Original Article

Yonago Acta medica 2016;59:188–195

Rice Bran Dietary Supplementation Improves Neurological Symptoms and Lossof Purkinje Cells in Vitamin E-Deficient Mice
Toru Takahashi,* Kazuhiro Nakaso,* Yosuke Horikoshi,* Takehiko Hanaki,*† Miho Yamakawa,*
Masato Nakasone,‡ Yoshinori Kitagawa,‡ Taisuke Koike§ and Tatsuya Matsura**Division of Medical Biochemistry, Department of Pathophysiological and Therapeutic Science, School of Medicine, Tottori University
Faculty of Medicine, Yonago 683-8503, Japan, †Division of Surgical Oncology, Department of Surgery, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8503, Japan, ‡Division of Anesthesiology and Clinical Care Medicine, Department of Surgery,
School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8503, Japan and §Planning & Development Department, Eisai
Food & Chemical Co., Ltd., Tokyo 103-0027, Japan

ABSTRACT

ation of Purkinje cells and decreased GFAP-immunoreactivity of Bergmann glia in the cerebellum. In addition,
activated astrocytes and microglia were observed inmice fed the VE-deficient diet. Mice fed the RB dietshowed improvement in these histological abnormalities.
Conclusion A VE-deficient diet induced motor dysfunction in mice due to the degeneration of Purkinjecells in the cerebellum. Oral supplementation of RB
increases VE in the brain and improved the motor dysfunction caused by VE deficiency. Thus, RB or unpolished rice may be a promising VE supplement.

Vitamin E (VE, α-tocopherol) is a
fat-soluble vitamin and is well known as an antioxidant. A deficiency in VE induces oxidative stress in thebrain and causes motor and memory dysfunction. The
consumption of a VE-rich diet has been given much attention in recent years, in regards to anti-aging and theprevention of age-related neuronal disorders.
Methods A VE-deficient mouse model was preparedby feeding the animals a diet lacking VE. In addition,
to evaluate the effect of VE-containing rice bran (RB)on VE deficiency, a diet including RB was also provided. VE levels in the brain tissue, as well as in the RB,
were measured using an HPLC system. Behavioral tests,
including rotarod, wheel running activity, Y-maze, andelevated plus maze were performed. To clarify the effectof VE deficiency and RB, we investigated the inductionof heme oxygenase-1 (HO-1). Histological studies were
performed using HE staining and immunohistochemical
studies were performed using antibodies against glial
fibrillary acidic protein (GFAP) and ionized calcium
binding adaptor molecule 1 (Iba1).
Results VE in the mouse brain under a VE-deficient
diet was decreased, and recovered α-tocopherol levels
were observed in the brain of mice fed an RB diet. Motor behavioral scores were decreased in VE-deficientconditions, while the supplementation of RB improved
motor function. HO-1, a marker of oxidative stress, wasupregulated in the mouse brain under VE deficiency,
however, RB supplementation inhibited the increase of
HO-1. Histological analyses showed neuronal degenerBackgroundKey words

antioxidant; Bergmann glia; oxidative
stress; rice bran; vitamin E

Vitamin E (VE) is a fat-soluble vitamin and is wellknown as a cell membrane antioxidant that protects cells
and organs against oxidative stress. α-Tocopherol (αTOC)
is the most studied form of VE, however, there are 8
identified vitamin E derivatives: α-, β-, γ- and δ-tocopherol (αTOC, βTOC, γTOC and δTOC, respectively) and
α-, β-, γ- and δ-tocotrienol (αT3, βT3, γT3 and δT3,
respectively).1, 2 These classes, TOCs and T3s, can bedifferentiated by an unsaturated side chain with threedouble bonds in the farnesyl isoprenoid tail of TOCs
and an isoprenoid tail with 3 double bonds in T3s.1, 2
Furthermore, differences between α-, β-, γ- and δ- arecharacterized by the number and location of methylgroups on the chromanol ring. Numerous reports havesuggested that all types of VE scavenge reactive oxygen
species and protect cells and organs against oxidative
stress.3–5 Furthermore, it has been reported that VE derivatives also stimulate intracellular signal transduction
and protect cells in antioxidant independent manner.2, 6
Epidemiological and clinical studies suggest that VE
is important for the prevention of oxidative stress-related
neurodegenerative disorders such as Parkinson’s disease,7–9 Alzheimer’s disease10 and amyotrophic lateralsclerosis.11 Moreover, it has been reported that αTOC
deficiency induced through the lack of αTOC transfer

