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.
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.
Dong and colleagues frame the study within the clinical problem of global cerebral ischemia, which occurs in scenarios such as cardiac arrest, drowning or profound systemic hypotension and is associated with high mortality and neurological impairment. Previous work indicates that cerebral ischemia alters vascular tone and impairs vessel responsiveness, so loss of autoregulation during hypercapnia and hypotension contributes to neuronal injury. The authors note that maintaining cerebrovascular autoregulation is therefore important for preserving cerebral blood supply and metabolism, but effective pharmacological strategies are lacking. The paper tests whether salvinorin A, a selective kappa opioid receptor (KOR) agonist that crosses the blood–brain barrier and has previously been reported to dilate vessels and reduce brain injury, can preserve cerebral pial artery autoregulation after transient forebrain ischemia. The investigators also set out to explore mechanisms, focusing on the PI3K/AKT pathway and downstream nitric oxide/cGMP signalling, because PI3K/AKT can regulate nitric oxide synthase (NOS) and cGMP-mediated vasodilation. Their primary aim was to determine if salvinorin A given at reperfusion preserves pial artery responses to hypercapnia and hypotension and whether PI3K/AKT/cGMP signalling mediates these effects.
The study used a rat model of transient global (forebrain) ischemia produced by bilateral common carotid artery occlusion (BCCAO) together with controlled haemorrhage to maintain arterial pressure at 25–30 mmHg for 10 minutes. The extracted text reports thirty Sprague–Dawley rats (250–300 g) were used and animals were acclimatised prior to surgery; however, the drug-treatment section lists group sizes of n=6 per group, and the text does not reconcile this apparent discrepancy. Anaesthesia was maintained with pentobarbital, and femoral artery and vein catheters were placed for monitoring and drug delivery. A closed cranial window permitted direct visualisation and topical treatment of cerebral pial arteries and allowed cerebrospinal fluid (CSF) sampling. Experimental groups included sham-operated controls, an ischaemia–reperfusion (IR) control receiving vehicle (DMSO, 1 mL/kg iv), two intravenous doses of salvinorin A (SA) administered at reperfusion (10 mg/kg and 20 mg/kg), and three groups receiving SA (20 mg/kg iv) plus one of the following agents applied topically via the cranial window: LY294002 (10 mM, PI3K inhibitor), L-NAME (10 mM, NOS inhibitor) or norbinaltorphimine (norBIN, 1 mM, KOR antagonist). The text describes these topical injections but does not detail the precise timing beyond administration after ischaemia. Primary physiological outcomes were dilation responses of cerebral pial arteries to hypercapnia (PaCO2 70–80 mmHg induced by 10% CO2 inhalation) and to hypotension (rapid withdrawal of 10–15 mL/kg blood to reduce arterial pressure by about 45%), measured before ischaemia and 1 hour after ischaemia using video microscopy through the cranial window. CSF was sampled at baseline and 1 hour post-ischaemia for cyclic GMP (cGMP) quantification by ELISA. Molecular and histological endpoints were assessed at 24 hours: phosphorylated AKT (p-AKT, Thr308) and total AKT by western blot in cortex/hippocampus/striatum, neuronal injury by haematoxylin–eosin staining, and apoptosis by TUNEL staining in the ischaemic-adjacent cortex. Motor function was evaluated on days 1, 2 and 5 post-ischaemia using a composite neuromotor score (screen clinging, horizontal bar, prehensile traction) with blinded assessment. Statistical analysis used one- or two-way ANOVA with Tukey multiple comparisons where appropriate; significance was set at P<0.05.
