Roles of Rufy3 in experimental subarachnoid hemorrhage-induced early brain injury via accelerating neuronal axon repair and synaptic plasticity

Experimental animals

A total of 460 8-week-old clean-grade adult male SD rats (weight: 300–350 g) were provided by Suzhou Zhaoyan New Drug Research Center Co., Ltd (Suzhou, China). The rats were reared at constant temperature (18–26 °C) and humidity (40–70%). The animal experiments were authorized, approved, and supervised by the Animal Ethics Committee of the First Affiliated Hospital of Soochow University, China. The animals were raised and used in strict accordance with the National Institutes of Health guidelines.

Neuron culture

As previously mentioned [22], we isolated and cultured primary neurons from the rat embryos on day 18. First, we separated and wiped the capillaries and meninges attached to the surface of the hemisphere. Then, the brain tissue was percussed and digested repeatedly after addition to 0.25% trypsin for 5 min. Then, the mixed liquid was centrifuged at 1000 rpm for 5 min. The deposit was added to neurobasal medium containing with 2% B27, 0.5 mM GlutaMAX TM-1, 50 U/ml penicillin and 50 U/ml streptomycin and mixed. Finally, neurons were inoculated into culture dishes, and 6-well or 12-well plates were precoated with poly-d-lysine (Sigma-Aldrich, St. Louis, MO, USA) containing fresh neurobasal medium at a density of 20,000 cells/cm2. The culture was kept in a 5% CO2 and 37 °C atmospheric incubator for 5 days. The corresponding lentivirus and enhanced infection enhancer were intervened simultaneously. Next, we incubated the neurons after oxyhemoglobin (OxyHb) stimulation under the same environment for 24 h. Finally, medium was siphoned from the plates, and primary neurons were scratched or fixed with 4% paraformaldehyde, after which relevant experiments were conducted.

SAH model

We established the SAH animal models using a stereotactic injection of autologous blood into the optic chiasm cistern, as described in our previous study [23]. After anesthesia with an intraperitoneal injection of 4% chloral hydrate (1 ml/100 g) (Sigma-Aldrich, St. Louis, MO, USA), the rats were fixed in a stereotaxic apparatus and a side needle (i.e., a needle with a round tip and a side hole located at the bottom) was stereoscopically inserted into the anterior skull base through a hole drilled in the skull. The skull was entered at the anterior midline of the sagittal point 7.5 mm away from the anterior fontanelle and the puncture direction was 45° to the coronal plane. The syringe was advanced until the tip reached the bottom of the skull and was then retracted by 0.5 mm. Then, a syringe pump was used to inject slowly 300 μl of fresh unheparinized autologous blood into the prechiasmatic cistern over 20 s. In the sham group, 300 μl of physiological saline was injected instead of blood. Twenty-four hours after SAH, 60 mL of ice-cold PBS was injected into the hearts of deeply anesthetized animals. The brain tissue of rats covered by blood clots was obtained for analysis (Fig. 1a).

Fig. 1figure 1

Experimental design. a Schematic representation of the areas selected in our study. b Experiment one was designed to determine the effect of Rufy3 on neuronal axons. c Experiment two was designed to determine the involvement of the Rufy3 in EBI under vivo SAH conditions. d Experiment three was designed to determine the involvement of neuroprotection through the Rufy3/Rap1 complex formation via accelerating neuronal axon repair and synaptic plasticity. e Experiment four was designed to evaluate the effect of Rufy3 expression on neurocognitive function

