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Research Report
Neuroprotective effect of nobiletin on cerebral ischemia–reperfusion injury in transient middle cerebral artery-occluded rats Nodoka Yasudaa,1, Takayuki Ishiia,1, Dai Oyamaa, Tatsuya Fukutaa, Yurika Agatoa, Akihiko Satoa, Kosuke Shimizua, Tomohiro Asaia, Tomohiro Asakawab, Toshiyuki Kanb, Shizuo Yamadac, Yasushi Ohizumid,e,f, Naoto Okua,n a Department of Medical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan b Department of Synthetic Organic and Medicinal Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan c Department of Pharmacokinetics and Pharmacodynamics, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan d Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan e Department of Anti-dementia Functional Food Development, Graduate School of Engineering, Tohoku University, Sendai, Japan f Yokohama College of Pharmacy, Yokohama, Japan
ar t ic l e in f o
abs tra ct
Article history:
Nobiletin, a citrus polymethoxylated flavone, is reported to possess various pharmacolo-
Accepted 3 February 2014
gical activities such as anticancer, anti-inflammation, and antioxidant effects. Recently,
Available online 15 February 2014
nobiletin was shown to provide therapeutic benefit for the treatment of Alzheimer's disease by activating cAMP-response element-binding protein (CREB). In the present study,
Keywords:
we investigated whether nobiletin could protect the brain against ischemia–reperfusion
Nobiletin Citrus polymethoxylated flavone Ischemic stroke
(I/R) injury and improve functional outcome in cerebral I/R model rats, since CREB activation is known to protect neuronal cells in cerebral ischemia. Nobiletin was injected twice at 0 and 1 h after the start of reperfusion in transient middle cerebral artery occlusion
Neutrophil infiltration
(t-MCAO) rats. Cerebral I/R induced prominent brain damage in the ischemic hemisphere
Motor function deficit
of t-MCAO rat brains; however, nobiletin treatment significantly reduced the infarct
Neuroprotectant
volume and suppressed the brain edema. Immunohistochemical and TUNEL staining indicated that nobiletin treatment significantly suppressed neutrophil invasion into the
Abbreviations: CREB, MPO,
cAMP-response element-binding protein; I/R,
myeloperoxidase; I/R,
ischemia–reperfusion; t-MCAO,
TTC, 2,3,5-triphenyltetrazolium chloride n Corresponding author. Fax: þ81 54 264 5705. E-mail address: [email protected] (N. Oku). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.brainres.2014.02.007 0006-8993 & 2014 Elsevier B.V. All rights reserved.
HCO-60, polyoxyethylene (60) hydrogenated castor oil;
transient middle cerebral artery occlusion; TG,
TokyoGreen;
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ischemic region and significantly decreased apoptotic brain cell death in ischemic hemisphere, suggesting that the anti-inflammatory effect and anti-apoptotic effect should be regarded as the neuroprotective mechanism of nobiletin. Moreover, nobiletin treatment ameliorated motor functional deficits in the ischemic rats compared with those deficits of the vehicle-treated group. These results indicate that nobiletin is a potential neuroprotectant for the treatment of cerebral I/R injury. & 2014 Elsevier B.V. All rights reserved.
1.
Introduction
Ischemic stroke is caused by a reduction in the blood supply to a part of the brain, resulting in fatal brain damage. Thrombolysis with tissue plasminogen activator (t-PA) is approved in many countries for the treatment of ischemic stroke (Young et al., 2007). Although this therapy leads to the temporal survival of cerebral cells in the ischemic region by recovering the oxygen and nutrient supply, a secondary impairment, namely, cerebral ischemia/reperfusion (I/R) injury, often occurs after recovery from ischemia (Eltzschig and Eckle, 2011; Wong and Crack, 2008). This injury is a complex disorder caused by oxidative damage, inflammation, glutamate neurotoxicity, and cerebral edema (GursoyOzdemir et al., 2004; Huang et al., 2006). The suppression of brain damage from this injury is essential to obtain a good stroke outcome and to prevent a decrease in the quality of life of stroke patients. However, a therapeutic strategy for cerebral I/R injury has not been established yet (Ginsberg, 2009; Hishida, 2007; Tuma and Steffens, 2012). Nobiletin, a flavonoid present in the peel of citrus fruits, possesses several biological activities (Nakajima et al., 2007; Onozuka et al., 2008; Matsuzaki et al., 2008; Ishiwa et al., 2000; Kandaswami et al., 1991). We previously reported that intraperitoneal treatment with nobiletin for 7 consecutive days suppresses neuronal cell death induced by 20 min ischemia in the mouse hippocampus (Yamamoto et al., 2009). Moreover, learning memory deficits following 5 min ischemia is improved by the consecutive treatment with nobiletin through stimulated phosphorylation of calcium/calmodulindependent protein kinase II (CAMK II) and cyclic-AMP-responsible-element-binding protein (CREB). Another report demonstrated that nobiletin shows an anti-neuroinflammatory effect by suppressing microglial activation in a microglial cell culture model (Cui et al., 2011). In addition pretreatment with nobiletin decreases H2O2-induced cytotoxicity in PC12 cells by several mechanisms, including those causing an increase in superoxide dismutase and glutathione activity (Lu et al., 2010). Thus, nobiletin acts as an antioxidant. In light of the above facts, nobiletin has the potential to improve cerebral I/R injury by these multiple mechanisms. In the present study, we assessed the therapeutic effect of intravenously injected nobiletin on cerebral I/R injury as an adjunctive drug after t-PA treatment in cerebral I/R model rats, namely, transient middle cerebral artery occlusion (t-MCAO) rats. The intravenous injection of drugs is considered as a suitable route of administration for the treatment of cerebral I/R injury both because neuronal cell death
progresses rapidly after I/R and because stroke patients cannot be administered drugs orally. However, nobiletin is hard to dissolve in water because of its hydrophobic property. Therefore, polyoxyethylene hydrogenated castor oil 60 (HCO60) was used as the vehicle for nobiletin in this study. HCO-60 is an emulsifier for the formulation of some drugs and is employed clinically (Hanawa et al., 2003; Ali et al., 2010). Also, some studies have shown that HCO-60 has no effect on the outcome of ischemia in animal stroke models (Sharkey and Butcher, 1994; Zhang et al., 2005).
2.
Results
2.1.
Nobiletin reduced cerebral damage in t-MCAO rats
At first, we investigated the therapeutic effect of nobiletin on cerebral I/R injury in the t-MCAO rats. Ischemic brain damage and brain swelling were evaluated at 24 h after the start of reperfusion (Fig. 1). As assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining, nobiletin greatly reduced brain damage compared with the vehicle (Fig. 1A and B). This reduction in brain cell death by nobiletin was observed in the ischemic region. Brain swelling was calculated by the increase in the size of the right hemisphere as compared with that of the left one (Fig. 1C). One hour of ischemia and 24 h of reperfusion increased the volume of the ischemic hemisphere compared with that of the non-ischemic one. The degree of brain swelling was significantly reduced by the administration of nobiletin. Brain swelling was not observed in the sham-operated group. In addition, nobiletin treatment did not induce hemorrhage.
