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Miconazole stimulates post-ischemic neurogenesis and promotes functional restoration in rats
Accepted Manuscript Title: Miconazole stimulates post-ischemic neurogenesis and promotes functional restoration in rats Authors: Ning Li, Xiubao Song, Liangmiao Wu, Tao Zhang, Chen Zhao, Xifei Yang, Luchen Shan, Pei Yu, Yewei Sun, Yuqiang Wang, Gaoxiao Zhang, Zaijun Zhang PII: DOI: Reference:
S0304-3940(18)30633-5 https://doi.org/10.1016/j.neulet.2018.09.035 NSL 33824
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
30-6-2018 12-9-2018 13-9-2018
Please cite this article as: Li N, Song X, Wu L, Zhang T, Zhao C, Yang X, Shan L, Yu P, Sun Y, Wang Y, Zhang G, Zhang Z, Miconazole stimulates post-ischemic neurogenesis and promotes functional restoration in rats, Neuroscience Letters (2018), https://doi.org/10.1016/j.neulet.2018.09.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Miconazole stimulates post-ischemic neurogenesis and promotes functional restoration in rats Ning Li a,†, Xiubao Song b,†, Liangmiao Wu a, Tao Zhang a, Chen Zhao a, Xifei Yang c, Luchen Shana, Pei Yu a, Yewei Sun a, Yuqiang Wang a, Gaoxiao Zhang a, *, Zaijun Zhang a, * a
Institute of New Drug Research and Guangzhou Key Laboratory of Innovative Chemical Drug Research in
Cardio-cerebrovascular Diseases, Jinan University College of Pharmacy, Guangzhou, 510632, China. b
Department of Rehahilitation, the First Affiliated Hospital, Jinan University, Guangzhou 510630, China.
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Key Laboratory of Modern Toxicology of Shenzhen, Center for Disease Control and Prevention, No. 8,
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Longyuan Road, Nanshan District, Shenzhen 518055, China.
Corresponding author: Dr. Zaijun Zhang or Dr. Gaoxiao Zhang, Institute of New Drug Research and
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Guangzhou Key Laboratory of Innovative Chemical Drug Research in Cardio-cerebrovascular Diseases, Jinan University College of Pharmacy, Guangzhou 510632, China.
E-mail address: [email protected] (Z. J. Zhang) and [email protected] (G. X. Zhang). Equal contribution.
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HIGHLIGHTS
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Miconazole could promote neurobehavioral recovery of rats after t-MCAO surgery. Miconazole could stimulate neuroregeneration in rat stroke model. Miconazole could increase the level of BNDF in the peri-infarct region after stroke.
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ABSTRACT
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Miconazole, a frequently used antifungal drug, has been identified with new functions to promote oligodendrocyte progenitor cells differentiation and to enhance remyelination. However, the neuroregenerative and therapeutic benefit of miconazole on ischemic stroke model have not been tested. In the present study, the effects of miconazole on a rat model of transient middle cerebral artery occlusion were evaluated. Rats received miconazole (10 mg/kg) or saline by intravenous administration for 7 days after stroke. A battery of neurobehavioral assessments, including rotarod test, open-field test, neurological severity score and novel object recognition task were evaluated. The results revealed a significant functional improvement in miconazole-treated rats compared with vehicle-treated control. Animals were sacrificed at 7 and 28 days after stroke. Double immunofluorescence staining for NeuN+/BrdU+, DCX+/BrdU+ and Nestin+/BrdU+ cells indicated miconazole significantly promoted neurogenesis. Western blotting analysis revealed miconazole upregulated the protein expression of brain derived neurotrophic factor, myocyte enhancer factor 2D, synaptophysin, and postsynaptic density protein 95, while downregulated the expression of cyclin-dependent kinase 5. Taken together, miconazole promoted functional recovery on ischemic stroke model via stimulating post-ischemic neurogenesis. Keywords: Miconazole; Neuroregeneration; Ischemic stroke; Brain derived neurotrophic factor.
