Neurochem Res (2014) 39:59–67 DOI 10.1007/s11064-013-1190-1
ORIGINAL PAPER
Geissoschizine Methyl Ether, an Alkaloid from the Uncaria Hook, Improves Remyelination After Cuprizone-Induced Demyelination in Medial Prefrontal Cortex of Adult Mice Shoko Morita • Kouko Tatsumi • Manabu Makinodan Hiroaki Okuda • Toshifumi Kishimoto • Akio Wanaka
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Received: 25 July 2013 / Revised: 10 October 2013 / Accepted: 26 October 2013 / Published online: 6 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Accumulating evidence indicates that the medial prefrontal cortex (mPFC) is a site of myelin and oligodendrocyte abnormalities that contribute to psychotic symptoms of schizophrenia. The development of therapeutic approaches to enhance remyelination, a regenerative process in which new myelin sheaths are formed on demyelinated axons, may be an attractive remedial strategy. Geissoschizine methyl ether (GM) in the Uncaria hook, a galenical constituent of the traditional Japanese medicine yokukansan (Yi-gan san), is one of the active components responsible for the psychotropic effects of yokukansan, though little is known about the mechanisms underlying the effects of either that medicine or GM itself. In the present study, we employed a cuprizone (CPZ)-induced demyelination model and examined the cellular changes in response to GM administration during the remyelination phase in the mPFC of adult mice. Using the mitotic marker 5-bromo-20 -deoxyuridine (BrdU), we demonstrated that CPZ treatment significantly increased the number of BrdUpositive NG2 cells, as well as microglia and mature oligodendrocytes in the mPFC. Newly formed oligodendrocytes were increased by GM administration after CPZ exposure. In addition, GM attenuated a decrease in myelin basic protein immunoreactivity caused by CPZ administration. Taken together, our findings suggest that GM
administration ameliorated the myelin deficit by mature oligodendrocyte formation and remyelination in the mPFC of CPZ-fed mice. The present findings provide experimental evidence supporting the role for GM and its possible use as a remedy for schizophrenia symptoms by promoting the differentiation of progenitor cells to and myelination by oligodendrocytes. Keywords Schizophrenia Yokukansan (Yi-gan san) Remyelination Myelin Immunohistochemistry Abbreviations BrdU 5-Bromo-20 -deoxyuridine CPZ Cuprizone GM Geissoschizine methyl ether GS Glutamine synthetase GSTpi Glutathione S-transferase-pi MBP Myelin basic protein mPFC Medial prefrontal cortex NF Neurofilament PB Phosphate buffer PBS Phosphate-buffered saline PBST PBS containing 0.3 % Triton X-100
Introduction S. Morita (&) K. Tatsumi H. Okuda A. Wanaka Department of Anatomy and Neuroscience, Faculty of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan e-mail: [email protected] M. Makinodan T. Kishimoto Department of Psychiatry, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan
Oligodendrocyte progenitor cells differentiate into myelinforming oligodendrocytes and myelin is essential for proper function and maintenance of neurons in the brain [for review see 1]. Many studies have implicated the medial prefrontal cortex (mPFC) as a site of myelin and oligodendrocyte abnormalities in schizophrenia [2–4], while a reduction in grey matter volume and changes in glucose metabolism and blood flow have also been
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reported in the mPFC of schizophrenia patients [5, 6]. In another study, loss of social experiences in juvenile mice resulted in myelination deficits in the mPFC and led to neuropsychological disorders that persisted into adulthood [7], indicating that myelination in the mPFC is pivotal in the development of complex emotional and cognitive behaviors. Remyelination has been demonstrated in animal models and some patients to be mediated by oligodendrocyte progenitor cells, which migrate into the lesion, proliferate, and differentiate into mature oligodendrocytes and then ensheath the demyelinated axons, though it is variable and insufficient. Although the reasons for remyelination failure in myelin disorders are unknown [8], the identification of oligodendrocyte progenitor cells within demyelinated lesions suggests a critical regulatory mechanism in the inhibition of oligodendrocyte progenitor cell differentiation, preventing their maturation to myelin-forming oligodendrocytes [9]. Therefore, development of therapeutic approaches to enhance remyelination is an attractive strategy for the treatment of schizophrenia patients. A variety of effective antipsychotics have been developed for schizophrenia, but because some induce extrapyramidal symptoms, hyperprolactinemia, drowsiness, dizziness, and unwanted weight gain [for review see 10], safer treatments are desirable. Yokukansan (Yi-gan san), a traditional Japanese medicine, has been shown to inhibit degeneration of oligodendrocytes and myelin sheaths in rats with thiamine deficiency-induced encephalopathy [11]. Moreover, this medicine is useful in treating neuropsychological disorders such as behavioral and psychological symptoms of dementia [12], borderline personality disorder [13], and psychotic symptoms of schizophrenia [14, 15]. Geissoschizine methyl ether (GM), a galenical constituent of yokukansan, isolated from the Uncaria hook, is one of the active components responsible for the psychotropic effects of yokukansan [16, 17], though little is known about the mechanisms underlying the effects of either this traditional medicine or GM. Cuprizone [oxalic acid bis (cyclohexylidenehydrazide, CPZ)], a selective and sensitive copper chelator, can be used to generate an animal model of toxic demyelination. Interestingly, mice treated with 0.2 % CPZ display behaviors reminiscent of schizophrenia symptoms. For example, CPZ-fed mice showed increased CNS activity, reduced anxiety in a novelty challenge test, and altered sensorimotor reactivity [18]. CPZ treatment also results in impairments in spatial working memory, social interaction, and prepulse inhibition [19]. In addition, these animals show loss of the myelin sheath and oligodendrocytes in the brain regions such as the corpus callosum, the cerebellum, the hippocampus, the caudate putamen, and the mPFC [20, 21], while such demyelinating lesions may be partially remyelinated with thinner myelin sheaths [22]. Therefore,
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CPZ has been found useful for studies of experimental remyelination [22]. These observations suggest that CPZtreated mouse can be used as an animal model of demyelination/remyelination suitable for testing the effects of GM administration. In the present study, we examined whether GM ameliorates demyelination, particularly promotion of remyelination in CPZ-treated mice. We initially investigated cell proliferation or survival using the thymidine analog 5-bromo-20 -deoxyuridine (BrdU) in combination with immunohistochemistry with cell markers. Next, we examined the expression level of myelin basic protein (MBP) using semi-quantitative morphometric analysis following MBP immunohistochemistry.
