The

impact

of

maternal

separation

on

adult

mouse

behaviour

and

on

the

total

neuron

number

in

the

mouse

hippocampus

The

maternal

separation

paradigm

has

been

applied

to

mice

as

an

animal

developmental

model

for

understanding

structural

deficits

leading

to

abnormal

behaviour

A

maternal

separation

-LRB-

MS

-RRB-

model

was

used

on

postnatal

day

-LRB-

PND

-RRB-

where

the

pups

were

removed

from

their

mother

for

h

-LRB-

-RRB-

When

the

pups

were

weeks

old

the

level

of

anxiety

and

fear

was

measured

with

two

behavioural

tests

an

open

field

test

and

an

elevated

plus

maze

test

The

Barnes

platform

maze

was

used

to

test

spatial

learning

and

memory

by

using

acquisition

trials

followed

by

reverse

trial

sessions

The

mice

spent

more

time

in

the

open

arms

of

the

elevated

plus

maze

compared

to

controls

but

no

other

treatment

differences

were

found

in

the

emotional

behavioural

tests

However

in

the

reverse

trial

for

the

Barnes

maze

test

there

was

a

significant

difference

in

the

frequency

of

visits

to

the

old

goal

the

number

of

errors

made

by

the

mice

compared

to

controls

and

in

total

distance

moved

The

mice

were

subsequently

sacrificed

and

the

total

number

of

neurons

estimated

in

the

hippocampus

using

the

optical

fractionator

We

found

a

significant

loss

of

neurons

in

the

dentate

gyrus

in

MS

mice

compared

to

controls

Apparently

a

single

maternal

separation

can

impact

the

number

of

neurons

in

mouse

hippocampus

either

by

a

decrease

of

neurogenesis

or

as

an

increase

in

neuron

apoptosis

This

study

is

the

first

to

assess

the

result

of

maternal

separation

combining

behaviour

and

stereology

Introduction

Developing

an

animal

model

of

mental

disorders

is

controversial

due

to

the

human

nature

of

the

symptoms

such

as

hallucinations

delusions

and

poverty

of

speech

These

symptoms

can

only

be

adequately

assessed

by

psychological

assessments

and

therefore

can

not

be

modulated

in

animals

A

way

to

try

to

circumvent

these

problems

is

studying

certain

psychological

or

psychophysiological

aspects

of

mental

disorders

such

as

latent

inhibition

prepulse

inhibition

or

gating

-LRB-

Ellenbroek

and

Cools

Geyer

and

Markou

Ellenbroek

et

al.

-RRB-

However

it

is

still

unclear

how

-LRB-

and

if

-RRB-

these

abnormalities

are

linked

to

the

symptoms

of

mental

illness

In

rodent

models

prepulse

inhibition

-LRB-

PPI

-RRB-

of

the

acoustic

startle

response

is

a

model

of

sensorimotor

gating

mechanisms

in

the

brain

while

an

equivalent

reaction

in

humans

is

eye

blinking

-LRB-

Braff

et

al.

Ellenbroek

et

al.

-RRB-

It

has

been

shown

that

rat

pups

that

underwent

h

maternal

separation

on

postnatal

day

-LRB-

PND

-RRB-

or

expressed

reduced

prepulse

inhibition

-LRB-

PPI

-RRB-

on

postnatal

day

and

had

hyperactivity

of

the

dopamineric

system

involving

the

dopamine

neurotransmitter

system

via

the

hypothalamic

pituitary

adrenal

-LRB-

HPA

-RRB-

axis

-LRB-

Ellenbroek

and

Cools

-RRB-

Further

the

PPI

deficits

could

be

reversed

with

typical

antipsychotic

drugs

like

haloperidol

and

were

not

detected

prior

to

puberty

-LRB-

Ellenbroek

et

al.

-RRB-

Due

to

the

changes

seen

in

the

HPA

axis

the

dopamine

system

hippocampus

and

long-term

behavioural

effects

modelling

deficits

seen

in

mental

patients

has

lead

Ellenbroek

and

co-workers

to

hypothesis

the

maternal

deprivation

model

to

be

a

schizophrenia-like

neurodevelopmental

animal

model

-LRB-

Ellenbroek

and

Cools

Ellenbroek

et

al.

-RRB-

One

of

the

striking

characteristics

of

the

developing

neuroendocrine

stress

system

in

the

mouse

and

rat

is

a

period

of

reduced

stress-responsiveness

the

so-called

stress

hypo-responsive

period

-LRB-

SHRP

-RRB-

-LRB-

Schapiro

et

al.

Cirulli

et

al.

Schmidt

et

al.

-RRB-

From

about

postnatal

day

-LRB-

PND

-RRB-

in

the

rat

and

PND

in

the

mouse

the

animals

neuroendocrine

system

is

characterized

by

a

low

basal

corticosterone

level

and

by

the

inability

of

a

mild

stressor

e.g.

exposure

to

novelty

to

induce

a

corticosterone

response

-LRB-

Cirulli

et

al.

Schmidt

et

al.

-RRB-

In

this

study

we

used

the

inbred

mouse

Due

to

a

similar

postnatal

neurodevelopmental

course

in

the

mouse

and

rat

-LRB-

Clancy

et

al.

-RRB-

we

used

PND

as

the

day

of

separation

equivalent

to

Ellenbroek

and

co-workers

day

of

choice

in

the

rat

We

tested

if

a

maternal

separation

on

PND

can

cause

an

adult

phenotype

characterized

by

altered

levels

of

activity

and

anxiety

learning

and

memory

dysfunction

deficits

in

behavioural

flexibility

-LRB-

reversal

deficits

-RRB-

as

well

as

changes

in

number

of

neurons

in

the

hippocampus

and

its

subregions

in

the

mouse

brain

Material

and

methods

Animals

The

offspring

of

male

and

female

mice

-LRB-

weeks

old

-RRB-

obtained

from

Taconic

Europe

-LRB-

Taconic

Farms

Inc.

DK

-RRB-

were

used

in

this

study

The

animals

were

acclimatized

to

the

animal

facility

for

week

Multiparous

females

were

used

since

there

is

a

higher

rate

of

offspring

survival

The

animals

were

housed

under

a

h

light/dark

cycle

-LRB-

lights

on

at

a.m.

-RRB-

with

constant

temperature

-LRB-

°C

-RRB-

and

humidity

-LRB-

-RRB-

in

Macrolon

type

III

cages

with

environmental

enrichment

in

the

form

of

wood

splints

bedding

-LRB-

aspen

-RRB-

wood

shavings

piece

of

Aspen

Corner

standard

mouse

house

made

of

recycled

cardboard

aspen

chewing

stick

size

medium

and

pads

of

nesting

material

-LRB-

all

obtained

from

Brogaarden

DK

-RRB-

Food

-LRB-

Altromin

pills

NR

-RRB-

and

tap

water

were

available

ad

libitum

Two

females

were

placed

in

a

male

's

cage

for

a

period

of

week

to

ensure

conception

followed

by

separation

of

the

two

females

to

their

own

cages

Maternal

separation

Pregnant

females

were

checked

for

litters

daily

at

a.m.

