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Journal of Central South University Medical Sciences logoLink to Journal of Central South University Medical Sciences
. 2023 May 28;48(5):648–662. doi: 10.11817/j.issn.1672-7347.2023.220380

Endogenous FGF21 attenuates blood-brain barrier disruption in penumbra after delayed recanalization in MCAO rats through FGFR1/PI3K/Akt pathway

内源性FGF21通过FGFR1/PI3K/Akt通路减轻MCAO大鼠延迟血管再通后半暗带的血脑屏障损伤（英文）

ZHENG Wen 1,2, LI Wenjun 1, ZENG Yini 1, YUAN Hui 1, YANG Heng 1, CHEN Ru 1, ZHU Anding 1, WU Jinze 1, SONG Zhi 1, YAN Wenguang 2,✉
Editor: PENG Minning
PMCID: PMC10930414  PMID: 37539567

Abstract

Objective

Restoration of blood circulation within “time window” is the principal treating goal for treating acute ischemic stroke. Previous studies revealed that delayed recanalization might cause serious ischemia/reperfusion injury. However, plenty of evidences showed delayed recanalization improved neurological outcomes in acute ischemic stroke. This study aims to explore the role of delayed recanalization on blood-brain barrier (BBB) in the penumbra (surrounding ischemic core) and neurological outcomes after middle cerebral artery occlusion (MCAO).

Methods

Recanalization was performed on the 3rd day after MCAO. BBB disruption was tested by Western blotting, Evans blue dye, and immunofluorescence staining. Infarct volume and neurological outcomes were evaluated on the 7th day after MCAO. The expression of fibroblast growth factor 21 (FGF21), fibroblast growth factor receptor 1 (FGFR1), phosphatidylinositol-3-kinase (PI3K), and serine/threonine kinase (Akt) in the penumbra were observed by immunofluorescence staining and/or Western blotting.

Results

The extraversion of Evans blue, IgG, and albumin increased surrounding ischemic core after MCAO, but significantly decreased after recanalization. The expression of Claudin-5, Occludin, and zona occludens 1 (ZO-1) decreased surrounding ischemic core after MCAO, but significantly increased after recanalization. Infarct volume reduced and neurological outcomes improved following recanalization (on the 7th day after MCAO). The expressions of Claudin-5, Occludin, and ZO-1 decreased surrounding ischemic core following MCAO, which were up-regulated corresponding to the increases of FGF21, p-FGFR1, PI3K, and p-Akt after recanalization. Intra-cerebroventricular injection of FGFR1 inhibitor SU5402 down-regulated the expression of PI3K, p-Akt, Occludin, Claudin-5, and ZO-1 in the penumbra, which weakened the beneficial effects of recanalization on neurological outcomes after MCAO.

Conclusion

Delayed recanalization on the 3rd day after MCAO increases endogenous FGF21 in the penumbra and activates FGFR1/PI3K/Akt pathway, which attenuates BBB disruption in the penumbra and improves neurobehavior in MCAO rats.

Keywords: middle cerebral artery occlusion, fibroblast growth factor 21, delayed recanalization, ischemia/reperfusion injury, blood-brain barrier disruption

Ischemic stroke is still one of the leading causes of disability and death worldwide[1-5]. Intravenous thrombolysis with recombinant tissue-type plasminogen activator (rt-PA) and endovascular thrombectomy have been proven to be more effective therapy for acute ischemic stroke[4-5]. However, basing on the hypothesis of penumbra and ischemia/reperfusion (I/R) injury, intravenous thrombolysis and endovascular thrombectomy were administrated only in patients within “time window”[2, 6]. Previously studs[6] revealed that delayed recanalization (beyond 6 h “time window” of acute ischemic stroke) may result in serious I/R injury, increase hemorrhagic transformation, and worsen functional outcomes. Recently, clinical trials confirmed that endovascular thrombectomy in 6-24 h after stroke onset led to favorable clinical outcomes in selected patients with acute ischemic stroke, without increasing symptomatic intracranial hemorrhage[7-8]. It was also reported that intravenous thrombolysis with alteplase (within 4.5-9.0 h after stroke onset or wake-up stroke) and tenecteplase (4-24 h after stroke onset) improved neurological outcomes in selected patients with salvageable brain tissue[9-11]. Additionally, spontaneous recanalization beyond 6 h “time window” in patients with ischemic stroke usually associates with improving long-term disability[6, 12-16]. All these evidences showed that delayed recanalization after “time window” may restore cerebral reperfusion and improve neurological outcomes in acute ischemic stroke. But all these studies did not clarify the I/R injury following recanalization.