Materials and Methods Animals Adult male mice (C57BL/6J) aged 57-113 days were used in the experiments. The animals were housed in a colony room with a 12-h light/dark cycle, and given access to commercial chow and tap water ad libitum. Animal care and the experiments were conducted in accordance with the guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals and the Guidelines for Proper Conduct of Animal Experiments of the Science Council of Japan. The Animal Care Committee of Nara Medical University approved the experimental protocol. Experimental Design Figure 1a depicts the experimental schedule used in the present study. Mice were given powdered standard rodent chow containing 0.2 % (w/w) CPZ (Sigma-Aldrich, Tokyo, Japan) for 4 weeks until perfusion. Control mice were fed with powdered standard rodent chow. We divided 20 C57BL/6J mice into 4 groups of 5 each. Two groups (CPZ ? vehicle and CPZ ? GM) were given powdered standard rodent chow containing 0.2 % (w/w) CPZ for 4 weeks, while the other 2 groups (normal ? vehicle and normal ? GM) received powdered standard chow without CPZ. After that 4-week period, all groups were fed standard chow, and the CPZ ? GM and normal ? GM groups received GM (0.3 mg/kg, kindly supplied by Tsumura & Co., Ibaraki, Japan) dissolved in a vehicle (0.5 % Tween80), while the CPZ ? vehicle and normal ? vehicle groups received the vehicle alone, via oral gavage once daily for the next 2 weeks. To monitor the proliferative activity of cells, mice received BrdU (Sigma-Aldrich, 1 mg/ml) in drinking water. BrdU administration via drinking water has been established for labeling of proliferating cells [23].
Neurochem Res (2014) 39:59–67 Fig. 1 Experimental design, and effects of CPZ treatment and GM administration on body weight of adult mice. a Mice were given a 0.2 % CPZcontaining or normal diet for 4 weeks, and thereafter were administrated GM (0.3 mg/kg) or the vehicle orally once daily for 2 weeks. BrdU was administered in drinking water (1 mg/ml). b Body weights were measured once a week. Representative data were obtained from 5 animals in each group (mean ± SEM; ***P \ 0.001 vs. normal chowfed mice, two-way ANOVA with Tukey–Kramer’s post hoc test). c Schematic of all bregma regions included for quantification
61 1.98 Bregma
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b 1.78
1.70
Antibodies We employed the following primary antibodies for lightmicroscopic immunohistochemistry: mouse monoclonal IgG against neurofilament (NF; clone 2H3; Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA; dilution 1:20); rabbit polyclonal IgG against MBP (A0623, DAKO, Cambridgeshire, UK; dilution 1:500), glutathione S-transferase-pi (GSTpi; 312, Medical & Biological Laboratories Co., Nagoya, Japan; dilution 1:500), glutamine synthetase (GS; G2781, Sigma-Aldrich; dilution 1:18,000), NG2 (Millipore-Chemicon, Temecula, CA; dilution 1:5,000), Iba1 (019-19741, Wako Pure Chemical Industries, Ltd., Osaka, Japan; dilution 1:200); rat monoclonal IgG against BrdU (OBT0030, Serotec, Raleigh, NC; dilution 1:100); and Armenian hamster monoclonal IgG against CD31 (clone 2H8, DSHB; dilution 1:20). Immunohistochemistry Mice were perfused with saline followed by 4 % paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB; pH 7.4) under deep anesthesia using pentobarbital. The dissected brains were post-fixed with 4 % PFA in 0.1 M PB (pH 7.2) for 6 h at 4 °C. Fixed brains were cryo-protected with 30 % sucrose in phosphate-buffered saline (PBS; pH 7.4) and
frozen at -80 °C in Tissue-Tek OCT compound (Sakura Finetechnical, Tokyo, Japan). Coronal sections were cut at a thickness of 20-lm with a cryostat (Bright Instrument, Huntingdon, UK). For immunofluorescence detection, we processed free-floating sections as previously described [24, 25]. In brief, sections were washed with PBS and treated with 25 mM glycine in PBS for 30 min, then incubated with 5 % normal goat serum in PBS containing 0.3 % Triton X-100 (PBST) for 24 h at 4 °C, followed by incubation with a primary antibody for 48–72 h at 4 °C. Thereafter, the sections were incubated with Alexa 488- or Alexa 546-conjugated secondary goat IgG (Invitrogen, Carlsbad, CA: dilution 1:1,000), or DyLight 405-conjugated secondary goat IgG (Thermo Scientific, Rockford, IL, dilution 1:400). For MBP immunohistochemistry, sections were pretreated with 0.3 % H2O2 in methanol for 15 min. The sections were permeablized by methanol for the MBP antibody to penetrate the subsequent layers of membrane. For BrdU immunolabeling, we performed antigen retrieval by incubating sections in Na-citrate buffer (10 mM, pH 6.0) for 10 min at 95 °C, and then incubated sections in 2 N HCl at 37 °C for 20 min. Confocal Observation and Quantification Specimens were placed on slides and coverslips sealed with Vectashield (Vector Labs), then observations were
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performed using a laser-scanning confocal microscope (FV1000, OLYMPUS, Tokyo, Japan, or C2, Nikon Corporation, Tokyo, Japan). We used 3–5 animals from each group for quantification, and at least 7 sections per animal were chosen from the mPFC region (between bregma ?1.70 and ?1.98 mm) regions according to the mouse brain atlas [26]. To minimize observation biases among different fields and sections, the parameters of the confocal microscope (such as pinhole size, brightness, and contrast setting) were maintained. Images were obtained (512 9 512 pixels) and saved as TIF files using OLYMPUS FV10-ASW Ver 1.7 Viewer or Nikon Nis-Elements AR software. Data Analysis For semi-quantitative analyses, the intensities of MBP and NF immunofluorescence were measured in saved images using ImageJ software (National Institutes of Health). The image analyst was blind to the treatment groups to eliminate biases. Data were analyzed by two-way ANOVA, followed by Tukey–Kramer’s multiple comparisons. Difference was assessed at a significance level of P \ 0.05.