If

litters

were

found

the

day

of

birth

was

defined

as

PND

for

that

litter

On

PND

litters

were

randomly

assigned

to

maternal

separation

-LRB-

MS

-RRB-

-LRB-

N

males

and

females

-RRB-

or

to

standard

facility

rearing

-LRB-

SFR

-RRB-

For

the

behavioural

testing

MS

animals

-LRB-

males

and

females

-RRB-

and

SFR

animals

-LRB-

males

and

females

-RRB-

were

included

while

of

the

MS

animals

-LRB-

males

and

females

-RRB-

and

control

animals

-LRB-

males

and

females

-RRB-

were

used

for

cell

counting

Litters

were

not

culled

or

sexed

at

birth

to

minimize

the

handling

of

the

pups

but

male

and

female

pups

were

separated

at

weaning

-LRB-

PND

-RRB-

and

group

housed

with

their

siblings

which

resulted

in

mice

per

cage

The

environmental

enrichment

applied

to

the

mothers

was

also

applied

to

the

MS

and

control

animals

The

deprivation

was

carried

out

on

PND

starting

at

a.m.

The

pups

remained

in

the

home

cage

but

were

placed

in

a

separate

room

with

the

same

temperature

humidity

and

lighting

conditions

as

the

home

stable

The

cage

was

placed

on

a

heating

pad

which

had

a

constant

temperature

of

°C

No

food

or

water

was

available

during

the

separation

The

dam

was

placed

in

a

cage

with

similar

facilities

as

the

home

cage

in

the

home

stable

The

pups

were

checked

every

h

using

a

red

light

during

the

night

Body

weights

were

recorded

before

and

after

separation

Immediately

after

h

the

dams

were

returned

to

the

home

cage

and

reunited

with

the

pups

Test

for

anxiety

and

fear

related

behaviour

When

the

pups

reached

weeks

of

age

they

were

subjected

once

to

the

open

field

test

-LRB-

Hall

-RRB-

and

the

elevated

plus

maze

test

which

is

based

on

the

procedure

used

by

Montgomery

-LRB-

-RRB-

and

later

validated

by

Pellow

et

al.

-LRB-

-RRB-

Behaviour

was

analysed

using

EthoVision

-LRB-

Noldus

Groeningen

The

Netherlands

-RRB-

All

behavioural

testing

took

place

between

a.m.

and

p.m.

Open

field

test

-LRB-

OFT

-RRB-

The

open

field

consisted

of

a

circular

wooden

platform

-LRB-

diameter

cm

-RRB-

surrounded

by

a

cm

high

wall

with

a

camera

mounted

directly

above

A

central

circle

of

cm

diameter

was

defined

in

the

behaviour

analysis

software

Three

light

bulbs

illuminated

the

arena

On

the

day

of

testing

each

animal

was

transported

in

a

cardboard

box

to

the

centre

of

the

open

field

and

behaviour

was

recorded

for

min

After

the

trial

the

maze

was

cleaned

with

a

solution

of

acetic

acid

and

soap

water

and

faecal

boli

were

counted

The

following

parameters

were

calculated

total

distance

moved

-LRB-

cm

-RRB-

time

spent

in

central

circle

and

time

spent

in

peripheral

zone

-LRB-

expressed

as

of

session

duration

-RRB-

Elevated

plus

maze

The

plus

maze

was

elevated

cm

above

the

ground

and

consisted

of

two

opposing

open

arms

-LRB-

cm

cm

-RRB-

connected

by

a

central

square

-LRB-

cm

cm

-RRB-

to

two

opposing

enclosed

arms

of

the

same

size

with

cm

high

walls

A

video

camera

placed

above

the

maze

recorded

the

animals

behaviour

On

the

day

of

testing

the

animal

was

placed

in

the

centre

of

the

maze

and

behaviour

was

recorded

for

min

Between

trials

the

maze

was

cleaned

as

described

for

the

OFT

The

following

parameters

were

calculated

total

duration

-LRB-

s

-RRB-

in

open

and

closed

arms

the

number

of

entries

into

the

open

and

closed

arms

and

the

total

distance

moved

-LRB-

cm

-RRB-

From

these

parameters

the

ratio

of

entries

into

the

open

arms

to

the

total

number

of

arm

entries

and

the

ratio

of

time

spent

in

the

open

arms

to

time

spent

in

both

open

and

closed

arms

was

calculated

Test

for

spatial

memory

Barnes

maze

The

Barnes

maze

-LRB-

Barnes

-RRB-

consisted

of

a

circular

white-coated

platform

cm

in

diameter

and

elevated

cm

over

the

ground

Sixteen

cm

wide

holes

were

evenly

distributed

around

the

perimeter

cm

from

the

edge

A

pair

of

rails

was

placed

under

two

opposing

holes

to

hold

the

hidden

escape

box

The

escape

box

was

a

dark

plastic

storage

box

with

a

cm

diameter

hole

in

the

lid

A

dark

cylindrical

cardboard

tube

-LRB-

cm

cm

high

-RRB-

with

a

lid

was

used

as

the

transport

and

start

chamber

Three

bulbs

illuminating

the

maze

and

high

irregular

rock

and

techno

music

played

from

a

computer

in

a

random

manner

provided

the

aversive

stimuli

As

with

the

previous

test

a

digital

video

camera

mounted

above

the

maze

recorded

animal

's

behaviours

Shaping

For

days

before

testing

commenced

the

animals

were

trained

to

enter

the

hidden

escape

box

Using

the

transport

cylinder

the

mouse

was

placed

near

the

edge

of

the

target

hole

with

the

hidden

escape

box

underneath

Two

cardboard

walls

blocked

entry

to

adjacent

holes

Only

dim

lightning

and

no

noise

was

used

during

this

phase

of

the

experiment

The

animal

was

allowed

min

to

enter

the

escape

box

and

if

this

failed

it

was

placed

manually

inside

When

the

animal

entered

the

box

it

was

quickly

carried

to

the

home

cage

Acquisition

trials

Six

consecutive

trials

were

given

one

per

day

The

animal

was

placed

in

the

transport

cylinder

oriented

in

a

random

direction

in

the

centre

of

the

maze

The

aversive

stimuli

were

turned

on

and

the

cylinder

removed

Recording

in

the

behaviour-observation

software

began

immediately

after

the

experimenter

had

left

the

room

The

trial

ended

after

min

or

when

the

animal

entered

the

hidden

escape

box

If

the

animal

failed

to

enter

the

box

or

re-entered

the

maze

after

recording

was

stopped

the

aversive

stimuli

were

turned

back

on

and

the

animal

was

allowed

min

more

to

enter

the

escape

box

If

that

also

failed

the

animal

was

manually

placed

in

the

box

After

completion

of

each

trial

the

box

was

placed

in

the

home

cage

Reversal

trials

Three

days

after

acquisition

trials

the

escape

box

was

placed

underneath

the

hole

opposite

to

the

hole

that

had

been

the

target

during

acquisition

training

Reversal

training

was

conducted

for

seven

consecutive

days

as

described

above

Parameters

Each

of

the

escape

holes

in

the

Barnes

maze

was

defined

as

a

separate

zone-of-interest

in

the

behaviour

analysis

software

The

following

parameters

were

analysed

latency

to

target

-LSB-

time

from

start

of

the

trial

to

first

entry

into

the

target

hole

zone

-LRB-

s

-RRB-

-RSB-

total

distance

moved

-LRB-

cm

-RRB-

and

error

frequency

-LRB-

number

of

visits

to

other

holes

than

the

target

hole

-RRB-

During

reversal

training

two

additional

parameters

were

analysed

the

mean

number

of

visits

to

the

old

target

hole

over

the

seven

trials

-LRB-

the

hole

where

the

escape

box

was

located

during

acquisition

training

-RRB-

and

the

mean

number

of

visits

to

the

two

holes

adjacent

to

the

old

target

hole

Three

different

search

strategies

could

be

distinguished

and

were

scored

manually

The

random

search

strategy

is

characterized

by

a

non-systematic

exploration

of

the

maze

with

many

centre

maze

crossings

and

some

perseverations

Perseverations

were

defined

as

repeatedly

searching

the

same

hole

or

two

adjacent

holes

-LRB-

Bach

et

al.