Experimental design and intervention

Experiment one was designed to explore the roles of Rufy3 in SAH in vitro. OxyHb was used to stimulate primary cultured neurons, and lentivirus-negative control 1 (LV-NC1), lentivirus-Rufy3 shRNA (LV-shRNA), lentivirus-negative control 2 (LV-NC2), and lentivirus-Rufy3 (LV-Rufy3) were used as the interventions. Cells were divided into seven groups: Control, OxyHb, OxyHb + HA, OxyHb + HA + LV-NC1, OxyHb + HA + LV-shRNA, OxyHb + HA + LV-NC2, and OxyHb + HA + LV-Rufy3 groups. The neurons received LV-NC1, LV-shRNA, LV-NC2, and LV-Rufy3 treatments at 24 h before OxyHb stimulation. The cells were lysed or fixed for western blot and immunofluorescence (IF) detection respectively. The experimental process is depicted in Fig. 1b. Experiment two was designed to determine the involvement of Rufy3 in EBI after SAH. In particular, 42 rats were assigned to seven point-in-time groups (n = 6 in each group): the sham group, the 6-, 12-, 24-, 48-, and 72-h SAH groups, or the 1-week SAH group. Brain tissue below the blood clot was obtained at different time points from SAH rats for western blot, reverse transcription-polymerase chain reaction (RT-PCR), and IF analysis. The experimental process is depicted in Fig. 1c. Experiment three was designed to examine the mechanisms underlying brain injury due to Rufy3 in EBI induced by SAH. Rats received intracerebroventricular injections of LV-NC1, LV-shRNA, LV-NC2, and LV-Rufy3 7 days before the blood injection and an intraperitoneal injection of 8-pCPT-2′-O-Me-cAMP (8p-CPT) 6 h after SAH. In total, 216 rats were randomly divided into nine groups (n = 24 in each): sham, SAH, SAH + LV-NC1, SAH + LV-shRNA, SAH + LV-NC2, SAH + LV-Rufy3, SAH + 8p-CPT, SAH + 8p-CPT + LV-shRNA, and SAH + 8p-CPT + LV-Rufy3 groups. First, brain tissue samples from 12 rats in each group were cut into slices for IF, Fluoro-Jade C (FJC), and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Next, brain tissue samples of the remaining 12 rats in each group were euthanized, perfused, and collected for western blot assay, immunoprecipitation (IP), Rap1 activation assay, and brain edema evaluation. The experimental process is depicted in Fig. 1d. Experiment four was designed to assess the effect on cognitive and motor disorders by regulating Rufy3 expression. In total, we randomly divided 108 adult male rats into eight groups (n = 12 in each), including sham, SAH, SAH + LV-NC1, SAH + LV-shRNA, SAH + LV-NC2, SAH + LV-Rufy3, SAH + 8p-CPT, SAH + 8p-CPT + LV-shRNA, and SAH + 8p-CPT + LV-Rufy3 groups. Neurological scores, rotarod test, adhesive-removal test, and Morris water maze were conducted on rats from different groups (Fig. 1e).

Lentiviral construction and in vivo injection

The expression of Rufy3 was downregulated by transfection with lentiviral vectors expressing Rufy3-specific shRNA. Three lentiviral Rufy3 shRNAs (83759-1, 83580-1, and 83578-1) and a negative control virus (LVCON313, LV-NC1) were purchased from Genescript (Nanjing, China). To establish and maintain overexpression of Rufy3, a lentiviral vector of LV-Rufy3-overexpression was designed, synthesized, and constructed by Genescript. In addition, a corresponding negative control virus (LVCON55, LV-NC2) was created. The sequence elements of the lentiviral vectors were Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin (LV-Rufy3) and hU6-MCS-CBh-gcGFP-IRES-puromycin (LV-shRNA). The viral titers of LV-shRNA-Rufy3 and LV-Rufy3 were 4 × 108 and 5 × 108 TU/ml, respectively. In vitro SAH, primary neurons were transfected with the corresponding LV using 20 μl HA (HitransGA) (GeneChem) to enhance the transduction efficiency after 4 days of extraction and culture. We calculated the virus usage based on the following formula: virus volume = MOI * cell count/virus titer (multiplicity of infection [MOI] = 10). Two days after the transduction, neurons were added to OxyHb (10 μM). The experimental rat SAH model was established 7 days after lentiviral injection. The selection of LV-shRNA-Rufy3 and doses of LV-shRNA-Rufy3 and LV-Rufy3 were based on the western blot analysis in normal rats. Based on the detection of the lentivirus infection effect, we selected LV-shRNA2 as the downregulated expression of Rufy3 (Fig. 2a). In addition, 18 µl/Kg of LV-shRNA-Rufy3 (Fig. 2b, d) and 15 µl/kg of LV-Rufy3 (Fig. 2c, e) were injected into the lateral ventricles using a 10-µl Hamilton microsyringe and stereotaxic apparatus, as described in previous studies. The needle was left in place for 1 min to avoid lentivirus reverse flow after infusion [24].

