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Intravenous administration of human adipose-derived stem cells ameliorates motor and cognitive function for intracerebral hemorrhage mouse model
Brain Research 1711 (2019) 58–67
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Intravenous administration of human adipose-derived stem cells ameliorates motor and cognitive function for intracerebral hemorrhage mouse model
T
Yoji Kuramotoa, Toshinori Takagia, Kotaro Tatebayashia, Mikiya Beppua, Nobutaka Doeb,c, ⁎ ⁎ Mitsugu Fujitad, , Shinichi Yoshimuraa, a
Department of Neurosurgery, Hyogo College of Medicine, Hyogo 663-8501, Japan Laboratory of Neurogenesis and CNS Repair, Hyōgo College of Medicine, Hyogo 663-8501, Japan c General Education Center, Hyogo University of Health Science, Hyogo 650-8530, Japan d Department of Microbiology, Kindai University, Faculty of Medicine, Osaka 589-8511, Japan b
H I GH L IG H T S
adipose-derived stem cells improve ICH-induced neurological deficits. • Human therapeutic effects are mediated by CD11b CD45 subpopulations. • The • Human adipose-derived stem cells can be served as a novel strategy for ICH treatment. +
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A R T I C LE I N FO
A B S T R A C T
Keywords: Human adipose-derived stem cell Intracerebral hemorrhage Neurobehavior Macrophage
Even today, intracerebral hemorrhage (ICH) is a major cause of death and disabilities. Rehabilitation is preferentially applied for functional recovery although its effect is limited. Recent studies have suggested that intravenous administration of mesenchymal stem cells would improve the post-ICH neurological deficits. Human adipose-derived stem cells (hADSCs) have been established in our laboratory. We aimed to evaluate the therapeutic efficacy of the hADSCs on the post-ICH neurological deficits using a clinical-relevant ICH mouse model. We also evaluated immune responses to clarify the underlying mechanisms. The hADSCs expressed MSC markers at high levels. The hADSCs administration into the ICH-bearing mice improved the neurological deficits during the subacute phases, which was shown by neurobehavioral experiments. Besides, the hADSC administration decreased the number of CD11+CD45+ cells and increased the proportion of CD86+ and Ly6C+ cells in the ICH lesions. In summary, intravenous administration of hADSCs during the acute phase improved ICH-induced neurological deficits during the subacute phase because of the suppression of acute inflammation mediated by CD11+CD45+ subpopulations. Our data suggest that hADSCs can be served as a novel strategy for ICH treatment.
1. Introduction Intracerebral hemorrhage (ICH) accounts for 10% of all stroke and causes death and disabilities (van Asch et al., 2010); its mortality rate at 1 month after onset is about 40% and disability rate at 3 months after onset is about 60% (Anderson et al., 2013). Surgical intervention is ineffective to improve post-ICH neurological functions (Mendelow et al., 2013). No acute medication is available to improve the post-ICH neurological functions. Rehabilitation is preferentially applied for functional recovery although its effect is limited (Saulle and Schambra,
2016). The effectiveness of stem cell therapy has been reported in animal models of ICH (Chen et al., 2012; Yang et al., 2012; Xie et al., 2016). Among various stem cells, mesenchymal stem cells (MSCs) exhibit unique biological effects such as the production of growth factors and cytokines, immunomodulation, neurogenesis, and angiogenesis (Caplan and Dennis, 2006; Lee et al., 2014). Several animal studies have shown that intravenous administration of MSCs improves the post-ICH neurological deficits (Jeong et al., 2003; Lee et al., 2007; Yang et al., 2012; Xie et al., 2016). Nevertheless, its underlying mechanism is yet unclear.
⁎ Corresponding authors at: Department of Microbiology, Kindai University, Faculty of Medicine, 377-2 Ohno-Higasi, Osakasayama, Osaka 589-8511, Japan (M. Fujita). Department of Neurosurgery, Hyogo College of Medicine, 1-1 Mukogawa, Nishinomiya, Hyogo 663-8501, Japan (S. Yoshimura). E-mail addresses: [email protected] (M. Fujita), [email protected] (S. Yoshimura).
https://doi.org/10.1016/j.brainres.2018.12.042 Received 9 November 2018; Received in revised form 19 December 2018; Accepted 28 December 2018 Available online 04 January 2019 0006-8993/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Human adipose-derived mesenchymal stem cells (hADSCs) share the surface phenotypes with mesenchymal stem cells (MSCs). A, a representative photograph of the semi-confluent state showing spindle-shaped cells attached to a culture dish. B, flow cytometry data showing MSC positive markers (CD105, CD73, and CD90) and MSC negative markers (CD11b, CD19, CD34, CD45, and HLA-DR).
2. Results
Human adipose-derived stem cells (hADSCs) have been established in our laboratory (Ikegame et al., 2011). We have recently reported the protective effects of hADSCs for cerebral infarction (Egashira et al., 2012). hADSCs secrete various cytokines at higher levels than bone marrow-derived (MSCs) (Ikegame et al., 2011). Based on these findings, hADSCs appear promising as a novel strategy for the clinical use in ICH patients. Based on these findings, we aimed to evaluate the therapeutic efficacy of hADSCs on the post-ICH neurological deficits using a clinically-relevant mouse model in this study. Besides, we evaluated immune responses to clarify the underlying mechanisms.
2.1. hADSCs share the surface phenotypes with MSCs To induce hADSCs, we extracted cell fraction from human adipose tissues and cultured the cells using the method described in the method section. The cultured cells became adherent and spindle-shaped (Fig. 1A). These cells expressed MSC surface markers CD73, CD90, and CD105 at high levels (all > 97%) whereas the cells did not express hematopoietic surface markers CD11b, CD19, CD34, CD45, and HLADR (all < 1%) (Fig. 1B). These findings led us to consider that these cells possessed the characteristics of hADSCs.
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These data suggest that the hADSC administration suppressed ICH-induced hyperactivity during the chronic phase. The wire hand test evaluates neuromuscular strength and stamina that is measured by hanging time on wire plates (latency to fall; Fig. 3B). One-way ANOVA suggested significant effects of groups (F2, 27 = 6.092, P = .0007). Post-hoc analyses revealed that the hADSC group exhibited significantly longer latency to fall than the ICH group (P = .0447). These data suggest that hADSC administration improved motor function of the ICH-bearing mice. The hotplate test evaluates the sensitivity to thermal nociception (Fig. 3C). Two-way repeated measures ANOVA suggested a significant effect of temperature (F7, 189 = 151.927, P = .0001) but not for groups (F2, 27 = 0.173, P = .842). The Y-maze test evaluates spatial working memory that is measured by the frequency to alternate turning directions (i.e., spontaneous alternation behaviors; Fig. 3D). One-way ANOVA suggested significant effect of groups (F2, 27 = 7.358, P = .00281). Post-hoc analysis revealed there was no difference between the ICH group and the hADSC group (P = .842) although both groups exhibited significant decreases in spontaneous alternation behaviors compared with the sham group (P = .004 and P = .012, respectively). These data suggest that ICH-induced spatial working memory deficits in mice. The Morris water maze learning test evaluates spatial recognition by two means. One is escaping latency that is measured by the time for mice to swim to a platform (Fig. 3E left). Two-way repeated measures ANOVA suggested significant effects of group (F2, 27 = 3.909, P = .032). Post-hoc analyses revealed a significant difference between the hADSC group and ICH group on days 4 and 5 (P = .0012 and .0001, respectively). Another measurement is the time to stay in the platformlocated quadrants of the swimming pool (Fig. 3E right). One-way ANOVA suggested significant effect of group (F2, 27 = 3.792, P = .035). Post-hoc analyses revealed hADSC group exhibited an increase in the time compared with the ICH groups (P = .037). These data suggest that the hADSC administration improved spatial recognition of the ICHbearing mice. The passive avoidance-learning test evaluates long-term memory function that is measured by passive avoidance latency to 24 h-interval electrical shocks (Fig. 3F). Two-way repeated measures ANOVA suggested significant effects of group (F2, 27 = 12.785, P = .00012). Posthoc analysis revealed that the hADSC group exhibited longer latency than the ICH group in 24 h and 48 h (P = .021 and P = .0002, respectively). These data suggest that the hADSC administration improved ICH-impaired long-term memory function. The elevated plus-maze test evaluates anxiety-like behaviors of mice that are measured by their preference of open arms to well-sheltered, closed arms (Fig. 3G). Two-way repeated measures ANOVA suggested no significant differences in the spending time in the closed arms (F2, 27 = 0.405, P = .671) and the open arms (F2, 27 = 0.146, P = .865). The open space swimming test evaluates depression-like symptoms that are measured by swimming length (Fig. 3H upper) and the immobility time of mice in a swimming pool (Fig. 3H lower); it has been used to evaluate the efficacy of antidepressant drugs in murine (Sun and Alkon, 2003; Stone and Lin, 2011). Two-way repeated measures ANOVA suggested significant effects of group on swimming length (F2, 27 = 9.943, P = .001) and immobility time (F2, 27 = 11.947, P = .000). Post-hoc analysis revealed that the hADSC group swam longer than that of the ICH group (P = .032) and that the hADSC group kept swimming longer than the ICH group (P = .022). These data suggest that the hADSC administration improved ICH-induced depression-like symptoms. Taken together, these neurobehavioral data suggest that intravenous administration of hADSCs at the acute phage improved ICHinduced neurological deficits during the chronic phases.
