- Email: [email protected]
Neuroprotective effects of saffron on the late cerebral ischemia injury through inhibiting astrogliosis and glial scar formation in rats
Biomedicine & Pharmacotherapy 126 (2020) 110041
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Neuroprotective effects of saffron on the late cerebral ischemia injury through inhibiting astrogliosis and glial scar formation in rats
T
Zhong Kaia,1, Wang Rou-Xina,1, Qian Xiao-Dongb, Yu Pinga, Zhu Xin-Yinga, Zhang Qia, Ye Yi-Lua,* a b
School of Basic Medical Sciences & Forensic Medicine, Hangzhou Medical College, Hangzhou, Zhejiang, China Huzhou Central Hospital, Huzhou, Zhejiang, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Saffron (Crocus sativus L.) Focal cerebral ischemia Astrogliosis Glial scar Inflammatory reaction
This study is to explore the neuroprotective effects and involved glial scar of saffron (Crocus sativus L.) on the late cerebral ischemia in rats. Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in Sprague Dawley rats that were randomly divided into sham group, MCAO group, edaravone group (as a positive control) and saffron groups (saffron extract 30, 100, 300 mg/kg). Saffron was administered orally at 2 h at the first day and once daily from day 2 to 42 after ischemia. Behavioral changes were detected from day 43 to 46 after ischemia to evaluate the effects of saffron. Infarct volume, survival neuron density, activated astrocyte, and the thickness of glial scar were also detected. GFAP, neurocan, phosphocan, neurofilament expressions and inflammatory cytokine contents were detected by Western-blotting and ELISA methods, respectively. Saffron improved the body weight loss, neurological deficit and spontaneous activity. It also ameliorated anxiety-like state and cognitive dysfunction, which were detected by elevated plus maze (EPM), marble burying test (MBT) and novel object recognition test (NORT). Toluidine blue staining found that saffron treatment decreased the infarct volume and increased the neuron density in cortex in the ischemic boundary zone. The activated astrocyte number and the thickness of glial scar in the penumbra zone reduced after saffron treatment. Additionally, saffron decreased the contents of IL-6 and IL-1β, increased the content of IL-10 in the ischemic boundary zone. GFAP, neurocan, and phosphocan expressions in ischemic boundary zone and ischemic core zone all decreased after saffron treatment. Saffron exerted neuroprotective effects on late cerebral ischemia, associating with attenuating astrogliosis and glial scar formation after ischemic injury.
1. Introduction Cerebral ischemia is a common clinical cerebrovascular disease characterized by a high rate of disability and imposes a heavy burden on society and patients [1]. So greater attention should be paid to improve strategies for neurological recovery after late cerebral ischemia injuries. The neurogenesis, astrogliosis/glial scar formation and angiogenesis are the most important morphologic changes (brain remodeling) in the late phase after ischemic or other brain injuries [2]. During the process of brain remodeling after ischemia, astrocytes are activated and become hypertrophic, hyperplastic. The activated astrocytes can up-regulate the glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycan, such as neurocan and phosphocan, which are the iconic protein of astrogliosis and glial scar [3–5]. Excessive expression of GFAP is closely related to the inflammatory response [6,7]. Microglia, astrocyte and macrophages release
inflammatory cytokines involved in the initial reaction, such as IL-β and TNF-α. These inflammatory cytokines can activate astrocytes, which then secrete more inflammatory cytokines and promote astrogliosis [8]. Aspects of the astrocytic inflammatory response to stroke may aggravate the ischemic lesion [8,9], but astrocytes also limit lesion extension via anti-excitotoxicity effects and release neurotrophins. Similarly, during the late phase after cerebral ischemia, the glial scar may block spontaneous axonal regeneration and subsequently reduce the functional outcome [10–12]. Thus, inhibition of the excessive astrogliosis and glial scar formation might be a therapeutic strategy for ischemic stroke [13,14]. Saffron, the dry stigmas of the plant Crocus sativus L., is a species belonging to the Iridaceae family and has been widely used as a herbal medicine and spice since ancient times. Pharmacological studies have demonstrated that saffron and its constituents protected against neuropsychiatric injury, especially including anxiety neurosis and
⁎
Corresponding author. E-mail address: [email protected] (Y.-L. Ye). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.biopha.2020.110041 Received 28 August 2019; Received in revised form 18 February 2020; Accepted 19 February 2020 0753-3322/ © 2020 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Biomedicine & Pharmacotherapy 126 (2020) 110041
K. Zhong, et al.
depression [15,16], ischemia [17,18], Alzheimer disease (AD) [19], Parkinson's disease (PD) [20,21] as well as other neurodegenerative diseases [22] in vivo and in vitro. Previous researches have reported that saffron and its active constituents, including crocin and safranal, both played neuroprotection against cerebral ischemic injury [23–27]. However, the neuroprotective effects of saffron and the involved astrogliosis and glial scar formation on the late cerebral ischemia are unknown. Thus, the purpose of the present study was to investigate the neuroprotective effect of saffron on the late cerebral ischemia and involved astrogliosis in the course of glial scar formation.
Table 1 Contents of active components in the decoction of saffron (%).
Contents (%) n
Crocin
Picrocrocin
Safranal
9.48 ± 0.07 6
5.06 ± 0.04 6
0.23 ± 0.06 6
2.3. Focal cerebral ischemia Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). After anesthesia with pentobarbital (700 mg/kg), a nylon filament (Guangzhou Jialing Biotechnology Co., Ltd., Guangzhou, China) was inserted into the internal carotid artery, and advanced approximately 20−25 mm distal to the carotid bifurcation to occlude the origin of the middle cerebral artery (MCA). The filament was carefully withdrawn 90 min after MCAO. In sham-operated rats, the same procedure was done without advancing of the filament into the MCA. Body temperature was maintained constantly at 37.0 ± 0.5℃ using a thermostatically controlled heating blanket during and after the surgery until rats recovered from anesthesia. Total 2 rats with intracranial hemorrhage were excluded, which were evaluated by neurological deficit score and brain dissection at 24 h after ischemia.
2. Materials and methods 2.1. Animals and treatment Male Sprangue-Dawley rats (250−280 g) were provided by Shanghai Slyke Laboratory Animal Limited Corporation (Certificate No. SCXK 2012-0002, China). All of them were kept in the same room under controlled environmental conditions (12:12 h light/dark cycle, ambient temperature of 22 ± 1 °C, relative humidity of 50 ± 10 %) with free access to food and water. The procedures were approved by the local committee for Animal Health, Ethics and Research of Hangzhou Medical college (No. 201713, Hangzhou, China). Rats were randomly divided into 6 groups: Sham group (saline, n = 7/7), MCAO model group (saline, n = 8/9), edaravone + MCAO group (edaravone 3 mg/ kg, n = 9/9) and saffron + MCAO groups (saffron extract 30, 100, 300 mg/kg, n = 8/9, 9/9, 8/9, respectively). Total 2 rats with intracranial hemorrhage were excluded, which were evaluated by neurological deficit score and brain dissection at 24 h after ischemia. They were administered with oral gavage at 2 h after ischemia in the first day and once daily from day 2 to 14 after ischemia. As a positive control, edaravone (3 mg/kg), a free radical scavenger, was used to improve the neurological symptoms and dysfunction caused by cerebral ischemia. The body weight was evaluated at 9 AM at day 1, 3, 7, 14, 21, 28, 35 and 42 after ischemia. Open field test (OFT), novel object recognition test (NORT), elevated plus maze (EPM) and marble burying test (MBT) were conducted from day 43 to 46 after ischemia, respectively. The experimental schedule was provided in the Fig. 1.
2.4. Neurological deficit evaluation The neurological deficit score was evaluated as described: 0, no deficit; 1, failure to fully extend left forepaw; 2, circling to the left; 3, falling to the left; 4, no spontaneous walking with a depressed level of consciousness [29]. Rats were evaluated at day 1, 3, and 7 in the first week after ischemia, and once every week thereafter. 2.5. OFT Each rat was gently put into the center of a brightly-lit open field arena (80 × 80 × 40 cm). The locomotor tracks were continuously recorded by Digbehv spontaneous activity system (Xinruan, Shanghai, China) for 5 min and analyzed by the EthoVision XT software (Noldus, Netherlands). Traveled total distance and traveled velocity were used to describe the spontaneous activity of rats. Time in the central zone was used to evaluate exploratory and anxiety-like behaviors.
2.2. Saffron extract preparation 2.6. NORT Saffron stigma 10 g was crushed and soaked with 75 % ethanol solution 200 ml for 20 min. Then they were treated with ultrasonic bath for 15 min for twice and refluxed with 70℃ water for 30 min. The filtrate was obtained by suction filtration. The residue was soaked with 75 % ethanol solution 200 ml again and repeated all the procedure until the residue turned white. At last, all the filtrates were mixed and concentrated in a rotary evaporator under reduced pressure to a concentration of 1.0 g/ml. The contents of active components were shown in the Table 1 by HPLC method [28]. (Table 1)
During habituation, rats were introduced into the empty chamber (80 × 80 × 40 cm) and left to freely explore it for 10 min. Afterwards, they were put into their home cages for 5 min, during which two identical objects (familiar object: cubic crude wood with 4 cm diameter) were placed in the chamber oppositely. The test was consisted of 3 phases: T1 phase, interval time, and T2 phase. In T1 phase, rats were allowed to explore the identical objects for 5 min. After 1.5 h interval time, rats were placed into the chamber again for 5 min, when one of
Fig. 1. Schedule of the experimental protocol. 2
Biomedicine & Pharmacotherapy 126 (2020) 110041
K. Zhong, et al.
the familiar objects was replaced by a novel one (novel object: cylindrical crude wood with 4 cm diameter and height), that’s T2 phase. Tracks were recorded and analyzed by a video analyzer (Ethovision XT, Noldus, Netherlands). The following parameters were analyzed: discrimination ratio (DR, DR = [N / (N + F)] × 100 %) and discrimination index (DI, DI = [(N - F) / (N + F)] × 100 %). N: exploration time with the novel object (min); F: exploration time with the familiar object (min). Exploration is defined as the distance between the nose of the rat facing the object and the object is within 2 cm.
performed with the polyclonal rabbit antibodies against GFAP (1:200, Santa Cruz). The brain cryo-sections preparation and immunohistochemical methods were based on our previous report [30]. Normal goat serum was used instead of the primary antibody in the control sections. The number of positive cells and the thickness of glial scar in the ischemic boundary zone were calculated with three different fields. The researcher, who was blind to the experimental groups.
2.7. EPM
Rats were decapitated and the ischemic boundary zone was separated on ice quickly. Then it was sonicated to obtain tissue homogenates. After removing particulars by centrifugation (2000×g, 4 °C, 20 min), assay was immediately detected. IL-1β, TNF-α, IL-6 and IL-10 were measured using respective ELISA systems (Shanghai Elisa Biotech Co., Ltd, China) according to the manufacturer’s instructions. The optical density was determined (absorbance at 450 nm) on a plate reader. Finally, the contents of IL-1β, TNF-α, IL-6 and IL-10 were calculated according to the corresponding standard curve.
2.11. Inflammatory cytokines contents detection
In the behavioral laboratory with diffuse and low lighting, EPM was conducted. At the beginning of the test, rats were placed at the center of the maze and faced an open arm. Then they were allowed to explore the maze freely for 5 min. Tracks were recorded and analyzed by a video analyzer (Ethovision XT, Noldus, Netherlands). Total distance traveled in all arms and central zone, distance ratio, total time and time ratio in open arms were measured to evaluate the anxiety-like behavior after late cerebral ischemia.
2.12. Western-blotting 2.8. MBT Samples of the ischemic boundary zone and ischemic core zone were homogenized on ice in PBS containing 1 % protease inhibitor and 1 % phosphatase inhibitor for Western-blotting. Proteins were obtained by centrifugation at 14,000 rpm at 4℃ for 15 min and quantified by Bradford assay. 50 μg sample of each rat was subjected to electrophone using 20 % SDS at 80 V. The proteins were transferred to polyvinylidene fluoride membranes at 250 mA for 2 h. Antibodies for antiGFAP (1:5000, Huabio), anti-neurocan (1:500, Abcam), anti-phosphocan (1:5000, Sigma), neurofilament H (1:500, Abcam) and GAPDH (1:10000, KangChen Bio-tech, Shanghai, China) were applied overnight. Then membranes were incubated with horseradish peroxidase (HRP) secondary antibody (KangChen Bio-tech, Shanghai, China). At last, the signal intensities of proteins were analyzed using Image J software.
Opaque cages (40 × 36 × 20 cm) were covered with 5 cm layer of corncob. Rats were placed individually to habituate the cage for 15 min. Then they were returned to their home cage. Meanwhile 20 clear blue glasses marbles (12 mm diameter) were evenly spaced in the habituation cages. After that, rats were reintroduced into the cage which they had been habituated. After 30 min, the test was terminated and the number of marbles was counted, which were covered with corncob by more than 2/3 vol. 2.9. Surviving neurons and infarct volume detection After anesthesia, rats were perfused transcardially with saline followed by 4 % paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) after ischemia. Then, the brains were post-fixed overnight in the same fixative, and immersed in 30 % sucrose solution in the phosphate buffer. The brains were dissected into 2 mm-thick coronal slices for 6 brain slices. The coronal sections (20 μm or 10 μm) were prepared by cryomicrotomy (CM1900; Leica, Germany) from each slice and stained with 1 % toluidine blue. The 20 μm-thick sections were used to detect the infarct volume, and the 10 μm-thick sections were used for microphotographic examination. For infarct volume, the 6 stained slices were photographed by a charge coupling device (CCD) camera and images were recorded on a computer. Infarct area and both hemisphere areas of each slice were determined by using an image analysis program (AnalyPower 2.0; Zhejiang University, Hangzhou, China) as reported elsewhere [30]. Infarct volume was calculated as total infarct area × thickness (2 mm). The summation of the infarct volumes of all brain slices were the total infarct volume. Then, the 10-μm-thick sections were used for histopathological examination and counting neuron densities in the temporal cortex in the ischemic boundary zone. Three different fields in each brain slice in bregma 0.0 mm and -4.0 mm were captured. The average numbers of healthy-looking neurons (cells with pyramidal-like appearance and larger sizes, but without shrunken appearance) in layers III-IV of the temporoparietal cortex were counted in sections stained with 1 % toluidine blue. The researcher, who was blind to the experimental groups, used Image J software to calculate the neuron number automatically.
