Aggf1 attenuates neuroinflammation and BBB disruption via PI3K/Akt/NF-κB pathway after subarachnoid hemorrhage in rats

All procedures and protocols for this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Loma Linda University in accordance with the guideline for the Care and Use of Laboratory Animals by National Institutes of Health. Two hundred forty-nine male Sprague-Dawley rats (Indianapolis, IN, USA) weighing 300–320 g were used, and they were housed in a light- and temperature-controlled room with unlimited access to food and water.

The endovascular perforation model was induced as previously described [20]. Briefly, rats were intubated and maintained with 3% isoflurane anesthesia in the air. Rodents were placed in a supine position, and the neck was opened with a sharp scalpel in the midline. After localization of the appropriate vessels, a sharpened 3-cm, 4–0 nylon suture was inserted into the left internal carotid artery through the external carotid artery and the common carotid bifurcation. The suture was advanced until resistance was reached, further advanced in order to puncture the vessel, and then immediately withdrawn after artery perforation. The sham-operated group underwent the same procedure without an endovascular puncture. After removal of the suture, the skin incision was sutured and the rats were housed individually in heated cages until recovery.

Animals were randomly assigned to five separate experiments as described (Fig. 1). The experimental group information was blinded to the surgeon and researchers who performed the neurobehavioral tests and Western blot/immunofluorescence as well as data analysis.

The assessment of SAH grading score was performed at 24 h after SAH by an independent investigator blinded to the experimental group information as previously described [21]. Briefly, the basal cistern was divided into six segments that were scored from 0 to 3 according to the amounts of subarachnoid blood. Rats with the grade < 8 at 24 h after SAH were excluded from this study.

The neurological status was assessed at 24 h after SAH using modified Garcia and beam balance as previously described [22]. The modified Garcia test (maximum score = 18) included vibrissae touch, trunk touch, spontaneous activity, spontaneous movement of the four limbs, forelimbs outstretching, and climbing capacity. The beam balance test (maximum score = 4) was conducted to assess the ability of rats to walk on a wooden beam for 1 min. High scores indicated better neurological function. The neurological assessment was performed by an investigator blind to experiment.

The foot-fault test was measured to assess the motor-sensory deficits by a partner blinded to the group information as previously described [23]. Briefly, rats were placed on a horizontal grid floor for 2 min. The foot-fault was defined as when the rat incorrectly placed a fore- or hindlimb, and it fell through one of the openings in the grid. The number of foot-fault was recorded and used for the statistical analysis.

The Morris water maze was performed to assess the spatial learning memory by an independent researcher blinded to the experimental group information as previously described [24, 25]. Briefly, rats were placed using a semi-random set of start locations to find a visible platform above the water level in 60 s. After that, the rats were guided to stay on the platform for 5 s. At the last day, the probe trial was performed in which the rats were allowed to swim to search the platform submerged in the water. Swim path, swim distance, escape latency, and probe quadrant duration were recorded by a computerized tracking system (Noldus Ethovision; Noldus, Tacoma, WA, USA).

Brain samples were collected at 24 h after surgery and separated into the left hemisphere, right hemisphere, cerebellum, and brain stem. Each part was weighed immediately after removal (wet weight) and then dried in an oven at 105 °C for 72 h (dry weight). After that, the percentage of brain water content was calculated as [(wet weight − dry weight)/wet weight] × 100%.

BBB permeability was evaluated by EB extravasation using spectrophotometry as previously described [20]. A 2% solution of EB in normal saline (5 ml/kg of body weight) was injected into the right femoral vein at 24 h after SAH. The stain was allowed to circulate for an additional 60 min before sacrifice. Then the rats were transcardially perfused with 120 ml of ice-cold PBS in deep anesthesia. The collected brain samples were subsequently removed and divided into four parts: right hemisphere, left hemispheres, cerebellar, and brain stem and stored in a − 80 °C freezer. After homogenization of each sample with PBS, the solution was centrifuged for 30 min at 14,000 r/min in 4 °C. The supernatant was collected, mixed with equal amount of trichloroacetic acid, and incubated at room temperature for 1 h. Then, the sample was centrifuged for 30 min at 15,000 r/min in 4 °C to separate the supernatant for measurements. EB stain results were measured by a spectrophotometer (Thermo Spectronic Genesys 10 UV, Thermo Fischer Scientific Inc., Waltham, MA, USA) at 610 nm and quantified with a standard curve. The results are presented as fold increase compared to sham group.

