Intranasal salvinorin A improves neurological outcome in rhesus monkey ischemic stroke model using autologous blood clot

Abstract

Salvinorin A (SA) exerts neuroprotection and improves neurological outcomes in ischemic stroke models in rodents. In this study, we investigated whether intranasal SA administration could improve neurological outcomes in a monkey ischemic stroke model. The stroke model was induced in adult male rhesus monkeys by occluding the middle cerebral artery M2 segment with an autologous blood clot. Eight adult rhesus monkeys were randomly administered SA or 10% dimethyl sulfoxide as control 20 min after ischemia. Magnetic resonance imaging was used to confirm the ischemia and extent of injury. Neurological function was evaluated using the Non-Human Primate Stroke Scale (NHPSS) over a 28-day observation period. SA significantly reduced infarct volume (3.9 ± 0.7 cm³ vs. 7.2 ± 1.0 cm³; P = 0.002), occupying effect (0.3 ± 0.2% vs. 1.4 ± 0.3%; P = 0.002), and diffusion limitation in the lesion (−28.2 ± 11.0% vs. −51.5 ± 7.1%; P = 0.012) when compared to the control group. SA significantly reduced the NHPSS scores to almost normal in a 28-day observation period as compared to the control group (P = 0.005). Intranasal SA reduces infarct volume and improves neurological outcomes in a rhesus monkey ischemic stroke model using autologous blood clot.

Introduction

Reestablishment of blood flow using thrombolysis and thrombectomy has been shown to improve the prognosis of stroke patients by reducing mortality and morbidity; however, only a small portion of patients can receive this therapeutic intervention.^1–3 An alternative strategy to offer neuroprotection during the ischemic period is essential to improve neurological outcome, and such a strategy could potentially be complementary to recanalization. Over a thousand substances or strategies to offer neuroprotection have been tested in animal models with various beneficial effects, but none have become clinically available, effective neuroprotectants.^4–6 As acute stroke occurs suddenly and deterioration in patients occurs quickly, rapid rescue is essential.⁷ The delivery of medication in the prehospital setting through an intranasal route is a very attractive alternative method for stroke, especially for the super acute phase of ischemic stroke when intravenous access has not yet been established, and it is difficult to route medication to the ischemic area.⁸

We have demonstrated that intranasal salvinorin A (SA) administration reduced the ischemic area and improved the neurological outcomes in a rodent middle cerebral artery occlusion (MCAO) model.⁹ SA is a highly selective and potent kappa opioid receptor (KOR) agonist that can easily pass through the blood–brain barrier (BBB). Intravenous SA has also been shown to preserve cerebral auto-regulation in a brain hypoxia-ischemia model in piglets.¹⁰ In this study, we hypothesized that intranasal SA administration in an acute phase of stroke could reduce infarct volume and improve neurological outcomes in a non-human primate (rhesus monkey) stroke model.

Materials and methods

Data availability

All data associated with this study are available in the main text or the Supplemental material.

Animal

This study was approved by the Animal Use and Care Board of the Institute of Laboratory Animal Sciences, Capital Medical University. Our experiments were based on the Guide for the Care and Use of Laboratory Animals and conducted according to the national guidelines.¹¹ The reporting of animal experiments complies with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. A total of eight healthy adult rhesus monkeys were included in this study (four in the SA group and four in the control group). To increase the homogeneity, the experimental animals we selected were all male animals with the age of 8–10 years and weighing of 9–11 kg.¹² Each monkey was housed individually in the same room and was given food, fruit, and water ad libitum.

Anesthesia and intraoperative management

Experimental animals were fasted for 12 h prior to anesthesia. All anesthesia and intraoperative management were conducted according to our previous report.¹³ In brief, experimental animals were anesthetized during the stroke model establishment, administration, and imaging examination. Ketamine (10 mg/kg) was administered intramuscularly to induce anesthesia, and propofol (300 μg/kg/min) was administered intravenously to maintain anesthesia. Additionally, all experimental animals received endotracheal intubation and mechanical ventilation during the stroke model establishment, and the relevant parameters of the ventilator were adjusted by an experienced anesthesiologist according to the actual situation. The anesthesia was concluded at the end of each procedure when anesthesia was no longer needed. Major physiological parameters were continuously monitored during the procedure.

