Diet

supplementation

for

weeks

with

polyphenol-rich

cereals

improves

several

functions

and

the

redox

state

of

mouse

leucocytes

Background

Cereals

naturally

contain

a

great

variety

of

polyphenols

which

exert

a

wide

range

of

physiological

effects

both

in

vitro

and

in

vivo

Many

of

their

protective

effects

including

an

improvement

of

the

function

and

redox

state

of

immune

cells

in

unhealthy

or

aged

subjects

come

from

their

properties

as

powerful

antioxidant

compounds

However

whether

cereal-based

dietary

supplementation

positively

affects

the

immune

function

and

cellular

redox

state

of

healthy

subjects

remains

unclear

Introduction

Nowadays

consumption

of

natural

products

is

a

matter

of

major

importance

as

regards

health

Indeed

consumption

of

fruit

and

vegetables

was

already

associated

with

a

reduced

risk

of

chronic

diseases

and

age-related

functional

decline

several

years

ago

-LSB-

-RSB-

Nevertheless

less

attention

has

been

focused

on

the

importance

of

phytochemicals

from

cereals

in

maintaining

health

It

is

well

known

that

the

nutritional

status

is

a

central

factor

contributing

to

immune

system

competence

-LSB-

-RSB-

and

that

an

adequate

function

of

the

immune

system

is

related

to

health

promotion

and

disease

prevention

-LSB-

-RSB-

Nutrients

that

have

traditionally

been

shown

to

be

required

for

correct

immune

system

function

include

different

types

of

vitamins

zinc

copper

and

selenium

However

there

is

an

increasing

amount

of

data

showing

the

implication

of

the

so-called

non-nutrients

i.e.

phytochemicals

in

the

immune

system

responses

-LSB-

-RSB-

Further

the

phytochemicals

found

in

natural

products

have

been

involved

in

protecting

against

a

wide

range

of

oxidative

stress-related

chronic

diseases

-LSB-

-RSB-

The

overproduction

of

oxidants

and

the

overwhelming

of

the

antioxidant

defence

lead

to

oxidative

stress

that

can

cause

oxidative

damage

to

large

biomolecules

such

as

proteins

DNA

and

lipids

resulting

in

an

increased

risk

of

disease

-LSB-

-RSB-

For

this

reason

to

forestall

an

excessive

production

of

reactive

oxygen

species

-LRB-

ROS

-RRB-

sufficient

amounts

of

antioxidants

need

to

be

consumed

However

the

generation

of

ROS

is

a

key

part

of

normal

immune

system

function

-LSB-

-RSB-

These

molecules

play

a

key

role

in

the

defence

against

infectious

agents

and

seem

to

be

involved

in

regulating

many

biological

processes

serving

as

second

messengers

-LSB-

-RSB-

Thus

it

is

important

to

maintain

a

balance

between

ROS

generation

and

antioxidant

defences

within

immune

cells

to

ensure

a

correct

functioning

of

the

immune

system

and

help

to

prevent

the

onset

of

chronic

diseases

Cereals

naturally

contain

a

wide

variety

of

polyphenols

such

as

the

hydroxycinnamic

acids

ferulic

vanillic

and

p-coumaric

acids

-LSB-

-RSB-

which

show

a

strong

antioxidant

power

and

may

help

to

protect

from

oxidative

stress

and

therefore

can

decrease

the

risk

of

contracting

many

diseases

Flavonoids

are

present

in

small

quantities

even

though

their

numerous

biological

effects

and

their

implications

for

inflammation

and

chronic

diseases

have

been

widely

described

-LSB-

-RSB-

The

mechanisms

of

action

of

polyphenols

go

beyond

the

modulation

of

oxidative

stress-related

pathways

-LSB-

-RSB-

Cereal

phytochemicals

exert

their

health

benefits

through

multifactorial

physiological

mechanisms

including

enhancement

of

the

immune

system

and

hormones

facilitation

of

substance

transit

through

the

digestive

tract

metabolism

modulation

and

antioxidant

antibacterial

and

antiviral

activities

-LSB-

-RSB-

It

is

known

that

unhealthy

or

aged

subjects

are

more

susceptible

to

infection

showing

an

impairment

of

function

and

redox

state

of

immune

cells

and

that

such

impairment

can

be

counteracted

by

antioxidant

administration

-LSB-

-RSB-

However

whether

dietary

antioxidant

supplementation

affects

the

functional

and

redox

state

of

immune

cells

of

healthy

subjects

remains

unclear

Not

all

subjects

are

likely

to

benefit

in

the

same

way

from

supplementation

i.e.

there

are

certain

groups

within

the

apparently

healthy

general

population

that

benefit

from

adequate

intakes

of

certain

compounds

-LSB-

-RSB-

Since

there

is

an

increasing

interest

in

consuming

products

naturally

rich

in

phytochemicals

able

to

provide

health

benefits

and

prevent

a

wide

variety

of

diseases

in

this

context

we

have

investigated

in

the

present

work

the

effects

of

diet

supplementation

for

weeks

with

different

types

of

cereal

fractions

naturally

rich

in

polyphenols

on

certain

parameters

of

immune

function

and

redox

state

of

peritoneal

leukocytes

from

adult

healthy

mice

Material

and

methods

Animals

One

hundred-fifty

female

ICR

-LRB-

-RRB-

mice

-LRB-

Mus

musculus

-RRB-

-LRB-

Harlan

Ibérica

Barcelona

Spain

-RRB-

which

were

weeks

old

on

arrival

to

our

laboratory

were

used

Mice

were

checked

periodically

by

animal

care

personnel

and

were

specific

pathogen

free

as

tested

by

Harlan

according

to

FELASA

recommendations

and

did

not

show

any

sign

of

malignancy

or

other

pathological

processes

The

animals

were

randomly

housed

in

polyurethane

boxes

at

a

constant

temperature

-LRB-

°C

-RRB-

in

sterile

conditions

inside

an

aseptic

air

negative-pressure

environmental

cabinet

-LRB-

Flufrance

Cachan

France

-RRB-

on

a

h

reversed

light/dark

cycle

All

animals

were

fed

water

and

standard

Sander

Mus

-LRB-

diet

from

Panlab

L.S.

Barcelona

Spain

-RRB-

pellets

ad

libitum

The

diet

was

in

accordance

with

the

recommendations

of

the

American

Institute

of

Nutrition

for

laboratory

animals

Mice

were

treated

according

to

the

guidelines

of

the

European

Community

Council

Directives

EEC

Although

we

have

previously

observed

that

the

oestrous

cycle

phase

of

mice

has

no

effect

on

this

experimental

assay

all

females

used

in

the

present

work

were

at

the

beginning

of

dioestrous

Experimental

groups

The

animals

were

marked

for

their

individual

follow-up

Five

experimental

groups

one

control

-LRB-

n

-RRB-

and

four

supplemented

-LRB-

mice

per

group

-RRB-

were

used

The

supplementation

started

at

weeks

of

age

Then

the

supplemented

groups

were

fed

for

a

period

of

weeks

and

at

the

age

of

weeks

samples

were

obtained

to

evaluate

certain

parameters

of

function

and

redox

state

of

peritoneal

leukocytes

The

experiments

were

carried

out

in

January

at

a

constant

laboratory

temperature

of

°C

Food

intake

was

measured

weekly

at

the

same

time

-LRB-

h

-RRB-

Each

animal

was

fed

weekly

g

of

diet

-LRB-

control

or

supplemented

-RRB-

and

after

week

the

uneaten

remains

were

weighed

Body

weights

were

determined

once

every

week

No

significant

differences

on

food

consumption

and

body

weight

among

diet

groups

during

the

study

were

observed

Cereal

products

and

preparation

of

extracts

All

cereal

products

-LRB-

wheat

germ

buckwheat

flour

fine

rice

bran

and

wheat

middlings

named

B

C

D

and

E

respectively

-RRB-

used

in

this

study

were

provided

by

Danone

Vitapole

-LRB-

France

-RRB-

The

samples

were

stored

and

kept

at

°C

prior

to

analysis

Moisture

content

of

the

cereal

samples

was

evaluated

by

drying

at

°C

for

min

The

analyses

were

done

in

duplicate

The

different

extracts

were

analyzed

by

Danone

Vitapole

-LRB-

France

-RRB-

Briefly

the

cereal

products

were

sequentially

extracted

with

water

at

°C

followed

by

methanol

and

ethyl

acetate

Extraction

with

each

solvent

was

carried

out

twice

for

min

under

in

the

dark

and

constant

agitation

-LRB-

rpm

-RRB-

The

ethyl

acetate

extracts

were

evaporated

under

vacuum

and

re-dissolved

in

methanol

The

final

insoluble

pellet

was

dried

The

soluble

fractions

and

the

insoluble

pellet

were

stored

at

°C

-LRB-

in

the

dark

and

under

-RRB-

prior

to

analysis

of

polyphenols

and

determination

of

antioxidant

activity

Analysis

of

total

polyphenols

and

antioxidant

activity

The

amount

of

total

phenolic

compounds

in

all

extracts

was

determined

by

Danone

Vitapole

-LRB-

France

-RRB-

according

to

the

Folin

Ciocalteu

method

-LSB-

-RSB-

The

total

phenolic

concentration

is

calculated

from

a

standard

curve

using

a

range

of

concentrations

of

gallic

acid

The

results

are

expressed

as

milligrams

of

gallic

acid

equivalents

per

gram

of

material

The

antioxidant

in

vitro

activity

of

the

extracts

obtained

from

selected

cereal

products

was

evaluated

using

an

Oxygen

Radical

Absorbance

Capacity

-LRB-

ORAC

-RRB-

procedure

-LRB-

Brunswick

Laboratories

Wareham

MA

USA

-RRB-

Values

are

expressed

as

μmol

of

Trolox

equivalents

-LRB-

TE

-RRB-

per

g

of

dry

matter

This

assay

was

conducted

in

triplicate

There

was

also

a

positive

correlation

between

ORAC

and

total

polyphenol

content

-LRB-

-RRB-

Preparation

of

diets

The

control

group

only

received

a

standard

maintenance

diet

-LRB-

diet

from

Panlab

L.S.

