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Donepezil promotes neurogenesis via Src signaling pathway in a rat model of chronic cerebral hypoperfusion
Brain Research 1736 (2020) 146782
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Research report
Donepezil promotes neurogenesis via Src signaling pathway in a rat model of chronic cerebral hypoperfusion
T
Jiang Mana, Kefei Cuib, Xiaojie Fuc, Di Zhanga, Zhengfang Lua, Yufeng Gaoa, Lie Yua, Nan Lid, ⁎ Jianping Wanga, a
Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China Department of Ultrasound, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China c Department of Neurointervention, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China d Department of Neurology, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China b
H I GH L IG H T S
used 2VO procedure to mimic chronic cerebral hypoperfusion in rats. • We promoted SVZ neurogenesis and neurologic outcome in 2VO rats. • Donepezil upregulated Src and EGFR/FGFR pathway within SVZ in 2VO rats. • Donepezil • Src inhibitor KX-01 abolished neurogenesis and neuroprotective effects of donepezil.
A R T I C LE I N FO
A B S T R A C T
Keywords: Chronic cerebral hypoperfusion Subventricular zone Neurogenesis Donepezil Src
Donepezil, a selective acetylcholinesterase (AchE) inhibitor, enhances stroke-induced neurogenesis within subventricular zone (SVZ). Src/Pyk-2 is one of the downstream pathways of acetylcholine receptors (AchRs), and has been shown to participate in the activation of fibroblast growth factor receptor (FGFR)/epidermal growth factor receptor (EGFR) signaling in cancer cells. In this study, we investigated whether donepezil could promote SVZ neurogenesis in chronic cerebral hypoperfusion (CCH) injury via Src signaling pathway. In the bilateral carotid artery occlusion (2VO) rat model, we observed more nestin/5-bromo-2′-deoxyuridine (BrdU)-positive cells and doublecortin (DCX)/BrdU-positive cells in the SVZ than that in the sham group. Further, donepezil obviously improved neurologic function after 2VO, induced the greater number of SVZ proliferative NSCs and neuroblasts, and elevated levels of Src, p-FGFR1, p-EGFR, p-Akt and p-Raf in ipsilateral SVZ. Lastly, Src inhibitor KX-01 abolished the beneficial effects of donepezil in 2VO rats. These results suggest that donepezil could upregulate Src signaling pathway to enhance CCH-induced SVZ neurogenesis.
1. Introduction Chronic cerebral hypoperfusion (CCH), a chronic state characterized as gradual and permanent cerebral blood flow reduction, is a common pathological process in vascular dementia (VD) (Qu et al., 2014; Santiago et al., 2018). CCH has been demonstrated to stimulate proliferation of SVZ neural stem cells (NSCs). Study has indicated that
increasing subventricular zone (SVZ) neurogenesis could improve outcomes after CCH (Shu et al., 2012). Cholinergic system regulates adult SVZ neurogenesis under physiological and pathological conditions (Paez-Gonzalez et al., 2014). Donepezil, an agonist of AchRs signaling pathway, can significantly enhance SVZ NSCs proliferation in the permanent middle cerebral artery occlusion mice (Wang et al., 2017a). As a widely used drug in VD
Abbreviations: CCH, chronic cerebral hypoperfusion; 2VO, bilateral carotid artery occlusion; SVZ, subventricular zone; NSCs, neural stem cells; AchE, acetylcholinesterase; FGFR, fibroblast growth factor receptor; EGFR, epidermal growth factor receptor; AchRs, acetylcholine receptors; BrdU, 5-bromo-2′-deoxyuridine; PVDF, polyvinylidenedifluoride; TBST, Tris-buffered saline-0.1% Tween 20; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PBS, phosphate-buffered saline; DCX, doublecortin; DAPI, 4′,6-diamidino-2-phenylindole; ChAT, choline acetyltransferase; GPCRs, G-protein-coupled receptors; PKC, protein kinase C; HB-EGF, heparin-binding epidermal growth factor ⁎ Corresponding author at: Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China. E-mail address: [email protected] (J. Wang). https://doi.org/10.1016/j.brainres.2020.146782 Received 4 August 2018; Received in revised form 15 February 2020; Accepted 12 March 2020 Available online 14 March 2020 0006-8993/ © 2020 Elsevier B.V. All rights reserved.
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3. Discussion
patients, donepezil has been proved to improve cognitive function via up-regulating cholinergic signaling pathways (Roman et al., 2010). But the role of donepezil in CCH and the mechanisms remain largely unclear. Previous studies revealed that up-regulation of Src/Pyk-2 signal pathway promoted phosphorylation of fibroblast growth factor receptor (FGFR), epidermal growth factor receptor (EGFR) and inhibitation of Src could down-regulate EGFR/FGFR signaling pathway (Sturla et al., 2011). FGFR and EGFR phosphorylated by Src participate in activation, proliferation and differentiation of NSCs (Annenkov, 2009). Donepezil increases SVZ neurogenesis in cerebral infarction by activating acetylcholine receptors (AchRs), which may cross talk with EGFR/FGFR signaling though the Src signaling pathway (Wang et al., 2017b). In this study, we investigated whether donepezil could promote SVZ neurogenesis in a rat model of CCH via Src signaling pathway.