Fig. 2figure 2

Selection of LV-Rufy3-shRNA and dose selection of LV-Rufy3-shRNA, LV-Rufy3, and 8p-CPT under normal conditions. a Representative bands of Rufy3 expression using three types of LV-Rufy3-shRNAs under the normal condition. LV-Rufy3-shRNA2 downregulated Rufy3 expression. b Representative bands of Rufy3 expression using three doses of LV-Rufy3-shRNAs (12, 18, and 24 µl/kg) under normal conditions. c Representative bands of Rufy3 expression using three doses of LV-Rufy3 (10, 15, and 20 µl/kg) under normal conditions. f Representative bands of Rap1 expression using three doses of 8p-CPT (2, 3, and 4 mg/kg) under normal conditions. d, e, g Quantitative analysis of Rufy3 and Rap1 expressions in different groups. The normal group was used as the standard. Data are shown as mean ± SEM (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001 vs. LV-NC or Vehicle group; #P < 0.05, ##P < 0.01, vs. 12 µl/kg-LV-shRNA group or 10 µl/kg-LV-Rufy3

Antibodies and reagents

Anti-ARAP3 antibody (MBS9401663) was supplied by MyBioSource (San Diego, CA, USA). Anti-Rufy3 antibody (PA5-54651), anti-ERK1 antibody (13-8600), and anti-p-ERK1 antibody (PA5-94966) were purchased from Invitrogen (ThermoFisher Scientific, Waltham, MA, USA). Anti-Rho antibody (ab40673), anti-fascin antibody (ab126772), anti-facin antibody (ab205), anti-synaspin I antibody (ab64581), anti-p-synaspin I antibody (ab32532), anti-Rap1 antibody (ab113480), anti-MBP antibody (ab40390), anti-hypophosphorylated neurofilament H[N52] antibody (ab82259), anti-NeuN antibody (ab177487), anti-NeuN (ab104224), and anti-β-Tubulin III (ab41489) antibody were purchased from Abcam (Cambridge, UK). Anti-GAPDH antibodies (AF7021, T0004) and anti-β-Tubulin antibodies (DF7967, T0023) were purchased from Affinity Biosciences (Cincinnati, OH, USA). Anti-MEK1 antibody (98195S), anti-p-MEK1 antibody (26975S), anti-mouse IgG (7076S), anti-rabbit IgG (7074s), horseradish peroxidase conjugated-linked-secondary antibody, and active Rap1 Detection Kit were purchased from Cell Signaling Technology (Beverly, MA, USA). Alexa Fluor 488 (A32790) and Alexa Fluor 555 (A32794) Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, and Alexa Fluor 488 (A-11001) and Alexa Fluor 555 (A-21424) Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody were purchased from Invitrogen. Situ Cell Death Detection Kit (12156792910) and 8p-CPT (C8988), the most common Rap1 agonist, were purchased from Sigma-Aldrich, and a dose of 2 mg/kg was used to promote the expression of Rap1 based on western blot results (Fig. 2f, g).

Western blot assay

We conducted a western blot assay 24 h after SAH, as described previously [22]. The brain samples and neurons were lysed by adding the western blot lysis buffer to phenylmethylsulfonyl fluoride. The protein concentration was determined by the bicinchoninic acid method. First, the protein samples of different groups (25 μg/lane) and molecular weight markers (8 μl/lane) were loaded on 10% and 12% sodium dodecyl-polyacrylamide electrophoresis gels. The samples were separated and electrophoretically transferred to nitrocellulose membranes. The nitrocellulose membranes were blocked with 5% skimmed milk for 1 h at room temperature and incubated with anti-ARAP3, anti-Rho, anti-Fascin, anti-Facin, anti-ERK1, anti-p-ERK1, anti-MEK1, anti-p-MEK1, anti-synaspin I, and anti-p-synaspin I antibodies at 4 °C overnight. GAPDH or β-tubulin was used as the loading control. Then, the membranes were incubated with anti-mouse IgG or anti-rabbit IgG horseradish peroxidase conjugated-linked secondary antibody (1:3000) at room temperature for 1 h. Next, the protein bands were detected using a luminescent image analyzer (Clinx ChemiScope5300, Clinx Science Instruments, Shanghai, China) after adding the developer solution to the membranes. Protein levels were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized to the relative density of the normal or sham group. The ratio of phosphoprotein to total protein was used to evaluate the phosphorylation level.