Stroke model establishment

The stroke model of using middle cerebral artery (MCA) M2 segment occlusion with an autologous blood clot was developed by the neuro-interventionalists based on our previous report.¹³ Briefly, we extracted 3–5 ml of femoral venous blood into a polyethylene catheter 24 h before the procedure. After the clot formation, a clot approximately 10 cm in length was intercepted and transferred to 4°C for standby.¹⁴ During the procedure, a Prowler-10 micro-catheter (Codman, Johnson, MA) with a Traxcess 0.010-inch guiding wire was introduced into the guiding catheter and navigated into the end of the M2 segment of the right MCA. Following catheter placement, the clot was transferred into the micro-catheter and flushed into the M2 segment with 1 ml of normal saline. Stroke was confirmed by vascular imaging and functional evaluations immediately following the clot placement.

Randomization and administration

After the establishment of the stroke model, randomization was performed by an independent experimenter. The experimental animals were then administrated SA or placebo according to the randomization results. Neuro-interventionalists who induced the stroke model were unaware of the randomization processes and results. SA (25 µg/kg; 0.5 µg/µl in 10% dimethyl sulfoxide) was administered intranasally 20 min after embolization to the experimental animals that were randomly assigned to the SA group. SA solution (1.25 µl/kg) was administered intranasally in each nostril, 2.5 µl/kg total every 30 s, until the full desired dose was reached. Total time of administration was approximately 10 min. We chose this dose based on our knowledge of protective effects of intranasal SA administration in a rodent stroke model.⁹ The 10% dimethyl sulfoxide is used as the solvent since SA can be dissolved readily, and dimethyl sulfoxide at the same concentration was used as the placebo and administered intranasally in the control group.

Imaging evaluation and neurological function evaluation

Successful embolization in a branch of the M2 segment was confirmed immediately after and an hour after the procedure by digital subtraction angiography. Magnetic resonance imaging was performed 24 h and 28 days following the procedure. To confirm cerebral infarction, we performed diffusion weighted imaging sequences. Additionally, we conducted T2-weighted imaging sequences to assess the space-occupying effect induced by cerebral infarction. Restricted diffusion in cerebral infarction was evaluated with diffusion weighted imaging sequences and derived apparent diffusion coefficient (ADC) sequences. We compared the mean ADC value of lesion with that of the contralateral side. The smaller the change, the closer the lesion was to normal brain tissue.¹⁵ Moreover, diffusion tensor imaging sequences were performed to detect white matter damage in experimental animals. The quantitative results of white matter damage were shown by relative changes in fractional anisotropy value in the lesion compared with that of contralateral side.

The neurological function of all animals was evaluated at 24 h, 48 h, 72 h, 7 days, 14 days, and 28 days after the procedure using the Non-Human Primate Stroke Scale (NHPSS), which is similar to the National Institutes of Health Stroke Scale in humans.¹⁶ Each assessment was performed by the same independent evaluator who was blinded to the animal grouping. Timeline for the interventional process during the procedure and the evaluation of imaging and neurological function throughout a 28-day follow-up period is shown in Figure 1(a).

Figure 1.

Study timeline and stroke model establishment. (a) Timeline for the interventional process during the procedure and the evaluation of imaging and neurological function throughout a 28-day follow-up period. (b) Before embolization, the middle cerebral artery M2 segment developed normally (left). Immediately after embolization, the M2 segment cannot be seen (middle). One hour after embolization, the vessel remained undeveloped (right). DSA: digital subtraction angiography; MRI: magnetic resonance imaging; NHPSS: Non-Human Primate Stroke Scale.

