Femoroacetabular

impingement

in

professional

athletes

associated

pathologies

and

return

to

sport

following

arthroscopic

decompression

Femoroacetabular

impingement

-LRB-

FAI

-RRB-

occurs

when

an

osseous

abnormality

of

the

proximal

femur

-LRB-

cam

-RRB-

or

acetabulum

-LRB-

pincer

-RRB-

triggers

damage

to

the

acetabular

labrum

and

articular

cartilage

in

the

hip

Although

the

precise

etiology

of

FAI

is

not

well

understood

both

types

of

FAI

are

common

in

athletes

presenting

with

hip

pain

loss

of

range-of-motion

and

disability

in

athletics

An

open

surgical

approach

to

decompressing

FAI

has

shown

good

clinical

outcomes

however

this

highly

invasive

approach

inherently

may

delay

or

preclude

a

high

level

athlete

's

return

to

play

The

purpose

of

this

study

was

to

define

associated

pathologies

and

determine

if

an

arthroscopic

approach

to

treating

FAI

can

allow

professional

athletes

to

return

to

high-level

sport

Hip

arthroscopy

for

the

treatment

of

FAI

allows

professional

athletes

to

return

to

professional

sport

Between

October

and

September

professional

athletes

underwent

hip

arthroscopy

for

the

decompression

of

FAI

Operative

and

return-to-play

data

were

obtained

from

patient

records

Average

time

to

follow-up

was

years

-LRB-

range

months

to

years

-RRB-

Forty

two

-LRB-

-RRB-

athletes

returned

to

professional

competition

following

arthroscopic

decompression

of

FAI

Three

athletes

did

not

return

to

play

however

all

had

diffuse

osteoarthritis

at

the

time

of

arthroscopy

Thirty-five

athletes

-LRB-

-RRB-

remain

active

in

professional

sport

at

an

average

follow-up

of

years

Arthroscopic

treatment

of

FAI

allows

professional

athletes

to

return

to

professional

sport

Introduction

In

publications

dating

back

several

decades

irregularities

in

the

morphology

of

the

femoroacetabular

articulation

have

been

implicated

as

a

possible

source

of

hip

pain

in

young

active

athletes

A

French

article

published

in

described

hip

pain

associated

with

structural

abnormalities

of

the

proximal

femoral

neck

in

athletes

participating

in

hockey

football

soccer

rugby

martial

arts

and

tennis

-LSB-

-RSB-

More

recently

developments

pioneered

by

open

hip

surgeons

have

shown

that

morphologic

abnormalities

of

both

the

femur

and

acetabulum

underlie

a

large

number

of

labral

and

chondral

injuries

in

the

hip

-LSB-

-RSB-

Wenger

et

al.

-LSB-

-RSB-

noted

the

presence

of

osseous

abnormalities

including

dysplasia

and

femoroacetabular

impingement

-LRB-

FAI

-RRB-

in

the

majority

of

patients

with

labral

tears

This

suggests

that

isolated

treatment

of

soft

tissue

pathologies

may

not

be

adequate

without

concomitantly

addressing

underlying

structural

abnormalities

The

concept

of

FAI

has

been

defined

by

Ganz

and

colleagues

-LSB-

-RSB-

In

this

condition

a

structural

or

spatial

abnormality

of

the

femur

-LRB-

cam

-RRB-

or

acetabulum

-LRB-

pincer

-RRB-

damages

the

chondrolabral

structures

during

normal

joint

movement

The

most

common

situation

is

a

mixed

cam

and

pincer

pathology

occurring

along

the

anterior

femoral

neck

and

the

anterior

superior

acetabular

rim

In

high

flexion

and

internal

rotation

movements

abutment

and

impingement

of

the

labrum

and

cartilage

occurs

The

precise

etiology

of

FAI

is

not

well

understood

Sub-clinical

slipped

capital

femoral

epiphyses

-LSB-

-RSB-

mal-union

of

femoral

neck

fractures

-LSB-

-RSB-

and

decreased

femoral

anteversion

-LSB-

-RSB-

are

described

causes

of

cam

impingement

Relative

posterior

opening

of

the

acetabulum

-LRB-

acetabular

retroversion

-RRB-

or

global

-LRB-

coxa

profunda

-RRB-

overcoverage

of

the

femur

by

the

acetabulum

are

described

causes

of

pincer

impingement

-LSB-

-RSB-

Cam

and

pincer

lesions

lead

to

distinct

patterns

of

labral

and

chondral

damage

-LSB-

-RSB-

and

long-standing

impingement

is

likely

a

significant

cause

of

previously

described

idiopathic

hip

joint

degeneration

-LSB-

-RSB-

Ganz

et

al.

