Shinshu Med J, 72⑶：169～182, 2024

New Emulsifying Oil Powder Extracted from Pacific Krill Protects
against Vascular Cognitive Impairment in a Mouse Model of Cerebral
Small Vessel Disease
Nanae Ishida1）2）＊, Hidetoshi Yamada3）, Masamichi Hirose4）
Eiichi Taira2） and Mitsuhiko Yamada1）
1） Department of Molecular Pharmacology, Shinshu University School of Medicine
2） Department of Pharmacology, Iwate Medical University, School of Medicine
3） Faculty of Life & Environmental Science, Teikyo University of Science4） Department of Internal Medicine, Specified Medical Corporation Jinseikai Asakura Hospital

Background : Marine n-3 polyunsaturated fatty acids (PUFAs) may be promising for reducing cognitive andfunctional decline in elderly patients with mild cognitive impairment. We examined the effects of a new emulsi­
fying oil powder including PUFAs extracted from Pacific krill on vascular cognitive impairment in a mouse modelof cerebral small vessel disease.
Methods : Small vessel disease was created using a mouse model of asymmetric common carotid artery stenosis
surgery (ACAS). Mice were fed an MF diet (MFD) or MFD supplemented with the new emulsifying Pacific krill oil
powder (EPKOP) from 4 weeks before ACAS to 5 weeks after ACAS. Two and/or from 4 to 5 weeks after ACAS,
all experiments were performed.
Results : Cerebral blood flow (CBF) was decreased in MFD-fed ACAS compared with MFD-fed sham-operated
mice. CBF was similar between MFD with EPKOP-fed ACAS and MFD-fed sham-operated mice. EPKOP re­versed the ACAS-induced decrease in the latency to fall in a rotarod test of ACAS mice. Moreover, it also re­versed the ACAS-induced longer escape latencies in the Morris water maze test of ACAS mice. Hematoxylin/
eosin staining demonstrated that EPKOP decreased the number of shrunken neurons in the ACAS mouse hip­pocampus. Interleukin 1β gene expression in the hippocampus was increased in MFD-fed ACAS compared with
MFD-fed sham-operated mice. In contrast, its gene expression in the hippocampus was similar between MFD
with EPKOP-fed ACAS and MFD-fed sham-operated mice.
Conclusion : These results suggest that the new emulsifying oil powder extracted from Pacific krill may beeffective to prevent vascular cognitive impairment. Shinshu Med J 72 : 169 ―182, 2024
(Received for publication November 6, 2023 ; accepted in revised form March 4, 2024)
Key words : vascular cognitive impairment, Pacific krill, n-3 polyunsaturated fatty acids
Abbreviations : ACAS, asymmetric common carotid artery surgery ; CBF, cerebral blood flow ; VCI, vascular cog­
nitive impairment ; CCA, common carotid artery ; MCP-1, monocyte chemotactic protein 1 ; IL-1β, interleukin-1β ;TNF-α, tumor necrosis factor α ; EPKOP, emulsifying Pacific krill oil powder

Ｉ＊

Corresponding author : Nanae Ishida
Department of Molecular Pharmacology, Shinshu University
School of Medicine, 3-1-1 Asahi, Matsumoto, Nagao
390-8621, Japan
E-mail : lauantai0318@gmail.com

No. 3, 2024

Introduction

Several types of vascular dementia have been re­ported worldwide1）2）and cerebral blood vessel disease(i.e., vascular cognitive impairment, VCI)-associatedcognitive impairment is one of the recent major public169Ishida・Yamada・Hirose et al.

health concerns3）. At present, vascular diseases andcerebral small vessel disease which was created using

other risk factors for VCI have mainly been treateda mouse model of asymmetric common carotid arteryto prevent VCI. In fact, the management of cardio­

surgery (ACAS)14）.

vascular risk factors, such as hypertension, diabetes,Ⅱdyslipidemia, obesity, and smoking might be effective
to reduce the development of VCI. The effective con­Ａ

Material and Methods

Ethicstrol of cardiovascular risk factors prevents VCI-The study was performed in strict accordance

related disease and may be more effective than cur­with the recommendations in the Guide for Care and4）5）rent pharmacological treatment for VCI.

