Genome-wide

microRNA

profiling

in

human

fetal

nervous

tissues

by

oligonucleotide

microarray

Objects

Our

objective

was

to

develop

an

oligonucleotide

DNA

microarray

-LRB-

OMA

-RRB-

for

genome-wide

microRNA

profiling

and

use

this

method

to

find

miRNAs

which

control

organic

development

especially

for

nervous

system

Introduction

To

date

microRNAs

-LRB-

miRNAs

-RRB-

a

ubiquitous

family

of

about

noncoding

regulatory

RNAs

have

been

identified

in

the

human

genome

and

published

in

a

database

-LRB-

-RRB-

-LSB-

-RSB-

Although

more

than

distinct

miRNAs

are

predicted

in

the

human

genome

little

is

known

about

their

biological

functions

especially

during

embryonic

development

MiRNAs

regulate

target

genes

at

the

post-transcriptional

level

and

play

important

roles

in

development

and

cell

lineage

decisions

However

in

vertebrates

neither

the

targets

of

miRNAs

nor

their

expression

profiles

during

development

are

well

understood

Some

miRNAs

are

specifically

expressed

in

individual

tissues

and

at

particular

developmental

stages

The

developmental

or

tissue-specific

patterns

of

miRNA

expression

observed

may

suggest

analogous

roles

in

regulating

human

development

or

cellular

differentiation

-LSB-

-RSB-

A

major

obstacle

in

the

study

of

miRNA

function

is

the

lack

of

methods

for

quantitative

expression

profiling

The

miRNA

microarray

method

a

powerful

tool

for

global

analysis

of

miRNA

expression

first

reported

by

Krichevsky

et

al.

-LSB-

-RSB-

is

based

on

a

membrane

array

spotted

with

specific

antisense

mature

miRNA

oligonucleotides

Since

then

other

types

of

oligonucleotide

array

methods

have

been

published

including

arrays

on

glass

slides

and

on

beads

-LSB-

-RSB-

In

this

study

a

robust

microarray-based

technique

was

established

and

used

to

identify

the

expression

of

miRNAs

in

human

fetal

organs

The

oligonucleotide

microarray

-LRB-

OMA

-RRB-

was

designed

according

to

the

method

of

Liu

-LSB-

-RSB-

with

modifications

We

used

miRNA

microarrays

to

study

the

profiles

of

miRNA

expression

in

nervous

tissue

samples

and

other

organic

samples

from

two

fetuses

Some

clusters

of

miRNA

families

were

co-expressed

providing

clues

about

the

maturation

processes

of

miRNAs

At

the

same

time

we

found

a

high

concordance

between

our

array

results

and

those

from

Northern

blots

The

microarray

described

here

offers

more

comprehensive

coverage

and

higher

throughput

than

other

methods

represents

a

powerful

tool

to

better

understand

miRNA

expression

profiles

in

human

tissues

and

provides

clues

to

the

mechanisms

for

regulating

protein

translation

Materials

and

methods

Samples

and

RNA

extraction

Two

fetal

abortuses

-LSB-

weeks

-LRB-

-RRB-

and

weeks

-LRB-

-RRB-

gestational

age

-RSB-

tissues

were

obtained

from

the

National

Infrastructure

Program

of

the

Chinese

Genetic

Resources

after

obtaining

informed

consent

The

tissues

were

liver

kidney

cerebrum

lung

and

heart

and

the

tissues

were

the

same

plus

ovary

spleen

hypothalamus

pancreas

and

cervical

thoracic

lumbar

and

sacral

spinal

cord

Total

RNA

was

isolated

using

TRIZOL

reagent

-LRB-

Invitrogen

-RRB-

according

to

the

manufacturer

's

standard

protocol

and

mRNA

was

purified

by

oligotex

-LRB-

Qiagen

-RRB-

Microarray

design

The

miRNA

oligonucleotide

microarray

design

was

based

on

that

of

spotted

OMAs

by

Liu

et

al.