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1. Introduction Stroke is a public health and socioeconomic issue worldwide [1]. In recent years, attention for stroke treatment has gradually shifted from rescuing the injured tissue to boosting post-stroke tissue repair [2]. Laboratory and clinical researches have indicated that the adult brain has the capability to self-repair in response to stroke. Upon ischemia, subventricular zone (SVZ)-derived neural progenitor cells (NPCs) can proliferate and migrate toward the damaged brain areas [3], where they differentiate into mature neurons and newborn granule cells integrated into the local neural network [4]. However, the spontaneous brain restoration process is constrained with limited improvement of neurological outcome [5]. Accumulating evidence suggests that it might be a promising therapeutic approach to stimulate post-ischemic structural and functional restoration by using exogenous pharmacological or cell-based treatments [6]. We have recently identified that tetramethylpyrazine nitrone promoted neurogenesis and oligodendrogenesis in the transient middle cerebral artery occlusion (t-MCAO) rats, consequently restored neurological function[7]. Miconazole has potent activities against various fungi through cytochrome P450 inhibition and reactive oxygen species (ROS) promotion [8]. A recent study identified miconazole, or structurally modified derivatives, with new functions to modify oligodendrocyte progenitor cells differentiation and to enhance remyelination in multiple sclerosis mice [9]. We wonder if moconazole also has neurogenesis promoting effect, and is beneficial in ischemic stroke model.
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2. Material and methods 2.1. Animals
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All animal procedures were carried out according to a protocol approved by Institutional Animal Ethical Committee of Sun Yat-sen University (Guangzhou, China). Adult male Sprague-Dawley (SD) rats weighing 250-280 g were used to establish focal ischemic cerebral injury. All animals were kept in a lighting regimen of Light-Dark 12:12 hours. 2.2. Experimental groups and animal surgery
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All animals were randomly assigned into three groups: Sham control, Stroke + saline and Stroke + miconazole. The ischemia-reperfusion stroke model in SD rats reproduced by transient MCAO was conducted according to our published procedure [7]. Reperfusion was achieved by withdrawal of the suture thread 2 hours after the onset of occlusion. Anesthesia was discontinued and animals were placed back into their cages. Sham control animals received midline neck incisions and the left common carotid artery was isolated, but not occluded. 2.3. Drug treatment
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Miconazole (10 mg/kg; Sigma-Aldrich, USA) was dissolved in 0.9% saline and administered intravenously (i.v.) through the tail vein. The concentration of miconzole used in the present study referred to the dosage used in the previous publication, in which miconzole (10 mg/kg, daily administrated by i.p.) significantly increased the number of new oligodendrocytes and enhanced remyelination in a lysolecithin-induced mouse model of focal demyelination [9]. Miconazole or vehicle (0.9% saline) was given at 3 h and then 6 h after MCAO on the first day, and then given twice daily at 9 am and 4 pm for another 6 days. The 5’-bromo-2’-deoxyuridine (BrdU, 50 mg/kg; Sigma-Aldrich, USA) for identification of proliferating cells was administered intraperitoneally (i.p.) at 21 days after MCA occlusion and then twice
daily for 7 consecutive days. Behavioral assessments were performed by investigators who were blind to treatment assignment throughout this trial. 2.4. Sensorimotor measurements
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All animals were trained for 3 days before ischemia and sensorimotor measurements were performed at 3 hours, 7, 14, 21, and 28 days after MCAO. Tests of rotorod, open-field, and neurological severity score (NSS) were carried out as described in our previous publication [7]. The NSS is a composite of motor, sensory, and reflex tests, including beam balance and graded on a scale of 0 to 18 (normal score= 0, maximal deficit score=18) [7]. 2.5. Novel object recognition task
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2.6. Histological and immunohistochemical assessment
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Rats were habituated to an open-field box (a PVC square arena, length × width × height, 50 × 50 × 40 cm) 28 days after MCAO. In the first phase, for 10 min two of the same objects (left and right: circle) were placed symmetrically 20 cm away from the wall. In the second phase, for 10 min two dissimilar objects, one circle and a square were presented in the same box 1 h after the first phase. The amount of time that a rat spent exploring novel object (TA) or familiar object (TB) during test was recorded. Discrimination index was calculated according to the following expression: (TA − TB) / (TA + TB).
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Animals were sacrificed at day 28 after occlusion and a series of 20 m thick brain coronal sections were cut every sixth section per rat between bregma levels +0.96 mm and −0.24 mm. For double staining, sections were incubated with anti-BrdU (1:500; Cell Signaling Technology, USA), anti-NeuN (1:500; Abcam, USA), anti-DCX (1:500; Cell Signaling Technology, USA), anti-Nestin (1:500; Novus, USA) antibodies. Brain sections were cover slipped and evaluated under a fluorescence microscope (Olympus, Japan). The images of NeuN+/BrdU+, DCX+/BrdU+ cells in the peri-infarct cortex and Nestin+/BrdU+ cells in ipsilateral lateral ventricular (LV) zone were acquired with fluorescence microscope. Results were obtained as the average areas of NeuN+/BrdU+, DCX+/BrdU+ and Nestin+/BrdU+cells in per section by an investigator blinded to each groups.