Results Effects of CPZ and GM Administrations on Body Weight of Adult Mice For these experiments, mice were fed chow with or without 0.2 % CPZ for 4 weeks and then administered 0.3 mg/kg GM or the vehicle alone for 2 weeks (Fig. 1a). In addition, mice received BrdU administrations for 2 weeks concurrently with GM. During the first 4 weeks, the CPZ-exposed mice showed significantly lower body weight than controls, which displayed a mild weight gain during the same period (Fig. 1b). Body weights of mice that received the CPZcontaining diet became indistinguishable from that of the controls at the end of fifth week, while GM administration did not have effects on body weight. For histological analysis, all mPFC subregions were included in rostrocaudal levels of the mPFC (between bregma ?1.70 and ?1.98 mm, Fig. 1c). Changes in the Number of Mature Oligodendrocytes and MBP Immunoreactivity in mPFC of Adult Mice After CPZ Exposure We quantified the number of mature oligodendrocyte using an antibody to GSTpi after a 4-week CPZ treatment. The number of GSTpi? cells was decreased after the CPZ treatment (30.70 ± 0.73/mm2) as compared with the
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normal chow-fed group (17.70 ± 0.84/mm2) (Fig. 2b). Thereafter we performed immunohistochemical analysis using an antibody to MBP as a marker for mature myelination and NF as a marker of axons after a 4-week CPZ treatment. A distinct decrease in MBP immunoreactivity was visible in the mPFC of CPZ-fed mice, while no difference could be observed in NF staining between control and CPZ (Fig. 2d–d’’). Also, semi-quantitative morphometric analysis demonstrated that CPZ treatment significantly decreased the MBP intensity (% of the control; 50.57 ± 4.67) and the ratio of MBP to NF intensity (normal, 0.31 ± 0.07; 0.15 ± 0.05) (Fig. 2e). These findings showed that CPZ treatment for 4 weeks dramatically decreased MBP immunoreactivity in the mPFC of adult mice. Changes in Number of BrdU-Labeled Cells in mPFC of CPZ-Fed Mice by GM Administration Next, we focused on BrdU-labeled cells that responded to demyelination in the mPFC of adult mice. We administered BrdU for 2 weeks and examined the identities of BrdU-positive cells by double immunolabeling for the cell markers including NG2 proteoglycan and GSTpi (for oligodendrocyte lineage), Iba1 (for microglia), GS (for astrocytes), and CD31 (for endothelial cells). BrdU-labeled cells were more abundant in the mPFC of CPZ-fed mice than in the normal mice (Fig. 3a– d). We also observed BrdU-labeled nuclei in NG2? oligodendrocyte progenitor cells, GSTpi? mature oligodendrocytes, Iba1? microglial cells, GS? astrocytes, and CD31? endothelial cells (Fig. 3e–i), with varying frequency. Results of quantitative analysis further revealed that CPZ treatment significantly increased the number of BrdU-labeled NG2? oligodendrocyte progenitor (normal ? vehicle, 22.1 ± 2.6; normal ? GM, 34.4 ± 7.5; CPZ ? vehicle, 50.5 ± 4.9; CPZ ? GM, 50.1 ± 4.0/mm2) and Iba1? microglial (normal ? vehicle, 17.1 ± 1.1; normal ? GM, 19.4 ± 2.1; CPZ ? vehicle, 44.2 ± 3.3; CPZ ? GM, 48.3 ± 2.7/mm2) cells, compared with those of the normal chow-fed group (Fig. 3j). GM administration alone had no significant effect on BrdU-labeled nuclei of the NG2? oligodendrocyte progenitor or Iba1? microglial cells. In contrast, a significant increase in BrdU-labeled GSTpi? mature oligodendrocytes (normal ? vehicle, 0.5 ± 0.5; normal ? GM, 1.5 ± 1.5; CPZ ? vehicle, 5.8 ± 1.9; CPZ ? GM, 21.1 ± 4.8/mm2) was detected with GM administration after CPZ treatment. On the other hand, CPZ and subsequent GM administration did not change the number of BrdU-labeled nuclei in GS? astrocytes or CD31? endothelial cells. The total number of GSTpi? mature oligodendrocytes tended to increase with GM administration after CPZ treatment, though it did not reach statistical significance (Fig. 4). These results indicate that newly formed oligodendrocytes were increased by GM administration after CPZ treatment in the mPFC of adult mice.
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Fig. 2 Loss of oligodendrocytes and MBP expression level in mPFC after a 4-week of CPZ treatment. Sections were prepared from untreated normal and CPZ-fed mice, and then examined by immunohistochemistry with the anti-GSTpi, MBP, and NF antibody. a An atlas-based image depicting the area of the representative confocal images (gray square). b The number and density of GSTpi? cells were measured. Quantitative analysis showed that the number of GSTpi? cells was significantly decreased by CPZ exposure. Immuno-signal intensity was determined using image analysis and the ratio of MBP to NF was calculated. Representative confocal images show immunoreactivity for c, d MBP and c’, d’ NF, and merged images c’’, d’’ for each group. MBP immunoreactivity was weaker in the mPFC after CPZ treatment than in the mPFC of normal mice. Scale bar = 50 lm. e Semiquantitative morphometric analysis showed that MBP immunoreactivity and rerative ratio of MBP/NF intensity were significantly decreased by CPZ treatment as compared with the control. Representative data were obtained from 5 animals in each group (mean ± SEM; *P \ 0.05, ***P \ 0.001, twoway ANOVA with Tukey– Kramer’s post hoc test)
GM-Induced Changes in MBP Expression in mPFC of CPZ-Exposed Mice To investigate the effect of GM administration on myelination in the mPFC of CPZ-fed mice, we performed immunohistochemical analysis using MBP and NF. In the mPFC of these mice, we observed a high proportion of MBP immunoreactivity overlapping with NF signals
(Fig. 5a–a’’). The intensity of MBP immunoreactivity was reduced considerably in the mPFC by CPZ treatment (Fig. 5c–c’’), whereas subsequent administration of GM ameliorated this CPZ-induced decrease (Fig. 5d–d’’). Mice treated with GM alone showed MBP immunoreactivity (Fig. 5b–b’’) at a level comparable to that of the control (Fig. 5a–a’’). Also, semi-quantitative morphometric analysis demonstrated that CPZ treatment significantly
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Fig. 3 Effects of CPZ treatment and GM administration on the number of BrdU-labeled cells. Mice were given a 0.2 % CPZ or normal diet for 4 weeks, and thereafter orally administered GM or vehicle once daily for 2 weeks. Newborn cells were detected by BrdU immunohistochemistry after administration of BrdU via drinking water (1 mg/ml). Two weeks later, coronal sections of the brains were double-labeled for BrdU and the cell markers (NG2, GSTpi, Iba1, GS, and CD31) in the mPFC. a–d Representative confocal images show large numbers of BrdU-labeled nuclei in the mPFC of CPZ-treated mice, whereas few were seen in the mPFC of control mice (arrowheads in a-d). e–i BrdU-labeled nuclei in NG2?, GSTpi?,
Iba1?, GS?, and CD31? cells in the mPFC. Scale bars = 50 lm in a and 10 lm in e. j Quantitative analysis of numbers of BrdU-labeled nuclei in NG2? and Iba1? cells showed significant increases after CPZ treatment relative to that of the control. The number of BrdUlabeled GSTpi? cells was significantly increased by GM administration as compared to the vehicle after CPZ treatment. Representative data were obtained from 5 animals in each group (mean ± SEM; **P \ 0.01, ***P \ 0.001 vs. normal ? vehcle, ##P \ 0.01, ### P \ 0.001 vs. normal ? GM, P \ 0.05, P \ 0.001 vs. CPZ ? vehicle, two-way ANOVA with Tukey–Kramer’s post hoc test)
decreased the MBP intensity, whereas repeated subsequent administrations of GM for 2 weeks significantly ameliorated the CPZ-induced decrease in that ratio (% of control; normal ? GM, 85.91 ± 8.59; CPZ ? vehicle, 58.64 ± 6.92; CPZ ? GM, 85.19 ± 8.05) (Fig. 5e). CPZ treatment significantly decreased the ratio of MBP to NF intensity, whereas subsequent GM administrations for 2 weeks significantly ameliorated the CPZ-induced decrease in that ratio (normal ? vehicle, 0.31 ± 0.03; normal ? GM, 0.32 ± 0.02; CPZ ? vehicle, 0.22 ± 0.02; CPZ ? GM, 0.28 ± 0.02) (Fig. 5f).