-LSB-

-RSB-

with

significant

modifications

The

mature

miRNA

sequences

which

ranged

from

to

nt

were

too

short

so

two

probes

were

designed

for

several

miRNAs

one

containing

active

sites

that

detected

both

the

mature

and

the

precursor

forms

-LRB-

probe

a

-RRB-

while

the

other

did

not

contain

the

active

sites

located

in

the

precursor

-LRB-

probe

b

-RRB-

An

oligo

probe

was

designed

that

was

not

homologous

to

any

human

sequence

and

was

used

as

normalization

control

-LRB-

control

oligo

-RRB-

the

sequence

was

ATGTCATCTTGCGCGGCAGCTCGTCGACCGTGGCGAGAGT

GTCTCGTCGATCATC

All

oligos

were

nt

long

In

theory

the

mature

miRNA

expression

level

can

be

determined

by

subtracting

the

-LRB-

a

-RRB-

signal

from

the

-LRB-

b

-RRB-

signal

Nine

probes

for

nine

tRNA

genes

were

used

as

positive

controls

and

six

probes

for

six

types

of

miRNA

genes

from

Arabidopsis

thaliana

were

used

as

negative

controls

Microarray

fabrication

All

probes

were

dissolved

in

mM

phosphate

acid

buffer

-LRB-

pH

-RRB-

to

a

final

concentration

of

pmol/μl

Then

the

control

oligo

was

added

to

each

miRNA

probe

at

pmol/μl

The

OMAs

were

spotted

by

a

GeneMachine

OmniGrid

Microarrayer

in

a

pin

and

spot

configuration

for

each

subarray

with

triplicates

The

spotting

conditions

were

humidity

and

°C

After

spotting

slides

were

hydrated

overnight

in

saturated

salt

solution

and

then

crosslinked

with

UV

light

at

-LRB-

UVP

-RRB-

Sample

labeling

The

cDNA

was

labeled

during

first

strand

synthesis

by

using

a

fluorescence-tagged

-LRB-

-RRB-

random

octameric

primer

Briefly

μg

total

RNA

and

μg

random

primer

were

mixed

and

incubated

at

°C

for

min

then

first-strand

buffer

M

DTT

mM

unlabeled

dNTP

mix

RNA

inhibitor

and

Superscript

II

-LRB-

U/μl

-RRB-

were

added

and

incubated

at

°C

for

h

then

denatured

at

°C

for

min

NaOH

was

added

to

hydrolyze

RNA

to

stop

the

reaction

of

reverse

transcription

and

then

HEPES

was

added

to

neutralize

it

The

labeled

cDNA

was

purified

by

using

the

QIAquick

Nucleotide

Removal

Kit

-LRB-

Qiagene

-RRB-

Microarray

hybridization

The

labeled

cDNAs

and

oligonucleotides

complementary

to

the

control

probes

were

dissolved

in

Denhardt

hybridization

buffer

and

were

hybridized

with

the

miRNA

oligonucleotide

microarray

for

h

at

°C

Then

the

slides

were

washed

with

buffer

I

-LRB-

SDS

-RRB-

for

min

at

°C

buffer

II

-LRB-

SDS

-RRB-

for

min

at

°C

buffer

III

-LRB-

SSC

-RRB-

for

min

at

room

temperature

dipped

in

double-distilled

water

for

min

at

room

temperature

and

then

dried

The

slides

were

scanned

by

an

Agilent

scanner

-LRB-

-RRB-

at

and

nm

Statistical

analysis

The

images

were

split

into

two

and

channels

and

each

channel

was

imported

into

the

Imagene

Software

to

read

the

signal

value

The

signal

was

used

as

reference

for

the

spot

size

of

each

miRNA

oligo

on

the

slides

The

expression

level

of

each

miRNA

in

the

sample

labeled

by

was

normalized

by

a

median

method

according

to

the

signal

between

two

microarrays

So

the

signal

after

normalization

gave

the

expression

level

of

each

miRNA

Clustering

was

carried

out

by

Genespring

Software

according

to

intensity

Northern

blot

analysis

Forty

micrograms

of

total

RNA

from

each

sample

was

separated

on

acrylamide

denaturing

gels

-LRB-

M

urea

-RRB-

and

then

transferred

to

Hybond

N

membranes

-LRB-

Amersham

-RRB-

by

electrophoresis

for

Northern

blots

The

filters

were

crosslinked

with

mJ

of

UV

-LRB-

Bio-Rad

-RRB-

and

baked

at

°C

for

h

The

specific

oligo

probes

complementary

to

the

corresponding

miRNAs

were

labeled

at

the

end

by

using

polynucleotide

kinase

with

-LRB-

Amersham

-RRB-

The

sequence

list

of

probes

for

Northern

blots

were

and

-LSB-

-RSB-

on

the

membrane

served

as

loading

control

Prehybridization

and

hybridization

were

carried

out

using

ExpressHyb

Hybridization

Solution

-LRB-

Clontech

-RRB-

according

to

the

manufacturer

's

instructions

Membranes

were

washed

at

room

temperature

twice

with

SSC

SDS

and

twice

with

SSC

SDS

The

blots

were

exposed

on

Molecular

Dynamics

Phosphorimager

screens

and

signals

were

quantified

using

ImageQuant

-LRB-

Molecular

Dynamics

-RRB-

For

reuse

blots

were

stripped

by

boiling

in

SDS

for

min

and

scanned

on

Phosphorimager

screens

Blots

without

radioactive

signals

were

re-hybridized

and

re-used

up

to

six

times

without

influencing

the

quality

of

radioactive

signals

Results

MiRNA

oligo

nucleotide

microarray

construction

We

designed

oligonucleotide

probes

and

covered

human

miRNAs

from

and

from

publications

Nine

tRNAs

served

as

positive

controls

The

oligonucleotide

probes

were

nt

in

length

For

miRNAs

two

different

probes

were

designed

one

containing

the

active

site

and

the

other

specific

for

the

precursor

Using

this

strategy

we

were

able

to

analyze

the

miRNA

and

pre-miRNA

in

the

same

experiment

A

control

oligo

probe

which

was

not

homologous

to

any

human

gene

sequence

was

designed

for

spotting

and

quality

control

All

miRNA

probes

were

dissolved

in

phosphate

buffer

at

μmol/ml

and

the

control

probe

was

mixed

with

each

miRNA

probe

at

μmol/ml

The

microarray

slides

were

printed

on

an

Ominigride

machine

-LRB-

Gene

Machine

-RRB-

Each

oligo

probe

was

spotted

in

triplicate

To

develop

the

optimal

conditions

for

the

OMA

we

compared

mRNA

and

total

RNA

as

the

starting

sample

Two

micrograms

of

mRNA

and

μg

of

total

RNA

were

separately

labeled

during

the

reverse

transcription

reaction

and

hybridized

to

the

OMA

The

results

indicated

that

mRNA

had

a

relatively

low

signal

compared

to

the

total

RNA

suggesting

that

mRNA

had

little

influence

on

the

results

-LRB-

Fig.

-RRB-

so

total

RNA

could

be

used

for

the

microarray

experiment

The

microarray

hybridization

time

was

tested

at

and

h

The

hybridization

signals

at

h

were

considerably

higher

than

those

at

h

while

the

signals

at

h

were

only

slightly

higher

than

those

at

h

-LRB-

Fig.

-RRB-

To

test

the

OMA

sensitivity

quantities

of

total

RNA

ranging

from

to

μg

were

labeled

and

then

hybridized

to

the

OMA

-LRB-

Fig.

