Fingolimod protects against neurovascular unit injury in a rat model of focal cerebral ischemia/reperfusion injury

Abstract

Recent research on the underlying mechanisms of cerebral ischemia indicates that the neurovascular unit can be used as a novel subject for general surveys of neuronal damage and protein mechanisms. Fingolimod (FTY-720) is a newly developed immunosuppressant isolated from Cordyceps sinensis that exhibits a wide range of biological activities, and has recently attracted much attention for the treatment of ischemic cerebrovascular diseases. In the current research, the role of FTY-720 and its possible mechanisms were assessed from an neurovascular unit perspective using a rat cerebral ischemia model. Our results revealed that FTY-720 markedly decreased infarct volume, promoted neurological function recovery, and weakened the blood-brain barrier permeability of ischemic rats. The protective roles of FTY-720 in ischemic stroke are ascribed to a combination of sphingosin-1-phosphate receptor-1 and reduced expression of sphingosin-1-phosphate receptor-1 in microvessels and reduction of interleukin-17A protein levels. These findings indicate that FTY-720 has promise as a new therapy for neurovascular protection and functional recovery after ischemic stroke.

Introduction

Ischemic stroke, the major type of stroke, is listed among the major causes of disability globally. The clinical treatment of ischemic stroke typically involves vascular modalities such as intravenous thrombolysis and mechanical thrombus retrieval. However, revascularization after thrombolysis may further aggravate ischemic brain injury (Eltzschig and Eckle, 2011; Lambertsen et al., 2019). Therefore, in 2001, the first Round Table of the National Institute of Neurology and Stroke (NINDS) proposed the idea of a neurovascular unit (NVU) with major structures including neurons, endothelial cells, astrocytes, microglia, and extracellular matrix (Zhao et al., 2020). Therapies that can efficiently target all NVU aspects are likely to gain acceptance as promising agents for treatment of ischemic stroke.

Fingolimod (FTY-720)—a sphingosine analogue that serves as a potent immunosuppressive agent for the central nervous system—has been used to treat relapsed remitting multiple sclerosis. FTY-720 is a high-affinity agonist for the sphingosine-1-phosphate receptor (S1PR) (Huwiler and Zangemeister-Wittke, 2018), with S1PR being widely distributed within the central nervous system. Sphingosin-1-phosphate receptor-1 (S1PR1) is expressed mainly in endothelial cells (Hugh Rosen et al., 2014), revealing the role of S1P/S1PR1-dependent signaling in improving the barrier integrity of endothelial cells in pulmonary artery and human umbilical vein (Garcia et al., 2001; Moccia et al., 2019). In addition, FTY-720 can induce depletion of circulating lymphocytes and exert an immunosuppressive effect, thereby reducing reperfusion injury to the heart (Ahmed et al., 2020), brain (Shang et al., 2020), and kidney (Shi et al., 2021) in mice, and protecting against inflammatory injury to various organs of the body. FTY-720 has demonstrated neuroprotective properties in experimental models of stroke, Alzheimer’s disease, and Parkinson’s disease (Zhao et al., 2017; Salas-Perdomo et al., 2019; Jesko et al., 2020). However, the molecular mechanisms regulating the neuroprotective properties in ischemic stroke have rarely been studied with a focus on the NVU.

Stroke is accompanied by a loss of neuronal function and damage to multiple tissues, followed by induction of inflammatory responses. Via their endothelial cells, cerebral microvessels play a significant role in sustaining the structural integrity of the blood-brain barrier (BBB) and NVU (Hawkins and Davis, 2005). When endothelial cells are damaged because of ischemic stroke, the tight junction proteins (TJP) occludin and claudin-5 degrade quickly, leading to destruction of the BBB and the triggering of relevant dysfunctions (Zhang et al., 2016). Cerebral edema was recently reclassified as cytotoxic, ionic, or angioedema on the basis of the changes observed in the brain (Salman et al., 2022b). Of the cells in the brain that contribute to the formation of the BBB and NVU, astrocytes are the most widely distributed (Zhao et al., 2020). Stroke is accompanied by activation of astrocytes, disappearance of the foot process, metamorphosis, and aggravated damage to the NVU (Huang et al., 2019). Microglia, which form the major immune cells of the brain, are essential for brain homeostasis and immune responses to different neurological disorders. Nonetheless, activated microglia can cause neurotoxicity via several neurotoxic reactions (Li and Barres, 2018). Notably, stroke patients have a high level of interleukin-17 (IL-17) in the peripheral blood compared with healthy individuals (Wo et al., 2020). Accumulating evidence suggests that IL-17A is the key inflammation-modulating molecule from the IL-17 family following ischemia/reperfusion injury, and that it further damages the structure of the NVU (Zhang et al., 2014).