RT-PCR assay

RNA was extracted from brain tissue samples using TRIzol (ThermoFisher Scientific, 15596026) and transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, 4368813). The abundance of target gene RNA was detected by real-time PCR using PowerUp SYBR Green Master Mix (ThermoFisher Scientific, A25742). The results of the quantitative PCR are presented relative to the mean values of housekeeping genes (ΔΔCt method). The mRNA levels were normalized for gene expression in brain tissue samples. The Rufy3 and GAPDH primer sequences were as follows: Rufy3 Forward Primer TGCAGCCGGTCCTTAGAAATG, Rufy3 reverse Primer AGGCTAGTCTGACCCCACAG. GAPDH Forward Primer 5′-ACCCACTCCTCCACCTTTGAC-3′, GAPDH Reverse Primer 5′-TGTTGCTGTAGCCAAATTCGTT-3′.

IF assay

IF staining was performed on cultured primary cortical neurons and brain tissue paraffin-embedded sections 24 h after SAH [22]. In experiments one and two, neurons and brain tissue specimens were fixed with 4% paraformaldehyde. Then the tissue was paraffin-embedded and sectioned into slices of 4 μm. The dewaxed sections and neurons were incubated with Rufy3 (1:200), MBP (1:300), N52 (1:300), β-tubulin III (1:250) and NeuN (1:300) antibodies overnight at 4 °C. In experiment three, the dewaxed sections were incubated with Rufy3 (1:200), Fascin (1:300), MBP (1:300) antibodies, and β-tubulin III (1:300) overnight at 4 °C. They were then incubated with the donkey anti-rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody or Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody at 37 °C for 1 h. Then, the sections and neurons were washed three times on the next day. Next, the water-soluble mounting medium, 4,6-diamino-2-phenylindole (SouthernBiotech, Birmingham, AL, USA), was added to the sections for cover slipping. Finally, the fluorescence of brain regions and neurons was observed under a fluorescence microscope (OLYMPUS BX50/BX-FLA/DP70; Olympus Co., Tokyo Japan), and ImageJ software was used to quantify the fluorescence intensity.

IP assay

IP detection was performed 24 h after SAH, as described previously [25]. First, the radioimmunoprecipitation assay lysis buffer was added to lyse the brain samples. Then, the lysate was incubated with a rabbit monoclonal antibody against Rap1 or rabbit IgG overnight at 4 °C with orbital shaking. Then, protein A + G Sepharose beads were added to every immunocomplex. Simultaneously, the pyrolysis mixture was incubated at 4 °C for 4 h with orbital shaking. Ultimately, the immunoblotting method was used to isolate and detect the protein.

Rap1 activation assay

An Active Rap1 Detection Kit was used for the accurate determination of the level of activated Rap1 [26]. In brief, brain tissue samples were lysed on ice and the obtained lysis solution was mixed and incubated with glutathione agarose beads coupled to GST-RalGDS (Ral guanine dissociation stimulator) to detect activated Rap1 levels. After washing the beads, the samples were loaded onto SDS-polyacrylamide gels, separated, and electrophoretically transferred to the nitrocellulose membranes. The membranes were probed with anti-Rap1 antibody after blocking in 3% bovine serum albumin (BSA) for 1 h at 4 °C overnight. Then, the membranes were incubated with an anti-rabbit IgG horseradish peroxidase (HRP) conjugate. Total Rap1 levels were measured using brain tissue sample lysates that were not subjected to coincubation with beads. Immunoreactive proteins were visualized by enhanced chemiluminescence using a luminescent image analyzer (Clinx ChemiScope 5300, Clinx Science Instruments), and protein intensities were analyzed using Image J software.

Measurement of neuronal axon lengths

To measure the lengths of neuronal axons, β-tubulin III positive neurite lengths were measured as previously described [27]. Briefly, six random microscopic fields (× 400 magnification) were chosen and photographed under a fluorescent microscope. All β-tubulin III immunofluoresce-positive cells in the microscopic field were selected to measure neuronal axon length. Neuronal axon length was defined as the distance between the body and the farthest tip of the neurite and the measurement of neuronal axon length was performed by the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Brain edema

As mentioned previously, wet and dry weighing methods were used to assess the cerebral edema index at 48 h after SAH [28]. We collected fresh brain tissue and promptly weighed it to record the wet weight. Next, the sample was dried at 100 °C for 24 h, and weighed to calculate the dry weight. The cerebral edema index was calculated as ([wet weight − dry weight]/wet weight) × 100%.