Barcelona

Spain

see

Table

-RRB-

and

the

remaining

groups

received

of

control

diet

plus

of

cereal

fractions

naturally

rich

in

polyphenols

-LRB-

B

C

D

and

E

see

Table

-RRB-

Control

and

supplemented

diets

were

prepared

by

mixing

the

standard

maintenance

diet

or

one

of

the

cereal

fractions

with

water

forming

a

biscuit-shape

preparation

Both

types

of

preparations

were

daily

turned

upside

down

and

maintained

in

a

dark

room

for

days

to

obtain

a

dry

preparation

Then

the

humidity

of

the

different

diets

was

measured

and

each

of

them

was

stored

at

°C

for

less

than

weeks

avoiding

exposure

to

light

in

order

to

prevent

their

oxidation

Table

composition

of

standard

-LRB-

control

-RRB-

and

experimental

-LRB-

B

C

D

and

E

-RRB-

dietsDiet

ingredientsa

-LRB-

-RRB-

ControlBCDEHFLFbHFLFbLFbLFbHFLFbPolyphenols

Gallic

acid

Catechin

p-HB

acid

Vanillic

acid

p-coumaric

acid

Sinapic

acid

Ferulic

acid

Quercetin

a

a

Rutin

a

a

Oryzanol

a

Vitamins

Vitamin

Vitamin

Vitamin

Pantothenic

Fatty

acids

Palmitic

Palmitoleic

Stearic

Oleic

Linoleic

Linolenic

Minerals

Each

control

-LRB-

non-supplemented

-RRB-

animal

received

a

of

maintenance

diet

-LRB-

Panlab

Barcelona

Spain

-RRB-

Each

supplemented

animal

received

an

of

maintenance

diet

plus

of

cereal

fractions

-LRB-

B

C

D

or

E

-RRB-

naturally

rich

in

polyphenols

B

wheat

germ

C

buckwheat

flour

D

fine

rice

bran

E

wheat

middlings

ORAC

-LRB-

μTrolox

Equivalents/g

-RRB-

B

C

D

E

Total

polyphenols

content

-LRB-

eq

mg

gallic

acid/g

-RRB-

B

C

D

E

HF

-LRB-

-RRB-

hydrosoluble

fraction

LF

-LRB-

-RRB-

lyposoluble

fraction

p-HB

p-hydroxybenzoic

g.

mg

g.

g

Collection

of

peritoneal

leukocytes

Peritoneal

suspensions

were

obtained

between

and

h

without

sacrificing

the

animals

following

the

method

previously

described

-LSB-

-RSB-

Peritoneal

leukocytes

-LRB-

macrophages

and

lymphocytes

-RRB-

were

collected

identified

quantified

by

their

morphology

using

optical

microscopy

-LRB-

-RRB-

and

their

concentration

was

adjusted

depending

on

the

function

assayed

-LSB-

-RSB-

The

cellular

viability

determined

in

each

experiment

using

the

trypan-blue

exclusion

test

was

in

all

cases

higher

than

Leukocyte

functions

The

chemotaxis

assays

were

performed

according

to

a

technique

previously

described

-LSB-

-RSB-

The

results

were

expressed

as

chemotaxis

index

-LRB-

C.I.

-RRB-

by

counting

the

number

of

macrophages

and

lymphocytes

at

the

lower

face

of

the

filters

of

the

Boyden

chambers

-LSB-

-RSB-

The

intracellular

superoxide

anion

-LRB-

-RRB-

levels

were

evaluated

as

described

elsewhere

-LSB-

-RSB-

The

results

were

expressed

as

nmol

of

per

cells

The

lymphoproliferation

assay

and

the

determination

of

-LRB-

-RRB-

levels

were

carried

out

following

the

methods

previously

described

-LSB-

-RSB-

The

proliferation

results

were

expressed

as

percentage

of

stimulation

by

the

mitogens

Concanavalin

A

-LRB-

Con

A

-RRB-

or

lipopolysaccharide

-LRB-

LPS

-RRB-

with

respect

to

spontaneous

proliferation

that

was

considered

to

be

In

the

case

of

the

results

were

expressed

as

pg/ml

Leukocyte

redox

state

Total

glutathione

and

its

oxidized

form

-LRB-

GSSG

-RRB-

were

spectrophotometrically

evaluated

according

to

-LSB-

-RSB-

The

results

are

expressed

as

To

calculate

the

GSSG/GSH

ratio

the

GSH

-LRB-

reduced

form

-RRB-

was

obtained

by

substracting

the

GSSG

values

from

the

total

glutathione

values

The

secretion

of

mouse

tumor

necrosis

factor

alpha

-LRB-

TNFα

-RRB-

was

measured

in

culture

supernatants

stimulated

with

LPS

using

an

ELISA

kit

-LRB-

HyCult

Biotechnology

Uden

The

Netherlands

-RRB-

The

results

were

expressed

as

pg/ml

being

the

minimum

detectable

value

pg/ml

Catalase

activity

-LRB-

CAT

-RRB-

was

carried

out

according

to

-LSB-

-RSB-

slightly

modified

Samples

of

peritoneal

suspension

-LRB-

adjusted

to

cells/ml

-RRB-

were

resuspended

in

mM

phosphate

buffer

sonicated

and

centrifuged

at

g

-LRB-

°C

-RRB-

for

min

The

reaction

was

initiated

by

the

addition

of

and

spectrophotometrically

measured

at

nm

-LRB-

°C

-RRB-

for

s

The

results

were

expressed

as

cells

Malondialdehyde

-LRB-

MDA

-RRB-

levels

were

evaluated

according

to

a

technique

previously

described

-LSB-

-RSB-

slightly

modified

and

quantified

by

high

performance

liquid

chromatography

-LRB-

HPLC

-RRB-

Aliquots

of

peritoneal

suspension

-LRB-

adjusted

to

cells/ml

-RRB-

resuspended

in

mM

-LRB-

Sigma

-RRB-

pH

were

sonicated

and

added

to

a

mixture

reaction

that

contained

M

ortophosphoric

acid

-LRB-

Fluka

Steinheim

Germany

-RRB-

acid

-LRB-

-RRB-

-LRB-

TBA

Sigma

-RRB-

and

mM

butylated

hydroxytoluene

-LRB-

BHT

Sigma

-RRB-

It

was

heated

at

°C

for

min

and

after

cooling

on

ice

the

samples

were

extracted

adding

n-butanol

-LRB-

Panreac

-RRB-

centrifugated

at

rpm

-LRB-

°C

-RRB-

for

min

and

μl

of

supernatants

-LRB-

organic

phase

-RRB-

were

injected

in

the

HPLC

column

-LRB-

Novapack

cm

mm

Waters

-RRB-

As

mobile

phase

mM

pH

methanol

-LRB-

v/v

-RRB-

was

used

The

flow

rate

of

the

mobile

phase

was

adjusted

to

ml/min

MDA

was

monitored

at

λ

nm

in

a

WATERS

ultraviolet

detector

Standard

curves

were

prepared

fresh

daily

using

MDA

bis

-LRB-

dimethyl

acetal

Sigma

-RRB-

The

results

were

expressed

as

Statistical

analysis

Statistical

analysis

was

performed

using

the

software

SPSS

-LRB-

Chicago

IL

USA

-RRB-

The

data

are

expressed

as

the

mean

standard

deviation

-LRB-

S.D.

-RRB-

of

the

eight

values

corresponding

to

the

number

of

experiments

Each

value

is

the

mean

of

the

data

from

assays

performed

in

duplicate

The

normality

of

the

samples

was

confirmed

by

the

Kolmogorov

Smirnov

test

and

the

homogeneity

of

variances

by

the

Levene

test

The

data

were

analysed

by

the

one-way

analysis

of

variance

-LRB-

ANOVA

-RRB-

followed

by

the

Tukey

test

The

Kruskall

Wallis

test

for

non-parametric

data

was

used

The

minimum

level

considered

statistically

significant

was

P

Results

Immune

system

response

Figure

shows

the

chemotaxis

capacity

lymphoproliferative

response

to

the

T-cell

mitogen

Concanavalin

A

-LRB-

ConA

-RRB-

or

the

B-cell

mitogen

LPS

and

release

of

of

peritoneal

lymphocytes

from

adult

female

mice

supplemented

for

weeks

with

cereal

fractions

naturally

rich

in

polyphenols

Mice

supplemented

with

cereals

showed

a

strongly

significant

increase

of

lymphocyte

chemotaxis

compared

with

that

of

control

mice

-LRB-

P

with

B

D

and

E

and

P

with

C

-RRB-

-LRB-

Fig.