-RRB-

Secondly

there

is

the

serial

search

strategy

defined

as

systematic

consecutive

hole

searching

in

a

clockwise

or

counter

clockwise

manner

and

finally

a

spatial

search

strategy

defined

as

searching

less

than

three

holes

from

the

location

of

the

goal

-LRB-

Barnes

Bach

et

al.

Inman-Wood

et

al.

Zhang

et

al.

Raber

et

al.

-RRB-

-LRB-

Fig.

-RRB-

Fig.

different

search

strategies

could

be

applied

Random

a

pattern

that

include

many

hole

examinations

in

a

random

manner

Serial

a

relative

systematic

search

either

clockwise

or

counterclockwise

Spatial

a

pattern

where

the

target

box

is

found

within

relative

short

time

and

with

high

accuracy

Fixation

and

embedding

Approximately

week

after

all

tests

were

concluded

the

animals

were

scarified

by

asphyxiation

and

within

h

the

brains

were

dissected

out

and

placed

in

a

formalin

solution

-LRB-

Fixatin

-RRB-

The

brains

were

split

through

corpus

callosum

separating

the

two

hemispheres

After

systematic

random

assignment

of

left

or

right

hemisphere

they

were

coloured

on

the

outer

surface

to

preserve

a

coded

sequence

and

embedded

in

paraffin

with

an

automatic

vacuum

tissue

processor

-LRB-

Leica

-RRB-

The

hemispheres

were

then

mounted

horizontally

balancing

on

needles

and

embedded

in

a

paraffin

block

with

up

to

four

hemispheres

in

one

block

Sectioning

The

whole

hemisphere

was

cut

horizontally

with

a

Leica

-LRB-

model

SM

-RRB-

microtome

with

a

microtome

setting

of

μm

thickness

All

sections

were

mounted

on

silicone-coated

glass

-LRB-

Frost

-RRB-

After

a

minimum

of

h

in

a

°C

heating

cupboard

the

sections

were

sampled

systematic

uniformly

randomly

-LRB-

SURS

-RRB-

The

first

section

was

randomly

selected

from

a

random

number

table

hereafter

every

section

was

sampled

systematically

for

staining

and

counting

This

provided

a

total

of

sections

containing

the

hippocampus

per

specimen

To

be

able

to

account

for

block

advance

-LRB-

BA

-RRB-

the

block

height

was

measured

for

every

section

The

BA

determines

the

hitting

probability

of

the

particles

within

the

block

-LRB-

see

later

for

calculation

-RRB-

Furthermore

the

paraffin

shrinkage

effect

was

calculated

and

amounted

to

about

The

sections

were

stained

with

a

modified

Vogt

's

Cresyl

violet

acetate

-LRB-

Armed

forces

-RRB-

Optical

design

equipment

The

optical

design

equipment

consisted

of

an

Olympus

microscope

with

an

oil

immersion

objective

of

high

numerical

aperture

-LRB-

-RRB-

which

allows

focusing

in

a

thin

focal

plane

inside

a

thick

section

A

camera

transmits

the

image

to

a

computer

screen

where

a

counting

frame

is

superimposed

using

the

computer-assisted

stereological

-LRB-

CAST

-RRB-

Grid

software

-LRB-

Visiopharm

Hørsholm

DK

-RRB-

A

motorized

automatic

stage

was

used

to

control

movement

in

the

x

y

plane

via

a

connected

joystick

Movement

in

the

z-axis

was

controlled

manually

with

the

focus

button

on

the

microscope

and

the

distance

between

the

upper

and

lower

surfaces

of

the

sections

was

measured

with

a

Heidenhein

microcator

-LRB-

Heidenhain

Germany

-RRB-

with

a

precision

of

μm

Definitions

and

divisions

of

the

hippocampus

The

hippocampus

was

subdivided

into

five

regions

the

dentate

gyrus

-LRB-

DG

-RRB-

the

hilus

of

the

dentate

gyrus

-LRB-

-RRB-

regio

inferior

-LRB-

-RRB-

region

superior

-LRB-

-RRB-

and

subiculum

Neurons

were

counted

manually

in

the

sub-sampled

sections

containing

the

hippocampus

using

the

optical

disector

design

The

layers

comprising

the

hippocampus

were

defined

consistently

in

all

sections

using

the

terminology

of

Blackstad

-LRB-

-RRB-

It

was

not

considered

too

difficult

to

differentiate

the

neuronal

layers

in

horizontal

sections

-LRB-

Fig.

-RRB-

The

granule

cell

layer

and

the

pyramidal

cell

layers

also

contain

the

cell

bodies

of

basket

cells

and

glial

cells

The

glial

cells

can

be

easily

identified

and

counted

separately

but

the

basket

cells

are

so

similar

in

appearance

to

the

granule

and

pyramidal

cells

that

they

are

included

in

the

estimates

It

was

previously

shown

that

they

comprise

less

than

of

the

neurons

in

these

layers

-LRB-

review

West

et

al.

-RRB-

Fig.

schematic

drawing

of

the

hippocampus

with

the

five

subregions

identified

in

this

study

Dg

the

granule

cell

layer

of

the

dentate

gyrus

hilus

of

the

dentate

gyrus

ri

regio

inferior

rs

region

superior

s

subiculum

Stereological

equations

Estimation

of

the

total

neuron

number

N

For

the

estimation

of

total

neuron

numbers

neurons

were

counted

in

optical

disectors

and

sampled

according

to

the

so-called

fractionator

principles

In

a

fractionator

cells

are

counted

directly

in

a

known

fraction

of

the

different

hippocampal

subdivisions

and

the

total

neuron

number

N

is

estimated

by

multiplying

the

number

of

particles

counted

with

the

reciprocal

sampling

fractions

where

ssf

is

the

section

sampling

fractions

asf

the

area

sampling

fraction

and

hsf

the

height

sampling

fraction

The

bilateral

cell

number

is

estimated

by

multiplying

the

unilateral

number

Q

by

This

is

admissible

when

the

right

or

left

hippocampus

is

sampled

systematically

randomly

The

asf

is

known

as

the

area

of

the

counting

frame

of

the

disector

relative

to

the

area

associated

with

each

x,y-step

movement

of

the

disector

After

having

ascertained

that

the

cell

density

was

constant

within

the

disector

height

the

height

sampling

fraction

was

defined

as

is

the

q

weighted

mean

section

thickness

-LRB-

Gundersen

et

al.