Fig. 2. Acute intravenous administration of hADSCs improves ICH-induced neurological deficits during the acute phase based on mNSS. A, Protocol of animal experiments to evaluate mNSS. C57BL/6J male mice were divided into three group: hADSC group (n = 11), ICH group (n = 10) and sham group (n = 9). B, mNSS were evaluated at Days 1, 2, 8 and 15. Data are plotted in mean ± SEM. P values are based on repeated measures ANOVA and post-hoc tests. †P < .05 and ††P < 01 compared with the sham group.
2.2. Acute intravenous administration of hADSCs improves ICH-induced neurological deficits during the acute phase based on mNSS To evaluate the significance of hADSCs on ICH, we induced ICH in the mouse brain and treated these mice with hADSCs. Subsequently, we evaluated the neurological signs according to the modified Neurological Severity Score (mNSS), a neurological scale commonly used for rodents (Fig. 2A) (Chen et al., 2001). Two-way repeated measure analysis of variance (ANOVA) suggested significant effects of group (F 2,27 = 11.5477, P = .0002). Post-hoc analyses revealed that the hADSC group and the ICH group exhibited an increase in the mNSS of Days 1 and 2 compared with the sham group (p < .01) (Fig. 2B). These data suggest that ICH-bearing mice suffered from severe neurological deficits during the acute phase. On Days 8 and 15, however, there was no difference in the mNSS among the groups. 2.3. Acute intravenous administration of hADSCs improves ICH-induced neurological deficits during the subacute phase based on neurobehavioral tests Although the mNSS well reflected the differences of the neurological signs of ICH-bearing mice in response to the hADSC administration at Days 1 and 2 we were unable to observe any difference in mNSS at Days 8 and 15 (Fig. 2). To address whether the hADSC administration would improve the neurological deficits of the ICH-bearing mice even during the subacute phase, we applied the following multitasking behavioral tests: open field test, wire hang test, hotplate test, Y-maze task, water maze learning test, passive avoidance-learning test, elevated plus-maze test, and open space swimming test (Fig. 3). Open field test evaluates spontaneous locomotive activities of mice in a novel environment (Fig. 3A). In this test, hyperactivity and habituation can be measured as locomotion counts (Winter et al., 2005; Bolivar et al., 2000). In this study, two-way repeated measures ANOVA suggested significant effects of groups (F2, 27 = 15.048, P = .00004). Post-hoc analyses revealed that the hADSC group exhibited significantly smaller locomotion counts than the ICH group for 3 days (P = .034). 60
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Fig. 3. Acute intravenous administration of hADSCs improves ICH-induced neurological deficits during the subacute phase based on neurobehavioral tests. The mice were subject to the following neurobehavioral tests on Day 29 and later: open field test (A), wire hang test (B), hotplate test (C), Y-maze test (D), water maze learning test (E), passive avoidance learning test (F), elevated plusmaze test (G), and open space swimming test (H). Data are plotted in mean ± SEM. P values are based on repeated measures ANOVA and post-hoc tests. *P < .05 and ** P < .01 compared with the ICH group. † P < .05 and ††P < .01 compared with the sham group. ‡P < .05 and ‡‡P < .01 compared with the conditioning trials of each group.
2.4. hADSCs administration suppress the accumulation of CD11b+ cells in the ICH lesions
2.5. hADSC administration decreases the number of CD11b+CD45+ cells and increases the proportion of CD86+ and Ly6C+ cells in the ICH lesions
The findings of the neurobehavioral tests led us to hypothesize that ICH-induced local inflammation in the brain might enlarge edema around the ICH and in turn induce neurobehavioral deficits. To address this hypothesis, we performed immunostaining assays for CD11b+ cells using frozen sections of the mouse brain at Day 8 (Fig. 4A). As we anticipated, CD11b+ cells accumulated around the ICH. The number of CD11b+ cells in the hADSC group was smaller than those of the ICH group. These data suggest that the hADSCs administration suppressed the accumulation of CD11b+ cells into the ICH lesions.
Brain CD11b+ cells include monocytes, macrophages, microglia, granulocytes, and natural killer cells (Li and Barres, 2017). Among them, we decided to focus on macrophages in the ICH-involving brain because of its majority (Mracsko and Veltkamp, 2014). To this end, we performed flow cytometry for CD11b+ cells (Fig. 5). A Wilcoxon test revealed decreases in the total cell number (P = .0085, Fig. 5B) and CD11b+CD45+ cells (P = .0138, Fig. 5C) in the hADSC group. We further examined the subpopulations of CD11b+CD45+ cells for their polarization. The proportion of CD86+CD163− cell (Fig. 5D) and 61
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Fig. 4. hADSCs administration suppress the accumulation of CD11b+ cells in the ICH lesions. CD11b staining of the ICH lesions at Day 8 in the hADSC group (A, n = 1) and the ICH group (B, n = 1). C, the number of CD11b+ cells around the ICH lesions were enumerated.
Ly6G+ Ly6C− cells (Fig. 5F) significantly increased in the hADSC group compared with the ICH group. In contrast, there was no difference between the ratio of CD163+CD86− (Fig. 5E) and Ly6G+Ly6C− cells (Fig. 5G). These data suggest that the ADSC administration decreased the number of CD11b+CD45+ cells but increased the proportion of CD86+ and Ly6C+ cells in the ICH lesions.
et al., 2013). The mNSS has several advantages in practice; it requires no particular apparatus, and it is repeatable. The disadvantage of the mNSS is that it does not detect the moderate changes of neurological signs during the chronic phase (Zhang et al., 2002) although it well detects the neurological differences during the acute phase as our data demonstrated (Fig. 2). Consistent with our data, a previous report has shown that intravenous administration of hADSC to ICH-bearing mice exhibited a significant improvement in mNSS at Day 8 but that no significant difference was observed after Day 8 to Day 29 (Yang et al., 2012). Our neurobehavioral tests were conducted at least 4 weeks later and met the cell therapy guidelines as long-term neurobehavioral test. There is a high demand for methods that can evaluate neurological changes during the subacute phase with no particular apparatus. We chose a collagenase-induced ICH mouse model (Fig. 2), which is the most frequently used model at present. This model has several advantages. Its procedure is easy to do, it is reproducible, and it shares the pathological mechanisms (such as vascular necrosis) with the human ICH. In this regard, recent cell therapy guidelines suggested that 1) reproducible animal models that can survive for a certain period are recommended because such models might undergo appropriate behavioral and histological tests and that 2) a combination of several different measures are also recommended to assess neurological functions (Savitz et al., 2011). Our ICH model matched these recommendations. However, since recombinant collagenase is an exogenous material, immune responses may theoretically occur to the collagenase inoculated into the brain. Nevertheless, we were able to address CD11bmediated immune responses in this study (Figs. 4–6) because of the following reasons. First, we used collagenase that did not contain animal-derived materials (animal origin free: AOF) so that it exhibited practically no antigenicity. Second, the volume of the collagenase solution was less than 1% of the volume of the induced ICH (MacLellan et al., 2008) so that the effect of collagen was considered ignorable. However, it is still ideal to develop new animal models that require no exogenous materials such as recombinant collagenase. ICH volume is a key factor affecting functional outcome of ICH. In this regard, an ideal method is to use animal-compatible MRI system although it is not easily available. Alternatively, we have previously shown that 0.45 units of collagenase injection stably induces 15.3–20.9 μl of ICH and 15–18 of Garcia score (Takagi et al., 2017). Here, Garcia scoring system evaluates the following six tests: spontaneous activity, symmetrical movement of four limbs, forepaw outstretching, climbing, body proprioception, and response to vibrissae touch (Garcia et al., 1995). Garcia score and mNSS are similar in evaluating motor, sensory, and reflex functions. Based on these findings, we omitted to measure ICH volume directly but evaluated mNSS instead. In parallel with this, we are in the process of applying intracranial ultrasonography technique to visualize ICH in a non-invasive way.