It is well-known that marine n-3 polyunsaturated
6）

Use of Laboratory Animals published by the US Na­
tional Institutes of Health. This study was approved

fatty acids (PUFAs) have health benefits . N-3 PUFAs

by the Animal Care Committee of Iwate Medical

which include eicosapentaenoic acid (EPA) and doco­

University. The protocol was approved by the Com­sahexaenoic acid (DHA), reduce serum triacylglycerol

mittee on the Ethics of Animal Experiments of Iwate(TAG) levels and have anti-inflammatory effects, lead­

Medical University (Permit Number : 30-18).

ing to reduction of the incidence of mortality due to

Ｂ Contents of a new emulsifying oil powder ex-

7）

cardiovascular diseases . Moreover, n-3 PUFAs playtracted from Pacific krill

a positive role in the prevention and treatment of met­

E. pacifica is the most common krill species in the8）9）abolic syndrome including dyslipidemia. These re­North Pacific Ocean. The common term ‘krill’ refers

sults suggest that n-3 PUFAs can reduce the devel­to Euphausiids and they are widespread in oceans

opment of VCI through the reduction of cardiovascularworldwide. E. pacifica is a good source of marine n-3

risk factors. In fact, a clinical study demonstrated that

PUFAs, which include EPA and DHA15）. Both fish oil

PUFAs may be promising for reducing cognitive andand krill oil are sources of EPA and DHA ; however,functional decline in the elderly with mild cognitive

compositions of these oils are different. Fish oil is

impairment10）. Moreover, the association between ce­

mostly composed of TAG. Krill oil is mostly composed

rebral white matter lesions and the plasma omega-3

of TAG and phospholipids. As the phospholipid form

to omega-6 PUFA ratio to cognitive impairment de­of n-3 PUFA is incorporated into plasma faster than

11）

the TAG form, krill oil can increase the omega-3

velopment has been demonstrated .

Krill oil includes PUFAs, and the direct neuropro­

index at a lower dose in humans16）17）. Krill oil also

tective effects of E. superba oil against aging andcontains the antioxidant astaxanthin. The new EPKOP

Alzheimer’s disease-related cognitive impairment infor human health from E. pacifica is mainly composed

12）

animals and humans were demonstrated . In fact, theof protein including peptide (70 ％), neutral lipid (10 ％),protective effect of krill oil against Alzheimer’s disease

diacyl phospholipid (10 ％), and alkyl phospholipid (2has been confirmed in an animal model of senescence-％) (Fig. 1A). This powder contains 39.3 mg/g of EPA,

accelerated prone mouse strain 8 (SAMP8) mice13）.

1.6 mg/g of DHA, 22.4 mg/g of alkyl phospholipid,

However, few studies showed the protective effects

and 285 ppm of astaxanthin (Table 1).

of krill oil on cerebrovascular dementia, especially small

Ｃ Experimental animals and asymmetric common

vessel disease-related dementia. In addition, whether

carotid artery stenosis surgery (ACAS)

krill oil directly improves hypoperfusion-induced VCIC57BL/6J male mice (8 weeks old, 20-25-g bodyeven if cardiovascular risk factors do not exist is un­

weight) were purchased from Japan SLC. To produce

known. Recently, we and ADEKA Co., Ltd. developed

VCI, stenosis of both common carotid arteries (CCAs)

a new emulsifying Pacific oil powder (EPKOP) ex­

was surgically created using an ameroid constrictor

tracted from Pacific krill for human health. We stud­and a microcoil (ACAS), as previously described18）19）.

ied the protective effects of the new EPKOP on vas­

Briefly, mice were anesthetized with isoflurane (2-cular cognitive impairment using a mouse model of4 ％), then we exposed both CCAs, and these were170

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Krill oil powder reduces vascular cognitive impairment