Dorph-Petersen

-RRB-

Estimation

of

total

volume

the

Cavalieri

estimator

Besides

estimating

total

neuron

numbers

it

was

also

decided

to

estimate

total

volume

of

the

different

compartments

of

the

hippocampus

although

the

main

purpose

was

estimation

of

cell

numbers

Estimates

of

volume

were

obtained

according

to

the

principles

of

Cavalieri

's

basic

estimator

-LRB-

Gundersen

and

Jensen

-RRB-

and

corrected

for

shrinkage

Notice

that

these

volumes

were

not

used

for

the

estimation

of

total

neuron

number

The

estimates

of

total

neuron

number

obtained

by

the

fractionator

design

are

independent

of

the

containing

volume

Further

when

total

number

and

volume

is

estimated

the

density

NV

-LRB-

-RRB-

can

be

obtained

as

well

without

extra

work

Error

predictions

The

precision

of

the

two

different

estimates

-LRB-

neuron

number

and

volume

-RRB-

can

be

expressed

by

the

coefficient

of

error

CE

The

CE

for

the

Cavalieri

estimation

of

volume

was

first

formulated

and

described

by

Gundersen

and

Jensen

-LRB-

-RRB-

revised

in

Gundersen

et

al.

-LRB-

-RRB-

Table

gives

an

example

of

how

CE

for

both

volume

and

neuron

estimates

are

calculated

in

this

study

-LRB-

see

also

Gundersen

et

al.

-RRB-

Table

of

how

the

CE

is

calculated

for

neuron

number

and

volume

respectivelySectionQj

A

Qi

QiB

Qi

Qi

Qi

Qi

A

Pi

PiB

Pi

Pi

Pi

Pi

When

an

appropriate

number

of

sections

have

been

chosen

-LRB-

or

a

little

less

-RRB-

it

is

the

number

of

points

counted

-LRB-

the

noise

-RRB-

which

decides

the

precision

of

the

estimate

To

count

about

points

per

sample

is

usually

enough

to

obtain

a

CE

around

unless

the

object

is

very

irregular

-LRB-

Gundersen

and

Jensen

Gundersen

et

al.

West

and

Gundersen

-RRB-

When

CE

is

estimated

for

the

cell

counting

Noise

is

equal

to

Q

Statistical

analyses

All

data

were

analysed

with

the

statistical

software

packages

SPSS

-LRB-

Statistical

Package

for

the

Social

Sciences

version

-RRB-

or

SigmaStat

-LRB-

version

-RRB-

If

Levene

's

test

for

equality

of

variances

did

not

fail

a

t

test

was

applied

to

test

for

differences

between

the

two

experimental

groups

For

data

where

normality

tests

failed

the

non-parametric

tests

Mann

Whitney

U

test

and

Kruskal

Wallis

test

were

applied

The

animals

performance

over

the

six

acquisition

trials

and

the

seven

reversal

trials

in

the

Barnes

maze

was

analysed

by

mixed-model

analysis

of

variance

with

trial

as

within-subject

factor

and

experimental

group

as

between-subjects

factor

Homogeneity

of

the

variance-difference

scores

was

determined

by

Mauchly

's

test

of

sphericity

-LSB-

SPSS

Inc.

Chicago

IL

USA

SPSS

Base

version

User

Manual

-RSB-

When

the

assumption

of

sphericity

was

violated

degrees

of

freedom

were

adjusted

with

the

Huynh

Feldt

correction

Group

differences

in

strategy

selection

in

the

Barnes

maze

were

analysed

with

the

likelihood-ratio

chi-square

test

for

each

trial

separately

Group

differences

were

considered

significant

when

P

Results

Only

significant

behavioural

results

are

depicted

and

statistically

elaborated

Body

weights

A

significant

weight

loss

was

found

in

the

pups

after

separation

-LRB-

P

paired

t

test

-RRB-

However

weeks

after

separation

the

body

weights

of

MS

and

SFR

animals

were

similar

-LRB-

P

unpaired

t

test

-RRB-

Open

field

test

Both

experimental

groups

spent

more

time

in

the

periphery

of

the

open

field

than

the

centre

-LRB-

P

t

test

-RRB-

There

was

no

significant

difference

between

MS

and

SFR

groups

in

either

distance

moved

or

frequency

of

visits

to

and

time

spent

in

the

centre

or

periphery

of

the

open

field

-LRB-

data

not

shown

-RRB-

Elevated

plus

maze

The

MS

animals

spent

more

time

in

the

open

arms

than

the

SFR

animals

and

allotted

a

greater

percentage

of

time

spent

in

both

open

and

closed

arms

to

the

open

arms

-LRB-

P

and

P

t

test

respectively

Fig.

-RRB-

Fig.

mean

-LRB-

SEM

-RRB-

time

spent

in

both

the

open

and

closed

arms

There

was

a

significant

difference

between

MS

-LRB-

N

-RRB-

and

SFR

-LRB-

N

-RRB-

in

time

spent

in

open

arms

-LRB-

P

ANOVA

-RRB-

Time

spent

in

closed

arms

compared

with

time

spent

in

open

arms

-LRB-

P

t

test

-RRB-

In

trial

progress

there

was

a

significant

difference

in

both

frequency

and

duration

in

closed

and

open

arms

-LSB-

Closed

arms

frequency

-LRB-

F

-LRB-

-RRB-

P

-RRB-

and

duration

-LRB-

Wilk

's

λ

F

-LRB-

-RRB-

P

-RRB-

Open

arms

frequency

-LRB-

Wilk

's

λ

F

-LRB-

-RRB-

P

-RRB-

and

duration

-LRB-

F

-LRB-

-RRB-

P

-RRB-

-RSB-

There

was

no

significant

difference

in

any

of

the

other

parameters

analysed

Barnes

maze

Acquisition

trials

Latency

to

reach

the

target

hole

total

distance

moved

on

the

maze

and

error

frequency

all

decreased

significantly

over

the

course

of

the

six

acquisition

trials

-LRB-

F

-LRB-

-RRB-

P

F

-LRB-

-RRB-

P

F

-LRB-

-RRB-

P

respectively

-RRB-

indicating

that

the

animals

indeed

learned

the

task

There

was

however

no

significant

difference

between

the

two

experimental

groups

Reversal

trials

Latency

to

reach

the

new

target

hole

-LRB-

Fig.

left

-RRB-

total

distance

moved

on

the

maze

-LRB-

Fig.

-RRB-

and

error

frequency

-LRB-

Fig.

-RRB-

all

decreased

significantly

over

the

course

of

the

seven

reversal

trials

-LRB-

F

-LRB-

-RRB-

P

F

-LRB-

-RRB-

P

F

-LRB-

-RRB-

P

respectively

-RRB-

The

animals

made

significantly

more

errors

over

the

course

of

reversal

training

-LRB-

F

-LRB-

-RRB-

P

Fig.

-RRB-

and

travelled

longer

distances

-LRB-

Fig.

P

Mann

Whitney

U-test

-RRB-

This

might

indicate

that

the

MS

animals

did

not

learn

the

reversal

task

as

fast

as

the

controls

Fig.

Mean

-LRB-

SEM

-RRB-

latency

to

Goal

There

was

a

significant

difference

in

Trial

progress

and

a

significant

Trial

x

Group

interaction

-LRB-

P

-RRB-

Right

Mean

-LRB-

SEM

-RRB-

frequency

of

visits

to

Old

Goal

There

was

a

significant

difference

between

the

two

groups

-LRB-

P

Mann

Whitney

U

test

-RRB-

and

a

significant

decrease

over

time

-LRB-

P

RM

ANOVA

-RRB-

MS

-LRB-

N

-RRB-

and

SFR

-LRB-

N

-RRB-

Fig.

