FTY720 Attenuates Cerebral Vasospasm After Subarachnoid Hemorrhage Through the PI3K/AKT/eNOS and NF-κB Pathways in Rats
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.355128
Source of Support: None, Conflict of Interest: None
Keywords: FTY720, NF-κB, PI3K/AKT/eNOS, subarachnoid hemorrhage, vasospasm
Despite years of study, subarachnoid hemorrhage (SAH) is still a challenging neurological disease and is associated with high morbidity and mortality. Cerebral vasospasm (CVS) remains the most worrisome complication and relates to high mortality., Nimodipine, a calcium channel blocker, blocks the influx of calcium and suppresses CVS by increasing the production of nitric oxide (NO) from endothelial nitric oxide synthase (eNOS). It is thus far the only effective therapy approved by the U.S. Food and Drug Administration. Liu et al. elucidated that fasudil in the treatment of CVS after SAH was effective by continuous intravertebral artery injection. Thus, it is essential to gain insight into the pathogenesis of SAH to better understand the disease process and to identify more effective treatments. The first 3 days are critical window periods for the development of CVS, which causes severe reduction of cerebral blood flow. NO, which is mainly produced by the endothelium via activation of eNOS, acts as a critical endothelial-derived relaxing factor. Recently, the role of NO and inflammation after SAH was recognized as a key factor in the development of CVS., FTY720 has recently been paid much attention as a bioactive molecule for its anti-inflammatory, antiapoptosis, and vasodilatation effects. To date, FTY720 receptors have been identified as important factors in the central nervous system (CNS) diseases, including multiple sclerosis (MS), neuropathic pain, brain ischemic stroke, and neurotrauma.,,, Studies have shown that FTY720 reduces stroke-related neuroinflammation, brain edema, and neuronal death and improves neurological outcomes.,, SAH was associated with a reduction of 60% in S1P levels and a 40% increase in S1P-lyase activity. Xu et al. demonstrated that FTY720 improved neurological outcomes in rats subjected to SAH by decreasing the intravascular adhesion of leukocytes to pial venules. Based on these observations, we tested the hypothesis that FTY720 could attenuate CVS in a rat model of SAH.
Animals and experimental groups
Seventy-five male Sprague–Dawley rats weighing 300 to 350 grams each were obtained from the Animal Laboratory, Nanjing Medical University, Nanjing, China. They were randomly assigned to five groups of 15 rats each: (1) the sham group: rats not subjected to any intervention; (2) the sham + FTY720 (1 mg/kg) group: rats treated with FTY720 (1 mg/kg) only; (3) the SAH + saline group: a group treated with saline, which was used as a vehicle after SAH; (4) the SAH + FTY720 (0.5 mg/kg) group: rats treated with FTY720 at a concentration of 0.5 mg/kg after SAH; and (5) the SAH + FTY720 (1 mg/kg) group: rats treated with FTY720 (1 mg/kg) after SAH. Both saline and FTY720 were intraperitoneally injected 30 minutes after SAH surgery.
Experimental model of SAH and drug administration
Zibly et al. provided one method to establish a model of SAH-induced vasospasm in swine, which produced consistent mild to moderate vasospasm following SAH. In our study, endovascular perforation was used to induce the SAH model according to Schwartz et al. Briefly, rats were anesthetized with 1% pentobarbital (50 mg/kg, intraperitoneally), supplied with intubation and a respirator apparatus, and maintained at 37.0 ± 0.5°C rectal temperature with a heating pad. To perform SAH, the right external carotid artery was isolated and severed to leave a stump of 3 to 4 mm in length, which was reopened after the internal carotid artery and common carotid artery were clamped. A monofilament nylon suture of Size 4.0 was then inserted through the reopened internal carotid artery to approximately 20 mm, at which point a slight resistance is typically felt. The suture was then advanced slightly further to perforate the artery and was immediately withdrawn, allowing reperfusion to induce SAH. The duration of the operation was less than 30 seconds. A similar procedure was performed in the sham group but with a blunt suture that was unable to pierce the arterial wall. After surgery, rats were released to their cages with free access to food and water after recovery from anesthesia. FTY720 (Cayman Chemicals Europe, Tallinn, Estonia) was dissolved in dimethyl sulfoxide (DMSO) and subsequently diluted with saline at a final concentration of 125 μg/mL, yielding a clear liquid. The final concentration of DMSO was less than 0.1%. FTY720 or saline (200–250 μL) was intraperitoneally injected into rats in the proper groups twice daily for 3 days until all rats were euthanized for further study. We consistently found blood clots around the bifurcation of the middle–anterior cerebral artery on autopsy of each of the rats. All the animal procedures complied with the Guide for the Care and Use of Laboratory Animals.