Discussion
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Abnormalities of myelin and oligodendrocytes have been reported in schizophrenia [for reviews see 2, 27]. Remyelination by endogenous oligodendrocyte progenitor cells occurs in animal models and some patients, though its extent is insufficient. Therefore, remyelination-enhancing medications, which are currently used for treatment of multiple sclerosis [28], may contribute to schizophrenia therapy. Yokukansan and its constituent GM are likely to have good effects on schizophrenia, although their pharmacological properties are not yet been fully
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Fig. 4 Effects of CPZ and GM administrations on number of GSTpi? mature oligodendrocytes. Mice were given a 0.2 % CPZ or normal diet for 4 weeks, and thereafter orally administered GM or the vehicle once daily for 2 weeks. Coronal sections of the mPFC were immunolabeled for GSTpi. The number and density of GSTpi? cells were measured. Quantitative analysis showed that the number of GSTpi? cells was not significantly changed by CPZ exposure or GM administration. Representative data were obtained from 5 animals in each group (mean ± SEM; two-way ANOVA with Tukey– Kramer’s post hoc test)
investigated. In the present study, we examined the effects of GM administration on body weight, BrdU assay, as well as expression of the myelin marker MBP in a mouse model of demyelination/remyelination induced by CPZ. We observed that GM administration increased new oligodendrocyte formation, and attenuated the decrease in immunoreactivity of MBP in the mPFC of CPZ-treated mice without causing overt weight gain. There is evidence for oligodendrocyte alterations across most psychiatric conditions, including but not limited to attention deficit and hyperactivity disorder, autism, bipolar disorder, major depressive disorder, Alzheimer’s disease and schizophrenia [for review see 29]. This would have implications GM may be a novel remedy not only for schizophrenia but also for other established demyelinating diseases. The oligodendrogenesis is important for remyelination in demyelinating diseases as well as for normal myelination, thus we examined the influence of GM administration on the number of BrdU-labeled cells in the mPFC of CPZfed mice. The number of BrdU-labeled cells in the mPFC of CPZ-treated mice increased in the absence of GM administration relative to those in the mPFC of control mice. The significant increase in number of BrdU-labeled NG2? cells in the CPZ-treated mice was considered to be an endogenous response of oligodendrocyte progenitor cells to pathological insult in the brain. This corroborates a previous report that oligodendrocyte progenitor cells proliferate in demyelinated lesions during remyelination [27]. In our previous study, we found that Olig2 lineage cells proliferated and differentiated into NG2? and APC? oligodendrocyte lineage cells more efficiently in the external capsule of CPZ-fed mice than in a normal diet group [28].
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Interestingly, we found that significantly more BrdUpositive cells had differentiated into GSTpi? mature oligodendrocytes by GM administration after CPZ treatment than by vehicle administration, while that difference was not observed for the Iba1?-microglia, GS?-astrocytes, or CD31?-endothelial cells (Fig. 3j). Clearance of damaged myelin is necessary for demyelination, and it has been shown that depletion of microglia impairs myelin debris clearance, demyelination, and remyelination [29]. In the present study, the number of BrdU-labeled microglial cells was significantly increased by CPZ treatment, whereas that was not changed by GM administration. It is shown that the dividing cells did not differentiate into cells with an astrocytic phenotype in the frontal cortex of adult male Sprague–Dawley rats [30]. Although it did not reach statistical significance, the total number of mature oligodendrocyte tended to increase with GM administration after depletion by CPZ treatment. Taken together, these observations indicate that GM has specific differentiation-promoting effects on oligodendrocyte progenitor cells. CPZ treatment in mice causes not only cell death, but also demyelination in the brain [for review see 22]. As shown in Fig. 2, MBP immunoreactivity was significantly decreased by CPZ treatment. Removal of CPZ from chow caused rapid but insufficient remyelination, as previously reported [31]. In our study, we also found that MBP immunoreactivity was restored to control levels in the mPFC of CPZ-fed mice by daily administration of GM for 14 days, but not by vehicle administration. Since myelin is produced by mature oligodendrocytes [for reviews see 1], these results suggest that the increase in MBP expression by GM administration can be attributed to promotion of oligodendrocyte genesis and their differentiation into oligodendrocytes. Although it is unclear whether these oligodendrocytes function as efficiently as pre-existing oligodendrocytes, GM likely acts as a remyelination promoter. CPZ treated mice show loss of the myelin sheath and oligodendrocytes not only in the mPFC, but also in the corpus callosum, the cerebellum, the hippocampus, and the caudate putamen [20]. These data warrant further studies on the use of GM in remyelination-enhancing medications for other brain regions. CPZ-exposed mice showed reduced body weight and MBP immunoreactivity, as reported in previous research [32, for review see 20]. Although some currently prescribed antipsychotics for schizophrenia induce adverse effects such as weight gain [for review see 10], GM did not affect the weight of the present mice. Previous work also showed that yokukansan improved psychological symptoms [12, 33, 34] and sleep disturbance [35] in patients with dementia, with very few side effects. These findings suggest that GM may be beneficial for the treatment of schizophrenia without severe adverse events.
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Fig. 5 Effects of CPZ and GM administrations on MBP immunoreactivity in mPFC of adult mice. Mice were given a 0.2 % CPZ or normal diet for 4 weeks, and then orally administered GM or the vehicle once daily for 2 weeks. Coronal sections of the mPFC were immuno-labeled with anti-MBP or anti-NF antibodies. Immunosignal intensity was determined using image analysis and the ratio of MBP to NF was calculated. Representative confocal images show immunoreactivity for a–d MBP and a’–d’ NF, and merged images a’’–d’’ for each group. MBP immunoreactivity was weaker in the mPFC after CPZ treatment than in the mPFC of normal mice, and this decrement was seen in the GMadministered group. Scale bar = 50 lm. e Semiquantitative morphometric analysis shows that MBP immunoreactivity and rerative ratio of MBP/NF intensity was significantly decreased by CPZ treatment as compared with the control, while no decrement was seen in the GM-administered group. Representative data were obtained from 5 animals in each group (mean ± SEM; *P \ 0.05, ***P \ 0.001, twoway ANOVA with Tukey– Kramer’s post hoc test)
Acknowledgments Support for this study and the GM was provided by Tsumura Research Laboratory, Tsumura & Co., Ibaraki, Japan. The work was also supported in part by Scientific Research Grants from the Japan Society for the Promotion of Science (No. 23590221 to A. Wanaka and No. 23500416 to K. Tatsumi). The anti-NF and anti-CD31 hybridoma was obtained from the DSHB, developed under the auspices of the National Institute of Child Health, and Human Development and maintained by the University of Iowa.