-RRB-

The

strongest

signal

was

obtained

at

μg

total

RNA

To

find

the

optimal

temperature

we

tested

hybridization

at

and

°C

The

signal

was

very

low

at

and

°C

-LRB-

Fig.

-RRB-

From

the

above

results

we

chose

μg

total

RNA

and

°C

hybridization

for

h

in

all

OMA

experiments

Fig.

oligonucleotiede

microarrary

construction

a

The

signal

at

hybridization

times

of

and

h

was

much

higher

than

that

at

h.

b

A

hybridization

temperature

of

°C

was

optimal

for

the

microarray

c

On

testing

quantities

of

total

RNA

from

to

μg

the

signal

was

highest

at

μg

d

Comparison

of

total

RNA

and

mRNA

as

the

starting

sample

The

highest

signal

pairs

were

chosen

and

signal

values

greater

than

were

shown

as

The

results

indicated

that

mRNA

had

little

influence

on

the

signal

so

total

RNA

could

be

used

as

the

starting

material

Distinct

miRNA

profiling

in

different

organs

during

human

development

Total

RNAs

of

samples

from

human

fetal

organs

at

and

were

labeled

during

reverse

transcription

The

labeled

cDNAs

hybridized

to

the

OMA

After

data

processing

we

found

that

of

the

miRNAs

were

expressed

in

human

fetal

organs

in

which

the

most

miRNAs

were

expressed

in

cerebrum

Distinct

miRNA

expression

patterns

were

found

in

different

organs

at

each

developmental

stage

-LRB-

Fig.

-RRB-

Some

miRNAs

were

seen

in

all

samples

from

both

embryonic

stages

-LRB-

e.g.

and

among

others

-RRB-

they

represent

a

type

of

restricted

spatial

and

temporal

expression

pattern

These

miRNAs

were

uniformly

expressed

over

time

which

suggests

more

general

roles

in

gene

regulation

Fig.

of

miRNome

expression

in

human

and

fetal

organs

a

Distinct

patterns

of

miRNA

expression

in

different

human

fetal

organs

-LRB-

abbreviation

listed

below

-RRB-

All

of

the

data

represent

the

average

of

at

least

three

replicates

from

the

same

organ

b

The

same

heart

sample

triplicate

chip

results

showed

good

reliability

Each

result

was

from

three

replicates

of

the

array

c

Three

stage-specific

miRNAs

and

were

expressed

in

and

liver

and

lung

The

bar

represents

the

microarray

signal

intensity

from

to

The

blue

line

on

the

left

indicates

that

the

four

miRNAs

were

clustered

by

cluster

software

analysis

C

cerebrum

Hr

heart

K

kidney

Li

liver

Lu

lung

O

ovary

P

pancreas

S

spleen

Vc

cervical

spinal

cord

Vl

lumbar

spinal

cord

Vs

sacral

spinal

cord

Vt

thoracic

spinal

cord

To

test

the

replicability

of

the

microarray

the

heart

sample

was

labeled

and

hybridized

in

triplicate

The

results

from

different

detection

batches

were

highly

reproducible

-LRB-

Fig.

-RRB-

Similar

miRNA

expression

patterns

were

seen

in

samples

from

developmentally

proximal

organs

indicating

that

a

miRNA

expression

profile

can

be

a

marker

of

developmental

stage

in

organs

originating

from

the

same

endodermal

layer

Three

different

miRNAs

and

were

predominantly

expressed

in

liver

and

lung

at

compared

with

the

same

organs

at

-LRB-

Fig.

-RRB-

As

an

indicator

of

miRNA

expression

level

the

color

of

samples

from

organs

was

brighter

than

those

from

organs

So

the

miRNA

expression

profile

can

be

used

as

a

biomarker

of

developmental

stage

CNS

specifically

and

higher

expressed

microRNAs

In

our

study

we

found

four

CNS

specifically

expressed

microRNAs

and

in

which

and

are

expressed

higher

in

cerebrum

than

in

At

the

same

time

and

cluster

members

were

found

expressed

relatively

higher

in

CNS

tissues

than

other

tissues

To

validate

the

reliability

of

the

microarray

data

we

selected

seven

CNS

specifically

and

higher

expressed

miRNAs

and

designed

specific

probes

for

Northern

blot

analysis

The

miRNAs

were

and

cluster

members

including

and

which

are

located

in

the

genomic

site

of

both

terminals

and

in

the

middle

of

the

cluster

We

found

good

consistency

between

the

Northern

blot

and

microarray

data

as

shown

by

the

results

from

the

selected

and

-LRB-

Fig.

-RRB-

Fig.

of

and

detected

by

Northern

blot

were

in

accord

with

the

signals

detected

by

microarray

The

abbreviations

are

as

in

the

legend

of

Fig.

The

and

are

shown

on

the

left

indicating

the

corresponding

expression

detected

by

Northern

blot

The

microarray

results

are

presented

numerically

indicated

by

Oligo

snRNA

detected

by

Northern

blot

was

used

as

detection

control

and

known

to

be

specifically

expressed

in

rat

nervous

system

are

also

only

expressed

in

the

human

fetal

nervous

system

found

by

microarray

and

Northern

blot

at

the

same

time

-LRB-

Fig.

-RRB-

Analysis

of

the

array

results

indicated

that

and

probably

originated

from

the

expressed

pre-mi-RNA

of

chromosome

and

may

play

key

roles

in

human

nervous

system

development

So

it

can

be

concluded

that

miRNA

microarrays

can

identify

different

pre-miRNA

origins

by

using

different

oligo

probes

Fig.

and

were

expressed

specifically

in

the

human

nervous

system

a

and

can

be

expressed

from

human

chromosomes

and

and

form

three

types

of

pre-miRNA

The

secondary

structures

of

pre-miRNAs

from

the

Sanger

miRNA

register

website

were

predicted

with

mfold

software

The

pre-miRNAs

can

be

excised

by

Dicer

into

the

same

mature

miRNAs

and

b

Northern

blot

results

probed

with

and

The

abbreviations

are

as

in

the

legend

of

Fig.