-RRB-

As

regards

lymphoproliferative

response

to

Con

A

-LRB-

Fig.

-RRB-

mice

supplemented

with

any

of

the

cereals

B

-LRB-

P

-RRB-

C

-LRB-

P

-RRB-

D

-LRB-

P

-RRB-

or

E

-LRB-

P

-RRB-

showed

a

significantly

higher

capacity

compared

with

that

of

control

mice

The

supplementation

with

the

cereals

C

-LRB-

P

-RRB-

D

-LRB-

P

-RRB-

or

E

-LRB-

P

-RRB-

but

not

with

B

was

able

to

increase

lymphoproliferation

in

response

to

LPS

with

respect

to

that

of

non-supplemented

mice

-LRB-

Fig.

-RRB-

The

secretion

of

by

peritoneal

leukocytes

from

mice

supplemented

with

cereal

B

-LRB-

P

-RRB-

C

-LRB-

P

-RRB-

or

E

-LRB-

P

-RRB-

was

higher

than

that

of

control

mice

-LRB-

Fig.

-RRB-

The

chemotaxis

capacity

of

macrophages

increased

significantly

in

cereal

E-supplemented

mice

in

comparison

with

that

of

control

mice

-LRB-

versus

P

-RRB-

The

intracellular

superoxide

anion

-LRB-

-RRB-

levels

in

non-stimulated

peritoneal

leukocytes

significantly

increased

after

supplementation

for

weeks

with

cereal

B

and

C

with

respect

to

non-supplemented

mice

-LRB-

Controls

B

P

C

P

-RRB-

The

release

of

intracellular

from

peritoneal

leukocytes

stimulated

with

latex

beads

was

significantly

higher

in

mice

supplemented

with

cereals

B

C

and

E

-LRB-

Controls

B

P

C

P

E

P

-RRB-

Fig.

-LRB-

a

-RRB-

Chemotaxis

capacity

of

peritoneal

lymphocytes

-LRB-

b

-RRB-

lymphoproliferation

in

ConA

-LRB-

μg/ml

-RRB-

stimulated

h-culture

supernatants

-LRB-

c

-RRB-

lymphoproliferation

in

LPS

-LRB-

μg/ml

-RRB-

stimulated

h-culture

supernatants

and

-LRB-

d

-RRB-

release

of

-LRB-

-RRB-

in

supernatants

of

h-cultures

of

peritoneal

leukocytes

stimulated

with

Con

A

-LRB-

μg/ml

-RRB-

from

adult

female

ICR

-LRB-

-RRB-

mice

supplemented

for

weeks

with

cereal

fractions

-LRB-

B

C

D

or

E

-RRB-

naturally

rich

in

polyphenols

Bars

show

the

mean

SD

of

eight

values

corresponding

to

the

same

number

of

experiments

being

each

value

the

mean

of

duplicate

assays

P

P

and

P

versus

the

control

group

Lymphoproliferation

in

response

to

both

mitogens

Con

A

and

LPS

was

expressed

as

of

stimulation

with

respect

to

spontaneous

proliferation

the

latter

considered

as

Spontaneous

lymphoproliferation

-LRB-

in

counts

per

min

-RRB-

Control

B

C

D

E

Cellular

redox

state

The

content

of

oxidized

glutathione

-LRB-

GSSG

-RRB-

and

the

GSSG/GSH

ratio

the

levels

of

TNFα

CAT

activity

and

MDA

levels

in

peritoneal

leukocytes

are

shown

in

Fig.

All

cereal

fractions

studied

decreased

significantly

the

GSSG

levels

-LRB-

P

-RRB-

compared

to

those

of

control

mice

-LRB-

Fig.

-RRB-

The

GSSG/GSH

ratio

-LRB-

Fig.

-RRB-

was

significantly

decreased

after

supplementation

with

any

of

the

cereals

B

-LRB-

P

-RRB-

D

-LRB-

P

-RRB-

or

E

-LRB-

P

-RRB-

compared

with

that

of

non-supplemented

mice

Supplementation

for

weeks

with

any

of

the

cereals

did

not

cause

changes

in

the

GSH

levels

of

peritoneal

leukocytes

with

respect

to

the

values

of

controls

-LRB-

-RRB-

The

secretion

of

TNFα

decreased

in

leukocytes

from

mice

supplemented

with

cereal

C

-LRB-

P

-RRB-

D

-LRB-

P

-RRB-

and

E

-LRB-

P

-RRB-

in

comparison

with

that

of

non-supplemented

mice

-LRB-

Fig.

-RRB-

The

CAT

activity

of

peritoneal

leukocytes

from

mice

supplemented

with

cereal

C

-LRB-

P

-RRB-

D

-LRB-

P

-RRB-

and

E

-LRB-

P

-RRB-

was

higher

than

the

activity

of

this

antioxidant

enzyme

in

control

mice

-LRB-

Fig.

-RRB-

Finally

the

lipid

oxidative

damage

in

peritoneal

leukocytes

evaluated

as

MDA

levels

-LRB-

Fig.

-RRB-

was

lower

-LRB-

P

-RRB-

in

mice

supplemented

for

weeks

with

cereals

than

in

the

control

mice

Fig.

-LRB-

a

-RRB-

Oxidized

glutathione

-LRB-

GSSG

-RRB-

content

-LRB-

b

-RRB-

GSSG/GSH

ratio

-LRB-

c

-RRB-

tumor

necrosis

factor

-LRB-

TNFα

-RRB-

levels

in

LPS-stimulated

-LRB-

μg/ml

-RRB-

culture

supernatants

-LRB-

d

-RRB-

catalase

-LRB-

CAT

-RRB-

activity

and

-LRB-

e

-RRB-

malondialdehyde

-LRB-

MDA

-RRB-

levels

in

peritoneal

leukocytes

from

adult

female

ICR

-LRB-

-RRB-

mice

supplemented

for

weeks

with

cereal

fractions

-LRB-

B

C

D

or

E

-RRB-

naturally

rich

in

polyphenols

Bars

show

the

mean

SD

of

eight

values

corresponding

to

the

same

number

of

experiments

being

each

value

the

mean

of

duplicate

assays

P

P

and

P

versus

the

control

group

Discussion

It

is

widely

accepted

that

antioxidant

compounds

may

help

to

protect

against

oxidative

stress

with

resulting

health

promotion

and

disease

prevention

-LSB-

-RSB-

Moreover

an

adequate

intake

of

these

compounds

can

enhance

certain

aspects

of

immune

function

which

is

related

to

health

-LSB-

-RSB-

Thus

it

is

becoming

clear

that

diet

supplementation

with

appropriate

amounts

of

antioxidants

enhances

the

immune

function

and

improves

the

redox

state

of

unhealthy

subjects

-LSB-

-RSB-

However

whether

antioxidant

supplementation

has

these

effects

in

healthy

subjects

still

remains

unknown

In

the

present

study

the

cereal

fractions

used

were

able

to

improve

several

aspects

of

immune

function

Thus

important

events

of

innate

immune

response

i.e.

migration

capacity

-LRB-

chemotaxis

-RRB-

and

microbicidal

activity

-LRB-

intracellular

superoxide

anion

levels

-RRB-

appear

to

be

effectively

affected

by

the

supplementation

with

cereals

naturally

rich

in

polyphenols

Akbay

et

al.

-LSB-

-RSB-

showed

that

the

three

flavonoid

glycosides

from

Urtica

dioica

L.

and

increased

in

vitro

the

chemotaxis

and

the

intracellular

killing

activity

of

neutrophils

using

methods

similar

to

those

of

the

present

work

In

our

study

the

fraction

E

is

only

able

to

enhance

macrophage

chemotaxis

whereas

all

the

cereal

fractions

were

able

to

show

a

strong

stimulatory

effect

on

this

function

in

lymphocytes

This

fact

suggests

that

the

polyphenols

present

in

cereal

fractions

could

exert

a

cell-dependent

effect

on

migration

capacity

Since

a

higher

NF-κB

expression

is

linked

to

an

increased

production

of

migratory

inhibitory

factor

-LRB-

MIF

-RRB-

thus

being

responsible

for

a

decreased

migration

capacity

-LSB-

-RSB-

and

several

polyphenols

are

able

to

inhibit

NF-κB

activation

-LSB-

-RSB-

it

seems

plausible

that

some

of

the

polyphenols

present

in

the

cereals

used

in

our

study

could

enhance

the

chemotaxis

capacity

through

their

action

on

NF-κB

activation

All

cereal

fractions

used

in

the

present

work

with

the

exception

of

D

have

been

shown

to

increase

the

microbicidal

activity

of

peritoneal

leukocytes

Since

p-hydroxybenzoic

acid

is

not

present

in

fraction

D

this

compound

could

be

responsible

for

that

effect

However

to

date

there

is

no

evidence

of

a

microbicidal

activity

increase

following

p-hydroxybenzoic

acid

administration

Lymphoproliferation

is

a

crucial

event

of

the

acquired

immune

response

Several

studies

have

demonstrated

a

strong

stimulatory

effect

on

this

capacity

and

on

the

cytokine

well-known

as

enhancer

of

proliferative

response

namely

the

after

antioxidant

supplementation

Thus

Bub

et

al.