developed

an

open

surgical

dislocation

approach

to

decompress

FAI

-LSB-

-RSB-

This

technique

has

demonstrated

good

results

of

FAI

decompression

in

a

general

population

-LSB-

-RSB-

However

we

believe

that

an

arthroscopic

approach

involves

less

post-operative

morbidity

and

allows

patients

including

professional

athletes

to

return

to

high-functioning

lifestyles

-LSB-

-RSB-

The

purpose

of

this

study

was

to

define

associated

pathologies

and

determine

if

professional

athletes

could

return

to

high-level

athletics

following

arthroscopic

decompression

of

FAI

Methods

Patient

selection

A

retrospective

chart

review

was

performed

of

all

professional

athletes

presenting

for

arthroscopic

treatment

of

FAI

by

the

senior

author

between

October

and

September

All

athletes

presented

with

debilitating

hip

pain

and

an

inability

to

participate

in

their

sport

All

were

diagnosed

with

FAI

based

on

physical

and

radiographic

examination

Physical

examination

criteria

included

a

positive

impingement

test

-LSB-

-RSB-

or

flexion

abduction

external

rotation

-LRB-

FABER

-RRB-

test

A

positive

impingement

test

was

defined

as

groin

pain

with

degrees

of

hip

flexion

and

maximal

internal

rotation

A

positive

FABER

test

was

defined

as

asymmetry

in

the

distance

between

the

lateral

knee

and

the

exam

table

between

the

injured

hip

and

the

non-injured

hip

Radiographic

criteria

included

decreased

anterior

and

superior

femoral

head

neck

offset

acetabular

retroversion

-LRB-

as

defined

by

a

positive

cross-over

sign

-RRB-

or

coxa

profunda

-LRB-

as

defined

by

the

medial

acetabular

border

overlapping

the

ilioischial

line

-RRB-

For

these

athletes

with

documented

physical

and

radiographic

evidence

of

FAI

conservative

treatment

was

limited

to

only

weeks

Based

on

evidence

described

by

Beck

and

colleagues

-LSB-

-RSB-

our

thought

was

that

conservative

treatment

could

not

address

the

underlying

bony

abnormalities

and

any

further

treatment

delay

in

these

highly

active

patients

would

likely

precipitate

further

irreversible

damage

to

the

articular

cartilage

Inclusion

criteria

for

this

study

included

professional

athletes

with

at

least

one

positive

physical

exam

finding

at

least

one

positive

radiographic

finding

and

failure

of

at

least

weeks

of

conservative

therapy

Forty-five

professional

athletes

from

various

sports

met

the

inclusion

criteria

and

were

included

in

the

study

-LRB-

Table

-RRB-

There

were

male

and

female

patients

Table

sports

activities

of

the

professional

athletesSportNo

of

Arthroscopic

procedure

The

modified

supine

approach

to

hip

arthroscopy

was

used

-LSB-

-RSB-

Two

portals

-LRB-

anterior

anterolateral

-RRB-

were

established

and

a

third

portal

-LRB-

distal

lateral

accessory

-RRB-

was

established

as

needed

-LSB-

-RSB-

At

the

time

of

arthroscopy

the

central

compartment

of

the

hip

was

first

inspected

Pincer

lesions

were

typically

identified

in

the

superior

acetabular

quadrant

For

the

treatment

of

small

pincer

lesions

-LRB-

to

mm

-RRB-

osteoplasty

of

the

proximal

femoral

neck

was

occasionally

sufficient

to

relieve

the

impingement

-LSB-

-RSB-

Patients

with

moderate

or

large-sized

lesions

underwent

acetabular

rim

trimming

using

an

arthroscopic

osteotome

or

mm

round

motorized

burr

-LSB-

-RSB-

Acetabular

labral

tears

were

arthroscopically

identified

and

based

on

previous

research

on

the

vascularity

of

the

labrum

-LSB-

-RSB-

detached

and

peripheral

midsubstance

tears

were

typically

repaired

-LSB-

-RSB-

Care

was

taken

to

preserve

as

much

healthy

viable

labrum

as

possible

however

degenerative

or

frayed

tissue

was

debrided

to

a

stable

remnant

Labral

repair

was

performed

with

suture

anchors

to

repair

detached

labral

tears

or

to

re-fix

the

labrum

following

iatrogenic

detachment

for

complete

resection

of

pincer

lesions

The

previously

described

technique

involved

placement

of

the

anchors

high

on

the

acetabular

rim

in

the

area

of

detachment

-LSB-

-RSB-

Typically

one

bioabsorbable

anchor

-LRB-

BioRaptor

Smith

Nephew

Andover

MA

USA

-RRB-

was

placed

at

the

acetabular

position

and

re-inforcement

was

placed

either

anteriorly

or

posteriorly

of

this

area

as

needed

One

limb

of

suture

-LRB-

Ultrabraid

Smith

Nephew

Andover

MA

USA

-RRB-

was

passed

between

the

labral

tissue

and

the

rim

and

was

retrieved

through

the

substance

of

the

labral

tissue

In

cases

of

inadequate

availability

of

labral

substance

or

in

patients

with

highly

degenerative

friable

tissue

the

suture

was

passed

around

the

labral

tissue

Standard

arthroscopic

knots

fixed

the

repair

to

the

rim

For

midsubstance

labral

tears

a

suture

passer

looped

an

around

the

torn

tissue

to

approximate

the

edges

of

the

midsubstance

split

The

condition

of

the

articular

cartilage

was

evaluated

according

to

the

Outerbridge

Classification

System

-LSB-

-RSB-

For

this

study

the

Outerbridge

Classification

System

was

defined

as

follows

grade

normal

cartilage

grade

cartilage

with

softening

and

swelling

grade

a

partial

thickness

defect

with

fissures

on

the

surface

that

do

not

reach

the

subchondral

bone

or

exceed

cm

in

diameter

grade

fissuring

to

the

level

of

the

subchondral

bone

in

an

area

with

a

diameter

more

than

cm

and

grade

IV

exposed

subchondral

bone

-LSB-

-RSB-

Chondral

procedures

performed

in

this

group

included

chondroplasty

and

microfracture

of

lesions

of

the

femoral

head

and

acetabulum

as

needed

Following

inspection

of

the

central

compartment

traction

was

released

and

the

peripheral

compartment

was

evaluated

for

cam

lesions

A

dynamic

examination

of

the

hip

in

all

motion

planes

allowed

for

direct

visualization

of

impingement

at

the

femoroacetabular

interface

For

the

treatment

of

cam

lesions

an

osteoplasty

was

performed

with

a

mm

round

motorized

burr

restoring

the

anatomic

concavity

of

the

femoral

head

neck

junction

-LSB-

-RSB-

Burring

was

performed

from

superior

to

inferior

along

the

anterior

femoral

head

neck

junction

to

a

depth

of

approximately

mm

and

a

width

of

approximately

mm

Care

was

taken

to

avoid

resecting

too

distally

along

the

femoral

neck

to

avoid

the

lateral

epiphyseal

vessels

A

final

dynamic

examination

was

performed

to

confirm

adequate

decompression

and

a

lack

of

entrapment

of

the

labrum

during

joint

motion

In

general

the

above-described

procedure

took

approximately

h

depending

on

the

degree

of

labral

and

chondral

pathology

Traction

time

was

limited

to

less

than

h

to

reduce

the

risk

of

neurovascular

compromise

Post-operative

management

Post-operative

medical

prophylaxis

included

an

aspirin

or

a

low-molecular

weight

heparin

a

non-steroidal

anti-inflammatory

and

an

antibiotic

Each

of

the

athletes

underwent

post-operative

rehabilitation

according

to

the

following

generalized

protocol

that

was

devised

based

on

personal

experience

For

basic

procedures

involving

decompression

of

FAI

and

labral

treatment

weightbearing

was

restricted

to

pounds

flatfoot

for

weeks

A

brace

was

prescribed

to

be

worn

for

days

to

protect

the

hip

and

limit

abduction

and

rotation

Continuous

passive

motion

-LRB-

CPM

-RRB-

was

used

to

apply

degrees

of

hip

flexion

for

up

to

h

per

day

for

weeks

Night

boots

were

worn

for

ten

post-operative

days

to

limit

rotation

during

sleep

If

capsular

modification

procedures

-LRB-

thermal

capsulorrhaphy

or

capsular

plication

-RRB-

were

performed

rotation

precautions

were

extended

for

a

total

of

days

to

avoid

capsular

stress

If

microfracture

was

performed

weightbearing

restrictions

and

CPM

use

were

extended

for

a

total

of

weeks

Physical

therapy

exercises

were

implemented

within

four

post-operative

hours

It

is

our

belief

based

on

clinical

observation

that

this

in

addition

to

an

early

emphasis

on

passive

hip

motion

-LRB-

particularly

internal

rotation

-RRB-

reduces

the

incidence

of

adhesion

formation

Active

hip

flexion

was

limited

for