2.6. hADSC administration does not affect CD11b+ cells in the spleen To evaluate the systematic immune responses, we also analyzed splenic CD11+ cells in the hADSC group and the ICH group by flow cytometry (Fig. 6). Both the groups exhibited no differences in the total cell number (Fig. 6B) and CD11b+CD45+ cells (Fig. 6C). We also evaluated the proportion of CD86+CD163−, CD163+CD86−, Ly6C+Ly6G−, and Ly6G+Ly6C− cells (Fig. 6D–G) in the CD11b+CD45+ cells; there were no differences (Fig. 6F–G). These data suggest that the hADSCs administration did not affect systemic immune responses and that ICH-induced inflammation was limited locally in the brain. 3. Discussions In this study, we were able to induce hADSC in serum-free condition; they expressed MSC markers at high levels (Fig. 1B). To evaluate the therapeutic efficacy of the hADSCs for ICH, we administered the hADSCs to ICH-bearing mice by intracranial inoculation of recombinant collagenase (Fig. 2A). The hADSCs administration improved ICH-induced neurological deficits during the acute phase based on mNSS (Fig. 2B) as well as the subacute phase based on neurobehavioral tests (Fig. 3). As we hypothesized that ICH-induced local inflammation in the brain might enlarge edema around the ICH and in turn induce neurobehavioral deficits, we directed our focus to immune responses of the ICH-bearing mice. The hADSCs administration suppressed the accumulation of CD11b+ cells in the ICH lesions based on immunostaining assays (Fig. 4). Flow cytometry revealed that the hADSC administration also decreased the number of CD11+CD45+ cells and increased the proportion of CD86+ and Ly6C+ cells in the ICH lesions (Fig. 5). Next, to address whether the systemic immune response would also be involved, we evaluated immune cells in the spleen. The hADSCs administration did not affect the proportion of CD11+CD45+ subpopulations in the spleen (Fig. 6). These data collectively suggest that intravenous administration of hADSCs into ICH-bearing mice during the acute phase improved their neurobehavioral deficits during the subacute phase because of the suppression of acute inflammation mediated by the CD11+CD45+ subpopulations. We applied the mNSS to evaluate neurological signs of the ICHbearing mice (Fig. 2). Although the mNSS has been developed initially to evaluate stroke-bearing rodents (Chen et al., 2001; Chen et al., 2017; Li et al., 2000), it has also been used for other brain diseases (Zhang 62
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Fig. 5. hADSC administration decreases the number of CD11b+CD45+ cells and increases the proportion of CD86+ and Ly6C+ cells in the ICH lesions. A, gating strategies of the flow cytometry for CD11b, CD45, CD86, CD163, Ly6C, and Ly6G in the ICH lesions at Day 8. B, the total cell number. C, the number of CD11b+CD45+ cells. D, the proportion of CD11b+CD45+CD86+CD163− cells. E, the proportion of CD11b+CD45+CD86−CD163+ cells. F, the proportion of CD11b+CD45+Ly6C+Ly6G− cells. G, CD11b+CD45+Ly6C−Ly6G+ cells. Data are plotted in mean ± SEM. P values are based on the Wilcoxon test. *P < .05 compared with the ICH group.
space swimming test suggest that the hADSC administration improves the post-ICH depression, which is another serious problem in human ICH (Robinson and Jorge, 2016). The hotplate test showed no difference in the sensory impairment, whereas the passive avoidancelearning test showed significant differences in the long-term memory functions induced by ICH. These data suggest not only that the hADSC administration directly improves the long-term memory functions but
In this study, the neurobehavioral tests well demonstrated the ICHinduced neurological deficits and the therapeutic effects of hADSCs administration during the subacute phase (Fig. 3). In particular, the results of the open field test, the wire hang test, and the open space swimming test suggest that the hADSC administration mainly improves the post-ICH motor function, which is the most significant clinical problem in human ICH (Lai et al., 2002). Besides, the results of the open 63
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Fig. 6. hADSC administration does not affect splenocyte. A, gating strategies of the flow cytometry for CD11b, CD35, CD86, CD163, Ly6C, and Ly6G in spleens of ICH-bearing mice at Day 8. B, the total cell number. C, the number of CD11b+CD45+ cells. D, the proportion of CD11b+CD45+CD86+CD163− cells. E, the proportion of CD11b+CD45+CD86−CD163+ cells. F, the proportion of CD11b+CD45+Ly6C+Ly6G− cells. G, CD11b+CD45+Ly6C−Ly6G+ cells. Data are plotted in mean ± SEM. P values are based on the Wilcoxon test.
macrophages in the ICH lesions. A previous study has shown that M2 macrophages accumulate in ICH lesions (Min et al., 2016). In addition to this, we also demonstrated that the altered balance of M1 vs. M2 macrophages in the ICH lesions would correlate with the neurological signs during the subacute phase. However, biological functions of ICHassociated macrophages are yet unclear; they remain to be elucidated in the future. Moreover, microglia is another large population of CD11b+CD45+ cells in the brain (Li and Barres, 2017). It is necessary to distinguish macrophages from microglia and address their functions independently in the future studies. We observed no changes in the cell number and proportion of CD11b+CD45+ cells in the spleen in response to the hADSC administration (Fig. 6). Given that the ADSCs affected the kinetics of CD11b+CD45+ cells in the brain but not in the spleen (i.e., the systemic immune response), our data suggest that at least a part of systemically administered hADSCs reached the ICH lesions and affected the immune responses directly. Indeed, bone marrow-derived MSCs are known to migrate into the brain in vivo when they are systemically administered (De Becker and Riet, 2016). Although it is unclear whether and how hADSCs migrate into the brain in our model, we are currently in the
that motor functions, depression-like symptoms, and long-term memory functions are firmly related to each other (Leisman et al., 2016). Taken together, our neurobehavioral data suggest the hADSC-based treatment would provide a clue to improve post-ICH motor deficits, depression, and long-term memory disturbance. We addressed the immunomodulation in response to the hADSCs administration (Figs. 4–6). Regarding the polarization of macrophages, CD86+ and Ly6C+ macrophages have been categorized as M1 macrophage (Sica and Mantovani, 2012; Yang et al., 2014); they amplify and activate the inflammation (Swirski et al., 2007). In contrast, CD163+ and Ly6G+ macrophages have been M2 macrophages (Sica and Mantovani, 2012; Yang et al., 2014); they suppress inflammations (Cherry et al., 2014). In this regard, we observed an increase in both CD86+ cells and Ly6C+ cells in the hADSCs group (Fig. 5C and D), both of which are considered as M1 macrophages. In contrast, we observed no changes in the proportion of both CD163 + cells and Ly6G + cells (Fig. 5E and F), both of which are considered as M2 macrophages. However, given the difference in the cell number of CD11b+CD45+ cells in the ADSC group and the ICH group (Fig. 5B), our data suggest that the hADSC administration primarily decreased the number of M2 64
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4.5. Neurobehavioral tests
process of establishing a new culture method to label hADSCs to trace the distribution of hADSCs in vivo. In conclusion, we observed in this study that intravenous administration of hADSCs into ICH-bearing mice during the acute phase improved their neurobehavioral signs during the subacute phase probably because of the suppression of acute inflammation mediated by CD11b+CD45+ subpopulations. From a clinical viewpoint, our data also suggest that hADSCs can be served as a novel strategy for ICH treatment.
From Day 29 and later on, the hADSC, ICH, and sham mice were evaluated for phenotypical differences for the following neurobehavioral tests: an open field, wire hang, hotplate, Y-maze, water maze learning, passive avoidance learning, elevated plus maze, and open space swimming. These testing were also evaluated by researchers who were blinded to the experimental groups. 4.5.1. Open field test A cubic open-field box made of transparent acrylic plates without a ceiling (30 × 30 × 30 cm) was housed in a ventilated soundproof chamber (Taiyo Electric). An overhead incandescent light bulb provided room lighting intensity (approximately 110 lx) at the center of the test arena. A fan attached in the upper part of the wall at one end of the chamber provided a masking noise of 45 dB inside the chamber. On each X and Y lateral side of the open-field box, two infrared beams were attached 2 cm above the floor at a 10 cm distance. A flip-flop circuit was implemented between the two beams. The total number of circuit breaks was counted as a locomotive behavior. Each mouse was allowed to explore the open-field arena freely for 10 min per day for 3 consecutive days.
4. Experimental procedure 4.1. Cell preparation The study protocol was approved by the Ethics Committee of Hyogo College of Medicine (approval number: 1880). Abdominal subcutaneous adipose tissues were obtained from patients who underwent abdominal surgeries after the acquisition of written informed consent. The adipose tissues were rinsed with povidone-iodine solution, cut, homogenized, cultured at 37 °C with 0.45 Wünsch units per gram liberase (Roche) for 70 min, and centrifuged at 1800 rpm and 20 °C for 10 min. The supernatants were discarded, and 10 ml MEMα medium (Thermo Fisher Scientific) was added to the pellets. The cell suspension was sifted through a 100 μm filter and centrifuged at 1200 rpm and 20 °C for 5 min. The supernatant was removed again. Then, the cell suspension was cultured at a density of 5000 cells/cm2 and 37 °C in 10 ml of 50% STK1 medium (DS Pharma Biomedical). STK2 medium (DS Pharma Biomedical) was used after the second passage. The cells were stocked at −80 °C with STEM-CELLBANKER (Nippon Zenyaku Kogyo) after the fourth passage. When the cells were used, the cells were quickly thawed in a water bath at 37 °C. The cells were resuspended in Lactec Infusion (Otsuka Pharmaceutical) and centrifuge at 1100 rpm for 5 min and discard the supernatants. The cell pellet was resuspended in Lactec Infusion and filtered. The cell density was adjusted to 1 × 106 cells/100 μl and placed on ice. The cell preparation was completed within 1 h before administration.
4.5.2. Wire hang test A grid plate (30 × 30 cm) consisting of a square metal flame and rods (3 mm in diameter) placed at 10 mm intervals (center to center) was used. Each mouse was placed on the grid plate and allowed to acclimate to this environment for 10 s. The grid plate was then gently inverted and secured to the top of a cubic open-topped glass box (25 × 25 × 25 cm). Latency to fall was measured, with a maximum trial time of 180 s. The test was repeated twice with an interval of 60 s. 4.5.3. Hotplate test The hotplate test was conducted using a thermo-controllable aluminum plate (Model MK-350B, Muromachi Kikai). Each mouse was placed into a plexiglass cylinder on the heated plate. The temperature of the plate was raised from 50 °C to 64 °C with 2 °C intervals. Each temperature was tested with an interval of 10 min. The latency to jump or paw licking was recorded for 20 s.