2.13. Statistical analysis Data were expressed as mean ± SEM. Repeated measures of oneway analysis of variance (ANOVA) and Tukey post-hoc tests were performed to analyze the significance of any treatment effects among the groups by SPSS 19.0 for Windows except the effects of saffron on NORT. t-test was used to compare the difference of DR and DI between T2 phase and T1 phase. p < 0.05 was considered statistically significance. 3. Results 3.1. Saffron improved the body weight loss and neurological deficit As shown in Fig. 2A, the body weight in sham group grown quickly while the model group obviously decreased from day 14 to day 42 after ischemia (F(5,43) = 1.228, 1.641, 2.088, 2.580, 3.419 at day14, 21, 28, 35, 42 after ischemia, respectively. p < 0.05). The body weight loss was improved after treatment with saffron 100 mg/kg (F(5,43)=3.419, p = 0.040), 300 (F(5,43)=3.419, p = 0.015) at day 42 after ischemia. Edaravone also improved the body weight loss from day 35 (F(5,43) =2.580, p = 0.045) to day 42 (F(5,43)=3.419, p = 0.044) after ischemia. Fig. 2B showed there was severe neurological deficit after ischemia. Saffron 100 mg/kg and edaravone treatment improved the neurological deficit from day 28–42 after ischemia (F(5,43) = 5.458, 5.276, 6.438 at day 28, 35, 42 after ischemia, respectively. p < 0.05). Saffron 300 mg/ kg improved the neurological deficit from day 35–42 (F(5,43)= 5.276, 6.438 at day 35, 42 after ischemia, respectively. p < 0.05). Saffron (30
2.10. Astrocyte activation/glial scar measurement To investigate the astrocyte activation/glial scar formation in the ischemic boundary zone, immunohistochemical methods were 3
Biomedicine & Pharmacotherapy 126 (2020) 110041
K. Zhong, et al.
Fig. 2. Effects of saffron on the body weight loss and neurological deficit. Body weight (A) and neurological deficit score (B) were measured every other day in the first week and every week from the second week. Values are presented as the mean ± SEM. Statistical analysis were carried out by one-way ANOVA and Tukey posthoc test with *P < 0.05, **P < 0.01 against the sham group and #P < 0.05, ##P < 0.01 against the model group.
3.3. Saffron improved the cognitive function deterioration
mg/kg) improved the neurological deficit only at day 42 after ischemia (F(5,43)=6.438, p < 0.05).
Compared with the T1 phase in the same group, there was no difference of DR and DI in model group (Fig. 4B, F(1,14) = 2.873, p = 0.940; Fig. 4C, F(1,14) = 1.904, p = 0.480), edaravone group (Fig. 4B, F(1,16) = 0.007, p = 0.065; Fig. 4C, F(1,16) = 0.007, p = 0.0.065) and saffron 30 mg/kg (Fig. 4B, F(1,14) = 2.719, p = 0.054; Fig. 4C, F (1,14) = 3.735, p = 0.075) group in T2 phase compared with T1 phase. However, DR and DI all increased in sham group (Fig. 4B, F(1,12) = 3.817, p = 0.047; Fig. 4C, F(1,12) = 0.108, p = 0.045), saffron 100 (Fig. 4B, F(1,16) = 0.111, p = 0.030; Fig. 4C, F(1,14) = 0.121, p = 0.041) and 300 mg/kg (Fig. 4B, F(1,14) = 0.102, p = 0.040; Fig. 3C, F (1,14) = 0.069, p = 0.014) groups in T2 phase when compared with T1 phase.
3.2. Saffron improved locomotor activity After ischemia, rats showed lower traveled velocity (F(5, 43) = 3.128, p = 0.028) and total traveled distance (F(5, 43)=2.320, p = 0.009) in open field. Saffron 100 mg/kg (F(5, 43)=3.128, p = 0.003), 300 mg/kg (F(5, 43)=3.128, p = 0.014) and edaravone (F(5, 43) =3.128, p = 0.008) increased the velocity (Fig. 3C). Saffron 300 mg/ kg (F(5, 43) = 2.320, p = 0.028) and edaravone (F(5, 43)=2.320, p = 0.012) increased the total distance (Fig. 3D). There was no effect on the velocity (Fig. 3C, F(5, 43) = 3.128, p = 0.217) and total traveled distance (Fig. 3D, F(5, 43) = 2.320, p = 0.202) after saffron (30 mg/ kg) treatment. The central time decreased after ischemia (Fig. 3E, F(5, 43) = 5.063, p = 0.025). Saffron 300 mg/kg (F(5, 43)=5.063, p = 0.046) and edaravone (F(5, 43)=5.063, p = 0.035) increased the central time in OFT (Fig. 3E).
3.4. Saffron ameliorated anxiety-like behaviors In EPM, as Fig. 5B shown, there was no difference in the total distance in open and close arms among groups (Fig. 5B, F(5,43) = 0.716,
Fig. 3. Effects of saffron on traveled velocity, traveled total distance and central time in OFT. The heatmaps (A) and the tracks (B) in OFT were presented. Traveled velocity (C), traveled total distance (D) and central time (E) were measured to evaluate the locomotor activity in rats with OFT. Values are presented as the mean ± SEM. Statistical analysis were carried out by one-way ANOVA and Tukey post-hoc test with *P < 0.05 against the sham group and #P < 0.05, ##P < 0.01 against the model group. 4
Biomedicine & Pharmacotherapy 126 (2020) 110041
K. Zhong, et al.
Fig. 4. Effects of saffron on DR and DI in NORT. The heatmaps in all groups in NORT were presented in Fig. 4A. DR (B) and DI (C) were used to evaluate the cognitive function in NORT. Values are presented as the mean ± SEM. Statistical analysis were carried out by t-test with *P < 0.05 against T1 phase in the same group.
3.5. Saffron reduced the infarct volume after ischemia
p > 0.05). However, distance ratio (Fig. 5C, F(5,43) = 2.211, p=0.045), total time (Fig. 5D, F(5,43) = 2.047, p = 0.049) and time ratio (Fig. 5E, F(5,43) = 1.259, p = 0.013) in open arms significantly decreased after ischemia. Saffron 30, 100, 300 mg/kg (F(5,43)=2.211, p = 0.022, 0.010, 0.005) and edaravone (F(5,43)=2.211, p = 0.024) increased the distance ratio in open arms (Fig. 5C). Saffron 100, 300 mg/kg (Fig. 5D, F(5,43) = 2.047, p = 0.018, 0.032) increased the total time in open arms. Saffron 30, 100 mg/kg (F(5,43)=1.259, p = 0.040, 0.046) and edaravone (F(5,43)=1.259, p = 0.041) increased the time ratio in open arms (Fig. 5E). In MBT, the buried marbles number significantly increased when compared with the sham group (Fig. 5F, F(5,43) = 2.282, p = 0.048). Saffron 100, 300 mg/kg (Fig. 5F, F(5,43) = 2.282, p = 0.023, 0.006) and edaravone (Fig. 5F, F(5,43) = 2.282, p = 0.002) reversed the increase of buried marbles number, which manifested by the improvement of anxiolytic state with saffron treatment.
At day 46 after ischemia, an obvious brain atrophy in the ischemic hemispheres was shown in the gross photographs of whole brains and brain sections (bregma –4.0 mm) with toluidine blue staining, whereas it was improved with edaravone or saffron treatment (Fig. 6A). The results of toluidine blue showed the infarct volume hasn’t been found in the sham group. The obvious infarct volume in model group and it was significantly reduced by saffron 100 (F(5,30) = 18.385, p = 0.014), 300 (F(5,30)=18.385, p = 0.022) mg/kg and edaravone (F(5,30) =18.385, p = 0.000) after ischemia, but not saffron 30 mg/kg (F(5,30) =18.385, p = 0.106) (Fig. 6B).
Fig. 5. Effects of saffron on the distance, time in EPM and buried marble number in MBT. The typical tracks in EPM (A) were presented in rats. The total distance in open and close arms (B), distance ratio in open arms (C), total time in open arms (D), and time ratio in open arms (E) were measured to evaluate the anxiety-like behaviors in rats with EPM. Schematic and buried marbles number was measured to evaluate the anxiety-like behaviors in MBT (F). Values are presented as the mean ± SEM. Statistical analysis were carried out by one-way ANOVA and Tukey post-hoc test with *P < 0.05 against the sham group and #P < 0.05, ##P < 0.01 against the model group. 5
Biomedicine & Pharmacotherapy 126 (2020) 110041
K. Zhong, et al.
Fig. 6. Effects of saffron on the infarct volume after ischemia. Representative photographs of brains and brain sections with toluidine blue staining (bregma -4.0 mm) showed the infarct volume (A) and the quantity of infarct volume (B) in all groups. Values are presented as the mean ± SEM. Statistical analysis were carried out by one-way ANOVA and Tukey post-hoc test with #P < 0.05, ##P < 0.01 against the model group.
13.416, p = 0.000) and edaravone (F(5,30)=13.416, p = 0.000) significantly decreased the GFAP-positive cells number in the ischemic boundary zone (Fig. 8B). In addition, saffron 100 (F(5,30) = 13.399, p = 0.000), 300 (F(5,30)=13.399, p = 0.000) mg/kg and edaravone (F (5,30)=13.399, p = 0.000) reduced the thickness of the scar wall by 55.2 %, 61.6 % and 52.4 %, respectively (Fig. 8D).
3.6. Saffron increased survival neuron density in cortex in the ischemic boundary zone The penumbra zone surrounds the ischemic core of the stroke lesion and is comprised of cells that are stressed and vulnerable to death, which is due to an altered metabolic, oxidative and ionic environment within the penumbra. There is hope that appropriate therapies may allow potential recovery of cells within this tissue region, or at least slow the rate of cell death, therefore, slowing the spread of the ischemic infarct and minimising the extent of tissue damage. As such, preserving the penumbra to promote functional brain recovery is a central goal in stroke research [31]. Representative photographs showed the densities of apparently survival neurons in temporoparietal cortex in the ischemic boundary zone. Neuron density was substantially reduced in temporoparietal cortex in the ischemic boundary zone after ischemia (Fig. 7A–B, F(5,30) = 17.320, p = 0.000). Saffron 100 (F(5,30)=17.320, p = 0.000), 300 (F(5,30)=17.320, p = 0.000) mg/kg and edaravone (F(5,30)=17.320, p = 0.000) attenuated the neuron loss in the ischemic boundary zone. But saffron 30 mg/kg had no effect on the survival neuron density (Fig. 7B, F(5,30) = 17.320, p = 1.000).
3.8. Saffron changed GFAP, neurocan, phosphocan, and neurofilament expressions in the ischemic boundary zone and ischemic core zone The results of Western-blotting showed there were a few GFAP (Fig. 9B–C), neurocan (Fig. 9D, 9 G), and phosphocan (Fig. 9E, 9 H) expressions in the ischemic boundary zone and ischemic core zone in the sham group. While they all significantly increased after ischemia (Fig. 9B-E, G–H, p < 0.05). Saffron 300 mg/kg and edaravone treatment reversed the expressions of GFAP (F(3,20)=24.013 in the ischemic boundary zone and F(3,20)=50.208 in the ischemic core zone, p < 0.05), neurocan (F(3,20)=23.743 in the ischemic boundary zone and F(3,20)=13.903 in the ischemic core zone, p < 0.05), and phosphocan (F(3,20)=7.872 in the ischemic boundary zone and F(3,20) =4.007 in the ischemic boundary zone, p < 0.05) (Fig. 9B-E, G–H), except phosphocan expression in the ischemic core zone after edaravone treatment (Fig. 9H, F(3,20) = 4.007, p = 0.051). But there was no difference between groups of neurofilament H expression in the ischemic boundary zone and ischemic core zone (Fig. 9F, I, F(3,20) = 1.523 in the ischemic boundary zone and F(3,20) = 3.173 in the ischemic core zone, p > 0.05), except saffron 300 mg/kg (F(3,20) =1.523, p = 0.047) treatment increased neurofilament H expression in the ischemic boundary zone (Fig. 9F).