Fig. 1 Panel A, Contents of a new emulsifying oil powder extracted from Pacific krill. The new EPKOP for human healthfrom E. pacifica is mainly composed of protein, including : peptide (70 ％), neutral lipid (10 ％), diacyl phospholipid(10 ％), and alkyl phospholipid (2 ％). Panel B, Small vessel disease was created using a mouse model of asymmetriccommon carotid artery stenosis surgery (ACAS) in two of the three groups. An image showing an ameroid con­strictor (AC) around the right common carotid artery (CCA) and placement of a microcoil around the left CCA.
Panel C, The schema of experimental protocols for each of the three groups (MF diet fed-sham operation mice, MF
diet-fed ACAS mice, MF with EPKOP diet-fed ACAS mice). Each diet was fed from four weeks before to five
weeks after sham surgery and ACAS, respectively. EPKOP, emulsifying Pacific krill oil powder.
No. 3, 2024

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Ishida・Yamada・Hirose et al.
Table 1
Phospholipid

Components of the lipid fraction

Alkyl phospholipid

DHA

EPA

Astaxanthin

mg/g
100

ppm

22.4

1.6

39.3

285

DHA, docosahexaenoic acid ; EPA, eicosapentaenoic acidfreed from their sheaths. A spiral microcoil was wrapp­
ed around the left CCA. An ameroid constrictor

14）

was also fitted in the right CCA (Fig. 1B). The sametest was repeated five times with an interval of 5 min
between attempts.
ＦY-maze test

procedure was performed except for ACAS in sham-

To assess spatial working memory and spontaneous

operated mice. Finally, the midline cervical incision

activity, the Y-maze test was performed as previously

was closed using 7-0 nylon suture. After the surgery,described19）21）. The Y-maze test was conducted at 2

mice were maintained in an environment with day/and 4 weeks after ACAS or sham surgery (n＝10 fornight reversal throughout the experiments. Before theeach) (Fig. 1C). Mice were allowed to freely explore

brain tissue was excised, isoflurane inhalation and/or

the three arms during an 8-min session after intro­

sodium pentobarbital (30 mg/kg) application into theduction to the end of the start arm. A video camera

abdominal cavity was performed to anesthetize all(Logicool HD Webcam C615, Logicool, Tokyo, Japan)

animals. To confirm the adequacy of anesthesia, thewas used to record the numbers of arm entries anddegree of motion of the sternum and movement of

trials. A mouse was considered to have entered an

the extremities were monitored. The toe pinch reflex

arm when all four limbs were within the arm. Spatial

test was used to confirm the appropriate depth ofworking memory was elucidated by the percentage

anesthesia.

of alternation behavior. Actual alternation behaviorＤ

was defined as entry into all three arms on consecu­

Experimental groups and diet

Male mice were divided into three groups, as fol­

tive occasions. The maximum alternation behavior waslows : MF diet (Certified diet, Oriental Yeast Co., Ltd.,

calculated as the total number of arm entries minus

Tokyo, Japan) fed sham-operated mice, MF diet-fed

two, and the percentage of alternation was calculated

ACAS mice, MF diet with new EPKOP-fed ACAS

as the number of actual alternations divided by thatmice (Fig. 1C). EPKOP intake continued from 4 weeks

of maximum alternations multiplied by 100. Sponta­

before ACAS to 5 weeks after ACAS. All mice were

neous activity was elucidated by the total number ofallowed to access water ad libitum and housed underarms entered during a session.

a 12-h light/dark cycle. Feed was replaced every

Ｇ

Morris water maze test

three days. Body weight was measured weekly for

The Morris water maze test was performed con­

10 weeks during experiments and did not differ

secutive days from 29th to 34th after ACAS or shamamong the three different groups of mice (Fig. 2A).

surgery (n＝10 for each) (Fig. 1C), as previously de­Ｅ

scribed19）22）. Briefly, to learn the platform position, aRotarod test
Motor coordination and balance were tested withvisible platform task was performed using a trans­

the rotarod test. The rotarod test was performed 14

parent circular platform with a diameter of 8 cm threedays after ACAS or sham surgery (n＝10 for each)times per day from the first to sixth days. After the(Fig. 1C) by placing the mouse on a rotating drum

visible platform task, the circular platform was im­(Muromachi Kikai CO., Japan) and measuring the timemersed 1 cm below the water surface in the center

the mouse maintained its balance on the rod, as pre­of a quadrant of the pool to perform a hidden platform