4.2. Animals and ICH induction All experiments were approved and confirmed by the Institutional Animal Care Ethical Committee (Approval Numbers: 16-029 and 17035). Seven to nine-week-old male C57BL/6J mice were housed under 12-hours light-cycling conditions and had free access to waters and foods (CLEA Japan). Before ICH induction, the mice were anesthetized with 1.5–2.0% isoflurane. ICH was induced by inoculating 0.4 units of collagenase AOF type A (Worthington) into the brain, which located 2 mm to the left side and 3.5 mm depth from the bregma.
4.5.4. Y-maze test A Y-shaped maze with 3 arm runways (length: 40 cm, width: 3 cm, height: 20 cm) diverging at 120° angles from a central area was used. The arm runways were labeled A, B, and C. The Y-shaped maze was placed on a pedestal (height: 30 cm) and enclosed with four white walls (120 cm in height). The maze was illuminated by indirect lighting. The intensity of illumination was 50 lx on the maze floor. Each mouse was placed onto the central area and allowed to explore freely for 5 min. A mouse was considered to enter an arm runway when all 4 paws were in the runway. An alternation was defined as an entry of mice into all 3 different arms in sequence (e.g., ABC, CBA, BAC). The maximum alternation was calculated as the total number of arm entries minus 2, and the frequency of alternation was calculated as (actual alternation/ maximum alternation) × 100.
4.3. Administration of hADSC to ICH-bearing mice Mice were randomly divided into the following three groups: the hADSC group (n = 11), the ICH group (n = 10), and the sham group (n = 9). The sample size was determined according to previous our report (Sawano et al., 2015). The hADSC group received an intravenous injection of hADSCs (106 cells /100 μl) 24 h after the ICH induction; the ICH group received an intravenous injection of 100 μl Lactec infusion (Otsuka pharmaceutical) as a vehicle instead of the ADSCs. We also prepared the sham group with scalp incision and trepanation.
4.5.5. Water maze learning test The water maze learning test was conducted using a circular pool (inside diameter: 95 cm, depth: 35 cm) enclosed by white walls (width: 150 cm, height: 120 cm). The pool was made opaque by titanium oxide and filled with water to 22-cm depth. The temperature of the water was maintained at 24 ± 1 °C. The pool was divided into four virtual quadrants: north, south, east, and west. A round white platform (diameter: 10 cm) was located at the center of the north quadrant of the pool, submerged 0.5 cm below the water surface. Each mouse was subjected to 5 tests per day with an interval of 30 s for 5 consecutive days. In each test, the mouse was released into the water with its head
4.4. mNSS A mNSS were evaluated by one researcher who was blinded to the experimental groups at Days 0, 1, 8, and 15. The evaluation items included motor function, sensory function, reflex, and balance impairment. Each item was scored from 1 to 18 points (Chen et al., 2001). 65
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500G. Enriched immune cells were recovered at the Histopaque interface. Spleens were minced, filtrated by 70 μm mesh, suspended in Histopaque, and centrifuged for 20 min at 500 G. Enriched immune cells were recovered at the Histopaque interface. The cells were corrected and incubated with PerCP-Cy5.5-conjugated anti-mouse CD11b monoclonal antibody (BD Pharmingen), phycoerythrin (PE)-Cy7-conjugated anti-mouse CD45 monoclonal antibody (BD Pharmingen), phycoerythrin (PE)-conjugated anti-mouse CD86 antibody (BD Pharmingen), ALEXA FLUOR 488-conjugated anti-mouse CD163/M130 polyclonal antibody (Bioss), APC-conjugated anti-mouse Ly-6C monoclonal antibody (BD Phamingen), and BV421-conjugated anti-mouse Ly-6G monoclonal antibody (BD Horizon). Isotype controls were also used.
facing the edge of the pool in one of the south, east, or west quadrants at random. A test was terminated when the mouse reached the platform and remained on it for 10 s. If the platform was not found within 60 s, the mouse was guided to the platform by the experimenter and kept there for 10 s. On day 6, the platform was removed. Each mouse was released into the south quadrant of the pool and was allowed to swim freely for 60 s. All tests were recorded with an overhead CCD camera. The duration that the mice stayed in the north quadrant where the platform was placed was measured using a video-tracking system (Be Chase ver.1.3, ISONIX). 4.5.6. Passive avoidance learning test An apparatus consisted of two compartments, light and dark with the same dimension (15 × 15 × 15 cm) with a grid floor. A guillotine door separated the two compartments. In the conditioning test, each mouse was placed in the light compartment. Ten sec later, the guillotine door was opened. When the mouse moved into the dark compartment, the guillotine door was closed. Ten sec later, a scrambled electrical shock (120 v, 5 s) was delivered through the grid floor. Twenty-four hours and 48 h later, the retention test without any shock was conducted. Each mouse was placed in the light compartment and the latency to enter the dark compartment was recorded up to 180 s.
4.6.3. Flow cytometer The cells were analyzed by LSRFortessaX-20 (BD Biosciences) and BD FACSDiva software (BD Biosciences). Analysis of flow cytometry result was using FlowJo ver10.5 (BD Bioscience). 4.7. Immunostaining assays Animals were perfused with normal saline and then 4% paraformaldehyde in PBS under deep anesthesia with 3–4% isoflurane. Brains were fixed for 1 day with 4% paraformaldehyde after removal, cryoprotected in 30% sucrose solution at 4 °C, and frozen at −80 °C. The brain tissues were sectioned in 8 μm thickness using a cryostat (CM1950; Leica Biosystems). The primary antibodies used are as follows: CD11b BD Bioscience, 1:50. DAB staining was conducted by DAB peroxidase substrate KIT SK-4100 (Vector) which contain secondary antibodies. The stained sections were visualized using a fluorescence microscope (BZ-x710; Keyence).
4.5.7. Elevated plus maze test The plus-shaped maze was made of gray acrylic plates and consisted of a square center area (10 × 10 cm), two opposing open arms (length: 30 cm, width: 5 cm), and two opposing closed arms. The closed arms had transparent side-walls (height: 15 cm). The apparatus was raised 75 cm above the floor and illuminated by indirect lighting. The luminance was 150 lx in the center area. A fan supplied background noise at 40 dB. Each mouse was placed on the center area and allowed to explore freely for 10 min. The mice were recorded with an overhead CCD camera. The spending time on each arm was calculated using a videotracking system (Be Chase ver.1.3, ISONIX).
4.8. Statistical analyses
4.5.8. Open space swimming test The open space swimming test was conducted using a circular pool (inside diameter: 95 cm, depth: 35 cm) enclosed by white walls (width: 130 cm, height: 120 cm). The pool was made opaque by titanium oxide and filled with water to 20-cm depth. The temperature of the water was maintained at 22 ± 1 °C. Each mouse was placed in the pool with its head facing the outer edge of the pool and allowed to swim (or not swim) freely for 10 min. All tests were recorded with a CCD camera. The swimming length and immobility time were calculated by a videotracking system (Be Chase ver.1.3, ISONIX). The total amount of immobility was calculated by measuring the duration of time that the mouse traveled below the velocity of 3.25 cm/s.
The results are expressed as mean ± SEM. For the statistical analysis of the behavioral tests, a repeated measures ANOVA was conducted for groups (hADSC, ICH, or sham) as the between-subject factor and repeated measures (e.g., session, trial, or time) as the within-subject factor. When ANOVA found significant effects, Tukey’s post-hoc comparisons or the Fisher least-squares difference test were applied. For the statistical analyses of flow cytometry data, Wilcoxon test was applied. Significance levels were set at P < .05 (two-tailed). These statistical analyses were performed using SPSS ver. 22 (IBM Japan) and JMP version 13 (SAS Institute). Declaration
4.6. Flow cytometry assay
The authors have no conflict of interest to disclose.
4.6.1. Analyses of hADSC surface antigen The hADSCs were analyzed by Human MSC Analysis kit (BD Biosciences, Material number 562245). The hADSCs were incubated with FITC-conjugated anti-human CD90 antibody, PerCP-Cy5.5-conjugated anti-human CD105 antibody, APC-conjugated anti-human CD73 antibody, and PE-conjugated antibodies against human CD34, CD11b, CD19, CD45, and HLA-DR.
Acknowledgements The authors thank Shigeki Ohboshi, Yuki Takeda, and Hiromi Takeda for preparation of hADSCs and support of our experiments. The authors also thank the staffs in Joint-Use Research facilities, Hyogo College of Medicine for having allowed us to their resources such as flow cytometers.