The

microarray

results

are

presented

numerically

indicated

by

Oligo

snRNA

detected

by

Northern

blot

was

used

as

detection

control

Members

of

the

cluster

which

contains

six

pre-miRNAs

within

about

kb

on

human

chromosome

share

the

same

expression

profile

-LSB-

-RSB-

The

microarray

and

Northern

blot

results

showed

that

these

six

miRNAs

had

the

same

expression

pattern

-LRB-

Fig.

-RRB-

suggesting

that

clustered

miRNAs

have

the

same

transcript

and

share

the

same

promoter

element

Fig.

cluster

miRNAs

a

The

three

known

human

miRNA

clusters

The

cluster

contains

six

pre-miRNAs

within

about

kb

on

chromosome

b

The

and

expression

profiles

detected

by

Northern

blot

-LRB-

the

name

of

the

miRNA

and

pre-miRNA

are

given

on

the

left

and

the

abbreviations

are

as

in

the

legend

of

Fig.

-RRB-

and

microarray

-LRB-

results

given

numerically

and

marked

as

and

-RRB-

snRNA

detected

by

Northern

blot

was

used

as

detection

control

Discussion

The

small

size

of

miRNAs

requires

more

sensitive

tools

for

quantitative

analysis

Although

the

optimized

method

of

RT-Q-PCR

can

indirectly

detect

mature

miRNAs

-LSB-

-RSB-

the

efficiency

of

detection

is

relatively

low

Currently

the

most

reliable

method

for

the

study

of

miRNA

expression

is

Northern

blot

analysis

with

polyacrylamide

gels

This

method

can

distinguish

pre-miRNA

and

miRNA

at

the

same

time

although

the

technique

is

relatively

insensitive

owing

to

the

large

total

RNA

volume

needed

and

it

is

labor-intensive

The

miRNA

oligonucleotide

microarray

provides

a

novel

method

to

carry

out

genome-wide

microRNA

profiling

in

human

samples

We

used

total

RNA

as

the

sample

for

the

microarray

test

not

just

labeling

filtered

low

molecular

weight

RNA

which

could

change

the

ratio

of

pre-miRNA

to

miRNA

So

the

profile

we

generated

was

that

of

pre-miRNAs

and

miRNAs

Owing

to

its

high

throughput

and

small

sample

requirement

the

miRNA

OMA

can

be

used

as

screening

method

in

miRNA

research

Some

microRNAs

are

within

the

introns

of

host

genes

Intronic

miRNAs

are

usually

expressed

in

coordination

with

their

host

gene

mRNA

suggesting

that

they

are

generally

derived

from

a

common

transcript

-LSB-

-RSB-

Some

human

microRNAs

are

even

processed

from

capped

polyadenylated

transcripts

and

can

function

as

mRNAs

-LSB-

-RSB-

Therefore

recognition

of

these

miRNA

gene

families

should

help

in

the

identification

of

putative

mRNA

targets

and

in

understanding

the

pathways

of

miRNA

biogenesis

Through

GeneCluster

software

analysis

we

found

that

and

form

a

cluster

Based

on

the

bioinformatics

study

and

previous

work

we

attempted

to

verify

the

hypothesis

that

the

cluster

may

share

the

same

expression

unit

We

searched

for

the

genome

location

and

possible

co-expressed

mRNA

and

found

the

six

pre-miRNAs

within

about

kb

on

human

chromosome

the

possible

co-expressed

mRNA

is

human

chromosome

open

reading

frame

-LRB-

-RRB-

transcript

variant

mRNA

indicating

that

the

cluster

members

are

intronic

miRNAs

Analysis

of

clustered

miRNA

expression

profiles

suggested

that

the

six

clustered

miRNAs

may

have

the

same

promoter

element

In

our

study

we

found

that

four

miRNAs

were

only

expressed

in

the

human

fetal

nervous

system

indicating

that

they

may

play

important

roles

in

human

nervous

system

development

Before

our

study

Krichevsky

et

al.

found

and

specifically

expressed

in

mouse

brain

and

they

were

de-regulated

in

mice

which

exhibited

severe

developmental

defects

of

the

brain

-LSB-

-RSB-

and

may

modulate

critical

development

processes

in

human

brain

development

and

changes

in

stage

and

level

of

expression

may

induce

major

developmental

errors

Giraldez

et

al.

showed

that

regulates

brain

morphogenesis

in

zebrafish

and

MZdicer

mutants

undergo

axis

formation

and

differentiate

multiple

cell

types

but

display

abnormal

morphogenesis

during

gastrulation

brain

formation

somitogenesis

and

heart

development

Injection

of

rescues

the

brain

defects

in

MZdicer

mutants

revealing

essential

roles

for

miRNAs

during

morphogenesis

-LSB-

-RSB-

No

human

is

registered

online

at

present

but

and

have

the

same

sequence

at

nucleotides

Giraldez

thought

this

is

most

important

for

recognition

In

our

study

members

of

the

cluster

were

highly

expressed

in

the

nervous

system

but

no

human

miRNA

expression

homologous

to

was

detected

by

Northern

blot

with

a

probe

-LRB-

data

not

shown

-RRB-

In

humans

it

is

not

that

plays

a

critical

role

in

nervous

system

morphogenesis

So

these

results

indicated

that

there

are

differences

in

the

mechanisms

of

brain

morphogenesis

between

human

and

zebra

fish

This

raises

the

question

of

whether

the

specifically

expressed

and

higher

expressed

cluster

are

functionally

linked

or

perhaps

this

just

reflects

higher

nonspecific

Dicer

activity

He

et

al.