Corresponding author: Kazuhiro Nakaso, MD, PhD
kazuhiro@med.tottori-u.ac.jp
Received 2016 May 16
Accepted 2016 June 27
Abbreviations: αTTP, αTOC transfer protein; BCA, bicinchoninic acid; DAB, 3,3-diamiobenzidine; GFAP, glial fibrillary acidic
protein; HO-1, heme oxygenase-1; HPLC, high performance liquid
chromatography ; Iba 1, ionized calcium binding adaptor molecule 1;
NR, normal diet; T3, tocotrienol; TOC, tocopherol; RB, rice bran;
VE, vitamin E; VE-def, VE-deficient feed; WRA, wheel running
activity

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Rice bran prevents vitamin E deficiency

Behavioral test
Rotarod test

protein (αTTP) induces cerebellar ataxia in humans andmice.12-14 These findings suggest that VE is essential forthe maintenance of brain integrity.
αTTP is an abundant cytosolic liver protein withhigh binding affinity to αTOC, but not to other VE
derivatives. αTTP can transport αTOC between separate membranes.15 αTTP is present in the brain and isconcentrated in Purkinje cell layers of the cerebellum.16
Moreover, a detail examination of the rat brain revealed
that αTTP is expressed in Bergmann glia located between Purkinje cells, which are important in maintaining the functional stability of cerebellar Purkinje cells.16
These facts suggest that the cerebellum is more vulnerable to αTOC deficiency compared with other brain
regions.
Rice brain (RB), a byproduct of the rice milling
process, is known to be a rich source of antioxidants
including VE. Although the concentrations of TOCs andT3s in RB vary according to the rice species and milling
techniques, RBs have abundant TOCs and T3s.17, 18 It
has been reported that dietary RB is useful for intestinal
regulation, decreasing low density lipoprotein, and lowering blood pressure.19–21 In the present study, we investigated the effects of dietary RB on neuronal abnormalities such as cerebellar ataxia induced by VE deficiency.

The motor coordination of mice was investigated using
an accelerating rotarod apparatus (MK-630B treadmill;Muromachi, Tokyo, Japan). The mice were placed on
the rod for four successive trials. The two highest scoresof the four trials were used to calculate an average score,
which indicates the latency to fall. During each trial, therotating rod accelerates gradually from 0 to 40 rpm overa period of 5 min, and, thereafter, is maintained at the40 rpm speed.
Wheel running activity (WRA)

WRA was investigated using the MK-750PC (Muromachi). Locomotor and running activity was converted to
revolutions per day. The mice were allowed free exercise, rest, food and water for 24 hours.
Y-mazeThe Y-maze apparatus was composed of three arms withwalls of 10 cm wide, 13 cm high, and 35 cm long, allowing the mice to see distal spatial features. The insides of
the arms were identical to each other, and provided nointerior cues.
Continuous spontaneous alternation testing was
performed by placing the mice in the Y-maze for 8 minwith all three arms. The number and sequence of arms
entered were recorded manually.
One alternation was counted when mice visited
three different arms consecutively. Immediate reentries
were discounted. The percentage of alternation was
indicated as spatial working memory. The spontaneousalternative rate (%) was calculated as the number of alternations/(the number of total arm entries –2) × 100.

MATERIALS AND METHODS
Animals

C57BL/6 mice were obtained from CLEA Japan (Tokyo,
Japan). The mice were housed under standardized conditions of light (06:00–18:00), temperature (25 °C), andhumidity (approximately 50%), and were allowed freeaccess to food and water. The mice used in this study
were treated in accordance with the Guideline for Animal Experimentation of Tottori University (No. 14-Y-03).
VE-deficient feed (VEdef) (Funabashi Farm, Chiba,
Japan), normal feed and RB were prepared. In order toinvestigate an effect of RB, mice were fed 5 types offeed. The diets were as follows: i) VEdef alone (n = 4);ii) a mixture of VEdef and RB at a concentration 2%
RB (n = 4); iii) a mixture of VEdef and RB at a concentration 5% RB (n = 4); iv) normal diet alone (n = 6) andv) a mixture of normal diet and RB at a concentration
5% RB (n = 6). Hereafter, these diets are referred to as
VEdef, 2%RB, 5%RB, NR and NR5%RB, respectively.
Mice were supplied with the above-mentioned dietsfrom 8 to 20 weeks of age. Behavior analyses were performed at 0, 0.5, 1, 2, 3 months, and, after 3 months, thebrain was sampled.