Physiological monitoring indicated no significant between-group differences in arterial blood pressure, blood gas tensions or pH at the measured time points (before ischaemia, onset of reperfusion, and 1 hour after ischaemia). Baseline pial artery autoregulation did not differ among groups prior to ischaemia. Ischaemia impaired pial artery dilation responses to both hypercapnia and hypotension at 1 hour post-ischaemia. Intravenous salvinorin A at both 10 mg/kg and 20 mg/kg given at reperfusion preserved the dilation responses of cerebral pial arteries to hypercapnia and to hypotension. The preservation of autoregulatory responses by salvinorin A was abolished when LY294002, L-NAME, or norBIN were administered topically in combination with salvinorin A. Histological assessment showed that salvinorin A reduced ischaemia-induced neuronal death in hippocampus/cortex/striatum compared with the IR (DMSO) control; the protective effect on neuronal injury was reversed by LY294002, L-NAME or norBIN. TUNEL staining indicated that salvinorin A significantly reduced apoptosis in the ischaemic-adjacent cortex at 24 hours, and again the anti-apoptotic effect was abolished by each of the three inhibitors. At the molecular level, p-AKT levels in cortical tissue were not reduced by ischaemia compared with sham in the extracted text, but salvinorin A administration increased p-AKT expression: the lower dose (SA10) produced a statistically significant increase versus IR (P<0.05) and the higher dose (SA20) produced a larger increase (P<0.01 versus IR). The PI3K inhibitor LY294002 abolished the salvinorin A-induced increase in p-AKT; L-NAME and norBIN did not. CSF cGMP concentrations were reported to be attenuated 1 hour after ischaemia, although the IR versus sham difference was not statistically significant in the extracted text. Salvinorin A elevated CSF cGMP levels, and this increase was blocked by LY294002, L-NAME and norBIN. Functionally, salvinorin A improved composite motor scores on post-ischaemia days 1, 2 and 5 compared with IR control; the motor improvements were negated when LY294002, L-NAME or norBIN were co-administered. Throughout, the extracted text indicates statistical significance for these comparisons but does not present full numerical values for most endpoints. The extracted material also does not reconcile the reported total animal number with the per-group n values given in the drug-treatment description.
Using the BCCAO transient global ischaemia model, the investigators interpret their findings to indicate that salvinorin A administered at reperfusion preserves cerebral pial artery autoregulation to hypercapnia and hypotension and reduces ischaemia-associated neuronal death and apoptosis, with concomitant improvement in motor function. They emphasise that loss of autoregulation contributes to disturbed brain metabolism, oedema and worsened injury, and that maintaining vascular responsiveness is therefore neuroprotective. Mechanistically, the authors highlight increased AKT phosphorylation and elevated CSF cGMP after salvinorin A as key biochemical correlates of the protective effects. Because the PI3K inhibitor LY294002 abolished salvinorin A-induced increases in p-AKT and cGMP, and because both the NOS inhibitor L-NAME and the KOR antagonist norBIN blocked the salvinorin A-mediated elevation of cGMP and the physiological protection, the study team infers involvement of a PI3K/AKT/NOS/cGMP pathway downstream of KOR activation. They also note that LY294002 abolished the p-AKT increase while L-NAME and norBIN did not, suggesting that salvinorin A may regulate PI3K/AKT in ways not solely dependent on NOS or KOR signalling; the authors mention prior reports of salvinorin A acting via ERK/MAPK and propose additional hypotheses such as platelet-mediated effects, which they say require further study. The investigators acknowledge limitations: only two doses of salvinorin A were tested and potential adverse effects at higher doses were not assessed, and timing of administration after ischaemia was not varied so the optimal therapeutic window remains unknown. They call for further work to delineate upstream and downstream mechanisms for PI3K/AKT modulation by salvinorin A and to clarify how these molecular changes translate into preserved vessel function and decreased neuronal injury. In conclusion, the authors propose that salvinorin A can preserve cerebrovascular autoregulation after forebrain ischaemia via the PI3K/AKT/cGMP pathway, with associated reductions in neuronal death and improvements in motor outcome.