3.7. Saffron inhibited the astrocyte activation and reduced the glial scar formation In the boundary zone, the density of GFAP-positive astrocytes was greatly increased after ischemia (Fig. 8A–B, F(5,30) = 13.416, p = 0.001). The intense astrocytic fibers surrounded the ischemic boundary zone and glial scar was formed at day 46 after ischemia (Fig. 8C). The infarct tissue was transformed into the center cavitation of a scar. Intense GFAP-positive astrocytic fibers were present in the scar wall parallel to the cavitation. Reactive astrocytes with hyperplasia/hypertrophy were prominent in the scar. Saffron 300 mg/kg (F(5,30) =
3.9. Saffron changed the contents of inflammatory cytokines The results of ELISA showed the contents of IL-6 (Fig. 10A, F(5,12) 6
Biomedicine & Pharmacotherapy 126 (2020) 110041
K. Zhong, et al.
Fig. 7. Effects of saffron on the survival neuron density in cortex in the ischemic boundary zone. Typical photos showed the density and the morphology of the survival neuron in the ischemic boundary zone (A). And the survival neuron density was calculated in the temporoparietal cortex in the ischemic boundary zone (B). Values are presented as the mean ± SEM. Statistical analysis were carried out by one-way ANOVA and Tukey post-hoc test with **P < 0.01 against the sham group and ##P < 0.01 against the model group. Scale bar=50 μm in Fig. 6A.
effects of saffron on late ischemic injury in rats, which manifested by improvement of body weight loss, neurological deficit, spontaneous activity, anxiety-like behaviors, cognitive dysfunction. Also, it decreased the infarct volume and increased the survival neuron density in cortex. According to the results, the most effective dosage of saffron was 300 mg/kg. As a positive control, edaravone was proven neuroprotective effects on late cerebral ischemia, which had improvement of neurological deficit and behavioral deterioration, as well as inhibition of glial scar formation. In this study, the first treatment was conducted at 2 h after the MCAO surgery. It is extremely important to clarify the requirement for studies that explore novel treatments for cerebral ischemia. Based on a preliminary experiment, we have explored the time-dependent effect of saffron. It (300 mg/kg) was injected at 1, 2 and 3 h after ischemia. The infarct volume and neurological deficit score were evaluated (data not shown). The results showed the therapeutic time window was within 2 h after ischemia. So for evaluating the effect on the late ischemic injury, multiple doses of saffron (30, 100, 300 mg/kg) were injected at 2 h
= 3.236, p = 0.012) and IL-1β (Fig. 10C, F(5,12) = 6.617, p = 0.000) increased and IL-10 decreased (Fig. 10B, F(5,12) = 4.878, p = 0.013) after cerebral ischemia. But the content of TNF-α didn’t change after ischemia (Fig. 10D, F(5,12) = 0.280, p = 0.902). Saffron 100, 300 mg/ kg and edaravone treatment reversed the contents of IL-6 (Fig. 10A, F (5,12) = 3.236, p = 0.014, 0.007, 0.020) and IL-10 (Fig. 10B, F(5,12) = 4.878, p = 0.002, 0.001). Also Saffron (30, 100, 300 mg/kg) and edaravone treatment reversed the content of IL-1β (F(5,12)=6.617, p = 0.003, 0.006, 0.002, 0.000) (Fig. 10C). Whether saffron or edaravone treatment didn’t affect the content of TNF-α in the ischemic boundary zone (Fig. 10D, F(5,12) = 0.280, p > 0.05). 4. Discussion Several studies have demonstrated that saffron has protective effects against ischemia-reperfusion injury by antioxidant property [25] in acute cerebral ischemia and improving recognition and spatial memory dysfunction [23]. The present study investigates the neuroprotective
Fig. 8. Effects of saffron on the GFAP-positive cells number and the thickness of glial scar. Typical photo showed the astrocyte activation (A) and glial scar formation in the ischemic boundary zone (C). The GFAP-positive cells number (B) and the thickness of glial scar (D) were detected in the ischemic boundary zone. Values are presented as the mean ± SEM. Statistical analysis were carried out by one-way ANOVA and Tukey post-hoc test with **P < 0.01 against the sham group and ## P < 0.01 against the model group. Scale bar = 50 μm in Fig. 7A and 500 μm in Fig. 7C. 7
Biomedicine & Pharmacotherapy 126 (2020) 110041
K. Zhong, et al.
Fig. 9. Effects of saffron on GFAP, neurocan, phosphocan and neurofilament H expressions in the ischemic boundary zone and ischemic core zone. Typical photos showed the contents of GFAP, neurocan, phosphocan and neurofilament expressions in the ischemic boundary zone and ischemic core zone by Western-blottingt (A). The contents of GFAP expression were presented in the ischemic boundary zone (B) and in the ischemic core zone (C). The contents of neurocan expression were presented in the ischemic boundary zone (D) and in the ischemic core zone (G). The contents of phosphocan expression were presented in the ischemic boundary zone (E) and in the ischemic core zone (H). The contents of neurofilament expression were presented in the ischemic boundary zone (F) and in the ischemic core zone (I). Values are presented as the mean ± SEM. Statistical analysis were carried out by one-way ANOVA and Tukey post-hoc test with *P < 0.05, **P < 0.01 against the sham group and #P < 0.05, ##P < 0.01 against the model group.
after ischemia in the first day and once daily from day 2 to day 14 after ischemia. After ischemia, it was often accompanied with several behavioral changes, including neurological deficit [32], spontaneous activity impairment [33], cognitive deterioration [34], and anxiety-like behaviors [35]. It corroborated the beneficial effects of saffron after MCAO by demonstrating that it improved body weight loss and neurological deficit at day 46 after ischemia. Additionally, toluidine blue staining showed that treated animals had significantly more survival neurons in the ischemic boundary zone and decreased the infarct volume at the late cerebral ischemia. These survival neuron cells most likely to migrate towards the infarct to replenish cortical and striatal neurons [30]. So, it was indicated that the better neurological recovery observed in these animals correlate with the more survival neurons. We also found there was an advantage of saffron on the behavioral dysfunction after late cerebral ischemia/reperfusion injury in rats. The traveled total distance and velocity are often used to evaluate the spontaneous activity in OFT. Our results showed that the spontaneous activity declined after ischemia. Saffron (100, 300 mg/kg) increased the velocity, total distance and central time, especially at the dosage of 300
mg/kg in late cerebral ischemia. It was in line with the improvement of the neurological deficit and manifested that saffron could promote the rehabilitation of stroke. Furthermore it has been found that the spatial organization is the most important property of locomotor activity in addition to the total distance [36]. Animals with lower anxiety and exploratory behavior tend to spend more time in the central, open area of the box [37], which was in accordance with the results of NORT. Accumulated researches had reported that saffron or its active constituent could improve the neurological deficit and memory impairment [23,38]. In the present study, we used NORT (a common experimental method to assess cognitive skills) [39,40] just to find saffron (100, 300 mg/kg) improved the cognitive decline especially. These results verified the neuroprotective effects of saffron in behavioral improvement in rats. EPM and MBT were both used to evaluate the anxiety-state after cerebral ischemia [41,42] and further confirmed the anti-anxiolytic effects of saffron [43,44]. These battery of behavioral tests highlighting long-term deficits could be useful to study future neuroprotective strategies for stroke treatment. The other novel findings of the current study are that saffron attenuates astrogliosis and glial scar formation in response to cerebral 8
Biomedicine & Pharmacotherapy 126 (2020) 110041
K. Zhong, et al.
Fig. 10. Effects of saffron on the contents of inflammatory cytokines in ischemic boundary zone. IL-6 (A), IL-10 (B), IL-1β (C) and TNF-α (D) contents in the ischemic boundary zone were detected by ELISA methods. Values are presented as the mean ± SEM. Statistical analysis were carried out by one-way ANOVA and Tukey post-hoc test with *P < 0.05, **P < 0.01 against the sham group and #P < 0.05, ## P < 0.01 against the model group.
speculate that this phenomenon may be related to the different stage of cerebral ischemia and animal model. More than 150 compounds have been identified in saffron stigma. But the main active components of saffron were crocin, picrocrocin and safranal [63], which was consistent with our results. Saffron or Crocus sativus L. (C. sativus) has been widely used as a medicinal plant to promote human health, especially in Asia. So, in this study we only explore the neuroprotective effects of saffron extract. We will further explore the neuroprotective effect of isolated active components, such as crocin, picrocrocin and safranal, on the late cerebral ischemia. In conclusion, our findings reveal that saffron can attenuate astrogliosis and glial scar formation after ischemic brain injury, following with neurological deficit improvement, which provides a new insight into understanding of the protective effect of saffron for clinical treatment of ischemic stroke and may be developed into new therapeutics for ischemic stroke.
ischemic insult. It significantly decreased the expression of GFAP, neurocan, and phosphocan in the ischemic boundary zone at day 46 after ischemia. Astrocytes, which constitute almost a third of cells in the central nervous system (CNS), participate in circuit [45,46], metabolism of energy substrates [47], axonal regeneration [48], and behavioral functions [49]. After severe injury insult in the CNS, the potential mechanisms of the axons failure to regenerate across tissue lesions could include absence of external inhibitory factors [50], stimulating factors [51], or promotion of astrogliosis and astrocyte scar formation [52], etc. In late stage, the activated astrocytes can up-regulate the GFAP and chondroitin sulfate proteoglycan, such as neurocan and phosphocan, which are the iconic protein of astrogliosis and glial scar [5], and inhibit axonal regeneration and release axonal growth inhibitors [53,54]. Some traditional Chinese medicine exerted protective effects on late cerebral ischemia through inhibiting astrogliosis and glial scar formation [55,56], which was in line with our results of saffron. It may be a useful strategy to study the neuroprotective effects for stroke. However, there was no difference on neurofilament H among groups, except increased level of neurofilament H in saffron 300 mg/kg group in the ischemic boundary zone when compared with the model group. The neurofilament protein of the CNS consists of three subunits: neurofilament light (NFeL), neurofilament medium (NFeM) and neurofilament heavy (NFeH), which are named according to their molecular weights. Bianca Mages et al. found NFeL increased in the ischemic penumbra [57] and increased serum levels of phosphorylated NFeH correlated with the clinical outcome of stroke patients [58]. It is suggested that neurofilament H in the ischemic brain tissue may not be a sensitive indicator of glial scar. Recently, it has been shown that reactive astrocytes upregulated over 1000 genes, including cytokines, induced in response to injury and wound in peripheral and central nervous system [59]. Although GFAP expression is maintained in glial scars for a prolonged period after injury [60], most expression changes are transient. However, proinflammatory cytokines remain persistently elevated after both ischemic and inflammatory insults, which plays a major role in reactive gliosis and scar formation [61]. Saffron decreased the expression of IL-6, IL-1β and increased the expression of IL-10, but there was no effect on the expression of TNF-α. It was similar to the document reported [62], which manifested the difference of TNF-α level was not apparent. So we
Author contributions Zhong Kai and Wang Rou-Xin performed the experiments, collected the data and wrote the manuscript. Wang Rou-Xin, Yu Ping and Zhu Xin-Ying performed the animal experiments and analyzed the data. Qian Xiao-Dong contributed to the design of the study. Zhang Qi and Ye Yi-Lu interpreted the data and revised the manuscript. Declaration of Competing Interest All authors declare no conflicts of interests. All authors agree to submit the manuscript. Acknowledgments The work was supported by the Project of Health and Family Planning Commission of Zhejiang Province (2018239491). References [1] Z. Chen, B. Jiang, X. Ru, H. Sun, D. Sun, X. Liu, et al., Mortality of stroke and its subtypes in China: results from a nationwide population-based survey, Neuroepidemiology 48 (3–4) (2017) 95–102.