Elevated plus maze test

The elevated plus maze consisted of two arms without
walls (6 cm wide × 30 cm length) and two enclosed bywalls (6 cm wide × 30 cm length × 20 cm height), with acenter area of 6 × 6 cm. This apparatus was elevated toheight of 50 cm from the ground. The mice were placedat the center area, and allowed to explore the maze for5 min. The time spent in the open arms and the numberof open arm entries were counted and estimated as parameter of anti-anxiolytic behavior. All measuring wererecorded manually.
Quantification of vitamin E

Mice were decapitated under deep anesthesia (pentobarbital, 50 mg/kg, i.p.) and brain tissues were collected. A
portion of the brain tissue (approximately 200 mg) was
crushed by a polytron homogenizer in 1.5 mL phosphate189

T. Takahashi et al.

buffered saline (PBS). To prevent oxidation of the samples, this procedure was performed on ice and under anitrogen gas stream.
The homogenates were used for the measurement of
VE and quantification of protein concentration. Mixtureof the brain homogenate and 2.0 mL ethanol was transferred to a glass tube with a screw cap, then, after added
5.0 mL n-hexane, it was shaken vigorously for 20 min.
The tube was centrifuged at 750 × g for 5 min, andthe hexane layer was collected in another glass tube. After 5.0 mL of n-hexane was added again, re-extraction(shaking, centrifuging and collection of hexane layer)
was performed. The collected hexane layer was evaporated under nitrogen gas stream. The fat-soluble extractthat remained was dissolved in 0.5 mL ethanol, passed
through a 0.22 μm filter to remove impurities, and collected in a storage glass tube.
Similarly, 1.0 g of each feed (5 types of feed and the
RB used for the experiment) was treated with the sameprocedures as extraction of the brain.
αTOC, γTOC, δTOC, αT3, γT3 and δT3 in thefat-soluble fraction extracted from brain or feed were
quantified by a high performance liquid chromatography(HPLC) system. VE levels in the brain were measured
using an HPLC system equipped with an absorbance detector (295 nm) and a phenylhexyl column (COSMOSIL
πNAP; Nacalai Tesque, Kyoto, Japan). Methanol 90%/
H2O 10% delivered at a flow rate of 1 mL/min was used
as eluent. αT3, γT3 and δT3 purified from palm for standards in HPLC were donated by Eisai Food & Chemical(Tokyo, Japan). αTOC, γTOC and δTOC were obtained
from Sigma (St. Louis, MO).

ty of immunoblot signal (expressed as the ratio of HO-1/β-actin) was semi-quantified using Image J software.
Semi-quantitative data were assessed by ANOVA using
the Stat View software, and the criterion for statisticalsignificance was P < 0.05.
Histological study

Immunohistochemistry was performed on paraffin-embedded sections of mouse brain tissues. A portion ofbrain tissue was fixed by immersion in 4% paraformaldehyde (Wako, Osaka, Japan), and embedded in paraffin.
Paraffin sections (4 μm) were deparaffinized by placing
slides into three changes of xylene, followed by rehydration in a graded ethanol series. Sections were treated withmethanol with 3% H2O2 at room temperature for 30 min
to reduce endogenous peroxidase activity.
For glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba1) staining,
the sections were rinsed in water and subjected to antigen retrieval in 10 mM citrate (pH 6.0) at 121 °C for 10
min in an autoclave. GFAP was detected by incubating
the sections in GFAP antibody at a 1:1000 dilution in
PBS at 4 °C overnight. Iba1 antibody was used at a 1:1000
dilution. The labeled antigens were visualized using theHisto Fine kit (Nichirei, Tokyo, Japan) for GFAP and
Iba1, followed by 3,3-diamiobenzidine (DAB) reaction.
Finally, the sections were counterstained with hematoxylin and observed by microscopy (BZ-9000; Keyence,
Osaka, Japan). Primary antibody comprised goat antibody to GFAP and Iba1 were obtained from Abcam(Cambridge, UK).
RESULTS