2.2. Fluorescence-labeled nobiletin accumulated in the ischemic region of t-MCAO rats To confirm the delivery of nobiletin to the damaged region, we performed ex vivo fluorescence imaging of TokyoGreenconjugated nobiletin (TG-nobiletin) injected into t-MCAO rats after reperfusion (Fig. 2). Although the fluorescence of TGnobiletin was detected both in ischemic and non-ischemic hemispheres, a higher accumulation was observed in the ischemic region. These data suggest that nobiletin given immediately after the start of reperfusion rapidly accumulated in the ischemic region and provided a prompt therapeutic effect on I/R injury.
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MPO-positive cells were never observed in the brain section of sham-operated rats.
2.4. Nobiletin prevented neuronal apoptosis after transient cerebral ischemia in rats DNA fragmentation after t-MCAO was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (Fig. 4A and B). TUNEL-positive cells were not detected in the non-ischemic hemisphere. However, many of them were found in both the striatum and the cortex of the vehicle-treated group. Treatment with nobiletin resulted in significantly fewer TUNEL-positive cells in both regions compared with their numbers in the vehicle-treated group (Fig. 4C and D), indicating that nobiletin was associated with decreased apoptotic cell death following cerebral I/R injury.
2.5. Nobiletin ameliorated motor function deficit in tMCAO rats
Fig. 1 – Therapeutic effect of nobiletin on ischemia/ reperfusion injury in t-MCAO rats; (A) t-MCAO rats (n ¼ 5) were injected with vehicle or nobiletin (15 mg/kg) via a tail vein at 0 and 1 h after the start of reperfusion. The brains were then dissected and stained with TTC solution. (B) The infarct volume, and (C) the degree of brain edema were calculated by using Image J. Data are presented as the mean7S.D. Significant differences are indicated as follows: n po0.05, nnpo0.01 nnnpo0.001 vs. vehicle.
2.3. Nobiletin suppressed neutrophil invasion in the ischemic hemisphere of t-MCAO rats To evaluate the anti-inflammatory effect of nobiletin in t-MCAO rats, we investigated neutrophil infiltration into the ischemic region immunohistochemically. Representative photographs of neutrophils immunostained for myeloperoxidase (MPO) at 24 h after the start of reperfusion are shown in Fig. 3A. In the non-ischemic hemisphere, almost no neutrophil infiltration was observed in either the striatum or cerebral cortex. In the ischemic hemisphere of the vehicletreated group, a large number of MPO-positive cells were observed in the brain parenchyma (Fig. 3B). In contrast, nobiletin treatment significantly decreased the number of neutrophils in both the striatum and the cerebral cortex compared with that for the vehicle-treated group.
Finally to evaluate the outcome of nobiletin treatment on cerebral I/R injury in the t-MCAO rats, we monitored motor function of the animals for 7 days post-reperfusion (Fig. 5). The sham-operated rats underwent the same procedure as that of t-MCAO rats but without the insertion of a nylon suture, and the normal rats were not subjected to surgery or anesthesia. All three groups of rats scored 21 points, the maximum number by this analysis method. t-MCAO induced a deficit of motor functional ability, resulting in a much lower score. On the other hand, the nobiletin-treated group showed a higher score than that of the control (vehicle) group, thus indicating that it improved the neurological deficits at 24 h after reperfusion in t-MCAO rats. Moreover, during 7 days after injection, the nobiletin-treated group had a consistently high score, compared with the control. At 7 days after injection, the mortality rate in the vehicle-treated rats was 33%, and in the nobiletin-treated rats it was 17%.
3.
Discussion
Different kinds of mechanisms are involved in cerebral I/R injury, resulting in fatal neuronal damage (Eltzschig and Eckle, 2011; Amantea et al., 2009). In the present study, the intravenous injection of nobiletin resulted in a remarkable therapeutic effect on this injury in t-MCAO rats. We propose that its multiple effects acted additively or synergistically to protect cerebral cells in the I/R regions. Our results showed that nobiletin suppressed apoptotic cell death in the t-MCAO rats. The antioxidant properties of nobiletin could play a role in this anti-apoptotic effect, since up-regulated formation of reactive oxygen species after reperfusion is one of the major factors inducing apoptosis in neuronal cells (Olmez and Ozyurt, 2012; Li and Jackson, 2002). Nakajima et al. demonstrated that nobiletin promoted CREB phosphorylation in mice (Nakajima et al., 2007). Also, Riccio et al. showed that CREB phosphorylation resulted in expression of B-cell lymphoma 2 and brain-derived neurotrophic factor (Riccio et al., 1999), which suppress apoptotic cell death in nerve cells
brain research 1559 (2014) 46–54
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Fig. 2 – Intrabrain distribution of TokyoGreen-conjugated nobiletin (TG-nobiletin) in t-MCAO rats. t-MCAO rats (n ¼ 5) were intravenously injected with TG-nobiletin (0.1 μmol/rat) just after the start of reperfusion. Localization of TG-nobiletin in the brain sections was visualized with IVIS at 1 h after injection. The color bar shows the relative level of fluorescence intensity, ranging from low (blue), to medium (green), to high (yellow, red). The images represent a typical result from one rat, although similar results were obtained from another 4 rats.
(Han and Holtzman, 2000; Linnik et al., 1995). Therefore it is probable that the nobiletin suppressed I/R injury following the elevation of CREB phosphorylation and expression of these proteins. The present study also indicated that nobiletin treatment inhibited the infiltration of neutrophils into the brain parenchyma by its anti-inflammatory effect. Neutrophils invade into the cerebral parenchyma through brain endothelium after a cerebral ischemic event, and subsequently induce progressive inflammation by the generation of cytotoxic substances such as hypochlorous acid in inflammation sites (Tuttolomondo et al., 2009). This infiltration is closely related to the inflammatory cytokine production from glial and neuronal cells at an early stage after an I/R event. Nobiletin was found to suppress the production of proinflammatory cytokines such as tumor necrosis factor (TNF-α) and interleukin-1β in cultures of LPS-stimulated microglial cells (Cui et al., 2011). However, the anti-inflammatory effect of nobiletin in a cerebral stroke animal model has never been documented. The present study demonstrates this effect in terms of morphology, motor function and survival, and suggests that nobiletin exerted its protective effect on the cerebral cells at an early stage after injection.