In this study, we demonstrated that donepezil could upregulate Src signaling pathway to promote SVZ neurogenesis in 2VO rats, indicating a potential therapeutic strategy and the mechanism of action for CCH treatment. Donepezil currently focused on studies of stroke, Alzheimer's Disease, and traumatic brain injury, indicating the treatment could enhance survival, proliferation and migration of NSCs (Yang et al., 2017; Bhatnagar et al., 2016; Wang et al., 2017a). But there are very few researches on the role of donepezil in SVZ neurogenesis in the rat model of CCH, and the mechanisms remain unknown. We found donepezil promoted neurologic recovery of CCH and increased SVZ neurogenesis, which is consistent with previous studies. Enhancing activation of AchRs increased neurogenesis under physiologic conditions, possibly by upregulating EGFR/FGFR signaling pathway. Our previous researches have uncovered α7 nAChR regulate neurogenesis via the α7 nAChR-FGFR1 pathway in a mouse model of middle cerebral artery occlusion (Wang et al., 2017b). In present study, we found donepezil promoted SVZ neurogenesis in CCH via EGFR/ FGFR signaling pathway and revealed the mechanisms. A novel mechanism of AchRs signal pathway probably indirectly enhancing EGFR via Src is investigated (Resende and Adhikari, 2009). EGFR/FGFR signaling pathway might be activated by AchRs in NSCs via intracellular messenger (Cai et al., 2002). There are mainly two receptors of Ach: mAchRs (muscarinic AchRs) and nAchRs (nicotinic AchRs). mAchRs are G-protein-coupled receptors (GPCRs), and nAchRs are ligand-gated ion channels (Di Liberto et al., 2014). mAchRs possibly have cross-talk with tyrosine-kinase receptors (RTK), including EGFR and FGFR (Hodges et al., 2012). Studies indicate that activated GPCRs can upregulate Ca2+ and Protein Kinase C (PKC)-mediated Src/Pyk-2 signal pathway (Cattaneo et al., 2014). Previous findings reveal that upregulation of Src/Pyk-2 signal pathway straightly promotes phosphorylation of FGFR and EGFR (Schott and Grove, 2013). Perhaps PKC activates metalloprotease via reactive oxygen and Src, and then membrane-anchored form of heparin-binding epidermal growth factor (HBEGF) was transformed into mature secreted form of HB-EGF upregulating EGFR signal pathway, and ultimately activate PI3K/Akt, and Raf/ MRK/ERK/RSK signal pathway, which are essential for cell proliferation, migration, and survival (Xie et al., 2012). nAchRs are also pro{Xie, 2012 #61}ved to activate Src/Pyk and EGFR/FGFR signaling pathways by increasing intracellular concentration of Ca2+ or β-arrestin-1 (Kiryushko et al., 2006). Maybe increasing activation of mAchRs and nAchRs by donepezil stimulate Ca2+ current in NSCs, contributing to upregulated Src signaling pathway. To determine the role of cholinergic system in neurogenesis after CCH, we treated 2VO rats with donepezil. We found 2VO rats injected with donepezil had better neurologic function performance, larger number of proliferative NSCs and neuroblasts in SVZ, higher level of Src, and increased expression of p-FGFR1, p-EGFR and its downstream proteins such as p-Akt and p-Raf in SVZ than did the vehicle-treated 2VO rats (Fig. 3), indicating that donepezil can promote neurologic recovery and SVZ neurogenesis in CCH rat model, probably via increasing Src and EGFR/FGFR signaling pathway. To verify the effect of the Src signaling pathway and its effect on EGFR/FGFR signaling in donepezil-induced neurogenesis, we treated 2VO rats with Src inhibitor KX-01 and donepezil. Conversely, we found KX-01 treatment significantly decreased the expression of Src, p-FGFR and p-EGFR, along with its downstream proteins such as p-Akt and p-Raf in SVZ. While the experimental results conform to the previous hypothesis, it’s better to divide phosphorylation levels of FGFR, EGFR and its downstream proteins, for example Akt and Raf by the total amount of proteins respectively. KX-01 also abolished the neurogenic effects of donepezil on proliferation of NSCs and neuroblasts within SVZ, and abrogated the neuroprotective effects of donepezil on CCH rats. The results of this study indicate that Src plays a vital role in the donepezil-induced SVZ
2. Results 2.1. Donepezil alleviates 2VO-induced memory impairment The mortality of each group was 2/32 (6.3%) in the Sham group, 8/ 38 (21.1%) in the 2VO + vehicle group, 5/35 (14.3%) in the 2VO + DZ group and 9/39 (23.1%) in the 2VO + DZ + KX-01 group. Mortality rates did not differ significantly among three 2VO groups. The cerebral blood flow decreased to about 30% of the baseline level before 2VO (Fig. 1B). In the reference memory test, 2VO rats received donepezil had shorter latency to find the platform than that in the 2VO + vehicle and 2VO + DZ + KX-01 group (Fig. 1C). KX-01 reversed the improvement seen with donepezil, and no differences were observed between the 2VO + vehicle and 2VO + DZ + KX-01 group (Fig. 1C). In the probe trail, rats in the 2VO + DZ group spent more time in the target zone (52.83 ± 10.21 s) than did the other two 2VO groups (2VO + vehicle, 30.38 ± 7.39 s; 2VO + DZ + KX-01, 38.04 ± 11.26 s; Fig. 1D). Swimming speed did not differ among the four groups (Fig. 1E). 2.2. Donepezil increases NSC proliferation via Src signaling pathway Compared with the Sham group, rats that underwent 2VO had more DCX/BrdU positive cells and nestin/BrdU positive cells in SVZ 14 days after surgical procedure (Fig. 2A-D). 2VO rats treated with donepezil had more DCX/BrdU positive cells (148.50 ± 35.74 per section; Fig. 2A and C) and nestin/BrdU positive cells (145.17 ± 29.21 per section; Fig. 2B and D) than that from other two 2VO groups. This neurogenic effect of donepezil was significantly abolished by Src inhibitor KX-01 treatment (DCX/BrdU positive cells, 91.50 ± 42.32 per section; Fig. 2A and C; nestin/BrdU positive cells, 97.50 ± 37.30 per section; Fig. 2B and D). No significant difference of DCX/BrdU positive cells and nestin/BrdU positive cells was found between 2VO + vehicle (DCX/BrdU positive cells, 97.33 ± 24.95 per section; Fig. 2A and C; nestin/BrdU positive cells, 77.83 ± 23.92 per section; Fig. 2B and D) and 2VO + DZ + KX-01 groups. 2.3. Donepezil stimulates Src and EGFR/FGFR signaling pathway Three 2VO groups had similar body weight change during the experiment (Fig. 3A). The quantification of Western blot analysis showed that the expression of p-FGFR1 in 2VO + vehicle group was significantly higher than that in sham group. Compared with the 2VO + vehicle group, 2VO rats injected with donepezil had significantly higher levels of Src, p-FGFR1, p-EGFR, p-Akt and p-Raf. Expression or phosphorylation levels of these proteins in 2VO + DZ + KX-01 group was significantly lower than in 2VO + DZ group. (Fig. 3C-G). No significant difference in brain water content was present among the three groups (Fig. 3H). 2
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Fig. 1. Donepezil improves learning and memory in 2VO rats. (A) Schematic representation of experimental design. (B) Changes of cerebral blood flow (CBF) in sham and 2VO rats. #p < 0.05 vs. Sham group, n = 30/group. (C)Time latency to find the platform in the Morris water maze (MWM) test. The performance of 2VO rats was worse than that of the Sham group. 2VO rats treated with donepezil exhibited decreased latency to find the platform compared with the other two 2VO groups. This therapeutic effect was abolished by KX-01. *p < 0.05 vs. 2VO + vehicle and 2VO + DZ + KX-01 group; #p < 0.05 vs. Sham group, n = 12/group. (D) Quantification showed that the 2VO + DZ group spent more time in the target zone during the probe trial than the other two 2VO groups. No significant difference was found between the 2VO + vehicle and 2VO + DZ + KX-01 groups. *p < 0.05 vs. 2VO + vehicle and 2VO + DZ + KX-01 group; #p < 0.05 vs. Sham group, n = 12/group. (E) Swimming speed did not differ among the four groups during the five sessions. p > 0.05, n = 12/group. Values are mean ± SD.