Immunoblot

Vitamin E quantification of the brain and feeds

Brain tissues were lysed in SDS sample buffer (50mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, and 2 mM ethylenediaminetetraacetic acid). Aliquots (20 μg) were separated
on the basis of molecular size on 10% polyacrylamide
gels, transferred onto polyvinylidene difluoride membranes (Hybond-P; GE Healthcare, Buckinghamshire,UK), and hybridized with HO-1 or β-actin antibody in
PBS with 0.1%Tween 20 at room temperature for 1 h.
Primary antibodies against hemeoxigenase-1 (HO-1)
and β-actin were obtained from Santa Cruz Biotechnology (Dallas, TX) and Cell Signaling Technology (Danvers, MA), respectively. The immunoreactive signal was
detected using horseradish peroxidase-linked anti-rabbit
or mouse IgG and ECL detection reagents (GE Healthcare). The protein content of each sample was measured
using the bicinchoninic acid (BCA) protein assay system(Thermo Fisher Scientific Inc, Rockford, IL). The densi-

In order to investigate the effect of VE in the RB on VE
deficiency in the brain, quantification of VE homologs
in the brain tissue and feeds used in this study were performed by HPLC.
First, all VE homologs in the RB used in this study
were measured, except for βTOC and βT3 (Table 1). Although αTOC, αT3, γTOC, γT3 and δTOC were detected, detectable levels of δT3 were not observed. The RB
used in this experiment included predominately αTOC,
γTOC and δTOC. The concentrations of T3s were lower
than those of TOCs. As a next step, we measured theconcentration of αTOC in each feed used in this experiment. The concentration of αTOC in VEdef, 2%RB,
5%RB, NR and NR5%RB were 0.66 ± 0.83 nmol/g,
15.55 ± 25.14 nmol/g, 31.51 ± 59.05 nmol/g, 477.93 ±
82.74 nmol/g and 546.16 ± 33.24 nmol/g, respectively.
Furthermore, we measured the concentration of VE
homologs in the brain tissue at 3 months after the start
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Rice bran prevents vitamin E deficiency
Table 1. Concentration of tocopherols and tocotrienols in rice bran (nmol/g)
αTOC

γTOC

δTOC

αT3

γT3

δT3

60.86 ± 6.06
182.97 ± 16.36
125.05 ± 19.86
28.02 ± 2.02
3.94 ± 2.45
N.D.
αT3, α-tocotrienol; αTOC, α-tocopherol; δT3, δ-tocotrienol; δTOC, δ-tocopherol; γT3, γ-tocotrienol; γTOC, γ-tocopherol; N.D., not detected.

with 5% RB showed a recovery of spontaneous activity.
To investigate anxiety behavior or deficits in shortterm memory in these mice, the Y-maze and the elevated plus maze were performed at the same time as thelocomotor analysis. At 3 months after the start of theexperiment, we observed no difference between the VEdef mice and the other groups in the non-motility tests.
Briefly, spontaneous alternative scores in the Y-maze test
were 51.8 ± 5.14%, 57.7 ± 7.83% and 59.5 ± 2.78% in the
VEdef, 2%RB and 5%RB groups, respectively. Moreover, the average of the time spent on open arms were18.0 sec, 20.0 sec and 17.72 sec in the VEdef, 2%RB and
5%RB groups, respectively.

Table 2. Vitamin E concentration in each feed
Feed

αTOC (nmol/g)

NR
477.93 ± 82.74
NR5%RB
546.16 ± 33.24
VEdef
0.66 ± 0.83
2%RB
15.55 ± 25.14
5%RB
31.51 ± 59.05
2%RB, a mixture of vitamin E deficient feed and RB at a concentration 2% rice bran; 5%RB, a mixture of vitamin E deficient feed
and RB at a concentration 5% rice bran; αTOC, α-tocopherol;
NR, normal diet alone; NR5%RB, a mixture of normal diet andRB at a concentration 5% rice bran; VE, vitamin E; VEdef, vitamin E-deficient feed.

Measurement of oxidative stress markerof special feeds administration. As expected, the amountof αTOC in the VEdef mouse brain was notably lower,
while αTOC levels in 5%RB mice were comparable
to those in NR mice (Table 2). The concentrations of
αTOC in the brains of each feed group is shown in Table
2. Since αTOC levels in the brain from NR5%RB mice
was similar to that from NR mice, we omitted NR5%RB
mice by the later experiments. Other VE homologs, suchas γTOC, δTOC, αT3, γT3 and δT3, were not detected
because their concentrations in the brain were lower
than that detectable by our HPLC system.