weeks

based

on

clinical

observation

to

minimize

the

risk

of

hip

flexor

tendonitis

Athletes

were

typically

allowed

to

return

to

full

competitive

activity

between

and

post-operative

weeks

Return-to-play

statistics

were

determined

by

retrospective

chart

review

and

personal

follow-up

communication

with

the

athletes

Results

The

average

age

of

the

athletes

at

the

time

of

surgery

was

years

-LRB-

range

-RRB-

Eleven

athletes

-LRB-

-RRB-

previously

underwent

hip

arthroscopy

by

multiple

primary

surgeons

for

the

treatment

of

labral

and

chondral

pathologies

and

experienced

a

recurrence

of

hip

symptoms

The

average

time

to

follow-up

was

years

-LRB-

range

months

to

years

-RRB-

Twenty-two

athletes

-LRB-

-RRB-

were

treated

for

an

isolated

cam

lesion

and

three

athletes

-LRB-

-RRB-

were

treated

for

an

isolated

pincer

lesion

Twenty-one

athletes

-LRB-

-RRB-

had

a

mixed

pathology

of

both

cam

and

pincer

lesions

All

of

the

athletes

had

acetabular

labral

tears

Twenty-five

patients

-LRB-

-RRB-

underwent

either

labral

repair

or

re-fixation

following

rim

trimming

with

suture

anchors

-LRB-

average

anchors

per

patient

range

-RRB-

Twelve

patients

-LRB-

-RRB-

had

intra-substance

labral

repair

Five

patients

had

labral

debridement

one

patient

had

a

detached

tear

one

had

frayed

labral

tissue

and

three

patients

had

complex

tears

consisting

of

detached

frayed

and

flap

components

Two

patients

were

labral

deficient

from

a

previous

arthroscopic

debridement

They

underwent

labral

grafting

using

an

iliotibial

band

autograft

Twenty-one

patients

-LRB-

-RRB-

had

a

grade

IV

acetabular

chondral

defect

underwent

arthroscopic

microfracture

underwent

thermal

chondroplasty

and

patients

had

no

treatment

due

to

the

diffuseness

of

their

disease

Three

patients

-LRB-

-RRB-

had

a

grade

IV

femoral

head

chondral

defect

one

was

treated

with

microfracture

one

with

chondroplasty

and

one

patient

had

no

treatment

due

to

the

diffuseness

of

his

disease

Seventeen

-LRB-

-RRB-

had

a

grade

I

III

acetabular

chondral

defect

-LRB-

treated

with

chondroplasty

and

treated

with

microfracture

-RRB-

and

-LRB-

-RRB-

had

a

grade

I

III

femoral

head

chondral

defect

-LRB-

all

were

treated

with

chondroplasty

-RRB-

Four

patients

-LRB-

one

baseball

player

one

football

player

one

hockey

player

and

golfer

-RRB-

had

extensive

diffuse

OA

at

the

time

of

arthroscopy

but

opted

to

delay

arthroplasty

Twenty-six

patients

-LRB-

-RRB-

had

a

partial

tear

of

the

ligamentum

teres

and

patients

-LRB-

-RRB-

had

a

complete

ligamentum

teres

avulsion

Thirteen

-LRB-

-RRB-

patients

underwent

thermal

capsulorrhaphy

and

-LRB-

-RRB-

underwent

plication

for

capsular

redundancy

Seventeen

patients

-LRB-

-RRB-

had

loose

bodies

in

the

joint

and

two

patients

required

excision

of

myositis

ossificans

of

the

rectus

femoris

Three

patients

-LRB-

-RRB-

underwent

cheilectomy

of

a

stenotic

cotyloid

fossa

and

patients

-LRB-

-RRB-

underwent

arthroscopic

release

of

a

tight

iliopsoas

Figure

illustrates

restoration

of

femoral

head

neck

concavity

in

an

athlete

treated

for

cam

impingement

-LRB-

Fig.

-RRB-

and

Fig.

illustrates

successful

removal

of

the

anterior

acetabular

overhang

in

an

athlete

treated

for

pincer

impingement

-LRB-

Fig.

-RRB-

Fig.

professional

hockey

player

was

evaluated

for

right

hip

pain

a

A

pre-operative

cross-table

lateral

radiograph

demonstrated

convexity

at

the

anterior

femoral

head

neck

junction

b

The

presence

of

a

cam

lesion

was

verified

arthroscopically

The

femoral

head

-LRB-

FH

-RRB-

and

acetabulum

-LRB-

Ac

-RRB-

are

visualized

in

the

peripheral

hip

compartment

with

the

camera

in

the

anterior

portal

c

A

motorized

burr

was

used

to

restore

anterior

concavity

at

the

head

neck

junction

This

was

visualized

at

the

superior

acetabular

-LRB-

Ac

-RRB-

position

-LRB-

-RRB-

with

the

camera

in

the

anterior

portal

d

A

post-operative

film

verified

successful

decompression

of

the

cam

impingement

and

restoration

of

the

femoral

head

neck

junction

concavityFig

professional

football

player

was

evaluated

for

right

hip

pain

a

A

pre-operative

AP

radiograph

demonstrated

a

cross-over

sign

of

the

right

acetabulum

indicating

acetabular

retroversion

b

The

presence

of

a

pincer

lesion

was

verified

arthroscopically

with

the

camera

in

the

anterolateral

portal

with

excessive

bone

along

the

anterior

superior

acetabulum

-LRB-

Ac

-RRB-

c

A

motorized

burr

in

the

anterior

portal

resected

the

lesion

with

the

burr

shield

placed

against

the

labrum

-LRB-

L

-RRB-

for

protection

d

The

labrum

was

fixed

back

to

the

rim

using

two

suture

anchors

e

A

post-operative

film

verified

the

successful

removal

of

pincer

impingement

and

lack

of

retroversion

in

the

superior

region

of

the

acetabulum

Forty-two

athletes

-LRB-

-RRB-

returned

to

professional

sport

following

hip

arthroscopy

Three

players

-LRB-

football

player

hockey

player

and

baseball

player

-RRB-

did

not

return

to

play

following

arthroscopy

Each

of

these

patients

had

diffuse

osteoarthritis

at

the

time

of

arthroscopy

Five

athletes

-LRB-

-RRB-

required

re-operation

Three

underwent

lysis

of

adhesions

and

two

had

symptomatic

treatment

of

extensive

osteoarthritis

All

of

the

patients

who

underwent

revision

surgery

for

lysis

of

adhesions

returned

to

professional

play

and

the

two

with

extensive

osteoarthritis

did

not

return

to

play

Thirty-five

of

the

athletes

-LRB-

-RRB-

remained

active

at

the

professional

level

at

an

average

of

years

after

hip

arthroscopy

Discussion

Intra-articular

hip

injuries

are

a

recognized

cause

of

pain

mechanical

symptoms

and

disability

in

athletes

Traumatic

intra-articular

injury

results

from

acute

injury

including

hyperabduction

injuries

direct

hip

contact

and

joint

dislocation

or

subluxation

Atraumatic

hip

injury

however

is

less

well

understood

Various

motions

exerted

during

sport

particularly

flexion

combined

with

internal

rotation

-LRB-

hockey-goalie

stance

-RRB-

have

been

suggested

as

potential

causes

of

overuse

hip

injury

-LSB-

-RSB-

In

this

position

a

cam

lesion

extending

anteriorly

or

superiorly

from

the

proximal

femoral

neck

would

impinge

at

the

anterosuperior

acetabular

rim

Combining

this

understanding

with

recent

reports

by

Wenger

et

al.

-LSB-

-RSB-

and

Ganz

et

al.

-LSB-

-RSB-

morphological

abnormalities

of

the

femoroacetabular

joint

have

been

closely

examined

as

possible

underlying

sources

of

atraumatic

hip

injuries

in

athletes

Ganz

and

colleagues

described

FAI

resulting

from

structural

abnormalities

of

the

proximal

femoral

neck

-LRB-

cam

-RRB-

acetabular

rim

-LRB-

pincer

-RRB-

or

most

commonly

a

combination

of

the

two

pathologies

-LRB-

mixed

-RRB-

Damage

to

the

articular

cartilage

and

acetabular

labrum

results

from

this

pathologic

bony

contact

and

FAI

is

a

likely

trigger

of

early

hip

joint

degeneration

In

our

experience

FAI

is

a

common

problem

that

has

only

recently

been

recognized

in

athletes

with

a

primary

hip

complaint

An

open

surgical

dislocation

approach

was

first

described

to

treat

hip

impingement

-LSB-

-RSB-

This

technique

showed

good

midterm

results

in

a

general

population

-LSB-

-RSB-

However

increasing

recognition

of

FAI

in

a

high-demand

competitive

athletic

population

encouraged

the

development

of

a

less

invasive

arthroscopic

approach

to

facilitate

prompt

return

to

competitive

play

Various

studies

have

demonstrated

that

hip

arthroscopy

is

a

safe

and

effective

intervention

in

athletes

with

intra-articular

injuries

-LSB-

-RSB-

Philippon

reported

on

a

series

of

ten

elite

athletes

who

underwent

hip

arthroscopy

for

labral

debridement

with

thermal

capsulorrhaphy

-LSB-

-RSB-

All

of

the

athletes

returned

to

high-level

athletic

activities

McCarthy

et

al.

-LSB-

-RSB-

reported

excellent

results

following

hip

arthroscopy

in

elite

athletes

-LRB-

average

follow-up

of

months

-RRB-

Byrd

et

al.