The cerebral I/R injury is the secondary brain damage provoked by abrupt restoration of the cerebral blood after an ischemic period[17]. With a decrease in oxygen and glucose supply, the majority of the cells in “ischemic core” die after acute ischemic stroke. Subsequently, the injury extends from “ischemic core” to “penumbra”[18]. At this particular condition, especially beyond the “time window”, the restoration of blood circulation (reperfusion) and the resupply of oxygen/glucose may trigger I/R injury and accelerate neuronal cell death in the region both “ischemic core” and “penumbra”[18], which is considered to enlarge the infarct area and worsen neurological dysfunction[16-17, 19]. The underlying mechanisms of I/R injury involve in mitochondrial dysregulation, oxidative stress, reactive oxygen species (ROS), calcium overload, blood-brain barrier (BBB) disruption, inflammation, free radicals, excitotoxicity, and neuronal apoptosis, etc.[18, 20]. However, increasing clinical perfusion imaging or MR diffusion weighted imaging (DWI) revealed delayed recanalization after standard “time window” in selected patients with acute ischemic stroke did not enlarge infarct[17, 21]. Additionally, emerging evidences[16-17, 22] showed that greater reperfusion percentage after thrombectomy is associated with decreased brain edema and infarct. Therefore, the I/R injury of cerebral tissues after delayed recanalization is far beyond the scope of previous understanding. The important discrepancy between clinical results and preclinical pathophysiology needs more investigation.

Extensive efforts have been made to elucidate the upregulation of neuroprotective agents in serum after acute ischemic stroke. The restoration of blood flow and the reperfusion of cerebral tissue promote the expression of neuroprotective agents in hypoperfused penumbra from serum, such as fibroblast growth factor 21 (FGF21). FGF21 is a multifunctional stress-inducible hormone and predominately express in liver. Ischemic injury could induce FGF21 secreted promptly from liver, pancreas, heart, kidney, skeletal muscles, and adipose tissue[4]. Recent research[4] indicated that FGF21 attenuated cell apoptosis to reduce I/R injury in liver, heart, and H9c2 cells via activating fibroblast growth factor receptor 1 (FGFR1). Our previous study[4] found FGF21 was strongly expressed in neuron and attenuated neuronal apoptosis in the penumbra after recanalization in middle cerebral artery occlusion (MCAO) rats. At the same time, we found the expression of FGF21 on astrocytes and endothelial cells significantly increased in the penumbra after recanalization. Previous studies[23-24] revealed that recombinant human FGF21 protected against BBB damage induced by traumatic brain injury or Type 2 diabetes by PPARγ activation or Nrf2 upregulation, respectively. In this study, we hypothesized that endogenous FGF21 expressed on astrocytes and endothelial cells following recanalization may contribute to attenuating BBB disruption in the penumbra and improving neurological outcomes through FGF21/FGFR1/PI3K/Akt pathway in MCAO rats.

1. Materials and methods

1.1. Animals

All experimental protocols were approved by the institution review board of Third Xiangya Hospital, Central South University (No. 2009-s000). One hundred and seventy adult male Sprague-Dawley rats (weighing 260-280 g) were used. All animals were housed in a standard environment [(22±1) ℃, humidity: (60±5)%] with regular light/dark cycle (12 h day/night cycle) and ad libitum feeding.

1.2. Construction of MCAO rat model

Construction of MCAO rat model was accord to previous studies[4]. Ketamine (80 mg/kg) and xylazine (10 mg/kg) were administrated to anesthetize rats by intraperitoneal injection. After the explosion of the common carotid artery, external carotid artery, and internal carotid artery, external carotid artery was ligated and a suture was inserted into the internal carotid artery to occlude the middle cerebral artery. The skin of incision was sutured. Rats recovered separately in 37 ℃ containers after the operation. Recanalization was performed on the 3rd day after MCAO by taking out the suture.

1.3. Study design

MCAO rats with modified Garcia score ranging from 6 to 9 were randomly assigned to a permanent MCAO (pMCAO) group or a recanalization MCAO (rMCAO) group following neurological function test on the 3rd day after MCAO.