Because VE has anti-oxidative activity, chronic VEdef
may induce oxidative stress. We investigated the expression of HO-1 in the brain by immunoblot analyses as anoxidative stress marker.
The expression of HO-1 in the brain increased
significantly in VEdef mice compared with NR mice,
whereas the expression significantly decreased in 5%RB
mice compared with VEdef mice (Fig. 2).
These results suggest that the influence of VEdef
on the brain is reflected in increasing oxidative stress,
which may be prevented by ingestion of (at least 5%)RB.

Behavioral analyses

To evaluate the effects of oral supplementation of RB
on the improvement of behavioral disorders caused bydietary loss of VE, we utilized the rotarod test, WRA,
Y-maze test, and elevated plus maze test in all groups ofmice.
The rotarod test was performed in order to investigate motor coordination or movement memory of mice
(Fig. 1A). Although the rotarod score of normal miceis progressed with every test attempt, this progression
was not observed in VEdef mice. VEdef mice displayeda significant decrease of duration time on the rotarod
compared with NR mice at 3 months, while the 5%RB
mice showed a significant increase of the rotarod scorecompared with that of VEdef mice.
We measured the spontaneous activity levels ofmice managed under the VE-deficient feed with or without δT3 using an activity wheel for mice (Fig. 1B). Although the mice under VE-deficient conditions showeda decrease in activity, the mice managed under VEdef

Histological analysis

Histological analyses were performed by taking advantage of the brain tissue sampled at the time of HPLC
analysis. Degeneration of Purkinje cells was observedin the cerebellum of VEdef mice at 3 months after thestart of the experiment (Fig. 3B). On the other hand, in
the RB5% mice, degeneration of Purkinje cells was mild
(Fig. 3C) compared with the VEdef mouse cerebellum.
Furthermore, we investigated the effect of the VEdef
and RB diets on Bergmann glia, which help maintain
Purkinje cells (Figs. 3D–I). Although GFAP-positive
Bergman glia were observed around Purkinje cells in
NR mice (Figs. 3D and G), GFAP immunoreactivityof the Bergmann glia decreased in the VEdef mouse
cerebellum (Figs. 3E and H). On the other hand, GFAP
immunoreactivity was recovered in the cerebellum from
5%RB mice (Figs. 3F and I).
Next, we investigated the effect of VEdef and RB
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supplement on neuronal loss in the hippoccampus. In
our experimental conditions, a significant loss of pyramidal neurons in the hippocampus was not observed(Figs. 4A–C). Furthermore, to confirm the activity ofastrocytes and microglia in the hippocampus, we performed immunostaining with anti-GFAP (Figs. 4D–F),
as well as anti-Iba1 antibody (Figs. 4G–I). In the hippocampus of each group, although there was a consistentlevel of staining intensity of glial cells, the immunore-

10000

DISCUSSION
Traditionally, VE has been described as having antioxidant activity.3–5 The αTOC-mediated free radical
quenching reaction is extremely fast (~108/M/sec), andthe water soluble antioxidant ascorbic acid (vitamin C)
is thought to recycle the oxidized αTOC radical generated during a redox reaction back to reduced αTOC.5, 22
Therefore, VE is a powerful antioxidant in vivo.
VE derivatives are found in the cell membrane ofvarious foods. However, T3s are found only in a few
vegetable fats such as palm oil and RB oil.23, 24 RB is
byproduct of the rice milling process and has abundantnutrients including both TOCs and T3s. Therefore, RB
is a promising oral supplement to provide VE.
A mutation of the αTTP gene has been reported as autosomal recessive familial VE deficiency in human.25, 26 InNR
VEdef

7500

activity in VEdef mice was more intense than that of
the other feed group (Figs. 4E and H). The activation ofastrocyte and microglia, as indicated by complicating
spinous processes and swollen cell bodies was observedin the VEdef mouse hippocampus (Figs. 4E and H). In
the hippocampus of the brain from 5%RB mice, thedegree of activation of astrocytes and microglia was lessthan that observed in VEdef mice (Figs. 4F–I).