Peripheral leukocyte count
Peripheral blood samples were used to determine the effect of FTY720 on leukocytes. Blood samples were collected before and 72 hours after surgery. The sham + FTY720 group was used to confirm the immunomodulatory effect of FTY720. A blood sample of approximately 0.5 mL was obtained from the subclavian vein and analyzed using an Advia 120 automated hematology analyzer (Siemens, Dublin, Ireland).
CSF samples were obtained as follows. First, the hair between the two ears was shaved. Rats were fixed by a stereotaxic instrument, and their heads were held in a uniform position by adjusting the ear bars. The occipital muscles were dissected to reveal the atlanto-occipital membrane. CSF was then collected from the cisterna magna and stored at −80°C until the concentrations of cytokines were measured.
The enzyme-linked immunosorbent assay (ELISA) kits were purchased from R and D, USA. Standards and samples were added to the ELISA wells and incubated for 2 hours at room temperature. Each well was washed five times with wash buffer. Then, specific conjugates of cytokines were added, followed by incubation for another 2 hours at room temperature. After washing another five times, substrate solution and stop solution were added in turn. The microplate reader was set to 450 nm. Finally, the concentrations of the cytokines were calculated.
Western blot analysis
Following anesthesia, the basilar artery and hippocampus of the rats were isolated and gently homogenized into whole-cell lysates in 5 mL of buffer. Equal amounts of protein (50 μg) were loaded in each lane of polyacrylamide-sodium dodecyl sulfate gels, which were electrophoresed, followed by protein transfer to a nitrocellulose membrane. The membrane was subsequently blocked with a blocking solution and probed with primary antibodies overnight at 4°C. Protein expression, including eNOS and AKT in the basilar artery and tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), caspase-3, bax, bcl-2, and p-iκBα in the hippocampus, was detected by primary antibodies. Next, the immunoblots were processed using horseradish peroxidase–conjugated antigoat IgG with secondary antibodies for 1 hour at room temperature. The blotting bands were quantified by densitometry with ImageJ software. β-Actin was blotted on the same membrane as a loading control for the fractions. The quantified values were expressed as a percentage of β-actin.
Measurement of NO level
As stated above, the basilar artery of the rats, homogenized into whole-cell lysate, was then centrifuged, and the supernatant was collected. The generation of NO was measured by a commercial Nitrate/Nitrite Fluorometric Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA) using the nitrate reductase method. The determination of NO levels was performed following the manufacturer's protocols.
The rats were sacrificed, and the basilar arteries with brain stems were postfixed overnight. After fixation, the samples were embedded in paraffin, and coronal sections of 5 μm in thickness were cut.
H and E staining, diameters, and internal areas of the basilar arteries
Sections of the brain stem containing basilar arteries in each group were stained by hematoxylin and eosin (H and E; hematoxylin for 5 minutes and eosin for 2.5 minutes) and observed under a BX51 microscope. We then measured the diameters of the basilar arteries and internal areas of each slice using the Image-Pro Plus software package (Media Cybernetics). From each animal, two basilar artery slices were obtained from the proximal, distal, and middle portions of the vessel. Using the software, we calculated the average diameter and the internal area of each slice.
Mortality and neurological function assessment
Neurologic functions were evaluated by the Modified Garcia Scale method, an 18-point scoring system measured 72 hours after SAH, which includes spontaneous activity, symmetry in the movement of all four limbs, forepaw outstretching, climbing, body proprioception, and response to vibrissae touch.
Data were expressed as the mean ± standard deviation, which were analyzed by SPSS Version 16.0 software (IBM, USA). Statistical significance was verified by one-way analysis of variance (ANOVA) for multiple comparisons. Fisher's exact test was used for statistical analysis of mortality in each group. A P value of less than 0.05 was considered statistically significant.
FTY720 decreased circulating blood cell counts
There were no significant differences in the counts of leukocytes, lymphocytes, or neutrophils in each group before surgery [Figure 1]a. Treatment with FTY720 in nonsurgical animals resulted in a decrease in circulating leukocytes and lymphocytes at 72 hours, whereas the neutrophils did not change significantly. In comparison with SAH + saline animals, both doses of FTY720 were associated with a statistically significant reduction in total leukocytes and lymphocytes in the peripheral blood [Figure 1]b.