-LSB-

-RSB-

showed

an

increase

in

both

functions

in

peripheral

blood

mononuclear

cells

-LRB-

PBMC

-RRB-

from

healthy

subjects

after

diet

supplementation

with

fruit

juices

rich

in

polyphenolic

compounds

In

ethanol-fed

mice

Cheshier

et

al.

-LSB-

-RSB-

demonstrated

an

increased

production

by

mitogen-stimulated

splenocytes

after

in

vitro

administration

of

Pycnogenol

a

commercial

combination

of

phenolic

compounds

from

pine

bark

To

date

no

studies

on

the

effect

of

the

isolated

polyphenolic

compounds

of

the

cereals

used

in

the

present

research

on

these

functions

have

been

carried

out

Glutathione

-LRB-

GSH

-RRB-

plays

important

roles

in

antioxidant

defence

nutrient

metabolism

and

regulation

of

many

cellular

events

and

its

deficiency

contributes

to

the

oxidative

stress-related

pathogenesis

of

several

chronic

diseases

-LSB-

-RSB-

Both

the

oxidized

glutathione

-LRB-

GSSG

-RRB-

content

and

the

GSSG/GSH

ratio

the

latter

being

considered

a

sensitive

marker

of

cellular

oxidative

stress

-LSB-

-RSB-

were

significantly

decreased

after

supplementation

with

all

the

cereal

fractions

although

fraction

C

only

showed

a

tendency

to

diminish

the

GSSG/GSH

ratio

However

the

GSH

content

of

peritoneal

leukocytes

was

not

changed

after

cereal-based

supplementation

suggesting

that

the

lower

GSSG/GSH

ratio

observed

is

primarily

due

to

diminutions

of

the

GSSG

content

Rosenblat

et

al.

-LSB-

-RSB-

demonstrated

that

isoflavonoid

glabridin

-LRB-

μg/kg/day

-RRB-

supplementation

was

able

to

reduce

the

GSSG

content

in

murine

peritoneal

macrophages

However

the

same

authors

also

observed

increased

GSH

levels

in

these

immune

cells

Tumor

necrosis

factor

is

one

of

the

most

common

proinflammatory

cytokines

produced

predominantly

by

macrophages

that

has

been

reported

to

increase

in

many

oxidative

stress-related

chronic

diseases

-LSB-

-RSB-

Our

study

showed

that

each

of

the

cereal

fractions

except

B

was

able

to

decrease

the

secretion

of

TNFα

by

peritoneal

leukocytes

In

agreement

with

Wang

and

Mazza

-LSB-

-RSB-

catechin

at

a

concentration

of

μM

inhibited

the

TNFα

production

of

LPS/IFNγ-activated

RAW

macrophages

However

our

results

suggest

that

p-coumaric

acid

could

also

have

an

important

anti-inflammatory

role

because

this

common

phenolic

acid

is

present

in

each

cereal

fraction

except

the

B.

Moreover

in

rats

with

endotoxemia

two

different

doses

-LRB-

mg/kg

i.p.

-RRB-

of

a

water

extract

of

propolis

a

natural

product

rich

in

p-coumaric

acid

were

able

to

inhibit

TNFα

production

in

plasma

-LSB-

-RSB-

Nevertheless

other

authors

did

not

observe

any

effect

on

TNFα

production

in

whole

blood

cultures

in

the

presence

of

phenolic

compounds

like

p-coumaric

acid

at

the

concentrations

of

M

-LSB-

-RSB-

The

most

probable

mechanism

triggered

by

phenolic

compounds

for

blocking

LPS-induced

production

of

TNFα

by

macrophages

could

be

the

inhibition

of

NF-kB

activation

-LSB-

-RSB-

Catalase

plays

a

fundamental

role

in

protecting

cells

against

oxidative

damage

-LSB-

-RSB-

Decreased

CAT

activity

may

compromise

the

overall

enzymatic

defence

system

In

the

present

study

each

of

the

cereal

fractions

increased

CAT

activity

in

peritoneal

leukocytes

although

fraction

B

only

showed

a

tendency

to

increase

this

enzymatic

activity

In

other

study

the

renal

CAT

activity

was

significantly

increased

in

rats

fed

black

rice

one

of

the

most

important

cereals

rich

in

phenolic

acids

-LSB-

-RSB-

Khan

et

al.