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2.7. Western Blotting
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Thirty milligram protein supernatant were subjected to SDS-PAGE analysis, transferred onto a PVDF membrane and probed with primary antibodies against cyclin-dependent kinase 5 (CDK5), postsynaptic density protein 95 (PSD95), synaptophysin (SYP), myocyte enhancer factor 2D (MEF2D), β-actin (1:1000; Cell Signaling Technology, USA) or BDNF (1:1000; Abcam, USA). Finally, quantitative assessment of the bands density was determined using Carestream Molecular Imaging Software (Carestream Health, USA) 2.8. Statistical analysis All the data were expressed as mean ± SEM and analyzed by one-way or two-way analysis of variance (ANOVA). Except that animal NSS results were expressed as median with interquartile range, and analyzed by a non-parametric Kruskal-Wallis test followed by the Dunn’s multiple comparisons. A value of p< 0.05 was considered to indicate the least significant difference (LSD) between groups.
3. Results 3.1. Miconazole alleviated post-stroke behavior impairment in rats
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To determine whether miconazole can improve recovery of impaired motor function, rotarod test and NSS were evaluated at 3 hours and then once every week after ischemia. Rats in Stroke + miconazole group showed significant improvement in rotarod (Fig.1A) and NSS tests (Fig.1B) compared with the Stroke + saline group. Moreover, in weekly open-field tests, total moving distance showed considerably recovery in Stroke + miconazole group compared with Stroke + saline group (Fig.1C). The function of recognition and memory was evaluated at 28 days after ischemia by novel objective recognition test, and Stroke + miconazole rats displayed much better performance in discrimination index than Stroke + saline rats (Fig.1D).
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3.2. Miconazole promoted neurogenesis during stroke recovery
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We assessed cell proliferation by injecting BrdU for 7 days before the termination of experiment. The NeuN/BrdU, DCX/BrdU and Nestin/BrdU double-labeling could presume newborn mature neurons, migrating and immature neurons (neuroblasts, NPCs), respectively. There were almost no NeuN+/BrdU+ and DCX+/BrdU+ cells found in Sham control group. However, both the numbers of NeuN+/BrdU+ and DCX+/BrdU+ cells markedly increased in the peri-infarct zone of Stroke + vehicle rats (Fig. 2A, B, D and E). Importantly, the numbers of NeuN/BrdU and DCX/BrdU double-positive cells of Stroke + miconazole rats were significantly higher than those of the Stroke + saline group (Fig. 2A, B, D and E). In the LV zone of Stroke + saline rats, the Nestin+/BrdU+ (NPCs) markedly increased compared with the Sham control group. Furthermore, the number of Nestin+/BrdU+ cells in Stroke + miconazole group was significantly higher than that of Stroke + saline group (Fig. 2C and F).
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3.3. Effects of miconazole on neurogenesis-related proteins expression in the peri-infarct area of stroke rats
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Brain-derived neurotrophic factor (BDNF) is considered to be a key neurotrophic factor that enhances NPCs proliferation. Western blot quantitative analysis revealed that moconazole significantly enhanced BDNF expression both at 7 and 28 day post-ischemia (Fig. 3A, B). We also examined Cyclin-dependent kinase 5 (CDK5) and myocyte enhancer factor 2D (MEF2D) signaling which regulated the expression and activity of neurotrophic factors. The results indicated in Stroke + moconazole group a substantial increase of MEF2D but a decrease of CDK5 in the peri-infarct region (Fig. 3A, C, D). BDNF plays an important role in synaptic function and plasticity. We examined the expression of the major pre-synaptic scaffold protein synaptophysin (SYN) and post-synaptic density protein 95 (PSD95). We found that PSD95 and SYN profoundly decreased at both 7 and 28 days post-ischemia. Moconazole treatment exhibited a significantly higher expression of PSD95 and SYN compared with saline treatment (Fig. 3A, E and F). 4. Discussion Ischemic stroke could induce sustained sensorimotor deficiencies and recognition memory impairments [10]. Recent evidence indicated that new generated neurons played a crucial role in the recovery of motor and learning abilities after stroke [11,12]. Our present study demonstrated that miconazole could significantly stimulate neurogenesis, meanwhile reduce sensorimotor and learning