4.6.2. Analyses of inflammatory cells from ICH-bearing mice Animals were perfused with normal saline and then 4% paraformaldehyde in PBS under deep anesthesia with 3–4% isoflurane. Then, brains and spleens were extracted. The brains were cut into small pieces and passed by 18-gauge and 20-gauge needle. After washing, brain cells were resuspended in 25% Percoll (GE Healthcare) and centrifuged for 20 min at 500G. The supernatants were discarded. The cells pellets were resuspended in Histopaque (Sigma) and centrifuged for 20 min at
Funding resources This work was supported by MEXT-Supported Program for the Strategic Research Foundation, Japan at Private Universities [Grant Number S1511034], Grant-in-Aid for Scientific Research (C) [Grant Number JP17K10854], and Research Support Program in the Hatasaki Foundation. 66
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Brain Research journal homepage: www.elsevier.com/locate/brainres
Research report
Intravenous administration of human adipose-derived stem cells ameliorates motor and cognitive function for intracerebral hemorrhage mouse model
T
Yoji Kuramotoa, Toshinori Takagia, Kotaro Tatebayashia, Mikiya Beppua, Nobutaka Doeb,c, ⁎ ⁎ Mitsugu Fujitad, , Shinichi Yoshimuraa, a
Department of Neurosurgery, Hyogo College of Medicine, Hyogo 663-8501, Japan Laboratory of Neurogenesis and CNS Repair, Hyōgo College of Medicine, Hyogo 663-8501, Japan c General Education Center, Hyogo University of Health Science, Hyogo 650-8530, Japan d Department of Microbiology, Kindai University, Faculty of Medicine, Osaka 589-8511, Japan b
H I GH L IG H T S
adipose-derived stem cells improve ICH-induced neurological deficits. • Human therapeutic effects are mediated by CD11b CD45 subpopulations. • The • Human adipose-derived stem cells can be served as a novel strategy for ICH treatment. +
+
A R T I C LE I N FO
A B S T R A C T
Keywords: Human adipose-derived stem cell Intracerebral hemorrhage Neurobehavior Macrophage
Even today, intracerebral hemorrhage (ICH) is a major cause of death and disabilities. Rehabilitation is preferentially applied for functional recovery although its effect is limited. Recent studies have suggested that intravenous administration of mesenchymal stem cells would improve the post-ICH neurological deficits. Human adipose-derived stem cells (hADSCs) have been established in our laboratory. We aimed to evaluate the therapeutic efficacy of the hADSCs on the post-ICH neurological deficits using a clinical-relevant ICH mouse model. We also evaluated immune responses to clarify the underlying mechanisms. The hADSCs expressed MSC markers at high levels. The hADSCs administration into the ICH-bearing mice improved the neurological deficits during the subacute phases, which was shown by neurobehavioral experiments. Besides, the hADSC administration decreased the number of CD11+CD45+ cells and increased the proportion of CD86+ and Ly6C+ cells in the ICH lesions. In summary, intravenous administration of hADSCs during the acute phase improved ICH-induced neurological deficits during the subacute phase because of the suppression of acute inflammation mediated by CD11+CD45+ subpopulations. Our data suggest that hADSCs can be served as a novel strategy for ICH treatment.
1. Introduction Intracerebral hemorrhage (ICH) accounts for 10% of all stroke and causes death and disabilities (van Asch et al., 2010); its mortality rate at 1 month after onset is about 40% and disability rate at 3 months after onset is about 60% (Anderson et al., 2013). Surgical intervention is ineffective to improve post-ICH neurological functions (Mendelow et al., 2013). No acute medication is available to improve the post-ICH neurological functions. Rehabilitation is preferentially applied for functional recovery although its effect is limited (Saulle and Schambra,
2016). The effectiveness of stem cell therapy has been reported in animal models of ICH (Chen et al., 2012; Yang et al., 2012; Xie et al., 2016). Among various stem cells, mesenchymal stem cells (MSCs) exhibit unique biological effects such as the production of growth factors and cytokines, immunomodulation, neurogenesis, and angiogenesis (Caplan and Dennis, 2006; Lee et al., 2014). Several animal studies have shown that intravenous administration of MSCs improves the post-ICH neurological deficits (Jeong et al., 2003; Lee et al., 2007; Yang et al., 2012; Xie et al., 2016). Nevertheless, its underlying mechanism is yet unclear.
⁎ Corresponding authors at: Department of Microbiology, Kindai University, Faculty of Medicine, 377-2 Ohno-Higasi, Osakasayama, Osaka 589-8511, Japan (M. Fujita). Department of Neurosurgery, Hyogo College of Medicine, 1-1 Mukogawa, Nishinomiya, Hyogo 663-8501, Japan (S. Yoshimura). E-mail addresses: [email protected] (M. Fujita), [email protected] (S. Yoshimura).
https://doi.org/10.1016/j.brainres.2018.12.042 Received 9 November 2018; Received in revised form 19 December 2018; Accepted 28 December 2018 Available online 04 January 2019 0006-8993/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Human adipose-derived mesenchymal stem cells (hADSCs) share the surface phenotypes with mesenchymal stem cells (MSCs). A, a representative photograph of the semi-confluent state showing spindle-shaped cells attached to a culture dish. B, flow cytometry data showing MSC positive markers (CD105, CD73, and CD90) and MSC negative markers (CD11b, CD19, CD34, CD45, and HLA-DR).
2. Results
Human adipose-derived stem cells (hADSCs) have been established in our laboratory (Ikegame et al., 2011). We have recently reported the protective effects of hADSCs for cerebral infarction (Egashira et al., 2012). hADSCs secrete various cytokines at higher levels than bone marrow-derived (MSCs) (Ikegame et al., 2011). Based on these findings, hADSCs appear promising as a novel strategy for the clinical use in ICH patients. Based on these findings, we aimed to evaluate the therapeutic efficacy of hADSCs on the post-ICH neurological deficits using a clinically-relevant mouse model in this study. Besides, we evaluated immune responses to clarify the underlying mechanisms.
2.1. hADSCs share the surface phenotypes with MSCs To induce hADSCs, we extracted cell fraction from human adipose tissues and cultured the cells using the method described in the method section. The cultured cells became adherent and spindle-shaped (Fig. 1A). These cells expressed MSC surface markers CD73, CD90, and CD105 at high levels (all > 97%) whereas the cells did not express hematopoietic surface markers CD11b, CD19, CD34, CD45, and HLADR (all < 1%) (Fig. 1B). These findings led us to consider that these cells possessed the characteristics of hADSCs.
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These data suggest that the hADSC administration suppressed ICH-induced hyperactivity during the chronic phase. The wire hand test evaluates neuromuscular strength and stamina that is measured by hanging time on wire plates (latency to fall; Fig. 3B). One-way ANOVA suggested significant effects of groups (F2, 27 = 6.092, P = .0007). Post-hoc analyses revealed that the hADSC group exhibited significantly longer latency to fall than the ICH group (P = .0447). These data suggest that hADSC administration improved motor function of the ICH-bearing mice. The hotplate test evaluates the sensitivity to thermal nociception (Fig. 3C). Two-way repeated measures ANOVA suggested a significant effect of temperature (F7, 189 = 151.927, P = .0001) but not for groups (F2, 27 = 0.173, P = .842). The Y-maze test evaluates spatial working memory that is measured by the frequency to alternate turning directions (i.e., spontaneous alternation behaviors; Fig. 3D). One-way ANOVA suggested significant effect of groups (F2, 27 = 7.358, P = .00281). Post-hoc analysis revealed there was no difference between the ICH group and the hADSC group (P = .842) although both groups exhibited significant decreases in spontaneous alternation behaviors compared with the sham group (P = .004 and P = .012, respectively). These data suggest that ICH-induced spatial working memory deficits in mice. The Morris water maze learning test evaluates spatial recognition by two means. One is escaping latency that is measured by the time for mice to swim to a platform (Fig. 3E left). Two-way repeated measures ANOVA suggested significant effects of group (F2, 27 = 3.909, P = .032). Post-hoc analyses revealed a significant difference between the hADSC group and ICH group on days 4 and 5 (P = .0012 and .0001, respectively). Another measurement is the time to stay in the platformlocated quadrants of the swimming pool (Fig. 3E right). One-way ANOVA suggested significant effect of group (F2, 27 = 3.792, P = .035). Post-hoc analyses revealed hADSC group exhibited an increase in the time compared with the ICH groups (P = .037). These data suggest that the hADSC administration improved spatial recognition of the ICHbearing mice. The passive avoidance-learning test evaluates long-term memory function that is measured by passive avoidance latency to 24 h-interval electrical shocks (Fig. 3F). Two-way repeated measures ANOVA suggested significant effects of group (F2, 27 = 12.785, P = .00012). Posthoc analysis revealed that the hADSC group exhibited longer latency than the ICH group in 24 h and 48 h (P = .021 and P = .0002, respectively). These data suggest that the hADSC administration improved ICH-impaired long-term memory function. The elevated plus-maze test evaluates anxiety-like behaviors of mice that are measured by their preference of open arms to well-sheltered, closed arms (Fig. 3G). Two-way repeated measures ANOVA suggested no significant differences in the spending time in the closed arms (F2, 27 = 0.405, P = .671) and the open arms (F2, 27 = 0.146, P = .865). The open space swimming test evaluates depression-like symptoms that are measured by swimming length (Fig. 3H upper) and the immobility time of mice in a swimming pool (Fig. 3H lower); it has been used to evaluate the efficacy of antidepressant drugs in murine (Sun and Alkon, 2003; Stone and Lin, 2011). Two-way repeated measures ANOVA suggested significant effects of group on swimming length (F2, 27 = 9.943, P = .001) and immobility time (F2, 27 = 11.947, P = .000). Post-hoc analysis revealed that the hADSC group swam longer than that of the ICH group (P = .032) and that the hADSC group kept swimming longer than the ICH group (P = .022). These data suggest that the hADSC administration improved ICH-induced depression-like symptoms. Taken together, these neurobehavioral data suggest that intravenous administration of hADSCs at the acute phage improved ICHinduced neurological deficits during the chronic phases.