-LSB-

-RSB-

showed

that

following

oral

feeding

-LRB-

w/v

-RRB-

for

days

of

polyphenols

from

green

tea

a

popular

beverage

rich

in

catechin

CAT

activity

significantly

increased

in

the

small

bowel

liver

and

lungs

of

mice

In

fact

fractions

C

and

D

-LRB-

the

fractions

rich

in

catechin

-RRB-

showed

the

greatest

effect

The

present

data

suggest

that

p-coumaric

acid

which

is

present

in

almost

all

the

cereal

fractions

-LRB-

C

D

and

E

-RRB-

might

also

have

a

relatively

important

role

in

modulating

the

CAT

activity

Indeed

an

oral

administration

of

p-coumaric

acid

-LRB-

mg/kg

-RRB-

for

five

days

ameliorated

the

CAT

activity

in

the

heart

from

rats

treated

with

doxorubicin

an

anticancer

antibiotic

-LSB-

-RSB-

This

potential

protective

action

of

p-coumaric

acid

is

likely

to

be

due

to

its

free

radical

scavenging

power

ROS

generation

is

able

to

promote

oxidative

damage

to

fatty

acids

present

in

biological

membranes

via

an

autocatalytic

process

known

as

lipid

peroxidation

Malondialdehyde

is

one

of

the

end-products

of

this

process

-LSB-

-RSB-

which

is

related

to

the

onset

of

chronic

diseases

-LSB-

-RSB-

In

the

present

work

all

the

cereal

fractions

strongly

decreased

the

MDA

levels

in

peritoneal

leukocytes

It

has

been

observed

that

highly

reactive

and

destructive

hydroxyl

radicals

are

effectively

scavenged

by

oral

administration

of

p-coumaric

acid

-LRB-

mg/day

-RRB-

for

days

in

rats

-LSB-

-RSB-

which

might

decrease

the

potential

damage

to

biological

membranes

by

lowering

MDA

production

Several

authors

have

shown

that

rutin

quercetin

or

catechin

which

are

some

of

the

flavonoids

present

in

the

cereals

used

in

the

present

work

are

able

to

decrease

lipid

oxidative

damage

-LSB-

-RSB-

The

same

happens

with

ferulic

acid

the

most

common

phenolic

acid

in

cereal

cell

walls

which

inhibit

peroxidation

-LSB-

-RSB-

Red

wine

polyphenols

together

with

ascorbic

acid

inhibit

lipid

peroxidation

in

human

muscle

tissue

-LSB-

-RSB-

The

same

authors

demonstrated

that

in

the

presence

of

catechin

ascorbic

acid

at

pH

works

in

a

sinergystic

way

preventing

lipid

oxidative

damage

and

β-carotene

cooxidation

This

fact

might

explain

the

central

role

that

consuming

natural

products

rich

in

polyphenolic

antioxidants

like

cereals

has

in

promoting

health

and

preventing

the

onset

of

chronic

diseases

The

present

data

demonstrate

the

importance

of

polyphenol-rich

cereal

consumption

because

not

only

has

shown

to

be

very

effective

for

modulating

several

parameters

of

immune

function

but

also

is

able

to

improve

to

a

large

extent

the

redox

state

of

immune

cells

from

mice

More

importantly

we

have

demonstrated

that

cereal-based

antioxidant

supplementation

can

exert

beneficial

effects

in

healthy

mice

and

thus

we

suggest

that

normal

healthy

populations

may

benefit

from

this

supplementation

Indeed

a

recent

study

has

provided

evidence

for

potential

protective

effects

of

moderate

consumption

of

polyphenol-rich

red

wine

in

healthy

subjects

-LSB-

-RSB-

It

has

been

reported

that

subjects

susceptible

to

infections

or

suffering

from

chronic

inflammatory

processes

showing

an

impairment

of

function

and

redox

state

of

immune

cells

likely

to

benefit

from

antioxidant

administration

-LSB-

-RSB-

However

it

is

difficult

to

know

whether

there

is

changes

or

not

in

the

healthy

mice

from

the

present

study

may

be

a

reliable

predictor

for

the

nutrient-immune

requirements

in

disease-associated

unhealthy

mice

To

confirm

this

possibility

we

should

know

the

optimal

combination

and

quantities

of

nutrients

the

potential

interactions

that

might

occur

between

these

nutrients

and

the

most

appropiate

time

at

which

that

immunonutrition

should

be

provided

In

words

of

Berger

-LSB-

-RSB-

the

efficiency

of

supplementation

is

a

question

of

timing

Antioxidant

nutrients

can

not

cure

an

installed

disease

but

they

may

prevent

its

promotion

It

is

important

to

point

out

that

the

cereal

fractions

used

by

us

contain

other

compounds

both

nutrients

and

non-nutrients

such

as

vitamins

minerals

or

fibre

and

therefore

we

can

not

rule

out

that

they

can

result

in

health

benefits

However

the

present

cereal

fractions

show

a

good

correlation

coefficient

between

total

antioxidant

capacity

and

content

of

phenolic

compounds

thus

suggesting

that

polyphenols

are

largely

responsible

for

the

antioxidant

activity

of

the

different

cereal

fractions

which

ultimately

may

help

to

protect

from

oxidative

stress

and

consequently

can

decrease

the

risk

of

many

potential

diseases

Nevertheless

it

is

important

to

take

into

account

some

issues

First

the

doses

used

play

a

crucial

role

since

it

is

known

that

excessive

amounts

of

antioxidant

compounds

can

impair

the

immune

function

-LSB-

-RSB-

or

even

becomes

potentially

prooxidants

-LSB-

-RSB-

Yet

we

have

not

used

megadoses

but

nutritional

doses

of

polyphenols

Second

the

present

recommendation

is

not

a

single

polyphenol-based

supplementation

to

modulate

the

antioxidant

status

or

the

immune

system

but

it

is

the

combination

of

different

substances

as

the

best

way

to

provide

protective

action

-LSB-

-RSB-

Therefore

the

cereals

used

by

us

contain

a

complex

natural

mixture

of

polyphenols

of

different

size

polarity

and

solubility

Third

we

can

speculate

that

one

of

the

reasons

for

the

protective

effects

of

cereals

could

be

that

the

metabolism

of

polyphenols

carried

out

by

the

gut

microflora

-LSB-

-RSB-

is

able

to

provide

immune

protection

beyond

the

gut

Fourth

it

should

not

be

ruled

out

the

possibility

that

synergistic

and/or

additive

effects

occur

Several

studies

have

shown

that

polyphenols

can

work

synergistically

with

other

antioxidant

compounds

-LSB-

-RSB-

Thus

it

is

of

great

physiological

importance

that

many

polyphenols

of

different

solubility

will

interact

in

the

body

cells

and

compartments

with

antioxidants

recharging

adjacent

antioxidants

in

an

integrated

manner

-LSB-

-RSB-

Finally

we

consider

the

present

nutritional

intervention

in

animals

as

preventive

but

not

therapeutic

since

it

is

able

to

maintain

the

normal

functional

and

redox

status

of

the

body

cells

from

a

general

population

of

mice

Therefore

we

do

not

deliver

a

cereal-based

antioxidant

supplement

to

unhealthy

subjects

i.e.

in

conditions

caused

by

excessive

free

radicals

generation

In

conclusion

we

strongly

believe

that

polyphenols

naturally

present

in

cereals

may

have

important

implications

for

health

preservation

by

acting

al

least

partly

as

modulators

of

immune

function

and

redox

state

Although

we

emphasize

the

importance

of

further

studies

on

this

subject

we

suggest

the

use

of

this

type

of

supplementation

for

the

general

population

since

prevention

is

a

much

more

effective

approach

that

treatment

of

disease

O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

leukocyte functions
leukocyte redox state
peritoneal leukocytes
polyphenols
health promotion