-LRB-

SEM

-RRB-

distance

moved

in

reverse

trials

There

was

a

significant

decrease

over

time

-LRB-

P

RM

ANOVA

-RRB-

and

a

significant

difference

between

the

two

treatment

groups

-LRB-

P

Mann

Whitney

U

test

-RRB-

but

no

significant

Trial

x

Group

interaction

MS

-LRB-

N

-RRB-

and

SFR

-LRB-

N

-RRB-

Fig.

-LRB-

SEM

-RRB-

frequency

of

errors

in

MS

-LRB-

N

-RRB-

and

SFR

-LRB-

N

-RRB-

there

was

a

significant

difference

between

the

two

treatment

groups

and

a

significant

decrease

in

Trial

progress

-LRB-

P

-RRB-

MS

-LRB-

N

-RRB-

and

SFR

-LRB-

N

-RRB-

The

animals

made

significantly

more

visits

to

the

old

target

hole

-LRB-

Fig.

right

-RRB-

and

the

two

adjacent

holes

over

the

course

of

the

seven

reversal

trials

-LRB-

t

-LRB-

-RRB-

P

t

-LRB-

-RRB-

P

-RRB-

This

tendency

to

venture

to

the

old

goal

and

the

adjacent

holes

could

be

an

indication

of

perseveration

a

behavioural

pattern

also

found

in

other

animal

models

and

in

schizophrenic

patients

-LRB-

Bleuler

Bach

et

al.

review

Crider

-RRB-

Search

strategies

The

animal

's

search

pattern

evolved

over

time

-LRB-

Fig.

-RRB-

The

usual

behaviour

sequence

was

a

progression

from

a

random

to

a

serial

and

finally

to

a

spatial

search

strategy

This

sequence

indicates

that

the

animals

became

more

accurate

and

more

efficient

in

locating

the

correct

hole

Likelihood-ratio

chi-square

tests

showed

that

the

experimental

groups

differed

in

their

choice

of

search

strategy

on

days

and

of

acquisition

training

-LRB-

χ

-LRB-

-RRB-

P

χ

-LRB-

-RRB-

P

respectively

-RRB-

Already

at

this

early

time

the

SFR

animals

adopted

a

serial

search

strategy

while

the

MS

animals

maintained

a

random

search

strategy

On

all

other

acquisition

and

reversal

training

days

the

two

experimental

groups

did

not

differ

significantly

with

respect

to

their

choice

of

search

strategy

Fig.

of

mean

-LRB-

-RRB-

search

strategies

applied

in

the

acquisition

and

reverse

trial

sessions

The

search

pattern

evolved

over

time

from

a

random

to

a

serial

and

finally

to

a

spatial

search

strategy

From

acquisition

days

and

the

SFR

animals

adopted

a

serial

search

strategy

while

the

MS

animals

maintained

a

random

search

strategy

On

all

other

acquisition

and

reversal

training

days

the

two

experimental

groups

did

not

differ

significantly

with

respect

to

their

choice

of

search

strategy

SFR

-LRB-

N

-RRB-

animals

to

the

left

and

MS

-LRB-

N

-RRB-

animals

to

the

right

Neuron

number

The

stereological

sampling

is

shown

in

Table

Table

of

the

stereological

sampling

used

in

this

-LRB-

maternal

separated

-RRB-

Area

-LRB-

frame

-RRB-

-LRB-

-RRB-

Z

depth

-LRB-

μm

-RRB-

and

Y

step

-LRB-

μm

-RRB-

Q

Mean

section

thickness

tq

-LRB-

μm

-RRB-

Height

sampling

fraction

Area

sampling

fraction

Section

sampling

fraction

-LRB-

k

-RRB-

zone

-LRB-

μm

-RRB-

-LRB-

Control

-RRB-

Area

-LRB-

frame

-RRB-

-LRB-

-RRB-

Z

depth

-LRB-

μm

-RRB-

and

Y

step

-LRB-

μm

-RRB-

Q

Mean

section

thickness

tq

-LRB-

μm

-RRB-

Height

sampling

fraction

Area

sampling

fraction

Section

sampling

fraction

-LRB-

k

-RRB-

zone

-LRB-

μm

-RRB-

There

was

no

significant

difference

in

the

total

bilateral

neuron

number

in

hippocampus

-LRB-

Table

-RRB-

However

in

the

five

subregions

of

hippocampus

there

was

a

significant

difference

in

the

dentate

gyrus

-LRB-

P

Students

t

test

-RRB-

but

in

the

four

other

regions

-LRB-

see

Fig.

Table

-RRB-

The

neuron

loss

in

the

dentate

gyrus

is

equivalent

to

a

reduction

in

the

maternal

separated

animals

compared

with

controls

The

results

should

be

interpreted

with

caution

due

to

the

low

number

of

subjects

Table

estimated

neuron

number

in

five

subregions

of

the

hippocampusMaternally

separated

-LRB-

MS

-RRB-

N

-LRB-

-RRB-

CENeurons

-LRB-

-RRB-

CENeurons

-LRB-

-RRB-

CE

Neurons

-LRB-

-RRB-

CE

Neurons

-LRB-

-RRB-

CENeurons

-LRB-

-RRB-

.0842.90.132920.061900.076720.071.5290.09

.1034.80.132430.061890.075760.071.3070.09

.0818.60.183030.062260.064740.081.3930.09

.0720.60.082620.061740.076650.071.2540.07

.0720.20.071550.081630.085520.071.0280.07

.1267.90.132020.071340.084600.081.0530.10

.0523.10.072300.071710.075970.071.1660.07

.0520.90.072290.072030.074840.091.1350.07

.1067.20.122570.061910.074870.091.2390.09

.1037.40.132240.061860.077880.061.4880.09

.0833.20.112290.071740.075380.081.1820.08

CVcontrol

-LRB-

SFR

-RRB-

N

.0726.30.072850.071750.088040.071.4760.07

.0730.20.092400.071910.086580.081.2960.08

.0731.50.072730.072050.076550.081.3330.07

.0525.30.091820.081860.076170.061.2040.08

.0834.10.092780.071990.087250.071.3970.05

.0820.30.092060.081150.096130.091.1120.07

.0628.00.072850.071910.076440.091.3680.07

.0727.90.082500.071800.086740.081.3120.08

Student

's

t

testP

P

P

P

P

P

P

Fig.