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ORIGINAL PAPER
Geissoschizine Methyl Ether, an Alkaloid from the Uncaria Hook, Improves Remyelination After Cuprizone-Induced Demyelination in Medial Prefrontal Cortex of Adult Mice Shoko Morita • Kouko Tatsumi • Manabu Makinodan Hiroaki Okuda • Toshifumi Kishimoto • Akio Wanaka
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Received: 25 July 2013 / Revised: 10 October 2013 / Accepted: 26 October 2013 / Published online: 6 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Accumulating evidence indicates that the medial prefrontal cortex (mPFC) is a site of myelin and oligodendrocyte abnormalities that contribute to psychotic symptoms of schizophrenia. The development of therapeutic approaches to enhance remyelination, a regenerative process in which new myelin sheaths are formed on demyelinated axons, may be an attractive remedial strategy. Geissoschizine methyl ether (GM) in the Uncaria hook, a galenical constituent of the traditional Japanese medicine yokukansan (Yi-gan san), is one of the active components responsible for the psychotropic effects of yokukansan, though little is known about the mechanisms underlying the effects of either that medicine or GM itself. In the present study, we employed a cuprizone (CPZ)-induced demyelination model and examined the cellular changes in response to GM administration during the remyelination phase in the mPFC of adult mice. Using the mitotic marker 5-bromo-20 -deoxyuridine (BrdU), we demonstrated that CPZ treatment significantly increased the number of BrdUpositive NG2 cells, as well as microglia and mature oligodendrocytes in the mPFC. Newly formed oligodendrocytes were increased by GM administration after CPZ exposure. In addition, GM attenuated a decrease in myelin basic protein immunoreactivity caused by CPZ administration. Taken together, our findings suggest that GM
administration ameliorated the myelin deficit by mature oligodendrocyte formation and remyelination in the mPFC of CPZ-fed mice. The present findings provide experimental evidence supporting the role for GM and its possible use as a remedy for schizophrenia symptoms by promoting the differentiation of progenitor cells to and myelination by oligodendrocytes. Keywords Schizophrenia Yokukansan (Yi-gan san) Remyelination Myelin Immunohistochemistry Abbreviations BrdU 5-Bromo-20 -deoxyuridine CPZ Cuprizone GM Geissoschizine methyl ether GS Glutamine synthetase GSTpi Glutathione S-transferase-pi MBP Myelin basic protein mPFC Medial prefrontal cortex NF Neurofilament PB Phosphate buffer PBS Phosphate-buffered saline PBST PBS containing 0.3 % Triton X-100
Introduction S. Morita (&) K. Tatsumi H. Okuda A. Wanaka Department of Anatomy and Neuroscience, Faculty of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan e-mail: [email protected] M. Makinodan T. Kishimoto Department of Psychiatry, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan
Oligodendrocyte progenitor cells differentiate into myelinforming oligodendrocytes and myelin is essential for proper function and maintenance of neurons in the brain [for review see 1]. Many studies have implicated the medial prefrontal cortex (mPFC) as a site of myelin and oligodendrocyte abnormalities in schizophrenia [2–4], while a reduction in grey matter volume and changes in glucose metabolism and blood flow have also been
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reported in the mPFC of schizophrenia patients [5, 6]. In another study, loss of social experiences in juvenile mice resulted in myelination deficits in the mPFC and led to neuropsychological disorders that persisted into adulthood [7], indicating that myelination in the mPFC is pivotal in the development of complex emotional and cognitive behaviors. Remyelination has been demonstrated in animal models and some patients to be mediated by oligodendrocyte progenitor cells, which migrate into the lesion, proliferate, and differentiate into mature oligodendrocytes and then ensheath the demyelinated axons, though it is variable and insufficient. Although the reasons for remyelination failure in myelin disorders are unknown [8], the identification of oligodendrocyte progenitor cells within demyelinated lesions suggests a critical regulatory mechanism in the inhibition of oligodendrocyte progenitor cell differentiation, preventing their maturation to myelin-forming oligodendrocytes [9]. Therefore, development of therapeutic approaches to enhance remyelination is an attractive strategy for the treatment of schizophrenia patients. A variety of effective antipsychotics have been developed for schizophrenia, but because some induce extrapyramidal symptoms, hyperprolactinemia, drowsiness, dizziness, and unwanted weight gain [for review see 10], safer treatments are desirable. Yokukansan (Yi-gan san), a traditional Japanese medicine, has been shown to inhibit degeneration of oligodendrocytes and myelin sheaths in rats with thiamine deficiency-induced encephalopathy [11]. Moreover, this medicine is useful in treating neuropsychological disorders such as behavioral and psychological symptoms of dementia [12], borderline personality disorder [13], and psychotic symptoms of schizophrenia [14, 15]. Geissoschizine methyl ether (GM), a galenical constituent of yokukansan, isolated from the Uncaria hook, is one of the active components responsible for the psychotropic effects of yokukansan [16, 17], though little is known about the mechanisms underlying the effects of either this traditional medicine or GM. Cuprizone [oxalic acid bis (cyclohexylidenehydrazide, CPZ)], a selective and sensitive copper chelator, can be used to generate an animal model of toxic demyelination. Interestingly, mice treated with 0.2 % CPZ display behaviors reminiscent of schizophrenia symptoms. For example, CPZ-fed mice showed increased CNS activity, reduced anxiety in a novelty challenge test, and altered sensorimotor reactivity [18]. CPZ treatment also results in impairments in spatial working memory, social interaction, and prepulse inhibition [19]. In addition, these animals show loss of the myelin sheath and oligodendrocytes in the brain regions such as the corpus callosum, the cerebellum, the hippocampus, the caudate putamen, and the mPFC [20, 21], while such demyelinating lesions may be partially remyelinated with thinner myelin sheaths [22]. Therefore,
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CPZ has been found useful for studies of experimental remyelination [22]. These observations suggest that CPZtreated mouse can be used as an animal model of demyelination/remyelination suitable for testing the effects of GM administration. In the present study, we examined whether GM ameliorates demyelination, particularly promotion of remyelination in CPZ-treated mice. We initially investigated cell proliferation or survival using the thymidine analog 5-bromo-20 -deoxyuridine (BrdU) in combination with immunohistochemistry with cell markers. Next, we examined the expression level of myelin basic protein (MBP) using semi-quantitative morphometric analysis following MBP immunohistochemistry.