In the present study, a rat model of middle cerebral artery occlusion (MCAO) was established to explore the protective effects of FTY-720 on ischemia/reperfusion injury and the possible underlying mechanism from the point of view of the NVU.

Methods

Experimental animals

All experimental protocols received approval from the Animal Care and Use Committee of Jinzhou Medical University (approval No. 2020090901) on September 9, 2020. Each animal experiment was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (8^th ed., 2011). This study is reported in accordance with the ARRIVE 2.0 guidelines (Animal Research: Reporting of In Vivo Experiments) (Percie du Sert et al., 2020). Specific-pathogen-free (SPF) male Sprague-Dawley rats (n = 112, 260–280 g, 12–14 weeks) were purchased from Liaoning Changsheng Biotechnology Company, China (License No. SCXK (Liao) 2020-0001). Each animal was raised under a 12-hour/12-hour light/dark cycle and controlled temperature conditions, with free access to water and food. Stroke is a sexually dimorphic disease, with it being shown that estrogen replacement therapy confers beneficial neuroprotective effects (Ma et al., 2021). Therefore, in this study, to avoid possible interference of estrogen on experimental results and unnecessary sample waste, only male rats were selected as experimental subjects. The rats were randomly divided into the following groups using a random number table method: Sham group (n = 6), MCAO untreated group (n = 53), and MCAO FTY-720-treated group (n = 53).

Model of focal cerebral ischemia

The focal cerebral ischemia MCAO model was created according to a previous description (Hasegawa et al., 2003). Briefly, 4–5% isoflurane (3 L/min; CSPS Co., Ltd., Beijing, China) was used for anesthesia, followed by maintenance with 1–2% isoflurane inhalation with a mask. After exposing the right common carotid artery (CCA) and the right internal carotid artery, MCAO was conducted by inserting a monofilament nylon suture (Beijing Xinong Biotechnology Co., Ltd, China) to a depth of approximately 18 mm to generate focal cerebral ischemia. After 2 hours, the filament was withdrawn to achieve reperfusion. A homeothermic heating pad was used to maintain the intraoperative body temperature of the rats at 37.0 ± 0.5°C. The sham group underwent the same procedure without insertion of the nylon monofilament. After ischemia, the rats were observed for circling onto the unaffected side (left) to assess whether MCAO was successfully performed. MCAO was also validated by staining with 2,3,5-triphenyltetrazolium chloride (TTC, MilliporeSigma, Burlington, MA, USA) after perfusion with pre-chilled phosphate-buffered saline (PBS; 250 mL). The presence of unstained ischemic brain tissue indicated successful establishment of the model. At this stage, ten rats were sacrificed to demonstrate MCAO.

Drug administration

In the MCAO models, FTY-720 (dissolved in DMSO; 1 mg/kg; MilliporeSigma) or DMSO (dimethyl sulfoxide; 0.5% concentration; MilliporeSigma) were administered via intraperitoneal injection immediately after reperfusion and then daily for up to 7 days. Brain tissues for future experiments were collected at 1, 3, and 7 days after reperfusion.

Measurement of neurological deficits

At 1, 3, and 7 days after reperfusion, the rats’ neurological deficits were assessed using Zea-Longa (Longa et al., 1989) and Ludmila-Belayev scores (Belayev et al., 1996). Briefly, the Zea-Longa scores were evaluated as follows: 0 indicated the absence of neurological deficit, 1 indicated inability to fully extend the contralateral front claw, 2 indicated circling towards the contrary side, 3 indicated falling towards the contrary side, 4 indicated inability to walk autonomously accompanied by depressive-like behavior, and 5 indicated death. The Ludmila-Belayev scores were assessed using a scale of 0–12, where 12 points indicated maximum neurological functional losses and 0 represented normal function. The Ludmila-Belayev scores were obtained via two tests: (1) a postural reflex test was conducted to examine the upper body posture by suspending the rats by their tails; and (2) a forelimb placement test was used to examine sensory-motor integration in the presence of tactile, visual, and proprioceptive stimuli.