9
Biomedicine & Pharmacotherapy 126 (2020) 110041
K. Zhong, et al.
injuries, Eur. J. Pharmacol. 557 (1) (2007) 23–31. [31] E. Horvath, A. Hutanu, L. Chiriac, M. Dobreanu, A. Oradan, E.E. Nagy, Ischemic damage and early inflammatory infiltration are different in the core and penumbra lesions of rat brain after transient focal cerebral ischemia, J. Neuroimmunol. 324 (2018) 35–42. [32] H. Wang, H. Guo, Q. Lou, Q. Shi, Effects of cysteinyl leukotrienes receptor antagonists on chronic brain injury after global cerebral ischemia/reperfusion, Zhejiang Da Xue Xue Bao Yi Xue Ban 47 (1) (2018) 19–26. [33] T. Yamaguchi, M. Suzuki, M. Yamamoto, YM796, a novel muscarinic agonist, improves the impairment of learning behavior in a rat model of chronic focal cerebral ischemia, Brain Res. 669 (1) (1995) 107–114. [34] M.V. Avrov, V.M. Alifirova, A.V. Kovalenko, An effect of complex therapy on cognitive impairment in patients with chronic cerebral ischemia, Zh. Nevrol. Psikhiatr. Im. S. S. 119 (2) (2019) 23–27. [35] M. Zhang, H. Yan, S. Li, J. Yang, Rosmarinic acid protects rat hippocampal neurons from cerebral ischemia/reperfusion injury via the Akt/JNK3/caspase-3 signaling pathway, Brain Res. 1657 (2017) 9–15. [36] Q. Zhang, E.Q. Wei, C.Y. Zhu, W.P. Zhang, M.L. Wang, S.H. Zhang, et al., Focal cerebral ischemia alters the spatio-temporal properties, but not the amount of activity in mice, Behav. Brain Res. 169 (1) (2006) 66–74. [37] J.N. Crawley, Exploratory behavior models of anxiety in mice, Neurosci. Biobehav. Rev. 9 (1) (1985) 37–44. [38] N. Pitsikas, P.A. Tarantilis, Crocins, the active constituents of Crocus sativus L., counteracted apomorphine-induced performance deficits in the novel object recognition task, but not novel object location task, in rats, Neurosci. Lett. 644 (2017) 37–42. [39] S. Lee, H.J. Park, S.J. Jeon, E. Kim, H.E. Lee, H. Kim, et al., Cognitive ameliorating effect of Acanthopanax koreanum against scopolamine-induced memory impairment in mice, Phytother. Res. 31 (3) (2017) 425–432. [40] N. Baazaoui, K. Iqbal, Prevention of amyloid-beta and tau pathologies, associated neurodegeneration, and cognitive deficit by early treatment with a neurotrophic compound, J. Alzheimers Dis. 58 (1) (2017) 215–230. [41] M. Oz, E.A. Demir, M. Caliskan, R. Mogulkoc, A.K. Baltaci, K.E. Nurullahoglu Atalik, 3’,4’-Dihydroxyflavonol attenuates spatial learning and memory impairments in global cerebral ischemia, Nutr. Neurosci. 20 (2) (2017) 119–126. [42] M. Frechou, I. Margaill, C. Marchand-Leroux, V. Beray-Berthat, Behavioral tests that reveal long-term deficits after permanent focal cerebral ischemia in mouse, Behav. Brain Res. 360 (2019) 69–80. [43] A. Ghajar, S.M. Neishabouri, N. Velayati, L. Jahangard, N. Matinnia, M. Haghighi, et al., Crocus sativus L. versus Citalopram in the Treatment of Major Depressive Disorder with Anxious Distress: A Double-Blind, Controlled Clinical Trial, Pharmacopsychiatry 50 (4) (2017) 152–160. [44] H. Hosseinzadeh, N.B. Noraei, Anxiolytic and hypnotic effect of Crocus sativus aqueous extract and its constituents, crocin and safranal, in mice, Phytother. Res. 23 (6) (2009) 768–774. [45] J.L. Stobart, K.D. Ferrari, M.J.P. Barrett, C. Gluck, M.J. Stobart, M. Zuend, et al., Cortical circuit activity evokes rapid astrocyte calcium signals on a similar timescale to neurons, Neuron 98 (4) (2018) 726–735 e724. [46] R. Martin, R. Bajo-Graneras, R. Moratalla, G. Perea, A. Araque, Circuit-specific signaling in astrocyte-neuron networks in basal ganglia pathways, Science 349 (6249) (2015) 730–734. [47] N. Marina, E. Turovsky, I.N. Christie, P.S. Hosford, A. Hadjihambi, A. Korsak, et al., Brain metabolic sensing and metabolic signaling at the level of an astrocyte, Glia 66 (6) (2018) 1185–1199. [48] G. Li, Y. Cao, F. Shen, Y. Wang, L. Bai, W. Guo, et al., Mdivi-1 inhibits astrocyte activation and astroglial scar formation and enhances axonal regeneration after spinal cord injury in rats, Front. Cell. Neurosci. 10 (2016) 241. [49] Y. Han, H.X. Yu, M.L. Sun, Y. Wang, W. Xi, Y.Q. Yu, Astrocyte-restricted disruption of connexin-43 impairs neuronal plasticity in mouse barrel cortex, Eur. J. Neurosci. 39 (1) (2014) 35–45. [50] S. Quraishe, L.H. Forbes, M.R. Andrews, The extracellular environment of the CNS: influence on plasticity, sprouting, and axonal regeneration after spinal cord injury, Neural Plast. 2018 (2018) 2952386. [51] C.I. Lin, C.C. Chiao, Blue light promotes neurite outgrowth of retinal explants in postnatal ChR2 mice, eNeuro (2019). [52] K.E. Rhodes, L.D. Moon, J.W. Fawcett, Inhibiting cell proliferation during formation of the glial scar: effects on axon regeneration in the CNS, Neuroscience 120 (1) (2003) 41–56. [53] C. Heine, K. Sygnecka, H. Franke, Purines in neurite growth and astroglia activation, Neuropharmacology 104 (2016) 255–271. [54] J.J. Hill, K. Jin, X.O. Mao, L. Xie, D.A. Greenberg, Intracerebral chondroitinase ABC and heparan sulfate proteoglycan glypican improve outcome from chronic stroke in rats, Proc Natl Acad Sci U S A 109 (23) (2012) 9155–9160. [55] C.Y. Wu, M. Fang, A. Karthikeyan, Y. Yuan, E.A. Ling, Scutellarin Attenuates Microglia-Mediated Neuroinflammation and Promotes Astrogliosis in Cerebral Ischemia - A Therapeutic Consideration, Curr. Med. Chem. 24 (7) (2017) 718–727. [56] Z. Dong, D. Ma, Y. Gong, T. Yu, G. Yao, Salvianolic acid B ameliorates CNS autoimmunity by suppressing Th1 responses, Neurosci. Lett. 619 (2016) 92–99. [57] B. Mages, S. Aleithe, S. Altmann, A. Blietz, B. Nitzsche, H. Barthel, et al., Impaired neurofilament integrity and neuronal morphology in different models of focal cerebral ischemia and human stroke tissue, Front. Cell. Neurosci. 12 (2018) 161. [58] P. Singh, J. Yan, R. Hull, S. Read, J. O’Sullivan, R.D. Henderson, et al., Levels of phosphorylated axonal neurofilament subunit H (pNfH) are increased in acute ischemic stroke, J. Neurol. Sci. 304 (1–2) (2011) 117–121. [59] J.L. Zamanian, L. Xu, L.C. Foo, N. Nouri, L. Zhou, R.G. Giffard, et al., Genomic analysis of reactive astrogliosis, J. Neurosci. 32 (18) (2012) 6391–6410.
[2] H.M. Bramlett, W.D. Dietrich, Pathophysiology of cerebral ischemia and brain trauma: similarities and differences, J. Cereb. Blood Flow Metab. 24 (2) (2004) 133–150. [3] J.S. Zhan, K. Gao, R.C. Chai, X.H. Jia, D.P. Luo, G. Ge, et al., Astrocytes in migration, Neurochem. Res. 42 (1) (2017) 272–282. [4] X. Jin, Y.J. Shin, T.R. Riew, J.H. Choi, M.Y. Lee, Increased expression of Slit2 and its robo receptors during astroglial scar formation after transient focal cerebral ischemia in rats, Neurochem. Res. 41 (12) (2016) 3373–3385. [5] G.R. Choudhury, S. Ding, Reactive astrocytes and therapeutic potential in focal ischemic stroke, Neurobiol. Dis. 85 (2016) 234–244. [6] A. Justin, S. Divakar, M. Ramanathan, Cerebral ischemia induced inflammatory response and altered glutaminergic function mediated through brain AT1 and not AT2 receptor, Biomed. Pharmacother. 102 (2018) 947–958. [7] P.B. de la Tremblaye, S.M. Benoit, S. Schock, H. Plamondon, CRHR1 exacerbates the glial inflammatory response and alters BDNF/TrkB/pCREB signaling in a rat model of global cerebral ischemia: implications for neuroprotection and cognitive recovery, Prog. Neuropsychopharmacol. Biol. Psychiatry 79 (Pt B) (2017) 234–248. [8] J. Yuan, M. Zou, X. Xiang, H. Zhu, W. Chu, W. Liu, et al., Curcumin improves neural function after spinal cord injury by the joint inhibition of the intracellular and extracellular components of glial scar, J. Surg. Res. 195 (1) (2015) 235–245. [9] M.G. De Simoni, P. Milia, M. Barba, A. De Luigi, L. Parnetti, V. Gallai, The inflammatory response in cerebral ischemia: focus on cytokines in stroke patients, Clin. Exp. Hypertens. 24 (7–8) (2002) 535–542. [10] D. Wu, M.C. Klaw, T. Connors, N. Kholodilov, R.E. Burke, V.J. Tom, Expressing constitutively active rheb in adult neurons after a complete spinal cord injury enhances axonal regeneration beyond a chondroitinase-treated glial scar, J. Neurosci. 35 (31) (2015) 11068–11080. [11] M. Pekny, M. Pekna, Astrocyte reactivity and reactive astrogliosis: costs and benefits, Physiol. Rev. 94 (4) (2014) 1077–1098. [12] M.A. Anderson, J.E. Burda, Y. Ren, Y. Ao, T.M. O’Shea, R. Kawaguchi, et al., Astrocyte scar formation aids central nervous system axon regeneration, Nature 532 (7598) (2016) 195–200. [13] H. Zhao, G. Li, R. Wang, Z. Tao, S. Zhang, F. Li, et al., MiR-424 prevents astrogliosis after cerebral ischemia/reperfusion in elderly mice by enhancing repressive H3K27me3 via NFIA/DNMT1 signaling, FEBS J. 286 (24) (2019) 4926–4936. [14] Y.M. Zhu, X. Gao, Y. Ni, W. Li, T.A. Kent, S.G. Qiao, et al., Sevoflurane postconditioning attenuates reactive astrogliosis and glial scar formation after ischemiareperfusion brain injury, Neuroscience 356 (2017) 125–141. [15] S. Samarghandian, M. Azimi-Nezhad, F. Samini, T. Farkhondeh, The role of saffron in attenuating age-related oxidative damage in rat Hippocampus, Recent Pat. Food Nutr. Agric. 8 (3) (2017) 183–189. [16] A.L. Lopresti, P.D. Drummond, Efficacy of curcumin, and a saffron/curcumin combination for the treatment of major depression: a randomised, double-blind, placebo-controlled study, J. Affect. Disord. 207 (2017) 188–196. [17] G.H. Farjah, S. Salehi, M.H. Ansari, B. Pourheidar, Protective effect of Crocus sativus L. (Saffron) extract on spinal cord ischemia-reperfusion injury in rats, Iran. J. Basic Med. Sci. 20 (3) (2017) 334–337. [18] L. Mahmoudzadeh, H. Najafi, S.C. Ashtiyani, Z.M. Yarijani, Anti-inflammatory and protective effects of saffron extract in ischaemia/reperfusion-induced acute kidney injury, Nephrology (Carlton) 22 (10) (2017) 748–754. [19] O. Mirmosayyeb, A. Tanhaei, H.R. Sohrabi, R.N. Martins, M. Tanhaei, M.A. Najafi, et al., Possible role of common spices as a preventive and therapeutic agent for alzheimer’s disease, Int. J. Prev. Med. 8 (2017) 5. [20] S.V. Rao Muralidhara, S.C. Yenisetti, P.S. Rajini, Evidence of neuroprotective effects of saffron and crocin in a Drosophila model of parkinsonism, Neurotoxicology 52 (2016) 230–242. [21] P.K. Pan, L.Y. Qiao, X.N. Wen, Safranal prevents rotenone-induced oxidative stress and apoptosis in an in vitro model of Parkinson’s disease through regulating Keap1/ Nrf2 signaling pathway, Cell. Mol. Biol. (Noisy-Le-Grand) 62 (14) (2016) 11–17. [22] K. Hatziagapiou, E. Kakouri, G.I. Lambrou, K. Bethanis, P.A. Tarantilis, Antioxidant properties of Crocus sativus L. And its constituents and relevance to neurodegenerative diseases; focus on alzheimer’s and parkinson’s disease, Curr. Neuropharmacol. (2018). [23] H. Hosseinzadeh, H.R. Sadeghnia, F.A. Ghaeni, V.S. Motamedshariaty, S.A. Mohajeri, Effects of saffron (Crocus sativus L.) and its active constituent, crocin, on recognition and spatial memory after chronic cerebral hypoperfusion in rats, Phytother. Res. 26 (3) (2012) 381–386. [24] A. Vakili, M.R. Einali, A.R. Bandegi, Protective effect of crocin against cerebral ischemia in a dose-dependent manner in a rat model of ischemic stroke, J. Stroke Cerebrovasc. Dis. 23 (1) (2014) 106–113. [25] S. Saleem, M. Ahmad, A.S. Ahmad, S. Yousuf, M.A. Ansari, M.B. Khan, et al., Effect of Saffron (Crocus sativus) on neurobehavioral and neurochemical changes in cerebral ischemia in rats, J. Med. Food 9 (2) (2006) 246–253. [26] H. Hosseinzadeh, H.R. Sadeghnia, Safranal, a constituent of Crocus sativus (saffron), attenuated cerebral ischemia induced oxidative damage in rat hippocampus, J. Pharm. Pharm. Sci. 8 (3) (2005) 394–399. [27] G. Akbari, S. Ali Mard, A. Veisi, A comprehensive review on regulatory effects of crocin on ischemia/reperfusion injury in multiple organs, Biomed. Pharmacother. 99 (2018) 664–670. [28] D.D. Liu, Y.L. Ye, J. Zhang, J.N. Xu, X.D. Qian, Q. Zhang, Distinct pro-apoptotic properties of Zhejiang saffron against human lung cancer via a caspase-8-9-3 cascade, Asian Pac. J. Cancer Prev. 15 (15) (2014) 6075–6080. [29] E.Z. Longa, P.R. Weinstein, S. Carlson, R. Cummins, Reversible middle cerebral artery occlusion without craniectomy in rats, Stroke 20 (1) (1989) 84–91. [30] Y.L. Ye, W.Z. Shi, W.P. Zhang, M.L. Wang, Y. Zhou, S.H. Fang, et al., Cilostazol, a phosphodiesterase 3 inhibitor, protects mice against acute and late ischemic brain
10
Biomedicine & Pharmacotherapy 126 (2020) 110041
K. Zhong, et al.
deficits through increasing regional cerebral blood flow and alleviating inflammation in CCI rats, Evid. Complement. Alternat. Med. 2017 (2017) 5173168. [63] M.R. Khazdair, M.H. Boskabady, M. Hosseini, R. Rezaee, A.M. Tsatsakis, The effects of Crocus sativus (saffron) and its constituents on nervous system: a review, Avicenna J. Phytomed. 5 (5) (2015) 376–391.