Materials and Methods Animals Adult male mice (C57BL/6J) aged 57-113 days were used in the experiments. The animals were housed in a colony room with a 12-h light/dark cycle, and given access to commercial chow and tap water ad libitum. Animal care and the experiments were conducted in accordance with the guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals and the Guidelines for Proper Conduct of Animal Experiments of the Science Council of Japan. The Animal Care Committee of Nara Medical University approved the experimental protocol. Experimental Design Figure 1a depicts the experimental schedule used in the present study. Mice were given powdered standard rodent chow containing 0.2 % (w/w) CPZ (Sigma-Aldrich, Tokyo, Japan) for 4 weeks until perfusion. Control mice were fed with powdered standard rodent chow. We divided 20 C57BL/6J mice into 4 groups of 5 each. Two groups (CPZ ? vehicle and CPZ ? GM) were given powdered standard rodent chow containing 0.2 % (w/w) CPZ for 4 weeks, while the other 2 groups (normal ? vehicle and normal ? GM) received powdered standard chow without CPZ. After that 4-week period, all groups were fed standard chow, and the CPZ ? GM and normal ? GM groups received GM (0.3 mg/kg, kindly supplied by Tsumura & Co., Ibaraki, Japan) dissolved in a vehicle (0.5 % Tween80), while the CPZ ? vehicle and normal ? vehicle groups received the vehicle alone, via oral gavage once daily for the next 2 weeks. To monitor the proliferative activity of cells, mice received BrdU (Sigma-Aldrich, 1 mg/ml) in drinking water. BrdU administration via drinking water has been established for labeling of proliferating cells [23].
Neurochem Res (2014) 39:59–67 Fig. 1 Experimental design, and effects of CPZ treatment and GM administration on body weight of adult mice. a Mice were given a 0.2 % CPZcontaining or normal diet for 4 weeks, and thereafter were administrated GM (0.3 mg/kg) or the vehicle orally once daily for 2 weeks. BrdU was administered in drinking water (1 mg/ml). b Body weights were measured once a week. Representative data were obtained from 5 animals in each group (mean ± SEM; ***P \ 0.001 vs. normal chowfed mice, two-way ANOVA with Tukey–Kramer’s post hoc test). c Schematic of all bregma regions included for quantification
61 1.98 Bregma
a
c
1.94
b 1.78
1.70
Antibodies We employed the following primary antibodies for lightmicroscopic immunohistochemistry: mouse monoclonal IgG against neurofilament (NF; clone 2H3; Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA; dilution 1:20); rabbit polyclonal IgG against MBP (A0623, DAKO, Cambridgeshire, UK; dilution 1:500), glutathione S-transferase-pi (GSTpi; 312, Medical & Biological Laboratories Co., Nagoya, Japan; dilution 1:500), glutamine synthetase (GS; G2781, Sigma-Aldrich; dilution 1:18,000), NG2 (Millipore-Chemicon, Temecula, CA; dilution 1:5,000), Iba1 (019-19741, Wako Pure Chemical Industries, Ltd., Osaka, Japan; dilution 1:200); rat monoclonal IgG against BrdU (OBT0030, Serotec, Raleigh, NC; dilution 1:100); and Armenian hamster monoclonal IgG against CD31 (clone 2H8, DSHB; dilution 1:20). Immunohistochemistry Mice were perfused with saline followed by 4 % paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB; pH 7.4) under deep anesthesia using pentobarbital. The dissected brains were post-fixed with 4 % PFA in 0.1 M PB (pH 7.2) for 6 h at 4 °C. Fixed brains were cryo-protected with 30 % sucrose in phosphate-buffered saline (PBS; pH 7.4) and
frozen at -80 °C in Tissue-Tek OCT compound (Sakura Finetechnical, Tokyo, Japan). Coronal sections were cut at a thickness of 20-lm with a cryostat (Bright Instrument, Huntingdon, UK). For immunofluorescence detection, we processed free-floating sections as previously described [24, 25]. In brief, sections were washed with PBS and treated with 25 mM glycine in PBS for 30 min, then incubated with 5 % normal goat serum in PBS containing 0.3 % Triton X-100 (PBST) for 24 h at 4 °C, followed by incubation with a primary antibody for 48–72 h at 4 °C. Thereafter, the sections were incubated with Alexa 488- or Alexa 546-conjugated secondary goat IgG (Invitrogen, Carlsbad, CA: dilution 1:1,000), or DyLight 405-conjugated secondary goat IgG (Thermo Scientific, Rockford, IL, dilution 1:400). For MBP immunohistochemistry, sections were pretreated with 0.3 % H2O2 in methanol for 15 min. The sections were permeablized by methanol for the MBP antibody to penetrate the subsequent layers of membrane. For BrdU immunolabeling, we performed antigen retrieval by incubating sections in Na-citrate buffer (10 mM, pH 6.0) for 10 min at 95 °C, and then incubated sections in 2 N HCl at 37 °C for 20 min. Confocal Observation and Quantification Specimens were placed on slides and coverslips sealed with Vectashield (Vector Labs), then observations were
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performed using a laser-scanning confocal microscope (FV1000, OLYMPUS, Tokyo, Japan, or C2, Nikon Corporation, Tokyo, Japan). We used 3–5 animals from each group for quantification, and at least 7 sections per animal were chosen from the mPFC region (between bregma ?1.70 and ?1.98 mm) regions according to the mouse brain atlas [26]. To minimize observation biases among different fields and sections, the parameters of the confocal microscope (such as pinhole size, brightness, and contrast setting) were maintained. Images were obtained (512 9 512 pixels) and saved as TIF files using OLYMPUS FV10-ASW Ver 1.7 Viewer or Nikon Nis-Elements AR software. Data Analysis For semi-quantitative analyses, the intensities of MBP and NF immunofluorescence were measured in saved images using ImageJ software (National Institutes of Health). The image analyst was blind to the treatment groups to eliminate biases. Data were analyzed by two-way ANOVA, followed by Tukey–Kramer’s multiple comparisons. Difference was assessed at a significance level of P \ 0.05.