Intracerebroventricular drug administration was performed as previously described [24]. Briefly, rats were placed in a stereotaxic apparatus under anesthesia with 2.5% isoflurane in 70/30% medical air/oxygen. The needle of a 10-μl Hamilton syringe (Microliter701; Hamilton Company, Reno, NV, USA) was inserted into the right lateral ventricle through a burr hole using the following coordinates relative to bregma: 1.5 mm posterior, 0.9 mm lateral, and 3.3 mm below the horizontal plane of the bregma. For Aggf1 in vivo knockdown, two different rat Aggf1 siRNA duplexes (Thermo Fisher Scientific, Waltham, MA) (total 500 pmol) were dissolved in RNase free suspension buffer and then infused into the right lateral ventricle via a pump with the rate 1 μl/min at 48 h before SAH induction. The same volume of Scr siRNA (Thermo Fisher Scientific, Waltham, MA) was used as a negative control. PI3K-specific inhibitor LY294002 (Selleck Chemicals, Houston, USA) was prepared at 50 mmol/l in PBS (contains 25% DMSO) with a total volume 5 μl. ICV injection was performed. The same volume of DMSO was used as a negative control. After injection, the needle was kept in place for an additional 5 min and retracted slowly. Then, the burr hole was sealed with bone wax immediately, and the rats were allowed to recover after sutures.

Western blot analysis was performed as previously described [26]. After sample preparation, equal amounts of a sample protein (50 μg) were loaded onto an SDS-PAGE gel. First, electrophoresis and transfer of the samples to a nitrocellulose membrane were performed. Second, the membrane was blocked for 2 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: anti-Aggf1 (1:2000, Sigma-Aldrich, USA), anti-albumin (1:5000, Abcam, USA), anti-Claudin-5 (1:1000, Abcam, USA), anti-myeloperoxidase (MPO, 1:1000, Abcam, USA), anti-Occludin (1:2000, Abcam, USA), anti-PI3K (1:1000, Cell signaling, USA), anti-Akt (1:1000, Cell signaling, USA), anti-phospho-Akt (p-Akt, 1:1000, Cell signaling, USA), anti-NF-κB p65 (1:500, Santa Cruz, USA), anti-phospho-NF-κB p65 (p-NF-κB p65, 1:500, Santa Cruz, USA), anti-VE-cadherin (1:500, Santa Cruz, USA), anti-IL-1β (1:300, Santa Cruz, USA), anti-TNF-a (1:300, Santa Cruz, USA), and anti-β-actin (1:5000, Santa Cruz, USA). Appropriate secondary antibodies (1:5000, Santa Cruz, USA) were selected to incubate with the membrane for 2 h at room temperature. Then, blot bands were visualized with an ECL reagent (Amersham Biosciences UK Ltd., PA, USA). Non-saturated bands were selected to perform densitometry quantification using Image J software (Image J 1.4, NIH, USA).

Double fluorescence staining was conducted as described previously [27]. The rats were deeply anesthetized at 24 h post-SAH and were transcardially perfused with 60-ml ice-cold PBS followed by 60 ml of 10% paraformaldehyde. The whole brains were collected and then fixed in 10% paraformaldehyde for 24 h followed by 30% sucrose solution for another 72 h. After being frozen at − 80 °C, the brain was cut into 10-μm-thick coronal sections using a cryostat (LM3050S; Leica Microsystems, Bannockburn, III, Germany). To perform double immunohistochemistry staining, the brain sections were incubated with primary antibody of anti-neuronal nuclei (NeuN, 1:200, Abcam, USA), anti-glial fibrillary acidic protein (GFAP, 1:500, Abcam, USA), anti-ionized calcium-binding adaptor molecule 1 (Iba-1, 1:200, Abcam, USA), anti-von Willebrand factor (vWF, 1:100, FITC, Abcam, USA), anti-myeloperoxidase (MPO, 1:200, Santa Cruz, USA), and anti-Aggf1 (1:100, Sigma-Aldrich, USA) overnight at 4 °C. After being incubated with the appropriate secondary antibody (1:200, Jackson Immunoresearch, West Grove, PA, USA) at room temperature for 2 h, the sections were visualized and photographed with a fluorescence microscope (Leica Microsystems, Germany). Microphotographs were analyzed with LASX software. The numbers of Iba-1-positive cells and MPO-positive cells were identified and counted in three different fields in the ipsilateral cortex from five random coronal sections per brain, and data were expressed as cells/field [22].

The overall mortality of SAH rats was 16.36% (35/214); no rats died in the sham group. According to the SAH grading score, 13 rats were excluded from this study due to low-grade SAH (Additional file 1: Table S1). Subarachnoid blood clots were markedly shown around the circle of Willis (Additional file 2: Figure S1A). There was no statistical difference in SAH grading scores among the SAH groups (Additional file 2: Figure S1B).

The expression of endogenous Aggf1 in the ipsilateral (left) cerebral cortex was assessed by Western blots. As shown in Fig. 2a, there was a significant increase of Aggf1 level at 24 h, which peaked at 72 h after SAH when compared to the sham group. Double immunofluorescence staining revealed that Aggf1 was mainly expressed in the endothelial cells and astrocytes, as well as microglia in the ipsilateral basal cortex at 24 h after SAH (Fig. 2b).