4. Experimental procedure
neurogenesis, possibly via EGFR/FGFR signaling pathway. Further exploration is warranted to clarify potential causal relationships between Src and EGFR/FGFR signaling pathway by using selective inhibitors of EGFR and FGFR. The major limitation of this study was lacking use of mAchRs and nAchRs inhibitors to negatively verify cholinergic signaling regulates EGFR/FGFR signaling pathway via Src. It would be more interesting to study the molecular mechanism by which cholinergic system regulate Src signaling pathway. In conclusion, we found donepezil could promote SVZ neurogenesis in CCH rat model, possibly via the cross talk with Src and EGFR/FGFR signaling pathway. These results are important to reveal the mechanisms by which donepezil promotes SVZ neurogenesis after CCH.
4.1. Animals and ethics statement Male Sprague-Dawley rats (260–300 g, 3 months old) were supplied by the Animal Experimental Center of Zhengzhou University. All rats were housed in plastic cages (5 per cage) with free access to food and water and were maintained at a constant temperature of 22 ± 1 °C. All protocols were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Zhengzhou University. All efforts were made to minimize the number of animals used and their suffering.
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Fig. 2. Donepezil increases SVZ neurogenesis after CCH. (A-B) Immunofluorescence staining of DCX/BrdU positive cells and nestin/BrdU positive cells after donepezil injection. Images are shown at × 200 magnification. Scale bar = 50 μm. (C-D) Quantification showed that donepezil-treated 2VO rats had more DCX/BrdU positive cells and nestin/BrdU positive cells than did 2VO + vehicle and 2VO + DZ + KX-01 group. *p < 0.05 vs. 2VO + vehicle and 2VO + DZ + KX-01 group; # p < 0.05 vs. Sham group, n = 6/group. Values are mean ± SD.
et al., 2017c). Vehicle and drugs were respectively administered via intraperitoneal injection once daily on day 7 after 2VO surgery for 7 days. All rats used for immunofluorescence received intraperitoneal injections of 5-bromo-2′-deoxyuridine (BrdU, 50 mg/kg, Sigma-Aldrich, St Louis, MO, USA) once daily on day 7 after the surgery for 7 days (Speisman et al., 2013). Body weights were measured weekly for 28 days after treatment and are showed as percent change as follows: (body weight at each time point-body weight before surgery)/body weight before surgery × 100% (Li et al., 2015).
4.2. Chronic cerebral hypoperfusion model CCH model rat model was established by 2VO as described previously (Zhu et al., 2018). Briefly, we anesthetized rats with an intraperitoneal injection of 10% chloral hydrate (400 mg/kg) and made a midline incision in the ventral side of the neck to expose common carotid arteries. The arteries were carefully separated from the sheaths and adjacent vagus nerves and then permanently double-ligated with 5–0 silk suture under a surgical microscope. The neck wound was sutured closed and topical lidocaine applied. Successful 2VO was defined as an approximately 70% decrease in central blood flow and was confirmed by laser-Doppler flowmetry (Moor Instruments, Devon, UK). Sham-operated rats received the same surgical procedures except for the occlusion of the bilateral common carotid arteries.
4.4. Morris water maze Rats for measuring body weights from each group were also used for spatial memory in the Morris Water Maze on day 35 as previously described (Hu and Zhang, 2018). An SLY-WMS Water Maze automatic control recorder (Beijing Sunny Instruments Co. Ltd, Beijing, China) recorded escape latencies (sec) and pathlengths (cm). A black circular maze pool (150 cm in diameter, 50 cm in height) was filled with water (20 ± 1 °C, 30 cm in depth) (Vorhees and Williams, 2006). The escape platform (12 cm in diameter) was fixed in the middle of the northeast quadrant of the pool, and 2 cm below the water surface.
4.3. Treatment and group Rats were randomly divided into the following four groups: shamoperated rats treated with 1% DMSO in saline (Sham, n = 32), 2VO rats treated with 1% DMSO in saline (2VO + vehicle, n = 38), 2VO rats treated with donepezil (2VO + DZ, n = 35), and 2VO rats treated with donepezil and KX-01 (2VO + DZ + KX-01, n = 39). Donepezil (1 mg/ kg/day, Abcam, Cambridge, MA, USA) were dissolved in saline and KX01 (Src inhibitor, 5 mg/kg/day, MedChemExpress, Monmouth Junction, NJ, USA) in 1% DMSO in saline (Ihalainen et al., 2011; Wang
4.4.1. Reference memory protocol Trainings consisted of 5 sessions (one session/day) of 3–15 min trials (20 s inter-trial-interval). Each rat was placed in the pool and was 4
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Fig. 3. Donepezil may upregulate EGFR/FGFR signaling pathway via Src in SVZ after CCH. (A) Change in rat body weight over 4 weeks (n = 12/group). No significant difference was found between the three 2VO groups. p > 0.05, n = 12/group. (B) Western blot analysis of p-EGFR, p-FGFR1, p-Raf, Src, and p-Akt in SVZ. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (C-G) Quantification of band densities showed that p-EGFR, p-FGFR1, p-Raf, Src and p-Akt expression levels were significantly increased in the 2VO + DZ group. *p < 0.05 compared with 2VO + vehicle and 2VO + DZ + KX-01 group. # p < 0.05 compared with Sham group, n = 6/group. (H) No significant difference was found in the brain water content among the four groups. p > 0.05, n = 6/ group. Values are mean ± SD.
released randomly from three starting locations and given 120 s to find the hidden platform. It would be guided to stay in the platform for 10 s in case of failing within 120 s, and the escape latency was recorded as 120 s. Rats were dried between intervals. The escape latency and swimming speed of each rat were recorded by investigators blinded to the treatment and groups.