20）

viously reported . The speed of the rotarod was ac­

task. The platform was kept in the same position

celerated from 4 to 40 rpm over a 5-min period. The

throughout the experiment. We performed the hidden

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Krill oil powder reduces vascular cognitive impairment

Fig. 2 Body weight and cervical blood flow (CBF) in the three different groups of mice. Panel A, Body weight wasmeasured for 10 weeks during experiments and did not differ among the three different groups of mice. Sham
operation or ACAS was performed 4 weeks after the experiment started. Panel B, Representative CBF images oflaser speckle flowmetry. Panel C, Mean local CBFs in circular regions of ROI1 and ROI2 in the three different
groups of mice at five weeks after ACAS or the sham operation. Data are the mean±SE obtained from 10 micefor each group. Sham operation＋MF, MF diet-fed sham operation mice ; ACAS＋MF, MF diet-fed ACAS mice ;ACAS＋MF with EPKOP, MF diet with emulsifying Pacific krill oil powder-fed ACAS mice.

platform task five times with a 5-min interval between

at the platform in 90 sec we guided it to the platform

attempts each day. Mice were released into the waterand allowed it to remain there for 15 sec. In eachat the starting position in the three other quadrants.

mouse, we calculated the latency to reach the platform

We monitored each mouse tested and recorded the(escape latency) based on the average of the 5 times

time required until it arrived at the platform. When aof performing the hidden platform task each day.

mouse arrived at the platform in 90 sec we allowed itＨto stay there for 15 sec. When a mouse did not arrive
No. 3, 2024

Cerebral blood flow (CBF) measurement

We measured CBF at 35 days after ACAS or sham173

Ishida・Yamada・Hirose et al.

surgery using a 2D laser blood flow imager (OMEGA­
ZONE, Omegawave Inc., Tokyo, Japan), (n＝10 foreach), as previously described18）19）. Briefly, laser speck­le flowmetry with the average of highspeed 2D im­

Table 2
PrimerGAPDH

aging was used to measure the relative CBF. After
anesthesia (1-2 ％ isoflurane), we surgically removed

IL-1β

the mouse scalp to expose the skull. The periosteum,
which adheres to the skull, was widely removed with
fine-tip forceps. The skull surface was wiped withsaline-soaked gauze to prevent drying of the surface.
CBF recordings were performed through the skull.

Sequences of oligonucleotide primers

TNF-α

MCP-1

Array
S

TGTGTCCGTCGTGGATCTGAA

TTGCTGTTGAAGTCGCAGGAG

S

TCCAGGATGAGGACATGAGCACAGAACGTCACACACCAGCAGGTTAS

AAGCCTGTAGCCCACGTCGTAA

GGCACCACTAGTTGGTTGTCTTS

GGCTCAGCCAGATGCAGTTAACA

GCCTACTCATTGGGATCATCTTG

Circular regions in identically sized ROIs (900 pixels),
located 1 mm posterior and 2 mm lateral to the breg­
ma were defined on the image for quantitative mea­

for each) using ReliaPrepTM RNA Tissue Miniprep

surement (Fig. 2B). These data reflect the CBF of the

System (Promega, Madison, WI, USA), according tosubsurface microvessels in the brain with a depth ofthe manufacturer’s instructions. We used 500 ng of

23）

0.5 mm .

total RNA as a template for reverse transcription withＩ

the SuperScript® III First-Strand synthesis system (In­Histology
Hematoxylin/eosin staining for histopathologicalvitrogen, Carlsbad, CA, USA). We performed real-time

19）

analysis was performed as previously described .