The intravenously injected TG-nobiletin accumulated in I/R regions, which corresponded to the protected region as judged from TTC staining. This accumulation appears to have been due to disruption of the blood–brain barrier (BBB) after the I/R event, for cerebral vascular permeability in t-MCAO rats is known to be increased at an early stage after I/R (Yang et al., 2007). In fact, intravenously injected macromolecules leak into the brain parenchyma immediately after the start of reperfusion (Ishii et al., 2010). Therefore, our data suggest that the intravenously injected nobiletin passed through the disrupted BBB and exerted a pharmacological effect on cerebral cells. The mechanism and receptor(s) of nobiletin, if present, remain to be elucidated. Ischemic cerebral damage often leads to a negative outcome for the stroke patient, especially by causing language and motor function disorders. Therefore, the extent of these disorders is regarded as the major indicator of therapeutic efficiency in stroke experiments. Our present results indicate that nobiletin treatment improved the motor function deficit caused by cerebral I/R in comparison with the control group during 7 days after reperfusion. In addition, the mortality ratio of the nobiletintreated group was reduced in comparison with the control group.
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Fig. 3 – Histological analysis of anti-inflammatory effect of nobiletin. The t-MCAO rats were injected with vehicle (n ¼4) or nobiletin (15 mg/kg, n ¼5) at 0 and 1 h after the start of reperfusion. Frozen sections of the brain were prepared at 24 h after the injection of vehicle or nobiletin, or at 25 h after sham-operation. (A) MPO immunostaining was performed to visualize neutrophils. Cells indicated by black arrows are MPO-positive cells (brown). Hematoxylin staining was performed for counterstaining. (B) Quantitative data on neutrophil invasion in the striatum and cortex were also obtained. Five or 4 animals and 4 sections/rat were used to obtain the data. Scale bar, 20 μm. Data are presented as the mean7S.D. Significant differences are indicated as follows: npo0.05, nnpo0.01 vs. vehicle.
These results suggest that nobiletin may be useful as a neuroprotectant for the treatment of cerebral I/R injury. Functional outcome and mortality may be much improved if the appropriate timing, frequency, and dosage of nobiletin injections can be established by further studies. Notably, the t-MCAO rats treated with nobiletin showed alleviated hemiparesis in their hind legs. This recovery probably contributed to the high scores of the nobiletintreated animals. A traumatic insult to the cerebral cortex can lead to disruption of the corticospinal tract, resulting in a neuropathological disorder such as hemiparesis of the contralateral hand and leg (Ueno et al., 2012). Moreover, the striatum is the principal input nucleus of the basal ganglia receiving motor information from the cerebral motor cortex. Hence, the observed decrease in neuronal cell death in the striatum and cortex of the ischemic brain resulting from the treatment with nobiletin led to amelioration of the motor function deficits in the t-MCAO rats. In conclusion, intravenously injected nobiletin decreased the apoptotic cell death and neutrophil infiltration in the
cerebral I/R region of t-MCAO rats. Moreover, nobiletin treatment suppressed the cerebral cell death judged from the results of TTC staining, and ameliorated the motor function deficit induced by cerebral I/R. Therefore, nobiletin has the potential to be a novel agent for the treatment of cerebral I/R injury. Further study will hopefully reinforce the utility of nobiletin as an adjunctive drug for use after thrombolysis treatment of ischemic stroke patients.
4.
Experimental procedures
4.1.
Animals
Eight-week-old male Wistar rats (180–220 g) were used and purchased from Japan SLC, Inc. (Shizuoka, Japan). The animals were cared for according to the Animal Facility Guidelines of the University of Shizuoka. All animal procedures were approved by the Animal and Ethics Review Committee of the University of Shizuoka. All surgeries were performed
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Fig. 4 – Histological evaluation of anti-apoptotic effect of nobiletin. t-MCAO rats were injected with vehicle (n ¼4) or nobiletin (15 mg/kg, n ¼5) at 0 and 1 h after the start of reperfusion. Frozen sections of the brain were prepared at 24 h after the injection of vehicle or nobiletin and stained with TUNEL reagent and DAPI. The fluorescence images in (A) the striatum and (B) the cortex were observed by confocal laser scan microscopy. Quantitative data on TUNEL-positive cells in (C) the striatum and (D) the cortex were obtained. Five or 4 animals and 4 sections/rat were used to obtain the data. Isc side indicates the ischemic hemisphere; and Non-isc side, the non-ischemic hemisphere. Scale bar, 20 μm. Data are presented as the mean7S.D. Significant differences are indicated as follows: npo0.05, nnpo0.01 vs. vehicle.
under isoflurane anesthesia, and all efforts were made to minimize suffering.
4.2.
Transient middle cerebral artery occlusion of rats
t-MCAO rats were prepared as described previously (Kuller, 1989). In brief, animals were anesthetized with 3% isoflurane, and the isoflurane content was then decreased to 1.5% during the operation. A heating pad was used to maintain a rectal temperature of 37 1C. After a median incision of the neck skin, the right carotid artery, external carotid artery, and internal carotid artery (ICA) were carefully isolated. A 4-0
monofilament nylon filament coated with silicon was inserted into the right ICA and advanced to the origin of the MCA to occlude it. Silk thread was used to fix the filament in place. After the operation, the neck wound was closed; and the animal was allowed to recover from anesthesia. Reperfusion was performed by withdrawing the filament by about 10 mm at 1 h after the occlusion under isoflurane anesthesia. The success of occlusion was judged by the appearance of hemiparesis and an increase in body temperature to (37.8– 38.8 1C) immediately before reperfusion. Animals with no observable deficits (hemiparesis or increase in body temperature) at 60 min after ischemia, and those that died within 1 h
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Kikai, Tokyo, Japan) and then stained with TTC (Wako Pure Chemical Ind. Ltd., Tokyo, Japan) to determine the amount of brain cell death, as TTC is converted into a red dye when taken up into living cells. The volume of damaged area was calculated by using an image-analysis system (NIH Image J). The damage regions appeared as completely white areas. Brain swelling was calculated as the ratio of volumes of right (ischemic side) to left (non-ischemic side) brain hemisphere sections.
4.5.
Cerebral distribution of nobiletin
Fig. 5 – Motor activity score of t-MCAO rats treated with nobiletin. t-MCAO rats were treated with vehicle (n ¼5) or nobiletin (15 mg/kg, n¼ 7) as described in the legend of Fig. 3, and these rats were then assessed for motor function by conducting a neuropathological test using a 21-point scoring system. Data are presented as the mean7S.D. Significant differences are indicated as follows: npo0.05 vs. vehicle.
Fluorescence-labeled TG-nobiletin was synthesized as described in Supplemental Fig. 1. TG-nobiletin (0.1 μmol/rat) was intravenously injected into the t-MCAO rats (n¼ 5) just after the start of reperfusion. Their brains were dissected at 1 h after the injection and sliced into 2-mm-thick coronal sections. All sections were placed on glass slides, and the fluorescence of TG was measured with an in vivo imaging system (IVIS, Xenogen Corp., Alameda, CA).
after reperfusion, were excluded from the quantitative analysis. In this study, 49% of rats subjected to surgery had a successful surgical outcome, and thus were used experimentally. In addition, 7 rats that were used to evaluate motor function were not subjected to surgery. At 1 day after injection, the mortality rate among the vehicle-treated rats was 25%, and among the nobiletin-treated rats was 9%. At 7days after injection, the rate among vehicle-treated rats was 33%, and among nobiletin-treated rats was 17%. Subarachnoid hemorrhage was observed in these deceased rats. Shamoperated rats underwent the same procedure without the insertion of a nylon suture. Normal rats were not subjected surgery or anesthesia. All animals were randomly assigned to control and experimental groups, and all quantitative data were assessed by several evaluators (double-blinded test).