Postoperative management

Postoperative management was conducted according to our previous report.¹³ Briefly, experimental animals were treated with intravenous mannitol (1 g/kg) and ceftriaxone sodium (50 mg/kg) post procedure. In addition, buprenorphine (20 µg/kg) was administered intramuscularly to relieve pain when necessary

Statistical analysis

Previous study showed that infarct volume of the rhesus monkey model with MCA M2 segment occlusion was 7.1 ± 1.3 cm³.¹³ In this study, SA is expected to reduce infarct volume by 60%.⁹ The sample size ratio of monkeys in the two groups was 1:1, with a power of 0.9 and α = 0.05. Based on an assumed difference of 0.6 × 7.1 = 4.26 cm³ between the two groups and an assumed common standard deviation of 1.3 cm³ of the two groups, the sample size for the two-sample t-test was four per group. Therefore, a total of eight rhesus monkeys were enrolled in the present study. The sample size was calculated via PASS 11.0 (NCSS, LLC).

We presented the descriptive statistics as mean ± standard deviation for continuous variables. Referring to previous literature using the same outcome measures,^13,17 comparisons of continuous variables between the SA group and control group were conducted with the normal two-sample t-test (equal variance). Repeated measure analysis of variance was used to compare the NHPSS scores between the two groups over all time points at once (P ≤ 0.05 (two-sided) was considered significant), and additionally as a post hoc analysis, the individual time points were compared with a t-test based on a significance level adjusted for multiple comparisons (Bonferroni correction). The significance level was set at P ≤ 0.05 (two-sided) for imaging evaluation. Statistical analyses were performed using SPSS 23.0 (IBM Corp).

Results

The stroke model was successfully established in all animals as evidenced by both vascular imaging and functional evaluations immediately following clot placement. All animals survived and completed the subsequent imaging and neurological function evaluations as planned. Physiological parameters during the procedure are presented in Supplemental Figure 1.

Digital subtraction angiography images confirmed occlusion at the MCA M2 segment (Figure 1(b)). No spontaneous recanalization, vasospasm, perforation, or dissection occurred in any of the animals. Magnetic resonance angiography also demonstrated perfusion deficit at 24 h post ischemia in the right MCA M2 segment (Supplemental Figure 2).

In terms of magnetic resonance imaging 24 h after the procedure, as shown in the territorial hyper-intense lesions of diffusion weighted imaging sequences in Figure 2(a), SA significantly reduced the infarct volume compared to the control group (3.9 ± 0.7 cm³ vs. 7.2 ± 1.0 cm³; P = 0.002). The T2-weighted imaging sequences in Figure 2(b) show that intranasal SA administration decreased relative volume of the affected ipsilateral hemisphere significantly compared to that of the control group (0.3 ± 0.2% vs. 1.4 ± 0.3%; P = 0.002), indicating a smaller occupying effect. In addition, as shown in Figure 2(c), intranasal SA administration significantly promoted the relative ADC value in the lesion compared to that of the control group (−28.2 ± 11.0% vs. −51.5 ± 7.1%; P = 0.012), meaning less diffusion limitation and less injury. However, as shown in Figure 2(d), with diffusion tensor imaging sequence, there was no significant difference in change of fractional anisotropy values between the SA group and the control group (−18.4 ± 6.5% vs. −23.6 ± 5.9%; P = 0.288). See Supplemental Tables 1 to 4 for the original data of imaging evaluation. Additionally, the infarct volume in the SA group at 28 days post procedure was smaller than that in the control group (0.8 ± 0.6 cm³ vs. 2.0 ± 0.6 cm³; P = 0.037; Supplemental Figure 3), indicating that the initial neuroprotective effect of SA was maintained.

Figure 2.