4.5. Immunofluorescence analysis Six rats from each group were sacrificed for immunofluorescence analysis on day 14 after 2VO as described previously. After being deeply anesthetized by chloral hydrate, rats were perfused transcardially with saline and 4% paraformaldehyde in 0.01 M phosphatebuffered saline (PBS). Then brain was dissected out, post-fixed in 4% paraformaldehyde, dehydrated in 30% sucrose/0.01 M PBS, and finally serially cut into 20-μm-thick floating sections every 480 μm by cryoultramicrotomy (CM1100, Leica Biosystems, Germany). Tissue sections were stored in antifreeze buffer in 24-well plates at −20 °C for later use. Corresponding seven or eight serial brain sections of each mouse were incubated in PBST (0.1% Triton X-100 in 0.01 M PBS) for 30 min, blocked in 1% bovine serum albumin solution for 30 min, and incubated at room temperature for 2 h or 4 °C overnight with antibodies against BrdU (1:1000, Abcam), doublecortin (DCX; 1:600, Santa Cruz), or nestin (1:300, Abcam). Then sections were washed in 0.01 M PBS three times and incubated with donkey anti-sheep IgG H&L (FITC;
4.4.2. Probe trial A 2-min free probe trial was administered in the pool from which the platform was removed. Trial was conducted 15 min after the last hidden platform training block. The rats were released from the quadrant opposite and discrimination scores t(G) − t(O)/t(G) + t(O), where t(O) is time spent in the opposite quadrant and t(G) is time spent in the goal quadrant, served as our strength of learning measure. The time spent in the target zone of each rat was recorded and quantified.
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1:500, Abcam), or donkey anti-goat IgG H&L (Alexa Fluor 594; 1:300, Abcam), or donkey anti-mouse IgG H&L (Alexa Fluor 594; 1:300, Proteintech Group Inc., Chicago, IL, USA) for 1 h at room temperature. After three 5-min washes with 0.01 M PBS, the sections were mounted on cover slips with a drop of mounting medium that contained 1.5 μg/ mL 4′,6-diamidino-2-phenylindole (DAPI; Santa Cruz). For each section that contained SVZ, we randomly chose three 10 × fields in the SVZ areas to quantify BrdU/nestin-positive cells and BrdU/DCX-positive cells. The BrdU/nestin-positive or BrdU/DCX-positive cells within the SVZ were expressed as average cell number per section. An investigator blinded to animal treatment analyzed all stained sections under a fluorescence microscope (ZEISS Scope A1, ZEISS, Germany).
CRediT authorship contribution statement
4.6. Western blot analysis
Appendix A. Supplementary data
Six rats from each group were sacrificed for Western blot analysis on day 14 after 2VO as described previously (Wang et al., 2016). Rats were deeply anesthetized by chloral hydrate and sacrificed. The brain was removed to cold PBS, and SVZ tissue was quickly dissected out from the frontal slice extending between the crossing of the anterior commissure and the rostral opening of the third ventricle (Wang et al., 2018a). The SVZ tissue was homogenized in radioimmunoprecipitation assay lysis buffer containing a protease/phosphatase inhibitor mixture (Beyotime Institute of Biotechnology), and clarified by centrifugation (14,000 × g, 10 min, 4 °C) (Wang et al., 2018b). Protein samples from each group were separated on 4–12% SDS–polyacrylamide gels and transferred onto polyvinylidenedifluoride (PVDF) membranes. The membranes were blocked in 5% bovine serum albumin in Tris-buffered saline-0.1% Tween 20 (TBST) for 1 h at room temperature and incubated for 2 h at room temperature with one of the following primary antibodies: anti-Src (1:5000, Abcam), anti-phospho-EGFR (1:100, Santa Cruz Biotechnology, Dallas, TX, USA), anti-phospho-FGFR1 (1:500, Abcam), anti-phospho-rapidly accelerated fibrosarcoma (Raf, 1:500, Santa Cruz), anti-phospho-Akt (p-Akt, 1:100, Santa Cruz) or rabbit antiglyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:2000, Sangon Biotech, Shanghai, China). After 4 washes in TBST, membranes were incubated with appropriate secondary antibodies conjugated to horseradish peroxidase for 1 h at room temperature. Protein bands were visualized by enhanced chemiluminescence ECL kit (CWBIO, Beijing, China), and target proteins were normalized against GAPDH. An investigator blinded to the groups quantified the optical density of the protein bands using Gel Analysis V 2.02 software (Clinx Science Instruments, Shanghai, China).
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.brainres.2020.146782.
Jiang Man: Conceptualization, Methodology, Software, Writing original draft, Writing - review & editing. Kefei Cui: Validation, Formal analysis. Xiaojie Fu: Validation, Supervision. Di Zhang: Writing - review & editing. Zhengfang Lu: Formal analysis. Yufeng Gao: Visualization. Lie Yu: Methodology. Nan Li: Resources. Jianping Wang: Conceptualization, Funding acquisition, Project administration. Acknowledgement This work was supported by grants from the National Natural Science Foundation of China (81571137 and 81771247).
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4.7. Brain water content We measured the brain water content to test the effects of donepezil on cerebral edema. Six rats from each group (Sham, 2VO + vehicle, 2VO + DZ and 2VO + DZ + KX-01) were killed at 24 h after the treatment. Their brains were quickly removed, weighed, and dried at 95 °C for 48 h to obtain the dry weight. The brain water content was expressed as follows: (wet weight-dry weight)/wet weight × 100% (Wang et al., 2016).