compared

B-cell

lymphoma

samples

and

cell

lines

to

normal

tissues

and

found

that

the

levels

of

the

primary

or

mature

miRNAs

derived

from

the

locus

are

often

substantially

increased

in

these

cancers

implicating

the

cluster

as

a

potential

human

oncogene

-LSB-

-RSB-

O'Donnell

et

al.

found

that

c-Myc-regulated

and

modulate

expression

-LSB-

-RSB-

These

findings

indicate

that

the

cluster

may

be

a

common

channel

to

regulate

cell

differentiation

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

microrna
oligonucleotide microarray
expression profile
nervous system development


Tracheobronchomalacia due to amyloidosis in a patient with rheumatoid arthritis In this case report , we describe a patient with longstanding rheumatoid arthritis who developed tracheobronchomalacia with fatal outcome . Despite negative antemortem biopsies of abdominal fat and tongue , amyloid was found postmortem in the trachea and appeared to be associated with tracheobronchomalacia . Introduction Amyloidosis in longstanding chronically active rheumatoid arthritis -LRB- RA -RRB- is a well-known complication of this disease -LSB- 1 -RSB- . We present a RA patient with amyloidosis , albeit with a unique presenting symptom and unexpected organ involvement . Case report The patient was a 69-year-old female with seropositive erosive RA , who had not been treated with disease-modifying antirheumatic drugs since 1984 . In March 2005 , she presented to another hospital because of progressive dyspnea of several hours , due to a bronchopneumonia . After 2 days , she developed a stridor . Otolaryngological examination results showed diffuse swelling of the tongue , pharynx and neck , and an extensive tracheobronchomalacia . Treatment consisted of intubation , antibiotics , diuretic therapy , and corticosteroids . She was admitted to our Intensive Care Unit because of progressive swelling and respiratory insufficiency due to tracheobronchomalacia . Physical examination results revealed extensive swelling and a remarkable protrusion of the tongue . Typical rheumatoid joint deformities and rheumatoid nodules were present but no active arthritis . Laboratory investigations revealed a normocytic anemia and a renal insufficiency . The measured creatinine clearance was 12 ml/min , with a proteinuria of 0.35 g/24 h. Computed tomography scan of thorax and neck provided no explanation for the tracheobronchomalacia . Clinical suspicion of amyloidosis was not confirmed by biopsies of abdominal fat tissue and of the tongue . Ultimately , the patient succumbed to multiple organ failure . Autopsy revealed AA amyloid in the trachea -LRB- Fig. 1 -RRB- , spleen , liver , and perivascularly in the kidneys . Fig. 1Amyloidosis of the trachea in Congo red Discussion Amyloidosis of the trachea is a rare but known disorder , even rarer in patients with systemic diseases -LRB- such as Sjogren syndrome -RRB- but never associated with tracheobronchomalacia -LSB- 2 -- 4 -RSB- . We report the first case of an RA patient with tracheobronchomalacia due to AA amyloidosis of the trachea . The diagnosis was only confirmed at postmortem , although several biopsies had been done in life to confirm the clinical suspicion . We refrained from rectal and kidney biopsies because of the relatively high risk of complications associated with these procedures ; the absence of diarrhea and the low level of proteinuria suggested a relatively low pretest probability . Therefore , the finding of renal amyloid was surprising . Recently , Uda et al. -LSB- 5 -RSB- described a prospective cohort study of patients with RA without clinical signs of kidney disease and amyloidosis of the kidney . At follow-up , the group of patients with amyloid deposition confined to the perivascular region continued to have little proteinuria and a good renal prognosis , compared to those with amyloid deposition in the glomeruli who developed rapidly deteriorating renal function . This patient , however , does show that the disease course in RA patients with perivascular amyloid deposition in the kidney is not benign and may be associated with fatal complications elsewhere . In conclusion , tracheobronchomalacia can be due to amyloid deposition in patients with RA . Amyloidosis should be considered in RA patients with a small amount of proteinuria , and a renal biopsy should be performed , especially as accumulating evidence shows that patients with amyloidosis have a better prognosis when the ongoing inflammation is effectively suppressed -LSB- 1 -RSB- .

