Salvinorin A preserves cerebral pial artery autoregulation after forebrain ischemia via the PI3K/AKT/cGMP pathway

This animal study (n=30) investigated the role of salvinorin A (10 and 20µg/kg) in cerebral pial artery protection in a rat model of forebrain ischemia injury and found that salvinorin A treatment preserved the autoregulation of the cerebral pial artery, protected the brain tissues from ischemia injury by decreasing cell death, and hastened the recovery of their motor functions.

Abstract

“Introduction: This study aimed to investigate the protective effect of salvinorin A on the cerebral pial artery after forebrain ischemia and explore related mechanisms.

Methods: Thirty Sprague-Dawley rats received forebrain ischemia for 10 min. The dilation responses of the cerebral pial artery to hypercapnia and hypotension were assessed in rats before and 1 h after ischemia. The ischemia-reperfusion (IR) control group received DMSO (1 µL/kg) immediately after ischemia. Two different doses of salvinorin A (10 and 20 µg/kg) were administered following the onset of reperfusion. The 5th, 6th, and 7th groups received salvinorin A (20 µg/kg) and LY294002 (10 µM), L-NAME (10 μM), or norbinaltorphimine (norBIN, 1 μM) after ischemia. The levels of cGMP in the cerebrospinal fluid (CSF) were also measured. The phosphorylation of AKT (p-AKT) was measured in the cerebral cortex by western blot at 24 h post-ischemia. Cell necrosis and apoptosis were examined by hematoxylin-eosin staining (HE) and TUNEL staining, respectively. The motor function of the rats was evaluated at 1, 2, and 5 days post-ischemia.

Results: The dilation responses of the cerebral pial artery were significantly impaired after ischemia and were preserved by salvinorin A treatment. In addition, salvinorin A significantly increased the levels of cGMP and p-AKT, suppressed cell necrosis and apoptosis of the cerebral cortex and improved the motor function of the rats. These effects were abolished by LY294002, L-NAME, and norBIN.

Discussion: Salvinorin A preserved cerebral pial artery autoregulation in response to hypercapnia and hypotension via the PI3K/AKT/cGMP pathway.“

Authors: H. P. Dong, W. Zhou, X. X. Ma, Z. Z. He & Z. H. Wang

Summary

This study aimed to investigate the protective effects of salvinorin A on the cerebral pial artery after forebrain ischemia and explore related mechanisms. Salvinorin A significantly increased the levels of cGMP and p-AKT, suppressed cell necrosis and apoptosis, and improved the motor function of the rats.

Introduction

Global cerebral ischemia, usually seen in clinical conditions such as cardiac arrest, drowning, or systemic hypotension during surgery, is associated with high mortality rates and neuronal death and behavioral dysfunction. Salvinorin A, a bioactive substance derived from natural plants, can restore autoregulation of blood vessels.

Salvinorin A, a KOR agonist, can pass through the blood-brain barrier and exert its effect on brain function via BBB protection, apoptosis reduction, and inflammation inhibition. It may also be able to protect the brain and improve neurological outcome via PI3K/AKT pathway activation.

Experimental animals and forebrain ischemia model

Thirty Sprague Dawley rats were used to establish a forebrain ischemia model. After bilateral common carotid artery occlusion, the blood flow was blocked for 10 min, and the CSF was sampled according to blood pressure and arterial blood gas.

Drug treatment

Sham-operated animals underwent a similar procedure to the experimental group, with the exception of arterial occlusion. Salvinorin A, L-NAME, or norbinaltorphimine were administered after ischemia to inhibit PI3K, NOS, or KOR.

Cerebral pial artery responses

Cerebral pial artery responses to hypercapnia and hypotension were obtained before ischemia and 1 h after ischemia. CSF was collected at baseline and 1 h after ischemia for cGMP level measurement and the cerebral cortex was collected for TUNEL staining and p-AKT detection.

Western blot

After 24 hours of forebrain ischemia, the hippocampus, cortex and striatum were obtained and homogenized. Proteins were separated by SDS-PAGE and transferred to a PVDF membrane, which was then incubated with primary antibodies against phosphor-AKT and total AKT.

Salvinorin A preserved cerebral pial artery autoregulation after cerebral cortex ischemia

All animals had the same baseline values for blood gas and temperature, but the dilation responses of the pial artery were impaired after ischemia and preserved with salvinorin A treatment.

Salvinorin A increased cGMP levels after cerebral cortex ischemia

Salvinorin A administration increased cGMP levels compared to IR (DMSO) control, but LY294002, L-NAME, or norBIN administration abolished salvinorin A-increased cGMP levels.

Salvinorin A protected against ischemia-induced cerebral cortex cell apoptosis

Salvinorin A significantly suppressed cerebral cortex cell apoptosis after ischemia, while LY294002, L-NAME or norBIN abolished the salvinorin A-suppressed apoptosis effect.

Salvinorin A increased the expression of p-AKT after cerebral cortex ischemia

The molecular mechanism involved in the cerebral cortex after ischemia was also explored. Salvinorin A administration remarkably increased p-AKT levels.

Salvinorin A administration significantly improved motor function in rats after ischemia induction, but LY294002, L-NAME, or norBIN administration abolished the improvement.

Discussion

In our study, we used BCCAO to simulate transient global cerebral ischemia. This model resembles cardiac arrest, drowning or systemic hypotension during surgery.

Cerebral ischemia leads to neurological deficits in memory and executive function. Early prevention of cerebral vessel dysfunction is key to neuronal protection.

In the present study, salvinorin A protected the brain tissues from ischemia injury in a rat model by preserving the autoregulation of cerebral pial artery and by decreasing cell apoptosis around the ischemia area.

Salvinorin A treatment maintained normal cerebral pial artery autoregulation function in animals after cerebral ischemia, and both doses were effective in reducing brain edema and increasing motor function injury.

Salvinorin A increased the expression of AKT phosphorylation in the cerebral cortex of stroke-prone spontaneously hypertensive rats compared with Wistar-Kyoto rats, and the PI3K inhibitor LY294002 abolished the salvinorin A-increased AKT phosphorylation rather than NOS antagonist L-NAME and KOR antagonist norBIN.

NO is the key factor that regulates vessel tension, and NOS is the rate-limiting enzyme in NO generation. PI3K/AKT/eNOS was considered the key step for NO generation during vessel dilation, and increased eNOS level could result in vessel dilation and improved endothelial dysfunction.

Salvinorin A administration at a dose of 10 or 20 mg/kg after ischemia preserved autoregulation of the cerebral pial artery to hypercapnia and hypotension via the PI3K/AKT/cGMP pathway. This mechanism may be involved in the alleviation of inflammation and edema induced by cerebral ischemia.

This study showed that salvinorin A could preserve cerebrovascular autoregulation in response to hypotension and hypercapnia after forebrain ischemia via the PI3K/AKT/cGMP pathway, which could result in decreasing of neuronal death and improvement of motor function.