Fig. 2. Acute intravenous administration of hADSCs improves ICH-induced neurological deficits during the acute phase based on mNSS. A, Protocol of animal experiments to evaluate mNSS. C57BL/6J male mice were divided into three group: hADSC group (n = 11), ICH group (n = 10) and sham group (n = 9). B, mNSS were evaluated at Days 1, 2, 8 and 15. Data are plotted in mean ± SEM. P values are based on repeated measures ANOVA and post-hoc tests. †P < .05 and ††P < 01 compared with the sham group.
2.2. Acute intravenous administration of hADSCs improves ICH-induced neurological deficits during the acute phase based on mNSS To evaluate the significance of hADSCs on ICH, we induced ICH in the mouse brain and treated these mice with hADSCs. Subsequently, we evaluated the neurological signs according to the modified Neurological Severity Score (mNSS), a neurological scale commonly used for rodents (Fig. 2A) (Chen et al., 2001). Two-way repeated measure analysis of variance (ANOVA) suggested significant effects of group (F 2,27 = 11.5477, P = .0002). Post-hoc analyses revealed that the hADSC group and the ICH group exhibited an increase in the mNSS of Days 1 and 2 compared with the sham group (p < .01) (Fig. 2B). These data suggest that ICH-bearing mice suffered from severe neurological deficits during the acute phase. On Days 8 and 15, however, there was no difference in the mNSS among the groups. 2.3. Acute intravenous administration of hADSCs improves ICH-induced neurological deficits during the subacute phase based on neurobehavioral tests Although the mNSS well reflected the differences of the neurological signs of ICH-bearing mice in response to the hADSC administration at Days 1 and 2 we were unable to observe any difference in mNSS at Days 8 and 15 (Fig. 2). To address whether the hADSC administration would improve the neurological deficits of the ICH-bearing mice even during the subacute phase, we applied the following multitasking behavioral tests: open field test, wire hang test, hotplate test, Y-maze task, water maze learning test, passive avoidance-learning test, elevated plus-maze test, and open space swimming test (Fig. 3). Open field test evaluates spontaneous locomotive activities of mice in a novel environment (Fig. 3A). In this test, hyperactivity and habituation can be measured as locomotion counts (Winter et al., 2005; Bolivar et al., 2000). In this study, two-way repeated measures ANOVA suggested significant effects of groups (F2, 27 = 15.048, P = .00004). Post-hoc analyses revealed that the hADSC group exhibited significantly smaller locomotion counts than the ICH group for 3 days (P = .034). 60
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Fig. 3. Acute intravenous administration of hADSCs improves ICH-induced neurological deficits during the subacute phase based on neurobehavioral tests. The mice were subject to the following neurobehavioral tests on Day 29 and later: open field test (A), wire hang test (B), hotplate test (C), Y-maze test (D), water maze learning test (E), passive avoidance learning test (F), elevated plusmaze test (G), and open space swimming test (H). Data are plotted in mean ± SEM. P values are based on repeated measures ANOVA and post-hoc tests. *P < .05 and ** P < .01 compared with the ICH group. † P < .05 and ††P < .01 compared with the sham group. ‡P < .05 and ‡‡P < .01 compared with the conditioning trials of each group.
2.4. hADSCs administration suppress the accumulation of CD11b+ cells in the ICH lesions
2.5. hADSC administration decreases the number of CD11b+CD45+ cells and increases the proportion of CD86+ and Ly6C+ cells in the ICH lesions
The findings of the neurobehavioral tests led us to hypothesize that ICH-induced local inflammation in the brain might enlarge edema around the ICH and in turn induce neurobehavioral deficits. To address this hypothesis, we performed immunostaining assays for CD11b+ cells using frozen sections of the mouse brain at Day 8 (Fig. 4A). As we anticipated, CD11b+ cells accumulated around the ICH. The number of CD11b+ cells in the hADSC group was smaller than those of the ICH group. These data suggest that the hADSCs administration suppressed the accumulation of CD11b+ cells into the ICH lesions.
Brain CD11b+ cells include monocytes, macrophages, microglia, granulocytes, and natural killer cells (Li and Barres, 2017). Among them, we decided to focus on macrophages in the ICH-involving brain because of its majority (Mracsko and Veltkamp, 2014). To this end, we performed flow cytometry for CD11b+ cells (Fig. 5). A Wilcoxon test revealed decreases in the total cell number (P = .0085, Fig. 5B) and CD11b+CD45+ cells (P = .0138, Fig. 5C) in the hADSC group. We further examined the subpopulations of CD11b+CD45+ cells for their polarization. The proportion of CD86+CD163− cell (Fig. 5D) and 61
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Fig. 4. hADSCs administration suppress the accumulation of CD11b+ cells in the ICH lesions. CD11b staining of the ICH lesions at Day 8 in the hADSC group (A, n = 1) and the ICH group (B, n = 1). C, the number of CD11b+ cells around the ICH lesions were enumerated.
Ly6G+ Ly6C− cells (Fig. 5F) significantly increased in the hADSC group compared with the ICH group. In contrast, there was no difference between the ratio of CD163+CD86− (Fig. 5E) and Ly6G+Ly6C− cells (Fig. 5G). These data suggest that the ADSC administration decreased the number of CD11b+CD45+ cells but increased the proportion of CD86+ and Ly6C+ cells in the ICH lesions.
et al., 2013). The mNSS has several advantages in practice; it requires no particular apparatus, and it is repeatable. The disadvantage of the mNSS is that it does not detect the moderate changes of neurological signs during the chronic phase (Zhang et al., 2002) although it well detects the neurological differences during the acute phase as our data demonstrated (Fig. 2). Consistent with our data, a previous report has shown that intravenous administration of hADSC to ICH-bearing mice exhibited a significant improvement in mNSS at Day 8 but that no significant difference was observed after Day 8 to Day 29 (Yang et al., 2012). Our neurobehavioral tests were conducted at least 4 weeks later and met the cell therapy guidelines as long-term neurobehavioral test. There is a high demand for methods that can evaluate neurological changes during the subacute phase with no particular apparatus. We chose a collagenase-induced ICH mouse model (Fig. 2), which is the most frequently used model at present. This model has several advantages. Its procedure is easy to do, it is reproducible, and it shares the pathological mechanisms (such as vascular necrosis) with the human ICH. In this regard, recent cell therapy guidelines suggested that 1) reproducible animal models that can survive for a certain period are recommended because such models might undergo appropriate behavioral and histological tests and that 2) a combination of several different measures are also recommended to assess neurological functions (Savitz et al., 2011). Our ICH model matched these recommendations. However, since recombinant collagenase is an exogenous material, immune responses may theoretically occur to the collagenase inoculated into the brain. Nevertheless, we were able to address CD11bmediated immune responses in this study (Figs. 4–6) because of the following reasons. First, we used collagenase that did not contain animal-derived materials (animal origin free: AOF) so that it exhibited practically no antigenicity. Second, the volume of the collagenase solution was less than 1% of the volume of the induced ICH (MacLellan et al., 2008) so that the effect of collagen was considered ignorable. However, it is still ideal to develop new animal models that require no exogenous materials such as recombinant collagenase. ICH volume is a key factor affecting functional outcome of ICH. In this regard, an ideal method is to use animal-compatible MRI system although it is not easily available. Alternatively, we have previously shown that 0.45 units of collagenase injection stably induces 15.3–20.9 μl of ICH and 15–18 of Garcia score (Takagi et al., 2017). Here, Garcia scoring system evaluates the following six tests: spontaneous activity, symmetrical movement of four limbs, forepaw outstretching, climbing, body proprioception, and response to vibrissae touch (Garcia et al., 1995). Garcia score and mNSS are similar in evaluating motor, sensory, and reflex functions. Based on these findings, we omitted to measure ICH volume directly but evaluated mNSS instead. In parallel with this, we are in the process of applying intracranial ultrasonography technique to visualize ICH in a non-invasive way.