Tissue collection and measurement of infarction

At 1, 3, and 7 days after reperfusion, selected rats were transcardially perfused with pre-chilled phosphate-buffered saline (PBS; 250 mL). The brain tissues were then rapidly dissected, placed in a precooled rat brain mold (Kent Scientific Crop, USA), and sliced into five coronal sections (2-mm thickness) using a cryostat (CM1900; Leica, Wetzlar, Germany). Thereafter, the brain sections were soaked in 0.5% TTC solution at 37°C for a 10-minute period to measure the infarct volume.

ImageJ v1.80 (NIH, Bethesda, MD, USA; Schneider et al., 2012) was later used to analyze the infarct volume (%) according to the following formula: (volume of contralateral hemisphere – volume of non-infarct ipsilateral hemisphere)/volume of contralateral hemisphere × 100.

Evaluation of Evans blue leakage

In those animals subjected to Evans blue (EB) staining, the stain was administered via intravenous injection (2% w/v in PBS, MilliporeSigma) at 3 mL/kg via the tail vein immediately before sampling at 2 hours after reperfusion, when each rat received a transcardial perfusion of PBS. The brain tissues were then dissected, placed in a precooled rat brain mold (Kent Scientific Crop), sliced into five coronal sections (2-mm thick) using a cryostat (CM1900; Leica), and photographed with a digital camera (Leica VARIO-SUMMILUX) to visualize the EB leakage. The nonischemic and ischemic sides of the brain were then separately collected into sterile centrifuge tubes and the EB level within the ischemic hemisphere was measured to quantify the destruction of the BBB. Briefly, the DMSO group ischemic and non-ischemic (DMSO-I and DMSO-Non-I) and FTY-720 group ischemic and nonischemic (FTY-720-I and FTY-720-Non-I) cerebral tissues were collected separately into four Eppendorf tubes, 1 mL of 50% trichloroacetic acid was added, and the tissues were chopped and homogenized. After homogenization, the brain tissue was put into a high-speed centrifuge with pre-cooling at 4°C and centrifuged at 14,000 × g for 15 minutes, after which the supernatant was transferred to a new Eppendorf tube and four times the volume of anhydrous ethanol solution was added. The mixture was added to a 96-well plate and the fluorescence intensity of each tube was detected using a fluorescence enzyme labeling instrument (BioTek Instruments, Inc., Winooski, VT, ↱USA) with excitation of 620 nm and reading of emission at 680 nm. The quantity of EB was described in ng/g.

Measurement of cerebral edema

ImageJ was used to analyze the extent of cerebral edema. The 2-mm-thick brain tissue slices were visualized with a digital camera (Leica VARIO-SUMMILUX, Germany) and software was used to measure the hemispheric areas in all sections. Edema was described as the ratio of the area of the affected hemisphere to that of the unaffected contralateral hemisphere.

Isolation of cerebral microvessels

At the end of the reperfusion, the ischemic and nonischemic cerebral tissues were collected separately into two Eppendorf tubes, to which 2 mL of PBS was added. The tissues were than chopped and homogenized and the homogenate collected, followed by centrifugation at 4°C and 3500 × g for 10 minutes. Finally, the supernatant was discarded and 1 mL of PBS was added for resuspension. The microvessels retained on an aseptic nylon cell filter (40 μm) were washed down with PBS, followed by 10 minutes of centrifugation at 3000 × g and 4°C. After discarding the supernatants, the crude extracted microvessels were purified with 15% dextran T-500, followed by 10 minutes of centrifugation at 25,000 × g and under 4°C. Afterwards, 20% dextran T-500 was added and the samples were centrifuged for 10 minutes at 25,000 × g and 4°C. Finally, the pellets (which represented the cerebral microvessels) were harvested and preserved at –80°C for use in the subsequent assays.

Immunofluorescence staining

At the end of reperfusion, the brains were fixed by circulatory perfusion with PBS and 4% paraformaldehyde (PFA). Brains were rapidly removed and postfixed in 4% PFA and 30% sucrose for 72 hours, embedded in OCT (Product code 4583, Sakura, Tokyo, Japan), and frozen at –80°C. A cryostat (CM1900; Leica) was then used to cut 20-μm-thick coronal sections of frozen brain.