-LSB-

-RSB-

reported

a

series

of

hip

arthroscopies

in

recreational

high

school

collegiate

elite

and

professional

athletes

Post-operative

improvement

as

quantified

by

the

modified

Harris

Hip

Score

was

present

in

all

classes

of

athletes

and

in

athletes

undergoing

any

arthroscopic

procedure

-LRB-

removal

of

loose

bodies

debridement

of

ligamentum

teres

excision

of

osteophyte

labral

excision

microfracture

chondroplasty

-RRB-

Byrd

et

al.

reported

better

results

in

athletes

who

recalled

a

traumatic

onset

to

their

hip

symptoms

when

compared

to

those

with

an

acute

or

insidious

onset

The

authors

suggested

that

an

unaddressed

pre-disposition

to

injury

might

have

had

a

negative

impact

on

self-reported

outcomes

in

athletes

-LSB-

-RSB-

FAI

may

be

the

unaddressed

pre-disposition

to

which

Byrd

and

colleagues

were

alluding

While

these

above

articles

demonstrated

successful

outcomes

following

debridement

procedures

in

athletes

there

are

a

few

caveats

that

must

be

addressed

First

it

is

likely

that

simple

excision

of

damaged

or

diseased

tissue

like

with

meniscal

tears

or

loose

bodies

can

provide

short-term

abatement

of

pain

and

mechanical

symptoms

and

allow

an

athlete

to

return

to

professional

athletics

However

recent

studies

have

shown

that

simple

hip

debridement

procedures

failed

to

address

the

primary

pathology

in

a

vast

majority

of

cases

-LSB-

-RSB-

A

recent

unpublished

study

performed

by

the

senior

author

showed

that

a

major

cause

of

revision

hip

arthroscopy

in

all

patients

was

treatment

of

labral

and

chondral

re-injury

and

decompression

of

previously

unaddressed

FAI

Future

studies

examining

the

clinical

and

radiographic

long-term

outcomes

of

athletes

treated

with

debridement

procedures

need

to

be

compared

to

those

athletes

treated

for

FAI

at

an

early

stage

of

this

disease

The

purpose

of

this

study

was

to

simply

state

that

despite

the

fact

that

FAI

surgery

is

a

more

extensive

procedure

than

debridement

professional

athletes

can

still

return

to

play

following

this

procedure

An

arthroscopic

approach

to

treat

FAI

was

recently

reported

-LSB-

-RSB-

however

early

outcome

studies

are

lacking

The

results

of

this

study

show

that

professional

athletes

with

FAI

can

return

to

high-level

competitive

sport

following

this

procedure

Ninety-three

percent

of

athletes

returned

to

their

previous

level

of

sport

Three

athletes

did

not

return

to

sport

-LRB-

hockey

player

football

player

and

baseball

player

-RRB-

however

all

had

diffuse

hip

osteoarthritis

present

at

the

time

of

arthroscopy

One

patient

-LRB-

a

senior

PGA

golfer

-RRB-

was

able

to

return

to

sport

following

arthroscopy

despite

his

diffuse

osteoarthritis

Based

on

this

information

it

seems

that

arthroscopic

treatment

of

FAI

in

the

presence

of

OA

can

allow

patients

to

return

to

low-impact

however

not

likely

high-impact

professional

sports

A

larger

cohort

of

patients

is

needed

to

test

this

hypothesis

Avascular

necrosis

-LRB-

AVN

-RRB-

and

femoral

neck

fracture

are

recognized

risks

of

osteoplasty

A

recent

study

showed

that

up

to

of

the

femoral

neck

may

be

resected

before

it

is

structurally

compromised

-LSB-

-RSB-

In

this

series

of

patients

no

post-operative

cases

of

AVN

or

femoral

neck

fracture

were

diagnosed

Additional

potential

complications

include

adhesion

formation

at

either

the

capsulolabral

margin

or

between

the

capsule

and

the

site

of

bony

resection

In

this

series

of

athletes

patients

-LRB-

-RRB-

required

revision

arthroscopy

for

the

lysis

of

adhesions

-LRB-

LOA

-RRB-

All

athletes

returned

to

competitive

sport

following

revision

arthroscopy

for

LOA

Two

additional

athletes

had

extensive

OA

at

the

time

of

primary

arthroscopy

and

subsequently

underwent

revision

arthroscopy

for

symptomatic

treatment

of

OA

including

osteophyte

debridement

Both

did

not

return

to

professional

athletics

due

to

the

extent

of

their

disease

There

were

no

reported

cases

of

heterotopic

ossification

or

neurovascular

compromise

including

lateral

femoral

cutaneous

sciatic

and

pudendal

neuropraxia

reported

although

this

statistic

may

have

been

underestimated

due

to

the

nature

of

this

professional

athlete

cohort

A

limitation

of

this

study

was

the

inherent

selection

bias

involved

with

the

study

of

professional

athletes

This

patient

sub-group

was

financially

motivated

to

return

to

play

and

may

have

been

less

likely

to

report

post-operative

symptoms

and

complications

than

the

rest

of

patients

treated

for

FAI

Despite

this

we

believed

that

a

return-to-play

analysis

was

critical

to

assess

the

outcomes

of

FAI

treatment

in

a

high-demand

population

This

cohort

of

professional

athletes

was

debilitated

prior

to

hip

arthroscopy

and

following

intervention

was

able

to

perform

in

physically

intense

professional

sport

activities

Also

the

athletes

precise

reason

for

retirement

and

whether

it

was

related

to

their

hip

injuries

was

difficult

to

discern

For

this

reason

given

the

relatively

short

span

of

all

professional

athletes

careers

we

chose

to

include

all

athletes

at

a

minimum

of

months

post-operative

This

time

period

was

selected

as

an

ample

time

period

for

completion

of

post-operative

rehabilitation

and

return

to

sport

Another

limitation

of

this

study

was

the

lack

of

follow-up

subjective

and

objective

data

Current

outcomes

instruments

-LRB-

modified

Harris

Hip

score

and

the

Non-Arthritic

hip

score

-RRB-

have

not

been

validated

for

use

in

high-level

athletes

It

is

our

belief

that

these

scoring

systems

fail

to

address

the

activities

that

are

most

limiting

to

athletes

and

hence

underestimate

the

degree

of

debilitation

in

professional

athletes

Evaluating

the

applicability

of

current

scoring

systems

in

athletes

and

developing

new

outcomes

instruments

for

athletes

will

be

the

focus

of

future

studies

Other

limitations

of

this

study

included

unequal

distribution

of

gender

-LRB-

males

and

females

-RRB-

and

professional

sports

Because

different

sports

place

different

demands

on

the

hip

we

can

not

draw

conclusions

among

the

various

sports

The

arthroscopic

treatment

of

FAI

represents

the

evolution

of

hip

arthroscopy

In

the

past

soft

tissue

pathologies

were

treated

in

isolation

without

addressing

underlying

impinging

osseous

abnormalities

Recent

developments

particularly

by

Ganz

and

colleagues

have

enlightened

us

to

the

pathology

of

FAI

and

the

associated

treatment

options

In

order

to

treat

high-demand

patients

with

FAI

an

arthroscopic

technique

was

developed

This

study

has

demonstrated

that

full

return

to

professional

competitive

sport

is

possible

following

arthroscopic

treatment

of

FAI

Additional

studies

are

needed

to

determine

the

effect

on

long-term

joint

degeneration

of

early

surgical

intervention

to

treat

FAI

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