[60] M.V. Sofroniew, Molecular dissection of reactive astrogliosis and glial scar formation, Trends Neurosci. 32 (12) (2009) 638–647. [61] J.E. Burda, M.V. Sofroniew, Reactive gliosis and the multicellular response to CNS damage and disease, Neuron 81 (2) (2014) 229–248. [62] D. Han, Z. Liu, G. Wang, Y. Zhang, Z. Wu, Electroacupuncture improves cognitive
11
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Neuroprotective effects of saffron on the late cerebral ischemia injury through inhibiting astrogliosis and glial scar formation in rats
T
Zhong Kaia,1, Wang Rou-Xina,1, Qian Xiao-Dongb, Yu Pinga, Zhu Xin-Yinga, Zhang Qia, Ye Yi-Lua,* a b
School of Basic Medical Sciences & Forensic Medicine, Hangzhou Medical College, Hangzhou, Zhejiang, China Huzhou Central Hospital, Huzhou, Zhejiang, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Saffron (Crocus sativus L.) Focal cerebral ischemia Astrogliosis Glial scar Inflammatory reaction
This study is to explore the neuroprotective effects and involved glial scar of saffron (Crocus sativus L.) on the late cerebral ischemia in rats. Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in Sprague Dawley rats that were randomly divided into sham group, MCAO group, edaravone group (as a positive control) and saffron groups (saffron extract 30, 100, 300 mg/kg). Saffron was administered orally at 2 h at the first day and once daily from day 2 to 42 after ischemia. Behavioral changes were detected from day 43 to 46 after ischemia to evaluate the effects of saffron. Infarct volume, survival neuron density, activated astrocyte, and the thickness of glial scar were also detected. GFAP, neurocan, phosphocan, neurofilament expressions and inflammatory cytokine contents were detected by Western-blotting and ELISA methods, respectively. Saffron improved the body weight loss, neurological deficit and spontaneous activity. It also ameliorated anxiety-like state and cognitive dysfunction, which were detected by elevated plus maze (EPM), marble burying test (MBT) and novel object recognition test (NORT). Toluidine blue staining found that saffron treatment decreased the infarct volume and increased the neuron density in cortex in the ischemic boundary zone. The activated astrocyte number and the thickness of glial scar in the penumbra zone reduced after saffron treatment. Additionally, saffron decreased the contents of IL-6 and IL-1β, increased the content of IL-10 in the ischemic boundary zone. GFAP, neurocan, and phosphocan expressions in ischemic boundary zone and ischemic core zone all decreased after saffron treatment. Saffron exerted neuroprotective effects on late cerebral ischemia, associating with attenuating astrogliosis and glial scar formation after ischemic injury.
1. Introduction Cerebral ischemia is a common clinical cerebrovascular disease characterized by a high rate of disability and imposes a heavy burden on society and patients [1]. So greater attention should be paid to improve strategies for neurological recovery after late cerebral ischemia injuries. The neurogenesis, astrogliosis/glial scar formation and angiogenesis are the most important morphologic changes (brain remodeling) in the late phase after ischemic or other brain injuries [2]. During the process of brain remodeling after ischemia, astrocytes are activated and become hypertrophic, hyperplastic. The activated astrocytes can up-regulate the glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycan, such as neurocan and phosphocan, which are the iconic protein of astrogliosis and glial scar [3–5]. Excessive expression of GFAP is closely related to the inflammatory response [6,7]. Microglia, astrocyte and macrophages release
inflammatory cytokines involved in the initial reaction, such as IL-β and TNF-α. These inflammatory cytokines can activate astrocytes, which then secrete more inflammatory cytokines and promote astrogliosis [8]. Aspects of the astrocytic inflammatory response to stroke may aggravate the ischemic lesion [8,9], but astrocytes also limit lesion extension via anti-excitotoxicity effects and release neurotrophins. Similarly, during the late phase after cerebral ischemia, the glial scar may block spontaneous axonal regeneration and subsequently reduce the functional outcome [10–12]. Thus, inhibition of the excessive astrogliosis and glial scar formation might be a therapeutic strategy for ischemic stroke [13,14]. Saffron, the dry stigmas of the plant Crocus sativus L., is a species belonging to the Iridaceae family and has been widely used as a herbal medicine and spice since ancient times. Pharmacological studies have demonstrated that saffron and its constituents protected against neuropsychiatric injury, especially including anxiety neurosis and
⁎
Corresponding author. E-mail address: [email protected] (Y.-L. Ye). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.biopha.2020.110041 Received 28 August 2019; Received in revised form 18 February 2020; Accepted 19 February 2020 0753-3322/ © 2020 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Biomedicine & Pharmacotherapy 126 (2020) 110041
K. Zhong, et al.
depression [15,16], ischemia [17,18], Alzheimer disease (AD) [19], Parkinson's disease (PD) [20,21] as well as other neurodegenerative diseases [22] in vivo and in vitro. Previous researches have reported that saffron and its active constituents, including crocin and safranal, both played neuroprotection against cerebral ischemic injury [23–27]. However, the neuroprotective effects of saffron and the involved astrogliosis and glial scar formation on the late cerebral ischemia are unknown. Thus, the purpose of the present study was to investigate the neuroprotective effect of saffron on the late cerebral ischemia and involved astrogliosis in the course of glial scar formation.
Table 1 Contents of active components in the decoction of saffron (%).
Contents (%) n
Crocin
Picrocrocin
Safranal
9.48 ± 0.07 6
5.06 ± 0.04 6
0.23 ± 0.06 6
2.3. Focal cerebral ischemia Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). After anesthesia with pentobarbital (700 mg/kg), a nylon filament (Guangzhou Jialing Biotechnology Co., Ltd., Guangzhou, China) was inserted into the internal carotid artery, and advanced approximately 20−25 mm distal to the carotid bifurcation to occlude the origin of the middle cerebral artery (MCA). The filament was carefully withdrawn 90 min after MCAO. In sham-operated rats, the same procedure was done without advancing of the filament into the MCA. Body temperature was maintained constantly at 37.0 ± 0.5℃ using a thermostatically controlled heating blanket during and after the surgery until rats recovered from anesthesia. Total 2 rats with intracranial hemorrhage were excluded, which were evaluated by neurological deficit score and brain dissection at 24 h after ischemia.
2. Materials and methods 2.1. Animals and treatment Male Sprangue-Dawley rats (250−280 g) were provided by Shanghai Slyke Laboratory Animal Limited Corporation (Certificate No. SCXK 2012-0002, China). All of them were kept in the same room under controlled environmental conditions (12:12 h light/dark cycle, ambient temperature of 22 ± 1 °C, relative humidity of 50 ± 10 %) with free access to food and water. The procedures were approved by the local committee for Animal Health, Ethics and Research of Hangzhou Medical college (No. 201713, Hangzhou, China). Rats were randomly divided into 6 groups: Sham group (saline, n = 7/7), MCAO model group (saline, n = 8/9), edaravone + MCAO group (edaravone 3 mg/ kg, n = 9/9) and saffron + MCAO groups (saffron extract 30, 100, 300 mg/kg, n = 8/9, 9/9, 8/9, respectively). Total 2 rats with intracranial hemorrhage were excluded, which were evaluated by neurological deficit score and brain dissection at 24 h after ischemia. They were administered with oral gavage at 2 h after ischemia in the first day and once daily from day 2 to 14 after ischemia. As a positive control, edaravone (3 mg/kg), a free radical scavenger, was used to improve the neurological symptoms and dysfunction caused by cerebral ischemia. The body weight was evaluated at 9 AM at day 1, 3, 7, 14, 21, 28, 35 and 42 after ischemia. Open field test (OFT), novel object recognition test (NORT), elevated plus maze (EPM) and marble burying test (MBT) were conducted from day 43 to 46 after ischemia, respectively. The experimental schedule was provided in the Fig. 1.
2.4. Neurological deficit evaluation The neurological deficit score was evaluated as described: 0, no deficit; 1, failure to fully extend left forepaw; 2, circling to the left; 3, falling to the left; 4, no spontaneous walking with a depressed level of consciousness [29]. Rats were evaluated at day 1, 3, and 7 in the first week after ischemia, and once every week thereafter. 2.5. OFT Each rat was gently put into the center of a brightly-lit open field arena (80 × 80 × 40 cm). The locomotor tracks were continuously recorded by Digbehv spontaneous activity system (Xinruan, Shanghai, China) for 5 min and analyzed by the EthoVision XT software (Noldus, Netherlands). Traveled total distance and traveled velocity were used to describe the spontaneous activity of rats. Time in the central zone was used to evaluate exploratory and anxiety-like behaviors.
2.2. Saffron extract preparation 2.6. NORT Saffron stigma 10 g was crushed and soaked with 75 % ethanol solution 200 ml for 20 min. Then they were treated with ultrasonic bath for 15 min for twice and refluxed with 70℃ water for 30 min. The filtrate was obtained by suction filtration. The residue was soaked with 75 % ethanol solution 200 ml again and repeated all the procedure until the residue turned white. At last, all the filtrates were mixed and concentrated in a rotary evaporator under reduced pressure to a concentration of 1.0 g/ml. The contents of active components were shown in the Table 1 by HPLC method [28]. (Table 1)
During habituation, rats were introduced into the empty chamber (80 × 80 × 40 cm) and left to freely explore it for 10 min. Afterwards, they were put into their home cages for 5 min, during which two identical objects (familiar object: cubic crude wood with 4 cm diameter) were placed in the chamber oppositely. The test was consisted of 3 phases: T1 phase, interval time, and T2 phase. In T1 phase, rats were allowed to explore the identical objects for 5 min. After 1.5 h interval time, rats were placed into the chamber again for 5 min, when one of
Fig. 1. Schedule of the experimental protocol. 2
Biomedicine & Pharmacotherapy 126 (2020) 110041
K. Zhong, et al.
the familiar objects was replaced by a novel one (novel object: cylindrical crude wood with 4 cm diameter and height), that’s T2 phase. Tracks were recorded and analyzed by a video analyzer (Ethovision XT, Noldus, Netherlands). The following parameters were analyzed: discrimination ratio (DR, DR = [N / (N + F)] × 100 %) and discrimination index (DI, DI = [(N - F) / (N + F)] × 100 %). N: exploration time with the novel object (min); F: exploration time with the familiar object (min). Exploration is defined as the distance between the nose of the rat facing the object and the object is within 2 cm.
performed with the polyclonal rabbit antibodies against GFAP (1:200, Santa Cruz). The brain cryo-sections preparation and immunohistochemical methods were based on our previous report [30]. Normal goat serum was used instead of the primary antibody in the control sections. The number of positive cells and the thickness of glial scar in the ischemic boundary zone were calculated with three different fields. The researcher, who was blind to the experimental groups.
2.7. EPM
Rats were decapitated and the ischemic boundary zone was separated on ice quickly. Then it was sonicated to obtain tissue homogenates. After removing particulars by centrifugation (2000×g, 4 °C, 20 min), assay was immediately detected. IL-1β, TNF-α, IL-6 and IL-10 were measured using respective ELISA systems (Shanghai Elisa Biotech Co., Ltd, China) according to the manufacturer’s instructions. The optical density was determined (absorbance at 450 nm) on a plate reader. Finally, the contents of IL-1β, TNF-α, IL-6 and IL-10 were calculated according to the corresponding standard curve.
2.11. Inflammatory cytokines contents detection
In the behavioral laboratory with diffuse and low lighting, EPM was conducted. At the beginning of the test, rats were placed at the center of the maze and faced an open arm. Then they were allowed to explore the maze freely for 5 min. Tracks were recorded and analyzed by a video analyzer (Ethovision XT, Noldus, Netherlands). Total distance traveled in all arms and central zone, distance ratio, total time and time ratio in open arms were measured to evaluate the anxiety-like behavior after late cerebral ischemia.
2.12. Western-blotting 2.8. MBT Samples of the ischemic boundary zone and ischemic core zone were homogenized on ice in PBS containing 1 % protease inhibitor and 1 % phosphatase inhibitor for Western-blotting. Proteins were obtained by centrifugation at 14,000 rpm at 4℃ for 15 min and quantified by Bradford assay. 50 μg sample of each rat was subjected to electrophone using 20 % SDS at 80 V. The proteins were transferred to polyvinylidene fluoride membranes at 250 mA for 2 h. Antibodies for antiGFAP (1:5000, Huabio), anti-neurocan (1:500, Abcam), anti-phosphocan (1:5000, Sigma), neurofilament H (1:500, Abcam) and GAPDH (1:10000, KangChen Bio-tech, Shanghai, China) were applied overnight. Then membranes were incubated with horseradish peroxidase (HRP) secondary antibody (KangChen Bio-tech, Shanghai, China). At last, the signal intensities of proteins were analyzed using Image J software.
Opaque cages (40 × 36 × 20 cm) were covered with 5 cm layer of corncob. Rats were placed individually to habituate the cage for 15 min. Then they were returned to their home cage. Meanwhile 20 clear blue glasses marbles (12 mm diameter) were evenly spaced in the habituation cages. After that, rats were reintroduced into the cage which they had been habituated. After 30 min, the test was terminated and the number of marbles was counted, which were covered with corncob by more than 2/3 vol. 2.9. Surviving neurons and infarct volume detection After anesthesia, rats were perfused transcardially with saline followed by 4 % paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) after ischemia. Then, the brains were post-fixed overnight in the same fixative, and immersed in 30 % sucrose solution in the phosphate buffer. The brains were dissected into 2 mm-thick coronal slices for 6 brain slices. The coronal sections (20 μm or 10 μm) were prepared by cryomicrotomy (CM1900; Leica, Germany) from each slice and stained with 1 % toluidine blue. The 20 μm-thick sections were used to detect the infarct volume, and the 10 μm-thick sections were used for microphotographic examination. For infarct volume, the 6 stained slices were photographed by a charge coupling device (CCD) camera and images were recorded on a computer. Infarct area and both hemisphere areas of each slice were determined by using an image analysis program (AnalyPower 2.0; Zhejiang University, Hangzhou, China) as reported elsewhere [30]. Infarct volume was calculated as total infarct area × thickness (2 mm). The summation of the infarct volumes of all brain slices were the total infarct volume. Then, the 10-μm-thick sections were used for histopathological examination and counting neuron densities in the temporal cortex in the ischemic boundary zone. Three different fields in each brain slice in bregma 0.0 mm and -4.0 mm were captured. The average numbers of healthy-looking neurons (cells with pyramidal-like appearance and larger sizes, but without shrunken appearance) in layers III-IV of the temporoparietal cortex were counted in sections stained with 1 % toluidine blue. The researcher, who was blind to the experimental groups, used Image J software to calculate the neuron number automatically.