The frozen brain sections (20-μm thick) were blocked for 1 hour with 5% normal goat serum, followed by overnight incubation at 4°C with the following primary antibodies: rabbit anti-occludin antibody (tight junction protein, 1:100; Cat# 40-4700, Thermo Fisher Scientific), rabbit anti-claudin-5 polyclonal antibody (tight junction protein, 1:100; Cat# PA5-99415, Thermo Fisher Scientific); rabbit monoclonal anti-S1PR1 antibody (1:100; Cat# 55133-1-AP, Proteintech, Chicago, IL, USA); mouse monoclonal anti-CD31 antibody (a marker for microvascular endothelial cells; 1:50; Cat# sc-376764, Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse monoclonal anti-NeuN (neuronal nuclei) antibody (a marker for neurons; 1:100; Cat# 834501, Biolegend, San Diego, CA, USA); mouse monoclonal anti-GFAP (glial fibrillary acidic protein) antibody (a marker for astrocytes; 1:100; Cat# 80788, CST, Danvers, MA, USA); rabbit monoclonal anti-Iba1 (ionized calcium-binding adapter molecule 1) antibody (a marker for microglia; 1:100; Cat# ab178846, Abcam, Cambridge, MA, USA) and mouse monoclonal anti-IL-17A antibody (a proinflammatory cytokine; 1:100; Cat#PA5-106856, Thermo Fisher Scientific, Waltham, MA, USA). The sections were rinsed with PBS then further probed using Alexa Fluor 488-labeled or Cy3-labeled secondary antibodies (1:200, Thermo Fisher Scientific) at room temperature for 2 hours, followed by three PBS washes. The sections were then dyed with 4′,6-diamidino-2-phenylindole (DAPI, Solarbio Science & Technology Co., Ltd., Beijing, China) and mounted using ProLong Antifade Reagents (Thermo Fisher Scientific). Fluorescence photographs were obtained by automated fluorescence microscopy (Leica DM 4B). All images used for direct comparisons were taken at the same exposure and laser intensity. Three slices were taken from each sample, and three random visual fields were taken from each slice. The slices were observed with a 20× objective lens (20×, NA 0.55, Leica, DMC6200).

TUNEL staining

The terminal deoxynucleotidyl transferase-mediated dUTP nick 3′-end labeling (TUNEL) staining kit (In Situ Cell Death Detection Kit, TMR red, Roche, Shanghai, China) was used to assess the extent of cell apoptosis, in line with the manufacturer’s specified protocols. Briefly, the brain sections were incubated for 5 minutes in the permeabilization solution, followed by 30 minutes incubation with the TUNEL reaction mixture at 37°C in the dark. The cell nuclei were visualized with DAPI. An automatic intelligent fluorescence microscope system (Leica DM4B) was used to acquire the images. Thereafter, the number of apoptotic cells within the ischemic penumbra that separated the ischemic core from the surrounding healthy brain tissue (the number of cells/mm²) were determined in a blinded manner (three visual fields, 200× magnification per section) using ImageJ v1.80.

Western blotting

The extracted cerebral microvessels were added to 60 μL RIPA lysis buffer (brain tissue added at 1:4), homogenized, lysed for 30 minutes, centrifuged at 16,000 × g for 20 minutes, and the precipitate discarded. The protein concentration was then evaluated using a BCA protein assay (Cat# 23227, Thermo Fisher Scientific). After boiling using a constant temperature bath (Monad Biotech Co., Ltd., Wuhan, Hubei Province, China), 10–12% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Solarbio Science & Technology Co., Ltd.) was used to separate the total proteins (30 μg), which were then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked for 1 hour with 5% skimmed milk (Solarbio Science & Technology Co., Ltd.) in TBST (tris-buffered saline and Polysorbate 20, also known as Tween 20) at room temperature, followed by overnight incubation at 4°C with the following primary antibodies: rabbit anti-occludin antibody (tight junction protein, 1:1000; Cat# 40-4700, Thermo Fisher Scientific), rabbit anti-claudin-5 polyclonal antibody (tight junction protein, 1:1000; Cat# PA5-99415, Thermo Fisher Scientific), rabbit anti-S1PR1 polyclonal antibody (endothelial cell receptor; 1:1000; Cat# ab11424, Abcam), rabbit anti-IL-17A polyclonal antibody (an inflammatory cytokine; 1:1000; Cat# PA5-106856, Thermo Fisher Scientific), mouse anti-β-actin polyclonal antibody (1:3000; Cat# 3700, CST). After incubation with the primary antibody overnight, the membranes were further incubated with goat anti-rabbit IgG (1:15,000; Cat# SE134, Solarbio Science & Technology Co., Ltd.)/goat anti-mouse IgG (1:15,000; Cat# SE131, Solarbio Science & Technology Co., Ltd.) secondary antibodies at room temperature for 2 hours. The membranes were visualized with an enhanced chemiluminescence system (Beyotime, Shanghai, China) to display the protein bands and a ChemiDoc-It TS2 Imager (UVP, LLC, Upland, CA, USA) was used to capture the images. ImageJ was used to perform densitometric analysis. The relative protein expression was normalized to β-actin. The above experimental design is shown in Figure 1A.