1.3.1. Experiment I

Experiment I was the time course. Rats were divided into a sham group, a pMCAO 3 d group, a pMCAO 4 d group, and a pMCAO 7 d group (n=6). Western blotting was performed to identify the expression of FGF21, FGFR1, phosphorylated FGFR1 (p-FGFR1), Occludin, Claudin-5, and zona occludens 1 (ZO-1) in the penumbra.

1.3.2. Experiment II

Experiment II was performed to identify the colocalization of FGF21 and evaluate BBB disruption after MCAO and recanalization. Rats were divided into a sham group, a pMCAO group, and a rMCAO group. Immunofluorescence staining was used to identify the colocalization of FGF21 and FGFR1 with astrocytes and endothelial cells respectively on the 4th day after MCAO (n=3, 3 sections per slice were photographed). The extraversion of Evans blue was tested on the 4th day after MCAO and recanalization, as well as the extraversion of IgG and albumin (n=3, 3 sections per slice were photographed).

1.3.3. Experiment III

Experiment III was performed to explore the underlying mechanisms of FGF21 attenuating the BBB disruption after recanalization. Rats were divided into a sham group, a pMCAO group, a rMCAO group, a normal saline (NS) group, and a SU5402 group (n=6). SU5402 (a cell-permeable, reversible, and ATP-competitive inhibitor of the tyrosine kinase activity of FGFR1 by interacting with the catalytic domain of FGFR1) or NS was administered via intracerebroventricular injection 1 h before recanalization. Western blotting was performed on the 4th day (i.e. the 1st day after recanalization) after MCAO to identify the expression of FGF21, FGFR1, PI3K, Akt, Occludin, Claudin-5, and ZO-1 in the penumbra (n=6). Infarct volume, modified Garcia score, and beam walking score were used to evaluate the neurological outcome on the 7th day after MCAO (n=9).

1.4. Intracerebroventricular injection of NS or SU5402

Intracerebroventricular injection of NS or SU5402 was performed according to prior studied [25]. Following anesthetizing with 2.5% isoflurane, the rats were layed up stereotaxic apparatus. The injection site was 1.5 mm posterior to bregma, 0.9 mm right lateral to midline, and 3.3 mm in depth. The needle of Hamilton syringe (10 μL, Microliter 701; Hamilton Company, Reno, NV, USA) was planted into the injection site. NS or SU5402 (10 μL, 50 μmol/L, sc-204308, Santa Cruz Biotechnology Inc., Dallas, TX, USA) was injected 1 h before recanalization with a rate of 2 μL/min. The needle was withdrawn slowly at 5 min after injection to decrease leakage. Subsequently, the incision was sutured.

1.5. Assessment of cerebral infarct volume

Triphenyl tetrazolium chloride (TTC, Sigma-Aldrich, St. Louis, MO, USA) staining was only used to assess the infarct volume on the 4th, 7th, and 30th day after MCAO as previous described [26]. After anesthetizing and perfusing with 4 ℃ phosphate-buffered saline (PBS 0.1 mmol/L, pH 7.4, Sigma-Aldrich, St. Louis, MO, USA), rat brain was quickly sliced coronally with 2 mm thickness. Brain sections were immerged in 2% TTC for 15-20 min and then were taken pictures. ImageJ (ImageJ 1.4; NIH, Bethesda, MD, USA) was used to calculate the infarct area and ipsilateral hemisphere of every section. The cerebral edema after infarction was corrected by the strategy that whole contralateral hemispheric volume subtracted non-ischemic ipsilateral hemispheric volume. The ratio of the rectified infarct volume to contralateral hemispheric volume was used in statistically analysis.

1.6. Neurological score evaluation

Modified Garcia score, beam walking test, and Morris water maze were employed by 2 independent investigator blinded to the procedures as prior studies[27]. The modified Garcia score includes 6 items (such as spontaneous activity, symmetry in the movement of limbs, forepaw outstretching, climbing, body proprioception, and response to vibrissae touch), with a score ranging from 0 to 18. Beam walking test graded 0-5 point scales. The higher the score on both tests, the better the neurological behavior. Swim path, escape latency, swim distance, and the time spent in the platform-quadrant were assessed in Morris water maze to evaluate the spatial learning.

1.7. Evans blue extraversion after MCAO and recanalization

Evans blue dye was performed to evaluate brain vascular barrier permeability and TTC staining was performed to show infarct scope on the 4th day after MCAO as prior study[28]. After intravenous injection of 2% Evans blue solution for 1 h, rats were sacrificed following anesthetization with isoflurane. Then 0.1 mol/L PBS was trans-cardiac perfused and rat brain was quickly sliced coronally with 2 mm thickness. Brain sections were immerged in 2% TTC for 15-20 min and then taken pictures. Subsequently, all sections were rinsed in formamide overnight to extract the Evans blue dye. The supernatant was tested in a spectrophotometer at 620 nm.