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

amyloidosis
kidney
rheumatoid arthritis
tracheobronchomalacia


Subintimal Double-Barrel Restenting of an Occluded Primary Stented Superficial Femoral Artery In-stent re-stenosis is a frequent complication of endovascular stents , especially in the superficial femoral artery -LRB- SFA -RRB- . Endovascular re-intervention of in - or peri-stent occlusive disease consists of recanilization through the occluded stent . In our case report , we describe the endovascular treatment of a previously placed stent in the SFA . We unintentionally passed the affected stent subintimally , in a double barrel fashion next to the first stent . The procedure was without any complications and with a successfull angiographic result . At one year follow-up the patient still has no complaints and the stent is still patent . In-stent restenosis is a frequent complication , especially in the superficial femoral artery -LRB- SFA -RRB- . If in-stent restenosis occurs there are several therapeutic options available such as conservative therapy and endovascular or surgical intervention . Endovascular reintervention of in - or peri-stent occlusive disease is one of the less invasive options . Endovascular therapy means passage through the occluded stent -LRB- recanalization -RRB- by additional means and redilatation or restenting of the lesion . In some cases passage of the occluded stent is not possible . In this case report we describe the unsuccessful attempt to recanalize an occlusion in a stent in the SFA followed by the successful bypass of the stent using a subintimal technique . The concept of utilizing this secondary stent was to create enough radial force to keep open the neo-lumen next to the occluded stent . Although the per-interventional extraluminal recanalization -LRB- PIER -RRB- technique is usually performed for longer lesions of the SFA , in this case a shorter segment was successfully managed -LSB- 1 , 2 -RSB- . An internet search for articles describing equivalent cases revealed no results . No publications on subintimal passage of a reoccluded stent and double-barrel stenting were found in the literature . Case Report A 75-year-old man underwent several percutaneous transluminal angioplasty -LRB- PTA -RRB- procedures in both upper leg vessels as well as stenting of a right SFA lesion in the adductor canal after an insufficient PTA result in an other facility -LRB- Figs. 1 and 2 -RRB- . Fig. 1 . See text . Fig. 2 . See text . The patient presented in 2004 with progressive complaints of the right leg . The patient 's maximum walking distance was 800 m but he smoked 5 -- 10 cigarettes a day . As consistent medication the patient was on antihypertensive drugs , 40 mg simvastatin -LRB- 10 mg Zocor , MSD -RRB- as well as 100 mg carbasalaatcalcium -LRB- Ascal Cardio , Viatris -RRB- . There was no cardiopulmonary comorbidity . Right pedal pulses were absent with a normal pulse at the right groin . Blood pressure was 160/95 mmHg . The maximum walking distance was 160 m with a drop in the ankle/brachial index -LRB- ABI -RRB- after excercise of 0.62 to 0.23 on the right side and 0.98 to 0.55 on the left side . In conclusion , this patient had peripheral arterial occlusive disease -LRB- PaOD -RRB- staged as Fontaine class 2a . A color Doppler ultrasound -LRB- CCDS -RRB- examination of the right femoropopliteal arteries revealed in - and peri-stent restenosis with a peak systolic volume -LRB- PSV -RRB- ratio of 5.7 and distal in-stent occlusion noted over 4 cm . At that time the patient could not be persuaded to undergo any intervention and was managed conservatively . In March 2005 the patient presented again with unchanged symptoms . On physical examination the walking distance was now 240 m with an ABI drop after excercise of 0.44 to 0.22 on the right side and 0.91 to 0.52 on the left side . The patient and his attending physician discussed endovascular treatment such as recanalization of the affected vessel with PTA and/or additional stenting as a proper next option for his PaOD Fontaine class 2a lifestyle-limiting symptoms . In April 2005 , an endovascular percutaneous intervention of the right SFA was performed at the Department of Radiology in our hospital . Because the patient was overweight an antegrade puncture was not a good option and the right SFA was approached by a crossover technique from the contralateral side . The right leg vessels were visualized angiographically demonstrating a stenosis of approximately 70 % at the origin on the right SFA . More distally in the SFA an occlusion was noticed reaching from 4 cm proximal of the occluded stent to the upper border of the patella -LRB- The total length of this lesion was approximately 11 cm . -RRB- The 5 Fr Brite Tip sheath -LRB- Cordis , New York , USA -RRB- in the right femoral artery was changed for a long 6 Fr Balkin Up & Over flexorsheath -LRB- Cook , Bloomington , IN , USA -RRB- . The sheath tip was placed in the left external iliac artery , just above the hip joint . First a PTA was performed at the origin of the SFA with a 6.0 / 40.0 mm balloon -LRB- Pheron , Biotronik , Switzerland -RRB- after an intravenous -LRB- IV -RRB- injection of 2,500 IU heparin , with a satisfactory angiographic result . However , it proved impossible , after several catheter-guidewire manipulation efforts , to recanalize the occluded stent with a 0.035-inch J-shaped Terumo -LRB- Terumo , USA -RRB- guidewire . Next the occluded stent was bypassed subintimally PIER-wise and the native lumen successfully re-entered just distal to the occlusion . The neo-lumen thus created was predilated with an unsatisfactory result due to recoil and consequently stented with a self-expandable 6.0 / 60.0 mm and a 6.0 / 80.0 mm stent -LRB- Absolute , Guidant , Indianapolis , IN , USA -RRB- . Post-dilatation was performed with a 6.0 mm Pheron balloon . Angiographic verification of the treated arterial segment revealed an acceptable angiographic result and good runoff to patent vessels below the knee . The 2,500 IU heparin was antagonized by an IV injection of 2,500 IU protamine . The angiographic sheath was withdrawn and the puncture site managed by manual compression for 10 min without any problems . The procedure was carried out without any minor or major events or complications . One day after the procedure the patient underwent CCDS proving patency of the treated proximal SFA and newly inserted stents with no residual stenotic segments . The ABI on the right side was 1.0 and on the left side was 1.1 . The patient reported a slightly painful sensation on the upper-medial side of his lower -LRB- treated -RRB- limb . On his planned check-up at the department of surgery in May 2005 the patient reported himself very satisfied with the result . The pain sensations had disappeared and there were no more limitations to his walking distance.In August 2005 his ABI on the right side was 0.85 with a drop , after exercise , to 0.25 . On the left side his ABI was 0.88 with a drop , after exercise , to 0.62 . On color Doppler ultrasound investigation a patent secondary stent segment and no new stenotic lesions were observed and a normal flow pattern was described . Discussion Peri - and in-stent restenosis is a common and well-known complication in the SFA region . There are several therapeutic options to solve this problem including endovascular treatment and bypass surgery . Because of our patient 's physical condition and his lifestyle-hampering symptomatology we chose was endovascular treatment in an attempt to recanalize the affected stent . During the procedure it proved impossible after several attempts to recanalize the occluded stent in the SFA , probably because of already organized and fibrosed thrombus material in the stent . The crossover was not the cause of this failure , since the pushability and steerability of the guidewire-catheter assembly was sufficient . No angioplasty could be performed in the occluded stent . After several attempts we finally managed to pass the occluded stent subintimally , successfully re-entering the native lumen distally and creating a neo-lumen side-by-side with the primary occluded stent . The neo-lumen was stented to create enough radial force to prevent its elastic recoil . Two self-expandable stents were positioned in a double-barrel fashion alongside the previously occluded stent with proper clinical and angiographic result . A retrograde puncture was preferred because of the lower complication rate compared with an antegrade approach in this obese male . Moreover a crossover introducer sheath assembly creates more possibilities for easier catheter-guidewire manipulations . Conclusion If a patient presents with claudication symptoms caused by in-stent restenosis or occlusion , one can try to recanalize the occluded stent . This is often not possible , especially if the occlusion is of an older date and consists of fibrosed tissue , mainly because of reorganizing thrombus . Mechanical debridement is an option , but this is more successful in -LRB- sub -RRB- acute -LRB- re -RRB- stenosis -LSB- 3 -RSB- . Subintimal passage of a occluded stent using the PIER technique could be an option in such cases . The PIER technique is intended as a primary therapy , usually for longer lesions of the SFA and for patients with severe -LRB- critical -RRB- limb ischemia , and has a 1-year patency rate of 22 % and a success rate of 62 % -LSB- 1 , 2 -RSB- . As shown in our case , PIER appears to be good not only for recanalizing occlusions but also for providing options for the treatment of nonpenetrable in-stent occlusions by trespassing PIER-wise and subintimal stenting of the neo-lumen .

