sample size is warranted to determine GTN neoadjuvant efficacy.


Introduction
Modern-day endovascular therapy (ET) has improved the rates of vessel revascularization and patient outcome resulting in the expansion of time windows for acute ischemic stroke (AIS) patients with large vessel occlusion (LVO) [1].However, there remains an apparent discrepancy between the effectiveness of recanalization and return to pre-stroke functional baseline.The mortality rate for AIS due to LVO remains at 15.3% and functional independence after endovascular recanalization remains at 46% in AIS patients after successful reperfusion [2,3].A neuroprotective adjunct to thrombectomy could help close the gap between successful reperfusion and patient outcomes at 90 days.In the present study, we determined whether glyceryl trinitrate (GTN), a donor of nitric oxide (NO), can be safely used in AIS patients in the context of endovascular recanalization.Secondarily, we did assess efficacy although the sample size was underpowered.
Zhe Cheng and Jie Gao contributed equally to this work.
The neuroprotective effect of NO donors has been shown to prevent ischemia-reperfusion (I/R) injury through the reduction of ROS formation in preclinical stroke studies [4].GTN, a Food and Drug Administration-approved vasodilator, is one of the widely used exogenous NO donors used in the clinic and has been trialed for neuroprotection in AIS [5].Previously transdermal and sublingual GTN have been found to have actions on high blood pressure without affecting platelet function and regional cerebral blood flow [6][7][8][9].However, no improvement in functional outcome was noted following AIS [10].The neuroprotective effect of NO donors after I/R injury is demonstrated to be consistently dependent on the dose, location, source, and environment [11].Thus, the delivery route of NO donors may be a key factor in exploring its neuroprotective effect in AIS patients.
Similar to many neuroprotective treatments, basic bench studies have confirmed the potential neuroprotective effect of GTN, but no successful clinical utility has been demonstrated.GTN failure clinically thus far may be related to the lack of studies combining reperfusion and administration [12].Recently, arterial administration of GTN in the middle cerebral artery occlusion animal model demonstrated a neuroprotective effect in AIS [11].As a result, intra-arterial administration of GTN may be a more rapid and effective way to maximize the potential of this drug.To our knowledge, there exists a paucity of literature regarding intra-arterial GTN as a neuroprotective measure in AIS patients after endovascular therapy.Given the technique of mechanical thrombectomy has improved over the past decade, reperfusion can now be successfully reestablished in most LVO patients and can provide an avenue for neuroprotectants to reach at-risk tissue.In the current study, we evaluated the safety, feasibility, and potential efficacy of AGAIN as an adjuvant therapy.

Methods/Design
This study had been registered at www. chictr.org.cn in April 2021 (ID: ChiCTR2100045254) and approved by the ethics committee of Beijing Luhe Hospital, Capital Medical University, Beijing, China.

Study Design
AGAIN [13] was a single-center, prospective, and randomized controlled study that was designed to evaluate the safety and efficacy of GTN administration after endovascular therapy in AIS patients within 6 h of symptom onset.Eligible patients were enrolled consecutively after undergoing ET for AIS in the Beijing Luhe Hospital Stroke Center (the Stroke Intervention & Translational Center, SITC), Capital Medical University from May 2021 to January 2022.Informed consent, which included an explanation of potential benefits and risks, was obtained from each participant or an appointed legal representative.A physician unaffiliated with the study monitored the health and safety status of the participants.A safety quality control conference was held by the GCP committee twice a year and on an as-needed basis.The safety of subjects was continuously monitored by the Beijing Luhe Hospital Ethics Committee.

Randomization and Blindness
The participants were consented and randomly assigned 1:1 (n = 40) to the two treatment groups with the use of computer-generated randomization.Interventionalists, outcome assessors, and investigators involved in data collection and analysis for the trial were blinded to this assignment with the use of opaque envelopes.Pre-made envelopes were used to administer either the nitroglycerin injection (5 mg, 1 ml) or saline (0.9%, 10 ml), following a randomized sequence generation.Each envelope was labeled with a specific enrollment number (0-40) corresponding to the randomization details.During the intervention, an unblinded nurse diluted the nitroglycerin injection to 100 mg/ml using a 10-ml syringe or directly withdrew saline into an identical syringe.