FTY720 decreased the levels of TNF-α and IL-1β in both the CSF and the hippocampus
TNF-α and IL-1β are classical proinflammatory cytokines. To examine whether FTY720 decreased inflammatory reactions after SAH, we measured the concentrations of TNF-α and IL-1β in CSF and protein expression in hippocampal tissue of rats using ELISA and Western blot, respectively. As shown in [Figure 2]a, there was no significant difference in TNF-α and IL-1β levels between the sham and sham + FTY720 groups. The levels in the SAH + saline group were higher compared with those in the sham group (106.7 ± 20 vs. 50.2 ± 8, TNF-α; 77.3 ± 12 vs. 42 ± 7.2, IL-1β;; P < 0.05). FTY720 at both concentrations of 0.5 mg/kg and 1 mg/kg not only reduced the secretion of TNF-α and IL-1β in CSF (83.7 ± 10.5, 77.3 ± 8 vs. 106.7 ± 20; 54.7 ± 12.5, 38.7 ± 7 vs. 77.3 ± 12, respectively) but also decreased protein expression in the hippocampus (P < 0.05) [Figure 2]b and [Figure 2]c.
FTY720 inactivated the pathways of apoptosis and inflammation
Caspase-3 plays a central role in the process of cell apoptosis, which is governed by antiapoptotic Bcl-2 and proapoptotic Bax proteins. Proinflammatory cytokines, such as TNF-α, activate caspase-3 and induce cell apoptosis. As shown in [Figure 3]a and [Figure 3]b, the levels of caspase-3 and bax in the SAH + saline group were significantly increased compared with those in the sham group and were reduced by FTY720 intervention. The antiapoptotic protein Bcl-2 was upregulated (P < 0.05). In addition, FTY720 (1 mg/kg) attenuated the expression of p-IκBα by approximately 65% (P < 0.05) [Figure 3]c. The reduction of p-IκBα in the cytoplasm implies a decrease of NF-κB in the cell nucleus. All data were compared using one-way ANOVA. These results suggest the potential anti-inflammatory and antiapoptotic effects of FTY720 in SAH rats.
FTY720 promoted NO generation through the PI3K/AKT/eNOS pathway
As shown in [Figure 4]a and [Figure 4]b, there was marked downregulation of AKT and eNOS after SAH, which was significantly improved by FTY720 treatment (P < 0.05). AKT was inhibited by MK2206, an AKT kinase inhibitor, by approximately 68% (P < 0.05). Furthermore, l-NNA (nomega-nitro-l-arginine), an eNOS antagonist, inhibited the protein expression of eNOS by 91% (P < 0.05) [Figure 4]c and [Figure 4]d. NO has potent vasodilative activity. As shown in [Figure 4]e, there was no difference in NO levels in the sham and sham + FTY720 groups. The NO content decreased in the SAH + saline group, and FTY720 increased the SAH-induced decrease in NO (67 ± 6.6 vs. 53.3 ± 9.8 P < 0.05). Both MK2206 and l-NNA decreased NO content (P < 0.05).
FTY720 alleviated CVS after SAH
As shown in [Figure 5], severe CVS occurred after SAH. At the higher magnification [Figure 5]h, a typical corrugated appearance of the basal lamina was observed, with a thickened and spastic muscular wall. This appearance was attenuated in the FTY720 treatment groups [Figure 5]i and [Figure 5]j. By measuring representative photographs, we observed that the diameter of the basilar arteries in the SAH + saline group significantly decreased compared with that in the sham group (119 ± 5 vs. 153 ± 9 μm). FTY720 treatment at both 0.5 and 1 mg/kg increased the diameters to 144 ± 8 and 150 ± 9 μm, respectively [Figure 5]k. The internal areas of the basilar artery were also measured, which confirmed the above results. As shown in [Figure 5]l, the area in the SAH + saline group was significantly decreased compared with that in the control group (14,527 ± 1,110 μm2 vs. 19,577 ± 1,260 μm2; P < 0.05). FTY720 at both concentrations significantly increased the internal area of the basilar artery (17,678 ± 547 μm2 at 0.5 mg/kg and 18,510 ± 879 μm2 at 1 mg/kg; P < 0.05). A one-way ANOVA was used to analyze the data.
FTY720 decreased mortality and improved neurological function 72 hours after SAH
As shown in [Figure 6]a, one rat died both in the sham and sham + FTY720 groups each. In the SAH + saline group, 67% (10/15) of the rats died, whereas 27% (4/15) of the rats that were treated with both concentrations of FTY720 died. A modified Garcia Scale was used to examine the neurological functions of all surviving rats. As shown in [Figure 6]b, FTY720 at both concentrations reduced the neurological deficit of SAH rats (12 ± 2 and 14 ± 4 vs. 9 ± 3. 0.5 and 1 mg/kg FTY720 treatment group vs. SAH + saline group, P < 0.05.).