2%RB
5%RB

5000

＊	
＊	
 ＊	

＊
Figure2	

2500
0

B

0	
  

2	
  
3	
  
1	
  	
Dura6on	  (months)	
  NR
VEdef

2%RB

300

5%RB

＊	

＊	

＊	

＊	
Rela6ve	
  expression	
(HO-­‐1/β-­‐ac6n)	

WRA	
  (rpm/day)A

Rotarod	
  (sec)1

T. Takahashi et al.

200
100
0

0	
  

2	
  
3	
  
1	
  	
Dura6on	  (months)	
  

3	
2	
1	
0	

Fig. 1. Behavioral changes due to administration of VE-deficient
diet or RB-containing diet.
5%RB mice displayed increased movement behavior in the rotarod (A) and WRA tests (B) compared with the VEdef mice.
Each group of mice NR (n = 6), VEdef (n = 4), 2%RB (n = 4)
and 5%RB (n = 4) was subjected to the rotarod and WRA test.
(A) Performance of each group of mice in the rotarod test. Thisscore displays the mean of latency to fall. There is a significant
difference between the VEdef and 5%RB mice at 3 months. (B)WRA was measured each month after the experiment started.
There is a significant difference between VEdef and 5%RB miceat 3-months. *P < 0.05 vs. VEdef at same month (ANOVA). NR,
normal diet alone; RB, rice bran; 2%RB, a mixture of vitamin E
deficient feed and RB at a concentration 2% rice bran; 5%RB, amixture of vitamin E deficient feed and RB at a concentration 5%
rice bran; VE, vitamin E; VEdef, vitamin E-deficient feed; WRA,
wheel running activity.

＊	
＊	

NR	
 VEdef	
 2%RB	
 5%RB	

HO-­‐1	β-­‐ac6n	
Fig. 2. The expression of heme oxygenase-1 in the mouse brain.
(Above) Quantified data of the relative expression of HO-1 in thebrain normalized against β-actin expression. Homogenized wholebrains were used for this analysis. A graph indicates relative values against NR (a reference value of 1.0). (Below) A representative
example of an immunoblotting experiment. Upper panel shows
HO-1 (32kDa) and lower panel shows β-actin (45kDa). HO-1 isupregulated in the brain of VEdef mouse, whereas that in 5%RB
is significantly decreased. *P < 0.05 vs. NR, **P < 0.05 vs. VEdef(ANOVA). n = 4–6 (mean ± S.E.). HO-1, heme oxygenase-1; NR,normal diet alone; VEdef, vitamin E-deficient feed.

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Rice bran prevents vitamin E deficiency

this family, patients show phonotypes that include cerebellar ataxia similar to spinocerebellar ataxia, and highdose supplementation of VE early in the disease process
may, to some extent, reverse ataxia and mental deterioration. Since αTTP is expressed in Bergmann glia, which
help maintain cerebellar Purkinje cells,16 a loss of function of αTTP may cause cerebellar degeneration. Thissuggests that the cerebellum may be vulnerable against
VE deficiency, even with the normal αTTP gene productactivity. αTTP-deficient mice also show a phenotype
similar to human αTTP deficiency.12
In this study, we investigated the effect of oral supplementation with RB as a source of VE on motor andcognitive function in mice fed a VE-deficient diet. In
our experimental conditions, VE deficiency induced

decreased activity of motor function, but not cognitive
function and anxiety. The rotarod score, an index of
motor function including balance and exercise learning, showed a low latency in the VEdef group, and also
showed recovered latency in the RB5% group (Fig. 1A).
Furthermore, we observed Purkinje cell degeneration
and reduced GFAP immunoreactivity of Bergmann glia
in the cerebellum, suggesting that cerebellar dysfunction
by VE deficiency caused motor dysfunction and that RB
supplement rescued this cerebellar dysfunction (Fig. 3).
The WRA test showed mildly lowered scores in
VEdef mice and a recovery effect of RB against VE
deficiency (Fig. 1B). In order to investigate the causeof this lowered activity, we examined the loss of dopaminergic neurons in the substantia nigra, which has