Figure 1.

Fingolimod (FTY-720) reduces cerebral infarction volume and improves neurological deficits in middle cerebral artery occlusion rats.

(A) The experimental design. (B) Representative 2,3,5-triphenyltetrazolium chloride (TTC) staining images of rat brain tissues. (C) Quantification of the rat cerebral infarct volume. White represents infarcted brain tissue (n = 5 rats per group). (D) The Zea-Longa scores (n = 9 rats per group). (E) The Ludmila-Belayev scores (n = 9 rats per group). (F) Representative immunofluorescence staining of NeuN (green) and DAPI (blue). Scale bars: 50 μm. (G) NeuN-positive cell number in the peri-infarct penumbra (n = 5 brain samples from three rats per group). Data are represented as mean ± SD. *P < 0.05, vs. DMSO group (one-way analysis of variance followed by Bonferroni post hoc test). The experiment was repeated five times. DAPI: 4′,6-Diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; EB: Evans blue; NeuN: neuronal nuclei.

Statistical analysis

No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those reported in a previous publication (Ghergherehchi et al., 2021). SPSS 26.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. The results are presented as mean ± standard deviation (SD). Differences between three and more groups were compared by one-way analysis of variance followed by a Bonferroni post hoc test. P < 0.05 was taken to indicate statistical significance. GraphPad Prism v8.01 (GraphPad Software, San Diego, CA, USA, www.graphpad.com) was used for plotting the results.

Results

FTY-720 reduces cerebral infarction volume and improves neurological deficits in MCAO rats

To determine whether FTY-720 exerted a protective mechanism on cerebral ischemia/reperfusion injury in rats, TTC staining was performed at 2 hours after ischemia and 1, 3, and 7 days after reperfusion, and the volumes of cerebral infarction before and after FTY-720 treatment were compared. At 1, 3, and 7 days after reperfusion, the infarct volume in the ischemic hemisphere of the FTY-720 group was significantly lower than that in the DMSO group (P < 0.05; Figure 1B and C). Evaluation of the neurological deficits using the Zea-Longa (Figure 1D) and Ludmila-Belayev scores (Figure 1E) revealed that the neurological function of the rats was significantly better in the FTY-720 group than that in the DMSO group (P < 0.05).

To further determine whether FTY720 could reduce the severity of neuron injury, neurons were identified using NeuN staining for neurons (Figure 1F). The results showed that the neurons in the peri-infarct penumbra were higher in the FTY-720-treated group than in the DMSO group (P < 0.05; Figure 1G).

FTY-720 reduces ischemia-induced BBB disruption and edema

Next, we employed two different methods to evaluate the effect of FTY-720 on ischemia-induced BBB disruption: EB extravasation and relative cerebral edema volume. Figures 2A and B show EB extravasation at 1, 3, and 7 days after reperfusion. As expected, the EB contents of the nonischemic hemispheric tissues were low at all timepoints. Cerebral ischemia caused a remarkable increase in the EB contents of the ischemic hemispheric tissues in the DMSO group. This considerable EB leakage was accompanied by a longer reperfusion duration. At all three reperfusion timepoints, the FTY-720 treatment reduced the EB extravasation in comparison with the DMSO group (P < 0.05).

Figure 2.

Fingolimod (FTY-720) reduces ischemia-induced blood-brain barrier disruption and edema.