total

neuron

number

for

the

two

treatment

groups

in

five

sub-regions

of

the

mouse

hippocampus

for

the

pooled

data

in

MS

-LRB-

N

-RRB-

and

SFR

-LRB-

N

-RRB-

MS

SFR

P

Student

's

t

test

in

the

dentate

gyrus

filled

circle

MS

open

circle

SFR

Sub

Subiculum

CA

Hilus

DG

Dentate

Gyrus

Volume

After

Cavalieri

estimation

of

volume

and

correction

for

shrinkage

-LRB-

-RRB-

a

Student

's

t

test

did

not

show

any

significant

difference

in

either

the

total

hippocampal

volume

or

in

the

five

subregions

of

the

hippocampus

between

groups

-LRB-

Table

-RRB-

Table

estimated

volume

in

five

subregions

of

the

hippocampusMaternally

separated

-LRB-

MS

-RRB-

N

.330.050.690.081.460.051.520.051.800.039.790.05

.380.050.690.061.140.061.390.051.230.047.840.05

.250.050.590.071.540.051.720.041.180.049.290.05

.660.120.600.070.520.091.000.061.060.044.840.08

.400.090.860.060.790.081.260.051.290.046.590.07

.730.120.650.071.070.061.390.051.330.036.170.07

.260.100.690.060.680.091.420.051.220.046.260.07

.290.071.040.081.000.061.260.051.120.066.710.06

.150.070.790.060.910.071.450.051.130.036.430.06

.000.060.740.060.980.071.470.051.130.067.320.06

.980.061.010.061.070.061.390.051.150.057.000.06

.310.050.910.051.220.061.630.042.180.039.250.05

.810.080.770.071.030.071.410.051.320.047.340.06

CVContol

-LRB-

SFR

-RRB-

N

.970.080.780.071.260.061.690.041.560.038.250.06

.290.090.910.071.010.061.220.051.450.036.880.07

.510.090.870.061.160.061.880.041.380.047.820.06

.550.090.520.090.850.071.300.050.730.046.050.07

.100.110.860.070.980.071.480.051.300.046.730.07

.320.100.520.101.030.071.190.051.730.056.780.08

.830.080.700.071.170.051.460.051.580.047.720.06

.510.090.750.081.070.051.460.051.390.047.180.07

Student

's

t

testP

P

P

P

P

P

Discussion

In

the

present

study

h

maternal

separation

on

PND

in

mice

pups

resulted

in

a

neuron

decrease

in

the

dentate

gyrus

and

behavioural

perseverations

in

the

Barnes

maze

The

anxiety

and

thus

stress

related

behavioural

tests

conducted

did

not

show

any

indications

of

an

elevated

anxiety

level

On

the

contrary

the

MS

mice

showed

a

reduced

sign

of

anxiety

since

they

ventured

more

often

onto

the

open

arms

than

the

equivalent

control

group

This

could

indicate

that

the

SHRP

was

not

repressed

in

spite

of

our

expectations

The

results

were

thus

surprising

and

contradicted

some

but

not

all

other

findings

-LRB-

Pihoker

et

al.

Plotsky

and

Meany

Cirulli

et

al.

Wigger

and

Neuman

Boccia

and

Pedersen

Parfitt

et

al.

-RRB-

A

study

by

Lehman

et

al.

-LRB-

-RRB-

found

that

MS

at

PND

did

not

result

in

the

anxiety/fear

response

predicted

by

the

group

-LRB-

Lehmann

et

al.

-RRB-

Others

have

also

found

that

effects

of

a

manipulation

of

the

HPA

axis

can

not

always

be

used

to

predict

effects

of

the

same

manipulation

of

fear/anxiety

expression

at

the

behavioural

level

-LRB-

see

Lehmann

et

al.

Parfitt

et

al.

-RRB-

Furthermore

Parfitt

et

al.

-LRB-

-RRB-

reported

that

maternally

separated

male

mice

had

a

prolonged

increase

in

plasma

CORT

after

an

acute

stressor

but

in

adulthood

showed

no

increased

fear/anxiety

behavioural

response

-LRB-

Parfitt

et

al.

-RRB-

The

lack

of

fear

response

does

evidently

not

necessarily

mean

that

the

corticosterone

plasma

level

is

not

elevated

in

MS

animals

but

since

the

corticosterone

level

was

not

measured

this

question

remains

unanswered

Finally

a

study

by

Francis

et

al.

-LRB-

-RRB-

concluded

that

MS

rats

subjected

to

an

enriched

environment

could

reverse

the

effect

from

MS

on

the

HPA

function

and

anxiety

behaviour

-LRB-

Francis

et

al.

-RRB-

In

conclusion

we

found

that

the

MS

mice

in

our

study

did

not

show

behavioural

indications

on

a

repressed

SHRP

which

might

be

explained

by

the

enriched

environment

the

mice

were

kept

in

The

corticosterone

plasma

level

was

not

measured

In

the

data

presented

for

the

Barnes

maze

there

were

no

significant

differences

between

the

two

treatment

groups

during

the

acquisition

period

Latency

to

reach

the

target

hole

total

distance

moved

on

the

maze

and

error

frequency

all

decreased

significantly

over

the

course

of

the

six

acquisition

trials

and

seven

reversal

trials

indicating

that

the

animals

were

able

to

learn

the

task

with

time

which

is

in

agreement

with

other

studies

-LRB-

e.g.

Pompl

et

al.

-RRB-

One

consequence

of

hippocampal

dysfunction

is

perseveration

-LRB-

Devenport

et

al.

-RRB-

For

the

MS

animals

the

group

differences

in

the

number

of

errors

made

the

goal

parameter

and

the

distance

moved

were

therefore

of

interest

The

differences

found

in

the

parameter

Close

to

Old

Goal

the

significantly

higher

frequency

of

Errors

and

visits

to

Old

Goal

by

the

MS

animals

indicate

that

the

MS

animals

were

more

perseverant

which

could

point

to

a

hippocampal

lesion

-LRB-

Devenport

et

al.

Bach

et

al.

-RRB-

However

since

perseveration

is

also

a

feature

of

prefrontal

cortical

dysfunction

and

there

was

no

hippocampal

dysfunction

in

the

acquisition

trials

we

can

only

conclude

that

the

behaviour

in

reversal

trial

may

be

related

to

hippocampus

but

we

can

not

exclude

that

it

is

was

caused

by

e.g.

a

prefrontal

cortical

deficit

One

of

the

advantages

of

the

Barnes

maze

is

its

ability

to

reveal

the

search

strategies

applied

by

the

mice

The

search

strategies

can

be

an

indication

of

how

well

the

cognitive

abilities

in

the

mice

are

due

to

the

use

of

extra-maze

cues

-LRB-

Barnes

Holmes

et

al.

-RRB-

In

this

study

an

overall

search

pattern

evolved

with

time

On

initial

trials

the

mice

tended

to

use

a

random

pattern

and

explore

many

incorrect

holes

often

returning

to

the

centre

after

investigating

the

edge

of

the

platform

With

more

experience

the

number

of

centre

crossings

decreased

and

a

more

systematic

search

was

applied

Thus

in

later

trials

many

mice

went

directly

to

the

correct

hole

or

one

or

two

holes

away

before

locating

the

hidden

box

The

mice

learned

the

task

better

after

a

previous

introduction

to

the

platform

and

used

the

Spatial

search

strategy

earlier

Furthermore

the

SFR

mice

used

the

search

strategy

Serial

significantly

faster

than

the

MS

mice

in

the

acquisition

trials

but

this

was

not

true

in

the

reversal

trials

The

serial

search

strategy

requires

the

mouse

to

use

the

multiple

relationships

among

extra-maze

stimuli

to

find

the

escape

tunnel

-LRB-

Barnes

Bach

et

al.

-RRB-

So

apparently

the

MS

mice

needed

more

time

to

acquire

to

the

spatial

search

strategy

when

learning

the

task

which

could

indicate

a

learning

disability

However

since

the

MS

mice

did

not

show

difficulties

in

using

the

spatial

search

strategy

in

the

reverse

trials

no

firm

conclusions

can

be

made

on

this

point

Over

of

granule

cell

neurogenesis

are

previously

described

to

occur

postnatally

in

the

rodent

with

peak

neurogenesis

between

PND

and

and

total

cell

number

increasing

throughout

the

first

year

and

continuing

throughout

life

-LRB-

Altman

and

Das

Schlessinger

et

al.

Bayer

et

al.

Kuhn

et

al.

Kempermann

et

al.