TUNEL staining

We used the TUNEL method to evaluate cortical cell apoptosis using the In Situ Cell Death Detection Kit at 24 h after SAH [29]. Briefly, we incubated rat brain sections with 0.1% Triton X-100 for 8 min. After washing the sections three times, we added the sections to the TUNEL reaction mixture at 37 °C for 1 h. Then, 4,6-diamino-2-phenylindole was added to cover the brain sections after washing them with PBST. Finally, we observed the sections under a fluorescence microscope. To estimate the degree of cortical cell apoptosis, the ratio of TUNEL-positive cells (red fluorescence) was recorded as the apoptotic index for each section. In brief, the TUNEL-positive cells were counted by an observer who was blinded to the sham and experimental groups. The apoptotic index was defined as the average number of TUNEL positive cells in each section counted in six microscopic fields (× 400 magnification).

FJC staining

FJC, a highly specific and sensitive fluorescent dye used to mark neuronal degradation [30], was added to the sections at 24 h after SAH. Briefly, brain sections were dewaxed and soaked in 0.06% KMnO4 solution at room temperature for 15 min in the dark. The sections were incubated with FJC working solution and 0.1% acetic acid solvent for 1 h. The sections were air-dried at room temperature and sealed with a neutral balsam medium. Finally, three sections of each rat and six microscope fields in each tissue section were examined under a fluorescence microscope and photographed to count the FJC-positive cells.

Neurological scoring

We used an 18-point scoring system (Table 1) to evaluate the neurologic function of rats at 24 h after SAH.

Table 1 Neurological evaluation of rats post SAHAdhesive removal test

The adhesive removal test was used to assess sensory and motor coordination abilities after SAH [30]. First, the rats were placed in a glass box and a circular sticker was placed on the palm of each forepaw of the rats. The time taken by the rats to remove all the stickers was recorded. The rats were regularly trained daily for 3 days before the test. The test was conducted 1 day before modeling and on days 1, 3, 5, 7, 10, 14, 21, 28, and 35 after SAH.

Rotarod test

This test was used to evaluate the movement ability of rats by rotating a cylinder provided by Anhui Zhenghua Biological Equipment Co. Ltd (Hefei, Anhui, China) [30]. The rats were placed on a horizontal axis that had been set at a stationary rate of 4 to 30 r/min for 1 min. Notably, the test was terminated immediately once the rats fell to the ground or gripped the device for two cycles. The duration spent by the rats on the horizontal axis was recorded. As with the adhesive removal test, the rats were trained for 3 days prior to modeling. The test was also conducted 1 day before modeling and on days 1, 3, 5, 7, 10, 14, 21, 28, and 35 after SAH.

Morris water maze

The Morris water maze experiment has been described previously [31]. The Morris water maze device consisted of a circular pool with a diameter of 2 m and a height of 0.75 m. The circular pool was filled with water mixed with melanin at a depth of 0.4 m at an appropriate temperature. Four equidistant points were randomly designated as North (N), South (S), East (E), and West (W) to divide the pool into four equivalent quadrants (NW, NE, SE, and SW). The circular platform had a diameter of 30 cm. We placed a 10 × 10 cm transparent plexiglass platform at the confluence of the eight random equidistant lines (N, S, E, W, NW, NE, SE, and SW), 2 cm below the surface of the water. A vidicon was installed on the ceiling above the swimming pool and used to track the trajectories of the animals. The rats in the experimental groups were trained for 4 consecutive days before formal testing. The trial lasted for 60 s and the per-interval testing time of each rat was 5 min. If the rat arrived at the platform within 60 s, it would rest on the platform for 15 s. In contrast, if the rat did not arrive at the platform within 60 s, it would be guided to the platform. The time and distance required for the rat to enter the water to find the underwater concealed platform and stand on it were recorded as water maze latency and swimming distance. The Morris water maze tests were conducted one day prior to modeling and on days 10, 14, 21, 28, and 35 after SAH, simultaneously, their latency and swimming distance were recorded.

Statistical analysis

The data were analyzed using the GraphPad Prism 7.0 software (San Diego, CA, USA). Data are expressed as the mean ± SEM (standard error of the mean). One-way or two-way analysis of variance (ANOVA) was used for multiple comparisons, and Bonferroni’s or Tukey’s post hoc test was used for comparisons between two pairs in multiple groups. P < 0.05 was considered statistically significant.

留言 (0)

沒有登入
gif