In this study, we demonstrated the protective effect of FTY720 toward CVS due to SAH. The results showed that FTY720 improved neurological functions and decreased the mortality of SAH rats by downregulating the counts of leukocytes and lymphocytes and the levels of TNF-α and IL-1β in both the CSF and the hippocampus. Inflammatory and apoptotic pathways were inhibited. In addition, FTY720 activated the PI3K/AKT/eNOS pathway to promote NO generation, which may protect the brain cells from ischemic injury by dilating the arteries. The diameters and internal areas of the basilar arteries were enlarged. Thus, we conclude that FTY720 has vasodilatory, anti-inflammatory, and antiapoptotic effects, which can alleviate CVS and protect neurological function after SAH in rats. The PI3K/AKT/eNOS and NF-κB pathways are involved in this process.
SAH is characterized by acute headaches, resulting in patients seeking early medical attention. CVS, which leads to secondary cerebral ischemia after SAH, is the main complication of SAH and increases the mortality of patients. Studies have shown that early brain injury, which occurs within 72 hours after SAH, is highly correlated with a poor prognosis due to microcirculatory disturbance, blood–brain barrier disruption, and the death of brain cells., Inflammation plays an important role in this condition. NO, which dilates blood vessels, has a neuroprotective effect in SAH-induced vasospasm. Acute microvascular constriction after SAH is potentially mediated by hemoglobin suppressing the NO/cGMP signaling pathway. Excessive inflammation and apoptosis contribute to the damage caused by cerebral ischemic diseases., These studies emphasized the importance of NO and inflammation in the development of CVS. Furthermore, the inflammatory action after SAH mainly lies in the CSF, brain parenchyma, and cerebral arteries.
S1P has recently been paid much attention as a bioactive molecule. Extracellular S1P interacts with five G protein-coupled receptors (GPCRs), which are widely expressed on the surface of the cell membrane. A small lipid molecule, S1P transduces intracellular signals to regulate cellular behavior, such as migration, adhesion, survival, and proliferation. Studies have shown that S1P activates the PI3K/AKT/eNOS pathway to exert vasodilatation through the Gi protein. In the CNS, S1P has been shown to influence many responses through their receptors and may become the new target to human CNS disorders such as MS. FTY720, a structural homolog of S1P, serves as an agonist of four of the five GPCRs, including S1P1, 3, 4, and 5. Studies have shown that FTY720 can cross the blood–brain barrier and regulate critical cellular processes, such as the increased endothelial barrier permeability induced by apoptosis and inflammation. FTY720 has been shown to reduce stroke-related neuroinflammation, brain edema, and neuronal death, which exerted a protective role against cerebral ischemia and intraparenchymal hemorrhage.,, Interestingly, although FTY720 strongly reduced the number of circulating leukocytes, it did not increase the morbidity of bacterial lung infections after stroke. With regard to the relationship between FTY720 and SAH, a study has shown that FTY720 preserved arteriolar dilation functions by reducing leukocyte adhesion to pial venules and improved neurological function 48 hours after SAH. However, some opposite results have also been demonstrated. Activated platelets in blood clots may be the source of S1P due to SAH. S1P induced vasoconstriction in the canine basilar artery possibly through the Rho/Rho-kinase pathway and might be considered a novel spasmogenic substance. Also, there were reports that FTY720 induced arterial vasospasm., Based on the importance of eNOS/NO and inflammation in the pathogenesis of CVS, we hypothesized that FTY720 exerted protective effects toward CVS in SAH. Our current study demonstrated that vessel constriction, following the neurological deficit and increased mortality induced by SAH, was alleviated by FTY720. The application of FTY720 did not result in critical CVS. On the contrary, FTY720 markedly increased AKT, eNOS, and NO levels, which play vasodilator and neuroprotective roles. NF-κB is one of the most important factors in regulating proinflammatory gene expression, such as TNF-α and IL-1. This research demonstrated that FTY720 decreased the counts of leukocytes and lymphocytes and the inflammation action in CSF and hippocampus through the NF-κB pathway. The counts of leukocytes and lymphocytes declined in sham + FTY720 animals, but there was no increased mortality. Furthermore, the apoptosis pathway was inactivated in the hippocampus after FTY720 treatment. Increases in the diameters and internal areas of the basilar arteries confirmed the vasodilator effect of FTY720.
In conclusion, we demonstrated that inflammation and eNOS/NO deficiency played important roles in the pathogenesis of CVS. FTY720 alleviated SAH-induced CVS and showed neuroprotective effects via the PI3K/AKT/eNOS and NF-κB pathways. Thus, FTY720 may be effective in the clinical treatment of SAH patients.
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Conflicts of interest
There are no conflicts of interest.
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