4.8. Statistical analysis Statistical analysis was carried out with SPSS version 13.0. Results are expressed as mean ± SD. We used Fisher’s exact test to examine differences in mortality among the groups and one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test to analyze Immunofluorescence and Western Blot differences. Repeated measures ANOVA followed by the LSD test was used to determine changes in escape latency, swimming speed and body weight between groups. p < 0.05 was considered statistically significant. 6
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Research report
Donepezil promotes neurogenesis via Src signaling pathway in a rat model of chronic cerebral hypoperfusion
T
Jiang Mana, Kefei Cuib, Xiaojie Fuc, Di Zhanga, Zhengfang Lua, Yufeng Gaoa, Lie Yua, Nan Lid, ⁎ Jianping Wanga, a
Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China Department of Ultrasound, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China c Department of Neurointervention, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China d Department of Neurology, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China b
H I GH L IG H T S
used 2VO procedure to mimic chronic cerebral hypoperfusion in rats. • We promoted SVZ neurogenesis and neurologic outcome in 2VO rats. • Donepezil upregulated Src and EGFR/FGFR pathway within SVZ in 2VO rats. • Donepezil • Src inhibitor KX-01 abolished neurogenesis and neuroprotective effects of donepezil.
A R T I C LE I N FO
A B S T R A C T
Keywords: Chronic cerebral hypoperfusion Subventricular zone Neurogenesis Donepezil Src
Donepezil, a selective acetylcholinesterase (AchE) inhibitor, enhances stroke-induced neurogenesis within subventricular zone (SVZ). Src/Pyk-2 is one of the downstream pathways of acetylcholine receptors (AchRs), and has been shown to participate in the activation of fibroblast growth factor receptor (FGFR)/epidermal growth factor receptor (EGFR) signaling in cancer cells. In this study, we investigated whether donepezil could promote SVZ neurogenesis in chronic cerebral hypoperfusion (CCH) injury via Src signaling pathway. In the bilateral carotid artery occlusion (2VO) rat model, we observed more nestin/5-bromo-2′-deoxyuridine (BrdU)-positive cells and doublecortin (DCX)/BrdU-positive cells in the SVZ than that in the sham group. Further, donepezil obviously improved neurologic function after 2VO, induced the greater number of SVZ proliferative NSCs and neuroblasts, and elevated levels of Src, p-FGFR1, p-EGFR, p-Akt and p-Raf in ipsilateral SVZ. Lastly, Src inhibitor KX-01 abolished the beneficial effects of donepezil in 2VO rats. These results suggest that donepezil could upregulate Src signaling pathway to enhance CCH-induced SVZ neurogenesis.
1. Introduction Chronic cerebral hypoperfusion (CCH), a chronic state characterized as gradual and permanent cerebral blood flow reduction, is a common pathological process in vascular dementia (VD) (Qu et al., 2014; Santiago et al., 2018). CCH has been demonstrated to stimulate proliferation of SVZ neural stem cells (NSCs). Study has indicated that
increasing subventricular zone (SVZ) neurogenesis could improve outcomes after CCH (Shu et al., 2012). Cholinergic system regulates adult SVZ neurogenesis under physiological and pathological conditions (Paez-Gonzalez et al., 2014). Donepezil, an agonist of AchRs signaling pathway, can significantly enhance SVZ NSCs proliferation in the permanent middle cerebral artery occlusion mice (Wang et al., 2017a). As a widely used drug in VD
Abbreviations: CCH, chronic cerebral hypoperfusion; 2VO, bilateral carotid artery occlusion; SVZ, subventricular zone; NSCs, neural stem cells; AchE, acetylcholinesterase; FGFR, fibroblast growth factor receptor; EGFR, epidermal growth factor receptor; AchRs, acetylcholine receptors; BrdU, 5-bromo-2′-deoxyuridine; PVDF, polyvinylidenedifluoride; TBST, Tris-buffered saline-0.1% Tween 20; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PBS, phosphate-buffered saline; DCX, doublecortin; DAPI, 4′,6-diamidino-2-phenylindole; ChAT, choline acetyltransferase; GPCRs, G-protein-coupled receptors; PKC, protein kinase C; HB-EGF, heparin-binding epidermal growth factor ⁎ Corresponding author at: Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China. E-mail address: [email protected] (J. Wang). https://doi.org/10.1016/j.brainres.2020.146782 Received 4 August 2018; Received in revised form 15 February 2020; Accepted 12 March 2020 Available online 14 March 2020 0006-8993/ © 2020 Elsevier B.V. All rights reserved.
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3. Discussion
patients, donepezil has been proved to improve cognitive function via up-regulating cholinergic signaling pathways (Roman et al., 2010). But the role of donepezil in CCH and the mechanisms remain largely unclear. Previous studies revealed that up-regulation of Src/Pyk-2 signal pathway promoted phosphorylation of fibroblast growth factor receptor (FGFR), epidermal growth factor receptor (EGFR) and inhibitation of Src could down-regulate EGFR/FGFR signaling pathway (Sturla et al., 2011). FGFR and EGFR phosphorylated by Src participate in activation, proliferation and differentiation of NSCs (Annenkov, 2009). Donepezil increases SVZ neurogenesis in cerebral infarction by activating acetylcholine receptors (AchRs), which may cross talk with EGFR/FGFR signaling though the Src signaling pathway (Wang et al., 2017b). In this study, we investigated whether donepezil could promote SVZ neurogenesis in a rat model of CCH via Src signaling pathway.