2.6. hADSC administration does not affect CD11b+ cells in the spleen To evaluate the systematic immune responses, we also analyzed splenic CD11+ cells in the hADSC group and the ICH group by flow cytometry (Fig. 6). Both the groups exhibited no differences in the total cell number (Fig. 6B) and CD11b+CD45+ cells (Fig. 6C). We also evaluated the proportion of CD86+CD163−, CD163+CD86−, Ly6C+Ly6G−, and Ly6G+Ly6C− cells (Fig. 6D–G) in the CD11b+CD45+ cells; there were no differences (Fig. 6F–G). These data suggest that the hADSCs administration did not affect systemic immune responses and that ICH-induced inflammation was limited locally in the brain. 3. Discussions In this study, we were able to induce hADSC in serum-free condition; they expressed MSC markers at high levels (Fig. 1B). To evaluate the therapeutic efficacy of the hADSCs for ICH, we administered the hADSCs to ICH-bearing mice by intracranial inoculation of recombinant collagenase (Fig. 2A). The hADSCs administration improved ICH-induced neurological deficits during the acute phase based on mNSS (Fig. 2B) as well as the subacute phase based on neurobehavioral tests (Fig. 3). As we hypothesized that ICH-induced local inflammation in the brain might enlarge edema around the ICH and in turn induce neurobehavioral deficits, we directed our focus to immune responses of the ICH-bearing mice. The hADSCs administration suppressed the accumulation of CD11b+ cells in the ICH lesions based on immunostaining assays (Fig. 4). Flow cytometry revealed that the hADSC administration also decreased the number of CD11+CD45+ cells and increased the proportion of CD86+ and Ly6C+ cells in the ICH lesions (Fig. 5). Next, to address whether the systemic immune response would also be involved, we evaluated immune cells in the spleen. The hADSCs administration did not affect the proportion of CD11+CD45+ subpopulations in the spleen (Fig. 6). These data collectively suggest that intravenous administration of hADSCs into ICH-bearing mice during the acute phase improved their neurobehavioral deficits during the subacute phase because of the suppression of acute inflammation mediated by the CD11+CD45+ subpopulations. We applied the mNSS to evaluate neurological signs of the ICHbearing mice (Fig. 2). Although the mNSS has been developed initially to evaluate stroke-bearing rodents (Chen et al., 2001; Chen et al., 2017; Li et al., 2000), it has also been used for other brain diseases (Zhang 62
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Fig. 5. hADSC administration decreases the number of CD11b+CD45+ cells and increases the proportion of CD86+ and Ly6C+ cells in the ICH lesions. A, gating strategies of the flow cytometry for CD11b, CD45, CD86, CD163, Ly6C, and Ly6G in the ICH lesions at Day 8. B, the total cell number. C, the number of CD11b+CD45+ cells. D, the proportion of CD11b+CD45+CD86+CD163− cells. E, the proportion of CD11b+CD45+CD86−CD163+ cells. F, the proportion of CD11b+CD45+Ly6C+Ly6G− cells. G, CD11b+CD45+Ly6C−Ly6G+ cells. Data are plotted in mean ± SEM. P values are based on the Wilcoxon test. *P < .05 compared with the ICH group.
space swimming test suggest that the hADSC administration improves the post-ICH depression, which is another serious problem in human ICH (Robinson and Jorge, 2016). The hotplate test showed no difference in the sensory impairment, whereas the passive avoidancelearning test showed significant differences in the long-term memory functions induced by ICH. These data suggest not only that the hADSC administration directly improves the long-term memory functions but
In this study, the neurobehavioral tests well demonstrated the ICHinduced neurological deficits and the therapeutic effects of hADSCs administration during the subacute phase (Fig. 3). In particular, the results of the open field test, the wire hang test, and the open space swimming test suggest that the hADSC administration mainly improves the post-ICH motor function, which is the most significant clinical problem in human ICH (Lai et al., 2002). Besides, the results of the open 63
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Fig. 6. hADSC administration does not affect splenocyte. A, gating strategies of the flow cytometry for CD11b, CD35, CD86, CD163, Ly6C, and Ly6G in spleens of ICH-bearing mice at Day 8. B, the total cell number. C, the number of CD11b+CD45+ cells. D, the proportion of CD11b+CD45+CD86+CD163− cells. E, the proportion of CD11b+CD45+CD86−CD163+ cells. F, the proportion of CD11b+CD45+Ly6C+Ly6G− cells. G, CD11b+CD45+Ly6C−Ly6G+ cells. Data are plotted in mean ± SEM. P values are based on the Wilcoxon test.
macrophages in the ICH lesions. A previous study has shown that M2 macrophages accumulate in ICH lesions (Min et al., 2016). In addition to this, we also demonstrated that the altered balance of M1 vs. M2 macrophages in the ICH lesions would correlate with the neurological signs during the subacute phase. However, biological functions of ICHassociated macrophages are yet unclear; they remain to be elucidated in the future. Moreover, microglia is another large population of CD11b+CD45+ cells in the brain (Li and Barres, 2017). It is necessary to distinguish macrophages from microglia and address their functions independently in the future studies. We observed no changes in the cell number and proportion of CD11b+CD45+ cells in the spleen in response to the hADSC administration (Fig. 6). Given that the ADSCs affected the kinetics of CD11b+CD45+ cells in the brain but not in the spleen (i.e., the systemic immune response), our data suggest that at least a part of systemically administered hADSCs reached the ICH lesions and affected the immune responses directly. Indeed, bone marrow-derived MSCs are known to migrate into the brain in vivo when they are systemically administered (De Becker and Riet, 2016). Although it is unclear whether and how hADSCs migrate into the brain in our model, we are currently in the
that motor functions, depression-like symptoms, and long-term memory functions are firmly related to each other (Leisman et al., 2016). Taken together, our neurobehavioral data suggest the hADSC-based treatment would provide a clue to improve post-ICH motor deficits, depression, and long-term memory disturbance. We addressed the immunomodulation in response to the hADSCs administration (Figs. 4–6). Regarding the polarization of macrophages, CD86+ and Ly6C+ macrophages have been categorized as M1 macrophage (Sica and Mantovani, 2012; Yang et al., 2014); they amplify and activate the inflammation (Swirski et al., 2007). In contrast, CD163+ and Ly6G+ macrophages have been M2 macrophages (Sica and Mantovani, 2012; Yang et al., 2014); they suppress inflammations (Cherry et al., 2014). In this regard, we observed an increase in both CD86+ cells and Ly6C+ cells in the hADSCs group (Fig. 5C and D), both of which are considered as M1 macrophages. In contrast, we observed no changes in the proportion of both CD163 + cells and Ly6G + cells (Fig. 5E and F), both of which are considered as M2 macrophages. However, given the difference in the cell number of CD11b+CD45+ cells in the ADSC group and the ICH group (Fig. 5B), our data suggest that the hADSC administration primarily decreased the number of M2 64
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4.5. Neurobehavioral tests
process of establishing a new culture method to label hADSCs to trace the distribution of hADSCs in vivo. In conclusion, we observed in this study that intravenous administration of hADSCs into ICH-bearing mice during the acute phase improved their neurobehavioral signs during the subacute phase probably because of the suppression of acute inflammation mediated by CD11b+CD45+ subpopulations. From a clinical viewpoint, our data also suggest that hADSCs can be served as a novel strategy for ICH treatment.
From Day 29 and later on, the hADSC, ICH, and sham mice were evaluated for phenotypical differences for the following neurobehavioral tests: an open field, wire hang, hotplate, Y-maze, water maze learning, passive avoidance learning, elevated plus maze, and open space swimming. These testing were also evaluated by researchers who were blinded to the experimental groups. 4.5.1. Open field test A cubic open-field box made of transparent acrylic plates without a ceiling (30 × 30 × 30 cm) was housed in a ventilated soundproof chamber (Taiyo Electric). An overhead incandescent light bulb provided room lighting intensity (approximately 110 lx) at the center of the test arena. A fan attached in the upper part of the wall at one end of the chamber provided a masking noise of 45 dB inside the chamber. On each X and Y lateral side of the open-field box, two infrared beams were attached 2 cm above the floor at a 10 cm distance. A flip-flop circuit was implemented between the two beams. The total number of circuit breaks was counted as a locomotive behavior. Each mouse was allowed to explore the open-field arena freely for 10 min per day for 3 consecutive days.
4. Experimental procedure 4.1. Cell preparation The study protocol was approved by the Ethics Committee of Hyogo College of Medicine (approval number: 1880). Abdominal subcutaneous adipose tissues were obtained from patients who underwent abdominal surgeries after the acquisition of written informed consent. The adipose tissues were rinsed with povidone-iodine solution, cut, homogenized, cultured at 37 °C with 0.45 Wünsch units per gram liberase (Roche) for 70 min, and centrifuged at 1800 rpm and 20 °C for 10 min. The supernatants were discarded, and 10 ml MEMα medium (Thermo Fisher Scientific) was added to the pellets. The cell suspension was sifted through a 100 μm filter and centrifuged at 1200 rpm and 20 °C for 5 min. The supernatant was removed again. Then, the cell suspension was cultured at a density of 5000 cells/cm2 and 37 °C in 10 ml of 50% STK1 medium (DS Pharma Biomedical). STK2 medium (DS Pharma Biomedical) was used after the second passage. The cells were stocked at −80 °C with STEM-CELLBANKER (Nippon Zenyaku Kogyo) after the fourth passage. When the cells were used, the cells were quickly thawed in a water bath at 37 °C. The cells were resuspended in Lactec Infusion (Otsuka Pharmaceutical) and centrifuge at 1100 rpm for 5 min and discard the supernatants. The cell pellet was resuspended in Lactec Infusion and filtered. The cell density was adjusted to 1 × 106 cells/100 μl and placed on ice. The cell preparation was completed within 1 h before administration.
4.5.2. Wire hang test A grid plate (30 × 30 cm) consisting of a square metal flame and rods (3 mm in diameter) placed at 10 mm intervals (center to center) was used. Each mouse was placed on the grid plate and allowed to acclimate to this environment for 10 s. The grid plate was then gently inverted and secured to the top of a cubic open-topped glass box (25 × 25 × 25 cm). Latency to fall was measured, with a maximum trial time of 180 s. The test was repeated twice with an interval of 60 s. 4.5.3. Hotplate test The hotplate test was conducted using a thermo-controllable aluminum plate (Model MK-350B, Muromachi Kikai). Each mouse was placed into a plexiglass cylinder on the heated plate. The temperature of the plate was raised from 50 °C to 64 °C with 2 °C intervals. Each temperature was tested with an interval of 10 min. The latency to jump or paw licking was recorded for 20 s.