4.6.
4.3.
Drug administration
Nobiletin was synthesized as described previously (Asakawa et al., 2011). Nobiletin was dissolved in 200 mg/mL HCO-60 (Nikko Chemicals Co., Ltd., Tokyo, Japan) and ethanol was diluted 10 fold with PBS to give a final nobiletin concentration of 6 mg/mL. This solution was sonicated with a Branson sonifier 250 (Branson Ultrasonic Corp., Danbury, CT) for 2 min at output setting 3 with a 50% duty cycle. Nobiletin was administered intravenously at a dose of 15 mg/kg at the start of reperfusion and 1 h thereafter (total dose was 30 mg/kg). In the vehicle group (control group), the solvent for dissolving nobiletin was administered to these rats at the same volume and with the same schedule.
4.4.
Immunohistochemistry
t-MCAO rats were injected with nobiletin (n ¼5) or vehicle (n ¼4) and sacrificed at 24 h after the reperfusion. Before sacrifice, the rats and sham-operated rats were perfused intracardially with PBS under chloral hydrate anesthesia. The brain slices were embedded in OCT compound (Sakura Finetek, Torrance, USA) and then rapidly frozen in dry ice/ ethanol. The frozen brains were cut into 10 μm-thick slices by using a cryostatic microtome (HM 505E, Microm, Walldorf, Germany) and thereafter thaw-mounted onto glass slides. Immunostaining for myeloperoxidase (MPO), a neutrophil marker, was performed with a VECTASTAIN ABC rabbit IgG kit (Vector Laboratories, Burlingame, CA) using diaminobenzidine (DAB) as the chromogen. Brain sections were incubated in acetone for 1 min at 4 1C, washed under running tap water for 5 min, and incubated with 0.3% H2O2 for 30 min at room temperature. After 20 min of preincubation with normal goat serum, the sections were incubated with rabbit polyclonal anti-MPO antibody for 30 min, followed by biotinylated anti-rabbit IgG goat antibody for another 30 min. Next, the sections were incubated with avidin–biotin-peroxidase complex for 30 min. Subsequently, they were treated with the components of a DAB peroxidase Substrate Kit, ImmPACT (Vector Laboratories) and counterstained with Mayer's hematoxylin solution (Wako Pure Chemical Ind. Ltd., Tokyo, Japan) at 4 1C. Finally, the sections were mounted with VectaMount Permanent Mounting Medium (Vector Laboratories, Burlingame, CA) and observed by microscopy. Quantitative data were obtained from sets of 4 or 5 animals, with 4 brain sections taken per animal.
Therapeutic experiment 4.7.
Nobiletin or vehicle was intravenously injected into t-MCAO rats (n ¼5 in each group) at 0 and 1 h post-reperfusion. The volume of damaged area and the degree of brain swelling were assessed at 24 h after the start of reperfusion. For this assessment the brains were dissected and sliced into 2-mmthick coronal sections by using a rat brain slicer (Muromachi
TUNEL staining
Brain sections of 10 μm thickness were prepared as described above and stained with TUNEL reagents supplied in an ApopTags Plus Fluorescein in situ Apoptosis Detection Kit (Chemicon International, Inc., USA). The sections were first fixed with 4% paraformaldehyde for 15 min at room
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temperature and then with ethanol/acetic acid (2:1) solution for 5 min at 20 1C. DNA strand breaks were labeled with the digoxigenin-conjugated terminal deoxynucleotidyl transferase enzyme by incubation for 1 h at 37 1C. Then, the sections were incubated in anti-digoxigenin–fluorescein solution for 30 min at room temperature. Finally, the sections were mounted with Perma Fluor Aqueous Mounting Medium (Thermo Shandon, Pittsburgh, PA, USA) that included DAPI (1.0 μg/mL) for nuclear staining and observed for fluorescence with a microscopic LSM system (Carl Zeiss, Co., Ltd., Germany). Quantitative data were obtained from sets of 4 or 5 animals, with 4 brain sections taken per animal.
4.8.
Monitoring motor function
t-MCAO rats were treated similarly as described under “Therapeutic experiment.” The functional outcome of the rats was assessed during 7 days post-reperfusion. The rats underwent a 21-point neurological score analysis as described previously (Hunter et al., 2000). The analysis was used to evaluate the motor function of the normal, sham-operated, and t-MCAO rats.
4.9.
Statistical analysis
Statistical analysis was performed by unpaired Student's t-test or one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests after checking normality with the Shapiro–Wilk test. Data were presented as the mean7S.D.
Acknowledgments This work was supported in part by Grants-in-Aid for Scientific Research (Nos. 22659010 and 23249005) from the Japan Society for the Promotion of Science, and by grants for the research projects “Application in Promoting New Policy of Agriculture, Forestry and Fisheries” and “Development of Fundamental Technology for Analysis and Evaluation of Functional Agricultural Products and Functional Foods” from the Ministry of Agriculture, Forestry, and Fisheries, Japan.
Appendix A.
Supplementary material
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.brainres. 2014.02.007.
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Y., Ohizumi, Y., 2007. Nobiletin, a citrus flavonoid that improves memory impairment, rescues bulbectomy-induced cholinergic neurodegeneration in mice. J. Pharmacol. Sci. 105, 122–126. Olmez, I., Ozyurt, H., 2012. Reactive oxygen species and ischemic cerebrovascular disease. Neurochem. Int. 60, 208–212. Onozuka, H., Nakajima, A., Matsuzaki, K., Shin, R.W., Ogino, K., Saigusa, D., Tetsu, N., Yokosuka, A., Sashida, Y., Mimaki, Y., Yamakuni, T., Ohizumi, Y., 2008. Nobiletin, a citrus flavonoid, improves memory impairment and Abeta pathology in a transgenic mouse model of Alzheimer’s disease. J. Pharmacol. Exp. Ther. 326, 739–744. Riccio, A., Ahn, S., Davenport, C.M., Blendy, J.A., Ginty, D.D., 1999. Mediation by a CREB family transcription factor of NGFdependent survival of sympathetic neurons. Science 286, 2358–2361. Sharkey, J., Butcher, S.P., 1994. Immunophilins mediate the neuroprotective effects of FK506 in focal cerebral ischaemia. Nature 371, 336–339. Tuma, R.F., Steffens, S., 2012. Targeting the endocannabinod system to limit myocardial and cerebral ischemic and reperfusion injury. Curr. Pharm. Biotechnol. 13, 46–58. Tuttolomondo, A., Di Sciacca, R., Di Raimondo, D., Renda, C., Pinto, A., Licata, G., 2009. Inflammation as a therapeutic target in acute ischemic stroke treatment. Curr. Top Med. Chem. 9, 1240–1260.