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Changes in Glial Cell Line-derived Neurotrophic Factor Expression in the Rostral and Caudal Stumps of the Transected Adult Rat Spinal Cord Limited information is available regarding the role of endogenous Glial cell line-derived neurotrophic factor -LRB- GDNF -RRB- in the spinal cord following transection injury . The present study investigated the possible role of GDNF in injured spinal cords following transection injury -LRB- T9 -- T10 -RRB- in adult rats . The locomotor function recovery of animals by the BBB -LRB- Basso , Beattie , Bresnahan -RRB- scale score showed that hindlimb support and stepping function increased gradually from 7 days post operation -LRB- dpo -RRB- to 21 dpo . However , the locomotion function in the hindlimbs decreased effectively in GDNF-antibody treated rats . GDNF immunoreactivty in neurons in the ventral horn of the rostral stump was stained strongly at 3 and 7 dpo , and in the caudal stump at 14 dpo , while immunostaining in astrocytes was also seen at all time-points after transection injury . Western blot showed that the level of GDNF protein underwent a rapid decrease at 7 dpo in both stumps , and was followed by a partial recovery at a later time-point , when compared with the sham-operated group . GDNF mRNA-positive signals were detected in neurons of the ventral horn , especially in lamina IX . No regenerative fibers from corticospinal tract can be seen in the caudal segment near the injury site using BDA tracing technique . No somatosensory evoked potentials -LRB- SEP -RRB- could be recorded throughout the experimental period as well . These findings suggested that intrinsic GDNF in the spinal cord could play an essential role in neuroplasticity . The mechanism may be that GDNF is involved in the regulation of local circuitry in transected spinal cords of adult rats . Introduction Spinal cord injury -LRB- SCI -RRB- , a common result of car accidents , high-altitude falls and crashes , and other violent injuries is increasing yearly . It has been reported that there are about 15 -- 40 traumatic SCI cases per million persons annually population worldwide -LSB- 1 -RSB- . Once the spinal cord is damaged , it usually results in severe neurologic dysfunction and disability . Over the past several years , many extensive experiments have been done in order to find a way to promote the repair of the injured spinal cord . However , no good therapeutic approaches have yet been found . Recently , it has been shown that delivery of exogenous neurotrophic factors -LRB- NTFs -RRB- to the injured spinal cord has beneficial effects in improving functional recovery -LSB- 2 -RSB- , indicating the potential value of NTFs in the treatment of SCI . Glial cell line-derived neurotrophic factor -LRB- GDNF -RRB- , a distant member of the transforming growth factor-β -LRB- TGF-β -RRB- family , was originally purified using an assay based on its ability to maintain the survival and function of embryonic ventral midbrain dopaminergic neurons in vitro -LSB- 3 -RSB- . GDNF supports neural survival in several neuronal populations , including midbrain dopamine neurons and motoneurons in the central nervous system . It promotes the survival and the differentiation of many peripheral neurons , such as sympathetic , parasympathetic , and sensory neurons -LSB- 3 -- 10 -RSB- . Following injury , GDNF showed a benefit effect to rescue motoneurons degeneration after spinal root avulsion and distal nerve axotomy -LSB- 11 -RSB- , promotion of axonal regeneration -LSB- 12 -RSB- and enhancing regeneration of dorsal roots -LRB- DR -RRB- into the adult rat spinal cord -LSB- 13 -RSB- . In addition , the transplantation of GDNF-producing cells greatly enhances the survival of spinal cord motorneurons -LSB- 11 , 14 , 15 -RSB- and regeneration of several spinal systems , including dorsal column sensory , and regionally projecting propriospinal pathways -LSB- 16 , 17 -RSB- after SCI . The biological action of GDNF is mediated by a two-component receptor GFRα-1 and protein tyrosine kinase Ret -LSB- 18 -- 21 -RSB- . The GFRα-1 receptors expressed on a variety of neurons that project into the spinal cord , including supraspinal neurons , dorsal root ganglia , and local neurons . GDNF mRNAs are widely distributed in a variety of neuronal and non-neuronal tissues of embryos and adults -LSB- 4 , 22 -- 25 -RSB- . Although exogenous GDNF is available partially to promote propriospinal axonal regeneration and locomotion functional recovery , the role of intrinsic GDNF in injured spinal cords is largely unknown . Therefore , the present study investigated the possible role of GDNF at various time intervals following spinal cord transection injury in adult rats , so as to gain insights into the contribution of endogenous GDNF in spinal neuroplasticity . Materials and methods Characterization of antibodies To localize GDNF protein in the spinal cord , an affinity-purified rabbit polyclonal antibody -LRB- D-20 , Santa Cruz Biotechnology , Santa Cruz , CA , USA -RRB- was used in this study . The specificity of antibody for GDNF was confirmed by Western blots using rat spinal cord homogenates . Preparation of probe for in situ hybridization For detection of GDNF cellular expression in situ , we used Digoxigenin-labeled oligonucleotide probe designed by Primer premier 5.0 package which was complementary to the rats GDNF gene sequence -LRB- 33 mer , 5 ′ - GCCCTACTTTGTCAC -- TCACCAGCCTTCTATTTC-3 ′ -RRB- . This antisense DNA single-stranded oligonucleotide probe was synthesized by Takara Biotechnology Company . Animal grouping and surgery Sixty-five adult Sprague-Dawley rats of either sex -LRB- weighing 200 -- 220 g -RRB- were obtained from the Animal Experiment Center of Sichuan University of Medical Sciences . Fifty rats among all animals , designated as sham-operated group and spinal cord transection group that animals were allowed to survive 3 , 7 , 14 and 21 days post operation -LRB- dpo -RRB- , were used for immunohistochemistry and in situ hybridization -LRB- n = 5 in each group -RRB- , and Western blot analysis -LRB- n = 5 in each group -RRB- respectively . Another five rats were intraperitoneally injected anti-GDNF solution -LRB- 2.5 ml/kg -RRB- once every 2 days until 21 dpo , designed as antibody neutralization group . Five animals , subjected to cord transaction , were injected with distilled water as the distilled water group . The last five operated rats were used to investigate corticospinal tract sprouting . Every effort was taken to reduce the number of animals and suffering during the experiments . All the experiments using animals were carried out according to the guidelines of the NIH Guide for Care and Use of Laboratory Animal . For the cord transection operation , the rats were anesthetized with 3.6 % Chloral Hydrate -LRB- 1 ml/100 g -RRB- . A midline incision was made in the back , then the T7 -- T8 spinous processes and the vertebral laminae were removed to expose the spinal cord at T9 -- T10 level . The cord was transected between T9 and T10 segments with a pair of microscissors . After surgery , the superficial back muscles and the skin were sutured along the midline . Behavior tests All rats in each group were evaluated using the BBB -LRB- Basso , Beattie , Bresnahan -RRB- scale score -LSB- 26 -RSB- . The BBB score for evaluation of hindlimbs function after transected injury ranges from zero , which corresponds to flaccid paralysis , to 21 , which is normal gait . Animals were allowed to walk around freely in a circular field -LRB- 1.2 m in diameter -RRB- for 4 min while movements of the hindlimbs were closely observed . Ranking according to the scoring system described by Basso et al. -LSB- 26 -RSB- includes frequency and quality of hindlimb movement as well as forelimb/hindlimb coordination . Measurement of somatosensory