2.13. Statistical analysis Data were expressed as mean ± SEM. Repeated measures of oneway analysis of variance (ANOVA) and Tukey post-hoc tests were performed to analyze the significance of any treatment effects among the groups by SPSS 19.0 for Windows except the effects of saffron on NORT. t-test was used to compare the difference of DR and DI between T2 phase and T1 phase. p < 0.05 was considered statistically significance. 3. Results 3.1. Saffron improved the body weight loss and neurological deficit As shown in Fig. 2A, the body weight in sham group grown quickly while the model group obviously decreased from day 14 to day 42 after ischemia (F(5,43) = 1.228, 1.641, 2.088, 2.580, 3.419 at day14, 21, 28, 35, 42 after ischemia, respectively. p < 0.05). The body weight loss was improved after treatment with saffron 100 mg/kg (F(5,43)=3.419, p = 0.040), 300 (F(5,43)=3.419, p = 0.015) at day 42 after ischemia. Edaravone also improved the body weight loss from day 35 (F(5,43) =2.580, p = 0.045) to day 42 (F(5,43)=3.419, p = 0.044) after ischemia. Fig. 2B showed there was severe neurological deficit after ischemia. Saffron 100 mg/kg and edaravone treatment improved the neurological deficit from day 28–42 after ischemia (F(5,43) = 5.458, 5.276, 6.438 at day 28, 35, 42 after ischemia, respectively. p < 0.05). Saffron 300 mg/ kg improved the neurological deficit from day 35–42 (F(5,43)= 5.276, 6.438 at day 35, 42 after ischemia, respectively. p < 0.05). Saffron (30
2.10. Astrocyte activation/glial scar measurement To investigate the astrocyte activation/glial scar formation in the ischemic boundary zone, immunohistochemical methods were 3
Biomedicine & Pharmacotherapy 126 (2020) 110041
K. Zhong, et al.
Fig. 2. Effects of saffron on the body weight loss and neurological deficit. Body weight (A) and neurological deficit score (B) were measured every other day in the first week and every week from the second week. Values are presented as the mean ± SEM. Statistical analysis were carried out by one-way ANOVA and Tukey posthoc test with *P < 0.05, **P < 0.01 against the sham group and #P < 0.05, ##P < 0.01 against the model group.
3.3. Saffron improved the cognitive function deterioration
mg/kg) improved the neurological deficit only at day 42 after ischemia (F(5,43)=6.438, p < 0.05).
Compared with the T1 phase in the same group, there was no difference of DR and DI in model group (Fig. 4B, F(1,14) = 2.873, p = 0.940; Fig. 4C, F(1,14) = 1.904, p = 0.480), edaravone group (Fig. 4B, F(1,16) = 0.007, p = 0.065; Fig. 4C, F(1,16) = 0.007, p = 0.0.065) and saffron 30 mg/kg (Fig. 4B, F(1,14) = 2.719, p = 0.054; Fig. 4C, F (1,14) = 3.735, p = 0.075) group in T2 phase compared with T1 phase. However, DR and DI all increased in sham group (Fig. 4B, F(1,12) = 3.817, p = 0.047; Fig. 4C, F(1,12) = 0.108, p = 0.045), saffron 100 (Fig. 4B, F(1,16) = 0.111, p = 0.030; Fig. 4C, F(1,14) = 0.121, p = 0.041) and 300 mg/kg (Fig. 4B, F(1,14) = 0.102, p = 0.040; Fig. 3C, F (1,14) = 0.069, p = 0.014) groups in T2 phase when compared with T1 phase.
3.2. Saffron improved locomotor activity After ischemia, rats showed lower traveled velocity (F(5, 43) = 3.128, p = 0.028) and total traveled distance (F(5, 43)=2.320, p = 0.009) in open field. Saffron 100 mg/kg (F(5, 43)=3.128, p = 0.003), 300 mg/kg (F(5, 43)=3.128, p = 0.014) and edaravone (F(5, 43) =3.128, p = 0.008) increased the velocity (Fig. 3C). Saffron 300 mg/ kg (F(5, 43) = 2.320, p = 0.028) and edaravone (F(5, 43)=2.320, p = 0.012) increased the total distance (Fig. 3D). There was no effect on the velocity (Fig. 3C, F(5, 43) = 3.128, p = 0.217) and total traveled distance (Fig. 3D, F(5, 43) = 2.320, p = 0.202) after saffron (30 mg/ kg) treatment. The central time decreased after ischemia (Fig. 3E, F(5, 43) = 5.063, p = 0.025). Saffron 300 mg/kg (F(5, 43)=5.063, p = 0.046) and edaravone (F(5, 43)=5.063, p = 0.035) increased the central time in OFT (Fig. 3E).
3.4. Saffron ameliorated anxiety-like behaviors In EPM, as Fig. 5B shown, there was no difference in the total distance in open and close arms among groups (Fig. 5B, F(5,43) = 0.716,
Fig. 3. Effects of saffron on traveled velocity, traveled total distance and central time in OFT. The heatmaps (A) and the tracks (B) in OFT were presented. Traveled velocity (C), traveled total distance (D) and central time (E) were measured to evaluate the locomotor activity in rats with OFT. Values are presented as the mean ± SEM. Statistical analysis were carried out by one-way ANOVA and Tukey post-hoc test with *P < 0.05 against the sham group and #P < 0.05, ##P < 0.01 against the model group. 4
Biomedicine & Pharmacotherapy 126 (2020) 110041
K. Zhong, et al.
Fig. 4. Effects of saffron on DR and DI in NORT. The heatmaps in all groups in NORT were presented in Fig. 4A. DR (B) and DI (C) were used to evaluate the cognitive function in NORT. Values are presented as the mean ± SEM. Statistical analysis were carried out by t-test with *P < 0.05 against T1 phase in the same group.
3.5. Saffron reduced the infarct volume after ischemia
p > 0.05). However, distance ratio (Fig. 5C, F(5,43) = 2.211, p=0.045), total time (Fig. 5D, F(5,43) = 2.047, p = 0.049) and time ratio (Fig. 5E, F(5,43) = 1.259, p = 0.013) in open arms significantly decreased after ischemia. Saffron 30, 100, 300 mg/kg (F(5,43)=2.211, p = 0.022, 0.010, 0.005) and edaravone (F(5,43)=2.211, p = 0.024) increased the distance ratio in open arms (Fig. 5C). Saffron 100, 300 mg/kg (Fig. 5D, F(5,43) = 2.047, p = 0.018, 0.032) increased the total time in open arms. Saffron 30, 100 mg/kg (F(5,43)=1.259, p = 0.040, 0.046) and edaravone (F(5,43)=1.259, p = 0.041) increased the time ratio in open arms (Fig. 5E). In MBT, the buried marbles number significantly increased when compared with the sham group (Fig. 5F, F(5,43) = 2.282, p = 0.048). Saffron 100, 300 mg/kg (Fig. 5F, F(5,43) = 2.282, p = 0.023, 0.006) and edaravone (Fig. 5F, F(5,43) = 2.282, p = 0.002) reversed the increase of buried marbles number, which manifested by the improvement of anxiolytic state with saffron treatment.
At day 46 after ischemia, an obvious brain atrophy in the ischemic hemispheres was shown in the gross photographs of whole brains and brain sections (bregma –4.0 mm) with toluidine blue staining, whereas it was improved with edaravone or saffron treatment (Fig. 6A). The results of toluidine blue showed the infarct volume hasn’t been found in the sham group. The obvious infarct volume in model group and it was significantly reduced by saffron 100 (F(5,30) = 18.385, p = 0.014), 300 (F(5,30)=18.385, p = 0.022) mg/kg and edaravone (F(5,30) =18.385, p = 0.000) after ischemia, but not saffron 30 mg/kg (F(5,30) =18.385, p = 0.106) (Fig. 6B).
Fig. 5. Effects of saffron on the distance, time in EPM and buried marble number in MBT. The typical tracks in EPM (A) were presented in rats. The total distance in open and close arms (B), distance ratio in open arms (C), total time in open arms (D), and time ratio in open arms (E) were measured to evaluate the anxiety-like behaviors in rats with EPM. Schematic and buried marbles number was measured to evaluate the anxiety-like behaviors in MBT (F). Values are presented as the mean ± SEM. Statistical analysis were carried out by one-way ANOVA and Tukey post-hoc test with *P < 0.05 against the sham group and #P < 0.05, ##P < 0.01 against the model group. 5
Biomedicine & Pharmacotherapy 126 (2020) 110041
K. Zhong, et al.
Fig. 6. Effects of saffron on the infarct volume after ischemia. Representative photographs of brains and brain sections with toluidine blue staining (bregma -4.0 mm) showed the infarct volume (A) and the quantity of infarct volume (B) in all groups. Values are presented as the mean ± SEM. Statistical analysis were carried out by one-way ANOVA and Tukey post-hoc test with #P < 0.05, ##P < 0.01 against the model group.
13.416, p = 0.000) and edaravone (F(5,30)=13.416, p = 0.000) significantly decreased the GFAP-positive cells number in the ischemic boundary zone (Fig. 8B). In addition, saffron 100 (F(5,30) = 13.399, p = 0.000), 300 (F(5,30)=13.399, p = 0.000) mg/kg and edaravone (F (5,30)=13.399, p = 0.000) reduced the thickness of the scar wall by 55.2 %, 61.6 % and 52.4 %, respectively (Fig. 8D).
3.6. Saffron increased survival neuron density in cortex in the ischemic boundary zone The penumbra zone surrounds the ischemic core of the stroke lesion and is comprised of cells that are stressed and vulnerable to death, which is due to an altered metabolic, oxidative and ionic environment within the penumbra. There is hope that appropriate therapies may allow potential recovery of cells within this tissue region, or at least slow the rate of cell death, therefore, slowing the spread of the ischemic infarct and minimising the extent of tissue damage. As such, preserving the penumbra to promote functional brain recovery is a central goal in stroke research [31]. Representative photographs showed the densities of apparently survival neurons in temporoparietal cortex in the ischemic boundary zone. Neuron density was substantially reduced in temporoparietal cortex in the ischemic boundary zone after ischemia (Fig. 7A–B, F(5,30) = 17.320, p = 0.000). Saffron 100 (F(5,30)=17.320, p = 0.000), 300 (F(5,30)=17.320, p = 0.000) mg/kg and edaravone (F(5,30)=17.320, p = 0.000) attenuated the neuron loss in the ischemic boundary zone. But saffron 30 mg/kg had no effect on the survival neuron density (Fig. 7B, F(5,30) = 17.320, p = 1.000).
3.8. Saffron changed GFAP, neurocan, phosphocan, and neurofilament expressions in the ischemic boundary zone and ischemic core zone The results of Western-blotting showed there were a few GFAP (Fig. 9B–C), neurocan (Fig. 9D, 9 G), and phosphocan (Fig. 9E, 9 H) expressions in the ischemic boundary zone and ischemic core zone in the sham group. While they all significantly increased after ischemia (Fig. 9B-E, G–H, p < 0.05). Saffron 300 mg/kg and edaravone treatment reversed the expressions of GFAP (F(3,20)=24.013 in the ischemic boundary zone and F(3,20)=50.208 in the ischemic core zone, p < 0.05), neurocan (F(3,20)=23.743 in the ischemic boundary zone and F(3,20)=13.903 in the ischemic core zone, p < 0.05), and phosphocan (F(3,20)=7.872 in the ischemic boundary zone and F(3,20) =4.007 in the ischemic boundary zone, p < 0.05) (Fig. 9B-E, G–H), except phosphocan expression in the ischemic core zone after edaravone treatment (Fig. 9H, F(3,20) = 4.007, p = 0.051). But there was no difference between groups of neurofilament H expression in the ischemic boundary zone and ischemic core zone (Fig. 9F, I, F(3,20) = 1.523 in the ischemic boundary zone and F(3,20) = 3.173 in the ischemic core zone, p > 0.05), except saffron 300 mg/kg (F(3,20) =1.523, p = 0.047) treatment increased neurofilament H expression in the ischemic boundary zone (Fig. 9F).