-RRB-

Much

research

has

focused

on

the

neurogenesis

in

both

the

adult

and

newborn

hippocampus

of

mammals

-LRB-

Kempermann

et

al.

Gould

et

al.

Eriksson

et

al.

Tanapat

et

al.

Gould

et

al.

Lemaire

et

al.

Malberg

et

al.

Raber

et

al.

Mirescu

et

al.

Greisen

et

al.

-RRB-

but

only

a

few

of

these

studies

have

used

modified

stereological

methods

-LRB-

Kempermann

et

al.

Eriksson

et

al.

Gould

et

al.

Lemaire

et

al.

Malberg

et

al.

Greisen

et

al.

-RRB-

of

these

previous

studies

have

quantified

the

total

cell

numbers

in

the

hippocampus

using

stereology

in

early

trauma

animal

models

or

in

humans

The

model

can

apparently

induce

a

neuron

change

in

the

hippocampus

but

whether

the

lower

neuron

number

is

a

decrease

due

to

a

neuron

loss

or

because

neurogenesis

has

been

affected

in

the

peak

period

could

not

be

determined

in

this

study

Secondly

the

impact

of

the

neuron

loss

is

not

immediately

linked

to

a

possible

memory

deficit

Even

though

the

MS

animals

had

a

reduced

emotional

responds

in

the

elevated

plus

maze

and

showed

perseverance

in

the

Barnes

maze

the

cognitive

deficits

could

just

as

well

be

due

to

a

prefrontal

damage

and

thus

not

a

result

of

the

decreased

neuron

numbers

in

DG

We

tested

if

a

maternal

separation

on

PND

can

cause

an

adult

phenotype

characterized

by

altered

levels

of

activity

and

anxiety

learning

and

memory

dysfunction

deficits

in

behavioural

flexibility

-LRB-

reversal

deficits

-RRB-

as

well

as

changes

in

number

of

neurons

in

the

hippocampus

and

its

subregions

in

the

mouse

brain

We

found

that

a

single

h

maternal

separation

on

PND

could

elicit

a

reduced

stress

response

in

the

elevated

plus

maze

and

induce

perseveration

behaviour

in

the

Barnes

maze

Further

a

reduction

in

total

neuron

numbers

was

found

in

the

dentate

gyrus

of

the

hippocampus

Ethics

The

experiment

was

carried

out

in

accordance

with

the

European

Communities

Council

Directive

of

November

-LRB-

-RRB-

and

the

Danish

legislation

regulating

animal

experiments

-LRB-

Animal

care

and

housing

BEK

nr

from

-RRB-

The

Danish

Animal

Experiments

Inspectorate

approved

the

protocols

-LRB-

Journal

No.