evoked potentials -LRB- SEP -RRB- The right peroneal nerve of the rats was dissected and a stimulation electrode was placed on it while a hole -LRB- 3 -- 4 mm in diameter -RRB- was made on the skull -LRB- 2 mm to the left of the midline and 2 mm in front of the fonticulus posterior -RRB- . An electrode was placed on the dura of brain cortex to record the SEP. . The reference electrode was inserted at the nose epithelium . A ground electrode was inserted at the tail . The stimulus intensity was set high enough to produce a marked muscle twitch in the hind limb , about 1.1 mA , the amplitude was 0.2 ms and the frequency 3 Hz . The SEP Tracings represented the average of 200 responses . Immunohistochemistry After anesthesia with 3.6 % Chloral Hydrate -LRB- 1 ml/100 g -RRB- , rats were perfused with 500 ml of cold phosphate-buffered saline -LRB- PBS -RRB- for 5 min and 500 ml cold 4 % paraformaldehyde solution for 30 min . The T9 and T10 spinal cord segments in the sham-operated group , and the cord stumps rostral and caudal to the injury site in the experimental group were harvested . This was followed by a postfix for 6 -- 12 h , then immersed in 0.1 M PBS containing 20 % sucrose overnight . Sections of 20 μm thickness were cut on a freezing microtome and used for freefloating GDNF immunostaining , then rinsed with 0.01 M PBS and soaked in PBS containing 3 % H2O2 for 30 min at room temperature to quench the endogenous peroxidase activity . They were immersed in PBS containing 5 % goat serum and 0.3 % TritonX-100 solution at room temperature for 2 h , then incubated at 4 °C for 24 h with anti-GDNF -LRB- 1:1,000 , Chemicon -RRB- rabbit polyclonal antibody . Sections were incubated with biotinylated goat anti-rabbit IgG antibody -LRB- 1:100 dilution -RRB- for 1.5 h at room temperature , and by reaction with avidin-biotinylated peroxidase complexes -LRB- 1:250 , ABC Elite , Vector Labs -RRB- . GDNF immunoreactivity was visualized with brown staining using diaminobenzidine -LRB- DAB -RRB- and H2O2 as substrate for 5 min , and observed with a light microscope . Negative control experiments in which PBS was substituted for the primary antibody were performed to ascertain the specificity of antibody staining . In situ hybridization Sections from sham-operated rats of 25 μm thickness were also cut on a freezing microtome and used for in situ hybridization . Sections were fixed in 4 % paraformaldehyde in 0.1 M PBS , pH 7.2 -LRB- all treatments were performed at room temperature unless otherwise indicated -RRB- , and further treated with 0.3 % TritonX-100 solution for 10 min and proteinase K -LRB- 5 μg/ml -RRB- at 37 °C for 25 min , refixed with 4 % paraformaldehyde for 5 min , repeatedly immersed in 0.1 M PBS , then acetylated with 0.25 % acetic anhydride in 0.1 M triethanolamine -LRB- pH 8.0 -RRB- to prevent non-specific binding of the probes . The sections were washed with 2 × SSC -LRB- pH 7.0 -RRB- and then prehybridized in a hybridization solution -LRB- 50 % formamide , 10 % dextran sulfate , 1 × Denhardt 's solution , 0.2 mg/ml Herring sperm DNA , and 10 mM dithiothreitol -RRB- without probes at 37 °C for 2 h before hybridization , then hybridized in 100 μl hybridization solution containing 1 μl probes at 37 °C for 12 -- 16 h in a moist chamber . This was followed by washing in decreasing concentrations of SSC , from 4 × SSC -LRB- pH 7.0 -RRB- at 37 °C for 20 min , 2 × SSC -LRB- pH 7.0 -RRB- at 42 °C for 20 min , 1 × SSC -LRB- pH 7.0 -RRB- at 48 °C for 20 min and ending with 0.5 × SSC -LRB- pH 7.0 -RRB- at 50 °C for 20 min . Then sections were incubated at 37 °C in 1 % blocking buffer -LRB- Roche -RRB- for 1 h , subsequently reacted in 1:1,000 sheep anti-digoxygenin-alkaline phosphatase -LRB- AP -RRB- antibody in 1 % blocking buffer at 4 °C overnight . Lastly , AP activity was detected using nitroblue tetrazolium -LRB- NBT -RRB- / 5-bromo-4-chloro-3-indolyl phosphate -LRB- BCIP -RRB- substrate -LRB- Roche -RRB- . The sections were visualized with blue and purple sedimentation then observed with a light microscope . GFAP/GDNF double-label immunohistochemistry Two-color immunohistochemical staining for simultaneous detection of glial fibrillary acidic protein -LRB- GFAP -RRB- / GDNF expression was performed as described above . Briefly , sections were stained with anti-GDNF rabbit polyclonal antibody -LRB- 1:1,000 -RRB- , and using DAB solutions as a substrate . This was followed by second incubation with anti-GFAP rabbit polyclonal antibody -LRB- 1:1,000 , AB5804 , Chemicon -RRB- , and finally , reacted with TMB solutions . Double labeling showed a combination of brown and blue product . Western blotting The spinal cord from the rostral and caudal stumps was obtained . After carefully removing the spinal meninges , the cords were homogenized on ice in a Lysis Buffer containing 0.05 M Tris -- HCl -LRB- pH 7.4 , Amresco -RRB- , 0.5 M EDTA -LRB- Amresco -RRB- , 30 % TritonX-100 -LRB- Amresco -RRB- , NaCl -LRB- Amresco -RRB- , 10 % SDS -LRB- Sigma -RRB- and 1 mM PMSF -LRB- Amresco -RRB- , then centrifuged at 12,000 g for 30 min . The supernatant was obtained and stored at − 80 °C for late use . Protein concentration was assayed with BCA reagent -LRB- Sigma , St. Louis , MO , USA -RRB- . A 20 μl aliquot of the samples was loaded on to each lane and electrophoresed on 12 % SDS-polyacrylamide gel -LRB- SDS-PAGE -RRB- for 2.5 h at a constant voltage of 120 V. Proteins were transferred from the gel to a nitrocellulose membrane for 435 min at 24 V . The membrane was blocked with phosphate-buffered saline containing 0.05 % Tween-20 -LRB- PBST -RRB- with 10 % non-fat dry milk overnight at 4 °C . The membrane was rinsed with PBST and incubated with the primary antibody for GDNF -LRB- 1:1,000 -RRB- at 4 °C . The membrane was incubated with a HRP-conjugated goat anti-rabbit IgG -LRB- 1:5,000 ; Vector Laboratories , CA -RRB- for 2 h at room temperature . The membrane was developed in ECM kit and exposed against X-ray film in a darkroom . Densitometry analysis for the level of GDNF protein was performed by Bio-Gel Imagining system equipped with Genius synaptic gene tool software . β-actin -LRB- the primary antibody , 1:1,000 , the secondary antibody , 1:2,000 ; Santa Cruz Biotechnology -RRB- was used as an internal control . BDA anterograde tracing At 14 dpo , the animals for this part were anesthetized and fixed in a David Kopf Instruments -LRB- Tujunga , CA -RRB- stereotaxic head-holder device . Burr holes were made in the dorsal cranium , and biotinylated dextran amine -LRB- BDA -RRB- -LRB- 10 % BDA solution , Molecular Probes -RRB- was microinjected into eight sites at a depth of 0.7 mm from the cortical surface -LRB- 0.5 μl/site -RRB- to cover the hindlimb region . Animals were then sacrificed 2 weeks later to allow sufficient time for axonal transport of BDA in corticospinal tract . The spinal cords were removed and postfixed at 3 days in cold 4 % paraformaldehyde in 0.1 M PBS -LRB- pH 7.2 -RRB- . Transverse sections -LRB- 30 μm -RRB- of spinal cord at the injury site and neighboring rostral and caudal parts to the injury site were processed for the presence of BDA-labeled axons by incubation in avidin-HRP -LRB- Molecular Probes -RRB- . Lastly , DAB stain was performed to visualize the positive fiber , as brown color staining . GDNF antibody neutralization After 14 dpo , each rat was intraperitoneally injected with 0.5 ml -LRB- 30 mg/ml , 30 mg of anti-GDNF diluted in 1 ml of distilled water -RRB- anti-GDNF solution once every 2 days until 21 dpo . GDNF antibody was the distilled water replace as control in another five rats . The locomotion in hindlimbs by BBB score was evaluated at 3 , 7 , 14 , and 21 dpo . Statistical analysis All data were expressed