(A) Representative brain sections displaying Evans blue (EB) staining within the ischemic region. Blue represents the exudation of EB. (B) EB leakage (n = 5 rats per group, 5 coronal brain sections per sample were counted). *P < 0.05, vs. 3-day DMSO group; #P < 0.05, vs. DMSO group. (C) Ischemia-mediated brain edema ratio. *P < 0.05, vs. DMSO group at identical reperfusion time (n = 5 rats per group). One-way analysis of variance followed by Bonferroni post hoc test was used. The experiment was repeated five times. DMSO: Dimethyl sulfoxide.

Cerebral edema is another indicator used to assess BBB injury caused by cerebral ischemia. Figure 2C illustrates results using the ratio of the enlargement of the ischemic hemisphere to reflect the severity of brain edema, and it can be observed that brain edema was largest at 1 day after reperfusion, and that the edema of the ischemic hemisphere had disappeared on day 7. After FTY-720 treatment, the enlargement rate of the ischemic hemisphere was significantly lower than that in the DMSO group, and the atrophic brain tissue was also effectively improved on day 7 (P < 0.05).

FTY-720 restores the loss of TJPs in the ischemic microvessels of ischemic stroke rats

Next, to explore whether FTY-720 modulated TJP levels, occludin and claudin-5 immunoreactivities were estimated using immunohistochemistry methods. The results showed that occludin and claudin-5 immunoreactivities were weakened in ischemic rats (DMSO group), while immunoreactivities were obviously observed after FTY-720 treatment (Figure 3A and B). We also used western blotting to evaluate the occludin and claudin-5 protein levels of the isolated microvessels. The results indicated declines in occludin and claudin-5 protein expression post-ischemia, whereas FTY-720 increased occludin and claudin-5 protein expression in the microvessels of the ischemic stroke rats (Figures 3C–E).

Figure 3.

Fingolimod (FTY-720) reverses tight junction protein losses in ischemic microvessels of ischemic stroke rats.

(A) Representative immunofluorescence staining using DAPI (blue) and occludin (green). (B) Typical immunofluorescence staining with DAPI (blue) and claudin-5 (green). Scale bars: 50 μm. (C) Typical western blot bands for claudin-5 and occludin. (D–E) Quantification of claudin-5 (n = 3 rats per group) and occludin (n = 3 rats per group) protein expression. *P < 0.05, vs. DMSO group. Results are presented as mean ± SD (one-way analysis of variance followed by Bonferroni post hoc test). The experiment was repeated three times. DAPI: 4′,6-Diamidino-2-phenylindole; DMSO: dimethyl sulfoxide.

FTY-720 alleviates ischemia/reperfusion induced cerebral microvascular damage by acting on the microvascular endothelial S1PR1

To gain a deeper insight into the protective impact of FTY-720 on cerebral microvessels, we observed CD31 (microvascular endothelial cells marker) and S1PR1 co-staining using immunohistochemistry methods. We observed that the connectivity of the cerebral microvessels was markedly lost after ischemia and that colocalization with S1PR1 was reduced (Figure 4A–C). After FTY-720 treatment, the connectivity of the cerebral microvessels was intact and there were several small branches. These results revealed that FTY-720 exhibited a protective role on cerebral microvessels by acting on microvascular endothelial S1PR1.

Figure 4.

Fingolimod (FTY-720) alleviates ischemia/reperfusion-induced cerebral microvascular damage by acting on microvascular endothelial S1PR1.

(A–C) Representative immunofluorescence staining for CD31 (green) and S1PR1 (red) at 1, 3, and 7 days after reperfusion. Scale bars: 50 μm. (D) Representative western blot bands of S1PR1. (E) Quantification of S1PR1 protein expression (n = 5 rats per group, *P < 0.05, vs. DMSO group, one-way analysis of variance followed by Bonferroni post hoc test). The experiment was repeated five times. DMSO: Dimethyl sulfoxide; S1PR1: sphingosin-1-phosphate receptor-1.

We additionally used western blotting to assess the protein levels of S1PR1 in isolated microvessels, the results of which also indicated that FTY-720 treatment significantly reduced the expression of S1PR1 on cerebral microvessels compared with the DMSO group (P < 0.05; Figure 4D and E). The morphology and continuity of the cerebral microvessels in the ischemic hemisphere remained intact after FTY-720 treatment (Figure 4A–C), which may have maintained vascular continuity and promoted cerebral microvascular angiogenesis by acting with S1PR1 on the microvascular endothelial cells.