Long-term cognitive and behavioral consequences of neonatal encephalopathy following perinatal asphyxia : a review Neonatal encephalopathy -LRB- NE -RRB- following perinatal asphyxia -LRB- PA -RRB- is considered an important cause of later neurodevelopmental impairment in infants born at term . This review discusses long-term consequences for general cognitive functioning , educational achievement , neuropsychological functioning and behavior . In all areas reviewed , the outcome of children with mild NE is consistently positive and the outcome of children with severe NE consistently negative . However , children with moderate NE form a more heterogeneous group with respect to outcome . On average , intelligence scores are below those of children with mild NE and age-matched peers , but within the normal range . With respect to educational achievement , difficulties have been found in the domains reading , spelling and arithmetic/mathematics . So far , studies of neuropsychological functioning have yielded ambiguous results in children with moderate NE . A few studies suggest elevated rates of hyperactivity in children with moderate NE and autism in children with moderate and severe NE . Conclusion : Behavioral monitoring is required for all children with NE . In addition , systematic , detailed neuropsychological examination is needed especially for children with moderate NE . Introduction It has been widely accepted that severe NE following PA can lead to serious motor disabilities , mental retardation and seizure disorders -LSB- 59 -RSB- . Traditionally , research in this area has focused on developmental consequences at a very young age . In addition , the effects of NE have often been treated as an all-or-nothing phenomenon ; patients have been believed to show a completely normal development or become severely disabled . So far , few studies have looked at milder consequences of NE at school age . From perinatal asphyxia to neonatal encephalopathy PA can be defined as impaired respiratory gas exchange accompanied by the development of acidosis -LSB- 30 -RSB- . The pathway of PA can have its onset in the antepartum , intrapartum or postpartum period . Estimates of the incidence of PA vary from 1 to 8 per 1,000 live births -LSB- 4 -RSB- . This wide range can be largely attributed to problems in selecting indicators to identify children with PA. . As a consequence , researchers have used many different sets of inclusion criteria when studying the effects of PA. . For this reason , it is difficult to compare results regarding developmental outcome of infants with PA across studies . In the past , single-marker definitions were commonly used to diagnose infants with PA -LRB- e.g. , low Apgar score -RRB- . However , these single markers have relatively low sensitivity and specificity for accurate diagnosis -LSB- 54 -RSB- . Moreover , they lack predictive power -LSB- 3 , 20 , 35 -- 37 , 40 , 51 -RSB- . Therefore , a shift has been made towards multiple-marker definitions based on combinations of indicators -LSB- 16 , 35 -RSB- . Among the perinatal indicators are fetal distress -LRB- abnormal heart rate or meconium-stained amniotic fluid -RRB- , delay in onset of spontaneous respiration , low Apgar score -LRB- < 6 or 7 at 5 min -RRB- , need for resuscitation and/or ventilation , and metabolic acidosis -LRB- cord pH < 7.0 or 7.1 and base deficit > 12 mmol/l -RRB- . Postnatal indicators of PA include NE , multi-organ failure and abnormal findings on brain imaging . Of the above , the concept of NE is generally accepted as an essential criterion for the diagnosis of PA -LSB- 4 , 30 -RSB- . NE has been defined as a clinical syndrome of disturbed neurological function in the earliest days of life in term infants , manifested by difficulty with initiating and maintaining respiration , depression of tone and reflexes , subnormal level of consciousness and often by seizures -LSB- 30 -RSB- . Recent studies of the protective effects of head cooling and systemic hypothermia after NE following PA used a combination of clinical and physiological criteria of PA -LRB- including a pH of 7.0 or less or a base deficit of 16 mmol per liter or more -RRB- and a neurological examination to determine the severity of NE -LSB- 22 , 52 -RSB- . These inclusion criteria are closely related to a set of criteria postulated by The American College of Obstetricians and Gynecologists -LRB- ACOG -RRB- . There has been extensive debate about the relationships between PA , NE and serious possible consequences such as CP -LSB- 25 -RSB- . Contrary to what was once believed , only a small part of all cases of CP are caused by intrapartum events -LSB- 10 -RSB- . Likewise , many cases of NE are not associated with a history of PA -LSB- 6 , 38 -RSB- . However , NE has to be present in the pathway from PA to subsequent CP -LSB- 25 -RSB- . The ACOG and the International Cerebral Palsy Task Force proposed a set of criteria to define an acute intrapartum hypoxic event that could subsequently lead to CP -LSB- 4 , 30 -RSB- . The presence of moderate or severe NE and metabolic acidosis -LRB- pH < 7 and base deficit ≥ 12 mmol/l -RRB- are considered to be essential markers for hypoxic ischemic injury . In addition , these criteria underline the importance of excluding other identifiable causes of NE before assuming asphyxia as the underlying etiology . Both in clinical practice and for scientific purposes , patients suffering from NE are often assigned an encephalopathy score . Several classification systems can be identified . Leviton and Nelson provide an overview of qualitative and quantitative grading schemes -LSB- 27 -RSB- . In addition , scoring systems were developed that are applicable in low-income settings . Moreover , a new grading system has gained interest in the literature -LSB- 19 , 33 -RSB- . The classification system of Sarnat and Sarnat developed in 1976 -LSB- 50 -RSB- is still widely used and the basis for most modern classification systems . In this review , the Sarnat system is used to grade NE because in most studies that were included this system it had been applied . According to the Sarnat system children are assigned a score of 1 , 2 or 3 -LRB- 1 = mild NE ; 2 = moderate NE ; 3 = severe NE -RRB- . Stage 1 lasts less than 24 h and is characterized by hyperalertness , uninhibited Moro and stretch reflexes , sympathetic effects and a normal EEG . Stage 2 is marked by obtundation , hypotonia , strong distal flexion and multifocal seizures . Infants with the most severe stage of NE -LRB- stage 3 -RRB- are stuporous and flaccid , and brain stem and autonomic functions are suppressed . In addition , the EEG shows abnormal patterns of brain activity . Grades of neonatal encephalopathy in relation to developmental outcome Encephalopathy scores have often been used to predict neurodevelopmental outcome -LSB- 43 , 44 , 46 -RSB- . Although strong relationships exist between the categories of NE and developmental outcome , diversity of outcome is reported within individual grades -LSB- 41 -RSB- . This diversity may be partly explained by individual researchers using different neurological signs for category inclusion -LSB- 27 -RSB- . The Sarnat grading system has helped to clarify the NE categories and improved their usefulness in studies of prognosis -LSB- 45 -RSB- . Most outcome studies have focused on neurological functioning and severe deficits in young children -LRB- < 4 years -RRB- . In general , very few children with mild encephalopathy show neurological impairments or have developed severe mental or motor retardation at preschool age . In contrast , children with severe encephalopathy nearly always die or develop severe impairments such as CP , mental retardation , epilepsy and in some cases sensorineural hearing loss or cortical visual impairment -LSB- 4 , 45 , 47 -RSB- . While dyskinetic CP or quadriplegia is more commonly seen following `` acute near total type of asphyxia '' with MRI abnormalities present in the deep grey nuclei and in the perirolandic cortex , milder forms of CP , such as spastic diplegia , are more common among those with `` subacute partial asphyxia '' with MRI abnormalities in the watershed areas . Hemiplegia can be seen following `` perinatal arterial stroke . '' Although this condition will present with NE and seizures , it is less commonly associated with PA -LSB- 13 , 34 -RSB- . Children who have suffered moderate encephalopathy seem to form a more heterogeneous group ; research shows varying rates of infant death and morbidity -LSB- 16 -RSB- . Need for long-term follow-up and focus on cognitive and behavioral outcomes Because the majority of studies have focused on detecting major developmental abnormalities at a very young age , still little is known about the milder difficulties children may experience in the long run . Recently , however , research has started to focus more on the subtle problems of children without major neurological deficits -LSB- 8 , 23 , 32 , 35 , 43 , 44 , 46 -RSB- . Knowledge of the outcome for this group of children is important because these subtle problems can affect later scholastic achievement and behavioral adjustment . Cognitive and behavioral difficulties can be expected because of the patterns of brain injury that have been associated with NE . The hippocampus and striatum are among the brain structures that can be affected -LSB- 7 , 21 , 31 , 42 , 56 -RSB- . These structures have been associated with specific cognitive functions such as memory and attention and hypothesized to play a role in the pathogenesis of ADHD , autism and schizophrenia -LSB- 14 -- 16 , 28 , 57 -RSB- . To gain insight into the long-term effects of NE on development , follow-up is required throughout the school-age period . Specific cognitive functions continue to develop throughout childhood . As it is impossible to examine a function that has not developed yet , short-term follow-up can not exclude minor cognitive and behavioral difficulties at school age . Moreover , subtle functional deficits usually do not become apparent until a child faces increasing demands to master complex abilities in school . The aim of this review was to discuss the results of previously published studies on the possible effects of NE on general cognitive functioning , educational achievement , neuropsychological functioning and behavior from infancy through adolescence . A literature search was performed using Web of Science . Studies were screened that had been published before October 2006 . Search terms were ' asphyxia neonatorum , ' ` neonatal encephalopathy , ' ` newborn encephalopathy , ' ` hypoxic-ischemic encephalopathy ' and ` perinatal asphyxia ' combined with the terms ` cognition/cognitive ' and ` behavior -LRB- - al -RRB- ' . Reference lists of retrieved articles were screened for additional relevant publications . Studies were selected that reported the developmental outcome of NE or HIE following PA of children until the age of 18 years . The presence of NE was required for selection . When no additional indicators of PA were used , the asphyxial etiology of the NE had to be assumed by the authors . Studies were excluded when NE was partially caused by other conditions than PA , for example -LSB- 17 , 34 -RSB- . Also , studies were excluded in which outcome was presented in preset categories -LRB- e.g. , normal versus abnormal -RRB- , in which several outcome measures were combined , for example -LSB- 11 , 18 , 26 -RSB- . In these cases it was not possible to judge whether a child had been assigned to the category ` abnormal ' because of cognitive or behavioral problems or because of other problems , e.g. , motor difficulties . A third reason for excluding studies from this review was the selection of children from a larger group of eligible patients . For example , some researchers selected only those children with abnormal findings on MRI in order to examine the correlation with outcome -LSB- 61 -RSB- . In this review , outcome is reported according to grade of NE -LRB- mild , moderate or severe -RRB- . Most authors