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impairments after stroke in rats. Neuroregeneration is one of a major brain repair processes during stroke recovery. In the adult SVZ, neural precursors commit to a neuronal fate and become neuroblasts, which can either continue proliferating or exit the cell cycle and become immature neurons [13]. Our data demonstrated that the number of Nestin+/BrdU+, DCX+/BrdU+ and NeuN+/BrdU+ cells in the ipsilateral hemisphere increased in Stroke + miconazole group, showing a high degree of cells proliferation and differentiation. However, many of these newly generated neurons undergo apoptosis, the survival cells may have altered morphology and poor functional integration in adult brain. The neurotransmission-regulating factors, PSD95 and synaptophysin enhanced by miconazole in vivo, will promote neuroblasts differentiation into neurons and then integrating into neural networks in brain recovery after stroke. Miconazole treatment could up-regulate the expression of BDNF after cerebral ischemia. Previous studies demonstrated that increasing BDNF levels or activating BDNF-associated signaling pathways lead to brain repair and neurological function recovery after stroke [14–16]. BDNF, as one of the important regulators for neuroregeneration of the adults, is the most abundant neurotrophin in the brain and play a vital role in promoting neuronal plasticity and neurogenesis after brain injury. CDK5 has been implicated in regulation of the cell cycle, migration, maturation and neuronal integration [17]. The work efficiency of BDNF could be influenced by the activity of CDK5, because decreasing of CDK5 was directly beneficial to phosphorylate the cognate BDNF receptor TrkB [18]. Our study suggested that miconazole could significantly inhibit the expression of CDK5 at day 7 after ischemia. However, the level of CDK5 seemed no statistically significant difference in all groups at 28 day. That might be due to the seven days treatment course of miconazole. MEF2 proteins are highly enriched in neurons and exhibit distinct patterns of expression in different regions of the brain. Evidences reported MEF2 proteins appear to play an important role in synaptic plasticity, suggesting the importance of these proteins for learning and memory [19]. The expression of MEF2D significantly increased in Stroke + miconazole rats in our study. We deduced that miconazole increased the expression of BDNF via regulation of CDK5 and MEF2D. In conclusion, our study demonstrated that treatment with miconazole could increase neurogenesis and promoted functional recovery after ischemic stroke in rats. The beneficial effect of miconazole was possibly mediated by stimulating expression of BDNF. These results suggest that miconazole might be a new therapeutic candidate repurposing for ischemic stroke. Acknowledgements
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This study was supported in part by the National Science and Technology Major Project of China (Innovative Drug Project 2018ZX09301031-002); the National Science Foundation of China (81603106, 81502908); the Scientific Projects of Guangdong Province (2017A030313742, 2016A020217013, GD-HK
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Cooperative Project 2016A050503030); the Science and Technology Program of Guangzhou (201804010495, 201704020181); as well as the Fundamental Research Funds for the Central Universities (21617469 and 17817017).
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Figure Legends:
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Fig. 1. Miconazole treatment improved functional outcomes after t-MCAO. In weekly functional tests, the Stroke + miconazole animals showed a recovery trend of running function (rotarod test; A) and neurological performance (NSS test; B) compared with Stroke + saline rats, and in total moving distance of open field test (C). Rats in Stroke + miconazole group displayed better in recognition memory test than Stroke + saline group at 4 weeks after surgery (D). Values are mean ± S.E.M. ## p < 0.01, ### p < 0.001 (Stroke + saline VS. Sham control); * p < 0.05 (Stroke + miconazole VS. Stroke + saline) by One-way ANOVA (C), Two-way ANOVA (A, D) with Bonferroni post hoc tests and non-parametric Kruskal-Wallis test with Dunn’s multiple comparisons test (B). n = 8 in Sham control; n = 9 in Stroke + saline and Stroke + miconazole.
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Fig. 2. Miconazole stimulated neural regeneration in rats’ cerebral injury area at 28 days after t-MCAO. Representative images of ipsilateral hemisphere co-staining of NeuN+ (green; mature neuron marker) / BrdU+ (red, new generated cell marker) cells (A), DCX+ (green; neuroblast marker) / BrdU+ (red) cells (B) and Nestin+ (green; Neural progenitor cell marker) / BrdU+ (red) cells (C) in each group. Scale bar:
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100 m. Insets show a higher magnification view. Scale bar: 50 m. DAPI (blue), nuclear staining. Scatter plot of NeuN+ /BrdU+ (D), DCX+ / BrdU+ (E) and Nestin+ / BrdU+ (F) double-positive cells. In (A) and (B), white dotted lines bordered between ischemia-affected region and unaffected area. LV is lateral ventricle. Values are mean ± S.E.M. ns, no significant different; * p < 0.05 (Stroke + miconazole VS. Stroke + saline) by One-way ANOVA with Bonferroni post hoc tests. n=3 in each groups.
Fig. 3. Miconazole promotes neurogenesis related with BDNF, MEF2D and CDK5 protein expression in rats ischemic region. Representative western blots (A) and quantification of the levels of BDNF (B),
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MEF2D (C), CDK5 (D), PSD95 (E) and Synaptophysin (SYP, F). Values are mean ± S.E.M. ### p < 0.001 (Stroke + saline VS. Sham control); * p <0 .05 (Stroke + miconazole VS. Stroke + saline) by two-way ANOVA with Bonferroni post hoc tests. n=4 in each groups.