In this study, we demonstrated that donepezil could upregulate Src signaling pathway to promote SVZ neurogenesis in 2VO rats, indicating a potential therapeutic strategy and the mechanism of action for CCH treatment. Donepezil currently focused on studies of stroke, Alzheimer's Disease, and traumatic brain injury, indicating the treatment could enhance survival, proliferation and migration of NSCs (Yang et al., 2017; Bhatnagar et al., 2016; Wang et al., 2017a). But there are very few researches on the role of donepezil in SVZ neurogenesis in the rat model of CCH, and the mechanisms remain unknown. We found donepezil promoted neurologic recovery of CCH and increased SVZ neurogenesis, which is consistent with previous studies. Enhancing activation of AchRs increased neurogenesis under physiologic conditions, possibly by upregulating EGFR/FGFR signaling pathway. Our previous researches have uncovered α7 nAChR regulate neurogenesis via the α7 nAChR-FGFR1 pathway in a mouse model of middle cerebral artery occlusion (Wang et al., 2017b). In present study, we found donepezil promoted SVZ neurogenesis in CCH via EGFR/ FGFR signaling pathway and revealed the mechanisms. A novel mechanism of AchRs signal pathway probably indirectly enhancing EGFR via Src is investigated (Resende and Adhikari, 2009). EGFR/FGFR signaling pathway might be activated by AchRs in NSCs via intracellular messenger (Cai et al., 2002). There are mainly two receptors of Ach: mAchRs (muscarinic AchRs) and nAchRs (nicotinic AchRs). mAchRs are G-protein-coupled receptors (GPCRs), and nAchRs are ligand-gated ion channels (Di Liberto et al., 2014). mAchRs possibly have cross-talk with tyrosine-kinase receptors (RTK), including EGFR and FGFR (Hodges et al., 2012). Studies indicate that activated GPCRs can upregulate Ca2+ and Protein Kinase C (PKC)-mediated Src/Pyk-2 signal pathway (Cattaneo et al., 2014). Previous findings reveal that upregulation of Src/Pyk-2 signal pathway straightly promotes phosphorylation of FGFR and EGFR (Schott and Grove, 2013). Perhaps PKC activates metalloprotease via reactive oxygen and Src, and then membrane-anchored form of heparin-binding epidermal growth factor (HBEGF) was transformed into mature secreted form of HB-EGF upregulating EGFR signal pathway, and ultimately activate PI3K/Akt, and Raf/ MRK/ERK/RSK signal pathway, which are essential for cell proliferation, migration, and survival (Xie et al., 2012). nAchRs are also pro{Xie, 2012 #61}ved to activate Src/Pyk and EGFR/FGFR signaling pathways by increasing intracellular concentration of Ca2+ or β-arrestin-1 (Kiryushko et al., 2006). Maybe increasing activation of mAchRs and nAchRs by donepezil stimulate Ca2+ current in NSCs, contributing to upregulated Src signaling pathway. To determine the role of cholinergic system in neurogenesis after CCH, we treated 2VO rats with donepezil. We found 2VO rats injected with donepezil had better neurologic function performance, larger number of proliferative NSCs and neuroblasts in SVZ, higher level of Src, and increased expression of p-FGFR1, p-EGFR and its downstream proteins such as p-Akt and p-Raf in SVZ than did the vehicle-treated 2VO rats (Fig. 3), indicating that donepezil can promote neurologic recovery and SVZ neurogenesis in CCH rat model, probably via increasing Src and EGFR/FGFR signaling pathway. To verify the effect of the Src signaling pathway and its effect on EGFR/FGFR signaling in donepezil-induced neurogenesis, we treated 2VO rats with Src inhibitor KX-01 and donepezil. Conversely, we found KX-01 treatment significantly decreased the expression of Src, p-FGFR and p-EGFR, along with its downstream proteins such as p-Akt and p-Raf in SVZ. While the experimental results conform to the previous hypothesis, it’s better to divide phosphorylation levels of FGFR, EGFR and its downstream proteins, for example Akt and Raf by the total amount of proteins respectively. KX-01 also abolished the neurogenic effects of donepezil on proliferation of NSCs and neuroblasts within SVZ, and abrogated the neuroprotective effects of donepezil on CCH rats. The results of this study indicate that Src plays a vital role in the donepezil-induced SVZ
2. Results 2.1. Donepezil alleviates 2VO-induced memory impairment The mortality of each group was 2/32 (6.3%) in the Sham group, 8/ 38 (21.1%) in the 2VO + vehicle group, 5/35 (14.3%) in the 2VO + DZ group and 9/39 (23.1%) in the 2VO + DZ + KX-01 group. Mortality rates did not differ significantly among three 2VO groups. The cerebral blood flow decreased to about 30% of the baseline level before 2VO (Fig. 1B). In the reference memory test, 2VO rats received donepezil had shorter latency to find the platform than that in the 2VO + vehicle and 2VO + DZ + KX-01 group (Fig. 1C). KX-01 reversed the improvement seen with donepezil, and no differences were observed between the 2VO + vehicle and 2VO + DZ + KX-01 group (Fig. 1C). In the probe trail, rats in the 2VO + DZ group spent more time in the target zone (52.83 ± 10.21 s) than did the other two 2VO groups (2VO + vehicle, 30.38 ± 7.39 s; 2VO + DZ + KX-01, 38.04 ± 11.26 s; Fig. 1D). Swimming speed did not differ among the four groups (Fig. 1E). 2.2. Donepezil increases NSC proliferation via Src signaling pathway Compared with the Sham group, rats that underwent 2VO had more DCX/BrdU positive cells and nestin/BrdU positive cells in SVZ 14 days after surgical procedure (Fig. 2A-D). 2VO rats treated with donepezil had more DCX/BrdU positive cells (148.50 ± 35.74 per section; Fig. 2A and C) and nestin/BrdU positive cells (145.17 ± 29.21 per section; Fig. 2B and D) than that from other two 2VO groups. This neurogenic effect of donepezil was significantly abolished by Src inhibitor KX-01 treatment (DCX/BrdU positive cells, 91.50 ± 42.32 per section; Fig. 2A and C; nestin/BrdU positive cells, 97.50 ± 37.30 per section; Fig. 2B and D). No significant difference of DCX/BrdU positive cells and nestin/BrdU positive cells was found between 2VO + vehicle (DCX/BrdU positive cells, 97.33 ± 24.95 per section; Fig. 2A and C; nestin/BrdU positive cells, 77.83 ± 23.92 per section; Fig. 2B and D) and 2VO + DZ + KX-01 groups. 2.3. Donepezil stimulates Src and EGFR/FGFR signaling pathway Three 2VO groups had similar body weight change during the experiment (Fig. 3A). The quantification of Western blot analysis showed that the expression of p-FGFR1 in 2VO + vehicle group was significantly higher than that in sham group. Compared with the 2VO + vehicle group, 2VO rats injected with donepezil had significantly higher levels of Src, p-FGFR1, p-EGFR, p-Akt and p-Raf. Expression or phosphorylation levels of these proteins in 2VO + DZ + KX-01 group was significantly lower than in 2VO + DZ group. (Fig. 3C-G). No significant difference in brain water content was present among the three groups (Fig. 3H). 2
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Fig. 1. Donepezil improves learning and memory in 2VO rats. (A) Schematic representation of experimental design. (B) Changes of cerebral blood flow (CBF) in sham and 2VO rats. #p < 0.05 vs. Sham group, n = 30/group. (C)Time latency to find the platform in the Morris water maze (MWM) test. The performance of 2VO rats was worse than that of the Sham group. 2VO rats treated with donepezil exhibited decreased latency to find the platform compared with the other two 2VO groups. This therapeutic effect was abolished by KX-01. *p < 0.05 vs. 2VO + vehicle and 2VO + DZ + KX-01 group; #p < 0.05 vs. Sham group, n = 12/group. (D) Quantification showed that the 2VO + DZ group spent more time in the target zone during the probe trial than the other two 2VO groups. No significant difference was found between the 2VO + vehicle and 2VO + DZ + KX-01 groups. *p < 0.05 vs. 2VO + vehicle and 2VO + DZ + KX-01 group; #p < 0.05 vs. Sham group, n = 12/group. (E) Swimming speed did not differ among the four groups during the five sessions. p > 0.05, n = 12/group. Values are mean ± SD.