Magnetic resonance imaging of experimental animals 24 h after the procedure. (a) Representative diffusion-weighted imaging sequences and results indicating that SA significantly reduced infarct volume (3.9 ± 0.7 cm³ vs. 7.2 ± 1.0 cm³; P = 0.002). (b) Representative T2-weighted images showing SA significantly reduced occupying effect (0.3 ± 0.2% vs. 1.4 ± 0.3%; P = 0.002) when compared to the control group. (c) Representative ADC sequence results showing SA significantly reduced diffusion limitation in the lesion (−28.2 ± 11.0% vs. −51.5 ± 7.1%; P = 0.012). (d) Representative diffusion tensor images and results indicating SA failed to reduce the white matter damage (−18.4 ± 6.5% vs. −23.6 ± 5.9%; P = 0.288) when compared to the control group. Asterisk (*) indicates statistical significance. SA: salvinorin A; ADC: apparent diffusion coefficient; FA: fractional anisotropy.

SA significantly improved long-term neurological outcome as measured by NHPSS. While there is no immediate neurological outcome improvement seen within the first 72 h after clot placement, the neurological outcome was significantly better in SA-treated animals from day 7 to day 28, compared to the control group (F = 18.8; P = 0.005; Figure 3). A table of the analysis of the NHPSS scores in the two groups is shown in Supplemental Table 5.

Figure 3.

Neurological function evaluation. Neurologic function was measured for 28 days post stroke. SA significantly improved the neurological prognosis of experimental animals when compared to the control group during follow-up (F = 18.8; P = 0.005 for comparison between the two groups of all time points at once). Data are expressed as mean and standard deviation. Hash (#) indicates statistical significance for the specific time point comparison. SA: salvinorin A; NHPSS: Non-Human Primate Stroke Scale.

Discussion

This small-scale pilot study indicates that intranasal administration of SA significantly reduced infarct volume and improved neurological outcomes in the absence of reperfusion in a monkey stroke model.

This is the first study to use an intranasal administration route to test a potential rescue medication for stroke in the super acute phase in a monkey permanent ischemic model without reperfusion. We used autologous blood clots through the least invasive method possible to closely simulate the clinical scenario in a very reproducible manner, as we have discussed previously.¹³ We chose not to include thrombolysis in the model in order to simulate the majority of clinical situations where more than 90% of ischemic stroke patients do not receive the recanalization therapy.¹⁸ Any outcome improvement with a single-dose intranasal administration should be considered a successful achievement that is worth further exploration due to its potential benefits for a large population. We have demonstrated that SA selectively dilates brain vessels and protects the brain from hypoxia and ischemia in both rodent and piglet models.^9,10 Although the use of piglet models as large animal models is indeed believed to reveal meaningful insights in translational stroke research,¹⁹ it has been reported that the vascular supply and Willis artery circle of pigs are still quite different from those of humans,²⁰ so that same stroke may produce different ischemia and behavior in pigs and humans, potentially reducing the efficiency of clinical translation. Compared with piglets, non-human primates, particularly rhesus monkeys, have brains that are much closer to that of humans. Genetically, the genomic sequence similarity of non-human primates and humans is as high as 92%.²¹ Anatomically, non-human primates have developed a complete Willis artery circle and distributions of the internal carotid and vertebral arteries as those in humans,^22–24 not to mention the ability of non-human primates to perform some of the sophisticated tasks, ensuring that neurological function could be assessed.²⁵ As such, as a transition to a potential clinical trial, a rhesus monkey ischemic model is used in this study.

To deliver a medication to the brain is challenging due to the presence of the BBB, especially during ischemic stroke. Intranasal administration has significant advantages in the prehospital setting of ischemic stroke.²⁶ The medication concentration in the brain after intranasal administration can be more than 50-fold compared to the concentration after intravenous administration.²⁷ Such higher concentration could potentially help to passively diffuse the hydrophobic molecule(s) into the ischemic area, especially in the super acute phase, or offer better protective effects in ischemic areas where there is still some small amount of blood flow. SA is a very hydrophobic small molecule with an ester-linkage that will be hydrolyzed when administered via intravenous administration. Thus, SA is suitable for intranasal administration.