-RRB-

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

maternal separation
behaviour
hippocampus
stereology


Microsurgical third ventriculocisternostomy as an alternative to ETV : report of two cases Objective To describe a microsurgical alternative to endoscopic third ventriculocisternostomy . Introduction In the last two decades , endoscopic third ventriculocisternostomy -LRB- ETV -RRB- via a precoronal burr hole and transfrontal approach became a good therapeutic alternative to shunt placing and in non-communicating hydrocephalus , ETV has become the first choice of treatment -LSB- 29 -RSB- . Also , ETV is more and more offered to patients with shunt at the time of shunt malfunction or in case of slit-ventricle syndrome -LRB- SVS -RRB- as a means to become shunt independent . However , in a subpopulation of patients , ETV is technically not feasible due to small ventricular system or slit ventricles . Patients with such small ventricles and multiple shunt malfunctions possess a highly therapeutic challenge . Other means of internal shunting , as an alternative to ventriculoperitoneal or ventriculoatrial shunting , have been described in the decades before ETV became a routine procedure . Third ventriculocisternostomy -LRB- TVS -RRB- has been performed by subtemporal craniotomy -LSB- 5 -RSB- , anterior subfrontal approach -LSB- 6 , 7 , 24 -RSB- , Torkildsen ventriculocisternostomy -LSB- 26 , 27 -RSB- , microsurgical ventriculocisternostomy -LSB- 17 , 20 -RSB- , stereotactic ventriculocisternostomy -LSB- 4 , 13 , 19 -RSB- , fluoroscopic ventriculocisternostomy -LSB- 10 -- 12 -RSB- , microsurgical opening of the lamina terminalis -LSB- 21 -RSB- , reconstruction of or stent placement in the aqueduct -LSB- 15 -RSB- , and endoscopic aqueductoplasty -LSB- 22 -RSB- . TVS by lamina terminalis fenestration is still a routine procedure in aneurysm surgery for subarachnoid hemorrhage , especially in case of anterior communicating artery aneurysms . However , the possibility of a microsurgical TVS -LRB- MTV -RRB- for obstructive hydrocephalus seems to be lost to oblivion by the widespread use of ETV . In this report , we describe two patients in whom an ETV could not be performed because of slit ventricles and who became shunt independent after MTV by a minimally invasive supraorbital approach . Case reports Case 1 This eight-year-old boy was born with an occipital meningocele and a thoracolumbar myelomeningocele . Both of these congenital abnormalities were operated upon 1 day after birth . Within several days , the patient developed a hydrocephalus which was treated with a ventriculoperitoneal shunt . One month later , a Chiari type II malformation was treated by a suboccipital craniotomy and a duraplasty . Since then , the patient experienced multiple drain infections and both proximal and distal drain dysfunctions . Ventriculoperitoneal shunting and ventriculoatrial shunting with different types of valves were implanted but none of them led to problem-free shunting , largely due to stiff slit ventricles . In March 2003 , the patient experienced another drain dysfunction with an intracranial pressure -LRB- ICP -RRB- of 50 cm H2O while maintaining slit ventricles followed by infection necessitating external ventricular drainage and antibiotics treatment . In search for a more definitive treatment of his recurrent drain dysfunctions , we considered to perform an ETV . However , the very narrow slit-like lateral ventricles and third ventricle did not allow a safe execution of this procedure -LRB- Fig. 1a -- c -RRB- . As an alternative , a microsurgical fenestration of the lamina terminalis and Liliequist 's membrane was performed via a right-sided eyebrow incision and a small supraorbital craniotomy -LRB- Fig. 2 -RRB- . The postoperative course was uneventful . The patient did not experience any symptoms suspect for hydrocephalus or raised intracranial pressure during a follow-up of 3 years . MRI control examination did not reveal a significant increase in the size of the ventricular system -LRB- Fig. 1d -- f -RRB- . Fig. 1Case report 1 . a , b Axial T1 MRI depicting very small slit-like lateral and third ventricles , c sagittal T2 MRI showing small third ventricle and lamina terminalis , d , e postoperative axial T1 MRI showing minimal increase in size of the lateral ventricles ; f postoperative sagittal T2 MRI shows a minimal increase of the third ventricle , but no flow void phenomenon at the lamina terminalisFig . 2Postoperative photograph of case 1 showing the hardly visible scar of the eyebrow incision Case 2 This 16-year-old boy , born with a lumbosacral meningocele and shunt dependent from birth on , developed with 13 years of age an increasing overdrainage syndrome with positional headaches . In another hospital , many valve revisions were performed in order to solve this problem and to rule out underdrainage . The original differential pressure valve was exchanged over a period of 3 years for several different Delta valves and a Codman Medos programmable valve , with which practical all settings were tried . Also , a shunt explantation was tried but after initial improvement , this led to a secondary increase of headaches and papilledema within several weeks , after which again , a Codman Medos valve was inserted . From this procedure , we obtained the security that the patient was still shunt dependant at that age . A new ICP monitoring was performed in the other hospital which showed negative intracranial pressures in upright sitting position and an ICP of 5 -- 10 cm H2O in the supine position . The headache continued , even seemed to increase and became also chronic with positional increase of headaches in the upright position . The largest problem , however , became the frequent absenteeism from school because of these headaches . Chronic consultation of a pediatric neurologist and conservative treatment measures did not alter the symptoms either . At this time , he was referred to our department . As a first measure , a PaediGav 9/29 gravitational valve was applied because we had gained good experience with this type of valve but without any positive effect on the headaches . Although the patient did not improve after many shunt upgrading , it was felt that his headaches most likely were to be attributed to chronic overdrainage . Therefore , we sought for a means to have the patient shunt independent -LRB- after proven shunt dependency at the time -RRB- . Because an ETV was considered to be impossible , a right supraorbital craniotomy with a lamina terminalis fenestration and fenestration of Liliequist 's membrane was executed and the shunt was completely removed , except for the ventricular catheter that was fixated and therefore left behind . Again , no significant change of the headache could be observed . A 24-h continuous ICP monitoring , performed after 6 weeks because of unchanged symptoms , revealed a normal ICP in supine and upright positions with a maximum nightly increase to 13 mm Hg . Two years after surgery , the patient is still shunt independent with chronic headaches , no papilledema -LRB- contrary to the first shunt explantation before MTV -RRB- , and in a rehabilitation program . A computed tomography -LRB- CT -RRB- scan 1.5 years after surgery shows a minimal increase in the size of the ventricles -LRB- Fig. 3 -RRB- . Fig. 3Case report 2 . a , b Preoperative CT scan depicting slit-like ventricles ; c , d postoperative CT scan with minimal increase of ventricle size and a residual ventricular catheter that could not be removed Discussion There are many complications related to shunt dependency . In 10.9 -- 29 % of cases complications -LRB- other than mechanical obstruction -RRB- occur , the majority of which is a shunt infection -LSB- 9 , 14 , 18 -RSB- . Mechanical complications in shunts including late shunt malfunction may even occur in up to 45.9 % in the first postoperative year and up to 81 % of patients at 12 years follow-up -LSB- 8 , 9 , 18 -RSB- . Also , the long-term shunt-related mortality rate of the shunt-treated patient , despite many improvements in shunt technique , diagnostics , and follow-up , remains high -LRB- 2.9 % to 12.4 % at 10 years follow-up -RRB- -LSB- 10 , 16 , 30 -RSB- . Therefore , the ultimate goal in hydrocephalus therapy is to get the patient shunt free . Since the first experience by Guiot -LSB- 10 -RSB- , transcortical or transventricular endoscopic third ventriculocisternostomy has proved to be a valid alternative in the treatment of obstructive hydrocephalus . Also , several studies have shown that ETV is an effective treatment in patients with obstructive hydrocephalus who had undergone previous shunting procedures -LSB- 1 , 23 , 25 -RSB- . It can provide a definitive cure for patients with difficult-to-manage shunt dysfunction . The potential advantages of ETV over conventional shunting are clear . However , ETV requires a certain degree of lateral and third ventricle dilatation to be performed safely . In case of small ventricles , the potential risks of this procedure -LRB- injuring fornix , thalamostriate or internal cerebral veins , hypothalamus -RRB- outweigh the advantages . One might consider to enlarge the ventricles and , thus , to facilitate an ETV . This has been safely and successfully performed by Butler and Khan -LSB- 2 -RSB- with an external ventricular drain and by Chernov et al. -LSB- 3 -RSB- by using a programmable valve of which the pressure was increased in a stepwise fashion . In our department , we have practised both techniques for ventricular dilatation successfully , but we also encountered serious complications from this strategy . In both cases described here , we already knew from previous shunt malfunctions that the ventricles hardly dilate with increased intracranial pressure . A shunt revision in case 1 for proximal catheter obstruction only several months before the MTV revealed an intracranial pressure of 50 cm H2O with only minimal ventricular dilatation . Therefore , in our two patients , we opted to perform an open microsurgical TVS by opening the lamina terminalis via a small supraorbital craniotomy and did not consider achieving ventricular expansion . Both patients became shunt independent . Open surgical TVS once was a popular therapeutic technique for the treatment of obstructive hydrocephalus before the advent of VP shunt systems . The idea of third ventricular communication into the chiasmatic cisterns was initially proposed by Dandy -LSB- 7 -RSB- in 1922 as a surgical procedure to alleviate obstructive hydrocephalus . It was first performed by Dandy via a subfrontal approach and frequently required the sacrifice of one optic nerve and elevating the chiasm . Later , Dandy -LSB- 5 -RSB- described the alternative subtemporal approach . In 1936 , Stookey and Scarff -LSB- 24 -RSB- described a subfrontal anterior approach for TVS through the lamina terminalis and the hypothalamus to the lumen of the third ventricle . Scarff -LSB- 21 -RSB- published a review of 527 patients subjected to this operation by both the subfrontal and subtemporal procedures , as reported by many authors . The over-all operative mortality was 15 % and the permanent arrest of the hydrocephalus was obtained in 70 % of the survivors . Open surgical TVS for obstructive hydrocephalus was then abandoned due to better and reliable shunting systems and because the advent of endoscopic techniques of this procedure is not reported to be in widespread use . Nevertheless , it remained a routine procedure during aneurysm surgery in order to get more slack ventricles and to prevent , or decrease the number of , posthemorrhagic hydrocephalus . To the best of our knowledge , MTV , by opening the lamina terminalis for the treatment of hydrocephalus , was last reported in 1988 . In this report from Reddy et al. -LSB- 17 -RSB- , five shunted patients with aqueduct stenosis and slit ventricle syndrome described are treated successfully with MTV via opening of the lamina terminalis . The lamina terminalis was approached in all patients via a classic pterional craniotomy . Since then , reports on ETV dominate the literature . Operative technique Many neurosurgeons consider craniotomy and MTV too large and risky a procedure . However , the procedure described in this report is a truly minimally invasive procedure . The operation time is about 1 h , the wound and scar are minimal , recuperation is very fast , and the potential risks are very low . One might even suggest that this procedure is safer than an ETV because it has an extracerebral approach , in contrast to ETV , and that surgery is better controlled with more opportunities for hemostasis . The supraorbital craniotomy with eyebrow incision has been described previously -LSB- 28 -RSB- . The Sylvian fissure needs not to be dissected . The ipsilateral optic nerve and the optic chiasm , the ICA and the ipsilateral ACA as well as the ACoA are exposed after which the lamina terminalis is reached . Lamina terminals fenestration is a standard procedure easily performed with the bipolar forceps or an arachnoid diamond knife . This , however , is not sufficient . It is essential to open Liliequist 's membrane to allow free and sufficient CSF communication between the posterior fossa and the suprasellar and frontobasal cisterns . Recovery of this operative procedure is fast and without major side-effects . Usually , hospitalization time is 2 to 3 days . Conclusion In conclusion , open microsurgical third ventriculocisternostomy by opening the lamina terminalis should be kept in mind as an alternative technique for treating patients with multiple shunt failures and small ventricles not accessible for an endoscopic approach in an effort to make these patients shunt independent .

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supraorbital approach
third ventriculocisternostomy
lamina terminalis
minimally invasive