RT-PCR analysis with ABI Step One Real-Time PCR

Briefly, after anesthesia with isoflurane (2 ％ ), andSystem using Fast SYBR Green Master Mix (Applied

sodium pentobarbital (15 mg/kg i.p.) the mouse brain

Biosystems, Foster City, CA, USA) to detect mono­

was transcardially perfused and fixed with a 4 ％cyte chemotactic protein 1 (MCP-1), interleukin (IL)-1β,solution of paraformaldehyde in 0.01 mol/L PBS for

tumor necrosis factor α (TNF-α), and glyceralde­more than 5-8 min in the three different groups ofhyde-3-phosphate dehydrogenase (GAPDH). Table 2

mice at five weeks after ACAS or the sham opera­

shows the sequences of oligonucleotide primers. Astion (n＝4 for each). After brain fixation, each brain

the expression of GAPDH mRNA was constant be­

was quickly removed and divided coronally at thetween groups, the expression of each gene was nor­bregma, the bregma at 1 mm, and bregma at 2 mm

malized to that of GAPDH mRNA.

through the hippocampus. The brain samples wereＫ

Statistical analysis

embedded in paraffin, and then cut into 3-μm-thickAll data are shown as the mean±SE. We used anal­

coronal sections. For histopathological analysis, fiveysis of variance (ANOVA) with Dunnett’s test for sta­

sections stained with hematoxylin/eosin were pre­tistical analysis of multiple comparisons of data. Two-pared. After HE staining, the shrunken neurons withway and one-way ANOVA with Dunnett’s test were

pyknotic nuclei located within the square area (100×used for statistical analysis of the Morris water maze

100 μm) in hippocampus CA1 region were counted totest and other experiments, respectively. P＜0.05 was

24）assess neuropathy as previously reported .
Ｊ

Quantification of mRNA by real-time PCR
The amount of mRNA by real-time reverse tran­

scription-polymerase chain reaction (RT-PCR) was25）

considered significant.
Ⅲ

Results

Ａ CBF was not decreased on feeding emulsifying

quantified as previously described . Briefly, after the

Pacific krill oil powder (EPKOP) to ACAS micebrain tissue was resected, total RNA was prepared

To examine the effects of EPKOP on ACAS-induced

from the frontal lobe (F) and hippocampus area (HS)decreases in CBF, CBF was measured and local CBFs

of the three different groups of mouse brains (n＝7were calculated in circular regions of ROI1 and ROI2

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Krill oil powder reduces vascular cognitive impairment(Fig. 2B). ACAS decreased local CBF in both circulararea of the hippocampus showed an increased number

regions of MFD-fed ACAS compared with MFD-fed

of shrunken neurons in MFD-fed ACAS mice com­sham-operated mice (Fig. 2C). In contrast, the local

pared with the two other groups of mice (Fig. 4B).

CBFs in both circular regions were similar between

Ｆ EPKOP inhibited the increases in IL-1β mRNA

MFD with EPKOP-fed ACAS and MFD-fed sham-expressions in ACAS mice

operated mice.

mRNA expression of the IL-1β gene was signifi­

Ｂ EPKOP reversed ACAS-induced shortening ofcantly increased in MFD-fed ACAS compared withlatency to fall measured by rotarod test

MFD-fed sham-operated mice in the hippocampus

The rotarod test showed that the latency to fall(Fig. 5A). In contrast, it was similar between MFD

was shorter in MFD-fed ACAS compared with sham-with EPKOP-fed ACAS and MFD-fed sham-operated

operated MFD-fed mice. Moreover, ACAS significantly

mouse brains. Expressions of MCP-1 and TNF-α

shortened the latency to fall in MFD-fed comparedmRNA were similar among the three different groups

with MFD with EPKOP-fed mice (Fig. 3A). In con­of mice in the frontal lobe (Fig. 5B-C). In contrast,

trast, the latency to fall was similar between MFD

those gene expressions in the hippocampus tended towith EPKOP-fed ACAS and MFD-fed sham-operated

be increased in MFD-fed ACAS and decreased inmice.

MFD with EPKOP-fed ACAS mice (Fig. 5B-C).