S0304-3940(18)30633-5 https://doi.org/10.1016/j.neulet.2018.09.035 NSL 33824
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
30-6-2018 12-9-2018 13-9-2018
Please cite this article as: Li N, Song X, Wu L, Zhang T, Zhao C, Yang X, Shan L, Yu P, Sun Y, Wang Y, Zhang G, Zhang Z, Miconazole stimulates post-ischemic neurogenesis and promotes functional restoration in rats, Neuroscience Letters (2018), https://doi.org/10.1016/j.neulet.2018.09.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Miconazole stimulates post-ischemic neurogenesis and promotes functional restoration in rats Ning Li a,†, Xiubao Song b,†, Liangmiao Wu a, Tao Zhang a, Chen Zhao a, Xifei Yang c, Luchen Shana, Pei Yu a, Yewei Sun a, Yuqiang Wang a, Gaoxiao Zhang a, *, Zaijun Zhang a, * a
Institute of New Drug Research and Guangzhou Key Laboratory of Innovative Chemical Drug Research in
Cardio-cerebrovascular Diseases, Jinan University College of Pharmacy, Guangzhou, 510632, China. b
Department of Rehahilitation, the First Affiliated Hospital, Jinan University, Guangzhou 510630, China.
c
Key Laboratory of Modern Toxicology of Shenzhen, Center for Disease Control and Prevention, No. 8,
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Longyuan Road, Nanshan District, Shenzhen 518055, China.
Corresponding author: Dr. Zaijun Zhang or Dr. Gaoxiao Zhang, Institute of New Drug Research and
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Guangzhou Key Laboratory of Innovative Chemical Drug Research in Cardio-cerebrovascular Diseases, Jinan University College of Pharmacy, Guangzhou 510632, China.
E-mail address: [email protected] (Z. J. Zhang) and [email protected] (G. X. Zhang). Equal contribution.
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HIGHLIGHTS
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Miconazole could promote neurobehavioral recovery of rats after t-MCAO surgery. Miconazole could stimulate neuroregeneration in rat stroke model. Miconazole could increase the level of BNDF in the peri-infarct region after stroke.
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ABSTRACT
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Miconazole, a frequently used antifungal drug, has been identified with new functions to promote oligodendrocyte progenitor cells differentiation and to enhance remyelination. However, the neuroregenerative and therapeutic benefit of miconazole on ischemic stroke model have not been tested. In the present study, the effects of miconazole on a rat model of transient middle cerebral artery occlusion were evaluated. Rats received miconazole (10 mg/kg) or saline by intravenous administration for 7 days after stroke. A battery of neurobehavioral assessments, including rotarod test, open-field test, neurological severity score and novel object recognition task were evaluated. The results revealed a significant functional improvement in miconazole-treated rats compared with vehicle-treated control. Animals were sacrificed at 7 and 28 days after stroke. Double immunofluorescence staining for NeuN+/BrdU+, DCX+/BrdU+ and Nestin+/BrdU+ cells indicated miconazole significantly promoted neurogenesis. Western blotting analysis revealed miconazole upregulated the protein expression of brain derived neurotrophic factor, myocyte enhancer factor 2D, synaptophysin, and postsynaptic density protein 95, while downregulated the expression of cyclin-dependent kinase 5. Taken together, miconazole promoted functional recovery on ischemic stroke model via stimulating post-ischemic neurogenesis. Keywords: Miconazole; Neuroregeneration; Ischemic stroke; Brain derived neurotrophic factor.
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1. Introduction Stroke is a public health and socioeconomic issue worldwide [1]. In recent years, attention for stroke treatment has gradually shifted from rescuing the injured tissue to boosting post-stroke tissue repair [2]. Laboratory and clinical researches have indicated that the adult brain has the capability to self-repair in response to stroke. Upon ischemia, subventricular zone (SVZ)-derived neural progenitor cells (NPCs) can proliferate and migrate toward the damaged brain areas [3], where they differentiate into mature neurons and newborn granule cells integrated into the local neural network [4]. However, the spontaneous brain restoration process is constrained with limited improvement of neurological outcome [5]. Accumulating evidence suggests that it might be a promising therapeutic approach to stimulate post-ischemic structural and functional restoration by using exogenous pharmacological or cell-based treatments [6]. We have recently identified that tetramethylpyrazine nitrone promoted neurogenesis and oligodendrogenesis in the transient middle cerebral artery occlusion (t-MCAO) rats, consequently restored neurological function[7]. Miconazole has potent activities against various fungi through cytochrome P450 inhibition and reactive oxygen species (ROS) promotion [8]. A recent study identified miconazole, or structurally modified derivatives, with new functions to modify oligodendrocyte progenitor cells differentiation and to enhance remyelination in multiple sclerosis mice [9]. We wonder if moconazole also has neurogenesis promoting effect, and is beneficial in ischemic stroke model.