3.7. Saffron inhibited the astrocyte activation and reduced the glial scar formation In the boundary zone, the density of GFAP-positive astrocytes was greatly increased after ischemia (Fig. 8A–B, F(5,30) = 13.416, p = 0.001). The intense astrocytic fibers surrounded the ischemic boundary zone and glial scar was formed at day 46 after ischemia (Fig. 8C). The infarct tissue was transformed into the center cavitation of a scar. Intense GFAP-positive astrocytic fibers were present in the scar wall parallel to the cavitation. Reactive astrocytes with hyperplasia/hypertrophy were prominent in the scar. Saffron 300 mg/kg (F(5,30) =
3.9. Saffron changed the contents of inflammatory cytokines The results of ELISA showed the contents of IL-6 (Fig. 10A, F(5,12) 6
Biomedicine & Pharmacotherapy 126 (2020) 110041
K. Zhong, et al.
Fig. 7. Effects of saffron on the survival neuron density in cortex in the ischemic boundary zone. Typical photos showed the density and the morphology of the survival neuron in the ischemic boundary zone (A). And the survival neuron density was calculated in the temporoparietal cortex in the ischemic boundary zone (B). Values are presented as the mean ± SEM. Statistical analysis were carried out by one-way ANOVA and Tukey post-hoc test with **P < 0.01 against the sham group and ##P < 0.01 against the model group. Scale bar=50 μm in Fig. 6A.
effects of saffron on late ischemic injury in rats, which manifested by improvement of body weight loss, neurological deficit, spontaneous activity, anxiety-like behaviors, cognitive dysfunction. Also, it decreased the infarct volume and increased the survival neuron density in cortex. According to the results, the most effective dosage of saffron was 300 mg/kg. As a positive control, edaravone was proven neuroprotective effects on late cerebral ischemia, which had improvement of neurological deficit and behavioral deterioration, as well as inhibition of glial scar formation. In this study, the first treatment was conducted at 2 h after the MCAO surgery. It is extremely important to clarify the requirement for studies that explore novel treatments for cerebral ischemia. Based on a preliminary experiment, we have explored the time-dependent effect of saffron. It (300 mg/kg) was injected at 1, 2 and 3 h after ischemia. The infarct volume and neurological deficit score were evaluated (data not shown). The results showed the therapeutic time window was within 2 h after ischemia. So for evaluating the effect on the late ischemic injury, multiple doses of saffron (30, 100, 300 mg/kg) were injected at 2 h
= 3.236, p = 0.012) and IL-1β (Fig. 10C, F(5,12) = 6.617, p = 0.000) increased and IL-10 decreased (Fig. 10B, F(5,12) = 4.878, p = 0.013) after cerebral ischemia. But the content of TNF-α didn’t change after ischemia (Fig. 10D, F(5,12) = 0.280, p = 0.902). Saffron 100, 300 mg/ kg and edaravone treatment reversed the contents of IL-6 (Fig. 10A, F (5,12) = 3.236, p = 0.014, 0.007, 0.020) and IL-10 (Fig. 10B, F(5,12) = 4.878, p = 0.002, 0.001). Also Saffron (30, 100, 300 mg/kg) and edaravone treatment reversed the content of IL-1β (F(5,12)=6.617, p = 0.003, 0.006, 0.002, 0.000) (Fig. 10C). Whether saffron or edaravone treatment didn’t affect the content of TNF-α in the ischemic boundary zone (Fig. 10D, F(5,12) = 0.280, p > 0.05). 4. Discussion Several studies have demonstrated that saffron has protective effects against ischemia-reperfusion injury by antioxidant property [25] in acute cerebral ischemia and improving recognition and spatial memory dysfunction [23]. The present study investigates the neuroprotective
Fig. 8. Effects of saffron on the GFAP-positive cells number and the thickness of glial scar. Typical photo showed the astrocyte activation (A) and glial scar formation in the ischemic boundary zone (C). The GFAP-positive cells number (B) and the thickness of glial scar (D) were detected in the ischemic boundary zone. Values are presented as the mean ± SEM. Statistical analysis were carried out by one-way ANOVA and Tukey post-hoc test with **P < 0.01 against the sham group and ## P < 0.01 against the model group. Scale bar = 50 μm in Fig. 7A and 500 μm in Fig. 7C. 7
Biomedicine & Pharmacotherapy 126 (2020) 110041
K. Zhong, et al.
Fig. 9. Effects of saffron on GFAP, neurocan, phosphocan and neurofilament H expressions in the ischemic boundary zone and ischemic core zone. Typical photos showed the contents of GFAP, neurocan, phosphocan and neurofilament expressions in the ischemic boundary zone and ischemic core zone by Western-blottingt (A). The contents of GFAP expression were presented in the ischemic boundary zone (B) and in the ischemic core zone (C). The contents of neurocan expression were presented in the ischemic boundary zone (D) and in the ischemic core zone (G). The contents of phosphocan expression were presented in the ischemic boundary zone (E) and in the ischemic core zone (H). The contents of neurofilament expression were presented in the ischemic boundary zone (F) and in the ischemic core zone (I). Values are presented as the mean ± SEM. Statistical analysis were carried out by one-way ANOVA and Tukey post-hoc test with *P < 0.05, **P < 0.01 against the sham group and #P < 0.05, ##P < 0.01 against the model group.
after ischemia in the first day and once daily from day 2 to day 14 after ischemia. After ischemia, it was often accompanied with several behavioral changes, including neurological deficit [32], spontaneous activity impairment [33], cognitive deterioration [34], and anxiety-like behaviors [35]. It corroborated the beneficial effects of saffron after MCAO by demonstrating that it improved body weight loss and neurological deficit at day 46 after ischemia. Additionally, toluidine blue staining showed that treated animals had significantly more survival neurons in the ischemic boundary zone and decreased the infarct volume at the late cerebral ischemia. These survival neuron cells most likely to migrate towards the infarct to replenish cortical and striatal neurons [30]. So, it was indicated that the better neurological recovery observed in these animals correlate with the more survival neurons. We also found there was an advantage of saffron on the behavioral dysfunction after late cerebral ischemia/reperfusion injury in rats. The traveled total distance and velocity are often used to evaluate the spontaneous activity in OFT. Our results showed that the spontaneous activity declined after ischemia. Saffron (100, 300 mg/kg) increased the velocity, total distance and central time, especially at the dosage of 300
mg/kg in late cerebral ischemia. It was in line with the improvement of the neurological deficit and manifested that saffron could promote the rehabilitation of stroke. Furthermore it has been found that the spatial organization is the most important property of locomotor activity in addition to the total distance [36]. Animals with lower anxiety and exploratory behavior tend to spend more time in the central, open area of the box [37], which was in accordance with the results of NORT. Accumulated researches had reported that saffron or its active constituent could improve the neurological deficit and memory impairment [23,38]. In the present study, we used NORT (a common experimental method to assess cognitive skills) [39,40] just to find saffron (100, 300 mg/kg) improved the cognitive decline especially. These results verified the neuroprotective effects of saffron in behavioral improvement in rats. EPM and MBT were both used to evaluate the anxiety-state after cerebral ischemia [41,42] and further confirmed the anti-anxiolytic effects of saffron [43,44]. These battery of behavioral tests highlighting long-term deficits could be useful to study future neuroprotective strategies for stroke treatment. The other novel findings of the current study are that saffron attenuates astrogliosis and glial scar formation in response to cerebral 8
Biomedicine & Pharmacotherapy 126 (2020) 110041
K. Zhong, et al.
Fig. 10. Effects of saffron on the contents of inflammatory cytokines in ischemic boundary zone. IL-6 (A), IL-10 (B), IL-1β (C) and TNF-α (D) contents in the ischemic boundary zone were detected by ELISA methods. Values are presented as the mean ± SEM. Statistical analysis were carried out by one-way ANOVA and Tukey post-hoc test with *P < 0.05, **P < 0.01 against the sham group and #P < 0.05, ## P < 0.01 against the model group.
speculate that this phenomenon may be related to the different stage of cerebral ischemia and animal model. More than 150 compounds have been identified in saffron stigma. But the main active components of saffron were crocin, picrocrocin and safranal [63], which was consistent with our results. Saffron or Crocus sativus L. (C. sativus) has been widely used as a medicinal plant to promote human health, especially in Asia. So, in this study we only explore the neuroprotective effects of saffron extract. We will further explore the neuroprotective effect of isolated active components, such as crocin, picrocrocin and safranal, on the late cerebral ischemia. In conclusion, our findings reveal that saffron can attenuate astrogliosis and glial scar formation after ischemic brain injury, following with neurological deficit improvement, which provides a new insight into understanding of the protective effect of saffron for clinical treatment of ischemic stroke and may be developed into new therapeutics for ischemic stroke.
ischemic insult. It significantly decreased the expression of GFAP, neurocan, and phosphocan in the ischemic boundary zone at day 46 after ischemia. Astrocytes, which constitute almost a third of cells in the central nervous system (CNS), participate in circuit [45,46], metabolism of energy substrates [47], axonal regeneration [48], and behavioral functions [49]. After severe injury insult in the CNS, the potential mechanisms of the axons failure to regenerate across tissue lesions could include absence of external inhibitory factors [50], stimulating factors [51], or promotion of astrogliosis and astrocyte scar formation [52], etc. In late stage, the activated astrocytes can up-regulate the GFAP and chondroitin sulfate proteoglycan, such as neurocan and phosphocan, which are the iconic protein of astrogliosis and glial scar [5], and inhibit axonal regeneration and release axonal growth inhibitors [53,54]. Some traditional Chinese medicine exerted protective effects on late cerebral ischemia through inhibiting astrogliosis and glial scar formation [55,56], which was in line with our results of saffron. It may be a useful strategy to study the neuroprotective effects for stroke. However, there was no difference on neurofilament H among groups, except increased level of neurofilament H in saffron 300 mg/kg group in the ischemic boundary zone when compared with the model group. The neurofilament protein of the CNS consists of three subunits: neurofilament light (NFeL), neurofilament medium (NFeM) and neurofilament heavy (NFeH), which are named according to their molecular weights. Bianca Mages et al. found NFeL increased in the ischemic penumbra [57] and increased serum levels of phosphorylated NFeH correlated with the clinical outcome of stroke patients [58]. It is suggested that neurofilament H in the ischemic brain tissue may not be a sensitive indicator of glial scar. Recently, it has been shown that reactive astrocytes upregulated over 1000 genes, including cytokines, induced in response to injury and wound in peripheral and central nervous system [59]. Although GFAP expression is maintained in glial scars for a prolonged period after injury [60], most expression changes are transient. However, proinflammatory cytokines remain persistently elevated after both ischemic and inflammatory insults, which plays a major role in reactive gliosis and scar formation [61]. Saffron decreased the expression of IL-6, IL-1β and increased the expression of IL-10, but there was no effect on the expression of TNF-α. It was similar to the document reported [62], which manifested the difference of TNF-α level was not apparent. So we
Author contributions Zhong Kai and Wang Rou-Xin performed the experiments, collected the data and wrote the manuscript. Wang Rou-Xin, Yu Ping and Zhu Xin-Ying performed the animal experiments and analyzed the data. Qian Xiao-Dong contributed to the design of the study. Zhang Qi and Ye Yi-Lu interpreted the data and revised the manuscript. Declaration of Competing Interest All authors declare no conflicts of interests. All authors agree to submit the manuscript. Acknowledgments The work was supported by the Project of Health and Family Planning Commission of Zhejiang Province (2018239491). References [1] Z. Chen, B. Jiang, X. Ru, H. Sun, D. Sun, X. Liu, et al., Mortality of stroke and its subtypes in China: results from a nationwide population-based survey, Neuroepidemiology 48 (3–4) (2017) 95–102.