Ueno, M., Hayano, Y., Nakagawa, H., Yamashita, T., 2012. Intraspinal rewiring of the corticospinal tract requires targetderived brain-derived neurotrophic factor and compensates lost function after brain injury. Brain 135, 1253–1267. Wong, C.H., Crack, P.J., 2008. Modulation of neuro-inflammation and vascular response by oxidative stress following cerebral ischemia–reperfusion injury. Curr. Med. Chem. 15, 1–14. Yamamoto, Y., Shioda, N., Han, F., Moriguchi, S., Nakajima, A., Yokosuka, A., Mimaki, Y., Sashida, Y., Yamakuni, T., Ohizumi, Y., Fukunaga, K., 2009. Nobiletin improves brain ischemiainduced learning and memory deficits through stimulation of CaMKII and CREB phosphorylation. Brain Res. 1295, 218–229. Yang, Y., Estrada, E.Y., Thompson, J.F., Liu, W., Rosenberg, G.A., 2007. Matrix metalloproteinase-mediated disruption of tight junction proteins in cerebral vessels is reversed by synthetic matrix metalloproteinase inhibitor in focal ischemia in rat. J. Cereb. Blood Flow Metab. 27, 697–709. Young, A.R., Ali, C., Duretete, A., Vivien, D., 2007. Neuroprotection and stroke: time for a compromise. J. Neurochem. 103, 1302–1309. Zhang, N., Komine-Kobayashi, M., Tanaka, R., Liu, M., Mizuno, Y., Urabe, T., 2005. Edaravone reduces early accumulation of oxidative products and sequential inflammatory responses after transient focal ischemia in mice brain. Stroke 36, 2220–2225.
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Research Report
Neuroprotective effect of nobiletin on cerebral ischemia–reperfusion injury in transient middle cerebral artery-occluded rats Nodoka Yasudaa,1, Takayuki Ishiia,1, Dai Oyamaa, Tatsuya Fukutaa, Yurika Agatoa, Akihiko Satoa, Kosuke Shimizua, Tomohiro Asaia, Tomohiro Asakawab, Toshiyuki Kanb, Shizuo Yamadac, Yasushi Ohizumid,e,f, Naoto Okua,n a Department of Medical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan b Department of Synthetic Organic and Medicinal Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan c Department of Pharmacokinetics and Pharmacodynamics, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan d Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan e Department of Anti-dementia Functional Food Development, Graduate School of Engineering, Tohoku University, Sendai, Japan f Yokohama College of Pharmacy, Yokohama, Japan
ar t ic l e in f o
abs tra ct
Article history:
Nobiletin, a citrus polymethoxylated flavone, is reported to possess various pharmacolo-
Accepted 3 February 2014
gical activities such as anticancer, anti-inflammation, and antioxidant effects. Recently,
Available online 15 February 2014
nobiletin was shown to provide therapeutic benefit for the treatment of Alzheimer's disease by activating cAMP-response element-binding protein (CREB). In the present study,
Keywords:
we investigated whether nobiletin could protect the brain against ischemia–reperfusion
Nobiletin Citrus polymethoxylated flavone Ischemic stroke
(I/R) injury and improve functional outcome in cerebral I/R model rats, since CREB activation is known to protect neuronal cells in cerebral ischemia. Nobiletin was injected twice at 0 and 1 h after the start of reperfusion in transient middle cerebral artery occlusion
Neutrophil infiltration
(t-MCAO) rats. Cerebral I/R induced prominent brain damage in the ischemic hemisphere
Motor function deficit
of t-MCAO rat brains; however, nobiletin treatment significantly reduced the infarct
Neuroprotectant
volume and suppressed the brain edema. Immunohistochemical and TUNEL staining indicated that nobiletin treatment significantly suppressed neutrophil invasion into the
Abbreviations: CREB, MPO,
cAMP-response element-binding protein; I/R,
myeloperoxidase; I/R,
ischemia–reperfusion; t-MCAO,
TTC, 2,3,5-triphenyltetrazolium chloride n Corresponding author. Fax: þ81 54 264 5705. E-mail address: [email protected] (N. Oku). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.brainres.2014.02.007 0006-8993 & 2014 Elsevier B.V. All rights reserved.
HCO-60, polyoxyethylene (60) hydrogenated castor oil;
transient middle cerebral artery occlusion; TG,
TokyoGreen;
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ischemic region and significantly decreased apoptotic brain cell death in ischemic hemisphere, suggesting that the anti-inflammatory effect and anti-apoptotic effect should be regarded as the neuroprotective mechanism of nobiletin. Moreover, nobiletin treatment ameliorated motor functional deficits in the ischemic rats compared with those deficits of the vehicle-treated group. These results indicate that nobiletin is a potential neuroprotectant for the treatment of cerebral I/R injury. & 2014 Elsevier B.V. All rights reserved.
1.
Introduction
Ischemic stroke is caused by a reduction in the blood supply to a part of the brain, resulting in fatal brain damage. Thrombolysis with tissue plasminogen activator (t-PA) is approved in many countries for the treatment of ischemic stroke (Young et al., 2007). Although this therapy leads to the temporal survival of cerebral cells in the ischemic region by recovering the oxygen and nutrient supply, a secondary impairment, namely, cerebral ischemia/reperfusion (I/R) injury, often occurs after recovery from ischemia (Eltzschig and Eckle, 2011; Wong and Crack, 2008). This injury is a complex disorder caused by oxidative damage, inflammation, glutamate neurotoxicity, and cerebral edema (GursoyOzdemir et al., 2004; Huang et al., 2006). The suppression of brain damage from this injury is essential to obtain a good stroke outcome and to prevent a decrease in the quality of life of stroke patients. However, a therapeutic strategy for cerebral I/R injury has not been established yet (Ginsberg, 2009; Hishida, 2007; Tuma and Steffens, 2012). Nobiletin, a flavonoid present in the peel of citrus fruits, possesses several biological activities (Nakajima et al., 2007; Onozuka et al., 2008; Matsuzaki et al., 2008; Ishiwa et al., 2000; Kandaswami et al., 1991). We previously reported that intraperitoneal treatment with nobiletin for 7 consecutive days suppresses neuronal cell death induced by 20 min ischemia in the mouse hippocampus (Yamamoto et al., 2009). Moreover, learning memory deficits following 5 min ischemia is improved by the consecutive treatment with nobiletin through stimulated phosphorylation of calcium/calmodulindependent protein kinase II (CAMK II) and cyclic-AMP-responsible-element-binding protein (CREB). Another report demonstrated that nobiletin shows an anti-neuroinflammatory effect by suppressing microglial activation in a microglial cell culture model (Cui et al., 2011). In addition pretreatment with nobiletin decreases H2O2-induced cytotoxicity in PC12 cells by several mechanisms, including those causing an increase in superoxide dismutase and glutathione activity (Lu et al., 2010). Thus, nobiletin acts as an antioxidant. In light of the above facts, nobiletin has the potential to improve cerebral I/R injury by these multiple mechanisms. In the present study, we assessed the therapeutic effect of intravenously injected nobiletin on cerebral I/R injury as an adjunctive drug after t-PA treatment in cerebral I/R model rats, namely, transient middle cerebral artery occlusion (t-MCAO) rats. The intravenous injection of drugs is considered as a suitable route of administration for the treatment of cerebral I/R injury both because neuronal cell death
progresses rapidly after I/R and because stroke patients cannot be administered drugs orally. However, nobiletin is hard to dissolve in water because of its hydrophobic property. Therefore, polyoxyethylene hydrogenated castor oil 60 (HCO60) was used as the vehicle for nobiletin in this study. HCO-60 is an emulsifier for the formulation of some drugs and is employed clinically (Hanawa et al., 2003; Ali et al., 2010). Also, some studies have shown that HCO-60 has no effect on the outcome of ischemia in animal stroke models (Sharkey and Butcher, 1994; Zhang et al., 2005).