Ｃ EPKOP did not affect spatial working memoryor spontaneous activity measured by Y-maze test

Ⅳ

Discussion

The Y-maze test showed that both the percent­

We demonstrated that EPKOP : 1) improved theage of alternation behaviors and rates of arm entries

decreased cerebral blood flow in ACAS mice, 2) re­were similar among the three different groups ofversed the shortened latency to fall based on rotarod

mice at two and four weeks after ACAS (Fig. 3B).

testing in ACAS mice, 3) reversed the longer escapeＤ EPKOP reversed ACAS-induced prolongation

latencies in ASCS mice based on the Morris waterof escape latency measured by Morris water maze

maze test, and 4) decreased the number of shrunken

test

neurons with pyknotic nuclei in the hippocampal area

The Morris water maze test showed that the escapeof ACAS mice. EPKOP ameliorated the increased

latency was significantly longer in MFD-fed ACAS

IL-1β gene expressions in the ACAS mouse hippo­

mice than in sham-operated MFD-fed mice during

campus. These results suggest that EPKOP improves

the acquisition phase of 6 days. MFD with EPKOPVCI and neural injury through the improvement ofsignificantly shortened the prolonged escape latency

cerebral blood flow and IL-1β gene expressions in thein MFD-fed ACAS mice during the acquisition phasemouse model of ACAS-induced vascular dementia.

of 6 days (Fig. 3C). Mean swimming speeds in the

Cerebral small vessel disease causes blood-brain

acquisition phase did not differ among the three dif­barrier (BBB) functional damage, leukocyte infiltration,

ferent groups of mice (data not shown).

and increases in IL-1β and TNF-α expressions, lead­Ｅ EPKOP improved increases in number of shrunk-ing to the injury of oligodendrocytes and neurons26）.

en neurons in ACAS mice

A rat cerebral small vessel disease model demonstrat­

To examine the effects of EPKOP on ischemia-

ed that IL-1β and TNF-α expressions were increased

induced neuronal damage, shrunken neurons with py­

in the hippocampus27）. Moreover, the Morris waterknotic nuclei in the hippocampal area were observed.

maze test demonstrated that cerebral hypoperfusion

Representative images of the hippocampal area re­induced a longer escape latency in rats28）. Our previous

vealed shrunken neurons with pyknotic nuclei (white

study demonstrated that ACAS-induced decreases

arrows) in MFD-fed ACAS mice but not in the twoin cerebral blood flow (i.e., cerebral hypoperfusion)other groups of mice (Fig. 4A). Quantitative analysis

caused a longer escape latency based on the Morrisof shrunken neurons with pyknotic nuclei in the C1

water maze test, hippocampal neuronal injury shown

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Ishida・Yamada・Hirose et al.

Fig. 3 Behavioral performance of ACAS mice in Rotarod, Y-maze, and Morris water maze tests. Each dietwas fed from four weeks before to five weeks after sham operation and ACAS, respectively. Panel
A, Motor coordination and balance were tested with the rotarod test. The rotarod test was per­formed at two weeks after ACAS or sham operation in the three different groups of mice. Panels
B-C, Spatial working memory elucidated by alternation behavior and spontaneous activity elucidat­
ed by the number of arm entries in the Y-maze test at two and four weeks after ACAS or thesham operation in the three different groups of mice. Panel D, Time-course of escape latency as­
sessed with the Morris water maze test from days 1 to 6 (consecutive days from 29th to 34th) after
ACAS or sham operation in the three different groups of mice. Data are the mean±SE obtained
from 10 mice for each group. EPKOP, emulsifying Pacific krill oil powder.

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Krill oil powder reduces vascular cognitive impairment

Fig. 4 Historical analysis of ischemia-induced neuronal injury in the mouse hippocampus in the threedifferent groups of mice at five weeks after ACAS or the sham operation. Each diet was fed fromfour weeks before to five weeks after sham operation and ACAS, respectively. Panel A, Typicalimage of HE staining (Original magnification : 2× Scale bar＝500 μm). White arrow shows shrunken
neurons with pyknotic nuclei. Panel B, Quantitative analysis of shrunken neurons with pyknotic nuclei
calculated from neurons located within the white square area (100×100 μm, CA1 region) shown inpanel A. Data are the mean±SE obtained from 4 mice for each group. EPKOP, emulsifying Pacifickrill oil powder.