9
Biomedicine & Pharmacotherapy 126 (2020) 110041
K. Zhong, et al.
injuries, Eur. J. Pharmacol. 557 (1) (2007) 23–31. [31] E. Horvath, A. Hutanu, L. Chiriac, M. Dobreanu, A. Oradan, E.E. Nagy, Ischemic damage and early inflammatory infiltration are different in the core and penumbra lesions of rat brain after transient focal cerebral ischemia, J. Neuroimmunol. 324 (2018) 35–42. [32] H. Wang, H. Guo, Q. Lou, Q. Shi, Effects of cysteinyl leukotrienes receptor antagonists on chronic brain injury after global cerebral ischemia/reperfusion, Zhejiang Da Xue Xue Bao Yi Xue Ban 47 (1) (2018) 19–26. [33] T. Yamaguchi, M. Suzuki, M. Yamamoto, YM796, a novel muscarinic agonist, improves the impairment of learning behavior in a rat model of chronic focal cerebral ischemia, Brain Res. 669 (1) (1995) 107–114. [34] M.V. Avrov, V.M. Alifirova, A.V. Kovalenko, An effect of complex therapy on cognitive impairment in patients with chronic cerebral ischemia, Zh. Nevrol. Psikhiatr. Im. S. S. 119 (2) (2019) 23–27. [35] M. Zhang, H. Yan, S. Li, J. Yang, Rosmarinic acid protects rat hippocampal neurons from cerebral ischemia/reperfusion injury via the Akt/JNK3/caspase-3 signaling pathway, Brain Res. 1657 (2017) 9–15. [36] Q. Zhang, E.Q. Wei, C.Y. Zhu, W.P. Zhang, M.L. Wang, S.H. Zhang, et al., Focal cerebral ischemia alters the spatio-temporal properties, but not the amount of activity in mice, Behav. Brain Res. 169 (1) (2006) 66–74. [37] J.N. Crawley, Exploratory behavior models of anxiety in mice, Neurosci. Biobehav. Rev. 9 (1) (1985) 37–44. [38] N. Pitsikas, P.A. Tarantilis, Crocins, the active constituents of Crocus sativus L., counteracted apomorphine-induced performance deficits in the novel object recognition task, but not novel object location task, in rats, Neurosci. Lett. 644 (2017) 37–42. [39] S. Lee, H.J. Park, S.J. Jeon, E. Kim, H.E. Lee, H. Kim, et al., Cognitive ameliorating effect of Acanthopanax koreanum against scopolamine-induced memory impairment in mice, Phytother. Res. 31 (3) (2017) 425–432. [40] N. Baazaoui, K. Iqbal, Prevention of amyloid-beta and tau pathologies, associated neurodegeneration, and cognitive deficit by early treatment with a neurotrophic compound, J. Alzheimers Dis. 58 (1) (2017) 215–230. [41] M. Oz, E.A. Demir, M. Caliskan, R. Mogulkoc, A.K. Baltaci, K.E. Nurullahoglu Atalik, 3’,4’-Dihydroxyflavonol attenuates spatial learning and memory impairments in global cerebral ischemia, Nutr. Neurosci. 20 (2) (2017) 119–126. [42] M. Frechou, I. Margaill, C. Marchand-Leroux, V. Beray-Berthat, Behavioral tests that reveal long-term deficits after permanent focal cerebral ischemia in mouse, Behav. Brain Res. 360 (2019) 69–80. [43] A. Ghajar, S.M. Neishabouri, N. Velayati, L. Jahangard, N. Matinnia, M. Haghighi, et al., Crocus sativus L. versus Citalopram in the Treatment of Major Depressive Disorder with Anxious Distress: A Double-Blind, Controlled Clinical Trial, Pharmacopsychiatry 50 (4) (2017) 152–160. [44] H. Hosseinzadeh, N.B. Noraei, Anxiolytic and hypnotic effect of Crocus sativus aqueous extract and its constituents, crocin and safranal, in mice, Phytother. Res. 23 (6) (2009) 768–774. [45] J.L. Stobart, K.D. Ferrari, M.J.P. Barrett, C. Gluck, M.J. Stobart, M. Zuend, et al., Cortical circuit activity evokes rapid astrocyte calcium signals on a similar timescale to neurons, Neuron 98 (4) (2018) 726–735 e724. [46] R. Martin, R. Bajo-Graneras, R. Moratalla, G. Perea, A. Araque, Circuit-specific signaling in astrocyte-neuron networks in basal ganglia pathways, Science 349 (6249) (2015) 730–734. [47] N. Marina, E. Turovsky, I.N. Christie, P.S. Hosford, A. Hadjihambi, A. Korsak, et al., Brain metabolic sensing and metabolic signaling at the level of an astrocyte, Glia 66 (6) (2018) 1185–1199. [48] G. Li, Y. Cao, F. Shen, Y. Wang, L. Bai, W. Guo, et al., Mdivi-1 inhibits astrocyte activation and astroglial scar formation and enhances axonal regeneration after spinal cord injury in rats, Front. Cell. Neurosci. 10 (2016) 241. [49] Y. Han, H.X. Yu, M.L. Sun, Y. Wang, W. Xi, Y.Q. Yu, Astrocyte-restricted disruption of connexin-43 impairs neuronal plasticity in mouse barrel cortex, Eur. J. Neurosci. 39 (1) (2014) 35–45. [50] S. Quraishe, L.H. Forbes, M.R. Andrews, The extracellular environment of the CNS: influence on plasticity, sprouting, and axonal regeneration after spinal cord injury, Neural Plast. 2018 (2018) 2952386. [51] C.I. Lin, C.C. Chiao, Blue light promotes neurite outgrowth of retinal explants in postnatal ChR2 mice, eNeuro (2019). [52] K.E. Rhodes, L.D. Moon, J.W. Fawcett, Inhibiting cell proliferation during formation of the glial scar: effects on axon regeneration in the CNS, Neuroscience 120 (1) (2003) 41–56. [53] C. Heine, K. Sygnecka, H. Franke, Purines in neurite growth and astroglia activation, Neuropharmacology 104 (2016) 255–271. [54] J.J. Hill, K. Jin, X.O. Mao, L. Xie, D.A. Greenberg, Intracerebral chondroitinase ABC and heparan sulfate proteoglycan glypican improve outcome from chronic stroke in rats, Proc Natl Acad Sci U S A 109 (23) (2012) 9155–9160. [55] C.Y. Wu, M. Fang, A. Karthikeyan, Y. Yuan, E.A. Ling, Scutellarin Attenuates Microglia-Mediated Neuroinflammation and Promotes Astrogliosis in Cerebral Ischemia - A Therapeutic Consideration, Curr. Med. Chem. 24 (7) (2017) 718–727. [56] Z. Dong, D. Ma, Y. Gong, T. Yu, G. Yao, Salvianolic acid B ameliorates CNS autoimmunity by suppressing Th1 responses, Neurosci. Lett. 619 (2016) 92–99. [57] B. Mages, S. Aleithe, S. Altmann, A. Blietz, B. Nitzsche, H. Barthel, et al., Impaired neurofilament integrity and neuronal morphology in different models of focal cerebral ischemia and human stroke tissue, Front. Cell. Neurosci. 12 (2018) 161. [58] P. Singh, J. Yan, R. Hull, S. Read, J. O’Sullivan, R.D. Henderson, et al., Levels of phosphorylated axonal neurofilament subunit H (pNfH) are increased in acute ischemic stroke, J. Neurol. Sci. 304 (1–2) (2011) 117–121. [59] J.L. Zamanian, L. Xu, L.C. Foo, N. Nouri, L. Zhou, R.G. Giffard, et al., Genomic analysis of reactive astrogliosis, J. Neurosci. 32 (18) (2012) 6391–6410.
[2] H.M. Bramlett, W.D. Dietrich, Pathophysiology of cerebral ischemia and brain trauma: similarities and differences, J. Cereb. Blood Flow Metab. 24 (2) (2004) 133–150. [3] J.S. Zhan, K. Gao, R.C. Chai, X.H. Jia, D.P. Luo, G. Ge, et al., Astrocytes in migration, Neurochem. Res. 42 (1) (2017) 272–282. [4] X. Jin, Y.J. Shin, T.R. Riew, J.H. Choi, M.Y. Lee, Increased expression of Slit2 and its robo receptors during astroglial scar formation after transient focal cerebral ischemia in rats, Neurochem. Res. 41 (12) (2016) 3373–3385. [5] G.R. Choudhury, S. Ding, Reactive astrocytes and therapeutic potential in focal ischemic stroke, Neurobiol. Dis. 85 (2016) 234–244. [6] A. Justin, S. Divakar, M. Ramanathan, Cerebral ischemia induced inflammatory response and altered glutaminergic function mediated through brain AT1 and not AT2 receptor, Biomed. Pharmacother. 102 (2018) 947–958. [7] P.B. de la Tremblaye, S.M. Benoit, S. Schock, H. Plamondon, CRHR1 exacerbates the glial inflammatory response and alters BDNF/TrkB/pCREB signaling in a rat model of global cerebral ischemia: implications for neuroprotection and cognitive recovery, Prog. Neuropsychopharmacol. Biol. Psychiatry 79 (Pt B) (2017) 234–248. [8] J. Yuan, M. Zou, X. Xiang, H. Zhu, W. Chu, W. Liu, et al., Curcumin improves neural function after spinal cord injury by the joint inhibition of the intracellular and extracellular components of glial scar, J. Surg. Res. 195 (1) (2015) 235–245. [9] M.G. De Simoni, P. Milia, M. Barba, A. De Luigi, L. Parnetti, V. Gallai, The inflammatory response in cerebral ischemia: focus on cytokines in stroke patients, Clin. Exp. Hypertens. 24 (7–8) (2002) 535–542. [10] D. Wu, M.C. Klaw, T. Connors, N. Kholodilov, R.E. Burke, V.J. Tom, Expressing constitutively active rheb in adult neurons after a complete spinal cord injury enhances axonal regeneration beyond a chondroitinase-treated glial scar, J. Neurosci. 35 (31) (2015) 11068–11080. [11] M. Pekny, M. Pekna, Astrocyte reactivity and reactive astrogliosis: costs and benefits, Physiol. Rev. 94 (4) (2014) 1077–1098. [12] M.A. Anderson, J.E. Burda, Y. Ren, Y. Ao, T.M. O’Shea, R. Kawaguchi, et al., Astrocyte scar formation aids central nervous system axon regeneration, Nature 532 (7598) (2016) 195–200. [13] H. Zhao, G. Li, R. Wang, Z. Tao, S. Zhang, F. Li, et al., MiR-424 prevents astrogliosis after cerebral ischemia/reperfusion in elderly mice by enhancing repressive H3K27me3 via NFIA/DNMT1 signaling, FEBS J. 286 (24) (2019) 4926–4936. [14] Y.M. Zhu, X. Gao, Y. Ni, W. Li, T.A. Kent, S.G. Qiao, et al., Sevoflurane postconditioning attenuates reactive astrogliosis and glial scar formation after ischemiareperfusion brain injury, Neuroscience 356 (2017) 125–141. [15] S. Samarghandian, M. Azimi-Nezhad, F. Samini, T. Farkhondeh, The role of saffron in attenuating age-related oxidative damage in rat Hippocampus, Recent Pat. Food Nutr. Agric. 8 (3) (2017) 183–189. [16] A.L. Lopresti, P.D. Drummond, Efficacy of curcumin, and a saffron/curcumin combination for the treatment of major depression: a randomised, double-blind, placebo-controlled study, J. Affect. Disord. 207 (2017) 188–196. [17] G.H. Farjah, S. Salehi, M.H. Ansari, B. Pourheidar, Protective effect of Crocus sativus L. (Saffron) extract on spinal cord ischemia-reperfusion injury in rats, Iran. J. Basic Med. Sci. 20 (3) (2017) 334–337. [18] L. Mahmoudzadeh, H. Najafi, S.C. Ashtiyani, Z.M. Yarijani, Anti-inflammatory and protective effects of saffron extract in ischaemia/reperfusion-induced acute kidney injury, Nephrology (Carlton) 22 (10) (2017) 748–754. [19] O. Mirmosayyeb, A. Tanhaei, H.R. Sohrabi, R.N. Martins, M. Tanhaei, M.A. Najafi, et al., Possible role of common spices as a preventive and therapeutic agent for alzheimer’s disease, Int. J. Prev. Med. 8 (2017) 5. [20] S.V. Rao Muralidhara, S.C. Yenisetti, P.S. Rajini, Evidence of neuroprotective effects of saffron and crocin in a Drosophila model of parkinsonism, Neurotoxicology 52 (2016) 230–242. [21] P.K. Pan, L.Y. Qiao, X.N. Wen, Safranal prevents rotenone-induced oxidative stress and apoptosis in an in vitro model of Parkinson’s disease through regulating Keap1/ Nrf2 signaling pathway, Cell. Mol. Biol. (Noisy-Le-Grand) 62 (14) (2016) 11–17. [22] K. Hatziagapiou, E. Kakouri, G.I. Lambrou, K. Bethanis, P.A. Tarantilis, Antioxidant properties of Crocus sativus L. And its constituents and relevance to neurodegenerative diseases; focus on alzheimer’s and parkinson’s disease, Curr. Neuropharmacol. (2018). [23] H. Hosseinzadeh, H.R. Sadeghnia, F.A. Ghaeni, V.S. Motamedshariaty, S.A. Mohajeri, Effects of saffron (Crocus sativus L.) and its active constituent, crocin, on recognition and spatial memory after chronic cerebral hypoperfusion in rats, Phytother. Res. 26 (3) (2012) 381–386. [24] A. Vakili, M.R. Einali, A.R. Bandegi, Protective effect of crocin against cerebral ischemia in a dose-dependent manner in a rat model of ischemic stroke, J. Stroke Cerebrovasc. Dis. 23 (1) (2014) 106–113. [25] S. Saleem, M. Ahmad, A.S. Ahmad, S. Yousuf, M.A. Ansari, M.B. Khan, et al., Effect of Saffron (Crocus sativus) on neurobehavioral and neurochemical changes in cerebral ischemia in rats, J. Med. Food 9 (2) (2006) 246–253. [26] H. Hosseinzadeh, H.R. Sadeghnia, Safranal, a constituent of Crocus sativus (saffron), attenuated cerebral ischemia induced oxidative damage in rat hippocampus, J. Pharm. Pharm. Sci. 8 (3) (2005) 394–399. [27] G. Akbari, S. Ali Mard, A. Veisi, A comprehensive review on regulatory effects of crocin on ischemia/reperfusion injury in multiple organs, Biomed. Pharmacother. 99 (2018) 664–670. [28] D.D. Liu, Y.L. Ye, J. Zhang, J.N. Xu, X.D. Qian, Q. Zhang, Distinct pro-apoptotic properties of Zhejiang saffron against human lung cancer via a caspase-8-9-3 cascade, Asian Pac. J. Cancer Prev. 15 (15) (2014) 6075–6080. [29] E.Z. Longa, P.R. Weinstein, S. Carlson, R. Cummins, Reversible middle cerebral artery occlusion without craniectomy in rats, Stroke 20 (1) (1989) 84–91. [30] Y.L. Ye, W.Z. Shi, W.P. Zhang, M.L. Wang, Y. Zhou, S.H. Fang, et al., Cilostazol, a phosphodiesterase 3 inhibitor, protects mice against acute and late ischemic brain
10
Biomedicine & Pharmacotherapy 126 (2020) 110041
K. Zhong, et al.
deficits through increasing regional cerebral blood flow and alleviating inflammation in CCI rats, Evid. Complement. Alternat. Med. 2017 (2017) 5173168. [63] M.R. Khazdair, M.H. Boskabady, M. Hosseini, R. Rezaee, A.M. Tsatsakis, The effects of Crocus sativus (saffron) and its constituents on nervous system: a review, Avicenna J. Phytomed. 5 (5) (2015) 376–391.
[60] M.V. Sofroniew, Molecular dissection of reactive astrogliosis and glial scar formation, Trends Neurosci. 32 (12) (2009) 638–647. [61] J.E. Burda, M.V. Sofroniew, Reactive gliosis and the multicellular response to CNS damage and disease, Neuron 81 (2) (2014) 229–248. [62] D. Han, Z. Liu, G. Wang, Y. Zhang, Z. Wu, Electroacupuncture improves cognitive
11