of the included studies used the classification system of Sarnat when determining the severity of the NE . However , in three cases the authors significantly deviated from this system -LSB- 32 , 47 , 53 -RSB- . In order to increase comparability across studies , the patients in these studies were reclassified according to Sarnat criteria . Articles were reviewed that reported general intellectual ability , levels of specific cognitive functions and/or behavioral problems . Details of the samples , inclusion criteria , outcome measures and results are described in Tables 1 , 2 , 3 , 4 , 5 , 6 , 7 and 8 . Table 1Griffiths Mental Development Scale * at 1 year CPNo CPCarli et al. -LRB- 2004 -RRB- -LSB- 12 -RSB- Moderate NE , N = 13Moderate NE , N = 27 • N = 10 : < − 2 SD • N = 4 : between − 2 SD and − 1 SD • N = 1 : between − 2 SD and − 1 SD • N = 23 : > − 1 SD • N = 1 : > − 1 SD • N = 1 : untestableGray et al. -LRB- 1993 -RRB- -LSB- 24 -RSB- Moderate NE , N = 10Moderate NE , N = 9 • N = 9 : < 55Mean = 118 -LRB- N = 1 not assessed -RRB- • N = 1 : 87Thompson et al. -LRB- 1997 -RRB- -LSB- 55 -RSB- No grades of NE recorded , N = 16No grades of NE recorded , N = 24Mean = 47 , range : 7 -- 101Mean = 16 , range 94 -- 128Barnett et al. -LRB- 2004 -RRB- -LSB- 9 -RSB- No grades of NE recorded , N = 59Mean = 100.34 , SD = 15.05 , range : 55 -- 130Rutherford et al. -LRB- 1996 -RRB- -LSB- 49 -RSB- Moderate NE , N = 8Mild NE , N = 4All below normal -LRB- cut-off point not reported -RRB- All normalSevere NE , N = 1Below normal * Griffiths mean of standardization sample = 100.18 , SD = 12.76 Table 2IQ at 2 -- 6 years -LRB- no CP -RRB- Age -LRB- years -RRB- Mild NEModerate NESevere NEBarnett et al. -LRB- 2004 -RRB- b -LSB- 9 -RSB- 2N = 45 , f Griffiths : mean = 103.53 , SD = 12.33 -LRB- range : 81 -- 140 -RRB- Robertson & Finer -LRB- 1985 -RRB- a -LSB- 43 -RSB- 3.5 N = 66 , Stanford-Binet : mean IQ = 101.5 , SD = 14.0 N = 94 -LRB- 21.3 % handicapped -RRB- Stanford-Binet2 : mean IQ = 92.3 , SD = 23.2 N = 7 , Stanford-Binet2 : mean IQ = 37.1 , SD = 26.7 Shankaran -LRB- 1991 -RRB- cb -LSB- 53 -RSB- 5N = 14 , f McCarthy : 64 % : > − 1 SD , 86 % : > − 2 SDBarnett et al. -LRB- 2002 -RRB- bd -LSB- 8 -RSB- 5.5 -- 6.5 N = 20 , WPPSI-R : mean IQ = 109.7 , SD = 14.6 -LRB- N = 1 : 76 , rest : > 90 -RRB- N = 12 , WPPSI-R : -LRB- N = 1 : untestable -RRB- N = 11 : mean IQ = 106.2 , SD = 11.8 -LRB- N = 1 : 84 , rest : > 94 -RRB- Barnett et al. -LRB- 2004 -RRB- b -LSB- 9 -RSB- 5 -- 6N = 53 , f WPPSI-R 85 % : > − 1 SD , mean IQ = 101.98 , SD = 16.06 , range : 69 -- 139Robertson & Finer -LRB- 1988 -RRB- e -LSB- 44 -RSB- 5.5 N = 56 , Stanford-Binet : mean IQ = 106 , SD = 12N = 71 , Stanford-Binet : mean IQ = 99 , SD = 18 -RRB- Griffiths Mental Developmental Scales : mean of standardization sample = 100.18 , SD = 12.76 ; Stanford-Binet Intelligence Scales : mean of standardization sample = 100 , SD = 16 ; McCarthy Scales of Children 's Abilities : mean of standardization sample = 100 , SD = 16 ; Wechsler Preschool and Primary Scale of Intelligence-revised : mean of standardization sample = 100 , SD = 15 . aAll groups are significantly different from the other groups ; p = 0.001 . bNo significance tests performed . cPatients were reclassified by author -LRB- MH -RRB- according to the classification system of Sarnat -LSB- 50 -RSB- . dIn the original article , results are presented for individual cases . Group means and standard deviations have been calculated by one of the authors -LRB- MH -RRB- . eMild NE not significantly below comparison group ; moderate NE significantly below mild NE and comparison group . fCombined group of mild , moderate and severe NETable 3IQ at 7 -- 9 years -LRB- no CP -RRB- Age -LRB- years -RRB- Mild NEModerate NESevere NEMarlow -LRB- 2005 -RRB- b -LSB- 32 -RSB- 7N = 65 , BAS-II : Group 1 -LRB- N = 34 -RRB- : GCS = 112.3 , SD = 11.2 Group 2d -LRB- N = 31 -RRB- : GCS = 102.7 , SD = 13.2 Robertson et al. -LRB- 1989 -RRB- a -LSB- 46 -RSB- 8N = 56 , WISC-R : mean IQ = 106 , SD = 13N = 66 , WISC-R : mean IQ = 102 , SD = 175 , WISC-R : mean IQ = 36 , SD = 7Robertson -LRB- 1997 -RRB- c -LSB- 47 -RSB- 9N = 64 , WISC-R : mean IQ = 100 , SD = 14WISC-R : Wechsler Intelligence Scale for Children-revised : mean of standardization sample = 100 , SD = 15 ; BAS-II : British Ability Scales , GCS : General Cognitive Score : mean of standardization sample = 100 , SD = 15 . aIQ of mild NE is not significantly lower than IQ of comparison group . IQ of moderate and severe NE is significantly lower than IQ of mild NE and comparison group -LRB- p < 0.001 -RRB- . bGSC of group 1 is not significantly lower than GSC of comparison group . GCS of group 2 is significantly lower than GCS of comparison group -LRB- p < 0.01 -RRB- . cMean IQ is significantly lower than IQ of comparison group . dMarlow refers to this group as ` severe NE . ' This group is reclassified by one fo the authors -LRB- MH -RRB- according to the classification system of Sarnat -LSB- 50 -RSB- Table 4Scholastic abilities at 7 -- 13 years -LRB- no CP -RRB- Mild NEModerate NESevere NEReadingRobertson & Finer -LRB- 1989 -RRB- -LSB- 46 -RSB- 13 % > 1 grade level below expected level for age35 % > 1 grade level below expected level for agea100 % > 1 grade level below expected level for agebRobertson -LRB- 1997 -RRB- -LSB- 47 -RSB- 41 % > 1 grade level below expected level for agedMarlow et al. -LRB- 2005 -RRB- -LSB- 32 -RSB- Group 1 : < control groupcGroup 2f : < control groupdMoster et al. -LRB- 2002 -RRB- -LSB- 35 -RSB- NE < control groupdNE < control groupdNE < control groupdWritingMarlow et al. -LRB- 2005 -RRB- -LSB- 32 -RSB- Group 1 : = control groupGroup 2f : < control groupeMoster et al. -LRB- 2002 -RRB- -LSB- 35 -RSB- NE = control groupNE = control groupNE = control groupSpellingRobertson & Finer -LRB- 1989 -RRB- -LSB- 46 -RSB- 2 % > 1 grade level below expected level for age18 % > 1 grade level below expected level for agea100 % > 1 grade level below expected level for agebMarlow et al. -LRB- 2005 -RRB- -LSB- 32 -RSB- Group 1 : < control groupcGroup 2f : < control groupdMoster et al. -LRB- 2002 -RRB- -LSB- 35 -RSB- NE = control groupNE = control groupNE = control groupMathRobertson & Finer -LRB- 1989 -RRB- -LSB- 46 -RSB- 16 % > 1 grade level below expected level for age20 % > 1 grade level below expected level for agea100 % > 1 grade level below expected level for agebRobertson -LRB- 1997 -RRB- -LSB- 47 -RSB- 39 % > 1 grade level below expected level for agedMarlow et al. -LRB- 2005 -RRB- -LSB- 32 -RSB- Group 1 : = control groupGroup 2f : < control groupdMoster et al. -LRB- 2002 -RRB- -LSB- 35 -RSB- NE < control groupcNE < control groupcNE < control groupcRobertson & Finer -LRB- 1989 -RRB- -LSB- 46 -RSB- : age 8 years ; Marlow et al. -LRB- 2005 -RRB- -LSB- 32 -RSB- : age 7 years ; Moster et al. -LRB- 2002 -RRB- -LSB- 35 -RSB- : age 8 -- 13 yearsaSignificant difference with mild NE and comparison group , p < 0.01 ; bstatistical analyses are not given because of small group and low scores ; csignificant difference with comparison group , p < 0.05 ; dsignificant difference with comparison group , p < 0.001 ; esignificant difference with comparison group , p < 0.01 ; fMarlow refers to this group as ` severe NE ' . This group is reclassified by one of the authors -LRB- MH -RRB- according to the classification system of Sarnat -LSB- 50 -RSB- Table 5Neuropsychological functions -LSB- 43 , 44 , 46 -RSB- Tests3 .5 years5 .5 years8 yearsReceptive vocabularyMild NE : in the mean rangeMild NE = control groupMild NE = control groupModerate NE < mild NEaModerate NE < mild NE + control groupbModerate NE < mild NE + control groupaSevere NE < mild + moderate NEaSevere NE : 100 % < − 1 SD cSevere NE < mild NE + control group cVisual - motor integrationMild NE : in the mean rangeMild NE = control groupMild NE = control groupModerate NE < mild NEaModerate NE < mild NE + control groupaModerate NE < mild NE + control groupa Severe NE < mild NE + control group cSevere NE < mild + moderate NEaSevere NE : 100 % < − 1 SD cReceptive vocabulary : Peabody Picture Vocabulary Test , visual-motor integration : Developmental Test of Visual-Motor Integration . ap < 0.001 ; bp < 0.01 ; cstatistical analyses are not given because of small group and low scoresTable 6Neuropsychological functions -LRB- NEPSY -RRB- : moderate NE compared to control group -LSB- 32 -RSB- Age : 7 yearsModerate NE group 1Moderate NE group 2 * Attention and executiveGroup 1 = control group -LRB- p = 0.08 -RRB- Group 2 < control group -LRB- p < 0.01 -RRB- LanguageGroup 1 < control group -LRB- p = 0.01 -RRB- Group 2 < control group -LRB- p < 0.01 -RRB- SensorimotorGroup 1 < control group -LRB- p = 0.04 -RRB- Group 2 = control group -LRB- p = 0.10 -RRB- VisuospatialGroup 1 = control group -LRB- p = 0.14 -RRB- Group 2 < control group -LRB- p = 0.02 -RRB- Memory and learningGroup 1 = control group -LRB- p = 0.61 -RRB- Group 2 < control group -LRB- p < 0.01 -RRB- * Marlow refers to this group as ` severe NE . ' This group is reclassified by one of the authors -LRB- MH -RRB- according to the classification system of Sarnat -LSB- 50 -RSB- Table 7Verbal memory -LRB- Rey 's Auditory Verbal Learning Test -RRB- * -LSB- 31 -RSB- Age : 16 yearsModerate NETotal words learned trial 1 -- 5NE : 52.5 , SD = 8.7 Control group : 57 , SD = 5.8 Delayed recall -LRB- p = 0.034 -RRB- NE : 11.6 , SD = 2.2 Control group : 13.3 , SD = 1.6 Recognition -LRB- p = 0.011 -RRB- NE : 14.2 , SD = 1.0 Control group : 15 , SD = 0.0 * Raw scores -LRB- number correct -RRB- Table 8Verbal memory -LRB- Children 's auditory verbal learning test -RRB- * -LSB- 47 -RSB- Age : 9 yearsModerate NELevel of learningaNE : 98 , SD = 16Control group : 104 , SD = 15Immediate recallaNE : 96 , SD = 18Control group : 105 , SD = 15Delayed recallaNE : 95 , SD = 15Control group : 103 , SD = 16Recognition accuracyNE : 22 % delayedbControl group : 27 % delayedTotal intrusionsNE : 19 % delayedbControl group : 20 % delayedImmediate attentionNE : 97 , SD = 15Control group : 100 , SD = 15 * Mean of standardization sample = 100 , SD = 15 , ap < 0.01 ; bDelayed is below the 17th percentile of the standardization sample General cognitive development Infancy -LRB- 0 -- 24 months -RRB- Eight studies were identified that measured developmental levels between the ages of 12 and 24 months in children with NE -LSB- 2 , 9 , 12 , 24 , 39 , 49 , 50 , 55 -RSB- . Two of these were performed in low-income , non-western countries -LSB- 2 , 55 -RSB- . Standardized testing with the Griffiths Mental Development Scales showed that almost all children with moderate NE who had been diagnosed to have CP at the age of 12 months were severely developmentally delayed -LRB- Table 1 -RRB- . In these studies , the rates of CP among children with moderate NE varied between 23 % -LSB- 24 -RSB- and 82 % -LSB- 49 -RSB- . Children with moderate NE without CP at this age performed in the average range or above , although the range of scores varies widely across studies . In addition , other studies in which the Denver Developmental Screening Test or the Bayley Scales of Infant Development were used reported normal development in children with mild NE , none of whom had CP . Two to six years , without CP In all of the selected studies -LSB- 8 , 9 , 43 , 44 , 53 -RSB- , children with mild NE showed average general intellectual abilities that equaled those of a healthy comparison group -LRB- Table 2 -RRB- . Although children with moderate NE also performed in the average range , their abilities were significantly below those of children with mild NE and a reference group . The general intellectual abilities of children with severe NE were assessed in only one study . These children showed very low levels of functioning : a mean IQ of 37.1 was found -LSB- 43 -RSB- . In two studies , no grades of NE were recorded -LSB- 9 , 53 -RSB- , which hampers interpretation of these data . Rates of average or above-average functioning were 85 % and 64 % , respectively . Seven to nine years , without CP At school age , children with mild NE showed age-appropriate levels