FTY-720 attenuates ischemia/reperfusion induced glial cell activation and IL-17A expression

To further comprehend the NVU status following ischemia, astrocytes and microglia activation were assessed by immunohistochemistry. First, we co-stained GFAP and IL-17A and observed ischemia-induced astrocyte activation, morphological changes, and apparent colocalization with IL-17A (Figure 5A). Moreover, FTY-720 treatment improved the activation of astrocytes and decreased colocalization with IL-17A. Next, we performed fluorescent staining of Iba-1 and IL-17A and recorded similar results to those mentioned above (Figure 5B).

Figure 5.

Fingolimod (FTY-720) attenuates ischemia/reperfusion-induced glial cell activation and IL-17A expression.

(A) Representative immunofluorescence staining for IL-17A (red) and GFAP (green). (B) Representative immunofluorescence staining for IL-17A (red) and Iba-1 (green). Scale bars: 50 μm. (C) Representative western blot bands for IL-17A. (D) Quantification of IL-17A protein expression (n = 5 rats per group, *P < 0.05, vs. DMSO group, one-way analysis of variance followed by Bonferroni post hoc test). The experiment was repeated five times. DMSO: Dimethyl sulfoxide; GFAP: glial fibrillary acidic protein; Iba1: ionized calcium-binding adapter molecule 1; IL: interleukin.

To further confirm that FTY-720 inhibited the expression of inflammatory factors, we also used a western blot to assess the protein levels of IL-17A in brain tissue. This showed that FTY-720 effectively diminished the expression of IL-17A (Figure 5C and D).

FTY-720 decreases neuronal apoptosis in the peri-infarct penumbra of rats after ischemia/reperfusion

To elucidate the protective mechanism of FTY-720 on ischemia/reperfusion-induced NVU damage, we used TUNEL staining to explore how FTY-720 affected neuronal apoptosis. We discovered that compared with the DMSO group, TUNEL-positive cells (which are used to quantify apoptotic cells) and the amounts of apoptotic cells decreased significantly after FTY-720 treatment (P < 0.05; Figure 6A and B). TUNEL staining was negative in the brain sections of the sham group, whereas the amounts of TUNEL-positive neurons were greater in the DMSO group during ischemia/reperfusion than in the FTY-720 group, implying that FTY-720 treatment could effectively decrease the number of TUNEL-positive neurons compared with the DMSO group (P < 0.05; Figure 6C and D).

Figure 6.

Fingolimod (FTY-720) decreases neuronal apoptosis in the peri-infarct penumbra of rats after ischemia/reperfusion.

(A) Representative immunofluorescence staining of TUNEL (red) and DAPI (blue). (B) TUNEL-positive cell number. (C) TUNEL (red) and NeuN (green) co-staining. Scale bars: 50 µm. (D) Number of NeuN/TUNEL-positive cells (n = 5 brain samples from three rats per group, *P < 0.05, vs. DMSO group, one-way analysis of variance followed by Bonferroni post hoc test). The experiment was repeated five times. DAPI: 4′,6-Diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; NeuN: neuronal nuclei; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick 3′-end labeling.

Discussion

Cerebral ischemia/reperfusion injury is a complicated pathological and physiological process, and the specific mechanisms behind it are currently unclear. In this study, we examined the protective effects of FTY-720 on cerebral ischemia/reperfusion injury in rats from the perspective of the NVU, and investigated the possible mechanisms behind these effects. Our results show that FTY-720 may combine with S1PR1 on microvessels to protect against BBB damage. FTY-720 inhibited the secretion of IL-17A by glial cells and reduced the inflammatory reaction in the brain, thereby providing a neuroprotective effect. In addition, FTY-720 significantly reduced infarction volume, improved neurologic deficits, and reduced neural cell apoptosis, findings consistent with previous studies (Hasegawa et al., 2010; Wei et al., 2011). These results confirm the neuroprotective effects of FTY-720 following cerebral ischemia, effects that lasted for 7 days. Our findings also provide an insight into the manner-of-action of the protective effects of FTY-720 in cerebral injury after stroke.

The repair of neurological function following the onset of stroke is a long-term process, and analysis of a single timepoint is insufficient to explain the protective effect of FTY-720. Therefore, we chose 1, 3, and 7 days as multiple timepoints for this study. We found that the ischemic brain tissue of the rats showed significant atrophy at 7 days after reperfusion. Moreover, treatment with FTY-720 effectively reduced this atrophy, further confirming the effectiveness of FTY-720 and suggesting that the effects may be multifaceted rather than being focused on just reducing neuron injury. Our results also suggest that FTY-720 had a sustained protective effect throughout the acute phase of stroke, and that the long-term protective effects of FTY-720 are worthy of further investigation.