4.2. Animals and ICH induction All experiments were approved and confirmed by the Institutional Animal Care Ethical Committee (Approval Numbers: 16-029 and 17035). Seven to nine-week-old male C57BL/6J mice were housed under 12-hours light-cycling conditions and had free access to waters and foods (CLEA Japan). Before ICH induction, the mice were anesthetized with 1.5–2.0% isoflurane. ICH was induced by inoculating 0.4 units of collagenase AOF type A (Worthington) into the brain, which located 2 mm to the left side and 3.5 mm depth from the bregma.
4.5.4. Y-maze test A Y-shaped maze with 3 arm runways (length: 40 cm, width: 3 cm, height: 20 cm) diverging at 120° angles from a central area was used. The arm runways were labeled A, B, and C. The Y-shaped maze was placed on a pedestal (height: 30 cm) and enclosed with four white walls (120 cm in height). The maze was illuminated by indirect lighting. The intensity of illumination was 50 lx on the maze floor. Each mouse was placed onto the central area and allowed to explore freely for 5 min. A mouse was considered to enter an arm runway when all 4 paws were in the runway. An alternation was defined as an entry of mice into all 3 different arms in sequence (e.g., ABC, CBA, BAC). The maximum alternation was calculated as the total number of arm entries minus 2, and the frequency of alternation was calculated as (actual alternation/ maximum alternation) × 100.
4.3. Administration of hADSC to ICH-bearing mice Mice were randomly divided into the following three groups: the hADSC group (n = 11), the ICH group (n = 10), and the sham group (n = 9). The sample size was determined according to previous our report (Sawano et al., 2015). The hADSC group received an intravenous injection of hADSCs (106 cells /100 μl) 24 h after the ICH induction; the ICH group received an intravenous injection of 100 μl Lactec infusion (Otsuka pharmaceutical) as a vehicle instead of the ADSCs. We also prepared the sham group with scalp incision and trepanation.
4.5.5. Water maze learning test The water maze learning test was conducted using a circular pool (inside diameter: 95 cm, depth: 35 cm) enclosed by white walls (width: 150 cm, height: 120 cm). The pool was made opaque by titanium oxide and filled with water to 22-cm depth. The temperature of the water was maintained at 24 ± 1 °C. The pool was divided into four virtual quadrants: north, south, east, and west. A round white platform (diameter: 10 cm) was located at the center of the north quadrant of the pool, submerged 0.5 cm below the water surface. Each mouse was subjected to 5 tests per day with an interval of 30 s for 5 consecutive days. In each test, the mouse was released into the water with its head
4.4. mNSS A mNSS were evaluated by one researcher who was blinded to the experimental groups at Days 0, 1, 8, and 15. The evaluation items included motor function, sensory function, reflex, and balance impairment. Each item was scored from 1 to 18 points (Chen et al., 2001). 65
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500G. Enriched immune cells were recovered at the Histopaque interface. Spleens were minced, filtrated by 70 μm mesh, suspended in Histopaque, and centrifuged for 20 min at 500 G. Enriched immune cells were recovered at the Histopaque interface. The cells were corrected and incubated with PerCP-Cy5.5-conjugated anti-mouse CD11b monoclonal antibody (BD Pharmingen), phycoerythrin (PE)-Cy7-conjugated anti-mouse CD45 monoclonal antibody (BD Pharmingen), phycoerythrin (PE)-conjugated anti-mouse CD86 antibody (BD Pharmingen), ALEXA FLUOR 488-conjugated anti-mouse CD163/M130 polyclonal antibody (Bioss), APC-conjugated anti-mouse Ly-6C monoclonal antibody (BD Phamingen), and BV421-conjugated anti-mouse Ly-6G monoclonal antibody (BD Horizon). Isotype controls were also used.
facing the edge of the pool in one of the south, east, or west quadrants at random. A test was terminated when the mouse reached the platform and remained on it for 10 s. If the platform was not found within 60 s, the mouse was guided to the platform by the experimenter and kept there for 10 s. On day 6, the platform was removed. Each mouse was released into the south quadrant of the pool and was allowed to swim freely for 60 s. All tests were recorded with an overhead CCD camera. The duration that the mice stayed in the north quadrant where the platform was placed was measured using a video-tracking system (Be Chase ver.1.3, ISONIX). 4.5.6. Passive avoidance learning test An apparatus consisted of two compartments, light and dark with the same dimension (15 × 15 × 15 cm) with a grid floor. A guillotine door separated the two compartments. In the conditioning test, each mouse was placed in the light compartment. Ten sec later, the guillotine door was opened. When the mouse moved into the dark compartment, the guillotine door was closed. Ten sec later, a scrambled electrical shock (120 v, 5 s) was delivered through the grid floor. Twenty-four hours and 48 h later, the retention test without any shock was conducted. Each mouse was placed in the light compartment and the latency to enter the dark compartment was recorded up to 180 s.
4.6.3. Flow cytometer The cells were analyzed by LSRFortessaX-20 (BD Biosciences) and BD FACSDiva software (BD Biosciences). Analysis of flow cytometry result was using FlowJo ver10.5 (BD Bioscience). 4.7. Immunostaining assays Animals were perfused with normal saline and then 4% paraformaldehyde in PBS under deep anesthesia with 3–4% isoflurane. Brains were fixed for 1 day with 4% paraformaldehyde after removal, cryoprotected in 30% sucrose solution at 4 °C, and frozen at −80 °C. The brain tissues were sectioned in 8 μm thickness using a cryostat (CM1950; Leica Biosystems). The primary antibodies used are as follows: CD11b BD Bioscience, 1:50. DAB staining was conducted by DAB peroxidase substrate KIT SK-4100 (Vector) which contain secondary antibodies. The stained sections were visualized using a fluorescence microscope (BZ-x710; Keyence).
4.5.7. Elevated plus maze test The plus-shaped maze was made of gray acrylic plates and consisted of a square center area (10 × 10 cm), two opposing open arms (length: 30 cm, width: 5 cm), and two opposing closed arms. The closed arms had transparent side-walls (height: 15 cm). The apparatus was raised 75 cm above the floor and illuminated by indirect lighting. The luminance was 150 lx in the center area. A fan supplied background noise at 40 dB. Each mouse was placed on the center area and allowed to explore freely for 10 min. The mice were recorded with an overhead CCD camera. The spending time on each arm was calculated using a videotracking system (Be Chase ver.1.3, ISONIX).
4.8. Statistical analyses
4.5.8. Open space swimming test The open space swimming test was conducted using a circular pool (inside diameter: 95 cm, depth: 35 cm) enclosed by white walls (width: 130 cm, height: 120 cm). The pool was made opaque by titanium oxide and filled with water to 20-cm depth. The temperature of the water was maintained at 22 ± 1 °C. Each mouse was placed in the pool with its head facing the outer edge of the pool and allowed to swim (or not swim) freely for 10 min. All tests were recorded with a CCD camera. The swimming length and immobility time were calculated by a videotracking system (Be Chase ver.1.3, ISONIX). The total amount of immobility was calculated by measuring the duration of time that the mouse traveled below the velocity of 3.25 cm/s.
The results are expressed as mean ± SEM. For the statistical analysis of the behavioral tests, a repeated measures ANOVA was conducted for groups (hADSC, ICH, or sham) as the between-subject factor and repeated measures (e.g., session, trial, or time) as the within-subject factor. When ANOVA found significant effects, Tukey’s post-hoc comparisons or the Fisher least-squares difference test were applied. For the statistical analyses of flow cytometry data, Wilcoxon test was applied. Significance levels were set at P < .05 (two-tailed). These statistical analyses were performed using SPSS ver. 22 (IBM Japan) and JMP version 13 (SAS Institute). Declaration
4.6. Flow cytometry assay
The authors have no conflict of interest to disclose.
4.6.1. Analyses of hADSC surface antigen The hADSCs were analyzed by Human MSC Analysis kit (BD Biosciences, Material number 562245). The hADSCs were incubated with FITC-conjugated anti-human CD90 antibody, PerCP-Cy5.5-conjugated anti-human CD105 antibody, APC-conjugated anti-human CD73 antibody, and PE-conjugated antibodies against human CD34, CD11b, CD19, CD45, and HLA-DR.
Acknowledgements The authors thank Shigeki Ohboshi, Yuki Takeda, and Hiromi Takeda for preparation of hADSCs and support of our experiments. The authors also thank the staffs in Joint-Use Research facilities, Hyogo College of Medicine for having allowed us to their resources such as flow cytometers.
4.6.2. Analyses of inflammatory cells from ICH-bearing mice Animals were perfused with normal saline and then 4% paraformaldehyde in PBS under deep anesthesia with 3–4% isoflurane. Then, brains and spleens were extracted. The brains were cut into small pieces and passed by 18-gauge and 20-gauge needle. After washing, brain cells were resuspended in 25% Percoll (GE Healthcare) and centrifuged for 20 min at 500G. The supernatants were discarded. The cells pellets were resuspended in Histopaque (Sigma) and centrifuged for 20 min at
Funding resources This work was supported by MEXT-Supported Program for the Strategic Research Foundation, Japan at Private Universities [Grant Number S1511034], Grant-in-Aid for Scientific Research (C) [Grant Number JP17K10854], and Research Support Program in the Hatasaki Foundation. 66
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