Results Effects of CPZ and GM Administrations on Body Weight of Adult Mice For these experiments, mice were fed chow with or without 0.2 % CPZ for 4 weeks and then administered 0.3 mg/kg GM or the vehicle alone for 2 weeks (Fig. 1a). In addition, mice received BrdU administrations for 2 weeks concurrently with GM. During the first 4 weeks, the CPZ-exposed mice showed significantly lower body weight than controls, which displayed a mild weight gain during the same period (Fig. 1b). Body weights of mice that received the CPZcontaining diet became indistinguishable from that of the controls at the end of fifth week, while GM administration did not have effects on body weight. For histological analysis, all mPFC subregions were included in rostrocaudal levels of the mPFC (between bregma ?1.70 and ?1.98 mm, Fig. 1c). Changes in the Number of Mature Oligodendrocytes and MBP Immunoreactivity in mPFC of Adult Mice After CPZ Exposure We quantified the number of mature oligodendrocyte using an antibody to GSTpi after a 4-week CPZ treatment. The number of GSTpi? cells was decreased after the CPZ treatment (30.70 ± 0.73/mm2) as compared with the
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normal chow-fed group (17.70 ± 0.84/mm2) (Fig. 2b). Thereafter we performed immunohistochemical analysis using an antibody to MBP as a marker for mature myelination and NF as a marker of axons after a 4-week CPZ treatment. A distinct decrease in MBP immunoreactivity was visible in the mPFC of CPZ-fed mice, while no difference could be observed in NF staining between control and CPZ (Fig. 2d–d’’). Also, semi-quantitative morphometric analysis demonstrated that CPZ treatment significantly decreased the MBP intensity (% of the control; 50.57 ± 4.67) and the ratio of MBP to NF intensity (normal, 0.31 ± 0.07; 0.15 ± 0.05) (Fig. 2e). These findings showed that CPZ treatment for 4 weeks dramatically decreased MBP immunoreactivity in the mPFC of adult mice. Changes in Number of BrdU-Labeled Cells in mPFC of CPZ-Fed Mice by GM Administration Next, we focused on BrdU-labeled cells that responded to demyelination in the mPFC of adult mice. We administered BrdU for 2 weeks and examined the identities of BrdU-positive cells by double immunolabeling for the cell markers including NG2 proteoglycan and GSTpi (for oligodendrocyte lineage), Iba1 (for microglia), GS (for astrocytes), and CD31 (for endothelial cells). BrdU-labeled cells were more abundant in the mPFC of CPZ-fed mice than in the normal mice (Fig. 3a– d). We also observed BrdU-labeled nuclei in NG2? oligodendrocyte progenitor cells, GSTpi? mature oligodendrocytes, Iba1? microglial cells, GS? astrocytes, and CD31? endothelial cells (Fig. 3e–i), with varying frequency. Results of quantitative analysis further revealed that CPZ treatment significantly increased the number of BrdU-labeled NG2? oligodendrocyte progenitor (normal ? vehicle, 22.1 ± 2.6; normal ? GM, 34.4 ± 7.5; CPZ ? vehicle, 50.5 ± 4.9; CPZ ? GM, 50.1 ± 4.0/mm2) and Iba1? microglial (normal ? vehicle, 17.1 ± 1.1; normal ? GM, 19.4 ± 2.1; CPZ ? vehicle, 44.2 ± 3.3; CPZ ? GM, 48.3 ± 2.7/mm2) cells, compared with those of the normal chow-fed group (Fig. 3j). GM administration alone had no significant effect on BrdU-labeled nuclei of the NG2? oligodendrocyte progenitor or Iba1? microglial cells. In contrast, a significant increase in BrdU-labeled GSTpi? mature oligodendrocytes (normal ? vehicle, 0.5 ± 0.5; normal ? GM, 1.5 ± 1.5; CPZ ? vehicle, 5.8 ± 1.9; CPZ ? GM, 21.1 ± 4.8/mm2) was detected with GM administration after CPZ treatment. On the other hand, CPZ and subsequent GM administration did not change the number of BrdU-labeled nuclei in GS? astrocytes or CD31? endothelial cells. The total number of GSTpi? mature oligodendrocytes tended to increase with GM administration after CPZ treatment, though it did not reach statistical significance (Fig. 4). These results indicate that newly formed oligodendrocytes were increased by GM administration after CPZ treatment in the mPFC of adult mice.
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Fig. 2 Loss of oligodendrocytes and MBP expression level in mPFC after a 4-week of CPZ treatment. Sections were prepared from untreated normal and CPZ-fed mice, and then examined by immunohistochemistry with the anti-GSTpi, MBP, and NF antibody. a An atlas-based image depicting the area of the representative confocal images (gray square). b The number and density of GSTpi? cells were measured. Quantitative analysis showed that the number of GSTpi? cells was significantly decreased by CPZ exposure. Immuno-signal intensity was determined using image analysis and the ratio of MBP to NF was calculated. Representative confocal images show immunoreactivity for c, d MBP and c’, d’ NF, and merged images c’’, d’’ for each group. MBP immunoreactivity was weaker in the mPFC after CPZ treatment than in the mPFC of normal mice. Scale bar = 50 lm. e Semiquantitative morphometric analysis showed that MBP immunoreactivity and rerative ratio of MBP/NF intensity were significantly decreased by CPZ treatment as compared with the control. Representative data were obtained from 5 animals in each group (mean ± SEM; *P \ 0.05, ***P \ 0.001, twoway ANOVA with Tukey– Kramer’s post hoc test)
GM-Induced Changes in MBP Expression in mPFC of CPZ-Exposed Mice To investigate the effect of GM administration on myelination in the mPFC of CPZ-fed mice, we performed immunohistochemical analysis using MBP and NF. In the mPFC of these mice, we observed a high proportion of MBP immunoreactivity overlapping with NF signals
(Fig. 5a–a’’). The intensity of MBP immunoreactivity was reduced considerably in the mPFC by CPZ treatment (Fig. 5c–c’’), whereas subsequent administration of GM ameliorated this CPZ-induced decrease (Fig. 5d–d’’). Mice treated with GM alone showed MBP immunoreactivity (Fig. 5b–b’’) at a level comparable to that of the control (Fig. 5a–a’’). Also, semi-quantitative morphometric analysis demonstrated that CPZ treatment significantly
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Fig. 3 Effects of CPZ treatment and GM administration on the number of BrdU-labeled cells. Mice were given a 0.2 % CPZ or normal diet for 4 weeks, and thereafter orally administered GM or vehicle once daily for 2 weeks. Newborn cells were detected by BrdU immunohistochemistry after administration of BrdU via drinking water (1 mg/ml). Two weeks later, coronal sections of the brains were double-labeled for BrdU and the cell markers (NG2, GSTpi, Iba1, GS, and CD31) in the mPFC. a–d Representative confocal images show large numbers of BrdU-labeled nuclei in the mPFC of CPZ-treated mice, whereas few were seen in the mPFC of control mice (arrowheads in a-d). e–i BrdU-labeled nuclei in NG2?, GSTpi?,
Iba1?, GS?, and CD31? cells in the mPFC. Scale bars = 50 lm in a and 10 lm in e. j Quantitative analysis of numbers of BrdU-labeled nuclei in NG2? and Iba1? cells showed significant increases after CPZ treatment relative to that of the control. The number of BrdUlabeled GSTpi? cells was significantly increased by GM administration as compared to the vehicle after CPZ treatment. Representative data were obtained from 5 animals in each group (mean ± SEM; **P \ 0.01, ***P \ 0.001 vs. normal ? vehcle, ##P \ 0.01, ### P \ 0.001 vs. normal ? GM, P \ 0.05, P \ 0.001 vs. CPZ ? vehicle, two-way ANOVA with Tukey–Kramer’s post hoc test)
decreased the MBP intensity, whereas repeated subsequent administrations of GM for 2 weeks significantly ameliorated the CPZ-induced decrease in that ratio (% of control; normal ? GM, 85.91 ± 8.59; CPZ ? vehicle, 58.64 ± 6.92; CPZ ? GM, 85.19 ± 8.05) (Fig. 5e). CPZ treatment significantly decreased the ratio of MBP to NF intensity, whereas subsequent GM administrations for 2 weeks significantly ameliorated the CPZ-induced decrease in that ratio (normal ? vehicle, 0.31 ± 0.03; normal ? GM, 0.32 ± 0.02; CPZ ? vehicle, 0.22 ± 0.02; CPZ ? GM, 0.28 ± 0.02) (Fig. 5f).