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2. Material and methods 2.1. Animals
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All animal procedures were carried out according to a protocol approved by Institutional Animal Ethical Committee of Sun Yat-sen University (Guangzhou, China). Adult male Sprague-Dawley (SD) rats weighing 250-280 g were used to establish focal ischemic cerebral injury. All animals were kept in a lighting regimen of Light-Dark 12:12 hours. 2.2. Experimental groups and animal surgery
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All animals were randomly assigned into three groups: Sham control, Stroke + saline and Stroke + miconazole. The ischemia-reperfusion stroke model in SD rats reproduced by transient MCAO was conducted according to our published procedure [7]. Reperfusion was achieved by withdrawal of the suture thread 2 hours after the onset of occlusion. Anesthesia was discontinued and animals were placed back into their cages. Sham control animals received midline neck incisions and the left common carotid artery was isolated, but not occluded. 2.3. Drug treatment
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Miconazole (10 mg/kg; Sigma-Aldrich, USA) was dissolved in 0.9% saline and administered intravenously (i.v.) through the tail vein. The concentration of miconzole used in the present study referred to the dosage used in the previous publication, in which miconzole (10 mg/kg, daily administrated by i.p.) significantly increased the number of new oligodendrocytes and enhanced remyelination in a lysolecithin-induced mouse model of focal demyelination [9]. Miconazole or vehicle (0.9% saline) was given at 3 h and then 6 h after MCAO on the first day, and then given twice daily at 9 am and 4 pm for another 6 days. The 5’-bromo-2’-deoxyuridine (BrdU, 50 mg/kg; Sigma-Aldrich, USA) for identification of proliferating cells was administered intraperitoneally (i.p.) at 21 days after MCA occlusion and then twice
daily for 7 consecutive days. Behavioral assessments were performed by investigators who were blind to treatment assignment throughout this trial. 2.4. Sensorimotor measurements
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All animals were trained for 3 days before ischemia and sensorimotor measurements were performed at 3 hours, 7, 14, 21, and 28 days after MCAO. Tests of rotorod, open-field, and neurological severity score (NSS) were carried out as described in our previous publication [7]. The NSS is a composite of motor, sensory, and reflex tests, including beam balance and graded on a scale of 0 to 18 (normal score= 0, maximal deficit score=18) [7]. 2.5. Novel object recognition task
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2.6. Histological and immunohistochemical assessment
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Rats were habituated to an open-field box (a PVC square arena, length × width × height, 50 × 50 × 40 cm) 28 days after MCAO. In the first phase, for 10 min two of the same objects (left and right: circle) were placed symmetrically 20 cm away from the wall. In the second phase, for 10 min two dissimilar objects, one circle and a square were presented in the same box 1 h after the first phase. The amount of time that a rat spent exploring novel object (TA) or familiar object (TB) during test was recorded. Discrimination index was calculated according to the following expression: (TA − TB) / (TA + TB).
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Animals were sacrificed at day 28 after occlusion and a series of 20 m thick brain coronal sections were cut every sixth section per rat between bregma levels +0.96 mm and −0.24 mm. For double staining, sections were incubated with anti-BrdU (1:500; Cell Signaling Technology, USA), anti-NeuN (1:500; Abcam, USA), anti-DCX (1:500; Cell Signaling Technology, USA), anti-Nestin (1:500; Novus, USA) antibodies. Brain sections were cover slipped and evaluated under a fluorescence microscope (Olympus, Japan). The images of NeuN+/BrdU+, DCX+/BrdU+ cells in the peri-infarct cortex and Nestin+/BrdU+ cells in ipsilateral lateral ventricular (LV) zone were acquired with fluorescence microscope. Results were obtained as the average areas of NeuN+/BrdU+, DCX+/BrdU+ and Nestin+/BrdU+cells in per section by an investigator blinded to each groups.