2.
Results
2.1.
Nobiletin reduced cerebral damage in t-MCAO rats
At first, we investigated the therapeutic effect of nobiletin on cerebral I/R injury in the t-MCAO rats. Ischemic brain damage and brain swelling were evaluated at 24 h after the start of reperfusion (Fig. 1). As assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining, nobiletin greatly reduced brain damage compared with the vehicle (Fig. 1A and B). This reduction in brain cell death by nobiletin was observed in the ischemic region. Brain swelling was calculated by the increase in the size of the right hemisphere as compared with that of the left one (Fig. 1C). One hour of ischemia and 24 h of reperfusion increased the volume of the ischemic hemisphere compared with that of the non-ischemic one. The degree of brain swelling was significantly reduced by the administration of nobiletin. Brain swelling was not observed in the sham-operated group. In addition, nobiletin treatment did not induce hemorrhage.
2.2. Fluorescence-labeled nobiletin accumulated in the ischemic region of t-MCAO rats To confirm the delivery of nobiletin to the damaged region, we performed ex vivo fluorescence imaging of TokyoGreenconjugated nobiletin (TG-nobiletin) injected into t-MCAO rats after reperfusion (Fig. 2). Although the fluorescence of TGnobiletin was detected both in ischemic and non-ischemic hemispheres, a higher accumulation was observed in the ischemic region. These data suggest that nobiletin given immediately after the start of reperfusion rapidly accumulated in the ischemic region and provided a prompt therapeutic effect on I/R injury.
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MPO-positive cells were never observed in the brain section of sham-operated rats.
2.4. Nobiletin prevented neuronal apoptosis after transient cerebral ischemia in rats DNA fragmentation after t-MCAO was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (Fig. 4A and B). TUNEL-positive cells were not detected in the non-ischemic hemisphere. However, many of them were found in both the striatum and the cortex of the vehicle-treated group. Treatment with nobiletin resulted in significantly fewer TUNEL-positive cells in both regions compared with their numbers in the vehicle-treated group (Fig. 4C and D), indicating that nobiletin was associated with decreased apoptotic cell death following cerebral I/R injury.
2.5. Nobiletin ameliorated motor function deficit in tMCAO rats
Fig. 1 – Therapeutic effect of nobiletin on ischemia/ reperfusion injury in t-MCAO rats; (A) t-MCAO rats (n ¼ 5) were injected with vehicle or nobiletin (15 mg/kg) via a tail vein at 0 and 1 h after the start of reperfusion. The brains were then dissected and stained with TTC solution. (B) The infarct volume, and (C) the degree of brain edema were calculated by using Image J. Data are presented as the mean7S.D. Significant differences are indicated as follows: n po0.05, nnpo0.01 nnnpo0.001 vs. vehicle.
2.3. Nobiletin suppressed neutrophil invasion in the ischemic hemisphere of t-MCAO rats To evaluate the anti-inflammatory effect of nobiletin in t-MCAO rats, we investigated neutrophil infiltration into the ischemic region immunohistochemically. Representative photographs of neutrophils immunostained for myeloperoxidase (MPO) at 24 h after the start of reperfusion are shown in Fig. 3A. In the non-ischemic hemisphere, almost no neutrophil infiltration was observed in either the striatum or cerebral cortex. In the ischemic hemisphere of the vehicletreated group, a large number of MPO-positive cells were observed in the brain parenchyma (Fig. 3B). In contrast, nobiletin treatment significantly decreased the number of neutrophils in both the striatum and the cerebral cortex compared with that for the vehicle-treated group.
Finally to evaluate the outcome of nobiletin treatment on cerebral I/R injury in the t-MCAO rats, we monitored motor function of the animals for 7 days post-reperfusion (Fig. 5). The sham-operated rats underwent the same procedure as that of t-MCAO rats but without the insertion of a nylon suture, and the normal rats were not subjected to surgery or anesthesia. All three groups of rats scored 21 points, the maximum number by this analysis method. t-MCAO induced a deficit of motor functional ability, resulting in a much lower score. On the other hand, the nobiletin-treated group showed a higher score than that of the control (vehicle) group, thus indicating that it improved the neurological deficits at 24 h after reperfusion in t-MCAO rats. Moreover, during 7 days after injection, the nobiletin-treated group had a consistently high score, compared with the control. At 7 days after injection, the mortality rate in the vehicle-treated rats was 33%, and in the nobiletin-treated rats it was 17%.
3.
Discussion
Different kinds of mechanisms are involved in cerebral I/R injury, resulting in fatal neuronal damage (Eltzschig and Eckle, 2011; Amantea et al., 2009). In the present study, the intravenous injection of nobiletin resulted in a remarkable therapeutic effect on this injury in t-MCAO rats. We propose that its multiple effects acted additively or synergistically to protect cerebral cells in the I/R regions. Our results showed that nobiletin suppressed apoptotic cell death in the t-MCAO rats. The antioxidant properties of nobiletin could play a role in this anti-apoptotic effect, since up-regulated formation of reactive oxygen species after reperfusion is one of the major factors inducing apoptosis in neuronal cells (Olmez and Ozyurt, 2012; Li and Jackson, 2002). Nakajima et al. demonstrated that nobiletin promoted CREB phosphorylation in mice (Nakajima et al., 2007). Also, Riccio et al. showed that CREB phosphorylation resulted in expression of B-cell lymphoma 2 and brain-derived neurotrophic factor (Riccio et al., 1999), which suppress apoptotic cell death in nerve cells
brain research 1559 (2014) 46–54
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Fig. 2 – Intrabrain distribution of TokyoGreen-conjugated nobiletin (TG-nobiletin) in t-MCAO rats. t-MCAO rats (n ¼ 5) were intravenously injected with TG-nobiletin (0.1 μmol/rat) just after the start of reperfusion. Localization of TG-nobiletin in the brain sections was visualized with IVIS at 1 h after injection. The color bar shows the relative level of fluorescence intensity, ranging from low (blue), to medium (green), to high (yellow, red). The images represent a typical result from one rat, although similar results were obtained from another 4 rats.