SA is a short acting compound. According to a pharmacokinetic study of intraperitoneal injection in rodents, the half-life of SA in the brain is 36 min,²⁸ indicating that it can be metabolically cleared in several hours after a single administration. The pharmacokinetics of SA after intranasal administration is unknown. Intranasal administration allows SA to penetrate the BBB directly, thus avoiding blood transport in which SA can be metabolized quickly through hydrolysis.²⁹ Additionally, intranasal administration may form a medication reservoir in the nasal cavity that could potentially prolong the pharmacological effect of SA since it can be absorbed gradually. The long-term neurological behavioral function improvement is thought to be due to the protective effects that occurred during the acute phase of stroke by reducing infarct volume and preserving auto-regulation, and possibly preventing an irreversible cascade of deleterious effects. We have demonstrated that the infarct size decreased significantly when SA was administered in the acute phase in a rodent MCAO model.⁹ We have also demonstrated that SA preserved auto-regulation well in a piglet brain hypoxia ischemia model.^10,30 It is highly possible that there is other mechanism(s) involved such protective effect resulting in improved long-term outcome with just a single dose administration in the acute phase. Further studies to address this are needed.

Our early work using a piglet model indicated that SA could dilate brain vessels significantly via KOR activation.^10,30 Subsequently, we discovered that KORs play an important role in modulating brain ischemic injury.^9,31 Many classic opioids can activate KOR; however, they possess potential detrimental side effects such as respiratory depression, especially in the presence of brain ischemia. SA is derived from a natural source and is the only non-opioid KOR agonist that does not generate dysphoria effects like classic opioid KOR agonists do.³² SA has been consumed by humans for centuries and its safety was also demonstrated recently in a double-blinded clinical trial, indicating no systemic blood pressure changes.³³ The characteristics of easy passage through the BBB and quick onset make SA a potential rescue medication for neurological diseases.³⁴ In this study, intranasal SA reduced infarct volume significantly and preserved neurological outcomes almost to the point of complete recovery at 28 days after stroke. These results clearly boosted our confidence for future large-scale studies of dose-range, therapeutic windows, and small-scale human clinical trials. It is important to note that a selective KOR agonist has already been developed for clinical usage. The drug nalfurafine, a selective KOR agonist, was introduced into clinical practice for repeated long-term usage for pruritus.^35–37 While SA is very potent, it is being explored by us for rescue purposes and the known psychotropic side effects (euphoria and dissociative effects) of SA are very short-lived. We believe that the known side effects should not preclude the clinical development of SA for life-saving purposes.

The key limitation of this study is that the model used represents a stroke with a relatively small area. It is yet to be determined whether SA will have similar neuroprotective effects on major vascular occlusion strokes. In the era of reperfusion, the efficacy of SA combined with recanalization therapy remains to be explored. Furthermore, SA was only administered in the super acute phase, whereas the effectiveness of SA during prolonged ischemia time is yet to be explored. This initial pilot study included only a small number of monkeys, which limited our ability to perform sophisticated mechanism studies. Another limitation is the lack of assessment of cerebral blow flow,³⁸ which may increase further after administration. Future studies need to address these issues. Another limitation related to this study relates to the statistical analysis. Since the number of animals used in this study is small, it is not possible to confidently assume a normal distribution based on our data only. The statistical analysis is based on existing publications with the assumption that the outcome measurements do not have a biased distribution.^13,17

In conclusion, intranasal administration of SA reduced brain infarct volume and improved neurological outcome in a monkey model of ischemic stroke that uses an autologous blood clot. This study provides a solid basis to perform clinical trials to advance SA as a potential alternative medication for stroke rescue.

ORCID iDs

Longfei Wu https://orcid.org/0000-0002-0954-9126

Xunming Ji https://orcid.org/0000-0003-0293-2744