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

interventional radiology
endovascular stenting
reocclusion


Basic Erosive Wear Examination -LRB- BEWE -RRB- : a new scoring system for scientific and clinical needs A new scoring system , the Basic Erosive Wear Examination -LRB- BEWE -RRB- , has been designed to provide a simple tool for use in general practice and to allow comparison to other more discriminative indices . The most severely affected surface in each sextant is recorded with a four level score and the cumulative score classified and matched to risk levels which guide the management of the condition . The BEWE allows re-analysis and integration of results from existing studies and , in time , should initiate a consensus within the scientific community and so avoid continued proliferation of indices . Finally , this process should lead to the development of an internationally accepted , standardised and validated index . The BEWE further aims to increase the awareness of tooth erosion amongst clinicians and general dental practitioners and to provide a guide as to its management . Introduction The objective of tooth wear indices is to classify and record the severity of tooth wear or dental erosion in prevalence and incidence studies . There are a myriad of indices -LSB- 1 -RSB- which vary in type of assessment , scale , choice of teeth and other styles , resulting in non-comparability -LSB- 4 -RSB- . The main aim of these indices has been in research and health service planning , but since so many indices have been published it has not been possible to compare the outcomes of different studies and so provide an international overview of the present status of this condition . The variation in these indices , and for some their complexity , means that they remain a research tool of limited relevance . Further , there are no convenient and simple methods for general dental practitioners -LRB- GDPs -RRB- to record the level of tooth wear and erosion apart from using subjective terms such as mild , moderate and severe . The very nature of these terms means that their interpretation varies considerably between clinicians . These are valid reasons for the need to establish a simple , repeatable and convenient index that is a valid research tool for the dental academic community but also of use in day-to-day dental practice for screening . Ideally , an index would have a basic structure that would allow for more sophisticated categories to be developed for specific research purposes , which could then be broken down again to the simplified version for clinical needs or for screening procedures . An example is the FDI DDE index -LSB- 8 -RSB- for enamel defects that has a basic screening index and an expanded epidemiological index . The Basic Periodontal Examination -LRB- BPE -RRB- -LSB- 5 -RSB- or the Periodontal Screening Index adopted from the Community Periodontal Index -LSB- 14 -RSB- was developed to allow a convenient , repeatable method of recording periodontal diseases . Uniquely , these indices have been accepted not only by academics but also as part of the routine clinical examinations by GDPs . Development of these indices allows dentists not only to screen for periodontal diseases in a simple and effective manner but also to improve awareness and understanding of the disease . The same objectives are now needed for erosive tooth wear . Unlike caries and periodontitis , however , there is uncertainty about the validity of the criteria -LSB- 9 , 12 -RSB- to define and grade tooth wear , erosion , abrasion and attrition , and at present , there is no conclusive knowledge how much erosive wear is physiological or can be accepted in relation to age -LSB- 2 , 19 -RSB- . The Basic Erosive Wear Examination -LRB- BEWE -RRB- has therefore been designed to provide a simple scoring system that can be used with the diagnostic criteria of all existing indices aiming to transfer their results into one unit which is the BEWE score sum . The aim of the BEWE is to be a simple , reproducible and transferable scoring system for recording clinical findings and for assisting in the decision-making process for the management of erosive tooth wear . Basic Erosive Wear Examination -LRB- BEWE -RRB- The BEWE is a partial scoring system recording the most severely affected surface in a sextant and the cumulative score guides the management of the condition for the practitioner . The four level score -LRB- Table 1 -RRB- grades the appearance or severity of wear on the teeth from no surface loss -LRB- 0 -RRB- , initial loss of enamel surface texture -LRB- 1 -RRB- , distinct defect , hard tissue loss -LRB- dentine -RRB- less than 50 % of the surface area -LRB- 2 -RRB- or hard tissue loss more than 50 % of the surface area -LRB- 3 -RRB- . The differentiation between lesions restricted to enamel and dentine can be difficult particularly in the cervical area -LSB- 11 , 12 -RSB- . Buccal/facial , occlusal , and lingual/palatal surfaces are examined with the highest score recorded . Table 1Criteria for grading erosive wearScore 0No erosive tooth wear1Initial loss of surface texture2 * Distinct defect , hard tissue loss < 50 % of the surface area3 * Hard tissue loss ≥ 50 % of the surface area * in scores 2 and 3 dentine often is involved The examination is repeated for all teeth in a sextant but only the surface with the highest score is recorded for each sextant . Once all the sextants have been assessed , the sum of the scores is calculated as indicated on the grid that follows : BEWE scores Highest scoreHighest scoreHighest score 1 . Sextant -LRB- 17 -- 14 -RRB- 2 . Sextant -LRB- 13 -- 23 -RRB- 3 . Sextant -LRB- 24 -- 27 -RRB- Highest scoreHighest scoreHighest scoreScore sum4 . Sextant -LRB- 37 -- 34 -RRB- 5 . Sextant -LRB- 33 -- 43 -RRB- 6 . Sextant -LRB- 44 -- 47 -RRB- The result of the BEWE is not only a measure of the severity of the condition for scientific purposes but , when transferred into risk levels , also a possible guide towards management -LRB- Table 2 -RRB- . Table 2Risk levels as a guide to clinical managementRisk levelCumulative score of all sextantsManagementNoneLess