1.8. Immunofluorescence staining

Following anesthetizing with 5% isoflurane and sequential trans-cardiac perfusion with 0.1 mol/L PBS and 10% formalin, the brain of rats was taken out and rinsed sequentially into 10% formalin (4 ℃, 24 h) and 30% sucrose solution (4 ℃, 3 d). Cryosections of rat brain (8 μm thickness) was used to perform double immunofluorescence staining as prior studies[26]. Brain sections were washed with PBS for 3 times (5 min each time) and were rinsed in 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at room temperature. Following washing with PBS for 3 times (5 min each time) again, brain sections were blocked with 5% donkey serum (566460, Sigma-Aldrich, St. Louis, MO, USA) over 2 h. Sections were incubate at 4 ℃ moist chamber overnight in corresponding primary antibodies: Rabbit anti-FGF21 (1꞉100, MBS2027242, MyBioSource, San Diego, CA, USA), rabbit anti-FGFR1 (1꞉100, ab10646, Abcam, Cambridge, MA, USA), rabbit anti-FGFR1 (phospho Y654, 1꞉200, ab59194, Abcam, Cambridge, MA, USA), mouse anti-glial fibrillary acidic protein (GFAP, 1꞉150, ab16997, Abcam, Cambridge, MA, USA), mouse anti-Lectin (1꞉200, ab177487, Abcam, Cambridge, MA, USA). Brain sections were incubated with appropriate fluorescence-conjugated secondary antibodies (1꞉150, Jackson Immuno- Research, West Grove, PA) for 2 h at room temperature. The sections were photographed on fluorescence microscope (Leica DMi8, Leica Microsystems, Wetzlar, Germany).

1.9. Western blotting analysis

After anesthetizing and 4 ℃ PBS perfusing, the contralateral and ipsilateral cerebrum of rats were frozen in liquid nitrogen quickly, and subsequently stored at -80 ℃. As previously described[27], brain tissues were homogenized in radio-immunoprecipitation assay (RIPA) lysis buffer (sc-24948A, Santa Cruz Biotechnology Inc., Dallas, TX, USA) according to the manufacturer’s recommendation and centrifuged for 20 min (14 000 g, 4 ℃). The protein concentration of supernatant was identified by spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The equal amount of protein were separated by electrophoresis on 10%-12% SDS-PAGE gel and then transferred to nitrocellulose membranes (0.2 μm). Subsequently, all membranes were blocked with 5% non-fat blocking milk in Tris-buffered saline with 0.1% Tween 20 (1706531, Bio-Rad, Hercules, CA, USA) and incubated overnight in primary antibodies: Rabbit anti-FGF21 (1꞉1 000), rabbit anti-FGFR1 (1꞉2 000), rabbit anti-FGFR1 (Phospho Y654, 1꞉2 000), rabbit anti-PI3K (1꞉2 000, 4292S, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Akt (1꞉1 000, ab179463, Abcam, Cambridge, MA, USA), rabbit anti-Akt (phosphor Y312+ Y315+Y316, 1꞉3 000, ab131443, Abcam, Cambridge, MA, USA), rabbit anti-Claudin-5 (1꞉3 000, ab131259, Abcam, Cambridge, MA, USA), rabbit anti-Occludin (1꞉1 500, ab216327, Abcam, Cambridge, MA, USA), and rabbit anti-ZO-1 (1꞉2 000, ab96587, Abcam, Cambridge, MA, USA). Antibody against β-actin (1꞉2 000, Santa Cruz Biotechnology Inc., Dallas, TX, USA) was used as an internal control. All membranes were immerged in respective secondary antibodies (Santa Cruz Biotechnology Inc., Dallas, TX, USA) for 1 h at room temperature. Immunoblots were probed with the ECL plus kit (RPN2232, Amersham Bioscience, Arlington Heights, IL, USA). Blot bands were quantified by densitometry with ImageJ software and all results were evaluated as relative density to β-actin.

1.10. Statistical analysis

All data were analyzed with GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA) and was expressed as mean±standard deviation ( x¯ ±s). Statistical difference among groups were analyzed with one-way or two-way analysis of variance (ANOVA) followed by multiple comparisons (Tukey or Sidak test). P<0.05 was considered a statistically significant difference.