of general intellectual ability and performed as well as healthy comprison children on standardized testing -LSB- 32 , 46 , 47 -RSB- -LRB- Table 3 -RRB- . Although most children with moderate NE obtained significantly lower scores on intelligence measures than children with mild NE and comparison groups , their ability levels were in the average range . Educational achievement Relative strengths and weaknesses in cognitive functioning can become visible when children have to acquire specific scholastic abilities in school . A test of school readiness showed that 5.5-year-old children with mild and moderate NE were not significantly behind compared to comparison children -LSB- 24 -RSB- . However , children with moderate NE achieved lower scores in all domains , and 42 % of them scored more than 1 SD below the average of the standardization group . Children with severe NE showed low school readiness scores at the age of 5.5 years . Table 4 gives an overview of the results of four studies , which assessed reading , writing , spelling and mathematics/arithmetic in school-aged children -LSB- 32 , 35 , 46 , 47 -RSB- . Survivors of mild NE compared to a comparison group were not significantly more often more than one grade behind in reading , spelling and arithmetic . In these domains , all children with severe NE were more than one grade behind the expected level for their age . The group with moderate NE appeared to have distinct difficulties in reading , spelling and arithmetic/mathematics . Writing has been found to be less developed in only one subgroup of one of the studies . Neuropsychological functions Studies of specific neuropsychological functions yielded results that are similar to the findings on general intellectual ability and educational achievement -LRB- Tables 5 , 6 , 7 and 8 -RRB- . Children who suffered from mild NE tended to show normal receptive vocabulary and visual-motor integration at 3.5 , 5.5 and 8 years of age -LSB- 43 , 44 , 46 -RSB- . As is the case in other cognitive domains , the severe group was impaired , and their ability levels were below those of children with mild or moderate NE and comparison groups . The moderate group showed an ambiguous pattern of performance . Deficits were found in receptive vocabulary , language and visual-motor integration . In contrast , attention and executive functions , visuospatial ability , and memory and learning were normal in a subgroup of 7-year-old children -LSB- 29 -RSB- . Although memory and learning did not seem to be impaired in this subgroup , these children had difficulties in the sub-domains narrative memory and sentence repetition , compared to intact memory for faces , names , everyday events and intact orientation . In addition , auditory verbal learning and delayed recall was impaired in a group of 9 - and a group of 16-year-old children with moderate NE -LSB- 31 , 47 -RSB- . Tests of other auditory skills in the former showed intact auditory attention and concentration , language and binaural separation , in contrast to impaired binaural integration , and sequencing and labeling -LSB- 47 -RSB- . Behavioral problems So far , few researchers have addressed the issue of behavioral problems in children with a history of NE . Four studies were identified that met the selection criteria . In general , hyperactivity was more often present in children with moderate NE , but not in children with mild NE -LSB- 32 , 35 , 44 -RSB- . In addition , one study that used parent 's observations of their child 's behavior found more problems related to tractability , aggression , passivity and anxiety in a mixed group of children with NE compared to a control group -LSB- 35 -RSB- . No differences were found , however , related to attention , habituation and impulsivity . A recent study reported an unexpectedly high proportion of children with moderate and severe NE that developed an autism spectrum disorder -LSB- 5 -RSB- . However , the inclusion criteria differed considerably from those of Sarnat . Both groups can be classified as moderate NE when Sarnat 's criteria are applied . In addition , the authors explicitly did not exclude etiologies of NE other than PA. . Discussion and conclusion In the introduction section of this review , problems were mentioned with regard to the comparability of different outcome studies of NE . Even after careful selection on the basis of predetermined criteria , comparing studies remained difficult . The main reason for this was the way test results were presented . In many cases , outcome of the children was reported in two or more categories , e.g. , normal/mildly delayed/abnormal . One of the problems that arise due to such categorization , is the subjectivity involved in determining the cut-off points of the categories . Often , these cut-off points seemed to be chosen arbitrarily or not justified at all . Another issue that adds to this problem is the use of different tests of development , cognitive ability or behavioral problems . One can hardly compare , for example , the proportions of children with developmental delay in two studies when these are represented by a score below 85 on the Bayley Scales of Infant Development in one of the studies and by a score below 70 on the Griffiths Mental Developmental Scales in the other . To enable readers to compare studies , results of significance testing between subgroups of children with NE and comparison groups were added to the tables whenever available . Children with a history of NE who suffer from CP at the age of 12 months are often also severely developmentally delayed . Estimates vary between 77 % -LSB- 12 -RSB- and 90 % -LSB- 24 -RSB- . Of the children without CP , those with mild NE have an excellent prognosis . Their intelligence , educational achievement and neuropsychological functions are comparable to healthy peers at least until middle childhood . Children with severe NE appear to be impaired in every cognitive domain . They are less intelligent and perform worse at school and at neuropsychological tests than both healthy controls and children with mild or moderate NE . As was expected , the children with moderate NE seem to form the most heterogeneous group . In general , these children are significantly less intelligent than children with mild NE and healthy peers , but their scores are still in the average range . And although these children are equally ready to go to school as other children at the age of 5.5 years , when measured in middle childhood , they perform less well in the domains reading , spelling and mathematics . On the basis of these results , it is possible that children with moderate NE fall behind in school , but do not show clear deficits at standardized testing . Another issue is raised by the relatively broad range of performance of children with moderate NE . Calculating mean IQ scores masks individual differences , which are large in groups of children with moderate NE . Therefore , means do not describe this group well , and it would be more informative to look at the distribution of scores . The description of neuropsychological functions in this review is based on six studies , in which five different tests were used in five different samples of children with moderate NE . These studies do not provide sufficient and comparable data to draw reliable conclusions about patterns of neuropsychological strengths and difficulties . The heterogeneous nature of the group of children with moderate NE raises the question whether these children really form one group . Marlow et al. -LSB- 32 -RSB- divided a group of children with moderate NE according to the severity of the symptoms of the NE . The group with the least severe neonatal symptoms showed a few discrete neuropsychological difficulties , but was more intelligent and performed better in school than the group with the most severe neonatal symptoms . Marlow suggested a dose-response effect of hypoxia within this group with moderate NE . This idea of a continuum of casualty with regard to morbidity has already been proposed by Low -LSB- 29 -RSB- . He suggested a theory of a critical threshold of asphyxia beyond which brain damage occurs . Beyond this threshold , a spectrum of minor and major deficits would occur . So , although the classification system of Sarnat -LSB- 50 -RSB- appears to be very useful for the prediction of the outcome of children with mild or severe NE , in case of moderate NE this qualitative system lacks predictive power . Future research should therefore focus on developing a reliable , more gradual model of prediction . Another issue regarding prediction of outcome after NE also mainly concerns moderately affected children . It is still unclear whether NE has a general effect on intellectual abilities or leads to specific patterns of cognitive strengths and difficulties . This review shows that systematic , detailed research of neuropsychological function is still scarce . A few case reports , which are not included in this review , have shown specific impairments in episodic memory -LRB- memory for events -RRB- , with relatively intact semantic memory -LRB- memory for facts -RRB- -LSB- 21 , 58 -RSB- . However , these findings , which were based on small numbers of children , do not justify generalization to all children with NE and should therefore be replicated in larger , well-defined samples . The introduction section of this review referred to possible associations between brain areas that are vulnerable to perinatal hypoxia and psychopathology , such as ADHD and autism . Long-term behavioral outcome of survivors of NE has received very little attention compared to neurological outcome and general intellectual ability . Only a few studies looked at the behavioral consequences of NE . Those studies found elevated rates of hyperactivity and autism in children with moderate NE . Evidence of hyperactivity and impulsive behavior has also been found in animal studies of PA -LSB- 1 , 60 -RSB- . These findings suggest that , in addition to cognitive impairment , children with NE could be at risk of developing behavioral problems . Because of the clinical implications this would have , future research should include behavioral monitoring of all children with NE . Long-term follow-up of children with NE has been advocated before , including the optimal ages of assessment of specific functions -LSB- 48 -RSB- . Recommendations include assessment of general cognitive and adaptive behavioral functioning at preschool age , and , in some cases , school achievement tests and a neuropsychological screening at school age . The authors of this review would like to go one step further in recommending standard screening for behavioral problems in all children with NE , regardless of their level of cognitive functioning . In addition , detailed neuropsychological assessment is recommended especially in all children with moderate NE . From this review it is concluded that general intellectual , educational and neuropsychological outcomes are consistently positive for children with mild NE and negative for severely affected children . However , children with moderate NE form a more heterogeneous group with respect to outcome . On average , intelligence scores are below those of children with mild NE and age-matched peers , but within the normal range . Difficulties have been found in the domains reading , spelling and arithmetic/mathematics . So far , neuropsychological functioning of children with NE has received relatively little attention . The studies that were selected for this review have yielded ambiguous results in children with moderate NE . A few studies suggest elevated rates of hyperactivity in children with moderate NE and autism in children with moderate and severe NE . Therefore , behavioral monitoring is required for all children with NE . In addition , systematic , detailed neuropsychological examination is needed especially for children with moderate NE .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O

hypoxia, brain
hypoxia-ischemia, brain
neuropsychology