Global cerebral ischemia, usually seen in clinical conditions such as cardiac arrest, drowning, or systemic hypotension during surgery, is associated with high mortality rates. Neuronal death and behavioral dysfunction caused by brain injury-induced ischemia are common in patients with stroke. Studies have shown that ischemia could change the vascular tension and reduce response of vessels, thereby leading to disruption of the blood flow around the ischemic area. The accompanying hypercapnia and hypotension could result in the dysfunction of the vessel and final cell injury in the brain. Currently, autoregulation of blood vessels is considered to play a critical role in the neuronal protection after ischemia during the hypercapnia and hypotension condition. It is necessary to maintain the autoregulation function of the blood vessel to preserve the normal blood supply and cell metabolism. However, until now, no effective therapeutic drug or strategy is available. Salvinorin A, a bioactive substance derived from natural plants, is a highly selective and effective agonist targeting Kappa opioid receptors (KOR). Salvinorin A has an ability to pass through the blood-brain barrier (BBB) to exert its effect on brain function. Salvinorin A could potentially protect the brain and improve neurological outcome via BBB protection, apoptosis reduction, and inflammation inhibition. Compared with other KORs, salvinorin A has the advantages of having low circulation toxicity and causing no respiratory repression; therefore, it exhibits a more promising future for clinical application. Studies have shown that salvinorin A could exert vessel dilation function in the physiological condition and preserve the autoregulation of brain vessels, as well as protect against forebrain ischemia-induced brain injury. However, the detailed mechanisms of salvinorin A on the protection of cerebral pial artery after ischemia have not been fully explored. The activation of the PI3K/AKT pathway has been found to have a protective effect on ischemic BBB damageand induces BBB permeability after cerebral ischemia. The PI3K/AKT pathway is involved in the regulation of nitric oxide synthase (eNOS) and exerts its function in multiple pathological conditions. Nitric oxide (NO), generated by NOS, is considered the key factor in endothelial cell function regulation and participates in the regulation of vessel dilation and blood flow increase through elevating cGMP levels. Our preliminary data have shown that salvinorin A increased cGMP levels in the cerebrospinal fluid (CSF); thus, we aimed to evaluate if the PI3K/AKT pathway is involved in the effect of salvinorin A on cGMP levels.
The study protocol (B-2016-069) was approved by the Institutional Animal Care and Use Committee of Shanghai Jiaotong University School of Medicine, Shanghai, China following generally accepted international guidelines for animal experimentation.
Thirty Sprague Dawley rats (250-300 g), provided by the Experimental Animal Center of Shanghai Jiaotong University, were used. All animals were acclimatized for 7 days in humidity-controlled housing with natural illumination and allowed to eat and drink freely at room temperature (23±2°C). The protocol flowchart is shown in Figure. Bilateral common carotid artery occlusion (BCCAO) was performed to establish the forebrain ischemia model as previously described. Rats were intraperitoneally (ip) injected with 3% pentobarbital (2 mL/kg) for maintenance of anesthesia. After tracheotomy, bilateral common carotid arteries were isolated and bilateral femoral arteries were catheterized to monitor the blood pressure, blood gas tensions, and pH. The femoral vein was catheterized for medication administration. Heparin (50 U) was given intravenously. Forebrain ischemia was induced by colligation of the bilateral common carotid arteries for 10 min after drawing 6-10 mL arterial blood instead of venous blood to maintain the arterial blood pressure at 25-30 mmHg and guarantee shutdown of pial artery blood flow and the withdrawal of blood that refused to go back to the femoral vein as previously described. Blood flow blocking was observed, and a pale appearance was exhibited under microscopy after ischemia, indicating a poor perfusion (Figure). Clips were removed after 10 min, and the blood was reinjected. We infused some Lactated Ringer's solution (1B2 mL) and NaHCO 3 according to blood pressure and arterial blood gas. The ports attached to the cranial window ring fit 17-gauge hypodermic needles for CSF sampling.
Sham-operated animals underwent a similar procedure, with the exception of arterial occlusion. The ischemia reperfusion (IR) control group received DMSO (Amresco, USA, 1 mL/kg, iv) administration immediately after ischemia (n=6). Two different doses of salvinorin A (Sigma, USA, 10 and 20 mg/kg, iv) were administered with the onset of reperfusion (n=6). The 5th, 6th, and 7th groups received salvinorin A (20 mg/kg, iv) and LY294002 (Cayman Chemical, USA, inhibitor of PI3K, 10 mM), L-NAME (Cayman Chemical, inhibitor of NOS, 10 mM) or norbinaltorphimine (norBIN, Sigma, antagonist of KOR, 1 mM), injected topically through cranial window, after ischemia (n=6) as described in previous studies.