4. Experimental procedure
neurogenesis, possibly via EGFR/FGFR signaling pathway. Further exploration is warranted to clarify potential causal relationships between Src and EGFR/FGFR signaling pathway by using selective inhibitors of EGFR and FGFR. The major limitation of this study was lacking use of mAchRs and nAchRs inhibitors to negatively verify cholinergic signaling regulates EGFR/FGFR signaling pathway via Src. It would be more interesting to study the molecular mechanism by which cholinergic system regulate Src signaling pathway. In conclusion, we found donepezil could promote SVZ neurogenesis in CCH rat model, possibly via the cross talk with Src and EGFR/FGFR signaling pathway. These results are important to reveal the mechanisms by which donepezil promotes SVZ neurogenesis after CCH.
4.1. Animals and ethics statement Male Sprague-Dawley rats (260–300 g, 3 months old) were supplied by the Animal Experimental Center of Zhengzhou University. All rats were housed in plastic cages (5 per cage) with free access to food and water and were maintained at a constant temperature of 22 ± 1 °C. All protocols were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Zhengzhou University. All efforts were made to minimize the number of animals used and their suffering.
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Fig. 2. Donepezil increases SVZ neurogenesis after CCH. (A-B) Immunofluorescence staining of DCX/BrdU positive cells and nestin/BrdU positive cells after donepezil injection. Images are shown at × 200 magnification. Scale bar = 50 μm. (C-D) Quantification showed that donepezil-treated 2VO rats had more DCX/BrdU positive cells and nestin/BrdU positive cells than did 2VO + vehicle and 2VO + DZ + KX-01 group. *p < 0.05 vs. 2VO + vehicle and 2VO + DZ + KX-01 group; # p < 0.05 vs. Sham group, n = 6/group. Values are mean ± SD.
et al., 2017c). Vehicle and drugs were respectively administered via intraperitoneal injection once daily on day 7 after 2VO surgery for 7 days. All rats used for immunofluorescence received intraperitoneal injections of 5-bromo-2′-deoxyuridine (BrdU, 50 mg/kg, Sigma-Aldrich, St Louis, MO, USA) once daily on day 7 after the surgery for 7 days (Speisman et al., 2013). Body weights were measured weekly for 28 days after treatment and are showed as percent change as follows: (body weight at each time point-body weight before surgery)/body weight before surgery × 100% (Li et al., 2015).
4.2. Chronic cerebral hypoperfusion model CCH model rat model was established by 2VO as described previously (Zhu et al., 2018). Briefly, we anesthetized rats with an intraperitoneal injection of 10% chloral hydrate (400 mg/kg) and made a midline incision in the ventral side of the neck to expose common carotid arteries. The arteries were carefully separated from the sheaths and adjacent vagus nerves and then permanently double-ligated with 5–0 silk suture under a surgical microscope. The neck wound was sutured closed and topical lidocaine applied. Successful 2VO was defined as an approximately 70% decrease in central blood flow and was confirmed by laser-Doppler flowmetry (Moor Instruments, Devon, UK). Sham-operated rats received the same surgical procedures except for the occlusion of the bilateral common carotid arteries.
4.4. Morris water maze Rats for measuring body weights from each group were also used for spatial memory in the Morris Water Maze on day 35 as previously described (Hu and Zhang, 2018). An SLY-WMS Water Maze automatic control recorder (Beijing Sunny Instruments Co. Ltd, Beijing, China) recorded escape latencies (sec) and pathlengths (cm). A black circular maze pool (150 cm in diameter, 50 cm in height) was filled with water (20 ± 1 °C, 30 cm in depth) (Vorhees and Williams, 2006). The escape platform (12 cm in diameter) was fixed in the middle of the northeast quadrant of the pool, and 2 cm below the water surface.
4.3. Treatment and group Rats were randomly divided into the following four groups: shamoperated rats treated with 1% DMSO in saline (Sham, n = 32), 2VO rats treated with 1% DMSO in saline (2VO + vehicle, n = 38), 2VO rats treated with donepezil (2VO + DZ, n = 35), and 2VO rats treated with donepezil and KX-01 (2VO + DZ + KX-01, n = 39). Donepezil (1 mg/ kg/day, Abcam, Cambridge, MA, USA) were dissolved in saline and KX01 (Src inhibitor, 5 mg/kg/day, MedChemExpress, Monmouth Junction, NJ, USA) in 1% DMSO in saline (Ihalainen et al., 2011; Wang
4.4.1. Reference memory protocol Trainings consisted of 5 sessions (one session/day) of 3–15 min trials (20 s inter-trial-interval). Each rat was placed in the pool and was 4
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Fig. 3. Donepezil may upregulate EGFR/FGFR signaling pathway via Src in SVZ after CCH. (A) Change in rat body weight over 4 weeks (n = 12/group). No significant difference was found between the three 2VO groups. p > 0.05, n = 12/group. (B) Western blot analysis of p-EGFR, p-FGFR1, p-Raf, Src, and p-Akt in SVZ. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (C-G) Quantification of band densities showed that p-EGFR, p-FGFR1, p-Raf, Src and p-Akt expression levels were significantly increased in the 2VO + DZ group. *p < 0.05 compared with 2VO + vehicle and 2VO + DZ + KX-01 group. # p < 0.05 compared with Sham group, n = 6/group. (H) No significant difference was found in the brain water content among the four groups. p > 0.05, n = 6/ group. Values are mean ± SD.
released randomly from three starting locations and given 120 s to find the hidden platform. It would be guided to stay in the platform for 10 s in case of failing within 120 s, and the escape latency was recorded as 120 s. Rats were dried between intervals. The escape latency and swimming speed of each rat were recorded by investigators blinded to the treatment and groups.