(Han and Holtzman, 2000; Linnik et al., 1995). Therefore it is probable that the nobiletin suppressed I/R injury following the elevation of CREB phosphorylation and expression of these proteins. The present study also indicated that nobiletin treatment inhibited the infiltration of neutrophils into the brain parenchyma by its anti-inflammatory effect. Neutrophils invade into the cerebral parenchyma through brain endothelium after a cerebral ischemic event, and subsequently induce progressive inflammation by the generation of cytotoxic substances such as hypochlorous acid in inflammation sites (Tuttolomondo et al., 2009). This infiltration is closely related to the inflammatory cytokine production from glial and neuronal cells at an early stage after an I/R event. Nobiletin was found to suppress the production of proinflammatory cytokines such as tumor necrosis factor (TNF-α) and interleukin-1β in cultures of LPS-stimulated microglial cells (Cui et al., 2011). However, the anti-inflammatory effect of nobiletin in a cerebral stroke animal model has never been documented. The present study demonstrates this effect in terms of morphology, motor function and survival, and suggests that nobiletin exerted its protective effect on the cerebral cells at an early stage after injection.
The intravenously injected TG-nobiletin accumulated in I/R regions, which corresponded to the protected region as judged from TTC staining. This accumulation appears to have been due to disruption of the blood–brain barrier (BBB) after the I/R event, for cerebral vascular permeability in t-MCAO rats is known to be increased at an early stage after I/R (Yang et al., 2007). In fact, intravenously injected macromolecules leak into the brain parenchyma immediately after the start of reperfusion (Ishii et al., 2010). Therefore, our data suggest that the intravenously injected nobiletin passed through the disrupted BBB and exerted a pharmacological effect on cerebral cells. The mechanism and receptor(s) of nobiletin, if present, remain to be elucidated. Ischemic cerebral damage often leads to a negative outcome for the stroke patient, especially by causing language and motor function disorders. Therefore, the extent of these disorders is regarded as the major indicator of therapeutic efficiency in stroke experiments. Our present results indicate that nobiletin treatment improved the motor function deficit caused by cerebral I/R in comparison with the control group during 7 days after reperfusion. In addition, the mortality ratio of the nobiletintreated group was reduced in comparison with the control group.
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Fig. 3 – Histological analysis of anti-inflammatory effect of nobiletin. The t-MCAO rats were injected with vehicle (n ¼4) or nobiletin (15 mg/kg, n ¼5) at 0 and 1 h after the start of reperfusion. Frozen sections of the brain were prepared at 24 h after the injection of vehicle or nobiletin, or at 25 h after sham-operation. (A) MPO immunostaining was performed to visualize neutrophils. Cells indicated by black arrows are MPO-positive cells (brown). Hematoxylin staining was performed for counterstaining. (B) Quantitative data on neutrophil invasion in the striatum and cortex were also obtained. Five or 4 animals and 4 sections/rat were used to obtain the data. Scale bar, 20 μm. Data are presented as the mean7S.D. Significant differences are indicated as follows: npo0.05, nnpo0.01 vs. vehicle.
These results suggest that nobiletin may be useful as a neuroprotectant for the treatment of cerebral I/R injury. Functional outcome and mortality may be much improved if the appropriate timing, frequency, and dosage of nobiletin injections can be established by further studies. Notably, the t-MCAO rats treated with nobiletin showed alleviated hemiparesis in their hind legs. This recovery probably contributed to the high scores of the nobiletintreated animals. A traumatic insult to the cerebral cortex can lead to disruption of the corticospinal tract, resulting in a neuropathological disorder such as hemiparesis of the contralateral hand and leg (Ueno et al., 2012). Moreover, the striatum is the principal input nucleus of the basal ganglia receiving motor information from the cerebral motor cortex. Hence, the observed decrease in neuronal cell death in the striatum and cortex of the ischemic brain resulting from the treatment with nobiletin led to amelioration of the motor function deficits in the t-MCAO rats. In conclusion, intravenously injected nobiletin decreased the apoptotic cell death and neutrophil infiltration in the
cerebral I/R region of t-MCAO rats. Moreover, nobiletin treatment suppressed the cerebral cell death judged from the results of TTC staining, and ameliorated the motor function deficit induced by cerebral I/R. Therefore, nobiletin has the potential to be a novel agent for the treatment of cerebral I/R injury. Further study will hopefully reinforce the utility of nobiletin as an adjunctive drug for use after thrombolysis treatment of ischemic stroke patients.
4.
Experimental procedures
4.1.
Animals
Eight-week-old male Wistar rats (180–220 g) were used and purchased from Japan SLC, Inc. (Shizuoka, Japan). The animals were cared for according to the Animal Facility Guidelines of the University of Shizuoka. All animal procedures were approved by the Animal and Ethics Review Committee of the University of Shizuoka. All surgeries were performed
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Fig. 4 – Histological evaluation of anti-apoptotic effect of nobiletin. t-MCAO rats were injected with vehicle (n ¼4) or nobiletin (15 mg/kg, n ¼5) at 0 and 1 h after the start of reperfusion. Frozen sections of the brain were prepared at 24 h after the injection of vehicle or nobiletin and stained with TUNEL reagent and DAPI. The fluorescence images in (A) the striatum and (B) the cortex were observed by confocal laser scan microscopy. Quantitative data on TUNEL-positive cells in (C) the striatum and (D) the cortex were obtained. Five or 4 animals and 4 sections/rat were used to obtain the data. Isc side indicates the ischemic hemisphere; and Non-isc side, the non-ischemic hemisphere. Scale bar, 20 μm. Data are presented as the mean7S.D. Significant differences are indicated as follows: npo0.05, nnpo0.01 vs. vehicle.
under isoflurane anesthesia, and all efforts were made to minimize suffering.
4.2.
Transient middle cerebral artery occlusion of rats
t-MCAO rats were prepared as described previously (Kuller, 1989). In brief, animals were anesthetized with 3% isoflurane, and the isoflurane content was then decreased to 1.5% during the operation. A heating pad was used to maintain a rectal temperature of 37 1C. After a median incision of the neck skin, the right carotid artery, external carotid artery, and internal carotid artery (ICA) were carefully isolated. A 4-0
monofilament nylon filament coated with silicon was inserted into the right ICA and advanced to the origin of the MCA to occlude it. Silk thread was used to fix the filament in place. After the operation, the neck wound was closed; and the animal was allowed to recover from anesthesia. Reperfusion was performed by withdrawing the filament by about 10 mm at 1 h after the occlusion under isoflurane anesthesia. The success of occlusion was judged by the appearance of hemiparesis and an increase in body temperature to (37.8– 38.8 1C) immediately before reperfusion. Animals with no observable deficits (hemiparesis or increase in body temperature) at 60 min after ischemia, and those that died within 1 h
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Kikai, Tokyo, Japan) and then stained with TTC (Wako Pure Chemical Ind. Ltd., Tokyo, Japan) to determine the amount of brain cell death, as TTC is converted into a red dye when taken up into living cells. The volume of damaged area was calculated by using an image-analysis system (NIH Image J). The damage regions appeared as completely white areas. Brain swelling was calculated as the ratio of volumes of right (ischemic side) to left (non-ischemic side) brain hemisphere sections.