Cerebral pial artery responses to hypercapnia and hypotension were obtained before ischemia and 1 h after ischemia as previously described. Hypercapnia (PaCO 2 70 to 80 mmHg) was produced by inhalation of a high concentration of CO 2 mixture gas (10% CO 2 ; 21% O 2 ; 69% N 2 ). Hypotension was produced by the rapid withdrawal of 10B15 mL/kg of blood from the femoral artery reduced by 45%. A closed cranial window, consisting of a steel ring with a glass cover slip connecting to 3 ports was placed for direct cerebral pial artery visualization and diameter measurement. The normal blood vessel of the cerebral cortex and the blood vessel dilation were visualized on a monitor connected to the microscope (model A60H, Leica Microsystems Inc., Germany) and measured via a video microscale (model VPA 550, For-A-Corp., USA) (Figure). The ports attached to the cranial window ring fit 17-gauge hypodermic needles for CSF sampling, washout, and drug administration. CSF was collected at baseline and 1 h after ischemia for cGMP level measurement. The cerebral cortex after 24 h of ischemia was collected for TUNEL staining and p-AKT detection.
Fresh CSF samples were collected as described above. The levels of cGMP were measured by ELISA kits according to the manufacturer's instructions (Sigma-Aldrich, USA).
Twenty-four hours after forebrain ischemia, rats were anesthetized with 3% pentobarbital and intracardially perfused first with 250 mL saline and then with 250 mL 4% paraformaldehyde. Samples were processed by embedding in paraffin blocks, and 5-mm thick sections were stained using HE. Hippocampal damage was determined by calculating the number of injured neurons in the hippocampus, cortex and striatum at a magnification of 400 Â. The percentage of injured neurons was determined by dividing the number of injured neurons by the total number of neurons in five separate fields.
After 24 h of ischemia, the rats were anesthetized and perfused transcardially with 0.9% saline followed by icy 4% paraformaldehyde solution and fixed for 24-48 h for paraffin embedding. TUNEL staining was performed according to the manufacturer's protocol, and slices were labeled with streptavidin-horseradish peroxidase and TUNEL-positive cells emitted a green fluorescent color. The negative control was labeled without streptavidin-horseradish peroxidase. TUNEL-positive cells were quantified using light microscopy at 400 Â magnification, and 5 fields for each section were selected from the ischemic adjacent cortex. The average percentage of TUNEL-positive cells was determined using Image-Pro Plus 6.0 image analysis software (Media Cybernetics, USA) and reported through a scale calibration.
Twenty-four hours after forebrain ischemia, the hippocampus, cortex and striatum were obtained and homogenized. Then, protein samples were collected, and the concentration was determined. Proteins (40 mg/lane) were separated and resolved by SDS-PAGE on a 12%acrylamide gel and transferred to a polyvinylidene fluoride membrane (PVDF, Millipore, USA). The membrane was blocked with 5% skim milk (BD Bioscience, USA) for 1 h at room temperature and incubated with primary antibodies against phosphor-AKT (p-AKT, Thr308) and total AKT (Cell Signaling Technology, USA) overnight. After washing with TBS-T three times, the membranes were incubated with HRP-conjugated secondary antibody (1:2000, Jackson ImmunoResearch Laboratories, Inc., USA) for 1 h at room temperature. Beta-actin (Cell Signaling Technology) was used as an internal control. A Calibrated Densitometer (Bio-Rad, USA) was used to scan immunoblotting images.
On the 1st, 2nd, and 5th post-ischemia day, rats were subjected to neuromotor tests (screen clinging, horizontal bar, and prehensile traction; 9=best possible score) as previously described. The animal was placed on the screen (29 Â 30 cm) when horizontal, and the screen was then rotated into the vertical plane. The time that the rat held on to the vertical screen was recorded to a maximum of 15 s (screen clinging). Next, the animal was placed at the center of a horizontal wooden rod (2.5 cm in diameter), and the time that the rat was able to remain balanced on the rod was recorded for a maximum of 30 s (horizontal bar). Finally, the time that the animal was able to cling to a horizontal rope was recorded to a maximum of 5 s (prehensile traction). The tests were performed by a researcher who was blind to animal group assignment.
All statistical analyses were performed with GraphPad Prism 5.0 software (USA). Data are reported as means ± SD. One-way or two-way ANOVA was employed for multiple group comparison. Motor function tests were analyzed using Tukey's multiple comparison test. A P value less than 0.05 was considered statistically significant.
Blood pressure, gas tension, and PH The data of blood pressure, gas tension, and pH are shown in Table. At each time point (a, before ischemia; b, onset reperfusion; and c, 1 h after ischemia), there was no significant difference when all groups were compared with each other.