2. Results

2.1. Animal mortality

The overall mortality after MCAO and recanalization in this study was 40.4% (59/146). Fifteen rats were excluded due to modified Garcia score ≤5 or ≥10 on the 3rd day after MCAO. Seventy-two rats with modified Garcia score ranging from 6 to 9 were randomly assigned into the pMCAO group or the rMCAO group. None of rats died in the sham group.

2.2. Time course

Protein samples were collected from rats on the 1st, 3rd, 4th, and 7th day after MCAO. No statistical difference was observed between the sham group and the MCAO group in respect to the expression of FGF21, FGFR1, and p-FGFR1 in the penumbra surrounding ischemic core after MCAO (all P<0.05). However, compared with the sham group, the expression of Occludin, Claudin-5, and ZO-1 significantly decreased in the penumbra on the 1st, 3rd, 4th, and 7th day after MCAO (all P<0.05, Figure 1).

Figure 1. Expression of FGF21, p-FGFR1, FGFR1, Claudin-5, Occludin, and ZO-1 after MCAO.

Figure 1

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A: Representative Western blotting images; B-F: Quantitative analyses of FGF21, p-FGFR1/FGFR1, Claudin-5, Occludin, and ZO-1 in the penumbra after MCAO (n=6), *P<0.05 vs Sham, †P<0.05 vs 1 d, ‡P<0.05 vs 3 d, §P<0.05 vs 4 d. FGF21: Fibroblast growth factor 21; FGFR1: Fibroblast growth factor receptor 1; p-FGFR1: Phosphorylated FGFR1; ZO-1: Zona occludens 1; MCAO: Middle cerebral artery occlusion.

2.3. Recanalization increased the expression of endogenous FGF21 on astrocytes and endothelial cells on the 4th day after MCAO

Double immunofluorescence staining showed the expression of FGF21 on astrocytes and endothelial cells surrounding ischemic core (in penumbra) on the 4th day after MCAO (i.e. 1 d after recanalization) between the sham group and the pMCAO group had no statistic difference (all P>0.05). However, compared with the sham group and the pMCAO group, the number of FGF21-positive astrocyte and the relative ratio of FGF21 in endothelial cells significantly increased surrounding ischemic core in the rMCAO group (P<0.05, Figure 2).

Figure 2. Recanalization increased the expression of FGF21 on astrocytes and endothelial cells on the 4th day after MCAO.

Figure 2

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A and B: Representative microphotographs of FGF21 (Green) colocalized with astrocytes (GFAP, Red) and endothelial cells (Lectin, Red). DAPI (Blue) marked nuclei. C: Samples were obtained from ischemic penumbra. D and E: Quantitative analyses of FGF21-positive astrocytes and relative ratio of FGF21 on endothelial cells (n=3), *P<0.05 vs Sham, †P<0.05 vs pMCAO. pMCAO: Permanent middle cerebral artery occlusion (MCAO) group; rMCAO: Recanalization MCAO group; FGF21: Fibroblast growth factor 21; GFAP: Glial fibrillary acidic protein; DAPI: 4',6-Diamidino-2-phenylindole.

2.4. Recanalization increased the expression of p-FGFR1 on astrocytes and endothelial cells on the 4th day after MCAO

No statistical difference was observed between the sham group and the pMCAO group with respect to the expression of p-FGFR1 on astrocytes and endothelial cells surrounding ischemic core on the 4th day after MCAO. However, compared with the sham group and the pMCAO group, the number of FGFR1-positive astrocyte and the relative ratio of p-FGFR1 on endothelial cells significantly increased surrounding ischemic core in the rMCAO group (P<0.05, Figure 3).

Figure 3. Recanalization increased the expression of p-FGFR1 on astrocytes and endothelial cells on the 4th day after MCAO.

Figure 3

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A and B: Representative microphotographs of p-FGFR1 (Green) colocalized with astrocytes (GFAP, Red) and endothelial cells (Lectin, Red). DAPI (Blue) marked nuclei. C: Samples were obtained from ischemic penumbra. D and E: Quantitative analyses of p-FGFR1-positive astrocytes and relative ratio of p-FGFR1 on endothelial cells (n=3), *P<0.05 vs Sham, †P<0.05 vs pMCAO. pMCAO: Permanent middle cerebral artery occlusion (MCAO) group; rMCAO: Recanalization MCAO group; p-FGFR1: Phosphorylated fibroblast growth factor receptor 1; GFAP: Glial fibrillary acidic protein; DAPI: 4',6-diamidino-2-phenylindole.