Interventions
Stroke patients that meet the inclusion criteria were randomly assigned to the experimental group and control group.All patients also received standard stroke treatment in accordance with the current guidelines for stroke management.In the experimental group, an 800 μg dose of GTN was diluted with 10 cc of isotonic NaCl solution and infused through the catheter over a 10-min span.This was commenced as swiftly as 3 min following vessel recanalization.Similarly, the control group received a 10 cc isotonic NaCl infusion via the catheter over a 10-min period, initiated at the same time point.

Outcomes and Assessment Procedures
The demographic information, risk factors for stroke, occlusion site, and procedure of ET were recorded.ASPECTS (Alberta Stroke Program Early CT Score) and NIHSS (National Institute of Health Stroke Scale) scores at admission were determined and collected for further analysis.Magnetic resonance imaging (MRI) or computerized tomography (CT) was performed 24 h after ET.NIHSS and mRS were evaluated at baseline and at 24 h, days 7, 14, and 90 after stroke onset.
The primary outcome was the incidence of symptomatic intracranial hemorrhage (ICH).ICH was classified into 5 categories according to the European Cooperative Acute Stroke Study II (ECASS 2) [14]: no evidence of hemorrhage, hemorrhagic transformation type I or II (HT I or HT II) and parenchymal hemorrhage type I or II (PH I or PH II).Symptomatic intracerebral hemorrhage (sICH) was defined as PH I or PH II diagnosed in the clinical setting with a ≥ 4-point increase in the NIHSS score.
The secondary safety outcomes included the following: (1) episodes of hypotension requiring clinical treatment during GTN administration; (2) neurological deterioration as defined as NIHSS score increasing more than 4 points from baseline to day 7; (3) ICH, fatal ICH; (4) death regardless of cause during the study period; (5) headache, tachycardia, emesis, pneumonia, urinary infection, and seizures.The secondary efficacy outcomes included the following: (1) functional independence (as determined by mRS scores of 0 to 2 at 90 days, which are representative of functional independence), (2) mRS score at 90 days, (3) NIHSS scores at 24 h and 7 days, (4) infarct volumes.The infarct volume was measured using the Siemens Syngo with Diffusion-Weighted Imaging (DWI) by the image tool (ROI) (this was done by outlining the infarcted area on each section in order to calculate the area and subsequently by multiplying this value from each layer by its respective thickness); (5) blood nitrate index (NO x ) detection (NO x is the general term for nitrogen and oxygen compounds which includes NO and NO 2 ).

Estimation of Sample Size
Intra-arterial administration of GTN in AIS patients was our first attempt to address the effect of GTN in AIS patients with endovascular therapy.There was no data available for reference.This study was a pilot investigation to examine the study design, methods, procedures, inclusion criteria, and operational strategies.The results will be used to estimate the sample size and conduct a power analysis for a phase-2 trial.According to a previous study in determining sample size for pilot studies [15], 10 to 20 patients in each group may be adequate to evaluate the initial trend of safety and feasibility of intra-arterial administration of GTN.Therefore, a similar number of samples of a total of 40 patients were selected in this pilot study.

Statistical Analyses
The intention-to-treat (ITT) principle was applied to examine the endpoint events in the present study.In the event that no differences were found between the two study groups in terms of the incidence of adverse events and functional prognosis at 90 days, we would move forward with a phase-2 trial.
The Kolmogorov-Smirnov test for normality and the equal variance test were performed before any statistical analysis was used.For continuous variables with normal distribution, a 2-sided t-test for independent samples was applied in order to detect differences between the groups.A Mann-Whitney U test was performed for continuous variables that lack a normal distribution.For categorical variables, χ2 or Fisher's exact tests were used when appropriate.p < 0.05 was considered statistically significant.Statistical analysis was performed on SPSS 22.0.

Patients
A total of 49 patients were screened and 40 patients enrolled and randomized in the study.No participants were lost to follow-up, and all participants were analyzed (Fig. 1).Baseline demographic and characteristics of the patients are summarized in Table 1.Age, sex ratio, baseline NIHSS, vascular risk factors, ASPECTS score, location of stroke, IV rt-PA, stent utilization, time from onset to recanalization, time from treatment to recanalization, the number of passages of retriever stent, and TICI score were not significantly different between the GTN and control groups.