All animals were monitored with blood gas and temperature and no significant difference was found on the baseline value (results not shown). No significant difference was found in pial artery autoregulation among the different groups before ischemia (Figure). However, the dilation responses of the pial artery to hypercapnia or hypotension were impaired after ischemia and were preserved with different doses of salvinorin A treatment. The preservation of autoregulation was abolished when LY294002 or L-NAME or norBIN was administered, as seen in Figure.
Compared to IR (DMSO) control, salvinorin A significantly suppressed ischemia-induced neuronal death, while LY294002 or L-NAME or norBIN could abolish the protective effect of salvinorin A against neuronal death induced by ischemia (Figureand).
A cell apoptosis assay was performed to evaluate the protective effect of salvinorin A on the cerebral cortex after ischemia. Our results showed that compared to IR (DMSO) control, salvinorin A administration significantly suppressed the cerebral cortex cell apoptosis, while LY294002, L-NAME or norBIN abolished the salvinorin A-suppressed apoptosis effect (Figureand).
The molecular mechanism involved in the cerebral cortex after ischemia was also explored. p-AKT levels did Figure. Salvinorin A (SA) preserved cerebral pial artery autoregulation after cerebral cortex ischemia. SA10, SA20, SA20+LY294002, SA20+L-NAME, SA20+norBIN were administered, and the cerebral pial artery autoregulation was evaluated. A, No significant differences were found in the pial artery autoregulation among different groups before ischemia. B, Pial artery autoregulation among different groups after ischemia. Data are reported as means±SD for n=6. && Po0.01 compared to the sham control group, **Po0.01 compared to the IR (DMSO) group, ## Po0.01 compared to the SA2 treatment group. The pial artery autoregulation was analyzed by one-way ANOVA followed by Tukey's test. LY: LY294002 (10 mM, injected topically); LN: L-NAME (10 mM, injected topically); IR (ischemia reperfusion control, DMSO): 1 mL/kg (iv); SA10: salvinorin A (10 mg/kg, iv); SA20: salvinorin A (20 mg/kg, iv); norBIN: norbinaltorphimine 1 mM (injected topically). not change after ischemia compared to those after the sham control. However, salvinorin A administration remarkably increased p-AKT level (Po0.05, SA10 compared to IR; Po0.01, SA20 compared to IR), whereas LY294002 abolished salvinorin A-increased p-AKT levels (Figureand).
Motor function test (TMS) score system was used to evaluate the motor function on days 1, 2, and 5 after ischemia induction. Compared to IR (DMSO) control, salvinorin A administration significantly improved the motor function of the rats on days 1, 2, and 5. However, LY294002, L-NAME, or norBIN administration abolished salvinorin A-improved motor function of the rats on days 1, 2, and 5 (Figure).
In our study, BCCAO was used as a model of transient global cerebral ischemia according to previous reports. The BCCAO model simulates the effects produced by the systemic decrease in blood flow, affects the entire brain, and clinically resembles transient global cerebral ischemia in a number of pathological aspects such as cardiac arrest, drowning or systemic hypotension during surgery. Global cerebral ischemia leads to neurological deficits in memory and executive function. Injury of a brain vessel, which could further increase the severity of cerebral ischemia, is the most common phenomenon during the cerebral ischemia. Early prevention of cerebral vessel dysfunction and improvement of the cerebral damage is the key step in neuronal protection. In the present study, we investigated the role of salvinorin A in cerebral pial artery protection in a rat model of forebrain ischemia injury. We found no significant difference in the pial artery autoregulation among different groups before ischemia. However, the dilation responses of the pial artery to hypercapnia or hypotension were impaired after ischemia. Hypercapnia was produced by inhalation of high concentration CO 2 mixture gas. Hypotension produced by the rapid withdrawal of 10B15 mL/kg of blood from the femoral artery was reduced by 45%. Two different levels of treatments were used in the preexperiments, and we found that the results of treatment with low levels of hypercapnia and hypotension were not ideal. Thus, we eventually gave up on the experimental results of low level processing in our final research. We found that two doses of salvinorin A treatment preserved the autoregulation of cerebral pial artery. Salvinorin A treatment protected the brain tissues from ischemia injury, which is demonstrated by decreased cell apoptosis around the ischemia area and recovery of motor function at 1 to 5 days. Autoregulation function during cerebral ischemia is the main physiological activity to maintain normal brain metabolism. Loss of autoregulation could result in disruption of brain metabolism, induction of