Discussion
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Abnormalities of myelin and oligodendrocytes have been reported in schizophrenia [for reviews see 2, 27]. Remyelination by endogenous oligodendrocyte progenitor cells occurs in animal models and some patients, though its extent is insufficient. Therefore, remyelination-enhancing medications, which are currently used for treatment of multiple sclerosis [28], may contribute to schizophrenia therapy. Yokukansan and its constituent GM are likely to have good effects on schizophrenia, although their pharmacological properties are not yet been fully
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Fig. 4 Effects of CPZ and GM administrations on number of GSTpi? mature oligodendrocytes. Mice were given a 0.2 % CPZ or normal diet for 4 weeks, and thereafter orally administered GM or the vehicle once daily for 2 weeks. Coronal sections of the mPFC were immunolabeled for GSTpi. The number and density of GSTpi? cells were measured. Quantitative analysis showed that the number of GSTpi? cells was not significantly changed by CPZ exposure or GM administration. Representative data were obtained from 5 animals in each group (mean ± SEM; two-way ANOVA with Tukey– Kramer’s post hoc test)
investigated. In the present study, we examined the effects of GM administration on body weight, BrdU assay, as well as expression of the myelin marker MBP in a mouse model of demyelination/remyelination induced by CPZ. We observed that GM administration increased new oligodendrocyte formation, and attenuated the decrease in immunoreactivity of MBP in the mPFC of CPZ-treated mice without causing overt weight gain. There is evidence for oligodendrocyte alterations across most psychiatric conditions, including but not limited to attention deficit and hyperactivity disorder, autism, bipolar disorder, major depressive disorder, Alzheimer’s disease and schizophrenia [for review see 29]. This would have implications GM may be a novel remedy not only for schizophrenia but also for other established demyelinating diseases. The oligodendrogenesis is important for remyelination in demyelinating diseases as well as for normal myelination, thus we examined the influence of GM administration on the number of BrdU-labeled cells in the mPFC of CPZfed mice. The number of BrdU-labeled cells in the mPFC of CPZ-treated mice increased in the absence of GM administration relative to those in the mPFC of control mice. The significant increase in number of BrdU-labeled NG2? cells in the CPZ-treated mice was considered to be an endogenous response of oligodendrocyte progenitor cells to pathological insult in the brain. This corroborates a previous report that oligodendrocyte progenitor cells proliferate in demyelinated lesions during remyelination [27]. In our previous study, we found that Olig2 lineage cells proliferated and differentiated into NG2? and APC? oligodendrocyte lineage cells more efficiently in the external capsule of CPZ-fed mice than in a normal diet group [28].
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Interestingly, we found that significantly more BrdUpositive cells had differentiated into GSTpi? mature oligodendrocytes by GM administration after CPZ treatment than by vehicle administration, while that difference was not observed for the Iba1?-microglia, GS?-astrocytes, or CD31?-endothelial cells (Fig. 3j). Clearance of damaged myelin is necessary for demyelination, and it has been shown that depletion of microglia impairs myelin debris clearance, demyelination, and remyelination [29]. In the present study, the number of BrdU-labeled microglial cells was significantly increased by CPZ treatment, whereas that was not changed by GM administration. It is shown that the dividing cells did not differentiate into cells with an astrocytic phenotype in the frontal cortex of adult male Sprague–Dawley rats [30]. Although it did not reach statistical significance, the total number of mature oligodendrocyte tended to increase with GM administration after depletion by CPZ treatment. Taken together, these observations indicate that GM has specific differentiation-promoting effects on oligodendrocyte progenitor cells. CPZ treatment in mice causes not only cell death, but also demyelination in the brain [for review see 22]. As shown in Fig. 2, MBP immunoreactivity was significantly decreased by CPZ treatment. Removal of CPZ from chow caused rapid but insufficient remyelination, as previously reported [31]. In our study, we also found that MBP immunoreactivity was restored to control levels in the mPFC of CPZ-fed mice by daily administration of GM for 14 days, but not by vehicle administration. Since myelin is produced by mature oligodendrocytes [for reviews see 1], these results suggest that the increase in MBP expression by GM administration can be attributed to promotion of oligodendrocyte genesis and their differentiation into oligodendrocytes. Although it is unclear whether these oligodendrocytes function as efficiently as pre-existing oligodendrocytes, GM likely acts as a remyelination promoter. CPZ treated mice show loss of the myelin sheath and oligodendrocytes not only in the mPFC, but also in the corpus callosum, the cerebellum, the hippocampus, and the caudate putamen [20]. These data warrant further studies on the use of GM in remyelination-enhancing medications for other brain regions. CPZ-exposed mice showed reduced body weight and MBP immunoreactivity, as reported in previous research [32, for review see 20]. Although some currently prescribed antipsychotics for schizophrenia induce adverse effects such as weight gain [for review see 10], GM did not affect the weight of the present mice. Previous work also showed that yokukansan improved psychological symptoms [12, 33, 34] and sleep disturbance [35] in patients with dementia, with very few side effects. These findings suggest that GM may be beneficial for the treatment of schizophrenia without severe adverse events.
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Fig. 5 Effects of CPZ and GM administrations on MBP immunoreactivity in mPFC of adult mice. Mice were given a 0.2 % CPZ or normal diet for 4 weeks, and then orally administered GM or the vehicle once daily for 2 weeks. Coronal sections of the mPFC were immuno-labeled with anti-MBP or anti-NF antibodies. Immunosignal intensity was determined using image analysis and the ratio of MBP to NF was calculated. Representative confocal images show immunoreactivity for a–d MBP and a’–d’ NF, and merged images a’’–d’’ for each group. MBP immunoreactivity was weaker in the mPFC after CPZ treatment than in the mPFC of normal mice, and this decrement was seen in the GMadministered group. Scale bar = 50 lm. e Semiquantitative morphometric analysis shows that MBP immunoreactivity and rerative ratio of MBP/NF intensity was significantly decreased by CPZ treatment as compared with the control, while no decrement was seen in the GM-administered group. Representative data were obtained from 5 animals in each group (mean ± SEM; *P \ 0.05, ***P \ 0.001, twoway ANOVA with Tukey– Kramer’s post hoc test)
Acknowledgments Support for this study and the GM was provided by Tsumura Research Laboratory, Tsumura & Co., Ibaraki, Japan. The work was also supported in part by Scientific Research Grants from the Japan Society for the Promotion of Science (No. 23590221 to A. Wanaka and No. 23500416 to K. Tatsumi). The anti-NF and anti-CD31 hybridoma was obtained from the DSHB, developed under the auspices of the National Institute of Child Health, and Human Development and maintained by the University of Iowa.
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