2.5. Evans blue extraversion after MCAO and recanalization

Compared with the sham group and the rMCAO group, Evans blue extraversion increased surrounding ischemic core in the pMCAO group. Following recanalization, Evans blue extraversion decreased significantly surrounding ischemic core in the rMCAO group, however, which increased significantly in ischemic core after recanalization/reperfusion compared with the pMCAO group (P<0.05, Figure 4).

Figure 4. Evans blue (EB) extraversion after MCAO and recanalization.

Figure 4

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A: Representative images of EB dyed and triphenyl tetrazolium chloride (TTC) stained brain slices; B: EB extraversion in ischemia; C: EB extraversion in the penumbra (n=6), *P<0.05 vs Sham, †P<0.05 vs pMCAO. pMCAO: Permanent middle cerebral artery occlusion (MCAO) group; rMCAO: Recanalization MCAO group.

2.6. Extraversion of IgG and albumin after MCAO and recanalization

Double immunofluorescence staining showed that compared with the sham group, the extraversion of IgG and albumin increased surrounding ischemic core in the pMCAO group. Following recanalization, the extraversion significantly decreased surrounding ischemic core in the rMCAO group (P<0.05, Figure 5).

Figure 5. Extraversion of IgG and albumin after MCAO and recanalization.

Figure 5

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A and B: Representative microphotographs of IgG and albumin (Green) colocalized with endothelial cells (Lectin, Red). DAPI (Blue) marked nuclei. C: Samples were obtained from ischemic penumbra. D and E: Quantitative analyses of relative ratio of IgG and albumin (n=3), *P<0.05 vs Sham, †P<0.05 vs pMCAO. pMCAO: Permanent middle cerebral artery occlusion (MCAO) group; rMCAO: Recanalization MCAO group.

2.7. SU5402 weakened the effects of recanalization on neurological outcomes on the 7th day after MCAO

Following recanalization, the infarct volume decreased, and modified Garcia scores and beam walking scores enhanced in the rMCAO group on the 4th day after MCAO compared with the pMCAO group (all P<0.05). However, as well the infarct enlarged, compared with the rMCAO group and the NS group, modified Garcia scores and beam walking scores significantly reduced in the SU5402 group (all P<0.05, Figure 6). No statistical differences were observed in the infarct volume, modified Garcia scores, and beam walking scores between the rMCAO group and the NS group (all P>0.05).

Figure 6. SU5402 weakened the effects of recanalization on neurological outcomes on the 7th day after MCAO.

Figure 6

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A: Representative images of triphenyl tetrazolium chloride (TTC) stained brain slices. TTC staining was only used to calculate infarct volume. B: Quantified infarct volume. C: Modified Garcia score. D: Beam walking score (n=6), *P<0.05 vs Sham, †P<0.05 vs pMCAO, ‡P<0.05 vs rMCAO, §P<0.05 vs NS. pMCAO: Permanent middle cerebral artery occlusion (MCAO) group; rMCAO: Recanalization MCAO group; NS: Normal saline group.

2.8. Endogenous FGF21 attenuated BBB disruption surrounding ischemic core through FGFR1/PI3K/Akt pathway on the 4th day after MCAO

Western blotting results showed that Occludin, Claudin-5, and ZO-1 significantly decreased surrounding ischemic core (in penumbra) after MCAO, compared with the sham group (all P<0.05, Figure 7A, 7F-7H). No statistic difference was observed between the pMCAO group and the sham group in regard to the expression of FGF21, p-FGFR1, PI3K, and p-Akt (all P>0.05, Figure 7A-7E). After the recanalization, the expression FGF21, p-FGFR1, PI3K, p-Akt, Occludin, Claudin-5, and ZO-1 significantly increased in the rMCAO group compared with the pMCAO group (all P<0.05, Figure 7A-7H). There were no statistical differences between the rMCAO group and the NS group in regard to the expression of FGF21, p-FGFR1, PI3K, p-Akt, Occludin, Claudin-5, and ZO-1 (all P>0.05). However, after the administration of SU5402, the expressions of PI3K, p-Akt, Occludin, Claudin-5, and ZO-1 significantly decreased in the SU5402 group compared with the rMCAO group (all P<0.05, Figure 7A, 7D-7H).