brain edema, and increase the severity, which were consistent with our results on abnormal pial artery dilation, increasing cell apoptosis and severe motor function injury. The previous study has shown that salvinorin A treatment exerted vessel dilation function in normal brain tissue. In our study, we applied the salvinorin A immediately after ischemia, and it still exerted protective effects and maintained the normal response to cerebral pial artery autoregulation. We also observed the effect of different doses of salvinorin A on cerebral pial artery response, and the results showed that both doses were effective. The PI3K/AKT pathway is considered a critical signaling pathway in multiple diseases. AKT phosphorylation is an important part of this pathway. Yoshitomi et al. demonstrated that phosphorylation of eNOS and Akt was decreased in the cerebral cortex of stroke-prone spontaneously hypertensive rats compared with that in Wistar-Kyoto rats. After reperfusion, increased levels of p-AKT and p-eNOS were observed transiently from 0.5 to 2 h and from 4 to 12 h, respectively, whereas both of them were much lower at 24 h after reperfusion than the sham group. The regulatory effect of salvinorin A on the AKT phosphorylation is poorly understood. Therefore, we investigated the effect of salvinorin A on the phosphorylation level of AKT. Our results showed that salvinorin A significantly increased the expression of AKT phosphorylation. The PI3K inhibitor LY294002 abolished the salvinorin A-increased AKT phosphorylation rather than NOS antagonist L-NAME and KOR antagonist norBIN, suggesting that salvinorin A may regulate the PI3K/AKT pathway through other ways. Previous studies have shown that salvinorin A preserved cerebrovascular autoregulation to hypotension and hypercapnia after brain hypoxia/ischemia via ERK/MAPK in a piglet model. Recently, we proposed that salvinorin A might also regulate PI3K/AKT pathway through activating platelet release. We are currently conducting further studies to confirm this. NO is the key factor that regulates vessel tension, while NOS is the rate-limiting enzyme in NO generation. Diffusion of NO from vessel wall to the vascular smooth muscle cells could activate cGMP and result in the relaxation of smooth muscle cells that eventually leads to the dilation of the vessel. PI3K/AKT/eNOS was considered the key step for NO generation during vessel dilation. Previous studies have shown that increased eNOS level could result in vessel dilation, improvement of endothelial dysfunctionand decrease brain injury during ischemia. In the present study, the level of cGMP in CSF was attenuated 1 h after ischemia, although no significant difference was detected between the IR group and sham control group. Our results also showed that salvinorin A treatment elevated the levels of cGMP in CSF. The PI3K inhibitor LY294002, NOS antagonist L-NAME and KOR antagonist norBIN could abolish salvinorin A-increased levels of cGMP, suggesting that the PI3K/AKT/cGMP pathway was involved in the regulatory process of salvinorin A on cGMP. However, the detail mechanisms involved need to be further explored. KOR has been demonstrated to protect the brain against cerebral ischemia injury. We found here that 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. Multiple mechanisms have been proposed to be involved in the regulatory role of KOR. For example, BRL52537 protects against cerebral IR injury via a mechanism involving STAT3 signaling (34); U-50488H exerts its effect via regulation of Na(+), K(+) ATPase activity. Induction of NO could also be generated by KOR agonists. A previous study has shown that KOR agonist could decrease the cell apoptosis in the setting of myocardial ischemia through PI3K-dependent AKT phosphorylation, while NO could be generated by NOS (eNOS) phosphorylation during the same process. Although KOR agonists exhibit tremendous therapeutic value, most KOR agonists have not been applied in clinical settings because of their intrinsic characteristics as opioids (low selectivity and/or lack of an acceptable safety profile). However, the detailed mechanisms of salvinorin A on the dilation function of brain vessels have not been fully explored. Further studies are needed to elucidate the upstream and downstream mechanism for salvinorin A modulation of the PI3K/AKT pathway in brain tissues. Based on our present results, we proposed that salvinorin A may alleviate inflammation and edema induced by cerebral ischemia. There are some limitations in this study. Firstly, only two doses of salvinorin A were used in the treatment of ischemia; however, we are not sure that an increased dose would result in adverse reactions. Secondly, the application of salvinorin A at other time points after ischemia should also be tested to find the optimal choice. In conclusion, our findings have shown 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.
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