Efficacy Outcomes
There was an absolute difference between the two groups in the proportion of patients who were functionally independent, favoring GTN (75.0%vs 65.0%), although it did not reach a significant level (p = 0.731) (Fig. 2).Additionally, at 24 h after stroke onset, a slight reduction in infarct volume as determined by MRI was found in the  GTN group, and the mean infarct volume was 33.2 ml compared with 38.9 ml in the control group, although this was not statistically different (p = 0.779) (Fig. 3).Similarly, no significant difference was observed in the NIHSS score at 24 h, 7 days, and mRS score at 90 days between the two groups (Table 3).Interestingly, a significant decline in blood NO x concentration was found in all patients between before and after thrombectomy (22.2 μmol/l at 2 h after GTN vs 31.0 μmol/l at 10 min after GTN and 30.4 μmol/l prethrombectomy, p < 0.05).Additionally, the concentration of blood NO x in the GTN group was significantly higher than the control group at 2 h after GTN administration (26.2 vs 18.0 μmol/l, p < 0.05) (Fig. 4).

Discussion
This study was the first, to our knowledge, to reveal the safety and feasibility of the administration of intra-arterial GTN as an adjuvant therapy after endovascular recanalization in AIS patients with LVO.No increased risk of sICH, mortality, or related complications such as hypotension,  tachycardia, headache, and emesis, as well as pneumonia, urinary infection, or seizures, was observed following this therapy.In this study, intra-arterial GTN after endovascular recanalization for AIS patients appears safe.Additionally, a favorable trend in the rate of functional independence (75.0%vs 65.0%) at 90 days after stroke onset and reduction in final infarct volume (33.2 ml vs 38.9 ml) was observed in the GTN group despite no significance.This pilot study was not powered to definitively evaluate these endpoints, but the data can be used to power a future study.Moreover, a significant decline in blood NO x concentration was found in all thrombectomy patients.GTN administration significantly increased blood NO x concentration after 2 h, suggesting intraarterial GTN may rapidly and effectively inhibit the consumption of NO after AIS.

Neuroprotective Mechanism of NO Donors and Clinical Limitation
The efficacy of reperfusion after ischemic stroke is limited by post-ischemic inflammatory changes and I/R injury, including reactive oxygen species (ROS) formation and accumulation [16].NO has a pivotal role in hypoxic signaling and exerts potent cytoprotective effects after ischemia and reperfusion in the brain [17].ROS formation induced by reperfusion contributes to a decrease in the bioavailability of NO, thus limiting its ability to promote various protective mechanisms after stroke, such as vasodilation, inflammation inhibition, and preservation of endothelial integrity under hypoxic conditions [18].Several preclinical studies suggested that NO donors could safely reduce infarct size, increase cerebral blood flow, and improve functional outcome in AIS models of both transient and permanent stroke [19] through alleviating I/R injury by several mechanisms [4].However, transdermal and sublingual GTN as previously trialed methods have failed to confer improvements in functional outcome in AIS patients [6][7][8][9][10].The reason for the failure of NO donors as neuroprotectant was considered to be associated with the dose, location, source, and environment of application in clinical studies for AIS [11].

The GTN Dose Calculation
A total of 800 μg NTG diluted in 10 cc isotonic NaCl solution was infused through the catheter after vessel  recanalization in the study.This dose of GTN is close however slightly higher than previous coronary artery research in which 200-500ug GTN was given through a catheter [20][21][22][23][24].Our study suggests that 800 μg is safe for postrecanalization cerebral administration.Although the current study indicates a trend in favorable outcomes, further studies with a larger sample size are needed to confirm this conclusion and as well as optimize the dose of GTN.

The Route of GTN Administration
Due to a cascade of expansion in inflammatory responses during the whole process of I/R injury, early intra-arterial administration of GTN after vessel recanalization (during the early stage of I/R injury) may be the best method to prevent I/R injury and exert its clinical neuroprotective benefits.Additionally, owing to the limited bioavailability, GTN administration as a patch on the arm or chest may not reach the cerebrovascular system in a way that enables it to confer significant or reliable benefits.Arterial administration of GTN has been demonstrated to be neuroprotective in the middle cerebral artery occlusion animal model [11].Therefore, it is possible that intra-arterial GTN after vessel recanalization would most effectively enable this compound to reach target sites and have the greatest therapeutic effect.