4.5. Immunofluorescence analysis Six rats from each group were sacrificed for immunofluorescence analysis on day 14 after 2VO as described previously. After being deeply anesthetized by chloral hydrate, rats were perfused transcardially with saline and 4% paraformaldehyde in 0.01 M phosphatebuffered saline (PBS). Then brain was dissected out, post-fixed in 4% paraformaldehyde, dehydrated in 30% sucrose/0.01 M PBS, and finally serially cut into 20-μm-thick floating sections every 480 μm by cryoultramicrotomy (CM1100, Leica Biosystems, Germany). Tissue sections were stored in antifreeze buffer in 24-well plates at −20 °C for later use. Corresponding seven or eight serial brain sections of each mouse were incubated in PBST (0.1% Triton X-100 in 0.01 M PBS) for 30 min, blocked in 1% bovine serum albumin solution for 30 min, and incubated at room temperature for 2 h or 4 °C overnight with antibodies against BrdU (1:1000, Abcam), doublecortin (DCX; 1:600, Santa Cruz), or nestin (1:300, Abcam). Then sections were washed in 0.01 M PBS three times and incubated with donkey anti-sheep IgG H&L (FITC;
4.4.2. Probe trial A 2-min free probe trial was administered in the pool from which the platform was removed. Trial was conducted 15 min after the last hidden platform training block. The rats were released from the quadrant opposite and discrimination scores t(G) − t(O)/t(G) + t(O), where t(O) is time spent in the opposite quadrant and t(G) is time spent in the goal quadrant, served as our strength of learning measure. The time spent in the target zone of each rat was recorded and quantified.
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1:500, Abcam), or donkey anti-goat IgG H&L (Alexa Fluor 594; 1:300, Abcam), or donkey anti-mouse IgG H&L (Alexa Fluor 594; 1:300, Proteintech Group Inc., Chicago, IL, USA) for 1 h at room temperature. After three 5-min washes with 0.01 M PBS, the sections were mounted on cover slips with a drop of mounting medium that contained 1.5 μg/ mL 4′,6-diamidino-2-phenylindole (DAPI; Santa Cruz). For each section that contained SVZ, we randomly chose three 10 × fields in the SVZ areas to quantify BrdU/nestin-positive cells and BrdU/DCX-positive cells. The BrdU/nestin-positive or BrdU/DCX-positive cells within the SVZ were expressed as average cell number per section. An investigator blinded to animal treatment analyzed all stained sections under a fluorescence microscope (ZEISS Scope A1, ZEISS, Germany).
CRediT authorship contribution statement
4.6. Western blot analysis
Appendix A. Supplementary data
Six rats from each group were sacrificed for Western blot analysis on day 14 after 2VO as described previously (Wang et al., 2016). Rats were deeply anesthetized by chloral hydrate and sacrificed. The brain was removed to cold PBS, and SVZ tissue was quickly dissected out from the frontal slice extending between the crossing of the anterior commissure and the rostral opening of the third ventricle (Wang et al., 2018a). The SVZ tissue was homogenized in radioimmunoprecipitation assay lysis buffer containing a protease/phosphatase inhibitor mixture (Beyotime Institute of Biotechnology), and clarified by centrifugation (14,000 × g, 10 min, 4 °C) (Wang et al., 2018b). Protein samples from each group were separated on 4–12% SDS–polyacrylamide gels and transferred onto polyvinylidenedifluoride (PVDF) membranes. The membranes were blocked in 5% bovine serum albumin in Tris-buffered saline-0.1% Tween 20 (TBST) for 1 h at room temperature and incubated for 2 h at room temperature with one of the following primary antibodies: anti-Src (1:5000, Abcam), anti-phospho-EGFR (1:100, Santa Cruz Biotechnology, Dallas, TX, USA), anti-phospho-FGFR1 (1:500, Abcam), anti-phospho-rapidly accelerated fibrosarcoma (Raf, 1:500, Santa Cruz), anti-phospho-Akt (p-Akt, 1:100, Santa Cruz) or rabbit antiglyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:2000, Sangon Biotech, Shanghai, China). After 4 washes in TBST, membranes were incubated with appropriate secondary antibodies conjugated to horseradish peroxidase for 1 h at room temperature. Protein bands were visualized by enhanced chemiluminescence ECL kit (CWBIO, Beijing, China), and target proteins were normalized against GAPDH. An investigator blinded to the groups quantified the optical density of the protein bands using Gel Analysis V 2.02 software (Clinx Science Instruments, Shanghai, China).
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.brainres.2020.146782.
Jiang Man: Conceptualization, Methodology, Software, Writing original draft, Writing - review & editing. Kefei Cui: Validation, Formal analysis. Xiaojie Fu: Validation, Supervision. Di Zhang: Writing - review & editing. Zhengfang Lu: Formal analysis. Yufeng Gao: Visualization. Lie Yu: Methodology. Nan Li: Resources. Jianping Wang: Conceptualization, Funding acquisition, Project administration. Acknowledgement This work was supported by grants from the National Natural Science Foundation of China (81571137 and 81771247).
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4.7. Brain water content We measured the brain water content to test the effects of donepezil on cerebral edema. Six rats from each group (Sham, 2VO + vehicle, 2VO + DZ and 2VO + DZ + KX-01) were killed at 24 h after the treatment. Their brains were quickly removed, weighed, and dried at 95 °C for 48 h to obtain the dry weight. The brain water content was expressed as follows: (wet weight-dry weight)/wet weight × 100% (Wang et al., 2016).
4.8. Statistical analysis Statistical analysis was carried out with SPSS version 13.0. Results are expressed as mean ± SD. We used Fisher’s exact test to examine differences in mortality among the groups and one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test to analyze Immunofluorescence and Western Blot differences. Repeated measures ANOVA followed by the LSD test was used to determine changes in escape latency, swimming speed and body weight between groups. p < 0.05 was considered statistically significant. 6
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