Cerebral edema after ischemic stroke is caused by an increase in the permeability of the BBB, which is damaged by the initial ischemic injury. Many researchers have focused on the regulation of water balance after cerebral edema, and aquaporins contribute to the transport of water in diverse pathologies, including ischemic stroke-induced brain edema (Markou et al., 2022; Salman et al., 2022a; Wagner et al., 2022). The most important water channel protein is thought to be aquaporin 4, which is used as a therapy to regulate edema after brain injury (Kitchen et al., 2020; Salman et al., 2021, 2022b; Sylvain et al., 2021). In addition, breakdown of the BBB itself is another cause of brain edema. Treatments to help maintain BBB integrity are also effective measures to reduce brain edema in ischemic insult. S1P receptors are widely expressed in the central nervous system, and S1PR1, which regulates microvascular function, is mainly expressed in the microvascular endothelium (Hugh Rosen et al., 2014; Goi and Childs, 2016). We found that after ischemic onset, the protein level of S1PR1 was significantly enhanced in the vascular endothelium, and that this caused BBB damage in the neural units. FTY-720 treatment effectively reduced levels of S1PR1 protein, thereby reducing ischemia-induced cerebral edema.

Recently, inflammatory factors were found to play a very important role in the mechanism of cerebral ischemic stroke injury. Inhibition of inflammation in the brain has been regarded as an excellent strategy for assisting the repair of neurological function after stroke. The release of large quantities of cytokines after ischemic neuronal injury activates glial cells in the brain, making them further secrete proinflammatory cytokines (Volonte et al., 2012; Burnstock, 2016; Kong et al., 2019). Acting as an immunosuppressant, FTY-720 effectively inhibits the secretion of inflammatory factors. Its immunosuppressive effect has also been confirmed in clinical trials in multiple sclerosis (Chitnis et al., 2018) and renal transplantation (Gholamnezhadjafari et al., 2016). FTY-720 treatment also led to attenuation of IL-17A-positive T-cells, less proinflammatory cytokine in the brain, and better preservation of function following hypoxic-ischemic brain injury in newborns (Yang et al., 2014). In our experiments, we found significant colocalization of IL-17A with astrocytes and microglia after stroke. Previous studies demonstrated that IL-17A is a key factor in the regulation of the inflammatory response after ischemia/reperfusion injury, which may further aggravate NVU injury (Shichita et al., 2009; Zhang et al., 2014; Liu et al., 2019). We found that FTY-720 treatment effectively reduced IL-17A levels, and that the effect was most significant on day seven. These results suggest that FTY-720 reduced the protein level of IL-17A, thereby inhibiting the inflammatory response in the brain and providing a protective role.

We also found that administration of FTY-720 considerably reduced the amount of TUNEL-positive neurons and considerably enhanced the number of neurons in the ischemic penumbra after ischemia, probably through the protective effects of FTY-720 on the BBB and inhibition of inflammatory cytokine release from astrocytes and microglia in the brain. In addition, the cerebral infarct volume and neurological score were significantly improved, indicating that FTY-720 achieved neuroprotective effects on the overall NVU, rather than exerting a protective effect only on neurons.

There are some limitations to this study. First, we did not measure fluorescence intensity, but only used representative fluorescence pictures to observe changes in occludin and claudin-5 from the microscopic point of view. Second, whether FTY-720 can be used over the long term to improve neurological deficits was not explored. Our findings indicate that treatment with FTY-720 inhibited IL-17A secretion from astrocytes and microglia. However, the exact mechanism by which FTY-720 inactivates these two types of cells in ischemic stroke was not investigated in this study.

In conclusion, our data show that FTY-720 significantly reduced infarct volume, improved neurologic deficits, increased repair of brain microvascular injury, and reduced neuronal apoptosis. FTY-720 can play a comprehensive role in protecting the ultrastructure of the NVU and improving neurological function after cerebral ischemia. Our research provides new insights into the clinical treatment of stroke using FTY-720.

Additional file: Open peer review reports 1 ^{(99.5KB, pdf)} and 2 ^{(95.1KB, pdf)} .