Figure 7. Effects of FGF21 on protecting BBB through FGFR1/PI3K/Akt pathway in penumbra on the 4th day after MCAO.

Figure 7

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A: Representative Western blotting images; B-H: Quantitative analyses of FGF21, p-FGFR1, PI3K, p-Akt, Occludin, Claudin-5, and ZO-1 (n=6), *P<0.05 vs Sham, †P<0.05 vs pMCAO, ‡P<0.05 vs rMCAO, §P<0.05 vs NS. BBB: Blood-brain barrier; pMCAO: Permanent middle cerebral artery occlusion (MCAO) group; rMCAO: Recanalization MCAO group; NS: Normal saline group; Scr siRNA: Scrambled siRNA; FGF21: Fibroblast growth factor 21; FGFR1: Fibroblast growth factor receptor 1; p-FGFR1: Phosphorylated FGFR1; PI3K: Phosphatidylinositol-4,5-bisphosphate 3-kinase; ZO-1: Zona occludens 1.

3. Discussion

In this study, we investigated BBB disruption surrounding ischemic core (in penumbra) after delayed recanalization in pMCAO rats and revealed the potential mechanism of FGF21 protecting BBB. We demonstrated that delayed recanalization after MCAO decreased BBB disruption surrounding ischemic core. Meanwhile, we found that the delayed recanalization contributed to enhancing endogenous FGF21 expression on astrocytes and endothelial cells surrounding ischemic core after MCAO, which phosphorylated FGFR1 and activated PI3K/Akt signaling pathway to attenuate BBB disruption surrounding ischemic core after recanalization. Conversely, the administration of FGFR1 inhibitor SU5402 down-regulated the expression of PI3K, p-Akt, Occludin, Claudin-5, and ZO-1 and abolished the protecting effect of FGF21 on BBB.

Restoration of blood circulation (recanalization and reperfusion) and resupply of oxygen and glucose in “time window” are the principal treating goal of acute ischemic stroke. A number of previous researches revealed that delayed recanalization (exceeding “time window”) might result in serious reperfusion injury, which might aggravate neurological outcomes[6]. The underlying mechanisms of I/R injury involve in BBB disruption, mitochondrial dysregulation, oxidative stress, ROS, calcium overload, inflammation, free radicals, excitotoxicity, and neuronal apoptosis, etc[18, 20, 29]. BBB is a complex physical and metabolic barrier between brain and blood circulation system, which consists of astrocytes, endothelial cells, pericytes, microglia, and a number of transmembrane proteins[30-32]. BBB protects the cerebral micro-environment by regulating paracellular permeability and preventing against penetration of pathogens, noxious chemicals, and metabolic products into cerebral tissues from blood circulation[31, 33-34]. The disassembly of tight junction complex leads to disruption of BBB integrity in acute ischemic stroke, and the following I/R may exacerbate this crucial pathophysiological process. Therefore, the tight junction proteins, such as Claudin-5, Occludin, and ZO-1, decreased in ischemic core[35-38]. In present study[4], we found that the expression of Claudin-5, Occludin, and ZO-1 decreased surrounding ischemic core after MCAO. At the same time, the extraversion of Evans blue, IgG, and albumin increased surrounding ischemic core after MCAO. These results suggested that BBB integrity disrupted and paracellular permeability increased surrounding ischemic core after MCAO.

Base on the theory of I/R injury, damaged BBB integrity may aggravate the neurological outcomes after delayed recanalization. However, our previous study[4] showed that delayed recanalization (surgery on the 3rd day after MCAO) reduced infarct volume, increased the Modified Garcia scores and Beam Walking scores by attenuating neuronal apoptosis in the penumbra, and improved the performances in Morris Water Maze compared to permanent MCAO rats. In this study, we found the expression of Claudin-5, Occludin, and ZO-1 increased surrounding ischemic core after recanalization. The extraversion of Evans blue, IgG, and albumin decreased surrounding ischemic core after recanalization (the Evans blue extraversion increased in ischemic core at the same time). These results suggested that the delayed recanalization partly restored the BBB integrity and paracellular permeability surrounding ischemic core after MCAO, which might contribute to salvaging the cerebral tissues and improving the neurological outcomes after MCAO.