Figure	
  3	

A	

B	

C	

D	

E	

F	

H	

I	

PC	

GC	

G	

BG	

PC	

Fig. 3. Histological analyses of the cerebellum.
(A–C) HE staining of the cerebellum. Bar = 100 μm. (A) The cerebellum from NR mice. Arrows indicate Purkinjecells (PC). (B) VEdef mouse cerebellum shows neuronal degeneration of Purkinje cells (white arrow heads). (C) Addition of 5%RB inhibits the neuronal degeneration of Purkinje cells. Neurodegeneration (white arrow head) is reduced
compared to that in VEdef mice. (D–I) Immunohistochemistry using anti-GFAP antibody. (D–F) Low magnification.
Bar = 200 μm. (G–I) High magnification. Bar = 50 μm. GC: Granule cell layer. PC: Purkinje cells. BG: Bergmannglia. (D and G) GFAP positive Bergmann glia (black arrow heads) are observed around Purkinje cells. (E and H)
GFAP immunoreactivity in Bergmann glia is reduced in the cerebellum of VEdef mice compared to NR or RB5%
mice. (F and I) The GFAP immunoreactivity of Bergmann glia is recovered by 5%RB supplementation. GFAP, glial
fibrillary acidic protein; HE, hematoxylin-eosin; NR, normal diet alone; RB, rice bran; 5%RB, a mixture of vitamin E
deficient feed and RB at a concentration 5% rice bran; VE, vitamin E; VEdef, vitamin E-deficient feed.

193

T. Takahashi et al.

been shown to decrease activity.27 In our experimental
conditions, significant neuronal loss was not observed in
the substantia nigra (data not shown). Therefore, we concluded the reduced WRA score in VEdef mice is due tocerebellar dysfunction.
Since VE deficiency induces non-motor dysfunction and neuronal loss in the brain,28 we examined theeffect of VE deficiency and oral supplementation of RB
on cognitive function and anxiety. Although reactive
astrocytes and activated microglia were observed in
the hippocampus, a loss of pyramidal neurons was notobserved in the VEdef mouse brain (Fig. 4). This result
is consistent with the result of the cognitive and anxiety
examination using the Y-maze system and the elevatedplus maze test. Since our experimental procedures was

designed for only 3 months, longer-term experiments
using the VEdef diet may show similar results as thosedescribed by Fukui, et al.28
It has been reported that TOCs and T3s have cytoprotective function due to not only antioxidative activity,
but also antioxidant-independent activities including
intracellular signal transduction.6, 27 However, judging
from the induction and/or inhibition of HO-1, the protective effect of RB may due to an antioxidative function.
Several antioxidative ingredients, as well as VE, may
be included in RB. Therefore, the effect by other ingredients should also be considered, and further study isneeded.
The RB used in this study was rich in TOCs rather
than in T3s (Table 1). Therefore, oral supplementation

Figure	
  4	

A	

B	

C	

D	

E	

F	

G	

H	

I	

Fig. 4. Histological analyses of the hippocampus.
(A–C) HE staining of CA1 area in the hippocampus. Significant cell loss of the pyramidal neurons was not observed
even in VEdef mice. Bar = 100 μm. (D–F) Immunohistochemistry for astrocytes using anti-GFAP antibody. Bar= 100 μm. (D) GFAP positive astrocytes are observed in the hippocampus. (E) Numbers of reactive astrocyte withcomplicating spinous processes and swelling of the cell body are observed in VEdef mouse hippocampus. (F) Fewerreactive astrocytes are observed in the hippocampus of 5%RB mice than VEdef mice. (G–I) Immunohistochemistry
for microglia using the anti-Iba1 antibody. Bar = 10 μm. (G) Iba1 immunoreactive microglia. (H) Iba1 immunoreactive microglia shows a swollen cell body with complicated spinous processes. (I) Microglia with decreased spinous
processes compared with that in the VEdef mouse hippocampus are observed in the 5%RB mouse brain. GFAP, glial
fibrillary acidic protein; HE, hematoxylin-eosin; Iba1, ionized calcium biding adaptor molecule 1; 5%RB, a mixture ofvitamin E deficient feed and RB at a concentration 5% rice bran.

194

Rice bran prevents vitamin E deficiencyof RB increased αTOC in the brain, decreased oxidative
stress, and improved motor function and histologicalabnormalities induced by VE deficiency. These results
suggest that RB is useful as a supplement food for VE.

13 Yoshida Y. The role of α-tocopherol in motor hypofunction
with aging in α-tocopherol transfer protein knockout miceas assessed by oxidative stress biomarkers. J Nutr Biochem.
2010;21:66-76. PMID: 19157826.
14 Ulatowski L, Parker R, Warrier G, Sultana R, Butterfield DA,
Manor D. Vitamin E is essential for Purkinje neuron integrity.
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Acknowledgments: This work was supported by JSPS KAKENHI
24591262 (KN), 15K09315 (KN), 23580168 (TM), 15K00816(TM), Eisai Food & Chemical Co., Ltd. (KN) and The Tojuro Iijima Foundation for Food Science and Technology (KN).
The authors declare no conflict of interest.

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