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Fig. 5 Quantitative analyses of pro-inflammatory cytokines and chemokine gene expressions by real-time
RT-PCR in the three different groups of mouse brain tissues at five weeks after ACAS or the shamoperation. Each diet was fed from four weeks before to five weeks after sham operation and ACAS,
respectively. mRNA levels of interleukin 1β (IL-1β), monocyte chemotactic protein 1 (MCP-1), andtumor necrosis factor α (TNF-α) are shown. Data for IL-1β, MCP-1, and TNF-α were normalized tothose for GAPDH. Data are the mean±SE obtained from 7 mice for each group. EPKOP, emulsify­
ing Pacific krill oil powder ; F, frontal lobe ; HS, hippocampus area.

by shrunken neurons with pyknotic nuclei, and in­

microglia activation released IL-1β, leading to PC12

creased IL-1β and TNF-α gene expressions in mice,cell apoptosis29）. Thus, EPKOP might improve hippo­

suggesting that cerebral hypoperfusion-induced in­campal neuronal injury and VCI through reduction

creases in IL-1β and TNF-α gene expressions playof the increased IL-1β gene expression.

important roles in the development of neural injury

Our present study demonstrated that the latency

and VCI. The present study demonstrated that EPKOP

to fall tested with the rotarod test was significantly

reduced the increased gene expression of IL-1β inshorter in MFD-fed ACAS than in MFD-fed sham-

ACAS mice, and TNF-α gene expression was similar

operated mice, indicating that muscle strength, motiva­

between EPKOP-fed ACAS mice and sham-operated

tion not to fall, and/or CNS motor function were signifi­

mice. Moreover, EPKOP reversed the longer escapecantly impaired in ACAS mice. Interestingly, EPKOP

latency based on the Morris water maze test andimproved the shortened the latency to fall to the nor­decreased the number of shrunken neurons in themal level in ACAS mice, suggesting that EPKOP canhippocampal area in ACAS mice. A previous study

maintain muscle strength, motivation not to fall, and/or

demonstrated that lipopolysaccharide-induced BV2

CNS motor function in mice with small vessel disease.

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Krill oil powder reduces vascular cognitive impairment

CBF was similar between MFD with EPKOP-fed

increased MCP-1 gene expression in brain tissues.

ACAS mice and MFD-fed sham-operated mice. This

Although whether EPKOP inhibits ACAS-induced

indicates that EPKOP improves the decreases in ce­

increases in MCP-1 gene expression is still unclear,

rebral blood flow in ACAS mice. Moreover, almost all

it may participate in the suppression of MCP-1 geneof the factors tested were similar between MFD withexpression in small vessel disease.

EPKOP-fed ACAS mice and MFD-fed sham-operated
30）

mice. Our previous study

We demonstrated in this study that EPKOP de­

demonstrated that phlo­creased the number of shrunken neurons with pyk­

rizin ameliorated the decreased cerebral blood flow

notic nuclei in the hippocampal area of ACAS micein ACAS mice, and that cerebral blood flow was sim­

similar to sham-operated mice. Hattori et al.18）demon­

ilar between phlorizin-treated ACAS mice and sham-strated that in the hippocampus stained with H&E

operated mice. Moreover, phlorizin also reversed thestaining, neural marker NeuN antibody, and glial

increases in IL-1β and the number of shrunken neu­

marker Iba1 antibody, shrunken neurons and neuro­rons in the ACAS mouse brain. Therefore, EPKOP

nal loss in the hippocampus were observed only on

might ameliorate neural injury and VCI through thethe right ameroid constrictor side but not the left

improvement of cerebral blood flow as phlorizin did.

microcoil side in ACAS mice. Moreover, activated

However, the mechanism of EPKOP-induced improve­

microglia was also observed surround the area ofment of cerebral blood flow in ACAS mice is still un­neuronal loss in the right side of hippocampus18）. We

known. Several studies showed that EPA induced ce­

demonstrated that shrunken neurons were observed

31）

rebral arteriolar dilatation in rabbits . Moreover, N-3

in only the right side of hippocampus stained with

PUFAs are likely to improve vascular endothelial func­

H&E staining in ACAS mice. Based on these results,

32）
33）

it is considered that the dementia model using thetion and arterial stiffness, which are both important34）

mechanistic determinants of CBF . In fact, transgenic

ACAS procedure has been completed in this experi­

fat-1 mice that overproduce n-3 PUFAs showed func­

ment. Thus, EPKOP might improve neuronal injury

tional and histological protection against focal cere­shown by shrunken neurons with pyknotic nuclei in35）

bral ischemia . Interestingly, these mice exhibitedthe hippocampal area of ACAS mice.

improvements in revascularization and angiogenesis.