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2.7. Western Blotting
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Thirty milligram protein supernatant were subjected to SDS-PAGE analysis, transferred onto a PVDF membrane and probed with primary antibodies against cyclin-dependent kinase 5 (CDK5), postsynaptic density protein 95 (PSD95), synaptophysin (SYP), myocyte enhancer factor 2D (MEF2D), β-actin (1:1000; Cell Signaling Technology, USA) or BDNF (1:1000; Abcam, USA). Finally, quantitative assessment of the bands density was determined using Carestream Molecular Imaging Software (Carestream Health, USA) 2.8. Statistical analysis All the data were expressed as mean ± SEM and analyzed by one-way or two-way analysis of variance (ANOVA). Except that animal NSS results were expressed as median with interquartile range, and analyzed by a non-parametric Kruskal-Wallis test followed by the Dunn’s multiple comparisons. A value of p< 0.05 was considered to indicate the least significant difference (LSD) between groups.
3. Results 3.1. Miconazole alleviated post-stroke behavior impairment in rats
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To determine whether miconazole can improve recovery of impaired motor function, rotarod test and NSS were evaluated at 3 hours and then once every week after ischemia. Rats in Stroke + miconazole group showed significant improvement in rotarod (Fig.1A) and NSS tests (Fig.1B) compared with the Stroke + saline group. Moreover, in weekly open-field tests, total moving distance showed considerably recovery in Stroke + miconazole group compared with Stroke + saline group (Fig.1C). The function of recognition and memory was evaluated at 28 days after ischemia by novel objective recognition test, and Stroke + miconazole rats displayed much better performance in discrimination index than Stroke + saline rats (Fig.1D).
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3.2. Miconazole promoted neurogenesis during stroke recovery
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We assessed cell proliferation by injecting BrdU for 7 days before the termination of experiment. The NeuN/BrdU, DCX/BrdU and Nestin/BrdU double-labeling could presume newborn mature neurons, migrating and immature neurons (neuroblasts, NPCs), respectively. There were almost no NeuN+/BrdU+ and DCX+/BrdU+ cells found in Sham control group. However, both the numbers of NeuN+/BrdU+ and DCX+/BrdU+ cells markedly increased in the peri-infarct zone of Stroke + vehicle rats (Fig. 2A, B, D and E). Importantly, the numbers of NeuN/BrdU and DCX/BrdU double-positive cells of Stroke + miconazole rats were significantly higher than those of the Stroke + saline group (Fig. 2A, B, D and E). In the LV zone of Stroke + saline rats, the Nestin+/BrdU+ (NPCs) markedly increased compared with the Sham control group. Furthermore, the number of Nestin+/BrdU+ cells in Stroke + miconazole group was significantly higher than that of Stroke + saline group (Fig. 2C and F).
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3.3. Effects of miconazole on neurogenesis-related proteins expression in the peri-infarct area of stroke rats
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Brain-derived neurotrophic factor (BDNF) is considered to be a key neurotrophic factor that enhances NPCs proliferation. Western blot quantitative analysis revealed that moconazole significantly enhanced BDNF expression both at 7 and 28 day post-ischemia (Fig. 3A, B). We also examined Cyclin-dependent kinase 5 (CDK5) and myocyte enhancer factor 2D (MEF2D) signaling which regulated the expression and activity of neurotrophic factors. The results indicated in Stroke + moconazole group a substantial increase of MEF2D but a decrease of CDK5 in the peri-infarct region (Fig. 3A, C, D). BDNF plays an important role in synaptic function and plasticity. We examined the expression of the major pre-synaptic scaffold protein synaptophysin (SYN) and post-synaptic density protein 95 (PSD95). We found that PSD95 and SYN profoundly decreased at both 7 and 28 days post-ischemia. Moconazole treatment exhibited a significantly higher expression of PSD95 and SYN compared with saline treatment (Fig. 3A, E and F). 4. Discussion Ischemic stroke could induce sustained sensorimotor deficiencies and recognition memory impairments [10]. Recent evidence indicated that new generated neurons played a crucial role in the recovery of motor and learning abilities after stroke [11,12]. Our present study demonstrated that miconazole could significantly stimulate neurogenesis, meanwhile reduce sensorimotor and learning