4.5.
Cerebral distribution of nobiletin
Fig. 5 – Motor activity score of t-MCAO rats treated with nobiletin. t-MCAO rats were treated with vehicle (n ¼5) or nobiletin (15 mg/kg, n¼ 7) as described in the legend of Fig. 3, and these rats were then assessed for motor function by conducting a neuropathological test using a 21-point scoring system. Data are presented as the mean7S.D. Significant differences are indicated as follows: npo0.05 vs. vehicle.
Fluorescence-labeled TG-nobiletin was synthesized as described in Supplemental Fig. 1. TG-nobiletin (0.1 μmol/rat) was intravenously injected into the t-MCAO rats (n¼ 5) just after the start of reperfusion. Their brains were dissected at 1 h after the injection and sliced into 2-mm-thick coronal sections. All sections were placed on glass slides, and the fluorescence of TG was measured with an in vivo imaging system (IVIS, Xenogen Corp., Alameda, CA).
after reperfusion, were excluded from the quantitative analysis. In this study, 49% of rats subjected to surgery had a successful surgical outcome, and thus were used experimentally. In addition, 7 rats that were used to evaluate motor function were not subjected to surgery. At 1 day after injection, the mortality rate among the vehicle-treated rats was 25%, and among the nobiletin-treated rats was 9%. At 7days after injection, the rate among vehicle-treated rats was 33%, and among nobiletin-treated rats was 17%. Subarachnoid hemorrhage was observed in these deceased rats. Shamoperated rats underwent the same procedure without the insertion of a nylon suture. Normal rats were not subjected surgery or anesthesia. All animals were randomly assigned to control and experimental groups, and all quantitative data were assessed by several evaluators (double-blinded test).
4.6.
4.3.
Drug administration
Nobiletin was synthesized as described previously (Asakawa et al., 2011). Nobiletin was dissolved in 200 mg/mL HCO-60 (Nikko Chemicals Co., Ltd., Tokyo, Japan) and ethanol was diluted 10 fold with PBS to give a final nobiletin concentration of 6 mg/mL. This solution was sonicated with a Branson sonifier 250 (Branson Ultrasonic Corp., Danbury, CT) for 2 min at output setting 3 with a 50% duty cycle. Nobiletin was administered intravenously at a dose of 15 mg/kg at the start of reperfusion and 1 h thereafter (total dose was 30 mg/kg). In the vehicle group (control group), the solvent for dissolving nobiletin was administered to these rats at the same volume and with the same schedule.
4.4.
Immunohistochemistry
t-MCAO rats were injected with nobiletin (n ¼5) or vehicle (n ¼4) and sacrificed at 24 h after the reperfusion. Before sacrifice, the rats and sham-operated rats were perfused intracardially with PBS under chloral hydrate anesthesia. The brain slices were embedded in OCT compound (Sakura Finetek, Torrance, USA) and then rapidly frozen in dry ice/ ethanol. The frozen brains were cut into 10 μm-thick slices by using a cryostatic microtome (HM 505E, Microm, Walldorf, Germany) and thereafter thaw-mounted onto glass slides. Immunostaining for myeloperoxidase (MPO), a neutrophil marker, was performed with a VECTASTAIN ABC rabbit IgG kit (Vector Laboratories, Burlingame, CA) using diaminobenzidine (DAB) as the chromogen. Brain sections were incubated in acetone for 1 min at 4 1C, washed under running tap water for 5 min, and incubated with 0.3% H2O2 for 30 min at room temperature. After 20 min of preincubation with normal goat serum, the sections were incubated with rabbit polyclonal anti-MPO antibody for 30 min, followed by biotinylated anti-rabbit IgG goat antibody for another 30 min. Next, the sections were incubated with avidin–biotin-peroxidase complex for 30 min. Subsequently, they were treated with the components of a DAB peroxidase Substrate Kit, ImmPACT (Vector Laboratories) and counterstained with Mayer's hematoxylin solution (Wako Pure Chemical Ind. Ltd., Tokyo, Japan) at 4 1C. Finally, the sections were mounted with VectaMount Permanent Mounting Medium (Vector Laboratories, Burlingame, CA) and observed by microscopy. Quantitative data were obtained from sets of 4 or 5 animals, with 4 brain sections taken per animal.
Therapeutic experiment 4.7.
Nobiletin or vehicle was intravenously injected into t-MCAO rats (n ¼5 in each group) at 0 and 1 h post-reperfusion. The volume of damaged area and the degree of brain swelling were assessed at 24 h after the start of reperfusion. For this assessment the brains were dissected and sliced into 2-mmthick coronal sections by using a rat brain slicer (Muromachi
TUNEL staining
Brain sections of 10 μm thickness were prepared as described above and stained with TUNEL reagents supplied in an ApopTags Plus Fluorescein in situ Apoptosis Detection Kit (Chemicon International, Inc., USA). The sections were first fixed with 4% paraformaldehyde for 15 min at room
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temperature and then with ethanol/acetic acid (2:1) solution for 5 min at 20 1C. DNA strand breaks were labeled with the digoxigenin-conjugated terminal deoxynucleotidyl transferase enzyme by incubation for 1 h at 37 1C. Then, the sections were incubated in anti-digoxigenin–fluorescein solution for 30 min at room temperature. Finally, the sections were mounted with Perma Fluor Aqueous Mounting Medium (Thermo Shandon, Pittsburgh, PA, USA) that included DAPI (1.0 μg/mL) for nuclear staining and observed for fluorescence with a microscopic LSM system (Carl Zeiss, Co., Ltd., Germany). Quantitative data were obtained from sets of 4 or 5 animals, with 4 brain sections taken per animal.
4.8.
Monitoring motor function
t-MCAO rats were treated similarly as described under “Therapeutic experiment.” The functional outcome of the rats was assessed during 7 days post-reperfusion. The rats underwent a 21-point neurological score analysis as described previously (Hunter et al., 2000). The analysis was used to evaluate the motor function of the normal, sham-operated, and t-MCAO rats.
4.9.
Statistical analysis
Statistical analysis was performed by unpaired Student's t-test or one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests after checking normality with the Shapiro–Wilk test. Data were presented as the mean7S.D.
Acknowledgments This work was supported in part by Grants-in-Aid for Scientific Research (Nos. 22659010 and 23249005) from the Japan Society for the Promotion of Science, and by grants for the research projects “Application in Promoting New Policy of Agriculture, Forestry and Fisheries” and “Development of Fundamental Technology for Analysis and Evaluation of Functional Agricultural Products and Functional Foods” from the Ministry of Agriculture, Forestry, and Fisheries, Japan.
Appendix A.
Supplementary material
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.brainres. 2014.02.007.
r e f e r e n c e s
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