than or equal to 2aRoutine maintenance and observationRepeat at 3-year intervalsLowBetween 3 and 8aOral hygiene and dietary assessment , and advice , routine maintenance and observationRepeat at 2-year intervalsMediumBetween 9 and 13aOral hygiene and dietary assessment , and advice , identify the main aetiological factor -LRB- s -RRB- for tissue loss and develop strategies to eliminate respective impactsConsider fluoridation measures or other strategies to increase the resistance of tooth surfacesIdeally , avoid the placement of restorations and monitor erosive wear with study casts , photographs , or silicone impressionsRepeat at 6 -- 12-month intervalsHigh14 and overaOral hygiene and dietary assessment , and advice , identify the main aetiological factor -LRB- s -RRB- for tissue loss and develop strategies to eliminate respective impactsConsider fluoridation measures or other strategies to increase the resistance of tooth surfacesIdeally , avoid restorations and monitor tooth wear with study casts , photographs , or silicone impressionsEspecially in cases of severe progression consider special care that may involve restorationsRepeat at 6 -- 12-month intervalsaThe cut-off values are based on experience and studies of one of the authors -LRB- A. L. -RRB- and have to be reconsidered . The management would include identification and elimination of the main aetiological factor -LRB- s -RRB- , prevention and monitoring , as well as symptomatic and operative intervention where appropriate . It does not provide guidance for the most appropriate prosthodontic or operative technique as there is too much variation at this level of decision making between clinicians . However , for the highest risk levels special care is suggested . The repetition of the BEWE will vary according to the severity and the relative importance of aetiological and risk factors . For patients particularly exposed to intrinsic or extrinsic acids -LSB- 17 -RSB- , the procedure should be repeated at 6-month intervals , but for most other cases , annually is acceptable . Discussion The need for a standardised and internationally accepted index is obvious , but there continues to be research needs which may not be fulfilled with a simplified index . Over the past 20 to 30 years , different researchers have developed indices which suit their own research needs but do not allow comparison to assess the prevalence of tooth wear between countries and regions . Therefore , this new scoring system has been designed to allow existing and hopefully future indices to be collapsed and re-analysed . It will be important that this proposed system is validated against existing data or in field trials . In time , it should initiate a consensus within the scientific community and so avoid continued proliferation of indices . Finally , this process should lead to the development of an internationally accepted , standardised and validated index . The structure of the BEWE is designed to allow fulfilling of most formal requirements generally formulated for indices . The grading includes four levels which is neither too precise nor too crude , and the threshold values should be easy to learn and to calibrate . In addition by removing the clear distinction between `` enamel loss '' and `` dentine '' exposed , it will not only evade diagnostic uncertainties but will open a broad applicability beyond the clinical situation . It can be used with study models or photographs which appear suitable for erosive wear as a surface phenomenon . Erosion already has been documented or diagnosed on study models and on photographs -LSB- 10 , 13 , 15 -RSB- , and this could be of particular value in cross-sectional and incidence studies as well as for the monitoring of individual cases . It will further allow a more reliable estimation of the severity of tooth surface loss on an individual basis rather than the current way to give data as `` x % of the subjects had at least one tooth with grade x or grade y erosion '' , and so avoid an overestimate of the problem . The BEWE is ideal for screening studies , but a longer version is required for a more detailed investigation . As to the field of dental care , the clinical value of the BEWE , as with the BPE , should be highly significant as a convenient diagnostic tool for GDPs and as a model to increase awareness assuming that this relatively new entity is not well recognised . Even in UK , where the prevalence and incidence of erosive wear is relatively high -LSB- 7 , 18 -RSB- , a questionnaire survey has shown that only one third of the practitioners noted erosion on a frequent basis and the majority underestimated the prevalence of the condition -LSB- 6 -RSB- . The benefit of a system that encourages the more careful examination of the dental hard tissues other than for caries is therefore obvious and is one further aim of the BEWE . In addition to diagnosis and scoring , also a strategy for treatment is included in the BEWE . There are a number of reviews with respect to the prevention and therapy of erosive wear -LRB- e.g. -LSB- 3 , 16 -RSB- -RRB- , but it is not only the variety of recommendations which makes the management of the condition difficult for the GDP , but also the fact that these recommendations were not made with respect to the severity of tooth surface loss in an individual . The risk levels presented aim to guide the management of erosive wear and allow sufficient flexibility so that different countries can adapt the index without compromising their unique needs . The levels defined , however , are suggestions at the present stage and need an ongoing review process . Conclusion The BEWE is a basic structure to initiate the development of an internationally accepted , standardised and validated index . This is to provide on one hand a clear and defined structure for scientific and clinical use , but on the other to be amenable for further development . It will encourage clinicians , students and GDPs to pay more attention to erosive wear and hence will be beneficial for patient care .

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erosion
tooth wear
diagnosis
examination
index