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impairments after stroke in rats. Neuroregeneration is one of a major brain repair processes during stroke recovery. In the adult SVZ, neural precursors commit to a neuronal fate and become neuroblasts, which can either continue proliferating or exit the cell cycle and become immature neurons [13]. Our data demonstrated that the number of Nestin+/BrdU+, DCX+/BrdU+ and NeuN+/BrdU+ cells in the ipsilateral hemisphere increased in Stroke + miconazole group, showing a high degree of cells proliferation and differentiation. However, many of these newly generated neurons undergo apoptosis, the survival cells may have altered morphology and poor functional integration in adult brain. The neurotransmission-regulating factors, PSD95 and synaptophysin enhanced by miconazole in vivo, will promote neuroblasts differentiation into neurons and then integrating into neural networks in brain recovery after stroke. Miconazole treatment could up-regulate the expression of BDNF after cerebral ischemia. Previous studies demonstrated that increasing BDNF levels or activating BDNF-associated signaling pathways lead to brain repair and neurological function recovery after stroke [14–16]. BDNF, as one of the important regulators for neuroregeneration of the adults, is the most abundant neurotrophin in the brain and play a vital role in promoting neuronal plasticity and neurogenesis after brain injury. CDK5 has been implicated in regulation of the cell cycle, migration, maturation and neuronal integration [17]. The work efficiency of BDNF could be influenced by the activity of CDK5, because decreasing of CDK5 was directly beneficial to phosphorylate the cognate BDNF receptor TrkB [18]. Our study suggested that miconazole could significantly inhibit the expression of CDK5 at day 7 after ischemia. However, the level of CDK5 seemed no statistically significant difference in all groups at 28 day. That might be due to the seven days treatment course of miconazole. MEF2 proteins are highly enriched in neurons and exhibit distinct patterns of expression in different regions of the brain. Evidences reported MEF2 proteins appear to play an important role in synaptic plasticity, suggesting the importance of these proteins for learning and memory [19]. The expression of MEF2D significantly increased in Stroke + miconazole rats in our study. We deduced that miconazole increased the expression of BDNF via regulation of CDK5 and MEF2D. In conclusion, our study demonstrated that treatment with miconazole could increase neurogenesis and promoted functional recovery after ischemic stroke in rats. The beneficial effect of miconazole was possibly mediated by stimulating expression of BDNF. These results suggest that miconazole might be a new therapeutic candidate repurposing for ischemic stroke. Acknowledgements
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This study was supported in part by the National Science and Technology Major Project of China (Innovative Drug Project 2018ZX09301031-002); the National Science Foundation of China (81603106, 81502908); the Scientific Projects of Guangdong Province (2017A030313742, 2016A020217013, GD-HK
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Cooperative Project 2016A050503030); the Science and Technology Program of Guangzhou (201804010495, 201704020181); as well as the Fundamental Research Funds for the Central Universities (21617469 and 17817017).
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Figure Legends:
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Fig. 1. Miconazole treatment improved functional outcomes after t-MCAO. In weekly functional tests, the Stroke + miconazole animals showed a recovery trend of running function (rotarod test; A) and neurological performance (NSS test; B) compared with Stroke + saline rats, and in total moving distance of open field test (C). Rats in Stroke + miconazole group displayed better in recognition memory test than Stroke + saline group at 4 weeks after surgery (D). Values are mean ± S.E.M. ## p < 0.01, ### p < 0.001 (Stroke + saline VS. Sham control); * p < 0.05 (Stroke + miconazole VS. Stroke + saline) by One-way ANOVA (C), Two-way ANOVA (A, D) with Bonferroni post hoc tests and non-parametric Kruskal-Wallis test with Dunn’s multiple comparisons test (B). n = 8 in Sham control; n = 9 in Stroke + saline and Stroke + miconazole.
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Fig. 2. Miconazole stimulated neural regeneration in rats’ cerebral injury area at 28 days after t-MCAO. Representative images of ipsilateral hemisphere co-staining of NeuN+ (green; mature neuron marker) / BrdU+ (red, new generated cell marker) cells (A), DCX+ (green; neuroblast marker) / BrdU+ (red) cells (B) and Nestin+ (green; Neural progenitor cell marker) / BrdU+ (red) cells (C) in each group. Scale bar:
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100 m. Insets show a higher magnification view. Scale bar: 50 m. DAPI (blue), nuclear staining. Scatter plot of NeuN+ /BrdU+ (D), DCX+ / BrdU+ (E) and Nestin+ / BrdU+ (F) double-positive cells. In (A) and (B), white dotted lines bordered between ischemia-affected region and unaffected area. LV is lateral ventricle. Values are mean ± S.E.M. ns, no significant different; * p < 0.05 (Stroke + miconazole VS. Stroke + saline) by One-way ANOVA with Bonferroni post hoc tests. n=3 in each groups.
Fig. 3. Miconazole promotes neurogenesis related with BDNF, MEF2D and CDK5 protein expression in rats ischemic region. Representative western blots (A) and quantification of the levels of BDNF (B),
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MEF2D (C), CDK5 (D), PSD95 (E) and Synaptophysin (SYP, F). Values are mean ± S.E.M. ### p < 0.001 (Stroke + saline VS. Sham control); * p <0 .05 (Stroke + miconazole VS. Stroke + saline) by two-way ANOVA with Bonferroni post hoc tests. n=4 in each groups.