The neurological scores of modified Garcia and beam balance were significantly reduced at 24 h after SAH in the SAH+vehicle and SAH+rh-Aggf1 (1 μg/rat) groups. However, administration of rh-Aggf1 (3 μg/rat) and rh-Aggf1 (9 μg/rat) significantly improved the neurological deficits (Fig. 3a). SAH induction significantly increased the brain edema of both hemispheres in the SAH+vehicle and SAH+rh-Aggf1 (1 μg/rat) groups compared to the sham group at 24 h after SAH (Fig. 3b). Administration of rh-Aggf1 (3 μg/rat) and rh-Aggf1 (9 μg/rat) considerably reduced brain edema (Fig. 3b). Based on these results, the optimal dose of rh-Aggf1 was 3 μg/rat, which was used for the rest of the experiments.

BBB permeability was evaluated by EB extravasation in both cerebral hemispheres. Although EB extravasation in the SAH+vehicle group was markedly increased at 24 h after SAH, rh-Aggf1 treatment (3 μg/rat) significantly decreased EB dye leakage in both hemispheres compared with the SAH+vehicle group (Fig. 3c).

MPO activity, an indicator of neutrophil accumulation, was evaluated in our study. As shown in Fig. 4a, significant increase of neutrophil accumulation was observed in the SAH+vehicle group. However, treatment with rh-Aggf1 significantly reduced the extent of neutrophil accumulation. In addition, the expression of TNF-α and IL-1β were increased in the SAH+vehicle group, while administration of exogenous rh-Aggf1 inhibited TNF-α and IL-1β expression (Fig. 4b, c).

The rats in the SAH+vehicle group showed significant neurological deficits in foot-faults both in the left forelimb and right forelimb than in the sham group at days 7, 14, and 21 after SAH. However, administration of rh-Aggf1 significantly improved the neurological deficits in foot-fault test both in the left forelimb and right forelimb (Fig. 5a). In the water maze test, the travel distance and escape latency for the rats to find the platform were significantly increased in the SAH+vehicle group when compared to the sham group. However, a significantly shorter distance on block 2, 3, 4, and 5 and a decrease in time to find the platform at days 22, 23, 24, and 25 were observed in the SAH+rh-Aggf1 group (Fig. 5b). In probe quadrant trial, the rats in the SAH+vehicle group spent remarkably less time in the target quadrant when compared to the sham group, while the reference memory deficits were significantly improved with rh-Aggf1 treatment (Fig. 5b). There was no significant difference in swimming velocity among all three groups.

To further verify the protective role of Aggf1 post-SAH, Aggf1 siRNA was injected i.c.v. in order to silence endogenous Aggf1. Western blot showed that the Aggf1 expression was inhibited by Aggf1 siRNA at 72 h after injection (Fig. 6a). The knockdown of Aggf1 exacerbated neurological deficits (Fig. 6b) and increased brain edema (Fig. 6c) and BBB permeability (Fig. 6d) at 24 h post-SAH. Furthermore, the knockdown of Aggf1 increased MPO expression (Fig. 6e).

SAH decreased the expression of PI3K and p-Akt compared with the sham group, whereas rh-Aggf1 significantly increased the levels of PI3K, p-Akt, Occludin, Claudin-5, and VE-cadherin and decreased the expression of p-NF-κB p65, IL-1β, and TNF-α. By contrast, knockdown of Aggf1 using Aggf1 siRNA got opposite changes on the expression of downstream signaling molecules compared with the rh-Aggf1 treatment group (Fig. 7a–i).

We assessed whether the anti-inflammatory effect of rh-Aggf1 was related to a reduction of microglia activation in the ipsilateral cortex. As shown in Fig. 8, the numbers of Iba-1-positive cells were significantly increased in the ipsilateral cortex in the SAH+vehicle group at 24 h after SAH, and activated microglia showed shorter processes. Administration of rh-Aggf1 dramatically reduced the number of Iba-1-positive cells, while this effect was reversed by PI3K-specific inhibitor LY294002 (Fig. 8).

As shown in Fig. 9, LY294002 significantly abolished the protective effects of rh-Aggf1 on neurological functions (Fig. 9a) and brain edema (Fig. 9b) at 24 h after SAH. Consistently, LY294002 reversed the protection of rh-Aggf1 treatment on neuroinflammation and BBB integrity, as the significant increase in albumin leakage and MPO expression was observed at 24 h after SAH (Fig. 9c–e). Moreover, pretreatment with LY294002 sufficiently decreased the expression of p-Akt, VE-cadherin, Occludin, and Claudin-5 with an increase in p-NF-κB p65, TNF-α, and IL-1β expression (Fig. 9c, f–l).