Increasing clinical evidences have showed that the delayed recanalization and reperfusion after “time window” improved neurological outcomes in patients with acute ischemic stroke. Spontaneous recanalization may occur in 21.4% MCAO patients within 24 h after stroke onset and in 52.7% MCAO patients after a week, usually associated with improving long-term disability[12-13, 39]. As it well known that endovascular thrombectomy in 6-24 h after stroke onset contribute to favorable clinical outcomes in selected patients with acute ischemic stroke[7-8]. Another clinical trial[9-10] also showed that the administration of alteplase beyond 4.5 h “time window” (4.5-9.0 h from ischemic stroke onset or wake-up stroke) improved the neurological outcomes in patients with salvageable brain tissues. Additionally, the administration of tenecteplase in acute ischemic stroke patients with evidence of a penumbral tissue 4-24 h after onset received clinical improvement[11]. However, all these evidences did not reveal the underlying molecular mechanism. Basing on the findings in our previous study[4], we suggested that the reperfusion following delay recanalization partly restored BBB integrity surrounding ischemic core, which might be one of the leading causes of improving neurological outcome after delayed recanalization in patients with acute ischemic stroke.

FGF21 is a multifunctional metabolic stress hormone and robustly secretes from liver, adipose tissue, pancreas, kidney, skeletal muscles, and cardiac muscles to serum after acute ischemic stroke[40]. FGF21 is capable to attenuate I/R injury in liver, heart, and H9c2 cells[40-41].In MCAO models, the expression of FGF21 significantly decreased in ischemic core[41].In our previous study[4], we revealed that endogenous FGF21 significantly decreased in the penumbra, although which increased in serum after MCAO. Recanalization (3 d after MCAO) increased the expression of FGF21 in the penumbra and contributed to attenuating neuronal apoptosis by activating FGFR1/PI3K/caspase-3 signaling pathway, which improved the neurological function in MCAO rats. In this study, double immunofluorescence staining showed that FGF21 robustly expressed on astrocytes and endothelial cells surrounding ischemic core after the recanalization. At the same time, the results showed that the recanalization restored the BBB integrity surrounding ischemic core after MCAO (the extraversion of Evans blue, IgG, and albumin decreased). To investigate the potential mechanism of FGF21 protecting BBB after recanalization, Western blottings were performed on the 4th day after MCAO (1 d after recanalization). The results indicated that the tight junction proteins, such as Claudin-5, Occludin, and ZO-1, decreased in the penumbra before recanalization in MCAO rats. Following recanalization, corresponding to the increase of FGF21, p-FGFR1, PI3K, and p-Akt, the expression of Claudin-5, Occludin, and ZO-1 significantly increased surrounding ischemic core. After intracerebroventricular injection of FGFR1 inhibitor SU5402, the expression of PI3K down-regulated, followed by p-Akt, Occludin, Claudin-5, and ZO-1 decreased compared with the NS group and the rMCAO group. The beneficial effects of recanalization on neurological outcomes after MCAO decreased correspondingly (the infarct volume increased, modified Garcia scores and Beam walking scores decreased compared with the NS group and the rMCAO group). The findings suggested that the up-regulation of FGF21 following recanalization attenuates BBB disruption surrounding ischemic core via FGFR1/PI3K/Akt signaling pathway, which improves the neurological functions in MCAO rats.

In conclusion, our study revealed that delayed recanalization on the 3rd day after MCAO increases endogenous FGF21 expression on astrocytes and endothelial cells surrounding ischemic core. FGF21 phosphorylates FGFR1 and activates PI3K/Akt signaling pathway, which attenuates BBB disruption and improves the neurobehavior outcomes in MCAO rats. Delayed recanalization on the 3rd day after MCAO may be a selectable treatment strategy in a part of patients with acute ischemic stroke.

Funding Statement

This work was supported by the Natural Science Foundation of Hunan Province (2020JJ4865 and S2021JJMSXM2689) and the Hunan Provincial Clinical Medical Technology Innovation Guidance Project (2020SK53612), China.

Conflict of Interest

The authors declare that they have no conflicts of interest to disclose.

AUTHORS’CONTRIBUTIONS

ZHENG Wen, LI Wenjun Manuscript writing and revision; ZENG Yini, YUAN Hui, YANG Heng, ZHU Anding, WU Jinze Collected data; CHEN Ru, YAN Wenguang Manuscript design, guidance, and revision; SONG Zhi Paper supervision. All authors read and approved the final manuscript.

Note

http://xbyxb.csu.edu.cn/xbwk/fileup/PDF/202305648.pdf

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