GTN Administration After Thrombectomy
This pilot study demonstrates that intra-arterial GTN is safe for AIS patients after endovascular recanalization.
Combining such neuroprotective techniques with intravenous or endovascular therapy is considered to be an important future phase in the development of effective adjunctive treatment strategies in AIS [12,25].The recent advances in mechanical thrombectomy for ischemic stroke have paved the way for promising new opportunities to effectively apply supplementary neuroprotective therapies to patients after reperfusion [26,27].These include NA1 (excitotoxic post-synaptic density protein 95 (PSD-95)) [28], intra-arterial cooling infusion [29], and normobaric oxygen (NBO) [30].Many potential avenues of neuroprotection are possible and need trial with different combinations of reperfusion and neuroprotective approaches for AIS patients.In the present study, intra-arterial GTN combined with endovascular reperfusion therapy has shown a trend in improving functional outcome and reduction of infarct volume.Primary safety outcomes were met, and while this study was not powered for the efficacy outcomes, our results warrant further investigations.

Limitations
The proposed study has certain limitations.First, it is a single-center trial and has a small sample size aimed at only determining the safety of the intervention; second, the target dosage of GTN needs to be optimized after endovascular therapy in the future studies; third, substantially higher use of stenting/angioplasty (about 50%) was found in this study, implying atherosclerotic mechanism of a stroke may respond differently to NO than patients with embolic thrombus.We will fully explore the types of strokes that are more likely to benefit from NO donors.Greater analysis based on etiology will be designed and performed in future studies.Lastly, while the intra-arterial administration of GTN shows promise, its short half-life (~ 5 min) could limit its impact, particularly if its function is to inhibit downstream reperfusion injury and inflammation.Therefore, a combined approach utilizing continuous intravenous GTN infusion may warrant consideration in future studies to ensure a more sustained therapeutic effect.

Conclusion
The AGAIN study results have demonstrated that intraarterial GTN is safe and feasible for AIS patients after endovascular recanalization.GTN successfully raised NO x levels over controls at 2 h of administration.Although the current data was not powered to determine its efficacy, a multi-center RCT trial with a larger sample size is warranted to further determine the efficacy of intra-arterial GTN and optimal dose in the future studies.

Fig. 1
Fig. 1 Flow and timeline of participants enrolled

Fig. 2
Fig. 2 Modified Rankin scale score in AIS patients after vessel recanalization at 90 days.Images of Scores on the modified Rankin scale range from 0 to 6 in AIS patients after vessel recanalization at

Fig. 3
Fig.3Infarct volume at 7 days after vessel recanalization.Comparison of infarct volume at 7 days after vessel recanalization between the two groups, demonstrating a decreased tendency of infarct volume determined by MRI in the NBO group.The mean infarct volume was 33.2 ml in GTP group compared with 38.9 ml in the control group

Fig.
Fig.4 Blood NO x concentration.*p < 0.05, blood NO x concentration in GTN group compared to control group at 2 h after GTN administration; #^p < 0.05, blood NO x concentration of all thrombectomy patients at 2 h after GTN compared to 10 min after GTN administration and pre-thrombectomy; 18 patients in GTN group and 17 patients in control group; NO x is the general term of nitrogen and oxygen compounds, including NO and NO 2

Table 1
Demographic and clinical characteristics of all patientsNIHSS the National Institute of Health Scale Score, ASPECT Alberta Stroke Program Early Computed Tomography Score, mRS, modified Rankin score, MCA middle cerebral artery segment, ICA internal carotid artery, P-ICA proximal segment of internal carotid artery occlusion combined with intracranial large artery occlusion, IV rt-PA intravenous recombinant tissue plasminogen activator, TICI thrombolysis in cerebral infarction, IQR interquartile range

Table 2
Comparison of safety outcomesICH intracranial hemorrhage, sICH symptomatic intracranial hemorrhage, SBP systolic blood pressure *transient hypotension is defined as SBP < 100 Hg from the beginning of using GTN to the end in 10 min a neurological deterioration is defined as NIHSS score increasing more than 4 points from baseline to day 7

Table 3
Comparison of efficacy outcomesNO x is the general term for nitrogen and oxygen compounds and this usually includes NO and NO 2 mRS modified Rankin scale a 1 patients in GTN group did not complete the MRI scan, the mean final infarct volumes were 33.2 mL in the GTN group and 38.9 ml in the control group b 18 patients in GTN group and 17 patients in control group

4
Blood NO x concentration.*p < 0.05, blood NO x concentration in GTN group compared to control group at 2 h after GTN administration; #^p < 0.05, blood NO x