as the mean ± S.E.M. . They were analyzed using One-way ANOVA and LSD-q test by SPSS software package . The statistical significance was defined as P < 0.05 . Results Behavior tests The BBB score for locomotor function in sham-operated rats hindlimbs was 21 . Compared with the sham-operation group , the BBB score of animals in the group with only the cord transection and distilled water group increased gradually from 7 to 21 dpo . A significant decrease in the BBB score for the GDNF-antibody treated group was observed -LRB- P < 0.05 -RRB- -LRB- Table 1 -RRB- . Table 1Mean values of BBB scores in cord transected rats -LRB- mean ± S.E.M -RRB- Group3 Days7 Days14 Days21 DaysSham-operation group5 ± 0.621 ± 021 ± 021 ± 0Transection group0 ± 00.8 ± 0.32.4 ± 0.73.6 ± 0.5 Distilled water group0 ± 00.6 ± 0.72.0 ± 0.43.3 ± 0.3GDNF-antibody treated group0 ± 00.2 ± 0.40.6 ± 0.30.9 ± 0.5 BBB score in rats subjected to the sham operation was constant . Following transection injury , the BBB score of animals increased significantly from 7 to 21 dpo -LRB- P < 0.05 -RRB- . There had no statistical significance in BBB score between cord transection group and distilled water group -LRB- P > 0.05 -RRB- , while a significant decrease of BBB score in GDNF-antibody treated group was observed -LRB- P < 0.05 -RRB- Somatosensory evoked potentials -LRB- SEP -RRB- Evoked potentials are the electrical signals generated by the nervous system in response to sensory stimuli . Somatosensory evoked potentials consist of a series of waves that reflect sequential activation of neural structures along the somatosensory pathways following electrical stimulation of peripheral nerves . SEP -LRB- P1N1P2 type -RRB- was recorded in the control rats and shown in Fig. 1 . Throughout the experimental period , no SEP could be recorded in rats subjected to cord transection . Fig. 1Somatosensory evoked potential was recorded in normal adult rats -LRB- P1N1P2 type -RRB- . The latency of SEP : P1 -LRB- 10.3 ± 0.26 -RRB- ms , N1 -LRB- 8.81 ± 0.34 -RRB- ms , P2 -LRB- 15.5 ± 0.43 -RRB- ms , amplitude : P1 -LRB- 2.34 ± 0.02 -RRB- , N1 -LRB- 16.3 ± 0.14 -RRB- , P2 -LRB- − 5.06 ± 0.05 -RRB- The GDNF immunostained intensity changes In the control group , GDNF immunoreactivity was observed in the cytoplasm and nuclei of the neurons from the ventral horn in the gray matter -LRB- Fig. 3a -RRB- . Most of the small and medium-sized neurons in the intermediate zone and dorsal horn were stained faintly . GDNF positive profiles -LRB- Fig. 3b -RRB- simultaneously labeled with GFAP , were present in the white matter -LRB- Fig. 2b -RRB- . In the negative control group , no specific immunopositive staining was detected -LRB- Fig. 2a -RRB- . Fig. 2In the negative control group , no specific immunopositive staining was detected -LRB- a -RRB- . GDNF positive profiles , simultaneously label-GFAP , were present in the white matter -LRB- b -RRB- In the rostral stump , strong immunostaining for GDNF was observed in neurons from the ventral horn at 3 and 7 dpo -LRB- Fig. 3c , d ; Table 2 -RRB- . Moderate to intense immunostained neurons were seen at 14 and 21 dpo , as shown in Figs. 3g , 4a and Table 2 . In the white matter , astrocytes showed strong immunoreactivity of GDNF from 3 to 21 dpo -LRB- Figs. 3d -- f , 4b ; Table 2 -RRB- . Fig. 3Under higher magnification -LRB- 400 × -RRB- , GDNF immunoreactivity -LRB- IR -RRB- was observed in neurons from the ventral horn of the gray matter in the normal spinal cord -LRB- a , arrow -RRB- . GDNF -LRB- IR -RRB- in astrocytes was also seen in white matter -LRB- b , arrow -RRB- . In the rostral stump , strong GDNF IR was detected in neurons from the ventral horn at 3 and 7 dpo -LRB- c , e , 400 × , arrows -RRB- . Moderate to intense immunostained neurons were labeled at 14 dpo -LRB- g , 400 × , arrow -RRB- . Intensely immunostained IR in astrocytes of the white matter were observed from 3 to 14 dpo -LRB- d , f , h , 400 × , arrows -RRB- Table 2GDNF immunostaining intensity in neurons and astrocytesNormal 3 Days7 Days14 Days21 DaysNeurons + + + + + + + + + ∼ + + + + + ∼ + + + Rostral part Astrocytes + + + + + + + + + + + + + + Caudal part Neurons + + ∼ + + + + + + + + + Astrocytes + + + + + + + + + + + + Intensity : + weak , + + moderate , + + + strongNeurons in the ventral horn of the rostral stump was strongly stained for GDNF at 3 and 7 dpo , and in the caudal stump at 14 dpo . The GDNF positive staining in astrocytes was also stronger at all time-points after transection injury than seen in control groupFig . 4In the rostral stump , moderate to intense immunostained neurons were labeled at 21 dpo -LRB- a , 400 × , arrow -RRB- . Intensely immunostained IR in astrocytes of the white matter were also observed at 21 dpo -LRB- b , 400 × , arrow -RRB- . In the caudal stump , immunostained products were detected in neurons of the gray matter at 3 and 7 dpo -LRB- c , d , 400 × , arrows -RRB- . Strong immunostained IR for GDNF in neurons was observed at 14 dpo -LRB- e , 400 × , arrow -RRB- . Under higher magnification , immunoreactive structures of GDNF were localized in both cytoplasm and nuclei at 21 dpo -LRB- g , 400 × , arrow -RRB- , GDNF IR in astrocytes of the white matter was intensely immunostained at 14 and 21 dpo -LRB- f , h , 400 × , arrows -RRB- In the gray matter of the caudal stump , moderately immunostained neurons were detected at 3 , 7 and 21 dpo , and more so at 14 dpo -LRB- Fig. 4c -- e , g ; Table 2 -RRB- . The subcellular localization for GDNF was observed both in cytoplasm and nuclei at 21 dpo -LRB- Fig. 4g -RRB- . At both 14 and 21 dpo , astrocytes in the white matter strongly stain with GDNF immunoreactivity as shown in Fig. 4f , h and Table 2 . Localization of GDNF mRNA in spinal cord of rats Signals of hybridization for GDNF mRNA were markedly detected in the Central canal and lamina III , IV and V , in which small neurons were moderately NBT/BCIP stained -LRB- Fig. 5a -RRB- . Medium-sized neurons were seen in the field of the intermediomedial nucleus and nucleus dorsalis -LRB- Fig. 5a -RRB- . In the ventral horn , GDNF mRNA-positive large neurons were also observed in lamina IX -LRB- Fig. 5b -RRB- . A few scattered glial cells were probe-labeled with light density in white matter -LRB- Fig. 5c -RRB- . Fig. 5In the spinal cord of adult rats , GDNF mRNA-positive product were markedly detected in neurons from the lamina III , IV and V and the central canal -LRB- a -RRB- . Medium-sized neurons were seen in the field of intermediomedial nucleus and Nucleus dorsalis . In the ventral horn , GDNF mRNA-positive large neurons were also observed in lamina IX . Under higher magnification -LRB- 200 × -RRB- , positive products with blue and purple staining were localized in the cytoplasm -LRB- b , arrows -RRB- , also in white matter scattered glial cells were probe-labeled with light density -LRB- c , arrows -RRB- Western blotting A single positive band was observed at a molecular weight of about 34,000 , which corresponds to the molecular weight of GDNF . In the rostral stump , the level of GDNF protein increased at 3 dpo , when compared with the control group -LRB- P < 0.05 -RRB- , then decreased quickly to its lowest level at 7 dpo -LRB- P < 0.05 -RRB- which was less than that of the control level , and then began to increase again at 14 dpo . However , GDNF protein was higher than what was seen in the control group at 21 dpo -LRB- Figs. 6a , 7 -RRB- . In the caudal stump , GDNF protein immediately decreased at 3 dpo , reached its lowest level at 7 dpo -LRB- P < 0.05 -RRB- , then increased from 14 to 21 dpo , but it did not go back to the control level -LRB- Figs. 6b , 7 -RRB- . Fig. 6In the rostral stump , GDNF protein level increased at 3 dpo -LRB- P < 0.05 -RRB- , then reached its lowest point at 7 dpo which was below the control group -LRB- P < 0.05 -RRB- , then began to recover at 14 dpo , but it was less than that of normal level . This was followed by a continuous increase higher than the control group at 21 dpo -LRB- a -RRB- . In the caudal stump , GDNF protein decreased at 3 dpo , reached its lowest point at 7 dpo -LRB- P < 0.05 -RRB- , then started to increase at 14 dpo , but it was lower than the normal level at 21 dpo -LRB- b -RRB- Fig. 7GDNF levels were significantly different from each other -LRB- P < 0.05 -RRB- , except for the comparison between the 21 dpo and normal group , as well as the 21 and 3 dpo group in the rostral stump . The groups were significantly different from each other -LRB- P < 0.05 -RRB- , except for the comparison between the 3 and 14 dpo group in the caudal stump BDA tracing BDA-positive axons which stained as brown silkiness were detected in rostral cord sections of the injury site -LRB- Fig. 8a -RRB- , but not in the field of scar tissue -LRB- Fig. 8b -RRB- , and in the caudal segment near the injury site -LRB- Fig. 8c -RRB- . Fig. 8BDA-positive axons staining as brown silkiness were detected in rostral cord sections near the injury site -LRB- a -RRB- , but not in the field of scar tissue -LRB- b -RRB- , and caudal cord sections near the injury site -LRB- c -RRB- Discussion In the present study , GDNF immunoreactive products in neurons from the gray matter and in astrocytes from the white matter of the spinal cord were observed in the sham-operation group . This indicates that GDNF could be involved in either the physiological function or the maintenance of survival for all kinds of neurons and astrocytes . It has been shown that GDNF mRNAs widely distributed in a variety of neuronal and non-neuronal tissues of embryos and adults -LSB- 4 , 23 -- 25 -RSB- . In addition , GDNF mRNA positive signals were found in the ventral horns , the field of nucleus dorsalis , and the dorsal horns in the spinal cords of adult rats -LSB- 2 , 16 , 27 -RSB- and in the dorsal root ganglia in newborn rats -LSB- 23 -RSB- . Therefore , it raised the possibility that GDNF was synthesized by cells of the spinal cord and the GDNF expression may be changed after lesion , which implies that GDNF plays a potential role in both physiological and pathological conditions . Following cord transection , rats exhibited spinal cord dysfunction throughout the experimental period . There was an absence of SEP in surface recordings , indicating a loss of sensory function in the hindlimbs following transection injury . Comparatively , locomotion function recovery was observed from 7 to 21 dpo , which was parallel to the improvement in BBB score . It showed that locomotor functional plasticity in the hindlimbs of rats did occur following SCI . As corticospinal tract regeneration was not seen in the caudal part near the injury site and scar tissues , it appears impossible to reestablish connectivity between the areas rostral and caudal of the injury site . The possible reason for locomotor function improvement may be related to the modulation of spinal circuit activity from endogenous neurotrophins . In the present study , BBB scores in the GDNF-antibody treated rats was inferior to that of the group not receiving the antibody treatment , indicating intrinsic GDNF may be available for improving locomotor function recovery to some extent in the hindlimbs of rats -LSB- 15 , 28 -RSB- . It has been reported that GDNF can promote the repair or survival of spinal motoneurons -LSB- 4 , 7 , 29 -- 32 -RSB- . Several other studies have shown that the administration of GDNF could promote axonal regeneration , and enhance locomotion functional recovery -LSB- 2 , 15 , 30 , 33 -- 36 -RSB- . GDNF immunoreactivity products in neurons from the ventral horn of the rostral stump were strongly stained at 3 and 7 dpo , and in the caudal stump at 14 dpo , suggesting that the GDNF expression in neurons was up-regulated following injury . This may be related to the locomotion functional recovery of hindlimbs . Glazner et al. -LSB- 37 -RSB- showed that strong labeling for GDNFR-α and c-ret mRNA was observed in large neurons from the ventral horn . Our results also supported the mRNA expression for GDNF in neurons of the ventral horn , as well as astrocytes in white matter using in situ hybridization . RET expression was unregulated following peripheral axotomy in alpha-motoneurons -LSB- 38 -RSB- , and topical application of GDNF 30 min after SCI significantly improved motor function and reduced blood -- spinal cord barrier -LRB- BSCB -RRB- breakdown , edema formation , and cell injury at 5 h has been reported -LSB- 39 -RSB- . Therefore , GDNF is available to assist in locomotion functional plasticity by exerting behavioral and anatomic neuroprotection following SCI . In the present study , the expression patterns of endogenous GDNF indicated its possible role in maintaining motorneuron survival because GDNF mRNA-positive products were detected in neurons from the ventral horn by in situ hybridization . Of course , GDNF retrograde axonal transport from a target tissue to neuronal cell bodies -LSB- 40 , 41 -RSB- is not absolutely excluded . It has been reported that GDNF produced by skeletal muscle is taken up at the nerve terminals and retrogradely transported to motoneurons of the ventral horn by axons -LSB- 30 -RSB- . Comparing the results of immunostaining , the GDNF level varied throughout the spinal cord at 3 dpo . In the caudal stump , GDNF showed a significant decrease . This was probably due to the transection injury that triggers a process destructive to ascending and descending tracts conduction and that extends tissue loss . Differently , a rapid up-regulation of GDNF was observed in the rostral stump . This suggested that GDNF had accumulated around the transection site due to interruption of its transport within the axons . Satake et al. -LSB- 42 -RSB- reported GDNF transcription began to increase within 30 min after injury and peaked within 3 h after spinal cord contusion injury . Interestingly , the level of GDNF protein reached its lowest point at 7 dpo in both stumps , probably due to the consequences of cell death or apoptosis and/or interruption of the spinal cord pathways . Subsequently , GDNF protein levels gradually started to recover and surpassed the control level in the rostral stumps at 21 dpo , which may be explained by -LRB- 1 -RRB- GDNF transport within axons was strengthened , -LRB- 2 -RRB- inflammation leads to the increase of GDNF synthesis -LSB- 36 -RSB- , and -LRB- 3 -RRB- induction of de novo synthesis of GDNF by neurons and/or non-neuronal cells . Noticeably , the GDNF positive cells in the white matter were labeled by GFAP simultaneously , which identified the labeled cells as astrocytes . Indeed , an association between reactive astrocytes and regenerating nerve fibers in the white matter of the central nervous system in vivo has been reported -LSB- 43 , 44 -RSB- . Also , increased GFR-1 in astrocytes of degenerating white matter in adult rat spinal cords after mechanical injury has been shown -LSB- 2 -RSB- . These results indicated that GDNF expression in astrocytes may be involved in neuroplasticity following spinal cord transection injury . The present study provides new evidence to understand the role of intrinsic GDNF in the rostral and caudal stumps of injured spinal cords . It suggests that GDNF plays an essential role in the neuroplasticity of the local circuitry , especially in the caudal stump of the transected spinal cords .

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

gdnf
spinal cord transection
immunohistochemistry
in situ hybridization
western blot
antibody
rats