This study had limitations. In this experiment,

These results suggest that n-3 PUFAs induce cerebral

cerebral blood flow was measured only at 35 weeks

vasodilation and take part in cerebrovascular remod­

after ACAS. Hattori et al.18） demonstrated that theeling. Thus, EPKOP-induced improvement of cere­

decrease in cerebral blood flow due to ACAS graduallybral blood flow in ACAS mice may be caused throughprogressed until the 7th day after ACAS, with bloodn-3 PUFA-induced cerebral vasodilation and cere­

flow decreasing by 65-70 ％ on the 7th day. Subse­

brovascular remodeling.

quent cerebral blood flow remained at the same level

It is well-known that the increase in MCP-1 expres­after 14 and 28 days. EPKOP intake continued fromsion in brain and endothelial cells plays important roles

4 weeks before ACAS to 5 weeks after ACAS, so it

in cerebral ischemia-induced cognitive deficits36）-38）.

is thought that the blood concentration of EPKOP-

Moreover, MCP-1 can increase IL-1β and TNF-α ex­

containing components might reach a sufficient39）

pressions in microglia . This indicates that the cere­amount to maintain blood flow through n-3 PUFA-

bral ischemia increases MCP-1 expression in the

induced cerebral vasodilation and cerebrovascularbrain, leading to inflammatory responses such as in­remodeling. These suggest that EPKOP fed mice docreases in IL-1β and TNF-α expression. In this study,

not cause ACAS-induced decrease in cerebral blood

cerebral gene expression of MCP-1 tended to be in­flow since the beginning of ACAS. It revealed thatcreased in ACAS mice and was similar between MFD

EPKOP might ameliorate neural injury and VCIwith EPKOP-fed ACAS and MFD-fed sham-operated

through the improvement of cerebral blood flow as

mice. Our previous study demonstrated that ACAS

phlorizin did. However, a direct protective effect of

No. 3, 2024

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Ishida・Yamada・Hirose et al.

EPKOP on ischemic brain tissues is still unknown.

whether EPKOP has an effect on hemodynamics,

Our previous study demonstrated that phlorizin di-protective effects of EPKOP on hippocampal tissue

rectly improved brain tissue injury in addition to im-

damage and cognitive decline might not be effects on

30）

proving cerebral blood flow . N-3 PUFAs directly

hemodynamics.

protect against hypoxia and ischemia-induced brain

In conclusion, we are the first to demonstrate thedamage in neonates by activating the Akt pathway

possibility of using EPKOP as a functional food for40）

in compromised neurons . Moreover, DHA and EPA

the prevention of VCI in a mouse model of small ves-

directly ameliorated oxygen and glucose deprivation-

sel disease.

41）

induced neuronal death in primary neuronal cultures ,Disclosuresuggesting the possibility of a direct protective effectof EPKOP on ischemic brain tissues. Therefore, theeffects of EPKOP on brain tissue would need to be

No author has an actual or perceived conflict ofinterest regarding the contents of this article.

examined in future project. As we did not measure

Acknowledgements

effects of EPKOP on hemodynamics the effects arestill unknown. Hattori et al.18） previously measured

New Emulsifying Oil Powder Extracted from Pacificmean blood pressure and heart rate in the ACAS

Krill was kindly provided by ADEKA Corporation

model and showed that there were no differences in(Tokyo, Japan).

these values in the ACAS group compared to theFunding information

sham group. Therefore, it is unlikely that hemodynamic changes are involved in hippocampal tissue

This work received no external funding.

damage or cognitive decline. Although it is not clear
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